Pharmacokinetics and fibrin specificity of alteplase during accelerated infusions in acute myocardial infarction.

Pharmacokinetics and fibrin specificity of alteplase (recombinant tissue-type plasminogen activator) were determined in 10 patients with acute myocardial infarction undergoing an accelerated infusion regimen during the alteplase/anistreplase patency study (TAPS). Fifteen milligrams of alteplase was administered as an intravenous bolus injection, followed by infusions of 50 mg over 30 min and 35 mg over a further 60 min. Mean steady state plasma concentrations of alteplase during the initial 30 min were 3.2 +/- 0.84 micrograms/ml, measured immunochemically, and 2.1 +/- 0.23 micrograms/ml, measured using a functional activity assay. These values were 45% and 51% higher, respectively, than those during the standard infusion schedule (p less than 0.01). However, the predominant plasma half-life determined by model fitting based on either assay (3.3 to 3.5 min) was unaltered compared with the standard regimen. Maximal concentrations of fibrin and fibrinogen degradation products were 5.1 +/- 2.2 and 1.9 +/- 1.1 micrograms/ml, respectively. Plasminogen decreased to 70% and alpha 2-antiplasmin to 35% of values before infusion. The results indicate that 1) improved coronary patency rates during "front-loaded" infusions can be rationalized in terms of higher plasma concentrations of both free and immunoreactive alteplase, 2) kinetic variables are comparable with those of other dosing strategies, and 3) fibrin specificity is not diminished relative to that of the standard infusion regimen.


Fluent aphasia in children: definition and natural history.

We compared the course of a preschool child we followed for 4 years with published reports of 24 children with fluent aphasia. Our patient spoke fluently within 3 weeks of the injury. She was severely anomic and made many semantic paraphasic errors. Unlike other children with fluent aphasia, her prosody of speech was impaired initially, and her spontaneous language was dominated by stock phrases. Residual deficits include chronic impairment of auditory comprehension, repetition, and word retrieval. She has more disfluencies in spontaneous speech 4 years after her head injury than acutely. School achievement in reading and mathematics remains below age level. Attention to the timing of recovery of fluent speech and to the characteristics of receptive and expressive language over time will permit more accurate description of fluent aphasia in childhood.


Developmental manual dyspraxia: a lesson in mind and brain.

For decades, pediatricians assiduously documented "soft neurologic signs" in children referred for school learning difficulties, although pediatric neurologists, including Charles F. Barlow, pointed out the dissociation between most neuropsychological abnormalities and the motor findings in question. Later, large epidemiologic studies confirmed the independence of soft signs from other neuropsychological defects. We used a standardized dyspraxia battery to study 164 schoolchildren 5 to 12 years old. We found that in the group as a whole there was a positive correlation between motor performance in the dyspraxia battery and IQ on the Wechsler Intelligence Scale for Children--revised (Full-Scale, Verbal, and Performance). In terms of dyspraxia subscales, we found that Performance IQ and Full-Scale IQ correlated with imitation of nonsense gestures and use of actual objects, whereas Verbal IQ correlated positively only with pantomime on command. In contrast, for the group of 24 subjects whose scores on the battery designated them as dyspraxic, there was no correlation between dyspraxia scores and IQ. Together with the existence of specific, dramatically different, types of dyspraxia among the 24 dyspraxic subjects, present findings uphold the earlier neurologic opinion that "motor soft signs" are not evidence for fixed "brain damage" or even for other types of motor dysfunction, and that dissociation of different specific cognitive dysfunctions is the rule within individual patients.


Immunolocation analysis of glycosaminoglycans in the human growth plate.

Monoclonal antibodies were used in this study to immunolocate glycosaminoglycans throughout the human growth plate. Chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate were observed in the extracellular matrix of all zones of the growth plate and persisted into the cartilage trabeculae of newly formed metaphyseal bone. Also present in the extracellular matrix was an oversulfated chondroitin/dermatan sulfate glycosaminoglycan which appeared to be specific to the proliferative and hypertrophic zones of the growth plate. As with the other extracellular matrix molecules, this epitope persisted into the cartilage trabeculae of the metaphyseal bone. Zonal differences between the extracellular and pericellular or lacunae matrix were also observed. The hypertrophic chondrocytes appeared to synthesize chondroitin sulfate chains containing a non-reducing terminal 6-sulfated disaccharide, which were located in areas immediately adjacent to the cells. This epitope was not found to any significant extent in the other zones. The pericellular region around hypertrophic chondrocytes also contained a keratan sulfate epitope which was also observed in the resting zone but not in the proliferative zone. These cell-associated glycosaminoglycans were not found in the cartilage trabeculae of metaphyseal bone, indicating their removal as the terminal hypertrophic chondrocytes and their lacunae are removed by invading blood vessels. These changes in matrix glycosaminoglycan content, both in the different zones and within zones, indicate constant subtle alterations in chondrocyte metabolic products as they proceed through their life cycle of proliferation, maturation, and hypertrophy.


S-antigen in rods and cones of the primate retina: different labeling patterns are revealed with antibodies directed against specific domains in the molecule.

S-antigen (arrestin) is a soluble 48 KD protein of the retinal photoreceptor cells. It has been found to have a function in regulation of the phototransduction cascade. Previous labeling experiments with anti-S-antigen (SAg) antibodies have yielded conflicting reports as to the presence of SAg in cone photoreceptor cells. In the present study we employed five monoclonal anti-SAg antibodies (MAb) directed against different known domains in the SAg molecule. MAb A9C6, D9F2, and C10C10 are directed against sequences in the carboxy half of the SAg molecule. MAb 5C6.47 and F4C1 are directed against the amino terminal. Immunoelectron microscopy was used in the localization of SAg in LR Gold-embedded baboon retinas. Green/red and blue cones were identified with MAb COS-1 and OS-2, respectively. MAb A9C6, D9F2, and C10C10 densely labeled rods and blue cones but not green/red cones. MAb 5C6.47 and F4C1 labeled rods and both blue and green/red cones. It appears that, in the baboon retina, different SAg molecules are present in the blue and green/red cones. Whereas the blue cone SAg shares common antigenic determinants with rods, both in the amino and carboxy terminals, the green/red cone SAg contains different antigenic determinants at the carboxy half of the molecule.


Microwave-aided binding of gold-protein-ligand (GPL) complexes. Light microscopic observations in the rat brain.

We describe a rapid method for the preparation and binding site labeling of cryostat sections for use in light microscopy. Instead of using antibodies to bind to specific sites, substance P, delta-sleep-inducing peptide, oxytocin, and dopamine were covalently attached to BSA and then the BSA-ligand complex was adsorbed on 5-nm colloidal gold particles. Bioassays carried out on isolated organs indicated that the physiological activity of the ligand GPL complex was maintained. Most of the technical steps included use of an ordinary microwave oven (MWO), with tissues exposed for less than 1 min in any given step. Cryostat sections of unfixed rat brain were pre-incubated for 50 sec in the MWO in a Tris-buffered solution (pH 7.4) containing 1.5% BSA, then further incubated for 50 sec in the MWO in Tris-buffered solution containing 1% gelatin and the diluted colloidal gold suspension. After washing, the preparations were postfixed for 30 sec in the MWO in 5% formaldehyde solution, pH 7.4. Finally, the cell-bound gold particles were enlarged by a silver-enhancing process and counterstained. Preparations observed at high magnification provided excellent resolution of the cell binding sites. Positive and negative controls performed by addition of BSA-conjugated ligands to the pre-incubation and incubation medium, and displacement of the markers by an excess of unbound ligand in the pre-incubation or the incubation medium, showed the specificity of the tissue labeling.


UV lasers for flow cytometric analysis: HeCd versus argon laser excitation.

Applying flow cytometric single cell analysis, we compared the performance of UV excitation from argon ion and HeCd lasers using various UV-excitable fluorochromes of cell kinetic and cell physiological relevance. The AT-specific DNA fluorochromes DAPI, Hoechst 33258, and Hoechst 33342 showed no significant differences of G1-phase resolution and cell cycle distribution. With the HeCd laser, high-resolution cell kinetic analysis applying the novel BrdU/Hoechst-PI quenching technique showed superior resolution and an almost normalized G2M/G1 channel ratio of the first cell cycle. Indo-1 analysis for detection of intracellular free calcium gave similar results for both excitation sources, although the indo-1 ratio of activated cells was lower for HeCd excitation. Monochlorobimane as an indicator fluorochrome of glutathione content could not be excited sufficiently with the 325-nm line of the HeCd laser and exhibited poor resolution between positive and negative cells. However, the second glutathione-specific fluorochrome o-phtalaldehyde gave even better results with the HeCd laser. Our data indicate that air-cooled HeCd lasers are cheap and reliable UV-excitation sources for most UV-excitable fluorochromes, and might be an alternative to the expensive water-cooled argon and krypton laser.


Early and late replicative chromosomal banding patterns of Gallus domesticus.

Early and late replicating chromosomal banding patterns of Gallus domesticus were investigated by cell synchronization and incorporation of 5'-bromodeoxyuridine during early and late DNA synthesis. The early replicating chromosomal banding patterns observed, as revealed by either acridine orange or Hoechst 33258/propidium iodide staining, were similar to the structural G-banding patterns obtained by trypsin digestion and Giemsa staining. Late replicating chromosomal banding showed extensive reverse band complementarity to the G-banding pattern. Cell synchronization increased the number of prometaphase and metaphase plates available for analysis. G-banding obtained by Hoechst 33258/propidium iodide staining was investigated due to the fact that it is compatible with chromosomal in situ hybridization procedures that use nonisotopically-labeled DNA probes. Standard replicative G-banded and R-banded idiograms, as obtained after cell synchronization, are proposed.


Binding of an iodinated substance P analogue to cultured anterior pituitary prolactin- and luteinizing hormone-containing cells.

Several lines of anatomic, biochemical, and pharmacological evidence suggest that the neuropeptide substance P has a direct action on cells of the anterior pituitary lobe via a specific neurokinin-1 receptor. In the present study we confirmed this association by combining Bolton-Hunter iodinated substance P-receptor autoradiography with immunocytochemistry on cultured anterior pituitary cells. Radiolabeled substance P was bound to living cell cultures at 0 degrees C, and after a brief wash the cultures were fixed and processed immunocytochemically for prolactin and luteinizing hormone. A large proportion of cultured anterior pituitary cells possessed substance P binding sites. When receptor autoradiography was combined with immunocytochemistry, it was evident that both prolactin- and luteinizing hormone-immunoreactive cells were labeled with radiolabeled substance P. However, a small proportion of the radioligand-labeled cells were not stained by the immunocytochemical procedure, suggesting that additional cell types possess substance P receptors. The present study presents morphological evidence that substance P binds to prolactin- and luteinizing hormone-containing cells of the anterior pituitary lobe. Therefore, it is likely that substance P has a direct action on mammotrophs and gonadotrophs.


Lectin staining of cultured CNS microglia.

Carbohydrate binding proteins, known as lectins, bind to specific sugar groups on most membranes. We used fluorescent and light microscopy to study the interaction of various lectins with the membranes of microglia cultured from neonatal rat or fetal mouse cerebral cortices. Microglia stained intensely with GS-1, RCA, WGA, and ConA and slightly with DBA, UEA, BPA, and SBA. No staining was seen with GS-2, MPA, or PNA. Staining was specific for microglia in the mixed glial cultures and was dose dependent. In addition, microglial lectin binding could be reduced or blocked by competitive inhibition using specific sugars. Treatment of the microglia with agents such as dimethylsulfoxide (DMSO), interleukin-1 (IL-1), interferon (IFN), or lipopolysaccharide (LPS) did not eliminate lectin staining, although the degree of staining was altered. Positive staining of the microglia was also associated with a functional change for at least one lectin, i.e., ConA. Superoxide anion production by microglia was increased in the presence of ConA. Overall, binding of the lectins GS-1, RCA, WGA, and ConA can be used as an identifying tool for microglia in glial cultures, but intensity of staining varies depending on their functional state.


Characterization of antigenic determinants of Borrelia burgdorferi shared by other bacteria.

Three antigenic determinants of Borrelia burgdorferi that were shared with other spirochetes and gram-negative bacterial species, as measured by Western blot, ELISA, or both, were identified and characterized using monoclonal antibodies (MAbs). Two were associated with immunogenic integral membrane lipoproteins of 19- and 22-kDa, respectively, by [3H]palmitate incorporation and Triton X-114 phase partitioning. A third antigenic determinant was shown to be associated with a 72-kDa heat shock protein that was also immunogenic in human patients. MAb agglutination assays with B. burgdorferi and treatment of the spirochete with proteases indicated that none of the antigenic determinants were surface exposed. NH2-terminal sequence analysis revealed the 72-kDa protein to have 100% identity with the first 13 amino acid residues of the Escherichia coli dnaK heat shock protein. The presence of these and other shared antigenic determinants in ELISA antigen preparations could explain the high degree of serologic cross-reactivity in current diagnostic procedures.


Effect of endotoxin on serum granulocyte and granulocyte-macrophage colony-stimulating factor levels in dogs.

The biologic effects of endotoxin are attributed to the release of several cytokines, including interleukin-1, interleukin-6, tumor necrosis factor, and the colony-stimulating factors. To investigate the mechanism of endotoxin-induced neutrophilia in dogs, several cell lines known to proliferate selectively in response to recombinant human colony-stimulating factors were examined to determine their responses to recombinant canine granulocyte colony-stimulating factor (rcG-CSF) or recombinant canine granulocyte-macrophage colony-stimulating factor (rcGM-CSF). The murine cell line NFS-60 was found to respond well to rcG-CSF and the human cell line TALL-101 to rcGM-CSF, and these responses were neutralized by antibodies to these recombinant proteins. These bioassays were then used to determine G-CSF and GM-CSF levels in dogs after intravenous endotoxin administration. G-CSF levels increased by 2 h, peaked at 4 h, and had not returned to normal by 24 h after endotoxin. In contrast, GM-CSF was not detectible before or after endotoxin administration.


Pristanic acid and phytanic acid in plasma from patients with peroxisomal disorders: stable isotope dilution analysis with electron capture negative ion mass fragmentography.

A sensitive and selective stable isotope dilution method was developed for the accurate quantitation of pristanic acid and phytanic acid using electron capture negative ion mass fragmentography on pentafluorobenzyl derivatives. This technique allows detection of 1 pg of each compound and was applied to plasma from healthy controls and patients suffering from various peroxisomal disorders. The age-dependency of phytanic and pristanic acid levels in plasma from healthy controls was demonstrated. The involvement of peroxisomes in the beta-oxidation of pristanic acid was concluded from its accumulation in plasma from patients with peroxisomal deficiencies. Pristanic acid/phytanic acid ratios were markedly increased in bifunctional protein and/or 3-oxoacyl-CoA thiolase deficiency, indicating their role in the (differential) diagnosis of disorders of peroxisomal beta-oxidation.


The ECG and the single channel.

Our ability to understand and use the ECG has increased with the availability of tools to study the heart's electrical system. Great advances were achieved with direct electrical recordings from intact animal and human hearts. The microelectrode opened the way to recording of the cellular action potentials and their underlying currents. We now have two new and powerful methods to study cardiac electrophysiology--the patch clamp and molecular biology. We have begun to characterize the behavior of the elementary unit of membrane current, the single channel. The single channel can best be visualized as existing in a finite set of states related sequentially as in a Markov chain. The primary structures of several channels have now been determined by cloning, and the structural determinants of channel function can be explored. We have entered the molecular age of electrophysiology and can expect a greater understanding of the basic physiological and pathophysiological processes underlying the ECG. We can also expect powerful drugs to be designed based on the structures of their channel targets. The ECG remains a rich area for study and an ever better clinical tool.


The ionic basis of the receptor potential of frog taste cells induced by sugar stimuli.

The ionic mechanism underlying the receptor potential in frog taste cells induced by sugar stimuli was studied with conventional microelectrodes by replacing the superficial and interstitial fluids of the tongue with modified solutions. The taste cell generated a depolarizing receptor potential accompanying a remarkable reduction of input resistance in response to stimulation with galactose and sucrose. The magnitude of the receptor potential in response to galactose solution increased linearly with decreasing pH in the pH range 6-8, but remained constant above pH8. The reversal potential was increased by only 29 mV by a 10-fold increase in the H+ concentration of the stimulus, suggesting that there are pH-dependent and pH-independent components in the mechanism generating the receptor potential. The use of Na(+)-free, Ca(2+)-free and K(+)-free interstitial fluids did not affect the receptor potential, but the elimination of Cl- from the interstitial fluid largely abolished it. Interstitial 0.1 mmol l-1 N,N'-dicyclohexyl-carbodiimide (DCCD) completely inhibited the receptor potential and interstitial 0.1 mmol l-1 N-ethylmaleimide (NEM) decreased the potential to 40% of the control value. Lowering the pH of interstitial fluid from 7.2 to 6.3 decreased the receptor potential to 30% of the control value. It is concluded that part of the receptor potential in frog taste cells induced by sugar stimuli may be produced by an inflow of H+ through the taste-receptive membrane. The intracellular pH of the taste cell may be regulated by a Cl(-)-dependent H+ pump in the basolateral membrane.


Differential expression of secretory granule proteases in mouse mast cells exposed to interleukin 3 and c-kit ligand.

It is now established that the subclasses of mast cells (MC) that reside in mucosal and serosal environments can be distinguished from one another in terms of their expression of specific secretory granule-localized proteases and proteoglycans. Further, the hematopoietic- and connective tissue-derived cytokines that regulate expression of the genes that encode these constituents of the granule can now be identified using recently developed gene-specific probes and recombinant cytokines. When bone marrow-derived MC (BMMC) were developed with recombinant interleukin 3 (rIL-3) and maintained with this cytokine in the absence or presence of recombinant c-kit ligand (rKL), they remained safranin-, produced almost no 35S-labeled heparin proteoglycans, and contained greater levels of mouse MC protease (MMCP) -5 mRNA and mast cell carboxypeptidase A (MC-CPA) mRNA than MMCP-6 mRNA. They did not contain MMCP-4 or -2 mRNA, genes expressed late in the differentiation of progenitor cells into serosal and mucosal MCs, respectively. In contrast, BMMC developed with rKL alone or by sequential culture in medium containing rIL-3 followed by rKL expressed high levels of MMCP-4 and -6 mRNA, as well as the transcripts that encode MMCP-5 and MC-CPA. Although rKL-developed BMMC were safranin+ and produced substantial amounts of 35S-labeled heparin proteoglycans, they contained only minimal amounts of histamine and MC-CPA enzymatic activity relative to serosal MC. These are the first studies to characterize the transcriptional granule phenotype of a population of BMMC derived using any recombinant cytokine, to demonstrate a dissociation between histochemical staining and granule maturation, and to demonstrate antagonistic regulation of late expressed protease genes by a cytokine.


Ligation of VLA-4 on T cells stimulates tyrosine phosphorylation of a 105-kD protein.

The VLA/integrins are a family of heterodimeric adhesion receptors shown to be involved in cell-to-cell and cell-to-extracellular matrix (ECM) interactions. Given recent evidence that VLA molecules can synergize with the CD3/T cell receptor (TCR) pathway to activate T cells, it is important to identify biochemical event(s) generated by these molecules. Here, we report that the engagement of VLA-4 on T cells with specific antibodies or its ligand activates protein-tyrosine kinase (PTK) activity as detected by antiphosphotyrosine immunoblotting. The crosslinking of VLA-beta 1 (CD29) with a specific monoclonal antibody (mAb) (anti-4B4) plus anti-mouse immunoglobulin resulted in the rapid tyrosine phosphorylation of a 105-kD protein (pp105) in the human T cell line H9, as well as in peripheral resting T cells. The increase in tyrosine phosphorylation of pp105 was specifically mediated by VLA-4, since mAbs against alpha 4, but not against other VLA alpha chains, could induce this phosphorylation. In addition, the binding of T cells with the CS1 alternatively spliced segment of fibronectin (the binding site recognized by VLA-4) induced pp105 tyrosine phosphorylation. Crosslinking the CD3 complex or VLA-4 molecules with mAbs demonstrated that each of these molecules stimulated the tyrosine phosphorylation of unique sets of proteins with different kinetics, suggesting that these two receptor systems are coupled to distinct PTKs. Since tyrosine phosphorylation of cellular proteins has been shown to be a crucial biochemical event in cell growth, our findings suggest that the induction of pp105 tyrosine phosphorylation via VLA-4 may play a role in the transduction of activation signals through this molecule.


Ontogeny of human natural killer (NK) cells: fetal NK cells mediate cytolytic function and express cytoplasmic CD3 epsilon,delta proteins.

Natural killer (NK) cells have been defined as CD3 epsilon-, CD16+ and/or CD56+ lymphocytes that mediate major histocompatibility complex (MHC)-unrestricted cytotoxicity against certain tumors and virus-infected cells. Unlike T lymphocytes, NK cells do not rearrange or productively express T cell antigen receptor genes. Moreover, NK cells from adults have been reported to not express CD3 gamma, delta, or epsilon proteins on the cell surface or in the cytoplasm. Nonetheless, NK cells have been shown to share a number of antigenic and functional similarities to T cells that suggest the possibility of common origins. In this report, we demonstrate that functional NK cells exist in liver at early stages of human embryonic development. Freshly isolated fetal NK cells mediated MHC-unrestricted cytotoxicity against NK-sensitive targets and acquired the ability to lyse NK-resistant tumors after overnight culture in interleukin 2. Unlike adult NK cells, freshly isolated fetal liver NK cells and clones derived from these cells, as well as a subset of cord blood NK cells, express substantial levels of CD3 delta and CD3 epsilon proteins in the cytoplasm. Expression of CD3 epsilon and CD3 delta transcripts and cytoplasmic proteins in fetal NK clones was confirmed by polymerase chain reaction and Western blot analysis. These findings support the concept that NK and T cells may arise from a common progenitor that expresses components of the CD3 complex. Alternatively, it is possible that the cytoplasmic CD3 delta, epsilon+ fetal NK cells represent a distinct subpopulation of NK cells that is predominant in the fetus, but replaced by the cytoplasmic CD3 delta,epsilon- adult NK cell population after embryogenesis.


An acidic fibroblast growth factor protein generated by alternate splicing acts like an antagonist.

Polymerase chain reaction amplification of cDNA for acidic fibroblast growth factor in several lines of cultured human cells revealed two forms of mRNA. The novel smaller mRNA lacks the entire second coding exon of the acidic fibroblast growth factor gene, whereas the previously identified mRNA consists of three coding exons. The truncated variant of acidic fibroblast growth factor (aFGF') is only 60 amino acids long with an apparent molecular mass of 6.7 kD on sodium dodecyl sulfate gels in contrast to 18 kD for the full-length acidic fibroblast growth factor. aFGF' elicits only minimal fibroblast proliferation and antagonizes the effects of acidic fibroblast growth factor when added exogenously to or when coexpressed with aFGF in BALB/c/3T3 fibroblasts. Thus, the truncated variant of acidic fibroblast growth factor may provide fibroblasts with a unique mechanism for endogenous regulation of their responses to acidic fibroblast growth factor.


Antitopoisomerase I monoclonal autoantibodies from scleroderma patients and tight skin mouse interact with similar epitopes.

We have generated for the first time monoclonal antibodies (mAbs) specific for topoisomerase I (topo I) from scleroderma patients, and tight skin mice which develop a scleroderma-like syndrome. The epitope specificity of these antibodies has been determined using a series of fusion proteins containing contiguous portions of topo I polypeptide. Western blot analysis demonstrated that both human and mouse mAbs bound strongly to fusion protein C encompassing the NH2-terminal portion of the enzyme, and weakly to fusion proteins F and G containing regions close to the COOH-terminal end of the molecule. This crossreactivity is related to a tripeptide sequence homology in F, G, and C fusion proteins. It is interesting that a pentapeptide sequence homologous to that in fusion protein C was identified in the UL70 protein of cytomegalovirus, suggesting that activation of autoreactive B cell clones by molecular mimicry is possible. Both human and mouse mAbs exhibiting the same antigen specificity, also share an interspecies cross-reactive idiotope. These data suggest that B cell clones producing antitopo autoantibodies present in human and mouse repertoire are conserved during phylogeny, and are activated during the development of scleroderma disease.


Angiogenesis inhibition suppresses collagen arthritis.

Neovascularization is observed in a spectrum of diseases such as solid tumors, diabetic retinopathy, and rheumatoid arthritis. It is also evident in rat collage-induced arthritis (CIA), an animal model with histologic, clinical, and radiographic manifestations resembling rheumatoid arthritis. To evaluate the effects of angioinhibition in CIA, Louvain rats were immunized with type II collagen to induce arthritis and then administered an angiogenesis inhibitor, AGM-1470, in an attempt to either prevent arthritis or suppress established disease. Using clinical and radiographic criteria, AGM-1470 prevented CIA and significantly suppressed established disease without evidence of immunosuppression. Histologic sections from control ankle joints manifested pannus and neovascularization, which were absent in experimental animals. This is the first study to investigate this novel agent in an autoimmune disease, and additional evaluation of this promising compound in other diseases that are potentially angiogenesis dependent, such as rheumatoid arthritis, might be warranted.


Characterization of GMP-140 (P-selectin) as a circulating plasma protein.

GMP-140 is a 140-kD granule membrane protein, found in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells, that is surface expressed on cell activation and mediates neutrophil attachment. Cloning data for GMP-140 from an endothelial library predict a soluble form of the protein, the transcription message for which is also found in platelets. In this study, we report the detection by enzyme-linked immunosorbent assay of soluble GMP-140 in plasma centrifuged for 3 h at 100,000 g (to remove platelet microparticles) and confirm its identity by purification from plasma. Plasma concentrations were found to be 0.251 +/- 0.043 micrograms/ml (means +/- SD, n = 10) in normal male controls and 0.175 +/- 0.063 micrograms/ml (means +/- SD, n = 10) in normal female controls. The purified protein had an identical molecular mass (nonreduced) to platelet membrane GMP-140 (approximately 3 kD lower, reduced) and was immunoblotted by polyclonal anti-GMP-140, and the anti-GMP-140 monoclonal antibodies AK4 and AK6. Analytical gel filtration studies indicated that the plasma GMP-140 eluted as a monomer whereas detergent-free, platelet membrane GMP-140 eluted as a tetramer consistent with plasma GMP-140 lacking a transmembrane domain. Purified plasma GMP-140 bound to the same neutrophil receptor as the membrane-bound form, and when immobilized on plastic, bound neutrophils equivalently to immobilized platelet membrane GMP-140. Since it has been shown that fluid-phase GMP-140 is antiinflammatory and downregulates CD18-dependent neutrophil adhesion and respiratory burst, its presence in plasma may be of major importance in preventing the inadvertent activation of neutrophils in the circulation.


The in vivo cytotoxic activity of CD8+ T cell clones correlates with their levels of expression of adhesion molecules.

CD8+ T cell clones specific for a defined epitope present in the circumsporozoite protein of Plasmodium yoelii display striking differences in their in vivo antiplasmodial activity. The adoptive transfer of certain clones (YA23 and YA26) into naive mice inhibits by 90% or more the development of liver stages of malaria parasites and protects against malaria infection. The adoptive transfer of two other T cell clones (YB8 and YA15) results, respectively, in partial or no inhibitory activity on parasite development. We found that "protective" and "nonprotective" cytotoxic T lymphocyte (CTL) clones do not differ in their fine epitope specificity and display similar levels of lysis and DNA degradation of target cells in vitro. Their pattern of production of lymphokines and granule-associated proteins also failed to correlate with their in vivo antiplasmodial activity. Histological studies combined with autoradiography showed that, upon adoptive transfer, only T cells from the protective CTL clones are capable of "associating" with a significant percentage of parasitized hepatocytes. Fluorescence-activated cell sorter analysis of surface molecules revealed pronounced differences in the levels of CD44 and VLA-4 expression by the different clones, correlating closely with their in vivo protective activity. The correlation between in vivo antiparasite activity and the expression of CD44 was further corroborated by the results of sorting, from the partially protective YB8 clone, two sub-populations expressing high and low levels of CD44. These were protective and nonprotective, respectively. The clones also differed in their adhesive properties. Cross-linking of CD44, using specific antibodies, induced LFA-1-mediated homotypic aggregation of protective clones, while nonprotective cells failed to aggregate.


Depletion of alpha/beta T cells by a monoclonal antibody against the alpha/beta T cell receptor suppresses established adjuvant arthritis, but not established collagen-induced arthritis in rats.

The effects of treatment with a monoclonal antibody (R73 mAb) against T cell receptor alpha/beta (TCR-alpha/beta) on both established adjuvant arthritis (EAA) and established collagen-induced arthritis (ECIA) in rats have been investigated. Rats were treated with R73 mAb when arthritis reached a peak. Treatment with the anti-TCR-alpha/beta mAb markedly suppressed EAA, whereas ECIA was not affected by the mAb treatment. Histologically, R73 mAb-treated rats with EAA showed mild hyperplasia of synovial tissues, sparse infiltration of inflammatory cells, and minimal erosion of cartilage, whereas arthritic rats treated with PBS and an irrelevant control mAb against Giardia had marked hyperplasia of synovium with pannus, massive inflammatory cell infiltrate, and severe destruction of cartilage and subchondral bone. R73 mAb-treated rats with ECIA exhibited pronounced formation of pannus containing many inflammatory cells and marked cartilage and subchondral damage similar to those in arthritic rats that received the control treatments. Treatment with R73 mAb depleted markedly alpha/beta+ T cells in both peripheral blood and synovial tissues of rats with EAA and ECIA. R73 mAb treatment was associated with marked reduction in arthritogen-specific delayed-type hypersensitivity responses in both EAA and ECIA. The titers of antibodies against type II collagen produced in rats with ECIA were not affected by the mAb. Thus, alpha/beta+ T cells appear to have a central role in EAA, but not in chronic ECIA.


Antibody and B7/BB1-mediated ligation of the CD28 receptor induces tyrosine phosphorylation in human T cells.

CD28 is an adhesion receptor expressed as a 44-kD dimer on the surface of a major subset of human T cells. The CD28 receptor regulates the production of multiple lymphokines, including interleukin 2 (IL-2), by activation of a signal transduction pathway that is poorly understood. Here we show that ligation of CD28 by a monoclonal antibody (mAb) or by a natural ligand, B7/BB1, induces protein tyrosine phosphorylation that is distinct from T cell receptor (TCR)-induced tyrosine phosphorylation. CD28-induced protein tyrosine phosphorylation was greatly enhanced in cells that had been preactivated by ligation of the TCR, or by pretreatment with phorbol esters. Rapid and prolonged tyrosine phosphorylation of a single substrate, pp100, was induced in T cells after interaction with B7/BB1 presented on transfected Chinese hamster ovary (CHO) cells. Anti-B7 mAb inhibited B7/BB1 receptor-induced tyrosine phosphorylation, indicating that B7-CD28 interaction was required. CD28-induced tyrosine phosphorylation was independent of the TCR because it occurred in a variant of the Jurkat T cell line that does not express the TCR. Herbimycin A, a protein tyrosine kinase inhibitor, could prevent CD28-induced tyrosine phosphorylation and CD28-induced IL-2 production in normal T cells. The simultaneous crosslinking of CD28 and CD45, a tyrosine phosphatase, could prevent tyrosine phosphorylation of pp100. These results suggest that specific tyrosine phosphorylation, particularly of pp100, occurs directly as a result of CD28 ligand binding and is involved in transducing the signal delivered through CD28 by accessory cells that express the B7/BB1 receptor. Thus, this particular form of signal transduction may be relevant to lymphokine production and, potentially may provide a means to study the induction of self-tolerance, given the putative role of the costimulatory signal in the induction of T cell activation or anergy.


Identification of overlapping HLA class I-restricted cytotoxic T cell epitopes in a conserved region of the human immunodeficiency virus type 1 envelope glycoprotein: definition of minimum epitopes and analysis of the effects of sequence variation.

Although the immunologic basis of protective immunity in human immunodeficiency virus type 1 (HIV-1) infection has not yet been defined, virus-specific cytotoxic T lymphocytes (CTL) are likely to be an important host defense and may be a critical feature of an effective vaccine. These observations, along with the inclusion of the HIV-1 envelope in the majority of vaccine candidates presently in clinical trials, underscore the importance of the precise characterization of the cellular immune responses to this protein. Although humoral immune responses to the envelope protein have been extensively characterized, relatively little information is available regarding the envelope epitopes recognized by virus-specific CTL and the effects of sequence variation within these epitopes. Here we report the identification of two overlapping CTL epitopes in a highly conserved region of the HIV-1 transmembrane envelope protein, gp41, using CTL clones derived from two seropositive subjects. An eight-amino acid peptide was defined as the minimum epitope recognized by HLA-B8-restricted CTL derived from one subject, and in a second subject, an overlapping nine-amino acid peptide was identified as the minimal epitope for HLA-B14-restricted CTL clones. Selected single amino acid substitutions representing those found in naturally occurring HIV-1 isolates resulted in partial to complete loss of recognition of these epitopes. These data indicate the presence of a highly conserved region in the HIV-1 envelope glycoprotein that is immunogenic for CTL responses. In addition, they suggest that natural sequence variation may lead to escape from immune detection by HIV-1-specific CTL. Since the region containing these epitopes has been previously shown to contain an immunodominant B cell epitope and also overlaps with a major histocompatibility complex class II T cell epitope recognized by CD4+ CTL from HIV-1 rgp160 vaccine recipients, it may be particularly important for HIV-1 vaccine development. Finally, the identification of minimal CTL epitopes presented by class I HLA molecules should facilitate the definition of allele-specific motifs.


Excitatory amino acid receptors on isolated retinal ganglion cells from the goldfish.

1. Whole-cell currents activated by the excitatory amino acids L-glutamic acid (glutamate, Glu), L-aspartic acid (Asp), and their analogues N-methyl-D-aspartate (NMDA), kainic acid (KA), quisqualic acid (QA), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were recorded from ganglion cells enzymatically dissociated from goldfish retina and grown in culture. All agonists induced detectable whole-cell responses in the majority of cells cultured from 2 to 72 h. 2. Currents activated by each of the agonists were selective for cations (Na+) over anions (Cl-). The responses to Glu, NMDA, and Asp were each potentiated when 1 microM glycine was coapplied. Extracellular Mg2+ blocked completely the response to NMDA plus glycine in cells held at negative potentials, but the block was relieved when cells were more depolarized. 3. Dose-response measurements revealed a rank order of sensitivity to the Glu analogues in the presence of 1 microM glycine and zero extracellular Mg2+; QA greater than AMPA greater than NMDA greater than KA. Cells were not responsive to APB (L-2-aminophosphonobutyric acid). 4. Kynurenic acid (Ky) produced a noncompetitive block of the NMDA response in the presence of 1 microM glycine with a Ki of 40 microM. Responses to KA, QA, and AMPA were blocked competitively by Ky with Kis of 72, 148, and 656 microM, respectively. QA and AMPA competitively blocked the response to KA with Kis of 114 microM and 1 mM, respectively. NMDA single channels had a mainstate slope conductance of 27-30 pS and two subconductance levels of 5 and 24 pS at 13 degrees C in symmetrical Na+. 5. Whole-cell responses to QA and AMPA were highly correlated, suggesting that QA and AMPA activated the same receptor or class of receptors; whereas, responses to QA and KA were not well correlated, suggesting that these agonists at least in part activated separate receptor populations.


Enhancement of erythropoiesis by recombinant human erythropoietin in low birth weight infants: a pilot study.

We randomly assigned eight concurrently symptom-free premature infants (birth weight less than or equal to 1250 gm) at high risk of requiring erythrocyte transfusions for anemia of prematurity to 6 weeks of intensive treatment with either subcutaneous recombinant human erythropoietin (r-HuEPO group) or a placebo (control group). Treatment with r-HuEPO was initiated at a dose of 100 units/kg per day 5 days a week, and was increased to 200 units/kg per day after 2 or 3 weeks if target reticulocyte counts were not achieved. All patients were given supplemental oral iron therapy at a dose of 6 mg/kg per day, as tolerated. Mean reticulocyte counts in r-HuEPO-treated and control infants were 64,600 versus 67,500 cells/mm3 at entry; were 245,600 versus 78,000 cells/mm3 after 1 week; and averaged 262,600 versus 136,400 cells/mm3 during the study. Mean reticulocyte counts in r-HuEPO-treated infants were 251,200 cells/mm3 during the week when r-HuEPO, 100 units/kg per day, was given, and were 269,500 cells/mm3 after the dose was increased to 200 units/kg per day. Mean hematocrit values at entry were 33.4% in babies who received r-HuEPO versus 33.6% in the control subjects, and were 31.4% in r-HuEPO-treated and 25.2% in the control subjects at the end of treatment. One r-HuEPO-treated and three control babies received transfusions during the study; the total volume of blood given was 17 ml in the r-HuEPO group and 101 ml in the control subjects. The percentage of hemoglobin F increased in infants not given transfusions. We conclude that r-HuEPO stimulates endogenous erythropoiesis in small premature babies who are receiving supplemental oral iron therapy. A controlled multicenter trial has been undertaken to confirm these promising preliminary observations.


Relative proportions of pathogen-related oral spirochetes (PROS) and Treponema denticola in supragingival and subgingival plaque from patients with periodontitis.

The purpose of this study was to use monoclonal antibodies to enumerate spirochetes in dental plaque, including the newly recognized pathogen-related oral spirochete (PROS) and specific serovars of Treponema denticola. Plaque was collected from control subjects with no apparent periodontal disease and from sites of moderate to severe chronic periodontitis in patients with inflammatory periodontal disease. Individual monoclonal antibodies were used to determine whether spirochetes were present and then a double-staining protocol was employed to count total spirochetes and specific treponemes in individual microscopic fields. Results indicate that spirochetes are more common at diseased sites and in subgingival plaque than at healthy sites or in supragingival plaque. Together PROS and T. denticola comprised the majority of all spirochetes in all samples and PROS and T. denticola serovars "B" and D were most numerous in plaque from patients with periodontitis. PROS were the majority of all spirochetes in supragingival plaque (76.2% +/- 23.8%) and subgingival plaque (60.9% +/- 19.1%) from periodontitis patients, significantly larger than the percentage of T. denticola serovar "B" (P less than .001 for both supragingival and subgingival plaque) and serovar D (P less than .01 for supragingival and P less than .001 for subgingival plaque). These observations indicate that PROS are the predominant spirochete in plaque from sites of patients with periodontitis, but other analytical approaches are necessary to determine if PROS or T. denticola are pathogenic.


Regulation of steroid synthesis and metabolism in isolated binucleate cells of the placenta in sheep and goats.

Binucleate cells of sheep and goat fetal placentae comprise about one-fifth of the trophectodermal layer at the feto-maternal interface. When isolated and incubated in vitro they produce the steroids that are synthesized by the placenta in vivo (progesterone in sheep, 5 beta-pregnane-3 alpha,20 alpha diol in goats). This study demonstrates that progesterone synthesis in binucleate cell preparations in sheep was increased by prostaglandin (PG) E-2, nordihydroguaiaracetic acid (NDGA) and methylisobutylxanthine, but reduced by indomethacin, whereas in goats only NDGA produced any effect (an increase). None of the other compounds tested (luteinizing hormone, follicle stimulating hormone, prolactin, dibutyryl cAMP, A23187 or phorbolmyristic acetate) had any effect. Sheep binucleate cells also produced PGE-2 from arachidonic acid. These results suggest that, in sheep, products of both the cyclooxygenase (producing PGE-2) and lipoxygenase (inhibited by NDGA) pathways of arachidonic acid metabolism have regulatory roles in placental steroid synthesis, but only the lipoxygenase pathway is relevant in goats.


NG-allyl- and NG-cyclopropyl-L-arginine: two novel inhibitors of macrophage nitric oxide synthase.

NG-Methyl-L-arginine has recently been shown to inactivate the inducible murine macrophage nitric oxide (.NO) synthase (Olken, N. M.; Rusche, K. M.; Richards, M. K.; Marletta, M. A. Biochem. Biophys. Res. Commun. 1991, 177, 828-833). NG-Allyl-L-arginine and NG-cyclopropyl-L-arginine were synthesized as potential mechanism-based enzyme inhibitors to exploit the chemistry presumed to occur at the active site. NG-Cyclopropyl-L-arginine was found to be a potent reversible inhibitor with a Ki = 7.7 microM. NG-Allyl-L-arginine was found to be both a potent reversible (Ki = 2.1 microM) and irreversible inhibitor of the enzyme. This irreversible inhibition demonstrated pseudo-first-order inactivation kinetics with kinact = 0.026 min-1 and KI = 3.4 microM. Stereospecific protection of the inactivation was afforded by L-arginine, and saturability of the inactivation rate was observed. Our studies indicate that both reversible and irreversible inhibition of the inducible .NO synthase can be achieved with relatively simple modifications of the substrate L-arginine.


Spatial and temporal patterns of intracellular calcium in colonic smooth muscle.

Intracellular calcium [Ca2+]i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca2+]i transient followed by a steady-state increase as the characteristic [Ca2+]i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca2+]i signal in single cells. The distribution of [Ca2+]i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca2+]i present in the subplasmalemmal space and in one cell pole. [Ca2+]i gradients within these regions were not constant but showed temporal changes in the form of [Ca2+]i oscillations and spatial changes in the form of [Ca2+]i waves. [Ca2+]i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca2+] and had a mean frequency of 12.6 +/- 1.1 oscillations per min. The baseline [Ca2+]i was 171 +/- 13 nM and the mean oscillation amplitude was 194 +/- 12 nM. Generation of [Ca2+]i waves was also independent of influx of extracellular Ca2+. [Ca2+]i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +/- 3 microns per sec (n = 6). Increases of [Ca2+]i induced by different agonists were encoded into changes of baseline [Ca2+]i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca2+]i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca2+]i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca2+]i oscillations.


Cl- channels in intact human T lymphocytes.

We recently described a large, multiple-conductance Cl- channel in excised patches from normal T lymphocytes. The properties of this channel in excised patches are similar to maxi-Cl- channels found in a number of cell types. The voltage dependence in excised patches permitted opening only at nonphysiological voltages, and channel activity was rarely seen in cell-attached patches. In the present study, we show that Cl- channels can be activated in intact cells at physiological temperatures and voltages and that channel properties change after patch excision. Maxi-Cl- channels were reversibly activated in 69% of cell-attached patches when the temperature was above 32 degrees C, whereas fewer than 2% of patches showed activity at room temperature. Upon excision, the same patches displayed large, multiple-conductance Cl- channels with characteristics like those we previously reported for excised patches. After patch excision, warm temperatures were not essential to allow channel activity; 37% (114/308) of inside-out patches had active channels at room temperature. The voltage dependence of the channels was markedly different in cell-attached recordings compared with excised patches. In cell-attached patches, Cl- channels could be open at cell resting potentials in the normal range. Channel activation was not related to changes in intracellular Ca2+ since neither ionomycin nor mitogens activated the channels in cell-attached patches, Ca2+ did not rise in response to warming and the Cl- channel was independent of Ca2+ in inside-out patches. Single-channel currents were blocked by internal or external Zn2+ (100-200 microM), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonate (SITS, 100-500 microM) and 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS, 100 microM). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.


Spermatic cord beta-human chorionic gonadotropin levels in seminoma and their clinical implications.

Current opinions differ as to the biological significance and treatment of pure seminoma associated with the serological establishment of beta-human chorionic gonadotropin. Between December 1987 and April 1990, 147 patients with malignant testicular tumors were treated. Of these patients 47 (32%) had a pure seminoma. In 35 of the 47 patients we measured the tumor markers beta-human chorionic gonadotropins and alpha-fetoprotein in the cubital vein blood and testicular vein blood. There were elevated beta-human chorionic gonadotropin levels in the cubital veins of 26% of the patients, in agreement with the literature. However, elevated levels were found in the testicular veins of 80% of the patients, which reflects the high sensitivity of marker identification in testicular vein blood. Apparently, most seminomas produce beta-human chorionic gonadotropin even if it is not detectable in the cubital vein. We believe that the presence of this marker in patients with pure seminoma is not an indication of greater tumor aggressiveness but of tumor mass.


Variability and circadian changes in home uroflowmetry in patients with benign prostatic hyperplasia compared to normal controls.

We evaluated the variability and circadian changes in consecutive measurements of home uroflowmetry in 32 patients with symptomatic benign prostatic hyperplasia (BPH) and 16 healthy men. In the BPH group 476 uroflow measurements were recorded during 24 to 72 hours (mean 14.9 measurements per patient), and the controls produced 100 flow recordings (mean 6.25 measurements per participant). Great variability between consecutive peak flow rates was observed in the BPH group, ranging from at least 1 standard deviation in 28 of 32 patients (87.5%) to at least 2 standard deviations in 15 of 32 (47%). In 21 of 32 patients (65.6%) the highest recorded peak flow rate was greater than, while the lowest peak flow rate was less than the -2 standard deviations plot in voiding nomograms. In the control group variability between consecutive voiding episodes also was marked, namely at least 1 standard deviation in 8 of 16 men (50.0%) and at least 2 standard deviations in 2 of 16 (12.5%). However, in none of the control men was any peak flow rate measurement less than the -2 standard deviations line. Circadian changes in diurnal and nocturnal measurements of voided volume, interval to maximal flow, flow time, peak flow rate and adjusted peak flow rate were recorded in the BPH group, providing a urodynamic support to a well known clinical observation.


Localized hyperthermia versus the sham procedure in obstructive benign hyperplasia of the prostate: a prospective randomized study.

Hyperthermia was shown to cause improvement in 50 to 60% of the patients with benign prostatic hyperplasia (BPH) without considering placebo effects. We studied 68 patients randomly assigned to a treatment group (38) and a sham group (30) who underwent the same manipulation but without applying radio frequency power. The Biodan Prostathermer was used. Criteria for inclusion were based on objective and subjective symptoms. Treatment was performed 6 times at 43 +/- 0.5C for the treatment group. Followup evaluation was performed at 3 months, and the same objective and subjective symptoms were recorded. We observed a statistically significant subjective improvement in the sham group (33%) that was not accompanied by any significant objective improvement. In the treatment group the subjective response was significantly better regarding number of patients (68%) and response rate, and was substantiated by a significant improvement in all objective symptoms (53% of the patients) except voided volume. Therefore, hyperthermia treatment had a definite therapeutic effect on BPH in excess of placebo.


Effect of recombinant human granulocyte colony-stimulating factor in patients receiving chemotherapy for urogenital cancer. Urological rhG-CSF Study Group.

We evaluated the efficacy and safety of recombinant human granulocyte colony-stimulating factor (G-CSF) in patients with leukopenia after chemotherapy for urogenital cancers. Recombinant human G-CSF was administered at 100 micrograms./m.2 intravenously in 36 patients and at 75 micrograms. per body weight subcutaneously in 37 for 8 days after methotrexate, vinblastine, doxorubicin and cisplatin (M-VAC) therapy or for 14 days after other chemotherapies. Cycle 1 of chemotherapy was given without recombinant human G-CSF, while at cycle 2, recombinant human G-CSF was given additionally. Therefore, cycle 1 of the regimen served as a control to cycle 2 in each patient. An elevation in the median white blood cell nadir was noted: 1,500 versus 3,200 (intravenous) and 2,100 versus 3,200 (subcutaneous) on M-VAC therapy, and 1,800 versus 2,100 (intravenous) and 1,700 versus 2,500 (subcutaneous) on other chemotherapeutic regimens. A shortening of the leukopenic period was observed in cycle 2. There were no significant adverse side effects attributed to the use of recombinant human G-CSF. The results indicate that recombinant human G-CSF may be safely and effectively used against leukopenia after chemotherapy for urogenital cancer. This remedy will become useful for completion of the schedule and dose intensification of chemotherapy in the future. Subcutaneous administration is considered to be the more preferable route.


Prostate and bone fibroblasts induce human prostate cancer growth in vivo: implications for bidirectional tumor-stromal cell interaction in prostate carcinoma growth and metastasis.

Prostate cancer selectively metastasizes to the axial skeleton to produce osteoblastic lesions, which suggests that bidirectional paracrine interactions exist between prostate cancer and bone cells. To evaluate the role of tumor-stromal cell interaction and stromal-specific growth factors in prostate cancer growth and dissemination, we coinoculated nontumorigenic human prostate cancer cells (LNCaP) and various tissue-specific fibroblasts subcutaneously in athymic mice. LNCaP tumors were induced most consistently by human bone fibroblasts (62%), followed by two prostate fibroblast cell lines (31% and 17%), but not by lung, kidney, or embryonic 3T3 fibroblasts. Carcinomas formed preferentially in male hosts, demonstrating in vivo androgen sensitivity. Immunohistochemical and biochemical techniques confirmed the human prostate component of these tumors and were paralleled by elevations in serum prostate specific antigen. In vitro mitogenic assays revealed a two-to three-fold bidirectional stimulation between LNCaP and bone or prostate fibroblast conditioned media, but not lung, kidney, or 3T3 fibroblast conditioned media. A novel method developed to deliver concentrated bone or prostate fibroblast conditioned media in vivo using a slowly absorbed matrix (gelfoam) also induced tumor formation, emphasizing the importance of fibroblast growth factors in LNCaP tumor formation. Northern analysis identified the stromal compartment as the primary source of extracellular matrix (collagen, fibronectin), while only LNCaP cells expressed transforming growth factor alpha. Although LNCaP and stromal cells express basic fibroblast growth factor (bFGF), the bidirectional paracrine-mediated mitogenic activity between these cells is not inhibited by anti-bFGF antibodies, suggesting that other undefined growth factors may be involved in stimulating LNCaP growth. These observations illustrate the importance of stromal-epithelial interaction in prostate tumor growth and suggest that extracellular matrix and paracrine-mediated growth factors play a role in prostate cancer growth and metastasis.


Quantifying the smooth muscle content of the prostate using double-immunoenzymatic staining and color assisted image analysis.

The primary objective of the present study was to develop a method for quantifying the smooth muscle content of the prostate adenoma. A double immunoenzymatic staining technique was coupled with color assisted image analysis to determine the area density of the smooth muscle within the prostate adenoma. Eight males with symptomatic BPH underwent transrectal biopsy of the prostate. Four micron thick tissue sections were used for the double immunoenzymatic staining process. Rabbit anti-desmin and mouse anti-human prostatic acid phosphatase antibodies were used to selectively bind smooth muscle and prostatic epithelium, respectively. The two different tissue antigens were identified with peroxidase-antiperoxidase (PAP) and alkaline phosphatase-antialkaline phosphatase techniques. The alkaline phosphatase activity and peroxidase activity were developed with fast red and DAB chromogens. The BQ MEG IV Vista color system image analysis was used to discriminate color differences from the stained tissue sections. The thresholds were set to identify smooth muscle (dark brown), epithelium (red), fibrous tissue (pale brown), and glandular lumina (colorless). The mean area density of smooth muscle, fibrous tissue, glandular epithelium, and glandular lumina was 22%, 54%, 16%, and 9%, respectively. The present study suggests that a significant component of the prostate adenoma is smooth muscle. The application of this technique will be utilized to provide further insights into the role of smooth muscle in the pathogenesis and therapy of BPH.


[Influence of normovolemic hemodilution on the respiratory and circulatory systems].

We studied respiratory and circulatory kinetics of normovolemic hemodilution in adult mongrel dogs. The dogs were divided into two groups; Dextran-40 (group A) and SALIN-HES (group B). No difference in PaO2 and PaCO2 was noted between the two groups. MPA and CI during normovolemic hemodilution were higher in group A than in group B. However, MPA, Qs/Qt and ETVI during normovolemic hemodilution were largely influenced in group A. L/P during normovolemic hemodilution was more influenced in group B than in group A. The compensatory effect with increase of the cardiac output during normovolemic hemodilution was noted until S3 (Hct = 10%), but it was not noted at S4 (Hct = 5%) and S5 (just before death). SvO2 is the most sensitive index indicating the risk of normovolemic hemodilution.


[Anesthetic management of palliative surgery for hypoplastic left heart syndrome--a case report].

The first patient with hypoplastic left heart syndrome who was treated successfully by palliative surgery at our hospital is reported. Soon after birth, the female infant showed tachypnea and cyanosis, and was transferred to our institution under a presumptive diagnosis of HLHS. Although cardiologists confirmed the diagnosis by two-dimensional echocardiography, the surgery was postponed for one month because it was possible to keep the ductus arteriosus open without PGE1, and the patient showed no deterioration. Fortunately, an abnormal vessel connecting the left atrium with the superior vena cava relieved severe pulmonary venous congestion by diverting the blood flow. During the pre-CPB period, frequent adjustment of the oxygen concentration and ventilator setting was required in order to keep the blood gas values optimal compared with the values before surgery. After CPB, adequate blood pressure using catecholamines and hyperventilation with 100% oxygen was necessary to increase the pulmonary blood flow and to decrease the pulmonary vascular resistance. It is concluded that preservation of the balance between PVR and SVR during the perioperative period, and adequate systemic arterial pressure after CPB are crucial. Furthermore, constant and intense observation is mandatory to facilitate immediate treatment even after surgery in case of systemic hypoperfusion due to excessive pulmonary blood flow.


[Application of FDA/EB staining for the detection of viable or non-viable mycobacteria in clinical specimens].

Two-hundred sputum specimens from tuberculosis patients were examined for viable or non-viable mycobacteria by a combination of fluorescein diacetate ethidium bromide (FDA/EB) staining, Ziehl-Neelsen staining, and the use of cultures in 3% Ogawa egg medium. The sputum specimens were treated with 3% NaOH for 10 min and washed in PBS. The bacteria was then harvested by centrifugation at 6,000 rpm for 5 min. Each sample was subjected to FDA/EB staining, Ziehl-Neelsen staining and cultures to compare the staining -method results and the results of colony formation. Ziehl-Neelsen staining method revealed acid-fast bacteria in the specimens, distributed from Gaffky 1 to Gaffky 8. The number of FDA-positive specimens and culture-positive specimens were identical in all Gaffky grades, suggesting that the FDA staining method well reflected the presence of viable mycobacteria in the specimens. We concluded that FDA staining is a valuable method to detect viable mycobacteria in sputum specimens on the first day of examination. It is therefore advantageous for doctors and patients to be immediately informed of culture results rather than waiting for several weeks.


Immunocytochemical localization of proANF 1-30, proANF 31-67 and atrial natriuretic factor in the kidney.

ProANF [1-30 first 30 amino acids (a.a) of the 126 a.a atrial natriuretic factor (ANF) prohormone], ProANF 31-67 (a.a 31-67) as well as atrial natriuretic factor (a.a 99-126), of the ANF prohormone localized to the sub-brush border of the pars convoluta and pars rectus of the proximal tubules of hydrated and dehydrated rat kidneys with immunoperoxidase staining. Immunofluorescent studies revealed that each of these peptides and especially ProANF 31-67 had a strong predilection for the perinuclear region in the proximal and distal tubules. ProANFs 1-30, 31-67, and 99-126 (that is, ANF) also localized with both immunoperoxidase and immunofluorescent staining to the cortical collecting ducts, glomeruli, peritubular, and interstitial blood vessels. ProANF 31-67 immunoperoxidase staining was particularly striking in the elastica of the small and large interstitial arteries. The whole prohormone being present was suggested by immunological recognition in the rat kidney of both the N-terminus and C-terminus of the ANF prohormone by radioimmunoassays. The concentration of the N-terminus in the kidney was 1.0 +/- 0.03 ng/g of kidney weight, while the C-terminus of the ANF prohormone concentration was 0.4 +/- 0.01 ng/g of kidney tissue. These findings of a sub-brush border and perinuclear location of the N-terminal and C-terminal ANF prohormone peptides suggest that the atrial natriuretic factor prohormone may be synthesized in and/or the respective peptides are captured by the proximal tubules, but also to a lesser extent by the distal tubules of the kidney.


Modulation of granulocyte LAM-1 and MAC-1 during dialysis--a prospective, randomized controlled trial.

Hemodialysis with first-use cellulosic dialysis membranes results in activation of the alternative pathway of complement and profound neutropenia followed by rebound leukocytosis. The neutropenia has been shown to be associated with increased expression of adhesion receptors and pulmonary sequestration of granulocytes. However, the mechanism underlying the return of the granulocytes has not been elucidated. We determined simultaneously the changes in the granulocyte adhesion receptor MAC-1 (CD11b-CD18) and the selectin LAM-1 receptor during dialysis using a complement activating and a non-complement activating membrane, in a randomized, cross-over study. With initiation of dialysis with cellulosic membranes, there was a rapid and prominent increase in the expression of MAC-1 receptors. At the nadir of granulocyte count, 15 minutes after initiation of dialysis with the complement activating membrane, there was a four-fold increase in the MAC-1 receptor expression. At the same time, there was a two-fold decrease in LAM-1 expression. There were no changes in the expression of two other granulocyte receptors CD11a and CD15 which are known not to be modulated during granulocyte activation. Granulocytes harvested during dialysis and which had high MAC-1 and low LAM-1 expression had a significantly decreased adherence to endothelial cell monolayers. Dialysis of the same patients with non-complement activating membranes resulted in no significant change in the expression of these receptors on granulocytes nor in their adherence to endothelial cells. These results shed new light on the mechanism of the cyclical granulocytopenia and rebound granulocytosis during dialysis with new cellulosic membranes.


Self-renewal inhibition of acute myeloid leukemia clonogenic cells by biological inducers of differentiation.

We analyzed the maintenance of acute myeloid leukemia clonogenic cells (AML CFU-L) in liquid culture in the presence of five potential differentiation inducers: trans-retinoic acid, 1,25-dihydroxy vitamin D3, interferon gamma, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), used singly and with two combined. The culture medium contained either fetal bovine or human serum from normal donors. CFU-L recovery after 7 days was compared to that observed in control cultures. Of AML cases and HL60 cells, 11/15 displayed greater than 50% CFU-L reduction in response to one or more inducers. In 8/9 responsive cases that underwent the nitroblue tetrazolium (NBT) reduction test, an increase in the percentage of functionally mature (NBT-positive) cells was detected. The combination of retinoic acid with interferon gamma was most effective in reducing CFU-L recovering (8 responsive/15 AML cases), G-CSF and M-CSF displayed either inhibitory or stimulatory activity in different AML cases. The type of serum employed generally did not affect the response to inducers of differentiation. No significant inhibition of the recovery of granulocyte-macrophage colony-forming units was determined by the five inducers in experiments with three normal bone marrow samples. Our experiments indicate that biological differentiation inducers can reduce AML CFU-L self-renewal and increase the proportion of differentiated cells at concentrations that do not affect normal myelopoiesis and could be achieved during treatments in vivo.


Molecular investigation of the cytokines produced by normal and malignant B lymphocytes.

Different normal and malignant human B-cell populations were studied with a twofold aim: to define which cytokines are produced in vivo, and to assess the relationship between cytokine production and kinetic state. To analyse normal B-cells representative of different stages of activation and proliferation in vivo, we purified germinal centre (GC)-B blasts and mantle B (M-B) cells from tonsils. To compare malignant B lymphocytes with their closest normal equivalent cells, we separated malignant CD5+B lymphocytes from the peripheral blood of patients with B-chronic lymphocytic leukemia (B-CLL) and normal CD5+B lymphocytes from cord blood. The expression of interleukins (IL) IL-1 alpha, IL-1 beta, tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), IL-2, IL-4, and IL-6 genes was analysed using Northern and Western blotting techniques. TNF-alpha mRNA is produced by resting (M-B) and actively proliferating (GC-B) normal B lymphocytes. TGF-beta mRNA is present at high levels in resting normal M-B cells, while the transcript levels are lower in proliferating GC-B and in activated CD5+B lymphocytes. IL-2 production is limited to the actively proliferating GC-B blasts, IL-1 beta and IL-6 to resting M-B cells. The cytokine production profile of CD5+ malignant B-CLL cells differs from that of their putative normal counterparts and is more like the profile of M-B cells, since B-CLL cells produce IL-1 beta, TNF-alpha, TGF-beta, and IL-6. These observations lead to the following conclusions: among normal B lymphocyte populations, resting M-B lymphocytes are the most active cytokine producers, and B-CLL malignant B cells reflect the production pattern of normal resting B lymphocytes.


Suppression of nigrostriatal and mesolimbic dopamine release in vivo following noradrenaline depletion by DSP-4: a microdialysis study.

Pretreatment of rats with the noradrenergic neurotoxin DSP-4 selectively reduced regional levels of noradrenaline in the brain by more than 75%, and decreased the concentration of endogenous DA in microdialysates of the caudate nucleus and nucleus accumbens by 52% and 28%, respectively. Results support the hypothesis that central noradrenergic mechanisms facilitate nigrostriatal and mesolimbic dopamine transmission in vivo.


Control of functional mRNA stability in bacteria: multiple mechanisms of nucleolytic and non-nucleolytic inactivation.

Messenger RNA in bacteria may be inactivated by several parallel mechanisms acting independently on different target sites. For any species of mRNA the overall rate of inactivation is determined by the sum of the contributions from the different mechanisms. Transcripts may be inactivated directly by endonucleolytic attack or by processive nucleolytic degradation, which may proceed in the 3'-5' direction and probably also in the 5'-3' direction. Moreover, the functional lifetime of many mRNAs may be determined by processes that are not nucleolytic, such as the binding of translational repressors or the formation of secondary structures which prevent initiation of translation. These non-nucleolytic processes may also determine the chemical stability as chemical degradation frequently appears to be closely coupled to functional inactivation. The relative importance of the different mechanisms in the inactivation of bulk cellular mRNA, as well as the general prospects for engineering of stable mRNAs are discussed.


Retron-Ec107 is inserted into the Escherichia coli genome by replacing a palindromic 34bp intergenic sequence.

Some natural isolates of Escherichia coli have been shown to produce a unique branched RNA-linked single-stranded DNA called msDNA. These bacteria contain a retro-element called retron consisting of the msr-msd region and the gene for reverse transcriptase (RT). All three E. coli retrons characterized to date have been shown to be integrated into a prophage or to be associated with phage-related genes. In this report, we identified a new msDNA from an E. coli wild strain. Using the msDNA as a probe, the retron for the msDNA was cloned and its DNA sequence was determined. The retron was found to consist of a 1.3kb DNA fragment, making it the smallest retron isolated to date. The msDNA produced from the retron consists of a 107 base single-stranded DNA, which is considered to be branched out from the 18th G residue of a 75-base RNA molecule by a 2',5'-phosphodiester linkage. Thus, the msDNA and the retron were designated msDNA-Ec107 and retron-Ec107, respectively. Most significantly, retron-Ec107 was inserted into the E. coli genome by replacing a 34bp intergenic sequence between the pyrE and ttk genes located at 82 min on the E. coli chromosome. Interestingly, the retron contains palindromic structures at both ends and the E. coli 34bp intergenic sequence also contains a 10bp inverted repeat structure. These palindromic structures might have played a role in the integration of retron-Ec107 into the E. coli genome.


Sequence diversity of the 1.3 kb retron (retron-Ec107) among three distinct phylogenetic groups of Escherichia coli.

In the preceding paper, we showed that a new 1.3 kb retron (retron-Ec107) in Escherichia coli is responsible for the biosynthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA-Ec107). Here, we show that this retron occurs in strains from different branches, A, B1, and D of a well-defined phylogenetic tree of a collection of wild E. coli. Sequence comparisons of the retrons from these three branches were carried out. Sequence homology was well conserved among the strains within the same branch and the retron sequence from branch A was exactly the same with that from branch D, while there were 18 base substitutions between the retrons from branch B1 and A or D, resulting in seven amino acid substitutions in reverse transcriptase. No substitutions were found in the msDNA- and msdRNA-coding regions, and there was no difference in the ability of msDNA production between them. These results suggest that the retron has probably been integrated into at least one of the three branches at an early stage of evolution and subsequently transferred to the other two branches, and also that the msDNA-producing system has been conserved during evolution with some mutations in the retron.


Regulation of the expression of the cell-cycle gene ftsZ by DicF antisense RNA. Division does not require a fixed number of FtsZ molecules.

We show that the 53-nucleotide RNA molecule encoded by gene dicF blocks cell division in Escherichia coli by inhibiting the translation of ftsZ mRNA. Such a role for dicF had been predicted on the basis of the complementarity of DicF RNA with the ribosome-binding region of the ftsZ mRNA. An analysis of ftsZ expression at its chromosomal locus, and of an ftsZ-lacZ translational fusion controlled by promoters ftsZ1p and ftsZ2p only, indicates that ftsZ is not autoregulated. Partial inhibition of FtsZ synthesis leads to increased cell size. However, the number of FtsZ molecules per cell can be reduced threefold without affecting the division rate significantly. Our results suggest that septation is not triggered by a fixed number of newly synthesized FtsZ molecules per cell.


Regulatory mutants and transcriptional control of the Serratia marcescens extracellular nuclease gene.

The extracellular nuclease of Serratia marcescens is regulated in a complex fashion. Unlike most catabolic enzymes, it appears not to be substrate regulated. However we have shown it to be regulated by an SOS-like system in S. marcescens. Additionally nuclease expression is regulated in a growth-phase-dependent manner. In this work we demonstrate that growth-phase-dependent regulation is at the transcriptional level. The putative LexA-binding site which mediates SOS regulation is shown to act as an operator site in vivo. The boundaries of a minimal promoter, still regulated by growth phase and SOS regulation, are defined along with the transcriptional start site. However, a region upstream of the nuclease promoter is shown to enhance significantly the expression of nuclease.


High-dose combination alkylating agents with autologous bone-marrow support in patients with breast cancer: preliminary assessment of DNA damage in individual peripheral blood lymphocytes using the single cell gel electrophoresis assay.

The single cell gel (SCG) assay is a sensitive electrophoretic technique for detecting the presence of DNA single strand breaks and alkali-labile damage in individual cells. This technique was used to evaluate the levels of DNA damage in cryopreserved peripheral blood lymphocytes (PBLs) from 11 breast cancer patients treated with high doses of cyclophosphamide and cisplatin and provided autologous bone marrow transplantation after treatment. PBL specimens for the SCG study were obtained just prior to treatment, following the administration of cyclophosphamide and cisplatin for 2 days, and upon lymphocytic recovery. Based on a concurrent analysis of DNA damage in cryopreserved and non-cryopreserved PBL samples from three patients, the mean level of DNA migration or the dispersion of damage among cells was not affected by the process of cryopreservation. The pre-treatment samples of several patients contained PBL with increased levels of DNA damage, presumably reflecting persistent DNA damage induced by previous treatment regimens. Chemotherapy resulted in a significant but variable increase in DNA damage in PBL samples from all patients. In this limited study, the level of damage did not correlate with serum levels of cyclophosphamide or with lymphocyte toxicity. Among the post-treatment samples, increased levels of DNA damage were absent in most but not all patients. The presence of damaged cells in the post-treatment samples may be indicative of an inadequate therapy regimen or of DNA damage resulting from non-therapy related processes. Because of its simplicity and short processing time, the SCG assay can be used to evaluate levels of DNA damage during the course of therapy, allowing the dose schedule to be altered to achieve a desired effect level.


Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities.

The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (Mr 38,000, 341 amino acids). To identify antigenic determinants on Hib porin that might be exposed at the bacterial cell surface, seven mouse monoclonal anti-Hib porin antibodies were generated. The monoclonal antibodies were tested for their binding to intact cells by flow cytometry; all but one bound to the cell surface. Digestions of Hib porin with cyanogen bromide, hydroxylamine or trypsin generated fragments, the identities of which were confirmed by microsequencing of the amino termini. Following electrophoresis and immunoblotting of the fragments, the specificities of the monoclonal antibodies for their cognate sequences were determined. The porin gene ompP2 was expressed in the baculovirus expression vector system; the recombinant porin was recognized by all of the monoclonal antibodies. Deletions were created by omega mutagenesis of ompP2, generating proteins truncated after amino acids 139, 174, 182, and 264. These deletion proteins were tested for reactivities with the monoclonal antibodies, thereby establishing the boundaries of three antigenic determinants that were recognized by the monoclonals: domain (i), amino acids 104-139; domain (ii) amino acids 162-174; and domain (iii), amino acids 267-341. The biological activities of monoclonal antibodies that were representative of these three classes were tested for their bactericidal activity in complement-mediated lysis of whole cells. The monoclonal antibodies were also tested for their immunoprotective properties in the infant rat model of bacteraemia. Although the monoclonal antibodies were surface-binding, they were neither bactericidal nor protective.


The ability of liver extracts from different-aged rats to repair 'mis-instructive' and 'non-instructive' lesions of DNA.

The ability to repair 'mis-instructive', O6-methylguanine, and 'non-instructive', AP sites, DNA lesions in Fischer 344 rat livers at various ages was determined. Different behaviours were observed. While the AP-endodesoxyribonuclease enzymes displayed a high constant level throughout the animals' lifetime, the O6-methylguanine-DNA methyltransferase activity presented a stepwise modulation (DNA normalisation of results): the O6-MT activity significantly increased within the first month of animal life and enhanced again after 6 months reaching a maximum plateau in the 12-18-month-old animals. Thereafter a net significant decrease of O6-MT enzyme was detected in the 24-month-old group. While the repair of the widely formed AP sites appeared uniformly efficient like 'house keeping' functions, the removal of the rare precancerous O6-methylguanine is age-dependent indicating a decreased protection of the youngest and oldest animals against this 'mis-instructive' damage. However, any extrapolation of the age-associated cancer risk needs further assessment.


Demethylation of satellite I DNA during senescence of bovine adrenocortical cells in culture.

Over the finite proliferative life span of cultured bovine adrenocortical cells, satellite I DNA shows a progressive and extensive loss of methylation at CCGG sites. This was shown by Southern blotting after digestion with the methylation-sensitive enzyme HpaII alone, which provides a sensitive indicator of methylation loss, or digestion with the combination of EcoRI and HpaII, which provides a quantitative indication of loss of methylation. Bovine tissues, including adrenal cortex, all showed a much higher level of satellite methylation than cultured adrenocortical cells. After adrenocortical cells are placed in culture, some demethylation of satellite I is seen as early as 10 population doublings. By 80 population doublings, loss of satellite DNA methylation is extensive. The loss does not appear to prevent continued cell division, since an extended life span clone of bovine adrenocortical cells transfected with SV40 T antigen showed a similar pattern of extensive demethylation. Satellite demethylation has been reported in aging in vivo and the present cell culture system may provide an in vitro model for this form of genetic instability.


The effect of aging on cell-cycle kinetics and X-ray-induced chromosome aberrations in cultured lymphocytes from patients with Down syndrome.

To evaluate the effects of aging on cytogenetic characteristics of lymphocytes from Down syndrome (DS), cell-cycle kinetics after PHA stimulation and chromosome-type aberration frequencies after X-ray exposure were investigated in vitro in the lymphocytes derived from 4 (or 3 for X-ray treatment) age groups of DS patients and age-matched controls. The results clearly showed higher mitotic and proliferation index levels in younger groups compared to older groups at the various culture intervals, whether the lymphocytes were from the DS patients or controls. The age-related changes of the proliferation index were mainly attributed to a delayed response to PHA as age increased. The changes of PHA responses seemed to be particularly marked during adolescence. Nonetheless, no significant differences were observed between the DS patients and age-matched controls for each age group. In all age groups, frequencies of both chromosome-type exchanges and deletions were elevated in the DS patients by about 1.3 times in comparison with the controls. The magnitude of radiosensitivity, however, seemed to decrease slightly in the 40-49-year group. To our knowledge, the present study is the first report in the literature to deal with the effect of aging on the greater radiosensitivity of DS lymphocytes.


Effect of iron chelators on the cytotoxic and genotoxic action of hyperoxia in Chinese hamster ovary cells.

The iron chelators o-phenanthroline and desferrioxamine were tested for their ability to protect Chinese hamster ovary cells against the cytotoxic and genotoxic effects of normobaric hyperoxia. Desferrioxamine added at sub-toxic concentrations (up to 2.5 microM) over a period of several days had no protective effect on hyperoxia-induced clonogenic cell killing and growth inhibition. The clastogenic effect of hyperoxia was strongly potentiated by desferrioxamine, while the induction of sister-chromatid exchanges (SCEs) by hyperoxia was unaffected. Similarly, o-phenanthroline (up to 0.25 microM) had no protective effect on hyperoxia-induced cell killing, growth inhibition, and SCE induction, while also this compound potentiated the clastogenic effect of hyperoxia. These results do not support a critical role for cellular iron in the mechanism of toxicity by normobaric hyperoxia in CHO cells. However, the results may still be consistent with a critical involvement of particular iron fraction(s) not susceptible to the chelators used. Furthermore, our results show that concentrations of iron chelators known to protect against short-term (up to 1 h) toxic exposure to oxidative stress become toxic themselves when applied chronically, i.e., in the order of days.


Effects of mutagens on the clonal lifespan of Paramecium tetraurelia.

There has been interest in the phenomenon that a cell cannot undergo unlimited reproduction under adequate conditions and undergoes senescence. In holotrichous ciliates, Paramecium has a limit of vegetative reproduction without sexual reproduction but Tetrahymena does not always have a limited lifespan. Comparing the two species would increase our knowledge of the mechanism of cellular clonal aging. We previously showed that mutations induced by X-rays shorten clonal lifespan. In this study, we examined whether mutagens shorten the clonal lifespan of Paramecium tetraurelia. P. tetraurelia was exposed to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 0.045 mg/ml, for 30 min. The animal was exposed to MNNG 6 times in total while young (under 80 divisions from the start of a clonal life cycle) or 4 times during the senescent stage. MNNG shortened the clonal lifespan as expressed by the decrease in fission number from 186 +/- 55 (4 cell lines) to 136 +/- 21 (6 cell lines) with the first two treatments but with further exposures the lifespan increased to 182 +/- 15 (5 cell lines). MNNG had no effect when administered at the older age. Exposure of P. tetraurelia to 4-nitroquinoline-N-oxide at 0.021 mg/ml twice for 12 and 15 min at the younger age reduced the mean clonal lifespan from 143 +/- 28 to 125 +/- 21 and the maximum lifespan from 263 +/- 33 to 175 +/- 25.


Modulation of DNA modification (I-compound) levels in rat liver and kidney by dietary carbohydrate, protein, fat, vitamin, and mineral content.

I-compounds are DNA modifications detected by 32P-postlabeling that increase with age in rodents without known carcinogen exposure. Diet type (natural ingredient versus purified) greatly influences patterns and levels of I-compounds. To test the hypothesis that I-compound formation is affected, also, by dietary macro- and micronutrients, effects of carbohydrate, protein, fat, vitamin, and mineral content on rat liver and kidney I-compounds were determined. Female Sprague-Dawley rats were fed basic or modified AIN-76A purified diets for 3-6 months. High protein (HP) diet (50%, w/w) increased I-compound levels in liver but not kidney. High carbohydrate (HC) diet (78%) produced a significant increase in the polar as well as total I-compound levels in both tissues. High fat diets (20%) elicited significantly lower levels of liver I-compounds than HC, HP, and basic diets. There were few significant differences between high polyunsaturated (safflower oil) and saturated fat (lard) diet groups. No qualitative differences in I-compound profiles were observed in either tissue. In rats fed basic diet supplemented with vitamins and/or minerals, increased vitamin content reduced the levels of polar I-compounds in liver. No extra diet-induced adducts were observed; all effects were of a quantitative nature. These data provide direct evidence that nutrients significantly influence I-compound levels and support the hypothesis that normal metabolism of nutrients leads to the production of small amounts of DNA-reactive electrophiles. These observations suggest a novel mechanism where nutrient composition of the diet may play a role in development of neoplasia and other adverse health effects.


Different rate of chromosome breakage in human fibroblast strains after storage in liquid nitrogen.

Cytogenetic investigation was carried out on fibroblasts stored in liquid nitrogen during a period of 7-99 months. Cell strains were from 9 individuals, 2 of whom were affected by xeroderma pigmentosum group C (XPC), and 2 XPC heterozygotes. In cell samples from 3 normal subjects and from 1 patient, high frequencies of abnormal mitoses were observed at the first passage after thawing, which returned to normal values after a few subcultures. The most frequent lesions were chromosome gaps and breaks. The cells damaged the most were those from one XP patient. These findings indicate that cells from some individuals are hypersensitive to clastogenic factors acting during freezing and thawing procedures. This sensitivity could be related to the genetic constitution, although the XP homozygous condition is not an essential or sufficient factor.


The micronucleus test of methyl methanesulfonate with mouse peripheral blood reticulocytes using acridine orange-coated slides.

The usefulness of the micronucleus assay using mouse peripheral blood erythrocytes and acridine orange (AO)-coated slides was evaluated with methyl methanesulfonate (MMS). The micronucleus test was carried out at doses ranging from 20 to 80 mg/kg body weight in CD-1 mice by intraperitoneal injection. Peripheral blood cells were examined from 0 to 72 h after treatment at 12- or 24-h intervals. Bone marrow cells from other mice treated with 80 mg/kg MMS were also sampled at the same times. The frequency of micronucleated reticulocytes (MNRETs) increased dose-dependently at every sampling time except 72 h, and the maximum frequency of MNRETs was observed at about 36 h after treatment. Micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow after a dose of 80 mg/kg were significantly induced at 12 h to 36 h, and the maximum frequency of MNPCEs was observed at 24 h after treatment. The induction of MNRETs was delayed by about 12 h compared to that of MNPCEs in bone marrow, and the maximum frequencies of MNRETs were lower than those of MNPCEs, but the induction of MNRETs by MMS was significant and dose-dependent. It is concluded, therefore, that bone marrow cells could be replaced by peripheral blood cells as material for the micronucleus assay using AO-coated slides.


The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides with triethylenemelamine.

A micronucleus assay using mouse peripheral blood and supravital staining with acridine orange (AO) was validated by two laboratories on triethylenemelamine-treated mice. Dose- and time-dependent increases in micronucleated peripheral reticulocytes were observed. This new method can be used as an alternative to the conventional bone marrow micronucleus assay.


Simplified mouse peripheral reticulocyte micronucleus test with dimethylnitrosamine.

The induction of micronuclei by treatment with dimethylnitrosamine was evaluated and compared in peripheral blood and bone marrow cells of male CD-1 mice. Peripheral blood preparations were made on acridine orange (AO)-coated slides and scanned by fluorescence microscopy. A significant increase in micronuclei was observed 24 h after treatment in bone marrow polychromatic erythrocytes, and 24-48 h after treatment in peripheral reticulocytes. The peak frequency of micronuclei in peripheral reticulocytes was delayed by about 24 h relative to bone marrow polychromatic erythrocytes. This micronucleus test using peripheral blood was shown to be easy to do and as sensitive as the test using bone marrow cells. From this result, it is concluded that the method with AO-coated slides and peripheral blood is as suitable as bone marrow cells for the micronucleus assay.


The micronucleus assay with peripheral blood reticulocytes by acridine orange supravital staining with 1-beta-D-arabinofuranosylcytosine.

Dose-dependent induction of micronuclei with 1-beta-D-arabinofuranosylcytosine (ara-C) was clearly shown in CD-1 mouse peripheral blood reticulocytes (RETs) using an acridine orange (AO) supravital staining method, as well as in the conventional bone marrow assay. The maximum frequencies of micronucleated RETs (MNRETs) in peripheral blood and of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow were comparable, as shown in two laboratories independently. The maximum frequencies of MNRETs in peripheral blood lagged about 24 and 12 h behind those of MNPCEs in bone marrow in experiments with 24- and 12-h sampling intervals, respectively. The proportion of each type of RET was examined periodically after treatment with ara-C at doses ranging from 6.25 to 50.0 mg/kg. The proportion of type I RETs among total RETs decreased 24 or 48 h after treatment according to the dose level. This suggest that this ratio could be a good indicator of the bone marrow cell toxicity of test chemicals.


Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides.

A new method for the micronucleus test using peripheral blood reticulocytes stained supravitally using acridine orange-coated slides was evaluated in male CD-1 mice treated with ethyl methanesulfonate (EMS) at doses of 100, 200, 300, and 400 mg/kg. Peripheral blood samples were taken 0, 24, 48, 72, and 96 h after treatment from each mouse without killing. The frequencies of micronucleated reticulocytes increased dose-dependently with the peak at 48 h after treatment. These results indicate that, at least for EMS, the new method used here can be an alternative to the conventional method using bone marrow polychromatic erythrocytes.


Micronucleus tests on N-ethyl-N-nitrosourea with mouse peripheral blood reticulocytes using acridine orange-coated slides.

The micronucleus test with peripheral blood using acridine orange-coated slides was validated in male CD-1 mice treated once with N-ethyl-N-nitrosourea (ENU) at doses of 6.25, 12.5, 25.0, and 50.0 mg/kg body weight. Peripheral blood preparations were made 0, 24, 48, and 72 h after treatment. The frequencies of micronucleated peripheral reticulocytes in the ENU-treated groups increased dose-dependently, peaking at 48 h after treatment. The results indicate that the method used in the present study can be an alternative to the method using bone marrow polychromatic erythrocytes.


Micronucleus assays on 5-fluorouracil and 6-mercaptopurine with mouse peripheral blood reticulocytes.

As part of the 5th collaborative study of the Collaborative Study Group for the Micronucleus Test (CSGMT), the sensitivity and advantages of the micronucleus assay using mouse peripheral blood cells were evaluated using 5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP). The peripheral blood cells were collected from a tail vein of CD-1 male mice just before and 24-120 h after intraperitoneal injection. At 24-h intervals. The maximum incidence of micronucleated reticulocytes (MNRETs) at 50 mg/kg 5-FU was observed 96 h after injection; at 100 mg/kg, the peak was delayed to 120 h, and followed severe bone marrow depression. With 6-MP, maximum MNRETs were observed 48 h after treatment at all doses tested. At dose levels higher than 50 mg/kg, severe bone marrow depression was observed after maximum MNRETs. Though the appearance patterns of MNRETs and the bone marrow depression were different between 5-FU and 6-MP, the positive response of both chemical could be detected with this assay system as well as with the micronucleus test using femoral bone marrow cells.


The micronucleus test using peripheral blood reticulocytes from methotrexate-treated mice.

The induction of micronuclei by methotrexate (MTX) was examined in two laboratories using mouse peripheral blood reticulocytes. MTX was a weak inducer in the micronucleus test using bone marrow cells and single treatments, and was one of the few chemicals showing a multiple-treatment effect (CSGMT/JEMS.MMS, 1990). In our preliminary experiments, the ratio of reticulocytes to total erythrocytes decreased greatly after a single treatment with MTX at 100 mg/kg, so lower dose levels of MTX were selected to carry out the micronucleus test in peripheral blood. Full-scale tests were performed at dose levels of 0, 10, 20, 40, and 80 mg/kg, with five sampling times of 0, 24, 48, 72, and 96 h. Frequencies of micronucleated reticulocytes (MNRETs) increased dose-dependently at 72 h, to a maximum of approximately 1%; some preparations obtained from the animals at higher doses could not be examined because the ratio of reticulocytes to total erythrocytes had decreased severely. At doses of 0.5-4.0 mg/kg, the effect of multiple treatments vs. single treatments was not clear, nor was the maximum level of response much different. Since MTX induced a clear positive response in peripheral blood reticulocytes after a single treatment, the reticulocytes in peripheral blood seem a more sensitive target.