A comparison of the abilities of nitrobenzylthioinosine, dilazep, and dipyridamole to protect human hematopoietic cells from 7-deazaadenosine (tubercidin).

Nitrobenzylthioinosine, dilazep, and dipyridamole are potent inhibitors of equilibrative transport of nucleosides that may have pharmacological applications in modulating the therapeutic index of nucleoside antimetabolites used in cancer chemotherapy. We have compared the relative abilities of these inhibitors to reduce the toxicity of in vitro exposures to tubercidin against clonogenic progenitor cells of normal human bone marrow (CFU-GEMM, BFU-E, CFU-GM) and of two leukemic human cell lines (HL-60/C1, CCRF-CEM) that differ in their expression of transporter subtypes. Short (1-h) exposures to 1 microM tubercidin alone inhibited colony formation (a) of normal human hematopoietic progenitors (CFU-GEMM, BFU-E, CFU-GM) by 100%, and (b) of HL-60/C1 and CCRF-CEM cells by > 90%. Pretreatment (30 min) with nitrobenzylthioinosine, dilazep, or dipyridamole followed by simultaneous treatment (1 h) with these transport inhibitors during tubercidin exposures reduced toxicity against hematopoietic progenitors and cell lines. Greater reductions of toxicity were consistently seen with bone marrow progenitors and CCRF-CEM cells than with HL-60/C1 cells. For CFU-GEMM, BFU-E, and CFU-GM cells, reductions in tubercidin toxicity of 50-100% were achieved at these concentrations: > or = 0.1 microM (nitrobenzylthioinosine); > or = 0.1 microM (dilazep); and > or = 3.0 microM (dipyridamole). Pretreatment (30 min) followed by simultaneous treatment (1 h) with any of the transport inhibitors (> or = 0.1 microM) and 0.1 microM [3H]-tubercidin blocked the uptake of radioactivity completely in CCRF-CEM cells and only partially in HL-60/C1 cells. These effects, which were consistent with the nucleoside transport phenotypes of CCRF-CEM cells (inhibitor-sensitive) and HL-60/C1 cells (inhibitor-sensitive and inhibitor-resistant), suggested that protection was due to the inhibition of tubercidin uptake via equilibrative nucleoside transport system(s). Light-density mononuclear cells from human bone marrow, of which the clonogenic progenitors represented only a minor (< 0.01%) subpopulation, possessed far fewer nitrobenzylthioinosine-binding sites (2 x 10(4) sites/cell, Kd = 0.7 nM) than either HL-60/C1 cells (1.7 x 10(5) sites/cell, Kd = 0.9 nM) or CCRF-CEM cells (3.3 x 10(5) sites/cell, Kd = 0.5 nM). Initial rates of uptake of 1 microM [3H]adenosine (0-6 s, 20 degrees C) by human bone marrow mononuclear cells were reduced partially by 0.1 microM inhibitor (nitrobenzylthioinosine > dipyridamole > dilazep) and completely by 10 microM inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)


Epidermal growth factor receptor monoclonal antibody inhibits constitutive receptor phosphorylation, reduces autonomous growth, and sensitizes androgen-independent prostatic carcinoma cells to tumor necrosis factor alpha.

Results of recent studies indicate that cultured, androgen-independent prostatic carcinoma cells synthesize and secrete transforming growth factor alpha, which interacts with epidermal growth factor receptors (EGFRs) to promote autonomous growth. In the present study, we evaluated the expression and constitutive activation of EGFRs in normal prostatic epithelial cells and the androgen-independent prostatic carcinoma cell lines PC3 and DU145. Our studies showed that cultured normal epithelial cells and androgen-independent prostatic carcinoma cells actively synthesize and exhibit constitutive phosphorylation of the M(r) 170,000 EGFR. The addition of monoclonal anti-EGFR reduced receptor phosphorylation and significantly inhibited the proliferation of prostatic tumor cells. The observed reduction in EGFR phosphorylation could be partially attributed to an antibody-induced decrease in the expression of metabolically labeled EGFR. Results of further studies showed that anti-EGFR enhanced the sensitivity of PC3 cells to the cytotoxic and cytostatic effects of tumor necrosis factor alpha. These studies demonstrate that constitutive activation of EGFR in androgen-independent prostatic carcinoma plays a functional role in the regulation of cellular proliferation in vitro. In addition, the enhanced sensitivity of prostatic carcinoma cells to tumor necrosis factor alpha in the presence of anti-EGFR provides a rationale for the further investigation of combination therapy in the treatment of disseminated, androgen-independent disease.


Time dependence of DNA lesions and growth inhibition by ICI D1694, a new quinazoline antifolate thymidylate synthase inhibitor.

DNA single-strand breaks and associated growth inhibition induced by the thymidylate synthase inhibitor N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazoline-6-ylmethyl)-N -methylamino]-2 - thenoyl)-L-glutamic acid (ICI D1694) were quantitated using the human ileocecal adenocarcinoma cell line, HCT-8. The effects of different concentrations and schedules of [6R,S]-5-formyltetrahydrofolate ([6RS]LV) and 2'-deoxy-thymidine (dThd) on drug growth inhibition and DNA damage were also evaluated. The drug concentrations for 50% inhibition of cell growth in culture following 2-h and 72-h exposures were 0.073 and 0.003 microM, respectively. After a 2-h drug exposure, the occurrence of DNA single-strand breaks (SSBs) was time dependent. It was detectable at 8 h and reached a maximum at about 24 h, 34 +/- 3 (SD) and 305 +/- 34 rad equivalents with 0.1 microM (50% inhibition concentration) and 1.0 microM (90% inhibition concentration) ICI D1694, respectively. A significant level of DNA SSBs (101 +/- 13 rad equivalents) was still detectable at 72 h after the 2-h treatment with 1 microM ICI D1694. No significant level of DNA SSBs was detected when cells were exposed simultaneously to ICI D1694 and 20 microM [6RS]LV. Complete rescue of drug-induced DNA SSBs could be achieved when cells were exposed to 10 microM dThd starting no later than 4 h after drug treatment. The growth inhibition of ICI D1694 was abrogated by [6RS]LV in a concentration-dependent manner. Complete protection was achieved when cells were exposed simultaneously to 1 microM ICI D1694 and 5 microMs [6RS]LV or to 3 microMs dThd immediately after drug treatment. The results demonstrate that: (a) the growth inhibition of ICI D1694 is a function of time and schedule; (b) the growth inhibition is accompanied by extensive DNA single-strand breaks and slow repair; (c) at 1 microM ICI D1694, 3 microMs dThd and 5 microMs [6RS]LV can completely rescue cells from drug effects when dThd is added up to 4 h following drug treatment or when [6RS]LV is given in combination with the drug; (d) interference of [6RS]LV with ICI D1694 action may be occurring at the level of drug uptake and at intracellular targets, while dThd interferes with the drug action at intracellular targets.


Cytotoxicity of CD3-ricin A chain immunotoxins in relation to cellular uptake and degradation kinetics.

The cytotoxicity of WT32 (CD3)-ricin A immunotoxin (IT) to the acute lymphoblastic leukemia T-cell line Jurkat was compared with the rate of internalization and intracellular degradation of WT32 and WT32-ricin A during continuous exposure. Moreover, the influence of NH4Cl and monensin on these processes was studied. Based on protein synthesis inhibition ([3H]leucine incorporation), it appeared that cytotoxicity was not fully expressed directly after exposure to IT due to a delay in either the internalization of membrane-bound IT or the action of intracellular ricin A. Varying the duration of incubation and postponing [3H]leucine addition for up to 24 h after initiation showed that cytotoxicity occurred in two phases, rapid internalization of initially bound IT followed by a continuous but slower uptake, possibly due to reexpression of the CD3 antigen. No differences were found in the rate of internalization and degradation of 125I-labeled WT32 and WT32-ricin A. Internalization started rapidly after binding at 37 degrees C, was fastest during the first 12 h (+/- 360,000 molecules/cell), and continued for at least 24 h (+/- 420,000 molecules/cell). Exocytosis of intracellularly degraded molecules became measurable after 1 to 2 h of incubation at 37 degrees C and increased to approximately 400,000 molecules/cell in 24 h. After 4 h of incubation at 37 degrees C the number of internalized molecules exceeded the amount of WT32 that could maximally bind to the cell membrane (+/- 150,000 molecules/cell), confirming reexpression of antigen. The addition of NH4Cl and monensin enhanced the cytotoxicity of WT32-ricin A, probably due to an increased intracellular amount of IT. These agents appeared to reduce strongly the degradation of internalized WT32, resulting in an accumulation of intracellular molecules. NH4Cl was most effective during the first 12 h of incubation, whereas monensin increased the amount of intracellular WT32 molecules after 2 to 24 h. Our observations suggest that incubation conditions for the optimal cytotoxicity of IT treatment can be predicted by studying the internalization and degradation of the IT or respective monoclonal antibody.


Phase I and endocrine study of exemestane (FCE 24304), a new aromatase inhibitor, in postmenopausal women.

Aromatase inhibitors are a useful therapeutic option in the management of endocrine-dependent advanced breast cancer. A single-dose administration of exemestane (FCE 24304; 6-methylenandrosta-1,4-diene-3,17-dione), a new irreversible aromatase inhibitor, was investigated in 29 healthy postmenopausal female volunteers. The compound, given at p.o. doses of 0.5, 5, 12.5, 25, 50, 200, 400, and 800 mg (n = 3-4), was found to be a well tolerated, potent, long-lasting, and specific inhibitor of estrogen biosynthesis. The minimal dose which produced the maximum suppression of plasma estrogens was 25 mg, reducing plasma estrone, estradiol, and estrone sulfate to 35, 28, and 39% of basal values, respectively. This maximum suppression, observed at 3 days, persisted for at least 5 days after administration of a single dose. However, there was no interference on cortisol, aldosterone, 17-hydroxyprogesterone, or dehydroepiandrostenedione sulfate plasma levels. Peak plasma exemestane concentrations of 27, 221, 343, and 414 ng/ml were reached within 2 h after administration of 50, 200, 400, and 800 mg, respectively. Plasma concentrations declined rapidly and fell under the detection limit (10 ng/ml) at 4 (50 mg) or 24 h (200 and 400 mg). No clinically significant adverse events which could be attributed to the drug were reported. Apart from transient eosinophilia in 3 patients, all biochemical and hematological laboratory parameters were within 1.25-fold of the normal ranges.


Phase II preclinical drug screening in human tumor xenografts: a first European multicenter collaborative study.

In a European joint project carried out in 6 laboratories a disease-oriented program was set up consisting of a panel of 7 tumor types, each represented by 4 to 8 different human tumor lines, for secondary screening of promising anticancer drugs. Human tumor lines were selected on the basis of differences in histology, growth rate, and sensitivity to conventional cytostatic agents. Xenografts were grown s.c. in nude mice, and treatment was started when tumors reached a mean diameter of 6 mm in groups of mice where at least 6 tumors were evaluable. Drugs were given at the maximum tolerated dose. For evaluation of drug efficacy, median tumor growth curves were drawn, and specific growth delay and treated/control x 100% were calculated. Doxorubicin (8 mg/kg i.v. days 1 and 8) was effective (treated/control < 50%, and specific growth delay > 1.0) in 0 of 2 breast cancers, 1 of 3 colorectal cancers, 2 of 5 head and neck cancers, 3 of 6 non-small cell lung cancers, 4 of 6 small cell lung cancers, 0 of 3 melanomas, and 3 of 6 ovarian cancer lines. Amsacrine (8 mg/kg i.v. days 1 and 8) was not effective, while datelliptium (35 mg/kg i.p. days 1 and 8) was active against 2 of 6 small cell lung cancer lines. Brequinar sodium (50 mg/kg i.p. days 1-5) showed efficacy in 4 of 5 head and neck cancers, 5 of 8 non-small cell lung cancers, and 4 of 5 small cell lung cancer lines. The project has been shown to be a feasible approach. Clinical activity for doxorubicin and inactivity for amsacrine against solid tumor types was confirmed in the human tumor xenograft panel. Additional anticancer drugs will be studied in the European joint project to further define the reliability of this novel, promising screening approach.


A murine model for antibody-directed targeting of vascular endothelial cells in solid tumors.

An attractive approach to the therapy of solid tumors would be to target cytotoxic agents or coagulants to the vasculature of the tumor rather than to the tumor cells themselves. This strategy has 3 advantages: (a) it should be applicable to many types of solid tumors because all require a blood supply for survival and growth; (b) the target endothelial cells are directly accessible through the blood and are normal cells, making the outgrowth of resistant mutants unlikely; and (c) there is an in-built amplification mechanism because thousands of tumor cells are reliant on each capillary for nutrients and oxygen. Despite its theoretical attractions, the approach of tumor vascular targeting has not been testable because antibodies that recognize tumor vascular endothelial cell antigens with adequate specificity are currently not available. In this study, we developed a model system in which to investigate the antibody-directed targeting of vascular endothelial cells in solid tumors in mice. A neuroblastoma transfected with the mouse interferon-gamma gene, C1300(Mu gamma), was grown in antibiotic-treated BALB/c nude mice. The interferon-gamma secreted by the tumor induces the expression of major histocompatibility complex Class II antigens on the tumor vascular endothelium. Class II antigens are absent from the vasculature of normal tissues, although they are present on B-lymphocytes, cells of monocyte/macrophage lineage, and some epithelial cells. Anti-Class II antibody administered i.v. strongly stains the tumor vasculature, whereas an antitumor antibody directed against a major histocompatibility complex Class I antigen of the tumor allograft produces classical perivascular tumor cell staining. This model should enable the theoretical superiority of tumor vascular targeting over conventional tumor cell targeting to be tested.


Receptors for interleukin 2 on human squamous cell carcinoma cell lines and tumor in situ.

Several human head and neck squamous carcinoma cell lines were found to bind 125I-labeled or fluorescein-labeled interleukin 2 (IL-2). This binding was inhibited by an excess of cold ligand, IL-2, and by anti-p55 and anti-p70 monoclonal antibodies to the alpha and beta chains, respectively, of the IL-2 receptor (IL-2R). A small number (300/cell) of high-affinity IL-2R (2 x 10(-12) M) and a larger number (> 13,000/cells) of intermediate-affinity IL-2R (3 x 10(-10) M) were present on these tumor cells. By affinity cross-linking, tumor cells were shown to bind 125I-IL-2 to a M(r) 66,000 and 55,000 doublet peptide. The alpha and beta chains of the IL-2R also were detected on the surface of cultured tumor cells using the relevant monoclonal antibodies and flow cytometry. Immunoperoxidase staining with anti-p70 monoclonal antibody confirmed the expression of IL-2R on squamous cell carcinomas of the head and neck in situ. The presence of transcripts for p55/IL-2R-alpha and p70/IL-2R-beta in PCI-1 cells was confirmed by the polymerase chain reaction followed by hybridization to the IL-2R-alpha complementary DNA probe or IL-2R-beta complementary DNA probe, respectively. Our observations demonstrate that intermediate-affinity and high-affinity IL-2Rs are expressed on some human squamous cell carcinomas of the head and neck and that the receptors are functional, because growth of these tumor cell lines can be directly inhibited by exogenously supplied IL-2. The presence of IL-2R on human solid tumors could be important to consider, in addition to immunomodulatory effects of IL-2, in developing optimal therapeutic strategies for the administration of IL-2 to patients with cancer.


Mucin gene expression in colonic tissues and cell lines.

Complementary DNA clones encoding four different mucin core peptides have been isolated. However, the expression of these mucin genes in the colon has not been systematically studied. The present investigation used Northern blot analysis to study the expression of MUC1, MUC2, MUC3, and MUC4 mRNA in paired normal and cancerous colonic tissues, and nine colon cancer cell lines. Results were correlated with the clinicopathological features of the tumors and with the immunohistochemical expression of several carbohydrate tumor-associated antigens that may reside on mucins. MUC1 mRNA was expressed in all colonic tissues, and levels in paired normal and cancer tissues were similar in most cases. MUC2 and MUC3 mRNAs were expressed in both normal and cancer tissues, but levels were often decreased in the cancers. MUC4 mRNA was present in normal mucosa with comparable or sometimes greater expression in cancers. There was no apparent correlation between the expression of any particular mucin gene or pattern of mucin genes and the site, stage, or histological type of tumor. In addition, the expression of mucin-associated carbohydrate antigens did not correlate with any individual mucin gene or group of mucin genes. In colon cancer cell lines all four MUC genes were expressed rather weakly or not at all. These results indicate that the human colon expresses a broad repertoire of mucin genes which are differentially regulated in malignancy. Whether this differential regulation of mucin genes affect the behavior of the tumor and results in the altered glycosylation commonly seen in these requires further investigation.


Differential expression of ras protooncogenes during in vitro differentiation of human erythroleukemia cells.

We have compared the expression of the ras protooncogene family (H-, K-, and N-ras) in leukemia cell differentiation utilizing as a model K562 and HEL erythroleukemia cells treated either with 1-beta-arabinofuranosylcytosine or 12-O-tetradecanoylphorbol-13-acetate (TPA). 1-beta-D-Arabinofuranosylcytosine induced terminal erythroid differentiation of K562 cells, while TPA induced myeloid differentiation of K562 and HEL cells, resulting in myelomonocytic-like cells expressing macrophagic and megakaryocytic markers. H-ras mRNA levels showed a dramatic decrease in K562 cells subjected to erythroid and myelomonocytic differentiation. The same result was found at the protein level for p21H-ras. Expression of K-ras and N-ras in K562 cells also decreased with differentiation, although significant mRNA levels remained despite cessation of cell proliferation. The decrease in K-ras expression was greater for TPA-treated cells than for 1-beta-arabinofuranosylcytosine-treated cells. TPA-induced myelomonocytic differentiation in HEL cells also resulted in a dramatic down-regulation of H-ras mRNA levels. Thus, by using a leukemia cell line able to differentiate along two different lineages, our results reveal a lineage-specific modulation of ras gene family expression.


Frequent p53 mutations in head and neck cancer.

Squamous cell carcinomas of the head and neck (SCCHN) are associated strongly with the use of tobacco and alcohol, but little is known about the molecular pathogenesis of these tumors. In the present study, we analyzed SCCHN for mutations in the tumor suppressor gene p53 by immunocytochemistry and complementary DNA sequencing. Overexpression of p53 protein was detected in 13 (100%) of 13 SCCHN cell lines and in tumor cells cultured directly from 10 (77%) of 13 patients with SCCHN. Direct evidence for p53 mutations was obtained by sequencing p53 complementary DNA from eight SCCHN cell lines and two tumor xenografts. The genetic alterations included seven missense mutations resulting in single amino acid substitutions, a mutation encoding a stop codon, one 10-base pair deletion, and one 2-base pair addition. All seven missense mutations were G to T transversions, five of which were clustered at codons 245 and 248. A similar high frequency of G to T transversions predominates in lung cancer, another tobacco-related disease. Mutation of the p53 gene is the most common genetic alteration detected in SCCHN and implicates this gene locus as a critical site of specific damage by mutagenic carcinogens in tobacco, one of the important risk factors in the etiology of this disease.


2H-nuclear magnetic resonance imaging of tumor blood flow: spatial and temporal heterogeneity in a tissue-isolated mammary adenocarcinoma.

2H-Nuclear magnetic resonance imaging of deuteron accumulation in tissue following an i.v. bolus of deuterium oxide provides a noninvasive means of constructing maps of tissue perfusion. With a measured arterial input function and a simple model for tissue-capillary exchange, these data can provide quantitative estimates of local flow. This technique was tested in rat brain and then applied to the study of spatial heterogeneity and temporal variation of blood flow in the tissue-isolated R3230AC mammary adenocarcinoma. Global flow from the brain averaged 0.96 ml/min.g, in good agreement with results obtained from other methods; the perfusion of brain was relatively homogeneous. Global tumor blood flow averaged 0.32 ml/min.g, ranging from 0.11 to 0.96 ml/min.g. Imaging revealed variations in perfusion both within and between the tumors that far exceeded those expected from brain flow heterogeneity and uncertainty in the flow estimates. By obtaining repeated flow images at 30-min intervals, it was possible to show that the regional blood flow shifted with time in single pixels and in multipixel regions. These experiments show that 2H-nuclear magnetic resonance may be useful in obtaining noninvasive and quantitative measurement of temporal blood flow changes in a solid tumor in vivo.


Fibrosarcoma cells transduced with the IL-6 gene exhibited reduced tumorigenicity, increased immunogenicity, and decreased metastatic potential.

Murine fibrosarcoma cell lines transduced with retroviral vectors containing the murine interleukin 6 (IL-6) gene constitutively secreted IL-6. When injected s.c. into normal mice these IL-6-secreting tumors exhibited reduced tumorigenicity. This reduced tumorigenicity was not seen in nude or irradiated mice, implicating a T-cell-dependent, radiosensitive host response activated by the cytokine. Subcutaneous IL-6-secreting tumor did not retard the growth of distant deposits of wild-type tumor in the same host. However, animals rejecting IL-6-secreting tumors exhibited resistance to later challenge with wild-type tumor. When injected i.v. in an experimental metastasis model the IL-6-secreting tumors failed to or were extremely inefficient in giving rise to pulmonary nodules; this was observed in both normal and immunoincompetent mice, implicating a second, nonimmune mechanism affecting the growth of the tumor modified to secrete IL-6.


Targeted therapy of athymic mice bearing GW-39 human colonic cancer micrometastases with 131I-labeled monoclonal antibodies.

The therapeutic potential of radiolabeled antibodies is usually evaluated in experimental animal models bearing s.c. xenografts. We have established a micrometastatic model of the GW-39 human colonic carcinoma in the nude mouse lung (J. Natl. Cancer Inst., 83: 627-632, 1991) and presented preliminary findings on the efficacy of a 131I-anticarcinoembryonic antigen (CEA) antibody in this model. We now extend our observations on the use of radioiodinated labeled monoclonal antibodies (MAbs) to treat multiple small tumor nodules. Biodistribution and dosimetry analysis was performed for intact and F(ab')2 of NP-4 anti-CEA IgG, Mu-9 anti-colon-specific antigen IgG, isotype-matched irrelevant anti-AFP IgG, and intact MAb 34A anti-lung endothelial IgG antibody. Comparisons were made for rad dose delivered to small s.c. tumors, normal lung, lung with tumor nodules, and isolated tumor nodules. Survival curves were generated for tumor-bearing animals treated 1, 7, or 14 days after tumor cell implantation with these antibodies using the maximal tolerated dose for intact antibodies (275 microCi) and for F(ab')2 fragments (1.2 mCi). The studies established the following observations: (a) in contrast to previous results in a bulky tumor model in hamsters, intact antibodies are more therapeutic than MAb fragments for both NP-4 and Mu-9; (b) tumor nodule size, even on the microscopic level, affects therapeutic outcome; antibodies were more effective when administered 7 days postimplantation (mean nodule diameter, 150 microns) compared with treatment 14 days postimplantation (mean nodule diameter, 750 microns); (c) administration of radioiodinated Mu-9 was exquisitely effective on single avascular tumor cells that had seeded in lung; irrelevant antibody was minimally radiotoxic; (d) as in the bulky disease model, the anti-colon-specific antigen p antibody delivers a higher rad dose than the anti-CEA antibody and is significantly more therapeutic in the micrometastasis model; (e) a higher affinity anti-CEA antibody (MN-14) recognizing the same epitope on CEA as NP-4 was equally therapeutic; (f) the use of MAb directed against the lung endothelium was not as therapeutic as a tumor-associated antibody; and (g) all tumor-associated antibodies were more efficacious than administration of the maximal tolerated dose of 5-fluorouracil and leucovorin in this human tumor-xenograft model. These results provide further support for the use of radioimmunotherapy in the handling of minimal disease, probably as part of an adjuvant treatment regimen.


Membrane peroxidative damage enhancement by the ether lipid class of antineoplastic agents.

The ether lipid antineoplastic agents have no known interaction with DNA, but rather they appear to target membranes. The primary mechanism of action is unknown but effects on membrane biology are documented. We have studied the effect of two ether lipids on membrane lipids and examined the hypothesis that membrane peroxidative damage may be involved in their mechanism of action. With the use of cells having membranes enriched in polyunsaturated fatty acids of the omega-3 family of fatty acids, we have demonstrated that the prototypical ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine and a thioether lipid analogue, 1-O-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine , increase membrane lipid peroxidation and cytotoxicity in a time- and drug concentration-dependent manner. The oxidative cofactors Fe2+ and ascorbic acid were required. The pattern of cell death did not fully correspond to the peroxidation, since cofactors were required for peroxidation but not cytotoxicity. However, the rate of decrease in cell viability after exposure to the drug and cofactors corresponded to the peroxidation rate. In addition, when L1210 cells modified with the monounsaturated fatty acid oleic acid or unmodified cells were used, there was no ether lipid-enhanced peroxidation, and the cells were significantly less sensitive to the drug, with or without cofactors. The lipid-soluble antioxidant vitamin E inhibited 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine peroxidation and cytotoxicity in a concentration-dependent manner in the presence of cofactors but not consistently without them. Depletion of cellular glutathione content of L1210 cells using L-buthionine-(SR)-sulfoximine resulted in 40% augmentation of cofactor-facilitated cytotoxicity of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine and a borderline effect on peroxidation. Another ether lipid, the thio compound 1-O-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine , enhanced peroxidation in the presence of cofactors with kinetics corresponding to those of cytotoxicity. In the presence of ether lipid and cofactors the intensity of ascorbate free radical increased, consistent with oxidative stress. We conclude that the ether lipids stimulate membrane lipid peroxidation in a time- and drug concentration-dependent manner in the presence of oxidative cofactors. Even though peroxidation may not fully explain the cytotoxic effect of the ether lipid class of anticancer drugs, this observation provides further information on the nature of the membrane damage induced by the drugs. Since the ether lipids generate no known free radical intermediates directly, this suggests that membrane damage indirectly results in a process involving a peroxidative reaction.


Identification of the cross-link between human O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea-treated DNA.

Chloroethylnitrosoureas induce reactive O6-guanine adducts in DNA that can form either interstrand cross-links or a covalent complex with the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). To test our hypothesis that these end-products are formed from the common precursor, 1-O6-ethanoguanine, we compared the kinetics of interstrand cross-link formation with those of decay of MGMT complex forming capacity. The half-lives of these processes were identical. Our hypothesis also predicts that the linkage between DNA and MGMT is 1-(guan-1-yl)-2-(cystein-S-yl)ethane. This notion was tested by forming the complex with 35S-labeled recombinant human MGMT and a chloroethylnitrosourea-treated oligodeoxynucleotide. After degradation by depurination and proteolytic digestion, the identity of the [35S]cysteine-guanine linkage was confirmed by comparison with the synthetic marker compound using high performance liquid chromatography and UV spectrometry. These results strengthen the hypothesis that DNA interstrand cross-links and DNA-MGMT complex both arise from the same precursor. The data also suggest that 1-O6-ethanoguanine is a good substrate for MGMT such that, under certain conditions in vivo, DNA-MGMT complex formation may constitute a significant secondary lesion.


Interleukin 4 receptor expression and growth inhibition of gastric carcinoma cells by interleukin 4.

The expression of the interleukin 4 (IL-4) receptor (IL-4R) and effects of human recombinant IL-4 on human gastric carcinoma cell lines were studied. We demonstrated that IL-4 inhibited the growth of gastric carcinoma cells in a dose dependent manner (0.1-100 units/ml) in a [3H]thymidine incorporation proliferation assay. The gastric carcinoma cells varied in sensitivity to treatment with low dose IL-4. Treatment of cells with IL-4 altered the morphology of the cells to a "flattened" morphological shape resembling differentiation. The IL-4-mediated growth inhibition was significantly abrogated by neutralization of IL-4 with specific anti-IL-4 antibody. IL-4R expression on the cell surface was determined by assessing biotin-labeled IL-4 binding to cells using flow cytometry. IL-4R expression ranged from 5 to 85% of total cell population in the gastric carcinoma cell lines assessed. There was a positive correlation between the sensitivity to IL-4-mediated growth inhibition and IL-4R expression. By Northern blot analysis, we demonstrated that mRNA of IL-4R was expressed in the gastric carcinoma cells. Using in situ hybridization, we confirmed that IL-4R mRNA was expressed in the gastric carcinoma cell at the single cell level. By using a sensitive polymerase chain reaction technique, we demonstrated that gastric carcinoma cells expressed IL-4 mRNA, suggesting a possible autocrine loop. These studies indicate that IL-4 can significantly modulate gastric carcinoma cells that possess IL-4R. IL-4R on gastric carcinoma cells may be a potential therapeutic target site for IL-4-directed therapy.


Magnetic resonance diffusion imaging detects structural damage in biological tissues upon hyperthermia.

The use of quantitative nuclear magnetic resonance (MR) imaging to investigate the extent and mechanism of hyperthermic damage in biological tissues has been studied. By using the multiple delay-multiple echo and pulsed-gradient spin echo MR imaging sequences, multiple frame MR images of freshly harvested rabbit tissues (brain, kidney, and muscle) and intact duck embryos in shells were obtained before and after heat treatment (45 degrees C for 30 min) using a clinical 1.5-Tesla whole-body superconducting MR scanner. Based on the relaxation and diffusion models, maps of the proton spin density, relaxation times, and various self-diffusion parameters of tissue water were generated from these multiple frame MR images. Our results indicated that the values of the diffusion barrier size and fractal parameter of the tissues and the self-diffusion coefficient of tissue water increased significantly, i.e., approached that of free water, after the heat treatment. In comparison, only slight changes in the spin density and relaxation times of the tissue water were found after the identical heat treatment. We concluded that the significant changes in the self-diffusive behavior of the tissue water are due to the denaturation of macromolecules (e.g., protein and fiber) within the tissues at elevated temperatures. We further suggested that MR diffusion imaging represents a powerful tool to investigate the extent and mechanism of heat damage of biological tissues in vivo and therefore bears important potential in the clinical assessment of the therapeutic efficacy of hyperthermia in cancer therapy.


Somatostatin receptors in human renal cell carcinomas.

The presence of somatostatin receptors was evaluated in samples of 39 surgically removed human renal cell carcinomas with receptor autoradiography on tumor sections by using iodinated [Tyr3]octreotide as the radioligand. All types, grades and stages of tumors were represented. Twenty-eight of 39 renal cell carcinomas (72%) were shown to be somatostatin receptor positive. The receptors were saturable, of high affinity (KD = 0.8 nM), and were specific for somatostatin and bioactive somatostatin analogues. No evident correlations were found between the status of somatostatin receptors in the tumor and the age or sex of the patients, the histopathological type or grade of the tumor, or the tumor-node-metastasis stage of the disease. However, numerous cases considered to be of poor prognosis were somatostatin receptor positive. No functional correlates for these receptors have been established, although the presence of somatostatin receptors in human kidneys and somatostatin effects on renal tubular functions in normal human volunteers have been reported. In a patient scanned in vivo for islet cell carcinoma with an 123I-labeled somatostatin analogue, bilateral renal cell carcinomas were also visualized; multiple bilateral renal cell carcinomas were identified on the 1- and 4-h images taken after injection of 123I-labeled somatostatin analogue. In conclusion, the high incidence of somatostatin receptors in renal cell carcinomas may have diagnostic value when performing in vivo imaging of somatostatin receptors and it may have potential therapeutic implications.


Deletions of 17p and p53 mutations in preneoplastic lesions of the lung.

Cytogenetic and p53 mutation analysis in two cases of severe dysplasia of the bronchial epithelium in lung cancer patients and p53 immunostaining in a third one are reported. The finding of both chromosomal deletions of 17p and p53 mutation indicates that these changes may take place early in the process of lung carcinogenesis.


The RCK gene associated with t(11;14) translocation is distinct from the MLL/ALL-1 gene with t(4;11) and t(11;19) translocations.

We previously demonstrated that the 11q23 breakpoint region, designated the RCK locus, of the RC-K8 B-lymphoma cell line with t(11;14)(q23;q32) is centromeric to PBGD, while breakpoints of infantile leukemia cell lines with t(11;19)(q23;p13) are detectable by pulsed-field gel electrophoresis with the CD3D probe. In the present study, using a probe within 1.0 kilobase of the t(11;14) breakpoint, we isolated a partial complementary DNA clone for the putative RCK gene, which detects a 7.5-kilobase mRNA. Sequence analysis predicted a novel protein of 472 amino acids which demonstrated sequence homology to a translation initiation factor/helicase family. We also isolated a phage clone from the CD3D/G yeast artificial chromosome clone (yB22B2) which detects 11- and 12-kilobase mRNAs, most likely for the MLL/ALL-1 gene associated t(4;11)(q21;q23) and t(11;19)(q23;p13) translocations. By pulsed-field gel electrophoresis after NotI digestion, this recombinant clone is on a 96-kilobase fragment, while RCK and PBGD probes are on a more telomeric 690-kilobase NotI fragment. These results, altogether, suggested that two different genes, RCK and MLL/ALL-1, are associated with 11q23 translocation of hematopoietic tumors.


p53 mutation and protein accumulation during multistage human esophageal carcinogenesis.

Preinvasive lesions of squamous cell carcinoma are well defined morphologically and provide a model for multistage carcinogenesis. Since alterations in the p53 tumor suppressor gene occur frequently in invasive esophageal squamous cell carcinoma, we examined a set of preinvasive lesions to investigate the timing of p53 mutation. Surgically resected tissues from nine patients with esophageal squamous cell carcinoma contained precursor lesions which had not yet invaded normal tissues. Immunohistochemistry showed high levels of p53 protein in both preinvasive lesions and invasive carcinomas in six cases; sequence analysis of all invasive tumors identified p53 missense mutations in two cases. Preinvasive lesions from both tumors with mutations plus one wild-type tumor were microdissected and sequenced. In one patient there were different mutations in the invasive carcinoma (codon 282, CGGarg > TGGtrp) and a preinvasive lesion (codon 272, GTGval > T/GTGleu/val). In a second case, an invasive carcinoma had a mutation in codon 175 (CGCarg > CAChis), and adjacent preinvasive lesions contained a wild-type sequence. A carcinoma and preinvasive lesion from the third case contained high levels of protein and a wild-type DNA sequence. Therefore, p53 mutation may precede invasion in esophageal carcinogenesis, and multifocal esophageal neoplasms may arise from independent clones of transformed cells. The timing of p53 protein accumulation is favorable for an intermediate biomarker in multistage esophageal carcinogenesis.


Prognostic value of urokinase-type plasminogen activator in 671 primary breast cancer patients.

Urokinase-type plasminogen activator (uPA) may be responsible for the invasive and metastasizing capacity of tumor cells. Evidence has been presented that primary breast cancer patients with tumors containing high levels of uPA experience a worse prognosis. In the present study we have assessed uPA status in routinely prepared cytosols of 671 primary human breast tumors and have evaluated its association with disease-free and overall survival. Isotonic regression analysis with length of disease-free survival as an end point revealed 1.15 ng/mg protein as the best cutoff point to discriminate between uPA positive (32% of the tumors) and uPA negative. In both Cox univariate and multivariate regression analysis (including also patient's age, menopausal status, lymph node status, and the number of positive lymph nodes, tumor size, and estrogen and progesterone receptor status), uPA positivity was significantly associated with increased rates of relapse and death. Corrected for all relevant factors in multivariate analyses for subgroups of patients, uPA positivity was significantly associated with an increased relapse rate in the subgroups of node-negative (P = 0.002; relative failure rate, 2.33), node-positive (P < 0.0001; relative failure rate, 1.95), postmenopausal (P < 0.0001; relative failure rate, 2.59), and steroid receptor-positive patients (P < 0.0001, relative failure rate, 2.76). We conclude that uPA positivity of human primary breast tumors is an important independent variable for the identification of patients at high risk for recurrence, also in clinically important subgroups of patients.


Immunogenetic influences on the initiation stage of the cutaneous chemical carcinogenesis pathway.

While it is generally agreed that environmental exposure to solar radiation and to certain classes of chemicals are the major causes of nonmelanoma skin cancer, it is also believed that genetic polymorphisms regulating immunological responses are important determinants of individual susceptibility to skin cancer. However, little is known about their interactions with the chemical carcinogenesis pathway prior to the actual development of tumors. This issue was examined by comparing susceptibility to skin cancer in C3H/HeN and C3H/HeJ mice, two strains that differ only at the lipopolysaccharide genetic locus, which serves as a regulator of a number of immunological activities. When subjected to a two-stage cutaneous tumorigenesis protocol, C3H/HeJ mice, which have a mutation at the lipopolysaccharide genetic locus that renders them deficient in their capacity to produce cytokines and to activate macrophages, developed nearly three times as many tumors as did C3H/HeN mice, which do not have this mutation. Epidermal DNA binding of 7,12-[3H]dimethylbenz(alpha)anthracene, an index of tumor initiation, was also significantly greater in C3H/HeJ than in C3H/HeN mice. Immunological activities regulated by the lipopolysaccharide genetic locus thus confer resistance to DMBA-induced cutaneous tumorigenesis in mice and are associated with changes that occur early in the tumorigenesis pathway, prior to the development of tumors.


p53 mutations in formaldehyde-induced nasal squamous cell carcinomas in rats.

Formaldehyde induces squamous cell carcinomas in the nasal passages of rats following chronic inhalation exposure at concentrations of > or = 10 ppm. We have examined the complementary DNA of the tumor suppressor gene p53 from 11 primary formaldehyde-induced tumors for mutation using DNA sequence analysis. A polymerase chain reaction-amplified fragment of the rat p53 complementary DNA containing the evolutionarily conserved regions II-V was directly sequenced from each tumor. Point mutations in the p53 complementary DNA sequence were found in 5 of 11 of the tumors analyzed. These data demonstrate p53 point mutations in formaldehyde-induced squamous cell carcinomas and indicate a common alteration in certain rat and human squamous cell carcinomas of the respiratory tract.


Chemotherapy of malignant brain tumors in children.

Chemotherapy has become an important modality in the management of children with brain tumors. Factors that impede improved effectiveness of anti-neoplastic drugs are being identified and addressed. Specific agents have emerged that show anti-brain tumor activity alone or in combination therapy. Use of these agents requires facility regarding their dosing, routes of delivery, mechanisms of action, metabolism and toxicities. Multi-institution cooperative group trials provide the optimum means by which to evaluate the effectiveness of chemotherapy in children with brain tumors.


The neurobiology of the opsoclonus-myoclonus syndrome.

Opsoclonus-myoclonus is a pervasive neurological syndrome of children and adults. Although rare, it raises important clinical and neurobiological issues. This article provides an overview of the clinical and laboratory features, differential diagnosis, treatment, and outcome of opsoclonus-myoclonus. It pursues immunologic, genetic, electrophysiologic, neurochemical, and other clues to a pharmacologic model. Key questions include how and where the brain is injured, reversibility of the injury, possible targets for pharmacologic intervention, and which new studies are needed.


Effect of a macrolide (spiramycin) on the pharmacokinetics of L-dopa and carbidopa in healthy volunteers.

In one well-equilibrated parkinsonian patient treated with combined L-dopa and carbidopa (Sinemet), we have observed changes in treatment efficacy while receiving spiramycin (Rovamycine) for an intercurrent respiratory infection. A preliminary study of the pharmacokinetics of L-dopa and its main metabolites 3-O-methyldopa (3-OMD) and dihydroxyphenylacetic acid (dopac) in two parkinsonian patients treated with Sinemet has revealed a marked decrease in the AUC0-360 of these two metabolites after a 3-day course of Rovamycine. In order to confirm this interaction, we have studied the modifications of the pharmacokinetics of L-dopa, 3-OMD, dopac, and carbidopa in eight male healthy volunteers after a single dose of Sinemet 250 (L-dopa, 250 mg and carbidopa, 25 mg) before and after a 3-day course of Rovamycine. Our study confirms this interaction. After spiramycin, we observed a marked reduction in AUC0-360 for L-dopa (p less than 0.001), 3-OMD (p less than 0.001), and carbidopa (p less than 0.001), and an increase in AUC0-360 for dopac (p less than 0.01). The L-dopa elimination half-life was increased (p less than 0.012); differences in peak plasma concentrations did not attain statistical significance. We think that these modifications in L-dopa pharmacokinetics after spiramycin are due to nonabsorption of carbidopa secondary to modified gastrointestinal motility.


Hypotensive and bradycardiac responses to thyrotropin-releasing hormone in a comatose patient.

A 46-year-old female motorcyclist, who suffered injuries to the brain stem in a traffic accident, showed hypotensive and bradycardiac responses to thyrotropin-releasing hormone (TRH) given to counter consciousness disturbance. The cardiodepressive responses to TRH were reduced with i.v. pretreatment with atropine sulfate, suggesting an involvement of the vagal nervous system in the development of the responses. Furthermore, this patient had complicated impairments in the sympathetic nervous system, which were revealed by the results of testing baroreceptor reflex sensitivity to pharmacological alterations in blood pressure. We thus speculate that the hypotensive and bradycardiac effects of TRH observed in this patient may result from derangements of the sympathetic nervous system caused by the injuries. This case report is believed to be a novel description of the cardiodepressive effects of TRH.


Cerebral amyloid angiopathy with granulomatous angiitis ameliorated by steroid-cytoxan treatment.

We report a case of a 62-year-old black woman who, 8 months prior to death, developed confusion, apraxias, disorientation, and difficulties with her vision. There was no dementia. Computed tomography (CT) scan and magnetic resonance imaging (MRI) suggested a tumor in the right posterior parietal white matter. A biopsy of the lesion displayed granulomatous angiitis and severe cerebrovascular amyloidosis, but no tumor was identified. Chronic inflammation with an occasional multinucleated giant cell was seen about the amyloid-infiltrated vessels. The cortex demonstrated gliosis but no plaques or tangles. Subsequently, the patient was treated with steroids and Cytoxan, with an improvement in her neurologic status. She died of opportunistic bronchopneumonia 8 months after the initial onset of her symptoms. On postmortem examination, the biopsied area of the brain showed atrophy with gliosis. Amyloid angiopathy was present but in much lesser degree than in the biopsy. Scant perivascular inflammatory infiltrates were seen only focally, and no giant cells were observed. The amyloid, both in the biopsy and autopsy material, was of the Alzheimer A4 type. This case suggests that steroid and cytoxan treatment ameliorated the angiitis and the amyloid angiopathy as well. The pertinent literature is discussed.


Electrical stimulation and multichannel EMG recording for identification of functional neural tissue during cauda equina surgery.

Electrical stimulation of structures within the surgical field was used to identify functional neural elements during 25 cauda equina operations. EMG responses from anterior thigh, posterior thigh, and anal sphincter muscles were recorded simultaneously using a multichannel signal averager. During nine operations, stimulation of a presumed filum terminale or other tissue produced clear EMG responses, prompting modification of surgical procedures. In one patient, this resulted in preservation of a flattened spinal cord which resembled a band of scar tissue. Some EMG responses were restricted to a single muscle group; these neural structures would probably not have been identified if only a single-channel EMG recording was used. Visual examination alone was not adequate for identifying functional neural elements, or for determining whether atretic-appearing nerve roots were functional. Electrical stimulation with multichannel EMG recording facilitates the preservation of functional neural elements and the optimization of surgical results in cauda equina surgery.


Neurological outcome following neonatal post-haemorrhagic hydrocephalus: the effects of maximum raised intracranial pressure and ventriculo-peritoneal shunting.

The neuromotor outcome of 33 survivors of grade 3 or 4 neonatal post-haemorrhagic hydrocephalus born between 1975 and 1988 was assessed at a mean age of 4.7 years (9 months to 13 years). Two outcomes were determined: 12 patients were either normal (10 or had neurological signs without functional impairments (2), while 21/33 were moderately (16), severely (2), or profoundly impaired (3). Intracranial pressure (ICP) was measured in 26/33 patients (4-40 mm Hg): 2 had normal pressures (< 5.6 mm Hg) and were normal. Raised ICP was not significantly different between outcome groups. Twenty-seven children were shunted; 10/27 had five or more operations (up to 14) and all of these had abnormal neurological outcomes, whereas the number of children with 1-4 shunt procedures was equal in both outcome groups. The rise in morbidity after the fourth shunt procedure may be associated with the ventriculitis suffered by 9 of the 10 patients with more than four shunts (P < 0.01): this compares with 4/14 cases of ventriculitis in the children with 2-4 shunts and no cases of infection in the 3/27 who were shunted once. Outcome was independent of antenatal and perinatal factors including the age at or mode of presentation, and was unrelated to grade of intraventricular haemorrhage or parenchymal changes on ultrasound or CT scanning. for these small numbers, adverse outcome is statistically related to more than four shunt procedures and ventriculitis but independent of maximum ICP or other perinatal factors.


Surgical management of posthemorrhagic hydrocephalus in 22 low-birth-weight infants.

In order to assess the complication rates of cerebrospinal fluid diversion techniques used at our institution, a retrospective study of the surgical management of posthemorrhagic hydrocephalus was conducted from a population of 547 premature infants admitted to the neonatal intensive care unit from 1987 to 1989. The incidences of periventricular-intraventricular hemorrhage in the 3 years studied were 44%, 37%, and 27%, respectively. Thirty-nine of the infants developed posthemorrhagic hydrocephalus as determined by serial cranial ultrasonography; 22 required cerebrospinal fluid diversion. During the study period, we began using subcutaneous ventricular reservoirs and a low-pressure Neonatal Shunt (customized device) in infants weighing less than 1500 g at the time of instrumentation. This change in management was associated with a significant reduction (P < 0.005) in the morbidity and mortality compared to the use of external ventricular drainage devices. On the basis of these findings, the use of external ventricular drainage devices was discontinued.


Congenital arachnoid cyst of the lateral ventricles in children.

The authors report a series of three children with symptomatic congenital arachnoid cyst of the lateral ventricles. Presenting symptoms consisted of macrocephally, delay in psychomotor development, and seizures. CT findings were of a well-defined cystic lesion placed in the atrium of the lateral ventricle. One child was treated by direct cyst exposure and cysto-peritoneal shunt. The other two were treated with ventriculo-cysto-peritoneal shunts; in one of these, we used a ventriculoscope both for cyst fenestration and for accurate shunt placement. The origin of intraventricular arachnoid cysts seems to be secondary to the displacement of arachnoid cells by the vascular mesenchyma, through the choroid fissure, during the process of choroid plexus development.


Pseudotumor syndrome in treated arachnoid cysts.

We report three patients with arachnoid cysts treated by cyst-peritoneal shunting in whom intracranial hypertension occurred during episodes of shunt malfunction. In one case this was associated with re-expansion of the arachnoid cyst, whilst in the other two cases this did not occur. The similarities between these two cases and patients with pseudotumor cerebri suggest a common pathogenic mechanism--specifically, a disturbance of the cerebrospinal fluid circulation.


Description of two informative cases of occult spinal dysraphism with remarks on possible traits in the embryogenesis.

Two cases of spinal occult dysraphism are described. The association of a dermoid cyst and ectopic tissue foci of both ecto- and mesodermal origin at the junction zone between lipoma and nervous tissue is argued to support the disjunction theory on lipomeningomyelocele formation.


Hyperfractionated radiotherapy and polychemotherapy in brain stem tumors in children.

Between October 1989 and January 1991 five children with brain stem tumors were treated with sequential chemo- and radiotherapy. The polychemotherapy consisted of procarbazine, ifosfamide, etoposide, methotrexate, cisplatin and cytosine arabinoside. Locally, hyperfractionated radiotherapy was delivered at a total dose of 63.8 Gy (1.1 Gy twice daily, 10 fractions per week). After a median observation time of 11.8 (range 4-23) months from diagnosis three children are alive and without evidence of tumor progression. Two patients died from tumor progression 11 and 16 months respectively after initiation of therapy.


Single photon emission computerized tomography in childhood hydrocephalus.

Single photon emission computerized tomography (SPECT) is now widely used as one of the tools in evaluating cerebral blood flow (CBF). The authors report the CBF changes in childhood hydrocephalus. Five pediatric cases studied by 123I-IM SPECT in children are presented. The authors counted radioactivities both in early and delayed images in each patient, and calculated the reabsorption ratio (RR). Two negative-RR cases and three positive-RR cases were found. All of the negative-RR patients had a poor prognosis, while all of the positive-RR patients had a favorable outcome.


Occurrence and management of fractured peripheral catheters in CSF shunts.

A series of 716 children underwent 2065 cerebrospinal fluid shunt procedures. Shunt failure due to fracture of the peripheral drain occurred 60 times, 38 times in ventriculo-atrial and 22 times in ventriculo-peritoneal shunts. The break occurred most commonly 2-4 cm above the neck incision in cardiac and just cephalad to the clavicle in abdominal drains. Fifty-nine ruptures occurred in Pudenz catheters (which were used in 82% of the shunts) and 1 occurred in a Holter drain (used in 17%). The fractured atrial catheters remained in situ (5/38) or were dislodged into the right cardiac ventricle (14/38), pulmonary arteries (9/38), right atrium (5/38) or hepatic veins (3/38). Two of the ruptured drains could not be located. Removal by a percutaneous transvascular snare technique was attempted in 27 cases and was successful in 24.


Laminotomy: a technical note.

In order to restore spinal integrity following posterior exposures of the spinal canal in children, we describe modifications of Raimondi's laminotomy technique. The use of a pneumatic dissecting tool with foot-plate to create a hinged osteoplastic laminotomy is described, as are techniques for securing the laminotomy flap in place at the end of the procedure.


Repair of giant occipital encephaloceles with microcephaly secondary to massive brain herniation.

Giant occipital encephaloceles rarely contain large amounts of neural tissue that cannot be replaced in the abnormally small calvarium. Resection of neural elements is therefore often necessary in order to accomplish a closure. A technique is described wherein an extracranial compartment is prepared utilizing fine tantalum mesh to enclose the neural contents. The mesh is attached to the periphery of the skull defect providing a rigid extracranial compartment for the encephalocele. As intracranial pressure increases, the calvarium is forced to expand. The tantalum mesh is gradually imbricated into the calvarium by daily digital compression. If ventriculomegaly occurs, an interval ventriculoperitoneal shunt is placed. The encephalocele repair is reopened and the tantalum is surgically imbricated at that time. This allows for a satisfactory cosmetic result with preservation of all neural elements.


Abnormal cerebral hemodynamics during attacks of alternating hemiplegia.

Frequent episodes of bilateral weakness and apathy, followed later by hemiplegia of alternating sides were observed in a now 32-month-old girl. Transcranial Doppler ultrasonography showed reduced flow velocities in the middle cerebral artery of the affected side during a hemiplegic attack and increased flow velocities at different sites of the basilar artery during a bilateral episode. These abnormal cerebral hemodynamics appear to indicate that alternating hemiplegia and some forms of migraine have a similar pathophysiology.


Traumatic cervical syringomyelia related to birth injury.

A rare case of cervical syringomyelia related to breech delivery is reported. The initial diagnosis was bilateral brachial plexus palsy due to birth injury, which was revealed by magnetic resonance imaging (MRI) to be traumatic syringomyelia. The usefulness of MRI in the early diagnosis of cervical cord birth injury, especially in differentiating between brachial plexus palsy due to birth injury and spinal cord trauma due to birth injury in infancy, is emphasized.


Muscle phosphofructokinase deficiency in a myopathic child with severe mental retardation and aplasia of cerebellar vermis.

Muscle phosphofructokinase (PFK) deficiency in man is responsible for at least two forms of myopathy; one is characterized by painful contractures of muscles and typically occurs in adults, whereas the other is often disabling and typically occurs in childhood, with psychomotor and growth retardation. In this investigation, a young myopathic patient with severe mental retardation and aplasia of the cerebellar vermis presented with muscular hypotrophy of the limbs, generalized hypotonia, convergent strabismus and marked pain during passive movement. Biopsy of quadriceps femoris muscle showed variation in the fiber size with sarcoplasmic areas positive for periodic acid-Schiff stain. Histochemical qualitative reaction for PFK showed no staining of muscle fibers; ultrastructural studies showed abnormal accumulation of glycogen granules in both intermyofibrillar and subsarcolemmal areas. While some enzyme activities in the muscular crude extract were significantly lower than in controls, direct assay of PFK revealed no activity, thus demonstrating that the child's myopathy was due to the lack of PFK activity.


Catecholamine alterations in experimental hydrocephalus.

Experimental hydrocephalus was induced in rats by intracisternal injection of kaolin suspension. The amounts of norepinephrine and dopamine were determined in the whole brain and specific brain regions at 1 week (acute phase) and 4 weeks (chronic phase). The turnover of catecholamine, an index of the activity of catecholamine-containing neurons, was determined by measuring the decrease in catecholamine contents 2 h after intraperitoneal injection of alpha-methyl-p-tyrosine (250 mg/kg), an inhibitor of tyrosine hydroxylase. We observed that the catecholamine contents in kaolin-induced hydrocephalus were not significantly different from control values. Following injection of alpha-methyl-p-tyrosine, there was decrease in levels of catecholamines in both control and hydrocephalic rats. This decrease was, however, significantly less in induced hydrocephalus than in control animals. This result suggested that in hydrocephalus, the activities of norepinephrinergic and dopaminergic neurons are reduced.


The child with a mass on its head: diagnostic and surgical strategies.

A series of 65 pediatric patients with scalp or calvarial masses is reported on. The majority of children presented with a disfiguring or painful mass on the head. Clinical findings suggested the correct diagnosis in 39/65 cases, skull radiographs in 46/65, and CT in 49/65. Taking the combined results of clinical and radiological studies, 54/65 of the lesions were accurately diagnosed. Tumor excision was curative in 43 of 48 patients who were operated on. Most scalp and calvarial neoplasms were benign; only 5/65 children harboured a malignant lesion. There was no mortality related to surgery in the series. Surgical intervention seems to be indicated in most cases, both for diagnosis and for treatment.


Gangliogliomas in childhood.

Ganglioglioma is a tumour of the central nervous system composed of an admixture of dysplastic nerve cells resembling pleomorphic ganglion cells, and glial elements, which may be astrocytic and/or oligodendroglial in appearance. A series of 12 patients aged between 9 months and 15 years 9 months, all of whom had suffered epilepsy refractory to medical treatment for up to 8 years, is presented. Computed tomographic and magnetic resonance scans were of prime use in localisation of the tumours. Calcification was noted preoperatively in 4 of 12 cases. The majority of patients obtained at least partial relief from symptoms after complete or partial resection. Histologically, 11 of the tumours included grade 1 astrocytic elements and the remaining one exhibited grade 2 areas. The diagnosis of ganglioglioma should be suspected in a child with refractory, long-standing epilepsy. Prognosis of these tumours is determined by the astrocytic component; if this is of low grade, surgical excision may result in marked symptomatic improvement or cure.


Attempts to induce immune-mediated cerebral arterial injury for an experimental model of moyamoya disease.

To examine the possible role of immune complex-mediated reactions in moyamoya disease, a novel experimental system using a serum sickness vasculitis model combined with intracisternal administration of antibodies or antigens was developed. Twenty-eight male Japanese white rabbits were divided into four experimental groups. Group I was treated twice with intravenous injections of heterologous serum. In group II, intracisternal administration of antibodies or antigens was combined with the second injection of serum. Group III received a single intravenous injection of antigens simultaneously with intracisternal administration of antibodies. Group IV was a technical control group. Cerebral arteritis, although likely in the initial process, was induced only in groups II and III. This study suggests that the cerebral arteries rarely develop arteritis in a serum sickness model alone. The cerebral arteries may require additional intracisternal administration of antibodies or antigens to induce in situ deposition of immune complexes around them.


Predicting the recurrence of ependymomas from the bromodeoxyuridine labeling index.

The usefulness of histopathological grading in predicting the prognosis of patients with ependymomas is controversial. To clarify the discrepancy between the histological malignancy and the prognosis of these tumors, we estimated the proliferative potential of 32 intracranial and intraspinal ependymomas and correlated the findings with the clinical behavior. Each patient received an intraoperative infusion of bromodeoxyuridine (BUdR, 200 mg/m2 i.v.) before tumor removal; the BUdR labeling index (LI), or percentage of BUdR-labeled cells, was determined immunohistochemically in excised specimens. The mean BUdR LI (+/- SD) of intracranial malignant ependymomas was 4.1 +/- 2.8%. Nonmalignant intracranial and intraspinal ependymomas and subependymomas had mean LIs of 1.5 +/- 0.9%, 1.1 +/- 0.3%, and less than 1%, respectively. Overall, 44% of the tumors recurred. There were no statistically significant differences in the recurrence rates of intracranial and intraspinal ependymomas, including subependymomas (43% and 44%, respectively), or of intracranial ependymomas with LIs greater than 1.0% and less than 1.0% (67% and 44%, respectively). However, the early recurrence rate (within 24 months after treatment) of tumors with LIs greater than 1.0% was higher than that of tumors with LIs of less than 1.0% (100% vs. 25%, P less than 0.05). The BUdR LI also showed a statistically significant inverse correlation with the time to recurrence. These findings indicate that BUdR LI reflects the proliferative potential of individual ependymomas and can be used to help predict the recurrence and estimate the prognosis of these tumors.


Brain abscess in infants.

Brain abscesses are rare in infants and their clinical presentation is specific for this age group. Seven cases of brain abscess in infants aged 2-11 months are reported. The underlying cause was meningitis in four, sepsis in two, and unknown in one. Gram-negative organisms were cultured in 6 patients. The abscess size was 5 cm or more in five cases; in four there were multiple lesions. Two abscesses were aspirated and irrigated; four particularly large lesions were drained and repeatedly aspirated and irrigated. One craniotomy was done. There were two deaths, one in the postoperative period and the other 6 months after discharge. Follow-up information is available for four children, showing a good result in only one of them. Formation of an abscess should be diagnosed early, and close ultrasound monitoring or CT scanning in infants with bacterial meningitis and sepsis is essential. The prognosis in cases in which large/multiple abscesses develop is poor.


Arachnoid cysts in children: a European co-operative study.

The data on arachnoid cysts in children (0-15 years) operated upon between 1980 and 1988 were analysed in a retrospective, co-operative study. The results from 285 patients indicate a predominance of these lesions in boys (64%) more than girls (36%) and a mean age of 6 years at onset of symptoms. Focal EEG patterns corresponding to the cyst's location were encountered in 32%. About 40% of all cysts were located along the midline, the sylvian fissure representing the predominant location. Open surgery, i.e. total excision or marsurpialization (together 43.3%), emerged as the first-choice surgical procedure. The type of surgery switched somewhat to shunting procedures in cases of lesions in deeper locations (22.8%). Morphological results on follow-up revealed a reduction of the size of the cyst in a significant majority (61%); in 18% the cyst had disappeared completely on CT scans. There was an obvious correlation between postoperative morphological findings and clinical outcome.


Scalp aplasia cutis congenita: closure by the L-shaped flap.

A case of aplasia cutis congenita of the scalp with bony defect in a newborn treated by early excision and reconstruction is presented. A recently described versatile minor flap is employed to cover the resultant defect. The advantages of early surgical repair and the flap are highlighted.


Intramedullary spinal cord abscess: a case report.

An 11-year-old boy presented with pain in the back, urinary retention, paraplegia and loss of sensations below L1. Investigations revealed an intramedullary lesion. An intramedullary spinal cord abscess was found at surgery. The pus was evacuated and abscess was excised. Minimal recovery was seen following surgery. Early intervention and a high index of suspicion is required in such cases.


Periventricular hydatid cyst presenting with hemichorea.

A case of periventricular hydatid cyst presenting with only hemichorea is reported. The unusual clinical course is described.


Ascaris lumbricoides: an unusual cause of shunt infection.

Two cases of delayed shunt infection attributable to the abdominal complications of Ascaris lumbricoides infestation are reported. The first child presented with a peritoneal shunt catheter protruding through the anus and the second patient was found to have two live worms around the cephalic shunt tubing. Both had enteric shunt infections which responded well to therapy. The epidemiology and treatment of ascariasis are briefly discussed.


Orbital porencephalic cyst following penetrating orbitocranial trauma.

In a 10-year-old boy an orbitocranial penetrating wound produced by an umbrella tip caused an orbital roof bone fragment to penetrate up to the anterior part of the third ventricle behind the left foramen of Monro. Hemorrhages and encephalomalacia developed along the trajectory of the fragment and subsequently a porencephalic cyst was formed at this site. Six months after the trauma, increased pressure developed in the left ventricular system due to obstructive hydrocephalus and consequently the porencephalic cyst herniated into the orbit through the orbital roof fracture, producing intermittent diplopia, left exophthalmos, and palpebral swelling. A ventriculo-peritoneal shunt led to shrinkage of the orbital cyst content and resolution of the symptoms.


Classification of the cerebral edemas with reference to hydrocephalus and pseudotumor cerebri.

Cerebral edema is a common clinical disorder that results from an abnormal increase in water content within the extracellular (EC) compartment of the brain. It is distinguished from two other types of brain bulk enlargement: (1) vascular swelling, caused by arterial dilatation or venous obstruction; and (2) cellular swelling, caused by cytotoxic injuries or metabolic storage. Under normal conditions, the EC compartment has two fluids, the interstitial fluid (ISF) and the cerebrospinal fluid (CSF), and extends from the blood brain barrier (BBB) through a series of 100 to 150-A-wide intercellular spaces that are anatomically continuous with the CSF spaces. There are four primary types of EC edema: (1) vasogenic edema, which results from an increase in brain capillary permeability, the most common type, in which leakage of plasma constituents into the brain follows the pathways of ISF bulk flow and is governed by the interaction of systemic arterial pressure and tissue resistance; (2) osmotic edema, which results from an unfavorable osmotic gradient between the plasma and ISF across an intact BBB; (3) compressive edema, which results from obstruction of ISF bulk flow pathways; and (4) hydrocephalic edema, which results from obstruction of CSF bulk flow pathways. In this latter type of edema, distension of the collecting channels proximal to the block leads to retrograde flooding of the EC compartment with the formation of periventricular edema. The syndrome of pseudotumor cerebri includes several different types of brain bulk enlargement.


Occlusion of the sagittal sinus in craniectomized rabbits.

Most attempts at production of hydrocephalus in experimental animals by obstructing the venous sinuses have failed. In adult humans, venous sinus occlusion usually results in the clinical syndrome of pseudotumor cerebri with small or normal sized ventricles. However, in children less than 18 month old with venous sinus hypertension, ventriculomegaly has been reported. We examined the change in ventricular size in craniectomized animals (simulating children with open sutures) with occlusion of the superior sagittal sinus. New Zealand rabbits weighing 1500-1800 g were anesthetized with an intramuscular injection of 2 ml 7:3 ketamine (100 mg/ml): Rompun (xylazine) (20 mg/ml) solution. The scalp was shaved, prepped with Betadine, and infiltrated with 1% lidocaine, and a midline scalp incision made. The periosteum was reflected laterally and a craniectomy performed with microscopic magnification. The dura was exposed overlying both cerebral hemispheres and the superior sagittal sinus from its origin to the torcular. In five control animals, the scalp was then closed. In ten experimental animals, small incisions were made in the dura just lateral to the superior sagittal sinus with a no. 11 scalpel and then with microscopic magnification the sinus was coagulated with bipolar cautery and transected; the scalp was then closed. All animals were allowed 5-7 days to recover, then ultrasound was used to assess ventricular size. We observed a small but statistically significant increase in ventricular size in the experimental group compared to the control group. This model provides evidence that venous sinus occlusion in animals with expandable crania can produce ventriculomegaly.


Pathogenesis of diastematomyelia: can a surgical model in the chick embryo give some clues about the human malformation?

To reproduce diastematomyelia, a sagittal incision was carried out at the level of the rhomboidal sinus of 36- to 40-h-old chick embryos. A small piece of membrane shell, a small agar screen, or a piece of quail isochronous isotopic notochord was inserted into the gap. The embryos were killed and fixed after 9 days' incubation. Diastematomyelia was obtained in several embryos treated with interposition of a membrane screen or a piece of quail notochord. Microscopic examination revealed two hemicords, each containing its own central canal; in some cases one of the cords showed hydromyelia. Absence of the rump was seen in association with experimental diastematomyelia. The interposition of a resorbable agar screen did not succeed in reproducing diastematomyelia. The results of these surgical manipulations suggest that diastematomyelia cannot be explained by a primary disorder of neurulation. It supports the theory of noninvolution of a firm midline structure (probably the neurenteric canal, rapidly surrounded by mesodermal cells originating from the notochord), which prevents the fusion of the separated parts.


Retroflexion of holoprosencephaly: report of two cases.

Two cases of retroflexed holoprosencephaly are presented. Reports of nine cases of retroflexion were available in the literature. Case analysis indicates that retroflexion results from subdural fluid collection. The incidence of retroflexion in holoprosencephaly was apparently higher than that of ordinary subdural hygromas. Possible explanations for this high incidence are given. The clinical significance and management of retroflexion are touched upon.


Klippel-Feil syndrome revisited: diagnostic pitfalls impacting neurosurgical management.

Klippel-Feil syndrome in its most basic definition includes several anomalous conditions of the cervicomedullary junction and suboccipital region. Pediatric neurosurgeons are often involved in surgical palliation of this syndrome, without realizing how the accompanying anomalies may obfuscate management in the older child. A brief review of the embryology of the rhombencephalon helps to clarify the etiology of some of these symptoms which may cause confusion and, occasionally, inappropriate treatment. Illustrative cases will demonstrate some of these pitfalls. Appropriate early intervention, such as posterior fossa decompression, ventricular shunting, and fundal plication, may help to avoid needless morbidity. The advent of magnetic resonance scanning has helped to clarify the diagnosis and resulted in more appropriate treatment in these cases.


Controversy pertaining to therapeutic modalities for tumors of the pineal region: a worldwide survey of different patient populations.

The management of tumors of the pineal region differs between Western countries and Japan. This paper reports on a worldwide survey of individual experience and regimens for treating pineal region tumors in different patient populations. Fifteen pediatric neurosurgeons from nine different countries participated in the survey, and a total of 408 pineal region tumors were evaluated. Determination of tumor histology as an initial procedure was strongly supported by the majority of neurosurgeons in North and Central America and Europe (group A), whereas all but one from Asia and Egypt (group B) emphasized initial application of the radiation test. The analysis of patient populations clearly revealed racial differences in tumor type which explain this discrepancy. Germinoma, the most radiosensitive tumor, constituted 43-70% (mean: 53.7%) of tumors in group B, followed by teratoma, pineoblastoma, and others, whereas in group A the incidence of germinoma was only 21-44% (mean: 34.7%), followed by a variety of tumors, such as astrocytoma, pineoblastoma, etc. The age distribution among intracranial germ cell tumors (GCT) obtained from data from the Brain Tumor Registry in Japan also demonstrated clear differences in the incidence of tumor types in different age groups in Japan: while germinoma constituted 70-84% of GCT in patients between the ages of 15 and 35 years, the incidence was much lower before 15 years and after 35 years, being 24% of tumors under 4 years and 34% of tumors after 40 years of age. The therapeutic regimen for pineal region tumors should depend on the patient population concerned, because of the differences relating to race and age distribution.


A proposed grading and scoring system for spina bifida: Spina Bifida Neurological Scale (SBNS)

Neurological symptoms present in neonates with spinal dysraphism often progress with growth. A simple, objective scoring system for quantitative analysis of spinal neurological deficits, called the Spina Bifida Neurological Scale (SBNS), is proposed. Scoring is based on (1) motor function, (2) reflexes, and (3) bladder and bowel function. These are each divided into six, four, and five points respectively with respect to the level of spinal function. Motor function and reflexes are bilaterally analyzed, and the maximum SBNS score of 15 points reflects a normal spinal neurological state (grade I). This scoring system was correlated with the clinical condition of 89 patients with spina bifida who were graded from I to V. A total score of less than 5 was associated with a nonambulatory state (grade IV or V) in 84.0% of patients, and a score of 5-9 was associated with an ambulatory state (grade III) in 93.8% of patients. Scores of 10-14 reflected control of bladder and bowel function (grade II) in all patients. The application of a standardized scoring scheme will assist in the evaluation of patients' clinical status and will enable analysis of chronological changes in neurological function.


Acute spontaneous subdural hematoma in a teenager.

A teenager with a history of sudden onset of headache and vomiting is described. Computed tomography revealed an acute subdural hematoma in the right temporoparietal region, causing marked compression of the right ventricular system and a shift of midline structures to the left. No operation was carried out because the symptoms and neurological signs were slight enough to allow monitoring by means of close clinical and neuroradiological investigations. Within 18 days the hematoma resolved spontaneously and completely. There was no history of trauma or any objective sign of trauma about the face or head, and radiography of the skull showed no fracture. We are not aware of any other report of a spontaneous subdural hematoma which did not require surgery. This feature makes our case unique. In addition, comparable cases in the literature are reviewed and the etiological possibilities of spontaneous subdural hematoma are discussed.


Successful surgical obliteration of a huge intradural arteriovenous fistula of the spinal cord in a child.

We report the case of an 8-year-old boy with a huge intradural spinal arteriovenous fistula (AVF), which was successfully obliterated by surgery. The symptoms were episodic headaches and progressive motor and sensory deficits. He had suffered a subarachnoid hemorrhage twice before but no ensuing neurological deficits. Neuroradiological examinations revealed an intradural AVF with a huge venous aneurysm ventral to the spinal cord of C7 to T2, which was fed by the left 5th intercostal artery and the right thyrocervical artery. These two feeding arteries were occluded intradurally just at the venous aneurysm emerging point. MR images taken sequentially after surgery demonstrated complete thrombosis and subsequent disappearance of the thrombosed malformation. Neurological symptoms improved gradually. Treatment of such malformations is discussed.


Delayed intraventricular tension pneumocephalus complicating posterior fossa surgery for cerebellar medulloblastoma.

A child is described in whom intraventricular tension pneumocephalus developed 10 days after removal of a cerebellar medulloblastoma and 1 day after suture removal. The tension pneumocephalus was associated with hydrocephalus and CSF leakage from the suture line. The symptoms of the pneumocephalus were rapidly progressing loss of consciousness and hemiplegia which were promptly reversed upon aspiration of the intracranial air. A large amount of intraventricular air present in the immediate postoperative period was, however, clinically silent. The characteristics of this unusual presentation, its relation to asymptomatic pneumocephalus, hydrocephalus and the preventive and therapeutic measures required to deal with such conditions are discussed.


Acute severe combined demyelination.

We present a second case in which Guillain-Barré syndrome (GBS) and acute disseminated encephalomyelitis (ADEM) appeared simultaneously, both in acute and fulminant form. The patient, a 10-year-old girl, presented with acute onset of coma and flaccid, are-flexic quadriparesis. The elevated CSF protein levels and delayed F waves fulfilled the criteria of GBS and an MRI study revealed extensive multifocal demyelination compatible with a diagnosis of ADEM. Prompt clinical response followed by complete recovery was achieved by treatment with corticosteroids. It is suggested that acute severe combined demyelination might constitute a separate entity in which the demyelinating process, involving simultaneously the central and the peripheral nervous systems, indicates immune response against a component of the myelin of one system carrying cross-antigenicity with the other.


Spontaneous intracerebral hemorrhage from an unsuspected ependymoma in early infancy.

A case of spontaneous intracerebral hemorrhage from an occipital ependymoma grade 2 in a 3-month-old boy is reported. The infant died 3 days after surgery. The clinical and pathomorphological characteristics are described. Despite the usually rich vascularization of these tumors, hemorrhages from intracranial ependymomas are relatively uncommon. The different forms and probable causes of bleedings are discussed.


A conformational study of alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) by NMR nuclear Overhauser effect spectroscopy (NOESY) and molecular dynamics calculations.

The conformational preference of the disaccharide alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) (1) about the glycosidic torsion angles, phi and psi, was studied by NMR NOESY spectroscopy and molecular mechanics calculations. The NOE data were consistent with either of two distinct conformations close to minima on a calculated phi/psi potential energy surface. Starting from the lowest energy conformation, a 1-ns molecular dynamics (MD) trajectory was computed in vacuo, from which the NOE curves were simulated and compared to the experimentally observed NOESY data.


The use of neocarrabiose oligosaccharides with different length and sulphate substitution as model compounds for 1H-NMR spectroscopy.

The high-field 1H-NMR spectra of various carrageenan oligosaccharides at room temperature are given. The assignments were faciliated by the use of proton double-quantum coherence (DQCOSY) and 1H-13C chemical shift correlation 2D NMR spectroscopy, and by comparing high-field 1H-NMR spectra of various 4-sulphated oligosaccharides of the neocarrabiose type. The effects of anomeric configuration on the 1H resonances on the same or neighbouring units are discussed. The 13C-NMR shift data are given for the tetrasaccharide of kappa-carrageenan.


Synthesis of D-glucose 3- and 6-[2-(perfluoroalkyl)ethyl] phosphates: a new type of anionic surfactant for biomedical use.

D-Glucose 3- and 6-[sodium 2-(perfluoro-hexyl or -octyl)ethyl phosphates) have been synthesized by condensation of 1,2,3,4,-tetra-O-acetyl-beta-D-glucopyranose and 1,2:5,6-di-O-isopropylidene-alpha-D-glucofuranose with 2-(perfluoroalkyl)ethylphosphoroditriazolides followed by O-deacetylation or deacetalation. The structures of the compounds were established on the basis of 1H-, 19F-, 31P-, and 13C-NMR data. These salts display strong surface activities and appear to have good biocompatibility.


Interaction of linear manno-oligosaccharides with three mannose-specific bulb lectins. Comparison with mannose/glucose-binding lectins.

Three new mannose-binding lectins, isolated from daffodil (NPA), amaryllis (HHA), and snowdrop (GNA) bulbs, are capable of precipitating with a linear mannopentaose (Man alpha 1-3Man alpha 1-3Man alpha 1-3Man alpha 1-2Man). NPA and HHA reacted strongly with the mannopentaose whereas GNA gave a precipitate only at concentrations greater than 500 microM. A phosphate group at C-6 of the nonreducing terminal mannosyl group prevented precipitation in all three cases. The reduced (NaBH4) mannopentaose, Man4Man-ol, did not precipitate with GNA or NPA, but was active with HHA. This activity was lost when Man4Man-ol was converted (NaIO4 then NaBH4; mild acid hydrolysis of the reduced product) into trisaccharide derivatives. With alpha-D-Manp-OMe the three lectins gave UV difference spectra having large positive peaks at 292-293 and 283-284 nm, and a small positive peak at 275 nm, characteristic of tryptophanyl and tyrosyl residues. The association constants for the interaction with alpha-D-Manp-OMe were very low (NPA, 86; HHA, 66; and GNA, 41 M-1), but the lectins bound methyl (1----3)-alpha-mannobioside with increased affinity (K for NPA 540, for HHA 2400, and for GNA 200 M-1). The bulb lectins lack binding sites for hydrophobic ligands, as judged by their failure to interact with the fluorescent probes 8-anilino-1-napthalenesulfonic acid (ANS) and 6-p-toluidino-2-naphthalenesulfonic acid (TNS).


New mannose-specific lectins from garlic (Allium sativum) and ramsons (Allium ursinum) bulbs.

Two new mannose-binding lectins were isolated from garlic (Allium sativum, ASA) and ramsons (Allium ursinum, AUA) bulbs, of the family Alliaceae, by affinity chromatography on immobilized mannose. The carbohydrate-binding specificity of these two lectins was studied by quantitative precipitation and hapten-inhibition assay. ASA reacted strongly with a synthetic linear (1----3)-alpha-D-mannan and S. cerevisiae mannan, weakly with a synthetic (1----6)-alpha-D-mannan, and failed to precipitate with galactomannans from T. gropengiesseri and T. lactis-condensi, a linear mannopentaose, and murine IgM. On the other hand, AUA gave a strong reaction of precipitation with murine IgM, and good reactions with S. cerevisiae mannan and both synthetic linear mannans, suggesting that the two lectins have somewhat different binding specificities for alpha-D-mannosyl units. Of the saccharides tested as inhibitors of precipitation, those with alpha-(1----3)-linked mannosyl units were the best inhibitors of ASA, the alpha-(1----2)-, alpha-(1----4)-, and alpha-(1----6)-linked mannobioses and biosides having less than one eighth the affinity of the alpha-(1----3)-linked compounds. The N-terminal amino acid sequence of ASA exhibits 79% homology with that of AUA, and moderately high homology (53%) with that of snowdrop bulb lectin, also an alpha-D-mannosyl-binding lectin.


Anticoagulant and antithrombin activities of oversulfated fucans.

Three species of oversulfated fucans having different sulfate contents (the ratio of sulfate/total sugar residues, 1.38-1.98) were prepared by chemical sulfation of a fucan sulfate (sulfate/sugar ratio, 1.28) isolated from the brown seaweed Ecklonia kurome. The anticoagulant activities of the oversulfated fucans were compared with that of a parent fucan with respect to activated partial thromboplastin time (APTT) and thrombin time (TT) in plasma. The respective activities (for APTT and TT) of the oversulfated fucans increased to 110-119% and 108-140% of the original values with increase in their sulfate content. The anticoagulant activity with respect to APTT (173 units/mg) of an oversulfated fucan (sulfate/sugar ratio, 1.98) was higher than that (167 units/mg) of heparin used as a standard. The heparin cofactor II-mediated antithrombin activity of the oversulfated fucans also increased significantly with increase in sulfate content. The maximum activity was higher than those of the parent fucan and heparin. However, the increment of the anticoagulant and the antithrombin effects gradually decreased with increase in the sulfate content of the fucans. These results indicate that the effects of the fucan sulfate are dependent on its sulfate content until a plateau is reached.


The conformational behaviour of the cardiac glycoside digoxin as indicated by NMR spectroscopy and molecular dynamics calculations.

The 1H- and 13C-NMR spectra of digoxin in solution in Me2SO-d6 have been assigned completely. Measurement of the 3JC,H values has enabled estimation of the torsional angles involving the bonds linking the digitoxose residues, between the inner digitoxose and the genin unit, and for the unsaturated gamma-lactone ring. These values have been supplemented by 1H-1H NOE data. In general, there is good agreement between the conformations in solution (NMR data) and the solid state (X-ray data), and that derived from theoretical modelling which shows evidence of conformational flexibility. The major difference occurs for the torsion between the genin and the innermost digitoxose residue where molecular dynamics predict the presence of two conformations, one similar to that seen by NMR and the other similar to the X-ray structure.


Synthesis and X-ray crystal and solution structures of 2,5-anhydro-3,4-O-(1,2-ethanediyl)-D-mannitol: a locked 4T3 furanose conformer.

2,5-Anhydro-3,4-O-(1,2-ethanediyl)-D-mannitol (1) was prepared from 2,5-anhydro-D-mannitol (2) in three steps. The fused ring system was introduced by a phase-transfer alkylation using 1,2-dibromoethane. Its conformation in solution was determined by NMR studies at 500 MHz. Variable-temperature studies showed no lineshape change from 25 to 80 degrees in D2O. The data indicate that the five-membered ring is locked by the trans-fused six-membered 1,4-dioxane ring into a twist 4T3 conformation. A single-crystal X-ray study was carried out. The crystals are orthorhombic, C222(1), a = 4.7252 (6), b = 14.0364 (12), c = 13.268 (2) A, Z = 4, with R = 0.032 for 894 observations. The molecule lies upon a crystallographic two-fold axis, and thus the five-membered ring exists in a perfect 4T3 conformation with a pseudorotation angle of 0 degree and amplitude of 47.2 degrees, in agreement with the NMR results. We have shown earlier that, among twenty possible conformers, phosphofructokinase acts specifically on the 4T3 conformer of the beta anomer of D-fructose 6-phosphate.


Conformational analysis of the anomeric forms of kojibiose, nigerose, and maltose using MM3.

Energy surfaces were computed for relative orientations of the relaxed pyranosyl rings of the two anomeric forms of kojibiose, nigerose, and maltose, the (1----2)-alpha, (1----3)-alpha, and (1----4)-alpha-linked D-glucosyl disaccharides, respectively. Twenty-four combinations of starting conformations of the rotatable side-groups were considered for each disaccharide. Optimized structures were calculated using MM3 on a 20 degree grid spacing of the torsional angles about the glycosidic bonds. The energy surfaces of the six disaccharides were similar in many respects but differed in detail within the low-energy regions. The maps also illustrate the importance of the exo-anomeric effect and linkage type in determining the conformational flexibility of disaccharides. Torsional conformations of known crystal structures of maltosyl-containing molecules lie in a lower MM3 energy range than previously reported.


Synthesis and properties of sulfated alkyl glycosides.

Alkyl glycosides were sulfated with sulfur trioxide-pyridine. Dodecyl alpha- and beta-D-glucopyranoside gave the corresponding 6-sulfates in 75 and 51% yields, respectively. Separation from polysulfated compounds was carried out by reversed-phase HPLC. Tetradecyl beta-maltopyranoside (16) gave a 88: 12 mixture of 6'- and 6-sulfates. The sulfated compounds were characterized by 1H-, 13C-, and 2-dimensional NMR spectroscopy. Surfactant and thermotropic liquid-crystalline properties of the sugar derivatives were examined. All of the glycosides show smectic phases (SA), and the clearing points rise by introduction of sulfate groups. Even glycosides having no unprotected hydroxy groups may show SA-phases when bearing sulfate groups. The mesomorphic properties cannot be explained by formation of distinct aggregates, but rather must be interpreted by an effective intramolecular contrast.


Synthesis of the methyl thioglycosides of deoxy-N-acetyl-neuraminic acids for use as glycosyl donors.

Methyl 2-thioglycoside derivatives of 4-, 7-, 8-, and 9-deoxy-N-acetylneuraminic acids have been prepared as glycosyl donors for the synthesis of sialoglycoconjates. Reduction of a (phenoxy)thiocarbonyl group, selectively introduced at the 4 position of methyl [2-(trimethylsilyl)ethyl 5-acetamido-3,5-dideoxy-8,9-O-isopropylidene-D- glycero-alpha-D-galacto-2-nonulopyranosid]onate (1), gave the 4-deoxy compound, which was transformed via O-deisopropylidenation, acetylation, selective removal of the 2-(trimethylsilyl)ethyl group, subsequent acetylation, and displacement of the 2-acetoxy group by a methylthio group, into methyl (methyl 5-acetamido-7,8,9-tri-O-acetyl-3,4,5-trideoxy-2-thio-D-manno-2- nonulopyranosid)onate (17). Methyl [2-(trimethylsilyl)ethyl 5-acetamido-8,9-di-O-acetyl-4-O-benzoyl- 3,5,7-trideoxy-alpha-D-galacto-2-nonulopyranosid]onate, prepared from 1 in five steps, and methyl [2-(trimethylsilyl)ethyl 5-acetamido-4,7,9-tri-O-acetyl-3,5,8-trideoxy-alpha-D-galacto-2- nonulopyranosid]onate, prepared from 1 in six steps, were converted via selective removal of the 2-(trimethylsilyl)ethyl group, O-acetylation, and displacement of the 2-acetoxy group by a methylthio group as described for 17, into the corresponding methyl 7- and 8-deoxy-2-thioglycosides. Reductive dechlorination of methyl [2-(trimethylsilyl)ethyl 5-acetamido-4,7-di-O-benzoyl-9-chloro-3,5,9-trideoxy-D-glycero-alpha-D-g alacto- 2-nonulopyranosid]onate, prepared from methyl [2-(trimethylsilyl)ethyl 5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosid++ +]onate by selective 9-O-tert-butyldimethylsilylation, benzoylation, removal of the 9-silyl group, and selective chlorination, gave a 9-deoxy compound. This was transformed, via O-debenzoylation, O-acetylation, selective removal of the 2(trimethylsilyl)ethyl group, 2-O-acetylation, 2-chlorination, displacement with potassium thioacetate, selective S-deacetylation, and S-methylation, into the methyl 2-thio-alpha-glycoside of 9-deoxy-N-acetylneuraminic acid.


Synthesis of a series of ganglioside GM3 analogs containing a deoxy-N-acetylneuraminic acid residue.

Ganglioside GM3 analogs containing 4-, 7-, 8-, and 9-deoxy-N-acetylneuraminic acids in the place of N-acetylneuraminic acid (Neu5Ac) have been synthesized. Glycosylation of 2-(trimethylsilyl)ethyl O-(6-O-benzoyl-beta-D-galactopyranosyl)-(1----4)-2,6-di- O-benzoyl-beta-D-glucopyranoside with the methyl 2-thioglycoside derivatives of the respective deoxy-N-acetylneuraminic acids, using dimethyl(methylthio)sulfonium triflate as a promoter, gave the four required 2-(trimethylsilyl)ethyl alpha-sialosyl-(2----3b)-beta-lactosides. These were converted via O-acetylation, selective removal of the 2-(trimethylsilyl)ethyl group, and subsequent imidate formation, into the corresponding alpha-sialosyl-(2----3b)-alpha-lactose trichloroacetimidates 15, 17, 19, and 21. Glycosylation of (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol with 15, 17, 19, and 21 in the presence of boron trifluoride etherate afforded the expected beta glycosides, which were transformed in good yields, via selective reduction of the azido group, coupling with octadecanoic acid, O-deacylation, and de-esterification, into the target compounds.


Regioselectivity of the insertion reactions of some aromatic diazo compound complexes with cyclomaltoheptaose.

Pyrolysis of solid complexes of aromatic diazo compounds with cyclomaltoheptaose (beta-cyclodextrin) yields either derivatives via insertion of carbene into hydroxyl groups. The distribution of the 2-, 3-, and 6-O-isomers indicates that the regioselectivity is moderate. The guest geometry is not as important as its size in determining the ratios of regioisomers. The origins of the regioselectivity are discussed.


Synthesis and conformational and NMR studies of alpha-D-mannopyranosyl and alpha-D-mannopyranosyl-(1----2)-alpha-D-mannopyranosyl linked to L-serine and L-threonine.

alpha-D-Mannopyranosyl and alpha-D-mannopyranosyl-(1----2)-alpha-D- mannopyranosyl linked to L-serine and L-threonine have been synthesised as model substances for the linkage region in certain O-linked glycoproteins. Metropolis Monte Carlo simulations were performed with a modified version of the GESA program, to yield theoretical NOEs and interatomic distances as ensemble-average values, and these were compared with results from steady-state NOE experiments. The NOEs were determined as ensemble-average and as global minimum values. NMR chemical shift differences, obtained for signals of the glycopeptides relative to those of the respective monomers, were interpreted in terms of short inter-residue atomic distances as found within the global minima, and on the basis of averaged distances derived from Monte Carlo simulations.


Syntheses of branched-chain sugars with push-pull functionality.

The reaction of methyl 4,6-O-benzylidene-3(2)-deoxy-alpha-D-erythro- hexopyranosid-2(3)-ulose with carbon disulfide, alkyl iodide, and sodium hydride gave methyl 4,6-O-benzylidene-3(2)-[bis(alkylthio)methylene]-3(2)-deoxy-alpha-D- erythro-hexopyranosid-2(3)-uloses. Methyl 4,6-O-benzylidene-2-[bis(methylthio)-methylene]-2-deoxy-alpha-D- erythro-hexopyranosid-3-ulose (5) reacted with aromatic amines to give, in a rearrangement process, N-aryl-2-aryliminomethyl-4,6-O-benzylidene-2-deoxy- alpha-D-erythro-hex-1-enopy-ranosylamin-3-uloses. The reaction of 5 with hydrazine hydrate afforded 5-methylthio-(methyl 4,6-O-benzylidene-2,3-dideoxy-alpha-D-erythro-hexopyranosido)[3,2- c]pyrazole.