Effects of 5-aza-2'-deoxycytidine and interferon-alpha on differentiation and oncogene expression in HL-60 myeloid leukemic cells.

The antineoplastic effects of 5-aza-2'-deoxycytidine (5-AZA-CdR) and interferon-alpha (IFN-alpha) on human HL-60 myeloid leukemic cells were investigated. 5-AZA-CdR and IFN-alpha in combination produced a greater inhibition of DNA synthesis and cell growth than either agent alone. The co-treatment produced a synergistic reduction in the clonogenicity of the HL-60 leukemic cells. In addition, this combination also produced a greater reduction in mRNA expression for c-myc than each agent alone. This antileukemic action produced by 5-AZA-CdR and IFN-alpha in combination correlated with the increase in induction of differentiation of the HL-60 leukemic cells. These results suggest that 5-AZA-CdR and IFN-alpha in combination produces an enhancement of their antineoplastic action on HL-60 human myeloid leukemic cells.


Influence of laminin or fibroblasts upon colony formation in the mouse by B16 melanoma cell spheroids: a morphometric analysis.

By microscopical observation and using an original morphometric method, we analyzed on histological sections the rate of lung colony formation after the intravenous injection into the mouse of B16 melanoma cells previously cultivated in vitro as aggregates. After the injection of B16 pure spheroids, superficial lung colonies were more numerous than internal lung colonies. After the injection of mixed spheroids (B16 + 3T3 fibroblasts), the size of colony sections was increased. Addition of laminin to pure or mixed spheroids decreased the size of colony sections but increased the number of internal lung colonies.


Effect of 15-deoxyspergualin, a microbial angiogenesis inhibitor, on the biological activities of bovine vascular endothelial cells.

We found recently that 15-deoxyspergualin, an analog of spergualin, which is an antibiotic and includes a spermidine moiety in its structure, exhibits anti-angiogenic activity. We have now carried out in vitro experiments with bovine vascular endothelial cells to determine which events occurring during angiogenesis are affected by this microbial angiogenesis inhibitor. 15-Deoxyspergualin did not inhibit the production of urokinase-type plasminogen activator (u-PA) or type IV collagenase by vascular endothelial cells. The direct inhibition of u-PA activity by 15-deoxyspergualin was not observed either. The angiostatic antibiotic neither affected the migration of vascular endothelial cells nor inhibited the endothelial cell proliferation in a two-dimensional culture system. We also examined the effect of 15-deoxyspergualin on the proliferation of endothelial cells in a three-dimensional culture system involving collagen gel, in which cell growth resembles more closely the endothelial cell proliferation during in vivo angiogenesis than that in a two-dimensional culture system without collagen gel. The antibiotic inhibited cell proliferation in a dose-dependent manner, indicating that the three-dimensional culture system is useful for finding a new angiogenesis inhibitor with a different mode of action from those of angiogenesis inhibitors found by using a two-dimensional assay system; however, no cause-effect relationship has yet been established. Taken together, these results suggest the possible involvement of the inhibition of vascular endothelial cell growth by 15-deoxyspergualin in its angiogenesis-inhibitory effect. 15-Deoxyspergualin appears to be a promising candidate as an angiogenesis inhibitor for controlling aberrant angiogenic responses occurring in different states, including tumor development.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of some natural products as components of Chinese herbal medicines on mammary gland growth and function in mice.

As a possible step to evaluate the role in the mammary glands of the scales of Guanshanjia, the fruits of Lulutong and the seeds of Wangbuliouxing, which are the natural products used widely as the components of herbal medicine in China for the improvement of lactation and the therapy of breast disorders, the effects of these agents on mammary gland growth and function were studied in female mice. The chronic administration of each agent in drinking water improved several parameters; however, the mode of action differed markedly among agents. The results indicate the necessity of acquiring enough knowledge of the characteristics of each agent for the efficient prescription of herbal medicine as well as the single use of the agent.


Protein composition of follicular fluid and oocyte cleavage occurrence in in vitro fertilization (IVF).

The present study was carried out from in vitro fertilization (IVF) attempts to analyze further the total and specific protein contents of 47 follicular fluids yielding one oocyte. The aim was to find correlations between the follicular concentrations of such proteins and the occurrence of coupled oocyte cleavage. These findings would be used as markers of IVF outcome. Two groups of follicular samples were distinguished: one group with cleavage occurrence (25 cases) and another group without cleavage or even fertilization (22 cases). In the group with cleavage a significantly higher level was observed for six proteins: C3 complement fraction and ceruleoplasmin (P less than 0.02), alpha-antitrypsin and transferrin (P less than 0.01), and alpha 2-macroglobulin and beta 2-microglobulin (P less than 0.001). The data are discussed with respect to changes in follicle permeability with advancing maturity.


Changes in PSA and early diagnosis of cancer of the prostate.

During the last four years approximately 400 men were evaluated in the early detection program for prostate cancer at The Ohio State University. Of these 400 men, 25 were found to have cancer. Eleven of the 25 had normal PSAs at the time cancer was diagnosed. The rise with a Hybritech assay, PSA of 1.2 ng/ml per year, one to two and subsequent years of greater than 1.2 ng/ml in this study indicates a higher likelihood of having a diagnosis of prostate cancer. Of the 25 patients who have been evaluated, only 4 patients had a subsequent drop of PSA from year one to two when the value dropped less than 0.6 ng/ml. Prostate intraepithelial neoplasia grade III has also been associated in these patients.


The single-nucleotide addition cycle in transcription: a biophysical and biochemical perspective.

This review has summarized the known features of the single-nucleotide addition reaction cycle in transcription. The reader will have noted that the information available is very incomplete, and that, in some cases, related experiments seem to lead to contradictory conclusions. We have tried to point out these discrepancies as they occur and to indicate areas where more experimentation is needed. We look forward to the day when all the microscopic steps of the single-nucleotide addition cycle can be identified and defined in thermodynamic, kinetic, and structural terms. At that point, we can begin to understand the principles that relate these parameters to template position and to the pathway of formation of a specific complex. It should be possible to provide specific molecular interpretations for observed effects on activation barrier heights to elongation and termination (154, 155) and to begin to understand the molecular bases of the regulation in these phases of transcription. Much work remains before this happy situation can be totally realized, but we feel that now the problem can at least be approached at this level. We hope that this review helps to illuminate the difficulties that remain.


A cell line that produces the glycoprotein hormone alpha-subunit contains specific nuclear factors similar to those present in thyrotropes.

A unique characteristics of thyrotrope-specific gene expression is the coordinated expression and regulation of the alpha- and beta-subunits of TSH. A cell line (alpha TSH) derived from the transplantable mouse thyrotropic tumor MGH101A, which no longer expresses the TSH beta-subunit gene but continues to secrete large amounts of alpha-subunit, was used as a model to study alpha-subunit gene expression independent from the TSH beta-subunit gene and was compared with the expression in TSH-secreting TtT97 tumors. Transient transfection studies showed a striking similarity in the activity of 5' deletions of the mouse alpha-subunit gene promoter in both alpha TSH and TtT97 cells and localized two regions important for expression that spanned 100 base pairs, from -480 to -417 and from -417 to -381. These regions were found to have no activity in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Analysis of the alpha-subunit 5' flanking DNA interactions with alpha TSH and TtT97 nuclear extracts showed two DNase I protected sequences, from -474 to -452 and from -447 to -400, both of which colocalized with the functionally important regions. Gel retardation analysis demonstrated the specificity of these interactions, and a similar migration of the DNA-protein complexes suggested that protein factors were similar in the two cell types. We conclude that the nuclear factors necessary for alpha-subunit expression in thyrotropes are retained in alpha TSH cells. Moreover, since alpha TSH cells do not express the TSH beta-subunit gene, the factors that determine the expression of the alpha-subunit may not be sufficient for TSH beta-subunit gene expression.


Matrix mineralization in hypertrophic chondrocyte cultures. Beta glycerophosphate increases type X collagen messenger RNA and the specific activity of pp60c-src kinase.

The phenomenon of chondrocyte hypertrophy is accompanied by the expression of type X collagen and the appearance of matrix mineralization. These events are also associated with changes in the phosphorylation of intracellular proteins. In this study the addition of 10 mM beta-glycerophosphate to hypertrophic chondrocytes resulted in stimulation of type X collagen synthesis up to 10 days in culture and an increase in the expression of type X collagen mRNA. This was followed by the onset of mineralization and the appearance of calcium hydroxyapatite. In contrast, the addition of beta-glycerophosphate to non-hypertrophic chondrocytes failed to induce expression of type X collagen or to produce changes in calcium and phosphate. The increased formation of type X collagen and of mineral in hypertrophic chondrocytes was accompanied by changes in the tyrosine kinase pp60c-src. While the level of c-src protein decreased approximately 2.5-fold in hypertrophic chondrocytes after 17 days of beta-glycerophosphate treatment, the specific activity of pp60c-src kinase increased approximately 3-fold in the cells that could be induced to mineralize but remained unchanged in cells that did not exhibit this property. Regulation of kinase activity may be an important event in endochondral ossification.


Adhesion molecule expression in human hepatic graft-versus-host disease.

An immunohistological study of the distribution of three cellular adhesion molecules, ICAM-1, VCAM-1 and ELAM-1, was undertaken on normal liver and liver biopsies taken from allogeneic bone marrow transplant (BMT) recipients. In normal controls, ICAM-1 was seen on vascular endothelium and sinusoidal lining cells, and VCAM-1 on Kuppfer cells and dendritic macrophages in portal tracts. ELAM-1 staining was virtually absent. Biopsies from BMT recipients with histological evidence of hepatic graft-versus-host disease (GVHD) showed ICAM-1 expression on damaged bile duct epithelium in only one of five cases, in contrast to four of five showing epithelial HLA-DR expression. Increased numbers of VCAM-1 positive portal tract macrophages were seen in GVHD and also in non-GVHD pathology. No increase in vascular endothelial expression of VCAM-1 or ELAM-1 was seen. These findings contrast with previous studies on other target sites for GVHD, namely skin and gastrointestinal tract, where the expression of all three molecules is increased on various cells. Although the lack of adhesion molecule expression in the liver in GVHD in this study may be related to the timing of biopsies or immunosuppressive therapy, it is likely to represent to some extent variation in cell and molecular changes occurring in the different tissues affected by GVHD.


Cartilage oligomeric matrix protein: a novel marker of cartilage turnover detectable in synovial fluid and blood.

Cartilage oligomeric matrix protein (COMP) is a tissue specific non-collagenous matrix protein. We have developed an enzyme-linked immunosorbent assay for the detection of this protein in synovial fluid and serum. The protein has been quantified in these fluids in patients with rheumatoid arthritis (RA), reactive arthritis, juvenile chronic arthritis, osteoarthritis and in sera of control subjects. The protein was detectable in all fluids and the synovial fluid levels were always higher than in serum in paired samples. The highest knee joint synovial fluid levels were found in reactive arthritis patients and the lowest in RA patients with advanced destruction of the knee joint. However, the relative synovial fluid content of COMP was higher in these RA patients than in patients with advanced osteoarthritis. In patients with long-standing reactive synovitis the concentrations decreased. This decrease, however, was less marked than for proteoglycan concentrations. The serum concentrations were low in patients with juvenile chronic arthritis and in patients with RA with advanced cartilage destruction of the studied knee joint. In the other groups serum levels did not differ between groups or from controls.


Effects of COMT inhibitors on striatal dopamine metabolism: a microdialysis study.

In vivo microdialysis was used to examine the effect of two new catechol-O-methyltransferase (COMT) inhibitors, Ro 40-7592 and OR-611, on extracellular levels of dopamine, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in rat striatum. The interactions of the COMT inhibitors with nomifensine, clorgyline and deprenyl were also studied. Ro 40-7592 (3-30 mg/kg, i.p.) decreased dose-dependently the efflux of HVA, increased that of DOPAC, and tended to increase that of dopamine. Higher doses of OR-611 (30-100 mg/kg, i.p.) also decreased the dialysate level of HVA, increased that of DOPAC, and tended to increase that of dopamine. Ro 40-7592 was about ten-fold as potent as OR-611. Neither of the COMT inhibitors changed dialysate levels of 5-HIAA. An OR-611 dose of 10 mg/kg i.p. had no significant effect, in contrast to Ro 40-7592, on any of the parameters studied; this dose was thus used to differentiate between the effects of central and peripheral COMT inhibition. Both nomifensine (15 mg/kg, i.p.) and clorgyline (4 mg/kg, i.p.) alone elevated extracellular dopamine levels, and lowered those of DOPAC and HVA, though there were quantitative and temporal differences between the drugs. L-Deprenyl (1 mg/kg, i.p.) alone had no significant effect on any of the compounds measured. Ro 40-7592 (10 mg/kg, i.p.) potentiated the effect of nomifensine on dopamine efflux, and it tended to increase clorgyline-induced dopamine efflux.(ABSTRACT TRUNCATED AT 250 WORDS)


Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death.

The role of inflammatory cytokines in the pathogenesis of neurological diseases is not well understood. The neurotoxic effects of cytokines could be mediated by immunostimulation of glial cells to produce toxic concentrations of nitric oxide (NO) and reactive nitrogen oxides. Cultured microglia and meningeal fibroblasts, but not Type 1 astrocytes, were induced by lipopolysaccharides and cytokines to synthesize NO and reactive nitrogen oxides from L-arginine. In co-cultures of immunostimulated microglia and cerebellar granule neurons, neurotoxicity was blocked by an inhibitor of NO synthase, NG-nitroarginine, and by oxyhemoglobin, which inactivates NO. Microglial-induced neurotoxicity was also partially attenuated by the N-methyl-D-aspartate (NMDA) receptor antagonists, MK-801 and 2-amino-5-phosphovalerate (APV). Superoxide dismutase, which stabilizes NO through inactivation of superoxide anion, augmented microglial-mediated neurotoxicity either alone or in combination with MK-801 or APV. Hence, immunostimulated microglia mediate neurotoxicity by NO, reactive nitrogen oxides, superoxide anion and NMDA-like substances. These findings suggest a novel role for microglial-produced NO and reactive nitrogen oxides as a neurotoxic agent in neurodegenerative disease states.


Tachykinins preferentially excite certain complex cells in the infragranular layers of feline striate cortex.

Microiontophoretically administered substance P (SP) affected the visually evoked responses (VER) and the spontaneous firing of 22 (14%) of the 152 neurons recorded from the striate cortex of anaesthetised cats. Enhancing effects were seen in 14 neurons and suppressant actions in 8 neurons. Most of the cells excited by SP were located in infragranular layers and had complex receptive fields; a few belonged to the movement-sensitive class or responded only weakly to visual stimulation. Of the neurons recorded in layer V, about 70% were excited by SP; the respective proportions were 8% in layer VI, and 2% in layer IV. Cells suppressed by SP had either simple or unimodal receptive fields including hypercomplex varieties; most of them were located in layer IVc. The effects of other tachykinins (neurokinin A, neurokinin B) and of the NK-3 receptor agonist Senktide tested in 36 cells were identical to those of SP with respect to types, and intracortical locations, of cells affected. During the enhancement induced by the tachykinins functional parameters of the neurons such as orientation and direction sensitivity were not substantially affected. It seems likely therefore that the effect of tachykinins in the primary visual cortex is not a shaping of receptive field properties, but rather a modulation of the general excitability of neurons projecting to subcortical centers, in particular to the midbrain and pons.


Ultrastructural morphology of projections from the medial geniculate nucleus and its adjacent region to the basal ganglia.

The efferent projections of the medial geniculate nucleus (MG) and its adjacent nuclei to the basal ganglia were studied in the rat by the antero- and retrograde tracing methods. Injections of wheat germ agglutinin conjugated to horseradish peroxidase into the caudal parts of the striatum and globus pallidus produced retrograde neuronal labeling in the medial division of the MG (MGm) and its adjacent structures including the suprageniculate, posterior intralaminar and peripeduncular nuclei, and substantia nigra pars lateralis. Injections of [3H]leucine into the MG and its surroundings resulted in anterograde labeling not only in the striatum but also in the globus pallidus. The resulting labeling was distributed exclusively in the caudal parts of these two nuclei. The electron microscopic autoradiography showed preferential radiolabeling of terminals and myelinated axons in both the globus pallidus and striatum. Labeled terminals in the pallidum mostly made symmetrical synapses on somata and major dendrites, while labeled terminals in the striatum established asymmetrical synapses on dendritic spines. These morphological differences in the synapses of the efferent systems originating from the MGm and its surrounding region suggest functional/chemical differentiations at their target sites in the basal ganglia.


Distribution of substance P-like immunoreactivity in guinea pig central nervous system.

The distribution of substance P-like immunoreactivity (SP-LI) in the guinea pig brain has been studied by immunohistochemistry and the results compared with the distribution in similar regions in the rat brain. In both species, dense SP-LI staining was found in the median eminence, arcuate hypothalamic nucleus, substantia nigra, dorsal raphe and dorsal tegmental nuclei, nucleus of the solitary tract, substantia gelatinosa of the spinal trigeminal nucleus, and spinal cord. Less dense staining was found in the caudate putamen, globus pallidus, nucleus accumbens, habenula, hypothalamic areas, and central grey. SP-LI cell bodies were found in areas previously described for the rat brain including several hypothalamic areas, limbic areas, central grey, and dorsal raphe and solitary tract nuclei. The major difference between the two species was found in the cortex and hippocampus. The guinea pig cortex contained many more SP-LI cells and fibres, distributed in layers II-VI, than the rat cortex. The guinea pig hippocampus contained marked staining, particularly in the pyramidal cell layer of CA1-3 fields of Ammon's horn and in the granular layer of the dentate gyrus, and SP-LI cells in the hilus of the dentate gyrus, whereas rat hippocampus contained few cells and no regions of dense staining. It is concluded that because the guinea pig brain has an extensive distribution of SP-LI in the cortex and hippocampus it resembles the primate brain more closely than does the rat brain.


Supraoptic nucleus afferents from the accessory olfactory bulb: evidence from anterograde and retrograde tract tracing in the rat.

Our earlier electrophysiological work provided evidence of a direct input to the supraoptic nucleus (SON) from the olfactory bulbs; however, these experiments could not determine if the input originated in the main and/or accessory portions of the olfactory bulb. Here, a connection between the accessory olfactory bulb (AOB) and the SON of the rat was examined using a combination of anatomic techniques. We employed neurophysin immunocytochemistry to delineate the morphological boundaries of the SON and the proximal arborizations of supraoptic dendrites. Accessory olfactory bulb efferents to the SON were studied by injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the AOB. The distribution of retrogradely labeled cells within the AOB was also determined after injection of either rhodamine-labeled latex microspheres (rhodamine beads) or Fluoro-Gold (FG) into the SON. Neurophysin immunocytochemistry revealed that SON dendrites extended beyond the generally accepted boundaries of the nucleus, coursing ventrolaterally along the surface of the periamygdaloid cortex. Anterograde tract tracing with WGA-HRP labeled AOB efferents including a dense plexus of terminals and fibers around the ipsilateral SON along the path of the ventrally projecting dendrites. Injections of retrograde tracers into the SON resulted in rhodamine bead or FG labeling of mitral cells throughout the ipsilateral AOB. Taken together, these anatomic studies suggest a direct projection from the accessory olfactory bulb to the SON of the rat and thus a vomeronasal organ to SON pathway.


Hypothalamo-spinal pathways and responses to photoperiod in Syrian hamsters.

Descending projections from the hypothalamic paraventricular nucleus (PVN) to the spinal cord mediate the effects of photoperiod on the reproductive system of hamsters. To elucidate the course of these PVN efferent fibers, injections of horseradish peroxidase conjugated to either cholera toxin or wheat germ agglutinin were made into the T1-C7 region of the spinal cord of hamsters. Four sets of descending tracts were identified in males and females. Two sets of fibers originated from the medial PVN and exited the nucleus dorsally and ventrally, respectively. Most of the descending fibers, however, organized themselves into two tracts that exited the PVN laterally. In earlier experiments, destruction of these lateral pathways prevented photoperiodic responses in hamsters of both sexes.


Mineralization and bone formation on microcarrier beads with isolated rat calvaria cell population.

Using enzymatically isolated rat bone cells in the presence of cytodex microcarrier beads, osteoblastic cell differentiation and bone nodule formation were studied at the optical and electron microscopic level. Cytochemical method showed an intense alkaline phosphatase activity mainly around the microcarriers where the cells have formed multilayers on day 4 of cultures. On day 7 of experiment cultures, Von Kossa method stained positively only the cytodex microcarriers. During the following days, bone nodule formation was closely associated with cytodex microcarriers. In contrast, in control cultures with negatively charged glass beads, cells failed to pile up around the glass beads, and bone nodule formation occurred randomly in the culture dishes with 24 hour delay. Light microscopy observations of experiment cultures revealed the formation of nodular structures, with active osteoblastic cells forming a mineralized matrix in which osteocytes were present. Transmission electron microscopy revealed first, a mineralization process of the surface of the cytodex microcarriers which appeared like a granular electron-dense, collagen-free layer followed by the deposit of a collagenous matrix. These results indicated that cytodex microcarriers provided an excellent matrix for bone cell differentiation and mineralization.


Elevated serum alpha-fetoprotein levels in primary gallbladder carcinoma without hepatic involvement.

Elevated serum alpha-fetoprotein (AFP) levels, although frequently associated with hepatocellular carcinoma, have also been reported in cases of primary gallbladder carcinoma. The case of a 56-year-old man with a markedly elevated serum AFP level and primary gallbladder carcinoma without detectable hepatic involvement is reported. The patient had right upper quadrant abdominal pain and a palpable right upper quadrant mass. Computerized tomography scan of the abdomen showed a large, subhepatic mass consistent with the gallbladder, and normal liver parenchyma. Liver enzyme levels were normal. Management included cholecystectomy followed by postoperative radiation therapy to the gallbladder bed and portal areas and systemic chemotherapy. At the end of therapy, the AFP level had returned to normal. Gallbladder carcinoma is rarely diagnosed before surgery, which sometimes inhibits operative planning. AFP may be a useful preoperative tumor marker in differentiating the patient with primary gallbladder carcinoma from the patient with gallbladder hydrops.


Tumor-associated antigen 43-9F is of prognostic value in squamous cell carcinoma of the lung. A retrospective immunohistochemical study.

Squamous cell lung carcinoma (SLC), the most frequent type of lung cancer, generally is treated surgically and its prognosis is poor. The only current clinically useful prognostic criterion is lymph node staging (TNM classification). Expression of a novel tumor-associated carbohydrate epitope Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3 Gal beta 1-4Glc identified by the 43-9F monoclonal antibody (MoAb) is associated with the growth pattern of SLC cell lines in athymic mice and in vitro. This implies that the 43-9F epitope may be related to tumor progression in patients with SLC and that, as such, it could be of prognostic value. Primary tumor specimens from 231 patients with lung carcinoma (130 with SLC, 64 with adenocarcinoma, 10 with small cell carcinoma, 16 with large cell carcinoma, and 11 with adenosquamous carcinoma) were examined by immunohistochemical studies on formalin-fixed, paraffin-embedded tissue samples for immunoreactivity with an MoAb to the 43-9F antigen. Univariate and step-wise Cox regression analyses were used to compare survival time by histopathologic diagnosis, smoker status, TNM classification, and type of surgical treatment. Patients with 43-9F epitope-positive SLC tumors had a significantly (P less than 0.01) better prognosis than patients with epitope-negative tumors. In contrast, no association was seen between 43-9F epitope expression and survival time for patients with lung adenocarcinomas. Further, the prognostic value of 43-9F expression in SLC was found to be superior to the N-classification with the added advantage that it requires access only to primary tumor tissue and thus is available before therapy.


A phase II trial of biochemical modulation using N-phosphonacetyl-L-aspartate, high-dose methotrexate, high-dose 5-fluorouracil, and leucovorin in patients with adenocarcinoma of unknown primary site.

5-Fluorouracil (5-FU) has modest activity as a single agent in a number of human adenocarcinomas. The technique of biochemical modulation has been used preclinically to increase the activity of 5-FU. With doses based on a Phase I study, the authors performed a Phase II trial in patients with advanced metastatic adenocarcinoma of an unknown primary site using N-phosphonacetyl-l-aspartate (PALA), methotrexate (MTX), 5-FU, and leucovorin. In some patients, 5-phosphoriosyl-l-pyrophosphate (PRPP) and uridine triphosphate (UTP) metabolite pools were assayed before and after PALA and MTX to assess metabolite pool shifts. Twenty-one patients were treated in this Phase II trial. Toxicity was tolerable. Biopsy specimens of tumor tissue showed increases in PRPP and decreases in UTP levels in two of three and three of four patients, respectively. However, only one objective response was seen, which is not different from that expected with 5-FU alone. Expected PRPP and UTP metabolite pool shifts were seen when biochemical modulation was performed with these drugs according to this schedule. Because toxicity was mild, more intense dose schedules of 5-FU are planned for future studies.


Neuroblastoma. Correlation of neuropeptide expression in tumor tissue with other prognostic factors.

The neuropeptide contents in neuroblastomas were quantified by radioimmunoassay (RIA) to assess their possible biologic significance. Neuroblastoma tumor tissue was obtained from the primary tumor site before therapy in 16 patients with newly diagnosed neuroblastoma and in 7 patients with central nervous system medulloblastomas or gliomas. The tumor tissue was assayed for vasoactive intestinal peptide (VIP), somatostatin (SRIF), substance P, and neurotensin by both immunostaining and RIA techniques. Increased VIP levels of 1.2 pg/micrograms DNA or more correlated significantly with cellular differentiation (P = 0.003) and favorable disease stage (P = 0.002) in neuroblastomas. Increased SRIF contents (greater than 1.3 pg/micrograms DNA) correlated with differentiation of the tumor (P = 0.002). Increased VIP and SRIF content did not correlate with N-myc oncogene expression or ras oncogene protein immunostaining. No VIP was detectable in brain tumors, and other neuropeptides were variable in content. RIA of VIP and SRIF levels in primary tumor tissue may offer an independent objective assay of biologic behavior in neuroblastoma biopsy specimens.


Acute phase reactants in predicting disease outcome.

From the studies which are reviewed above, it is generally apparent that in terms of the acute phase response, the initial findings in early inflammatory arthritis (particularly rheumatoid arthritis, with which the majority of such studies are concerned) have little predictive value for either the functional outcome or mortality. The wide interindividual variability in these measurements is also likely to limit their clinical usefulness as predictors of disease outcome. The trend in certain acute phase reactants may be more useful in indicating disease activity, although the number of satisfactory studies in this area is very limited.


Transcription in maize mitochondria: effects of tissue and mitochondrial genotype.

Mitochondrial run-on assays were used to determine transcriptional rates for nine B37(N) maize mitochondrial genes. Quantitation by radiographic imaging detected a 15-fold range in transcriptional rates; the order of apparent promoter strength was rps12 greater than rrn26 greater than atp6 greater than rrn18 greater than cox2 greater than atp alpha greater than atp9 greater than cox3 greater than cob. By probing single-stranded DNAs of both polarities with the run-on-products we showed that gene-specific antisense transcription did not occur. We also tested whether relative transcriptional rates were dependent on either the mitochondrial genotype or the tissue from which the mitochondria were isolated. Although tissue-specific differences in transcriptional rates were not detected, significant variation in apparent promoter strength for at least one gene, rps12, was dependent on the cytoplasmic genotype; rps12 had a five-fold reduced transcriptional rate in B37(T), the Texas male cytoplasmic strain of maize. Pulse-chase experiments suggested that differential transcript stability was not a major determinant of steady state mitochondrial RNA levels. These results indicate not only that promoter strength is an important component of the regulation of transcript levels in maize mitochondria, but also that the strength of a specific gene promoter can be dependent on the cytoplasmic genotype. Finally, the high transcriptional rate of both ribosomal RNA genes and the one mitochondrially encoded ribosomal protein gene studied suggests coordinate transcriptional regulation of both RNA and protein components of the mitochondrial ribosome.


Amino acids and peptides. XXXIII. Synthesis of N-terminal epitope peptides of mammalian metallothioneins (MTs).

In order to determine the fine structure of the mammalian metallothionein (MT) epitope to a monoclonal anti-rat Zn-MT-II antibody (MT 189-14-7), N-terminal peptides of various lengths of mammalian metallothioneins (MTs) were synthesized by a conventional solution method using the newly developed beta-2-adamantylaspartate, and their immunological properties were examined. It was found that the N-terminal acetyl group was indispensable for the reaction with the monoclonal antibody and the N-terminally acetylated pentapeptide, Ac-Met-Asp-Pro-Asn-Cys-OH, was the smallest peptide which exhibited a significant reactivity with the antibody.


Synthesis and anti-human immunodeficiency virus type 1 (HIV-1) activity of 3-substituted derivatives of 3'-azido-3'-deoxythymidine (AZT), and inhibition of HIV-1 reverse transcriptase by their 5'-triphosphates.

Various 3-substituted 3'-azido-3'-deoxythymidine analogs (2a-i) were prepared by the reaction of 3'-azido-3'-deoxythymidine (1), AZT with N,N-dimethylformamide dialkylacetal or alkyl bromide in the presence of base and their activities against human-immunodeficiency virus type-1 (HIV-1) were evaluated. The corresponding 5'-triphosphate analogs (9) were also synthesized in order to examine inhibition of HIV-1 reverse transcriptase activity. Beyond expectation, some N3-derivatives of AZT were found to reserve the anti-HIV-1 activity to some extent. Among the compounds (2a-i) obtained, 3-allyl-AZT (2e) was the most active against HIV-1 replication in MT-4 cells in vitro with an EC50 value of 0.9 microM. 3-Allyl-AZT 5'-triphosphate (9e), however, exhibited no inhibition of HIV-1 reverse transcriptase activity.


Antiandrogen. I. 2-azapregnane and 2-oxapregnane steroids.

2-Azachlormadinone acetate (5a, 17-acetoxy-6-chloro-2-azapregna-4,6-diene- 3,20-dione), 2-oxachlormadinone acetate (6, 17-acetoxy-6-chloro-2-oxapregna-4,6-diene-3,20-dione) and the derivatives were prepared as potential antiandrogenic agents. Biological evaluation showed that 5a and 6 had a potent antiandrogenic activity when tested in the castrated male rat.


Flow cytometric measurement of intracellular bilirubin in human peripheral blood mononuclear cells exposed to unconjugated bilirubin.

Human peripheral blood mononuclear cells were incubated at 37 degrees C with bilirubin in bovine albumin solution. Histological analysis of bilirubin-treated cells demonstrated a prominent brown pigment deposited in the cytoplasm. Homogenates of these cells in chloroform-methanol solution showed an identical absorption spectrum with pure bilirubin dissolved in the same solution. When bilirubin-treated cells were excited at 488 nm, a significant autofluorescence was detected by flow cytometry at 585 nm in a dose-dependent manner, which had a significant correlation with the amount of bilirubin chemically extracted from the cells (r = 0.963, n = 34, p less than 0.001). Intraassay and interassay variability of the autofluorescence by flow cytometric analysis was small (both less than 5%). When bilirubin-treated cells were stained with fluorescein-labeled anti-CD14 antibody, the CD14 positive cell population can be fractionated without interference of autofluorescence derived from intracellular bilirubin. These results suggest that flow cytometric analysis of bilirubin-treated cells can quantitate intracellular bilirubin, and that bilirubin does not interfere with analysis of surface antigens.


Macroamylases: differences in activity against various-sized substrates.

Hyperamylasemia caused by macroamylases can lead to the overdiagnosis of acute pancreatitis. We examined whether interference from macroamylase is less in assays that use high-molecular-mass (high-M(r)) substrates rather than oligosaccharide substrates. We hypothesized that high-M(r) substrates would be sterically excluded from macroamylasemic complexes and thus would be hydrolyzed less efficiently. Eighteen macroamylasemic samples were assayed by using red-dyed amylopectin or blue-dyed starch as polysaccharide substrates or by using maltoheptaose or maltotetraose as oligosaccharide substrates. The oligosaccharide substrates gave comparable results (y = 0.81x + 83), but we observed consistently lower activities for amylopectin than for maltotetraose (y = 0.32x + 38). We observed no bias among methods when nonmacroamylasemic specimens were analyzed. The mechanism of this difference was examined by adding antihuman pancreatic amylase antibodies to hyperamylasemic serum samples from patients without macroamylasemia and to purified human pancreatic or salivary isoamylases. In each case, polyclonal and monoclonal antibodies lowered amylase activity more in assays with complex polysaccharides than in those with oligosaccharides. The use of high-M(r) substrates diminishes interference, and detection of suspected macroamylasemia may be possible through comparing activities determined from automated methods that use different substrates.


Influence of a serotonin- and dopamine-rich diet on platelet serotonin content and urinary excretion of biogenic amines and their metabolites.

Using high-performance liquid chromatography and gas chromatography, we reevaluated the 24-h influence of a serotonin- and dopamine-rich diet on platelet serotonin and serotonin, 5-hydroxyindoleacetic acid (5-HIAA), and major catecholamine metabolites in the urine of 15 healthy adults. Although there were significant responses in urinary free serotonin and catecholamine metabolites, their concentrations did not exceed the upper limits of the reference ranges for any of the participants. For urinary 5-HIAA, pronounced effects were observed within 2-4 h. After 6-8 h, results for 11 participants exceeded the upper limit of the reference range. The median recovery of dietary serotonin as urinary 5-HIAA was 20% and subject to a large range (1-50%). There was no significant change in platelet serotonin. We conclude that, using specific analytical methods, no dietary restrictions need be imposed to diagnose catecholamine (metabolite)-producing tumors. For diagnosis of carcinoids on the basis of urinary 5-HIAA it is appropriate to completely abstain from serotonin-containing foods for greater than or equal to 12 h before testing. Platelet serotonin is a more sensitive marker for carcinoids that produce only small amounts of serotonin, and is unaffected by dietary serotonin.


Detection of antithyroglobulin autoantibodies with defined epitopic specificity by a microparticle-enhanced nephelometric immunoassay.

To hydrophilic, polyfunctional spherical microparticles of predetermined diameter, produced by copolymerization of acrylic monomers, we covalently bound human thyroglobulin. The thyroglobulin-microsphere conjugate was agglutinated, in the presence of antimouse immunoglobulins antiserum, by four monoclonal antibodies, each recognizing a different antigenic domain on the thyroglobulin molecule. These agglutinations were quantified by measuring with a specially designed nephelometer the light scattered by clusters of the conjugates. Agglutination with the monoclonal antibody recognizing antigenic domain II of the thyroglobulin molecule was specifically inhibited by some human sera that contained antithyroglobulin autoantibodies. This allowed us to develop a microparticle-enhanced nephelometric immunoassay for these autoantibodies with defined epitopic specificity. Using this assay, we detected and quantified antithyroglobulin autoantibodies in serum samples from all eight patients examined with Hashimoto disease and from most (75%) patients with untreated Graves disease.


Rhabdomyosarcoma of the head and neck in children.

Twenty-two children, 9 male and 13 female, with a non-orbital rhabdomyosarcoma of the head and neck, treated between 1970 and 1988, have been reviewed. Since 1972, treatment has consisted of combination chemotherapy, and where necessary radiotherapy and/or surgery. Complete clinical remission after initial chemotherapy was observed in 21 children. Six children were cured after primary treatment but 15 developed recurrent disease. Thirteen children had a parameningeal localized tumour, with eventual meningeal involvement in 5. Survival in this group was worse than in the non-meningeal group. Lymph node metastasis at first presentation (5 patients) had no influence on prognosis, whereas development of lymph node metastases during follow-up resulted in 100% mortality. All patients were retrospectively classified according to both the IRS-classification and TNM-descriptive system. No correlation with either system could be established. Fourteen of 15 children with recurrent disease were treated, 4 of whom were cured. Thus, 10 out of 22 (45%) children were long-term survivors.


The effects of diuretics on nasal transmucosal potential difference.

The effects of application of frusemide, amiloride and bumetanide on nasal transmucosal potential difference (NTPD), at rest and during exercise, were studied in 8 normal subjects. In a double-blind placebo controlled study, 8 volunteers had NTPD recorded at 4-min intervals during 12-min periods of rest, before and after treatment, during 12 min of exercise, and recovery. Application of placebo, frusemide and bumetanide did not significantly alter NTPD at rest. Amiloride caused a significant reduction (P less than 0.025). During exercise there was a significant rise in NTPD with placebo (P less than 0.05), frusemide (P less than 0.05) and amiloride (P less than 0.05). There was no increase in NTPD during exercise with bumetanide.


Neurodevelopmental outcome in infants with bronchopulmonary dysplasia.

It appears that the survivor of BPD is at risk for neurodevelopmental compromise but not necessarily to any greater extent than are prematurely born infants in general. What appears to be true is that as the neonatal course becomes more complicated, and the birth weight drops, the risk increases. If a BPD survivor sustains a moderate to severe IVH, particularly if accompanied by periventricular leukomalacia, the risk of significant handicap increases substantially. Effects of socioeconomic status (SES) are also important, and become more evident over time with a direct correlation between SES and outcome. Maximizing the environmental conditions while in the special care nursery, and reducing stress on the infant during the illness, may help to reduce risk of a compromised developmental outcome in the survivors. Careful monitoring of oxygen saturation postdischarge may similarly have a positive effect on outcome. Although direct comparability among studies is not possible, it appears that most reports suggest about half the survivors are free of any handicapping condition at follow-up, and about half are either moderately or severely impaired. Cerebral palsy is the most frequently reported handicapping condition. Since the presence or absence of significant IVH is not consistently reported across studies, it is not possible to know if infants who subsequently develop CP sustained a bleed, in addition to BPD, in the neonatal period. In studies in which this is more carefully delineated, that appears to be the case. In studies in which infants with significant hemorrhage were either excluded or dropped out, no survivors with cerebral palsy are reported. Approximately 4% of survivors across all studies reviewed were blind as a result of severe retinopathy of prematurity. Several studies excluded infants who developed significant ROP from their follow-up, so this percentage may be an underestimate of the actual incidence across all BPD survivors. With a greater number of gestationally younger infants surviving, this is one area of handicap that may increase in the years to come. Not all studies report on the presence of sensorineural hearing loss in survivors, but there is the suggestion this may occur perhaps in up to 4% of survivors. This is an area that future research should address. For infants who fall in the moderately handicapped category, typically defined as more than one standard deviation below the mean on a standardized developmental examination, that rating may change over time in either direction.(ABSTRACT TRUNCATED AT 400 WORDS)


Effect of various polyamino acids and D- and L-amino acids on the blood fibrinolytic system.

1. Comparative additional effects of 24 commercially obtained amino acids and their derivatives were studied by the whole blood clot lysis time (WBCLT) method. D-Arg and L-Lys (greater than 50 micrograms/ml) activated the fibrinolytic system, and poly-L-Glu (mol. wt 6000-90,000) (less than 50 micrograms/ml) were less effective. 2. Poly-L-Arg, poly-L-Lys and poly-(Lys-Ala-Glu-Tyr) accelerated the TPA inhibition in the presence of human plasma. 3. For in vivo experiments, 5 mg of poly-L-Glu were given intravenously to 10 rats. 4. The shortening of WBCLT and elevation of EFA (P less than 0.01) were found after 1 hr administration. 5. The main enzymes which increased in plasma were proved to be the endogenous plasminogen activators with mol. wt higher than 70,000, by zymography.


Platelet immunology. ELISA for detection of platelet antibodies, platelet-specific antigens and platelet glycoproteins.

The development of an enzyme linked immuno-sorbent assay (ELISA) for detection of platelet antibodies and platelet specific-antigens has been described. In the assay, intact platelets fixed to the bottom of microplates have been used as targets. After incubation with serum, the attached antibody has been detected by antiglobulin serum labelled with enzyme followed by assay of the enzyme reaction with its substrate. The clinical significance of platelet antibodies in alloimmune neonatal thrombocytopenia (AINT), post-transfusion purpura (PTP), and refractoriness to platelet transfusion therapy has been described. The clinical significance of autoantibodies in autoimmune thrombocytopenic purpura (AITP) and the detection of platelet surface glycoproteins by a modification of the platelet ELISA has also been described. In addition, a survey of the present state of knowledge of human platelet immunology, especially platelet-specific alloantigens and alloantibodies has been given. The studies of AINT and PTP have revealed a human immune response gene located in the MHC region: The susceptibility to anti-Zwa alloimmunization is strongly associated with the HLA-B8 and even more DR3 and DRw52a tissue types. In the studies of PTP, the first example of PTP due to anti-Zwb (only detectable in the platelet ELISA) has been described, and an association of the presence of anti-Zw antibodies of IgG3 subclasses (defined in the platelet ELISA using monoclonal mouse antibodies) and destruction of autologous platelets has been described. In an investigation of Ig classes and IgG subclasses of platelet associated Ig (PAIg) in children suffering from autoimmune thrombocytopenic purpura, no correlation was found between the immunochemical properties of the PAIg and the clinical course of the disease. However, a correlation between increased amounts of PAIgG3 and very low platelet counts was observed. A modification of the platelet ELISA using monoclonal antibodies against glycoproteins (GP) has been described. Using antibodies against GPIb and GPIIb/IIIa, it has been possible to diagnose Glanzmann's thrombasthenia (GT) and Bernard-Soulier syndrome (BSS). Especially in the diagnosis of thrombocytopenic patients suspected for BSS where platelet aggregation studies has been impossible due to low platelet counts, the detection of decreased amounts of surface GPIb in ELISA has been of great value. In conclusion, the platelet ELISA has been found simple and sensitive, and suitable for routine investigations of AINT, PTP, AITP, and refractoriness to platelet transfusion.


Psychosis and idiopathic precocious puberty in two 7-year-old boys.

The authors present case reports of psychosis and idiopathic precocious puberty (IPP) with tall stature in two 7-year-old boys. Other similarities in the two cases are discussed. Review of the literature revealed only one report regarding the role of accelerated sexual maturation in the clinical picture of psychoses in children and one case report of Pervasive Developmental Disorder and IPP in a 5-year-old girl. Possible mechanisms by which psychosis and IPP with tall stature may be related are discussed.


Chromosome detection by in situ hybridization in cancer cell populations which were flow cytometrically sorted after immunolabeling.

We examined the feasibility of performing non-radioactive in situ hybridization (ISH) in flow cytometrically sorted (tumor) cells with a chromosome #1 specific centromere probe. The study was performed in a model system of HL60 cells mixed with different quantities of HeLa cells. These latter cells were sorted directly onto poly-l-lysine coated glass slides on the basis of their keratin content, a cytoskeletal component not present in HL60 cells. Overall morphology of the separated HeLa cells was excellent and, after the ISH procedure, the appropriate number of ISH spots was observed in more than 85% of the sorted cells. This percentage did not differ significantly in cell mixtures with different percentages of HeLa cells (down to 1%). Sorting of HeLa cells in different phases of the cell cycle, and subsequent ISH, revealed the same spot number for chromosome #1 in all cell cycle stages, including mitosis. In the latter phase of the cell cycle we did not find a duplication of the chromosome #1 centromere, not even after sorting of the mitotic cells on the basis of specific labeling with an antibody to mitotin. The early G2 mitotin negative fraction, however, showed a significant percentage of cells with a duplicate spot number, most likely representing a tetraploid cell fraction in this HeLa cell culture. The protocol that evolved from these model studies was applied to cell suspensions of malignant body cavity effusions as well as solid bladder carcinomas. In several of these cases numerical chromosome aberrations could be detected by ISH more evidently after sorting on the basis of keratin labeling.


Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.


Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.


Multiple myeloma. New treatment options.

Before proceeding to treatment selection for multiple myeloma, it is important to exclude monoclonal gammopathy of unknown significance or any similar smouldering or indolent type of myeloma not requiring immediate chemotherapy. In patients with active myeloma, pretreatment prognostic classification is important for appropriate balancing of the risks and benefits of particular treatment options. For example, conventional chemotherapy such as melphalan/prednisone may be appropriate for an elderly patient with active stage III myeloma, whereas high dose chemotherapy with or without bone marrow transplantation or cytokine support may be considered for the patient under age 45 with even earlier stage disease. A variety of options are now available for patients with relapsing or resistant disease, particularly using agents with potential for reversal of multi-drug resistance. The use of interferon-alpha as maintenance following initial response to chemotherapy is important for prolongation of remission, duration and potentially survival. A variety of supportive measures are also helpful including the use of epoetin (erythropoietin) to improve refractory anaemia and bisphosphonates for the inhibition of ongoing bone resorption. With the availability of various treatment options, it has become important for patients to be evaluated by a specialist in the field.


Newer neuromuscular blocking drugs. An overview of their clinical pharmacology and therapeutic use.

Four new nondepolarising muscle relaxants, pipecuronium bromide, doxacurium chloride, mivacurium chloride and Org 9426 (rocuronium) offer alternatives to the established agents atracurium besilate and vecuronium bromide. Pipecuronium and Org 9426 are steroidal compounds, the latter being a monoquaternary agent, whereas doxacurium and mivacurium are bisquaternary benzylisoquinolinium compounds. Pipecuronium and doxacurium have a relatively slow onset and a long duration of action. Pipecuronium produces maximum block in 3 to 6 min when given in a dose of 45 to 80 micrograms/kg, and a duration of clinical relaxation of between 40 and 110 min. Doxacurium is more potent, but is the least rapid and the longest acting relaxant currently available. When administered in doses of 37 to 80 micrograms/kg, it produces maximum block within 3.5 to 10 min, with a duration of clinical relaxation of 77 to 164 min. The advantage of both pipecuronium and doxacurium is their cardiovascular stability. Both agents are primarily eliminated via the kidneys and both have now been licensed for use in the US. Mivacurium is a muscle relaxant with a short duration of action. When administered in doses of 0.1 to 0.25 mg/kg it produces maximum block in 2 to 4 min, but the duration of clinical relaxation is less than 20 min. Higher doses which could induce a faster neuromuscular block are unfortunately associated with some histamine liberation. The drug is metabolised by plasma cholinesterase. Mivacurium has also been licensed for use in the US. Org 9426 is an agent with a rapid onset but an intermediate duration of action. A dose of 0.5 to 0.6 mg/kg induces maximum block in about 1.5 min and has a duration of clinical relaxation of about 30 min. The rapid onset of effect could be useful for early intubation as an alternative to suxamethonium chloride. However, much more clinical experience is needed with this agent, particularly with regard to duration of action of larger doses and occurrence of side effects. The drug is mainly eliminated via the liver.


Drug treatment of the irritable bowel syndrome.

Irritable bowel syndrome (IBS) is defined as a functional bowel disorder in which abdominal pain is associated with defecation or a change in bowel habit, and with features of disordered defecation and distension. The irritable bowel syndrome occurs in 10 to 20% of people worldwide and is very commonly encountered in clinical practice. This has encouraged the pharmaceutical industry to search for effective drug therapy. So far, a universally effective agent has not been found, and since this is a chronic, benign disorder, beginning in youth, long term drug use should be avoided. Nevertheless, if a specific IBS symptom, such as constipation or abdominal pain dominates, a specific drug may be helpful. However, tests and treatment should be minimised or even avoided in order to do no harm. A largely nonpharmaceutical approach to IBS should be taken. This approach employs drugs sparingly and then only targeted at specific and resistant symptoms.


Effective treatment of urethritis. A practical guide.

Most cases of urethritis can be readily treated using recommended regimens. The most important causes of urethritis are Chlamydia trachomatis and Neisseria gonorrhoeae, and initial treatment is directed at them. Optimal management requires obtaining a thorough sexual history, evaluation for objective clinical and laboratory evidence of infection, antimicrobial therapy directed towards the major aetiologies, and evaluation and treatment of sexual partners. Treatment of gonorrhoea requires a single-dose regimen active against N. gonorrhoeae, plus a regimen active against C. trachomatis and nongonococcal urethritis. The usually recommended treatment for N. gonorrhoeae is a single dose of ceftriaxone 250mg intramuscularly, but there are many alternatives, including oral ones. Only in very restricted geographical areas and under restricted situations are penicillins still reliable against N. gonorrhoeae. Recommended optimal treatment of C. trachomatis or nongonococcal urethritis currently requires 7 days' treatment with a tetracycline. Some guidelines now propose ofloxacin 300 mg orally twice daily for 7 days as an equivalent alternative, and there are very promising data with a single dose therapy with azithromycin, a long-acting macrolide antimicrobial. Using recommended regimens, microbiological failure is infrequent in compliant patients. Recurrent urethritis is, however, frequent. For patients who receive recommended treatment and do well, no follow-up cultures are needed. Patients with persistent or recurrent symptoms require careful re-evaluation of the patient, documentation of urethritis, and retreatment with antimicrobial agents a second time if urethritis is documented by positive cultures or increased numbers of polymorphonuclear leucocytes in urethral secretions.(ABSTRACT TRUNCATED AT 250 WORDS)


Diabetes insipidus. Current treatment recommendations.

Cranial diabetes insipidus (DI) arises when release of arginine vasopressin (AVP, antidiuretic hormone) in response to osmotic stimuli is inadequate. The correct diagnosis and management of cranial DI is particularly important when it arises as an acute complication of surgery, trauma or in subjects who lack thirst sensation. Desmopressin (1-desamino-8-D-arginine-vasopressin, DDAVP) provides an effective and convenient replacement therapy when given by the intranasal route. However, nasal administration is difficult for some patients, and in the future oral or transcutaneous desmopressin formulations may prove to be satisfactory alternatives. By contrast, treatments for nephrogenic DI, where there is failure of the antidiuretic response to endogenous or exogenous vasopressin, have been disappointing and water replacement remains the mainstay of therapy. An understanding of the physiology and pathophysiology of water homeostasis and correct interpretation of water balance and electrolyte data are essential for correct diagnosis and management of all cases of DI.


Lansoprazole. A review of its pharmacodynamic and pharmacokinetic properties and its therapeutic efficacy in acid-related disorders.

Lansoprazole is an effective acid pump inhibitor acting at the final enzymatic step of the acid secretory pathway of the parietal cell, decreasing gastric acid secretion regardless of the primary stimulus. Results of short term (less than 8 weeks) clinical trials have shown lansoprazole to be significantly superior to placebo and ranitidine in the treatment of duodenal ulcer, both in the rate of healing and in overall healing at 4 weeks. Lansoprazole appears to heal duodenal ulcer more quickly than famotidine, and demonstrates slightly greater efficacy at 4 weeks, although both drugs appear to have equivalent efficacy overall. Gastric ulcers and reflux oesophagitis are also healed by lansoprazole 30 mg/day for 4 to 8 weeks, with healing rates after 8 weeks of approximately 85 to 95% for both indications. Lansoprazole appears to be superior to ranitidine and comparable to omeprazole in treating reflux oesophagitis. Furthermore, lansoprazole has relieved reflux symptoms more quickly than either ranitidine or omeprazole. Preliminary data also indicate that lansoprazole may be effective in the treatment of peptic ulcer disease and reflux oesophagitis refractory to H2-receptor antagonists, and in patients with Zollinger-Ellison syndrome. While direct comparisons with omeprazole are limited, results suggest that lansoprazole, used for short term treatment, is at least as effective as omeprazole in the treatment of peptic ulcer and reflux oesphagitis. Lansoprazole has been well tolerated in short term clinical trials, with an incidence of adverse effects comparable with that of other agents in its therapeutic class. Trials assessing long term tolerability data are ongoing and will be required as part of the assessment of the safety profile, if lansoprazole is to be used prophylactically to prevent ulcer recurrence. Thus, by virtue of its ability to heal ulcers and rapidly relieve associated symptomatology, lansoprazole represents a useful alternative for the treatment of acid related disorders.


Effects of substance P on extracellular dopamine in neostriatum and nucleus accumbens.

Microdialysis was used to monitor changes in dopamine release in the neostriatum and nucleus accumbens after peripheral administration of substance P in freely moving rats. Substance P in a dose of 50 micrograms/kg produced a steady moderate increase in dopamine levels in the neostriatum, which persisted for at least 5 h. In contrast, a dose of 250 micrograms/kg caused an acute increase in dopamine levels in the nucleus accumbens, which lasted about 2 h. These data suggest that the peripheral administration of substance P can influence dopamine release in mesolimbic and mesostriatal terminals.


Felodipine. A review of the pharmacology and therapeutic use of the extended release formulation in cardiovascular disorders.

Felodipine is a vascular-selective, dihydropyridine calcium antagonist previously investigated as a conventional tablet formulation administered twice daily. More recently considerable experience has been gained with an extended release (ER) formulation which has the convenience of once daily administration. Felodipine ER has been well studied in patients with essential hypertension. As monotherapy in mild to moderate essential hypertension, felodipine ER is at least as effective in reducing blood pressure as other calcium antagonists, beta-blockers, diuretics and ACE inhibitors, with some results favouring felodipine ER at a statistically significant level at the dosages used. It is also effective combined with controlled release metoprolol or enalapril in patients with mild to moderate essential hypertension. In patients with more severe forms of essential hypertension uncontrolled by beta-blocker and/or diuretic therapy, felodipine ER was effective as an 'add-on' therapy in placebo-controlled trials, and, at the dosages used, more effective than either sustained release nifedipine or nitrendipine. Felodipine produces effective control of blood pressure without negative effects on cardiac performance. In addition to its antihypertensive action, results suggest that felodipine therapy is associated with significant regression of left ventricular hypertrophy. Furthermore, it appears suitable for use in patients with concomitant diabetes, renal dysfunction or asthma, and is also being investigated for use in patients with congestive heart failure or angina pectoris. Felodipine ER is an effective drug for the treatment of all grades of essential hypertension, and can be used both as monotherapy and in combination with other antihypertensive agents. Further clinical experience should fully establish the long term tolerability of felodipine ER and consequently its place in therapy relative to other accepted antihypertensive drugs. However, with the convenience of once daily administration, felodipine ER is a worthwhile innovation in the treatment of hypertension.


Characterization of cell selectivity of two novel inhibitors of nitric oxide synthesis.

We have assessed the capacity of two novel inhibitors to block cytokine-induced nitric oxide (NO) synthesis by macrophages and vascular smooth muscle cells, as well as NO production by the constitutive enzyme in central nervous system tissue. NG-Cyclopropyl-L-arginine selectively inhibited Ca2+/calmodulin-dependent NO synthesis, with an IC50 of 0.55 microM in brain versus 184 and 258 microM in macrophages and vascular smooth muscle cells, respectively. In contrast, NG-amino-L-homoarginine blocked NO production by all of the cell types examined, with IC50 values ranging from 6.6 to 26 microM. Both inhibitors were active in an in vivo model of endotoxic shock.


Nitric oxide releases acetylcholine in the basal forebrain.

In conscious rats, the basal forebrain was superfused through a push-pull cannula and the release of acetylcholine was determined in the superfusate. Superfusion with the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine, diminished the release of acetylcholine. Subsequent superfusion with the NO donor, 3-morpholino-sydnonimine, enhanced the release of the neurotransmitter. It is concluded that endogenous NO enhances the release of acetylcholine from its neurons.


Effect of a benzodiazepine (chlordiazepoxide) on a GABAA receptor from rat brain. Requirement of only one bound GABA molecule for channel opening.

Chlordiazepoxide (CDPX) enhanced the rate of chloride exchange mediated by the major GABAA receptor found on sealed native membrane vesicles from rat cerebral cortex. The initial rate constant for chloride exchange for this receptor, (JA), a measure of open channel, was determined from the progress of GABA-mediated influx of 36Cl-. The dependence of JA on GABA concentration was hyperbolic in the presence of CDPX (150 microM, sufficient to give maximum enhancement of chloride exchange rate) but sigmoid in its absence. Enhancement of channel opening (10-fold at 0.3 microM GABA) decreased with increasing GABA concentration. The maximal response, above 1,000 microM GABA, was unaltered. The half-response concentration was reduced from 80 microM to 50 microM. CDPX alone caused no measurable 36Cl- exchange. In the presence of CDPX, channel opening occurred with only one bound GABA molecule, whereas in its absence, channel opening with two bound GABA molecules was much more favorable. This could not be direct allosteric modulation of the channel opening conformational change by binding of CDPX at effector sites, but could be explained by an additional change of the receptor on binding CDPX to give a closed state which gave channel opening mediated by a single GABA binding site. Another possibility is that CDPX could act at one of the channel opening binding sites without a postulated, second closed conformational state.


Human interleukin-1 receptor antagonist is expressed in liver.

Using PCR and Northern blot analysis, an IL-1 receptor antagonist specific transcript was amplified from HepG2- and liver mRNA. cDNA clones coding for IL-1 receptor antagonist were isolated from a liver cDNA library and sequence comparison revealed complete identity with the secreted, monocytic form of IL-1 receptor antagonist.


1,4-Dihydropyridines modulate GTP hydrolysis by Go in neuronal membranes.

Several lines of evidence suggest that L-type Ca2+ channels (1,4-dihydropyridine receptors) are modulated by GTP-binding proteins. We have further examined this interaction by measuring the effect of 1,4-dihydropyridines on GTPase activity in brain membranes. Dihydropyridine agonists significantly increased GTPase, reflected by an increase in the maximal rate of GTP hydrolysis, without affecting the affinity for GTP or the binding of a non-hydrolysable analogue of GTP. The stimulating effect on GTPase was abolished by antisera raised against Go alpha but not Gi alpha. L-type Ca2+ channels may act as endogenous GTPase activating proteins (GAPs) to stimulate GTP hydrolysis by Go.


Glucagon-like peptide-1(7-37)/(7-36)amide is a new incretin.

Glucagon-like peptide-1 (GLP-1) is the main product of the intestinal processing of proglucagon. It is released from the intestinal K-cells into the circulation in response to the oral ingestion of food. At the pancreatic beta cell GLP-1 is a potent insulin secretagogue in the presence of elevated glucose levels, defining glucagon-like peptide-1 as a new incretin. Its action is mediated by specific receptors coupled to the adenylate cyclase system by a stimulatory G-protein. Finally, glucagon-like peptide-1 stimulates proinsulin gene expression and it is thus involved at several levels in the regulation of insulin synthesis and secretion.


Identification of assembled epitopes on the alpha-subunit of human follicle stimulating hormone.

In order to characterize further the antigenic structure of human follitropin (hFSH), BALB/c mice were immunized with hFSH and anti-hFSH monoclonal antibodies (mAbs) were generated. The hFSH subunit specificity of the mAbs was assessed by a solid-phase enzyme-linked immunosorbent assay (ELISA) and a solution-phase radioimmunoassay (RIA), each using hFSH, hFSH alpha, and hFSH beta. Five mAbs bound hFSH and hFSH alpha in the ELISA and the RIA. In addition, some mAbs recognized hFSH beta, albeit to a much lower degree, as demonstrated by displacement of [125I]hFSH binding to the mAbs by hFSH beta, in the solution-phase RIAs. Next, synthetic peptides corresponding to the hFSH alpha-subunit sequence were used to identify sequences specific to the epitopes of each of the five mAbs. Using this epitope mapping strategy, two assembled epitopes were identified. mAbs 3A and 4B distinguish one discontinuous epitope comprised minimally of sequences alpha-16-21 and alpha-66-92, whereas mAbs 5F and 2E distinguish a second discontinuous epitope comprised minimally of sequences alpha-40-50 and alpha-66-72.


Effects of calcitonin on human trophoblastic cells in culture: absence of autocrine control.

The purpose of this work was to investigate the effects of calcitonin (CT) on trophoblastic cells with respect to cAMP levels and human chorionic gonadotrophin (hCG) secretion in cultured cells from first-trimester and term placentas and in a choriocarcinoma cell line (JEG-3). The expression of the CT gene was investigated to elucidate a putative autocrine control of CT during pregnancy. The addition of salmon CT (10(-10) M and above) resulted in concentration-dependent increases in cAMP secretion by normal trophoblastic cells from term and first-trimester placentas. Moreover, CT was found to increase cAMP secretion preferentially in completely differentiated cells, i.e. after 4-7 days in culture. Addition to the culture medium of JEG-3 cells slightly increased cAMP secretion only at a concentration of 10(-8) M. The basal level of hCG in the medium was found to be higher in the first-trimester than in the term trophoblast culture, but salmon CT induced an increase in hCG secretion by term placenta cells only. CT gene expression in our experimental model was investigated to elucidate a putative autocrine control of CT action during pregnancy. It was not found to be expressed in syncytiotrophoblast cells from either first-trimester or term placenta cells by the method used. Our data demonstrate the absence of autocrine control of CT effects in trophoblastic cells, and suggest that CT is likely to exert its effect preferentially on differentiated cells.


Topographic analysis of human follicle-stimulating hormone-beta using anti-peptide antisera.

The purpose of this study was to identify peptide sequences of human follicle-stimulating hormone-beta (hFSH beta) which are accessible subsequent to association with hFSH alpha in heterodimeric hFSH. Antisera were raised against synthetic peptides (Abpep) corresponding to hFSH beta sequences 1-20, 16-36, 33-53, 49-67, 66-85, 81-100 and 98-111. The topography of hFSH beta was studied by testing the binding of these antisera to hFSH beta and hFSH captured by monoclonal antibodies (MAb) in an enzyme-linked immunosorbent assay (ELISA). When hFSH and hFSH beta were captured by the same MAb, binding of Ab16-36, Ab33-53, Ab81-100 and Ab98-111 to hFSH was significantly lower compared to hFSH beta. However, compared to other Abpep, binding of Ab35-53 to hFSH was strong. Similar results were obtained when hFSH was captured by an alpha-specific MAb (10.3A6). Using 10.3A6, it was also possible to demonstrate significant binding of Ab49-67 to hFSH. The data suggests that residues in regions 33-53 and 49-67 of hFSH beta appear to be accessible in the heterodimeric hFSH in addition to the glycosylated region of 1-15. Regions 16-36, 33-53, 81-100 and 98-111 of hFSH beta appear to contain subunit contact-associated sequences which are either masked or structurally altered subsequent to association with hFSH alpha in the heterodimeric hFSH.


A separating method for quantifying mucus glycoprotein localized in the different layer of rat gastric mucosa.

A method was devised for separating rat gastric mucosa into three layers each containing a different mucin species. The mucus gel (first layer) was removed by stirring the gastric mucosa in a solution of phosphate-buffered saline containing 2% N-acetylcysteine. The surface mucosa (second layer), rich in surface mucus cells, was then separated from the deep mucosa (third layer) containing mucus neck cells, by scraping with forceps. The effectiveness of this method was confirmed by light microscopical observation after GOCTS-PCS (dual staining by the galactose oxidase-cold thionin Schiff method and paradoxical concanavalin A method) and AB-PAS staining (dual staining with alcian blue and the periodic acid Schiff method). The fixed specimen of scraped mucus and cell debris was rich in AB-PAS and GOCTS positive mucus, but was hardly stained by PCS, indicating mucus derived from surface mucus cells to have been efficiently recovered from this preparation. The residual mucosa could be stained by PCS but hardly at all by AB-PAS or GOCTS. The lyophilized powder specimens obtained from the three different layers of rat gastric mucosa were used to extract and quantify mucus glycoprotein (mucin). This was done to examine changes in mucin content in the three layers of gastric mucosa one hour following the oral administration of 20% ethanol or 0.35 N hydrochloric acid, both mild irritants. Mucin content was noted to significantly increase in the first layer but hardly at all in the second layer. In the third layer, it decreased significantly by 0.35 N hydrochloric acid, but changed only slightly by 20% ethanol administration.(ABSTRACT TRUNCATED AT 250 WORDS)


Overlap and discrepancy between tests for anti-C100, anti-GOR and anti-CP9 in patients with chronic liver disease and inhabitants in Saga, Japan.

The authors evaluated the clinical significance of anti-C100, anti-GOR and anti-CP9 in hepatitis C virus (HCV)-related liver disease in two populations: 459 healthy subjects and 385 patients with chronic liver disease (CLD). Previously we reported high rates of mortality and morbidity (5.3%) of CLD in subjects in Saga, Japan. This was ascribed to the high prevalence (10.8%) of anti-HCV among randomized populations, as detected by the C100 ELISA test system, as compared with a finding of 2-3% in Japanese blood donors in the same decade. The incidence of anti-C100, anti-GOR and anti-CP9 detected by ELISA test system in the healthy population currently surveyed was 17.0%, 19.2% and 32.0% respectively, as compared with 75.3%, 60.3% and 73.0% respectively, in those with CLD. The incidence of positivity for at least one of the three antibodies was high (36.4%) among healthy subjects, and even higher (86.5%) among the patients with CLD. In the healthy subjects, incidence of positivity increased with age. The healthy and CLD populations differed in the proportion of cases positive for all three antibodies vs. those positive for at least one antibody: healthy subjects, 52/167, 31.1%, vs. CLD patients, 197/333, 59.2%; P less than 0.01. Among the anti-C100-positive healthy cases, these was a significantly high level of AST, ALT, ZTT and gamma GTP compared with negative cases, with or without anti-GOR and anti-CP9 (P less than 0.01-0.05). These observations suggest that the presence of anti-C100 may be related to the active state of HCV-related liver disease.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparative sequence analyses of the 16S rRNA genes of Lactobacillus minutus, Lactobacillus rimae and Streptococcus parvulus: proposal for the creation of a new genus Atopobium.

16S rRNA gene sequencing was performed on the species Lactobacillus minutus, Lactobacillus rimae and Streptococcus parvulus in order to clarify their taxonomic position. Based on comparative sequence analyses these organisms represent a hitherto unknown line of descent within the lactic acid group of bacteria for which a new genus, Atopobium gen. nov., is proposed.


Effect of human chorionic gonadotropin (hCG) on reverse transcriptase activity in HIV-1 infected lymphocytes and monocytes.

The majority of infants born to HIV-positive mothers are not infected in utero, suggesting that the pregnancy factors produced by fetal trophoblasts may provide protection against HIV-1 infection. Except for steroid female hormones, little is known of other pregnancy factors that may regulate either resistance or susceptibility to HIV-1. Human chorionic gonadotropin (hCG)--the major glycoprotein produced by the placental trophoblast throughout the pregnancy--was tested on reverse transcriptase activity in HIV-infected ACH-2 lymphocytes and U1 monocytes. The results suggest that low non-cytotoxic doses of hCG (0.01-1.0 IU range) may inhibit viral replication in maternal blood cells.


Arg276 of GseA, a Chlamydia trachomatis Kdo transferase, is required for the synthesis of the chlamydial genus-specific epitope in Escherichia coli.

GseA is an enzyme from Chlamydia trachomatis that can catalyse the addition of three 3-deoxy-D-manno-octulosonic acid (Kdo) residues onto lipid A precursors. GseA is similar, and in a few stretches identical, in its amino acid sequence to KdtA, an Escherichia coli Kdo transferase. In this study we altered an amino acid of GseA in a region that is identical between GseA and KdtA to test its importance in the structure or catalytic activity of GseA. We found that when Arg276 was changed to Lys, Ile or Ser, GseA activity was lost, suggesting an enzymatic role for this amino acid residue.


[Microcephalic children without mental retardation].

8 microcephalic children, mean age 3.06 years, with head circumferences -3.5 SD to -6 SD, achieved IQ scores of 79-115 on the Thermann, WPPSI, WISC-R, and/or Columbia tests. They had no dysmorphic features nor congenital anomalies, and their fundi were normal. 7 had congenital microcephaly, associated with proven autosomal dominant heritage in 3, 1 had suspected autosomal recessive heritage, 1 had intrauterine growth retardation (overall smallness), 1 had cerebral palsy, and in 1 there was an unknown prenatal cause. 6 of these children had 1 or 2 developmental disabilities, including neurosensory deafness, cerebral palsy, short attention span, perceptual dysfunctions, and learning disability. Microcephaly, most probably secondary to malnutrition in early infancy, was diagnosed in 1 child without developmental disabilities. However, our data support the concept that some microcephalic children with significantly small head circumference, who function without mental retardation, have a high incidence of other developmental disabilities.


[Evans syndrome in a child].

A Bedouin girl, aged 2 3/12, was first seen at the age of 9 months for sequential onset of Coombs-positive hemolytic anemia and immune thrombocytopenia. Comprehensive laboratory workup revealed no underlying disease. Evans' syndrome, relatively rare in childhood, was diagnosed and corticosteroid therapy initiated. She responded well and normal hemoglobin and thrombocyte counts were achieved within a month. All attempts at tapering-off the dose of corticosteroids resulted in relapses, until high-dose intravenous gamma-globulin was given.


Research in child language disorders: what do we know and where are we going?

Advances in understanding the nature of language impairment in children over the past decade have been abundant. In this paper, several recurring hypotheses about the nature of the clinical disorder of language impairment in children and underlying etiologies are explored and the ways in which we are moving forward in our understanding of them are considered. Specifically, in the first part, the recent thinking on the nature of specific language impairment as a clinical concept is examined. Characteristics of the population are reviewed and the search for causation is considered. Literature on the relations between language and cognition and between language and social cognition is probed for contributions to our understanding of developmental disorders of language. Two areas in which we have seen marked progress and which hold promise for development in the coming decade are the focus of the second part of the paper. The focal areas are: computer-assisted language sample analyses and neurophysiological contribution to understanding language impairment in children. Thoughts about future directions for work in the area of developmental disorders of language are offered in conclusion.


Inhibition of the alternative pathway of complement by glomerular chondroitin sulphate proteoglycan.

Cultured rat glomerular epithelial cells (GEC) synthesize and secrete a complement inhibitory chondroitin sulphate B proteoglycan (termed GCRF). As proteoglycans and their component glycosaminoglycans may affect several different steps of complement activation, the functional properties of GCRF were investigated in this study. GCRF inhibited preformed alternative pathway convertases (C3bBbP), but did not substantially accelerate their decay. In contrast to other polyanions, GCRF did not affect the activity of human or rat factor H, nor did it inhibit the terminal complement proteins. GCRF inhibited the effect of decay-accelerating factor (DAF) on C3bBbP when the two were incubated together and DAF was in excess, but it did not affect the decay of DAF of classical pathway convertases. However, when DAF was first incorporated into the erythrocyte membrane, the effect of GCRF on C3bBbP was additive to that of DAF. Thus, GCRF inhibits the activity of C3bBbP and blocks the action of DAF, but not that of factor H, perhaps by binding to factor B in the alternative pathway convertase.


Enhancement of trinitrophenyl-specific humoral response to TNP proteins as the result of carrier chlorination.

Trinitrophenyl (TNP)-specific humoral response was tested after immunization with TNP-protein conjugates composed of either native proteins or proteins modified by chlorination. Significant enhancement of the hapten-specific response was observed as a result of carrier chlorination. This effect was dose dependent and affected mainly T-helper-cell activation. Since stimulated neutrophils chlorinate in vivo proteins, we propose that they take part in the induction phase of immune response by facilitation of antigen processing and/or presentation.


Aphidicolin-induced proliferative arrest of murine mast cells: morphological and biochemical changes are not accompanied by alterations in cytokine gene induction.

Investigations of mast cell biology have often used immortalized cultured cells which are continuously proliferating. In vivo, however, only 2% or fewer tissue mast cells are actively dividing. We used aphidicolin, an inhibitor of DNA polymerase to induce a proliferative arrest of murine mast cells characterized by an inhibition of cell division and thymidine incorporation, with accumulation of cells in G1 and early S phase of the cell cycle. Uridine incorporation and cell viability were not significantly impaired. DNA synthesis and cell division both resumed rapidly upon removal of the drug. Morphometric analysis demonstrated that cell size, granule size, and number of granules per cell were all increased in aphidicolin-treated cells. Proliferative arrest also produced a 14-fold increase in cellular histamine content, but did not alter the proteoglycans synthesized by the cell. The level of c-myc mRNA was reduced in aphidicolin-arrested cells, but returned to the level observed in untreated cells within 1 hr of removal of the drug. In contrast, the constitutive steady-state RNA levels of tumour necrosis factor-alpha (TNF-alpha), B2-microglobulin, actin, and the c-Ha-ras and c-fes protooncogenes were not altered. Aphidicolin-induced proliferative arrest did not prevent the induction of TNF-alpha, interleukin-6 (IL-6) and c-fos genes in response to calcium ionophore. Both the magnitude and induction kinetics of these messages were similar in aphidicolin-treated and untreated cells. We conclude that proliferative arrest results in morphological and biochemical changes suggestive of cellular maturation, but inhibition of cell division alone is not sufficient to alter mast cell phenotype. Although optimal c-myc expression appears to require active proliferation, cytokine gene induction can occur in non-dividing cells. These data suggest that the proliferative quiescence of in vivo mast cells should not preclude their involvement in biological events via elaboration of multi-functional cytokines.


In vitro effects of drugs on production of mucins in rabbit tracheal epithelial cells expressing mucin gene: a model system for studying upper airway respiratory diseases.

Rabbit tracheal epithelial cells, cultured on collagen-coated dishes in serum-free and hormone-supplemented medium, were found to incorporate [3H]glucosamine into high-molecular-weight components that were secreted in the medium. The chemical analysis of the secreted products resulted in a profile that resembled that of mucous glycoproteins (mucins). When examined by dot blot analysis, the total RNA isolated from these cells hybridized to an antisense 30-mer oligonucleotide corresponding to a rat intestine mucin peptide sequence, indicating that mucin gene was expressed in these cell lines. Lung and liver tissues of rabbit did not express this gene. Transmission electron microscopy exhibited secretory granules in these cells. The incorporation of [3H]glucosamine into mucins was inhibited by three aryl-N-acetyl-galactosaminides and a chemical carcinogen, N-nitroso-N-ethyl urea, whereas 5-azacytidine enhanced the proliferation of cells as well as the radiolabeling of mucins. Parasympathetic agent (pilocarpine), cholinergic antagonist (atropine), and beta-adrenergic agonist (isoproterenol) alone have little effect on the secretion of mucins. The cholinergic agonist, methacholine, was found to increase the production of mucins and addition of atropine to the medium before methacholine blocked this stimulation. Histamine was found to stimulate mucin production in these cells.


Diminished epinephrine excretion in genetically obese (ob/ob) mice and monosodium glutamate-treated rats.

While numerous studies have examined sympathetic nervous system activity in experimental obesity, adrenal medullary function in this condition has received less attention. The experiments described herein evaluated adrenal medullary secretion by measurement of urinary epinephrine (Epi) excretion in genetically obese (ob/ob) mice and monosodium glutamate (MSG)-treated rats. In both male and female ob/ob mice Epi excretion was reduced 42% (P less than 0.015) and 47% (P less than 0.025), respectively, despite higher rates of urine output and excretion for other amines in obese compared to lean animals. In a similar fashion, urinary Epi was also lower in MSG-treated adult rats than in untreated controls; this reduction was out of proportion to group differences in body weight or excretion of other catecholamines. Administration of D,L-fenfluramine to mice, or dietary protein supplementation in rats, increased Epi excretion to the same extent in obese and lean animals. These findings indicate that secretion of Epi by the adrenal medulla is diminished, but is normally responsive to stimulation in these two models of animals obesity, and are thus consistent with accumulating evidence of a functional impairment in adrenal medullary secretion in animal and human obesity.


Transferrin receptor expression by retinal pigment epithelial cells in proliferative vitreoretinopathy.

Immunotoxins directed against a membrane marker of cell proliferation, transferrin receptor, were investigated to inhibit the growth of retinal pigment epithelial (RPE) cells in proliferative vitreoretinopathy (PVR). We undertook an immunocytological study in specimens of vitreous, subretinal fluid, and epiretinal membranes from patients with PVR to address the expression of transferrin receptor by proliferating pigment epithelial cells during the course of PVR and in normal human ocular structures. Thirty four specimens of vitreous and subretinal fluid, as well as seven epiretinal membranes, were immunocytologically examined using monoclonal antibodies to transferrin receptor. They showed a strong expression of this marker by a large majority of the cells in these two periretinal fluids (mean percentages 80 and 91% in vitreous and subretinal fluid, respectively). In contrast, only a few cells within epiretinal membranes were found to express transferrin receptor. In normal human eye sections conjunctival and corneal epithelial cells, subcapsular epithelium of the lens strongly expressed transferrin receptor, whereas RPE cells remained negative to antitransferrin receptor antibodies. A few iris or ciliary pigment epithelial cells reacted weakly. Thus, this study shows that most intravitreal and subretinal fluid proliferating cells strongly express transferrin receptor on their surface. Also confirmed is that immunotoxins to this membrane antigen could constitute potentially useful therapeutic agents in PVR.


Lithium inhibits substance P and vasoactive intestinal peptide-induced relaxations on isolated porcine ophthalmic artery.

Lithium is currently a major drug used as a treatment for affective disorders and cluster headache, among other conditions. Its mechanism of action remains unknown. The trigeminovascular system may be involved in the pathophysiology of cluster headache and related diseases by liberating neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) in the eye-forehead region. The objective of this study was to investigate whether or not a low concentration of lithium may interfere with peptidergic neuro-transmission at this level. Vasoactive intestinal peptide (VIP), SP, CGRP, capsaicin, and SP+CGRP concentration-response relaxation curves were obtained in the presence and absence of 2 x 10(-4) M lithium in isolated porcine ophthalmic arteries. Lithium inhibited the SP and VIP, but not the CGRP responses. Capsaicin-(a neurotoxin causing release of stored sensory neuropeptides) induced relaxations were partially inhibited by lithium. It is concluded that lithium may interfere with SP- and VIP-induced relaxation. If SP and VIP are of pathogenic significance in cluster headache, lithium may possibly work by counteracting unwanted effects of such peptides.


Reoxygenation in the RIF-1 tumor after chemotherapy.

The proportion of hypoxic cells in the RIF-1 tumor was observed for 24 hr after treatments with bleomycin, cisplatin, cyclophosphamide, and mitomycin C. The assays were based upon the paired survival curve method for determining the hypoxic fraction using irradiation of aerobic and artificially hypoxic tumors. It was observed that at 1/2 hr after bleomycin, hypoxic fraction was elevated but returned to baseline levels by 2 hr. Following cisplatin, hypoxic fraction did not rise above baseline. However, after cyclophosphamide, hypoxic fraction was elevated and did not return to baseline over the 24-hr observation period of this study. At 1/2 hr after mitomycin treatment, the hypoxic fraction was raised but within 1 hr returned to baseline. These results indicate that tumor reoxygenation varies after different drug treatments and that the determination of drug specificity for aerobic versus hypoxic cells may be strongly biased by the choice of the time after treatment for making the determination.


Location of motoneurons innervating the middle ear muscle of the chicken, (Gallus domesticus).

The motoneuron pool for the musculus columellae, the avian equivalent to the m. stapedius, was identified by retrograde labeling with WGA-HRP. It consists of a discrete group of approximately 65 neurons located along the dorsolateral border in the ventral subnucleus of the facial nuclear complex. Other facial motoneurons were only labeled when diffusion of the tracer into neighbor structures was not excluded. The dorsal subnucleus of the facial nerve innervates the m. depressor mandibulae.


Ca(2+)-activated nonselective cation, maxi K+ and Cl- channels in apical membrane of marginal cells of stria vascularis.

Patch clamp recordings on the apical membrane of marginal cells of the stria vascularis of the gerbil were made in the cell-attached and excised configuration. Marginal cells are thought to secrete K+ into and absorb Na+ from endolymph. Four types of channel were identified; the most frequently observed channel was a small, nonselective cation channel which was highly similar to that found in the apical membrane of vestibular dark cells (Marcus et al., (1992) Am. J. Physiol. 262, C1423-C1429). The small nonselective cation channel was equally conductive (26.7 +/- 0.3 pS; N = 49) for K+, Na+, Rb+, Li+ and Cs+, 1.6 times more permeable to NH4+, but not permeable to Cl-, Ca2+, Ba2+ or N-methyl-D-glucamine. This channel yielded linear current-voltage relations which passed nearly through the origin (intercept: -2.2 +/- 0.4 mV, N = 49) when conductive monovalent cations were present on both sides of the membrane in equal concentrations. Channel activity required the presence of Ca2+ at the cytosolic face but not the extracellular (endolymphatic) face; there was essentially no activity for cytosolic Ca2+ less than or equal to 10(-7) M Ca2+ and full activity for greater than or equal to 10(-5) M. Cell-attached recordings had a conductance of 28.6 +/- 2.2 pS (N = 6) and a reversal voltage of -2.2 +/- 5.2 mV (N = 3) which was interpreted to reflect the intracellular potential of marginal cells under the present conditions. The three other types of channel were a Cl- channel (approximately 50 pS; N = 2), a maxi-K+ channel (approximately 230 pS; N = 1), and another large channel, probably cation nonselective (approximately 170 pS; N = 1). The 27 pS nonselective cation channel may be involved in K+ secretion and Na+ absorption under stimulated conditions which produce an elevated intracellular Ca2+; however, consideration of the apparent channel density in relation to the total transepithelial K+ flux suggests that these channels are not sufficient to account for K+ secretion.