Basolateral distribution of fibronectin matrix assembly sites on vascular endothelial monolayers is regulated by substratum fibronectin. Endothelial cells exhibit binding sites for the amino terminus of fibronectin that participate in subendothelial fibronectin matrix assembly. These binding sites, termed matrix assembly sites, are localized on the basolateral surface of confluent endothelial monolayers (Kowalczyk et al. Blood, 75:2335, 1990). The present study investigates the role of cell-cell and cell-substratum interactions in the localization of matrix assembly sites to the basal surface of endothelial cells. Cells were cultured in Transwell culture inserts and matrix assembly sites were detected by binding assays using an iodinated 70 Kd amino-terminal fibronectin fragment. Integrity of intercellular junctions was monitored by measuring protein flux across Transwell filters. Time course experiments demonstrated that matrix assembly site expression on the basolateral cell surface preceded intercellular junction formation. Transfer of confluent monolayers to calcium-free medium resulted in the loss of junctions and in an increase in 125I-70 kD binding from the apical medium. The increased 125I-70 kD binding resulted from increased access of 125I-70 kD to basolateral matrix assembly sites and not from the relocation of binding sites to the apical membrane. To determine the effect of matrix composition on matrix assembly site expression and localization, cells were seeded onto vitronectin- or fibronectin-coated substrates. Fibronectin increased the expression of matrix assembly sites on the apical surface within 24 hours. By 48 hours, matrix assembly sites were located only on the basolateral surface. Vitronectin had no effect on the expression or localization of matrix assembly sites. These results indicate that the expression and localization of matrix assembly sites on the surface of vascular endothelial cells can be regulated by substratum fibronectin.


Candida albicans growth studies: a hypothesis for the pathogenesis of Candida infections in burns. The proteolytic environment in which Candida albicans exists strongly affects its virulence. To determine whether virulence might be related to C. albicans growth in different proteolytic environments, we measured renal fungal load in burned mice and found significantly greater Candida census in kidneys from mice that were challenged with a high proteinase-generating parent C. albicans (MY 1044) versus those that were challenged with its low proteinase-generating mutant (MY 1049). In vitro, MY 1044 cells grew faster than MY 1049 cells in media that contained sera from burned mice as the only nitrogen source. Augmentation of media with proteinase or a mixture of amino acids increased growth of MY 1049 cells, whereas augmentation with proteinase inhibitor decreased MY 1044 growth. In conclusion, in vitro growth of both the mutant and its parent strain was affected by the proteolytic environment in which they existed; thus, virulence differences for MY 1044 and MY 1049 could be due in part to growth differences between these two strains in different proteolytic environments. These results were combined with existing observations, and we proposed a theory for the pathogenesis of C. albicans in burns.


Structure of the axonal surface recognition molecule neurofascin and its relationship to a neural subgroup of the immunoglobulin superfamily. The chick axon-associated surface glycoprotein neurofascin is implicated in axonal growth and fasciculation as revealed by antibody perturbation experiments. Here we report the complete cDNA sequence of neurofascin. It is composed of four structural elements: At the NH2 terminus neurofascin contains six Ig-like motifs of the C2 subcategory followed by four fibronectin type III (FNIII)-related repeats. Between the FNIII-like repeats and the plasma membrane spanning region neurofascin contains a domain 75-amino acid residues-long rich in proline, alanine and threonine which might be the target of extensive O-linked glycosylation. A transmembrane segment is followed by a 113-amino acid residues-long cytoplasmic domain. Sequence comparisons indicate that neurofascin is most closely related to chick Nr-CAM and forms with L1 (Ng-CAM) and Nr-CAM a subgroup within the vertebrate Ig superfamily. Sequencing of several overlapping cDNA probes reveals interesting heterogeneities throughout the neurofascin polypeptide. Genomic Southern blots analyzed with neurofascin cDNA clones suggest that neurofascin is encoded by a single gene and its pre-mRNA might be therefore alternatively spliced. Northern blot analysis with domain specific probes showed that neurofascin mRNAs of about 8.5 kb are expressed throughout development in embryonic brain but not in liver. Isolation of neurofascin by immunoaffinity chromatography results in several molecular mass components. To analyze their origin the amino-terminal sequences of several neurofascin components were determined. The NH2-terminal sequences of the 185, 160, and 110-135 kD components are all the same as the NH2 termini predicted by the cDNA sequence, whereas the other neurofascin components start with a sequence found in a putative alternatively spliced segment between the Ig- and FNIII-like part indicating that they are derived by proteolytic cleavage. A combination of enzymatic and chemical deglycosylation procedures and the analysis of peanut lectin binding reveals O- and N-linked carbohydrates on neurofascin components which might generate additional heterogeneity.


Developmental regulation of insulin-like growth factor binding protein production: studies in fetal, postnatal, and pregnant sheep. To assess the roles of developmental factors in the regulation of sheep IGFBP production at the cellular level, we characterized and compared the IGFBPs released by fetal, postnatal, and maternal sheep skin fibroblasts in culture with those in fetal, postnatal, and maternal sheep plasma. Sheep fibroblasts produced seven IGFBPs: a 36.5-41 kDa protein induced in vitro by IGF-I, likely representing oIGFBP-3; a 28.5 kDa protein that reacted with antisera to human IGFBP-2, likely representing oIGFBP-2; 25 and 27 kDa proteins induced in fetal fibroblasts by IGF-I; a 22 kDa protein that was inhibited by IGF-I, likely representing oIGFBP-4; and 21 and 23 kDa proteins labelled only by IGF-II, suggesting their similarities to IGFBP-6. The developmental pattern of IGFBP production by sheep fibroblasts in culture was similar in several respects to that observed in sheep plasma. For example, relative amounts of the 21, 22, and 28.5 kDa IGFBPs exceeded that of the 36.5-41 kDa protein in early fetal fibroblast conditioned media and in fetal plasma, while the relative concentrations of the 36.5-41 kDa protein increased markedly during the perinatal period. Sheep plasma differed, however, in two major respects from fibroblast conditioned media: First, fetal, and to a far lesser extent maternal, plasma contained a 200 kDa IGF-II-selective BP, likely to be the circulating form of the IGF-II receptor; and second, plasma, unlike conditioned media, contained a 26 kDa IGFBP, likely to be oIGFBP-1. The results of our studies suggest that the production and release of IGFBPs by isolated sheep fibroblasts is regulated by developmental factors operative under in vitro culture conditions. The differences in the relative levels of IGFBPs in conditioned media from fetal, postnatal, and maternal sheep fibroblasts resemble in several respects the differences in the relative concentrations of the various IGFBPs in fetal, postnatal, and maternal sheep plasma. Thus, sheep fibroblasts provide a useful though imperfect model system by which to examine the nutritional and hormonal regulation of sheep IGFBP production at various developmental stages.


Production of granulocyte colony-stimulating factor and granulocyte/macrophage-colony-stimulating factor after interleukin-1 stimulation of marrow stromal cell cultures from normal or aplastic anemia donors. We have studied stromal cell function in naive or interleukin-1 (IL-1)-stimulated (100 pg/ml) long-term marrow cultures (LTC) from 12 normal donors and 21 patients with severe aplastic anemia (AA). Conditioned media (CM) from normal LTC contained levels of erythroid burst-promoting activity (BPA) and granulocyte/macrophage (GM) colony-stimulating activity (CSA) comparable to those previously described (Migliaccio et al., [1990] Blood, 75:305-312). The addition of IL-1 to these cultures increased the level of CSA and, specifically, of granulocyte colony-stimulating factor (G-CSF) released. Anti-GM-CSF antibody neutralized BPA and CSA in normal naive LTC CM but only the CSA in the CM from IL-1-stimulated LTC. Since the concentrations of GM-CSF, as detected with a specific immunoassay, did not increase after IL-1 treatment, these data suggest that IL-1-stimulated cultures contain an unidentified growth factor having BPA. CM from AA stromal cells contained levels of CSA comparable to those observed in normal stromal cell CM but had significantly lower levels of BPA. Neither anti-GM-CSF nor anti-IL-3 antibodies neutralized the BPA in AA stromal cell CM. This activity may be related to that found in the CM of IL-1-treated normal stromal cells. In nearly 50% of stromal cell cultures of AA patients, addition of IL-1 failed to increase the BPA, CSA, or G-CSF. The presence of an inhibitor in naive or IL-1-treated AA stromal cell CM was excluded by adding the CM to IL-3-stimulated cultures. These findings suggest that G-CSF and GM-CSF genes are differentially regulated in the marrow microenvironment. Furthermore, a marrow microenvironment, deficient in BPA production and, in some cases, unresponsive to IL-1 could contribute to marrow failure in some patients with AA.


Regulation of albumin expression in fetal rat hepatocytes cultured under proliferative conditions: role of epidermal growth factor and hormones. Sustained production of plasma proteins, notably albumin, is a reliable indicator of the differentiated state of hepatocytes. In this work, we have developed a fetal hepatocyte culture system where studying the regulation of albumin expression in proliferating liver cells. Our results show that under proliferative conditions (i.e., in the presence of EGF) fetal hepatocytes maintain albumin production above control quiescent non-treated cells. Glucagon and noradrenaline have no effect on the proliferation induced by EGF in cultured fetal hepatocytes; however, they act synergistically with the growth factor, increasing intracellular albumin levels. The maximum response is obtained by treatment of cells with EGF and noradrenaline. The stimulatory noradrenergic effect is mimicked by agents that increase cyclic AMP levels (forskolin plus IBMX). However, vasopressin or phorbol esters have no effect on albumin production, neither alone nor in combination with EGF. Dexamethasone, which does not alter the proliferative induction of EGF, increases albumin content. This effect is independent of the proliferative status of the cells and is not enhanced by glucagon, noradrenaline, or cyclic AMP increasing agents. The hormonal changes observed in albumin production partially correlate with changes in mRNA levels. This is the first time that cyclic AMP increasing agents are shown to act synergistically with EGF, increasing the expression of this liver specific gene.


Characterization of the human myeloid cell nuclear differentiation antigen: relationship to interferon-inducible proteins. The human myeloid cell nuclear differentiation antigen (MNDA) is expressed specifically in cells of the granulocyte/monocyte lineage. The MNDA has been isolated by using a monoclonal antibody affinity matrix and reversed-phase high performance liquid chromatography. Its NH2-terminal sequence has been obtained, as well as additional sequence information derived from peptides produced by cyanogen bromide and SV8 protease cleavages. Meaningful similarities were observed in extended regions between the MNDA and the reported beta interferon-inducible proteins, 202 and 204, from Ehrlich ascites mouse tumor cells. An amphipathic, basic alpha-helical region, showing no similarity to the 202 and 204 proteins, exhibited close similarity to a region in the interferon response factor-2, a protein which binds the interferon stimulated response element. The relatively high number of S(T)PXX motifs present in the partial amino acid sequence of the MNDA, described herein, suggests that the MNDA binds DNA and is a transcription factor.


Cloning and sequence determination of bovine insulin-like growth factor binding protein-2 (IGFBP-2): comparison of its structural and functional properties with IGFBP-1. Insulin-like growth factor binding proteins (IGFBPs) are secreted by several cell types and can modify IGF actions. Mandin-Darby Bovine Kidney (MDBK) cells have been shown to secrete a 34,000 Da form of IGF binding protein whose N-terminal sequence is similar to a form of IGFBP purified from rat BRL-3A cells that has recently been named IGFBP-2. These studies report the complete amino acid sequence of bovine IGFBP-2 and compare its functional properties with human IGFBP-1. The protein is 81% identical to rat IGFBP-2. When compared with both rat IGFBP-2 and human IGFBP-1, the positions of all 18 cysteine residues are conserved. Similarly an RGD sequence is present near the carboxyl terminus in both proteins. IGFBP-2 has a higher affinity for IGF-II than for IGF-I and its affinity for both forms of IGF is greater than for human IGFBP-1. Like IGFBP-1 the protein can enhance the DNA synthesis response of porcine aortic smooth muscle cells to IGF-I; however, IGFBP-2 was much less potent. The maximum potentiation of the IGF-mediated mitogenic response that could be achieved was approximately 42% that of IGFBP-1. This potentiation is dependent upon a factor contained in platelet poor plasma and if this factor is omitted from the incubation medium, IGFBP-2 inhibits DNA synthesis. The purification of IGFBP-2 will allow more detailed comparisons to be made between it and other forms of IGFBPs in physiologic test systems.


Identification of localized autoantibody epitopes in thyroid peroxidase. Recent reports have disagreed on the nature of the autoantibody epitopes in thyroid peroxidase (TPO). We used immunoprecipitation of recombinant human TPO constructs to determine if localized autoantibody binding sites exist in this autoantigen. In vitro transcription and translation of TPO cDNA fragments yielded 35S-labeled products consisting of either full-length protein (933 amino acids) or N-terminal peptides of 631, 455, and 120 amino acids. Immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the Hashimoto's sera consistently precipitated the full-length and the 631 amino acid products, but not the shorter N-terminal peptides. An additional construct resulting in a full-length TPO peptide with an internal deletion of amino acids 4-455 was also made, and this product was also precipitated by the Hashimoto's sera. A fusion protein consisting of maltose binding protein followed by amino acids 456-933 of human TPO was produced in Escherichia coli and subjected to Western blot analysis using the Hashimoto's sera. The Hashimoto's sera reacted with the MalTose binding protein TPO (MBP/TPO) fusion protein, but not a control fusion protein (MBP/LacZ alpha). Together, these results indicate the presence of localized autoantibody epitopes in the portion of the human TPO molecule from amino acids 456 to 933, with at least one binding site located between amino acids 456 and 631.


Basal and regulated secretion of insulin-like growth factor binding proteins in osteoblast-like cells is cell line specific. Local secretion of insulin-like growth factor binding proteins (IGFBPs) may modulate the effects of IGF-I and IGF-II on bone cell metabolism and proliferation. Several osteoblast-derived cell lines are currently used as interchangeable models to study IGFBP production, although it is unknown whether findings in one cell line can be extrapolated to another cell line or to normal human osteoblasts. In this study, we examined by Western ligand blotting both basal and regulated secretion of IGFBPs in vitro in 1) normal human osteoblast-like (hOB) cells cultured from explants of human trabecular bone; 2) an SV40-transformed hOB (HOBIT) cell line; and 3) several human (U-2, MG-63, TE-85) and rat (ROS 17/2.8 and UMR-106-01) osteosarcoma cell lines. Constitutively, hOB and HOBIT cells produced a similar pattern of IGFBPs, while all other cell lines produced their own unique pattern of IGFBPs. The two rat cell lines differed from the human cell lines as well as from each other. The response to hormonal stimulation also varied among the cell lines. Treatment of hOB and HOBIT cells with IGF-I resulted in a 2-fold increase in medium levels of IGFBP-3; IGF-I decreased levels of 24-kilodalton (kDa) IGFBP in hOB cell-conditioned medium. In addition, IGF-I markedly increased levels of the 29/32/34 kDa IGFBP triplet in U-2 cells, but had little or no effect in the other human and rat osteosarcoma cell lines. PTH increased a 29-kDa IGFBP apparent only in UMR 106-01 cell-conditioned medium, whereas GH had no direct effect on IGFBP secretion in any of the osteoblast-like cells tested. We conclude that basal and regulated secretion of IGFBPs from osteoblast-like cells is cell-line specific. Spontaneously transformed human or rat osteoblast-like cells provide unique model systems to study features of distinct IGFBPs and their regulation; however, hOB cells and their derivatives may be more appropriate models for understanding the regulation of IGFs in human bone.


Cytokine modulation of progesterone and estradiol secretion in cultures of luteinized human granulosa cells. To clarify the possible roles of cytokines in the regulation of luteal cell function, we examined the effects of interferon (IFN), interleukin-1 (IL-1), and tumor necrosis factor (TNF) on progesterone and estradiol secretion in cultures of luteinized human granulosa cells. IFN gamma reduced hCG-stimulated progesterone secretion in a concentration-dependent manner; at its maximal inhibitory concentration (10 ng/mL), IFN gamma reduced progesterone secretion to 20% of that in the hCG-stimulated controls. Whereas other IFN (alpha and beta) reproduced the inhibitory effect of IFN gamma, IL-1 and TNF had no effect on hCG-stimulated progesterone secretion at concentrations of 1 and 10 ng/mL. IFN gamma also markedly reduced FSH-stimulated estradiol secretion. Unlike their effects on hCG-stimulated progesterone secretion, IL-1 and TNF reproduced the inhibitory effect of IFN gamma on FSH-stimulated estradiol secretion. IFN gamma significantly reduced both hCG- and FSH-stimulated cAMP generation in granulosa cells. IL-1 and TNF inhibited FSH-stimulated cAMP generation, but they did not inhibit hCG-stimulated cAMP generation. None of these cytokines reduced forskolin-stimulated cAMP generation, thus suggesting that these cytokines affect steps proximal to cAMP generation without affecting cAMP generation itself. IFN gamma also reduced progesterone secretion in response to (Bu)2cAMP, suggesting that it also affects steps distal to cAMP generation. This study has demonstrated that cytokines modulate the steroidogenesis of luteinized human granulosa cells in vitro; the results suggest that cytokines may play permissive roles in regulating luteal cell function.


Effects of recombinant human insulin-like growth factor-I (rhIGF-I) on total and free IGF-I concentrations, IGF-binding proteins, and glycemic response in humans. To examine the effects of repeated administration of recombinant human insulin-like growth factor-I (rhIGF-I) on IGF-I levels, free IGF-I pharmacokinetics, glycemic response, and IGF-binding proteins (IGFBP), we administered rhIGF-I (0.03 mg/kg iv bolus) to 12 healthy males each morning for 5 consecutive days. Serum was collected over 24 h on days 1 and 5 for measurement of total and free IGF-I, glucose, insulin, and IGFBP. Total IGF-I was measured by RIA after acid/ethanol extraction. Free IGF-I was separated from binding protein-complexed IGF-I using size exclusion high performance liquid chromatography before measurement by RIA. IGFBP were quantitated by optical densitometry of Western ligand blots. Total IGF-I increased significantly from 0-24 h after administration on day 1 (mean +/- SD, micrograms/L: 120 +/- 44 to 166 +/- 51, P = 0.0002) but did not increase significantly from 24 h on day 1 to 0 h on day 5 (166 +/- 51 to 178 +/- 62) or from 0-24 h on day 5 (178 +/- 62 to 209 +/- 89). The area under the total IGF-I concentration curve was greater on day 5 than day 1 (311 +/- 99 min.g/L vs. 249 +/- 77, P = 0.0001). There were no significant differences in free IGF-I concentration or pharmacokinetic parameters or in the degree or timing of hypoglycemia between days 1 and 5. Plasma insulin levels decreased significantly following rhIGF-I administration (day 1 baseline: 53 +/- 11 pmol/L, nadir: 18 +/- 6 pmol/L at 30 min, P = 0.003); day 5 baseline: 47 +/- 15 pmol/L, nadir: 16 +/- 8 pmol/L at 30 min, P = 0.0003. Western ligand blotting revealed the transient appearance of a 30-kilodalton band which migrates in a manner similar to IGFBP-1. This band was undetectable at baseline, peaked between 150 and 210 min after rhIGF-I administration, and diminished by 480-600 min. The response was similar on days 1 and 5. There were no substantial changes in the serum levels of any other IGFBP. In summary, repeated iv bolus administration of rhIGF-I increased the level of total circulating IGF-I without changing free IGF-I disposition or glycemic response. A 30-kilodalton IGFBP band, most likely IGFBP-1, appeared transiently following rhIGF-I administration, probably as a result of suppression of insulin levels. IGFBP-2, -3, and -4 were unaffected.


Glycosylation changes in human chorionic gonadotropin and free alpha subunit as gestation progresses. Carbohydrate is important to the structure, function, and circulatory survival of the glycoprotein hormones. Human CG (hCG) and the related free alpha-molecule of pregnancy contain four and two asparagine-linked oligosaccharides, respectively. The present study analyzes changes in the glycosylation patterns of hCG and free alpha in early vs. late gestation. Five volunteers provided 24-h urine samples, weekly, throughout their pregnancies. Extracts of early pregnancy (weeks 7-12) and late pregnancy (weeks 28-32) urines were pooled. Early and late samples from each patient were subjected to gel filtration to separate hCG and free alpha, and the populations thus obtained were analyzed by lectin affinity chromatography on Concanavalin A-Sepharose (Con A) and Lens culinaris-agarose (Lch). Using Con A, free alpha and hCG were separated into an unbound fraction (eluted with Con A buffer), a weakly bound fraction (eluted with 10 mmol alpha-methyl-D-glucoside) and a tightly bound fraction (eluted with 500 mmol alpha-methyl-D-mannoside). For free alpha-molecule, a significant decrease in tightly bound Con A forms, was noted from early to late pregnancy with a mean difference of 17.0 +/- 2.4% (P less than 0.01). Concomitantly, in late pregnancy, an increase in Con A unbound forms of free alpha was noted with mean difference of 12.5 +/- 1.7% (P less than 0.01). These changes indicate the presence of more highly branched oligosaccharides on free alpha as gestation advances. No changes were noted in the Con A binding of intact hCG; nearly all hCG bound in both early and late pregnancy. Using Lch, free alpha and hCG were separated into an unbound fraction (eluted with Lch buffer) and a bound fraction (eluted with 500 mmol alpha-methyl-D-mannose). Both free alpha and intact hCG in late pregnancy exhibited increased binding to Lch, with mean differences from early to late pregnancy of 30.2 +/- 4.8% (P less than 0.01) and 11.4 +/- 4.5% (P less than 0.05), respectively. These data indicate increased incorporation of fucose into the carbohydrate moieties in late pregnancy. Taken together, these data derived by analysis using lectin specificity imply the presence of more highly branched, fucosylated oligosaccharides as gestation progresses.


Comparison of CNS homing pattern among murine TH cell lines responsive to myelin basic protein. A myelin basic protein (MBP)-reactive TH cell line capable of inducing experimental allergic encephalomyelitis (EAE), and a MBP-reactive TH cell clone that does not cause EAE were labeled with a fluorescent vital dye, and transferred into naive syngeneic SJL/J mice. Animals were killed before the appearance of symptoms (3 and 4 days post-injection). Sections obtained from the spleen, spinal cord and brain of both groups of animals were examined by fluorescence microscopy to localize labeled TH cells. At all time points examined, the spleens of both groups contained innumerable labeled cells. The spinal cords and brains of animals that had received EAE-causing cells had a basal level of 20 labeled cells/cm2 at 3 days; this number increased rapidly to 150 cells/cm2 in the spinal cord at 4 days. Perivascular infiltrates and small foci of astrogliosis were already apparent in this group 3 days after injection. The spinal cords and brains of animals that had received the non-EAE-causing TH cells contained 50 labeled cells/cm2 at 3 days. The density of these transferred cells, as compared to that of the EAE-causing cells, suggested that they have an unaltered CNS-homing capability. However, by 4 days, the number of non-EAE-causing labeled cells had returned to near basal level. Our findings suggest that discrimination between disease and non-disease causing MBP-responsive TH cells occurs within the first 3 days following transfer, requires the presence in the CNS of a limited number of TH cells, and depends on yet unidentified TH cell factor(s).


Suppression of experimental autoimmune encephalomyelitis by gallium nitrate. We examined the effect of gallium (Ga) nitrate on the development of the development of experimental autoimmune encephalomyelitis (EAE). Weekly subcutaneous injections of 10-30 mg/kg prevented clinical signs as well as histopathological changes of EAE. The optimal timing of a single injection of Ga was 6 days after induction of EAE, with amelioration also apparent following a single injection on day 3 or 9 but not day 12. Ga administered in vivo suppressed myelin basic protein (MBP) and purified protein derivative-specific lymphocyte proliferative responses in vitro. Addition of Ga to MBP-specific T lymphocyte line cultures at various times after initiation of culture revealed that Ga exerts an effect at an early stage of cellular activation.


Myelin proteolipid protein: minimum sequence requirements for active induction of autoimmune encephalomyelitis in SWR/J and SJL/J mice. Proteolipid protein (PLP) is the major protein constituent of mammalian central nervous system myelin. We have previously identified two different PLP encephalitogenic T cell epitopes in two mouse strains. Murine PLP peptides 103-116 YKTTICGKGLSATV and 139-151 HCLGKWLGHPDKF are encephalitogenic determinants in SWR/J (H-2q) and SJL/J (H-2s) mice, respectively. The purpose of the present study was to determine the minimum sequence requirements for each of these PLP encephalitogens. In SWR/J mice, at least two distinct overlapping peptides can induce experimental autoimmune encephalomyelitis (EAE). The eleven residue sequences PLP 105-115 TTICGKGLSAT and PLP 106-116 TICGKGLSATV are encephalitogenic in SWR/J mice, but PLP 106-115 TICGKGLSAT, the decapeptide indigenous to both sequences, is non-encephalitogenic. In contrast, the shortest PLP sequence capable of inducing EAE in SJL/J mice is the nonapeptide 141-149 LGKWLGHPD. These data indicate that encephalitogenic determinants of PLP are short contiguous peptide sequences similar in length and diversity to those of MBP.


Synthetic peptide specificity of anti-myelin basic protein from multiple sclerosis cerebrospinal fluid. Human myelin basic protein (h-MBP) purified from central nervous system (CNS) myelin has a molecular mass of 18.5 kDa and 170 residues. Eighteen synthetic peptides ranging from 8 to 25 residues and covering the length of h-MBP were prepared by the Fmoc method. Antibodies to h-MBP (anti-MBP) were isolated and purified from cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) by two-step affinity chromatography. Purified anti-MBP was reacted with increasing amounts of h-MBP as well as each of the 18 synthetic peptides in an initial liquid phase assay, and then titers of free anti-MBP in the resulting mixtures were determined by a solid phase radioimmunoassay. Purified anti-MBP was neutralized by h-MBP and 6 of the 18 synthetic peptides used in this study. The antibody was completely neutralized by peptides No. 12 (residues: 80-97), No. 15 (residues: 91-106) and No. 56 (residues: 75-95) and was partially neutralized by peptides No. 27 (residues: 61-75), No. 16 (residues: 64-78) and No. 21 (residues: 69-83). The 12 remaining synthetic peptides covering both the amino (residues 1-63) and carboxyl (residues 117-162) terminals of h-MBP did not neutralize purified anti-MBP. These results suggest that anti-MBP purified from CSF of patients with MS have affinity for discontinuous epitopes located between residues 61 and 106 on the h-MBP molecule. Alternatively anti-MBP may be polyspecific recognizing different amino acid sequences.


Galanin immunoreactivity in the primate central nervous system. Galanin-immunoreactive profiles were localized within the monkey and human central nervous system. In the monkey telencephalon, galanin-immunoreactive perikarya were seen within the anterior olfactory nucleus, basal forebrain, endopiriform nucleus, hippocampus, and bed nucleus of the stria terminalis. The caudate nucleus and putamen contained galanin-immunoreactive perikarya whereas the nucleus accumbens displayed only galanin-immunoreactive fibers. In the diencephalon, galanin-immunoreactive profiles were seen within the medial preoptic area, periventricular, suprachiasmatic, paraventricular, and arcuate nuclei as well as the lateral hypothalamic area. Within the thalamus, only galanin-immunoreactive fibers were seen within the midline paraventricular, reuniens, and rhomboid nuclei. In the mesencephalon, scattered galanin-immunoreactive fibers were seen in the periaquaductal gray, ventral tegmental area, and midbrain reticular formation. In the metencephalon, galanin-immunoreactive neurons were observed in the medial vestibular nucleus and nucleus prepositus. In the myelencephalon, galanin-immunoreactive perikarya were seen within the nucleus of the tractus solitarius and hypoglossal nucleus. Dense collections of galanin-immunoreactive fibers were found in the spinal descending tract of V, nucleus of the tractus solitarius, and dorsal motor nucleus of X. Galanin immunoreactivity was also observed within all circumventricular organs. Spinal anterior horn neurons expressed galanin immunoreactivity, and immunopositive fibers were seen within the tract of Lissauer and the substantia gelatinosa. Although the distribution of galanin immunoreactivity was generally similar between monkeys and humans, there were a few striking exceptions. The human supraoptic nucleus contained galanin-immunoreactive neurons, whereas the monkey supraoptic nucleus displayed only immunopositive fibers. Similarly, galanin-immunoreactive perikarya and fibers were seen in the human locus coeruleus and subcoeruleus, whereas in monkeys these regions contained only fibers. These data demonstrate a widespread distribution of galanin-containing profiles in primates, suggesting that galanin may modulate cognitive, sensory, motor, and autonomic processes.


Substance P-immunoreactive sensory axons in the rat respiratory tract: a quantitative study of their distribution and role in neurogenic inflammation. Substance P is one of the peptides released from sensory nerves that mediate "neurogenic inflammation." Although substance P-immunoreactive (SP-IR) axons are known to be present within the mucosa of the respiratory tract, the relative extent of the innervation of various components of the mucosa is not known. Therefore, we determined the distribution and number of SP-IR axons in the rat trachea and bronchi, by using immunohistochemistry on tissue whole mounts. Specifically, we sought to learn whether these axons directly innervate the postcapillary venules involved in neurogenic plasma extravasation, the arterioles involved in neurogenic vasodilatation, and the airway smooth muscle involved in bronchoconstriction in pathogen-free, adult male F344 rats. We found that 90% of the SP-IR axons were single axons, usually having varicosities. Eighty-five percent of these were in the epithelium, 6% innervated arterioles, and the remainder elsewhere in the lamina propria. Only 10% of the mediator-sensitive postcapillary venules (i.e., venules labeled with Monastral blue pigment after challenge with capsaicin or substance P) were within 10 microns of SP-IR axons. SP-IR axons were more than 10 times as frequent in the smooth muscle of the distal bronchi as in the trachea. Capsaicin pretreatment (168 mg/kg over 7 days) reduced the number of SP-IR axons in the trachea by 96%, which is consistent with their being sensory. Unilateral vagotomy reduced the number of SP-IR axons bilaterally in the trachea and ipsilaterally in the main stem bronchus. Using an antibody to Protein Gene Product 9.5 as a nonspecific marker for all nerves in the trachea, we determined that SP-IR axons constituted 90% of the axons in the epithelium, 32% of the axons on arterioles, and only 4% of the axons in the smooth muscle. We conclude that most SP-IR nerves in the trachea are sensory axons and most of these axons end in the epithelium. SP-IR axons innervate mucosal arterioles, but few innervate postcapillary venules. Therefore, the mechanism by which sensory axons evoke plasma extravasation from these venules is likely to involve the diffusion of the peptide or a secondary mediator from the epithelium or from the arterioles upstream.


Co-localization of tyrosine hydroxylase, GABA and neuropeptide Y within axon terminals innervating the intermediate lobe of the frog Rana ridibunda. Possible co-existence of gamma-aminobutyric acid (GABA), catecholamines, and neuropeptide Y (NPY) in the same nerve terminals of the frog intermediate lobe was investigated by immunocytochemistry at the electron microscopic level. Co-localization of GABA and tyrosine hydroxylase (TH) was studied by using a double immunogold labeling procedure. Co-localization of glutamate decarboxylase (GAD) and NPY was studied by combining, respectively, the peroxidase-antiperoxidase method and a radioimmunocytochemical labeling procedure. Catecholamines and GABA were systematically co-localized in nerve endings of the pars intermedia. Most of the NPY-immunoreactive fibers also contained GAD-like immunoreactivity. However, a few NPY-positive nerve terminals were not immunoreactive for GAD. These data provide evidence for co-existence of a regulatory peptide (NPY) and several neurotransmitters (i.e., GABA and catecholamines) within the same axon terminals in the intermediate lobe. Since GABA, dopamine, and NPY have all been shown to inhibit the activity of frog melanotrope cells, the present findings suggest that these neuroendocrine factors may interact either at the pre-synaptic or post-synaptic level.


Prefrontal cortical efferents in the rat synapse on unlabeled neuronal targets of catecholamine terminals in the nucleus accumbens septi and on dopamine neurons in the ventral tegmental area. Physiological and pharmacological studies indicate that descending projections from the prefrontal cortex modulate dopaminergic transmission in the nucleus accumbens septi and ventral tegmental area. We investigated the ultrastructural bases for these interactions in rat by examining the synaptic associations between prefrontal cortical terminals labeled with anterograde markers (lesion-induced degeneration or transport of Phaseolus vulgaris leucoagglutinin; PHA-L) and neuronal processes containing immunoreactivity for the catecholamine synthesizing enzyme, tyrosine hydroxylase. Prefrontal cortical terminals in the nucleus accumbens and ventral tegmental area contained clear, round vesicles and formed primarily asymmetric synapses on spines or small dendrites. In the ventral tegmental area, these terminals also formed asymmetric synapses on large dendrites and a few symmetric axodendritic synapses. In the nucleus accumbens septi, degenerating prefrontal cortical terminals synapsed on spiny dendrites which received convergent input from terminals containing peroxidase immunoreactivity for tyrosine hydroxylase, or from unlabeled terminals. In single sections, some tyrosine hydroxylase-labeled terminals formed thin and punctate symmetric synapses with dendritic shafts, or the heads and necks of spines. Close appositions, but not axo-axonic synapses, were frequently observed between degenerating prefrontal cortical afferents and tyrosine hydroxylase-labeled or unlabeled terminals. In the ventral tegmental area, prefrontal cortical terminals labeled with immunoperoxidase for PHA-L were in synaptic contact with dendrites containing immunogold reaction product for tyrosine hydroxylase, or with unlabeled dendrites. These results suggest that: (1) catecholaminergic (mainly dopaminergic) and prefrontal cortical terminals in the nucleus accumbens septi dually synapse on common spiny neurons; and (2) dopaminergic neurons in the ventral tegmental area receive monosynaptic input from prefrontal cortical afferents. This study provides the first ultrastructural basis for multiple sites of cellular interaction between prefrontal cortical efferents and mesolimbic dopaminergic neurons.


Patterns of antigenic expression in the thalamic reticular nucleus of developing rats. The present study describes the development of the thalamic reticular nucleus in rats with the use of Nissl staining and antibodies to parvalbumin and pro-alpha-thyrotropin-releasing hormone (alpha TRH). Two major subdivisions of the reticular nucleus are apparent: 1) the main body, which is itself heterogeneous and lies for the most part between the fibres of the internal capsule and external medullary lamina, and 2) the perireticular nucleus, which lies lateral to the main body and medial to the globus pallidus. In the main body of the reticular nucleus of adults, most cells in all regions are immunoreactive to parvalbumin and alpha TRH. During development there are two waves of parvalbumin and alpha TRH expression. The first wave occurs between postnatal day (P) 0 and P10, and labelled cells are apparent in rostrolateral areas of the main body of the nucleus only. At P10, such cells are not apparent. From P7 to adult, there is a second wave of parvalbumin and alpha TRH expression: labelled cells emerge first in central, then in caudal, and finally in rostral areas of the nucleus. In adults, the perireticular nucleus is made up of a few small cells which are immunostained for parvalbumin and alpha TRH. These cells are more frequent in areas of the internal capsule adjacent to the ventral regions of the main body of the reticular nucleus, rostrodorsal to the entopeduncular nucleus. From E (embryonic day) 17 to about P10, the perireticular nucleus consists of a surprisingly large population of neurones, many of which are parvalbumin and alpha TRH immunoreactive. By about P10, as in adults, there are few perireticular cells.


Glucose metabolism in freshly isolated Müller glial cells from a mammalian retina. Glucose metabolism was studied in isolated retinal Müller glial cells from the juvenile guinea pig. Cells, once enzymatically isolated and purified, were identified by morphological criteria, positive vimentin immunoreactivity, and histochemical staining for glycogen. Purified suspensions of Müller cells were obtained in quantities sufficient for biochemical analysis (approximately 2 x 10(5)/pair of retinas) and light microscopic autoradiography. In bicarbonate-buffered Ringer's medium containing 3H-2-deoxyglucose and no glucose, greater than or equal to 80% of the glucose analogue taken up intracellularly by Müller cells was phosphorylated to 3H-2-deoxyglucose-6-phosphate. In autoradiographs, this non-metabolized product provided visual evidence of glucose phosphorylation: the distribution of cell grains mirrored the morphology of individual Müller cells in situ. Exposure to the glycolytic inhibitor iodoacetate (500 microM) caused an 85% decrease in adenosine triphosphate (ATP) content; concomitantly, 3H-2-deoxyglucose-6-phosphate decreased by 90% and paralleled a dramatic decrease of cell labelling in autoradiographs, while levels of 3H-2-deoxyglucose did not change. In the continual absence of glucose, glycogen content decreased with time and this decrease was slowed by 36% in the presence of iodoacetate. This indicated that, in control conditions, glycosyl units from glycogen sustain cellular metabolism, and hence 3H-2-deoxyglucose phosphorylation. 3H-2-deoxyglucose-6-phosphate concentration was 43-fold less than that of ATP in the control conditions so that depletion of ATP during iodoacetic acid (IAA)-blocked glycolysis was not due to hexokinase activity. These results demonstrate that this preparation is adequate for quantitative studies of glucose metabolism at the cellular and molecular level in an important metabolic compartment of the mammalian retina.


Pigmented basal cell carcinoma: investigation of 70 cases. Pigmented basal cell carcinoma (PBCC) is a clinical and histologic variant of BCC. Our purpose was to identify the histologic subtypes of BCC that were most often associated with pigment and to determine whether this correlated with outcome after excision. A series of PBCC was identified and the histologic subtype noted. Margins of all excisions were examined for residual tumor. These results were then compared with a series of nonpigmented BCCs. In a series of 1039 consecutive BCCs, 70 (6.7%) contained pigment. The histologic growth pattern most frequently associated with pigment was the nodular/micronodular pattern (12.4%) followed by the nodular (7.7%), superficial (7.2%), micronodular (4.0%), and the nodular/micronodular/infiltrative (3.4%) patterns. Margins were examined for evidence of residual tumor in the 40 cases that were excised. In only one case (2.5%) was the margin positive for tumor. This was statistically significant (p less than 0.05) compared with 388 excisions of nonpigmented BCCs with comparable growth patterns in which 69 (17.7%) showed positive margins. PBCC, as a clinical variant, is more frequently excised with adequate margins than are tumors of comparable histologic subtypes that do not contain pigment.


Muir-Torre syndrome.

Muir-Torre syndrome. Muir-Torre syndrome is characterized by multiple sebaceous tumors, various internal malignancies and an autosomal dominant inheritance. We herein report a typical case. The patient was a 69-year-old man with sebaceous adenomas, a keratoacanthoma, and actinic keratosis in addition to carcinomas of the prostate, colon, duodenum, and larynx. His family members also suffered from multiple cancers.


Eruptive pruritic papular porokeratosis. Three cases of an unusual variant of porokeratosis (Mibelli) were described. Patients with disseminated superficial porokeratosis for some years suddenly developed intensively pruritic erythematous papules. Skin biopsies revealed that these papules contained cornoid lamellae on their tops. Pruritic papules subsided in several months, leaving slightly hyperkeratotic brown annular lesions which were shown to contain typical cornoid lamellae histopathologically. This type of porokeratosis has not been reported in the literature.


Primary systemic amyloidosis: a unique case complaining of diffuse eyelid swelling and conjunctival involvement. A 52-year-old Japanese woman with the chief complaint of marked swelling of her upper eyelids and a mass over the bulbar conjunctiva is reported. She previously noticed frequent purpura after minimal trauma, which was resolved shortly after taking some ascorbic acid. Laboratory data showed Ig-G kappa type M-protein in the serum and kappa type Bence-Jones protein in the urine by immunoelectrophoresis. Systemic examination showed mild hepatosplenomegaly, 1st degree of AV block, and a mild increase in plasma cells in the bone marrow biopsy. Histologically, the whole dermis of the eyelid skin and conjunctiva was replaced by a large quantity of amorphous, eosinophilic substances, which were diffusely positive with Direct Fast Scarlet 4BS. Immunohistochemical staining was positive for anti-amyloid P component antibody. Typical amyloid fibrils were proved by electron microscopy. She was finally diagnosed as primary systemic amyloidosis with diffuse swollen eyelids and conjunctival mass, symptoms which in primary systemic amyloidosis are very rare.


Molluscum contagiosum occurring in an epidermal cyst--report of 3 cases. We present three unusual cases of molluscum contagiosum occurring in epidermal cysts. All of them are asymptomatic, elevated, oval nodules diagnosed clinically as epidermal inclusion cyst or prurigo nodularis. Histology showed true epidermal cysts containing molluscum bodies throughout the cyst wall and some type of laminated material within the cyst itself. The lesion, in all three cases developed in the pubic area of young adult men.


An association of acanthosis nigricans and Crouzon syndrome. An 11-year-old Japanese female having acanthosis nigricans associated with Crouzon syndrome is reported. Crouzon syndrome is a craniostenotic craniofacial malformation associated with premature closure of selective calvarial sutures, exophthalmos, maxillary hypoplasia, and a beak-shaped nose. It is an autosomal dominant inherited disorder. Crouzon syndrome is one of the syndromes which may be associated with acanthosis nigricans. The association of acanthosis nigricans with Crouzon syndrome is assumed to be a rare abnormality, although the true frequency is uncertain. We have reviewed the reported cases of acanthosis nigricans associated with Crouzon syndrome and characteristics were discussed.


Epidermal keratinocytes of bullous pemphigoid express intercellular adhesion molecule-1 (ICAM-1). Intercellular adhesion molecule-1 (ICAM-1) is the ligand for lymphocyte function associated antigen-1 (LFA-1), mediating the adhesion of lymphocytes to vascular endothelium. Keratinocytes are known to express ICAM-1 in some inflammatory dermatoses. Using an indirect immunofluorescence method, we examined the patterns of ICAM-1 and LFA-1 staining in bullous pemphigoid (BP) lesions and compared them to pemphigus vulgaris (PV) cases. ICAM-1 was expressed on the cell surface and in the cytoplasm of epidermal keratinocytes at the sites of erythematous and bullous lesions of BP. LFA-1 molecules were expressed on T cells at the basement membrane zone. In addition, HLA-DR-positive keratinocytes were observed in the basal layer. ICAM-1 was not, however, expressed on epidermal keratinocytes in uninvolved skin from BP patients, PV or normal control skin. It is known that ICAM-1 is expressed on keratinocytes at the site of lymphoid infiltration in cutaneous dermatoses and that LFA-1-positive T cells can bind to interferon gamma-induced ICAM-1-positive keratinocytes. Our results suggest that cellular immunity involving ICAM-1 and LFA-1 may play a part in the pathogenesis of bullous pemphigoid.


Cutaneous leiomyosarcoma.

Cutaneous leiomyosarcoma. A child's head-sized tumor on the upper back developed in a 43-year-old man. In spite of an extensive operation for the tumor, he died of multiple metastasis 15 months after the operation. Histologically, tumor cells proliferated throughout the dermal and subcutaneous regions, and a boxed-in appearance was noted with silver staining. Because electron microscopic observations strongly suggested a smooth muscle origin, we diagnosed this case as cutaneous and subcutaneous leiomyosarcoma.


Attention deficit disorder in reading-disabled twins: evidence for a genetic etiology. In order to assess the genetic etiology of attention deficit hyperactivity disorder (ADHD), the basic regression model for the analysis of selected twin data (DeFries & Fulker, 1985, 1988) was fitted to questionnaire data (DICA: Diagnostic Interview for Children and Adolescents; Herjanic, Campbell, & Reich, 1982) for 37 identical and 37 fraternal twin pairs tested in the Colorado Reading Project. Results of this analysis suggest that ADHD is highly heritable. Moreover, adjusting DICA scores for either IQ or reading performance differences did not substantially change parameter estimates. In future analyses of larger data sets, distinguishing between possible subtypes of attentional problems (e.g., ADD with or without hyperactivity) may facilitate tests of more searching etiological questions.


Rostrocaudal differences in morphology and neurotransmitter content of cells in the subretrofacial vasomotor nucleus. The rostral ventrolateral medulla (RVLM) contains sympathoexcitatory neurons that exert a powerful control over the sympathetic outflow to the cardiovascular system. In the cat there is a concentration of such neurons (but not neurons subserving other functions) within a narrow longitudinal column in the RVLM termed the subretrofacial (SRF) nucleus. Furthermore, it has been suggested that there are subgroups of cells, located at different rostrocaudal levels of the SRF nucleus, that preferentially or exclusively control different vascular beds (e.g. in the kidney and hindlimb). The aim of this study was to map quantitatively the rostrocaudal distribution within the nucleus of different cell types, defined according to morphological and/or chemical criteria, and to correlate this with the regional vasomotor effects (in hindlimb and kidney) evoked by stimulation of SRF cells at the corresponding rostrocaudal levels. SRF cells were highly heterogeneous with respect to both their morphology and chemical properties. They varied greatly in size (equivalent diameter ranging from 10-40 microns) as well as in shape and orientation. An immunohistochemical examination using the avidin-biotin procedure revealed that many SRF cells (estimated 57% of all SRF cells) were immunoreactive for tyrosine hydroxylase (TH, a marker of catecholamine cells). In addition, there were SRF cells immunoreactive for neuropeptide Y (NPY, 11% of total), enkephalin (ENK, 16% of total), and serotonin (5HT, 10% of total), but not for substance P, galanin or somatostatin. Different cell types, defined according to their morphology and/or chemical properties, were unevenly distributed throughout the nucleus. In the most caudal part of the SRF nucleus, virtually all cells were TH-positive, and the large majority (estimated 80%) were NPY-positive, suggesting that many cells at this level contained both TH and NPY. In contrast, in the most rostral part of the SRF nucleus, only 30% of cells were TH-positive, and no NPY-positive cells were observed. Both 5HT- and ENK-positive cells were found throughout the rostrocaudal extent of the nucleus, but predominantly within its rostral part. Furthermore, TH-positive cells in the rostral SRF nucleus were on average significantly larger (mean equivalent diameter 18-43% greater) than TH/NPY-positive cells in the caudal part of the nucleus, but smaller than 5HT- or ENK-positive cells at the same level. Overall, rostral cells (regardless of their chemical type) were larger than caudal cells within the SRF nucleus (mean equivalent diameter 13-28% greater).(ABSTRACT TRUNCATED AT 400 WORDS)


Interaction of putative neurotransmitters in rostral ventrolateral medullary cardiovascular neurons. As recent immunohistochemical evidence has shown the coexistence of putative neurotransmitters in the rostral ventrolateral medulla (RVLM), we have investigated the possibility that there may be an interaction of putative transmitters on the firing frequency of cardiovascular neurons in the RVLM. Extracellular activity was recorded from 37 spontaneously firing units in the right RVLM of urethane anaesthetized and artificially ventilated rats. Nine of these units were classified as cardiovascular neurons because: (i) they were silenced by baroreceptor activation (1-3 micrograms phenylephrine i.v.); and (ii) they showed rhythmicity of their spontaneous activity in synchrony with the cardiac cycle. Microiontophoresis of combinations of near threshold amounts of L-glutamate (GLU; 10 nA), acetylcholine (Ach; 30 nA) and substance-P (SP; 60 nA) showed a synergistic interaction of these substances with one another in eliciting changes in firing frequency of cardiovascular neurons. These results show that GLU and Ach, GLU and SP and Ach and SP interact synergistically to influence the firing frequency of cardiovascular neurons in the RVLM and suggest that these substances play a physiological role in the neural control of the circulation.


Nervous control of the release of substance P and neurokinin A from the isolated perfused porcine ileum. Using isolated perfused porcine ileum we studied the release of substance P (SP) and neurokinin A (NKA) in response to electrical stimulation of the mixed periarterial nerves and to infusion of different neuroactive agents. Nerve stimulation (8 Hz) had no significant effect on the release of SP and NKA. Nerve stimulation also had no effect on the release of SP and NKA during infusion of atropine (10(-6) M) or phentolamine (10(-5) M), whereas a significant increase (from 8.2 +/- 1.9 to 20.1 +/- 4.6 pmol/l for SP and from 12.3 +/- 2.7 to 34.2 +/- 7.7 pmol/l for NKA, n = 7) was observed during nerve stimulation after pretreatment with both atropine and phentolamine. This increase was abolished by hexamethonium (3 x 10(-5) M). Also acetylcholine infusion causes a significant release of SP and NKA after infusion of both atropine and phentolamine (to 172 +/- 56% and 232 +/- 69% of basal release, n = 7), an effect that was abolished by hexamethonium infusion. Infusion of atropine alone increased the release of SP and NKA significantly (to 337 +/- 92% and 386 +/- 124% of basal output, n = 5). Norepinephrine (10(-6) M) inhibited the release of SP and NKA (to 69 +/- 6% and 80 +/- 6% of basal release, n = 7). Our results suggest that the SP- and NKA-producing neurons receive intrinsic tonic muscarinic inhibitory impulses, extrinsic nicotinic excitatory impulses, and extrinsic adrenergic inhibitory impulses.


Role of vitamin A in the haematotoxicity of hexachlorocyclohexane (HCH) in the rat. The haematotoxicity of technical hexachlorocyclohexane (HCH) (1000 ppm) was investigated in male albino rats fed with diet free of vitamin A or containing vitamin A at 2000 or 10(5) I.U./kg. Assessment of HCH-induced haematotoxicity at the end of the 7 weeks feeding period was done on the basis of haemoglobin content, total count of red blood cells and white blood cells and the differential counts of the white blood cells as well as by parameters such as packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin content, prothrombin time and clotting time. In the rats fed with vitamin A-free diet containing HCH, significant reductions were noticed in the total white blood cells count, clotting time and prothrombin time indicating severe haematotoxicity. Differential count of the white blood cells of these rats revealed a non-significant reduction in the lymphocyte count. The only indication of haematotoxicity caused by hexachlorocyclohexane in the vitamin A supplemented rats was a slight but statistically significant reduction of the total count of white blood cells. These results demonstrate that the haematotoxicity of hexachlorocyclohexane in the rats is enhanced by vitamin A-deficiency and its supplementation particularly in excess but not at hypervitaminotic level is protective against the toxicity.


Simultaneous immunostaining method for localization of bromodeoxyuridine and calcitonin gene-related peptide. A new simultaneous double immunostaining method has been optimized to localize the DNA synthesis marker bromodeoxyuridine (BrdU) and calcitonin gene-related peptide (CGRP) in endocrine cells of Bouin's-fixed, paraffin-embedded rat lung. Nuclease pre-treatment before immunostaining is compatible with optimal tissue morphology and CGRP antigenicity preservation. Nickel-enhanced development of avidin-biotin-peroxidase staining is used to show CGRP immunoreactivity in black and alkaline phosphatase-anti-alkaline phosphatase is applied to demonstrate incorporated BrdU in red. The present methodology could be useful for studies requiring detection of incorporated BrdU in cells producing regulatory peptides or other labile antigens.


Immunohistochemical study of basement membrane reconstruction by an epidermis-dermis recombination experiment using cultured chick embryonic skin: induction of tenascin. The production of extracellular matrix components such as laminin, Type IV collagen, fibronectin, and tenascin during the formation of basement membrane in cultured epidermis-dermis recombinant skin of 13-day-old chick embryo was analyzed immunohistochemically. The epidermis and dermis were separated from each other by treatment with EDTA and/or dispase. The basal lamina of the basement membrane was thus removed from both epidermis and dermis. The isolated epidermis was overlaid onto the isolated dermis, i.e., recombined, and then cultured for 1-7 days in a chemically defined medium (BGJb) on a Millipore filter. Immunofluorescence labeling was used for light microscopy and HRP or colloidal gold labeling for electron microscopy. In specimens from 2-day cultures, positive sites of anti-laminin and anti-fibronectin reaction were observed light microscopically as patches which, at the electron microscopic level, corresponded to fragments of the basal lamina located immediately beneath and in the vicinity of the attachment plaques of the hemidesmosomes. The staining pattern became continuous 7 days after recombination. Fluorescence labeling of laminin and fibronectin appeared somewhat earlier than that of Type IV collagen and tenascin. All of the four components were found localized primarily in the basal lamina. Furthermore, fibronectin and tenascin were also distributed in the extracellular matrix of the dermis. The expression of tenascin, which does not exist in the basement membrane of 13-day-old intact embryonic skin, was induced in vitro. These results suggest that hemidesmosomes may play an important role in the reconstruction of the basement membrane and that various components of the basement membrane appeared at different times during the reconstruction.


[3H]-adenine metabolism and radiation damage during in vitro development of the kidney. We followed the early post-induction changes in nucleic acid synthesis of the metanephric kidney anlage in vitro. Enhanced incorporation of [3H]-thymidine and [3H]-adenine was detected, but several factors were shown to influence the interpretation of such in vitro experiments. The incorporation is dependent not only on the stage of development of the target organ but also on its transfer to organ culture, as early rudiments require an "adaptive" pre-cultivation to stabilize their metabolism; at more advanced stages growth and DNA synthesis proceed without delay. Another potential artifact is radiation damage which is caused by the incorporated radioisotope and can be detected in prolonged cultures. A [3H]-adenine pulse of more than 1 microCi/ml for 2 hr leads to definite growth retardation, and a 10-microCi/ml pulse causes extensive cell death and atrophy on a 4- to 6-day subculture. The radiation damage is dose-dependent and of variable severity in the different cell lineages within the organ. Since the radioisotope doses were in the range of those commonly used for monitoring cell proliferation and metabolism, we stress the risk of obtaining artifactual results, especially in prolonged cultures after pulse-labeling.


Differential extractability of calcium and pectic substances in different wall regions of epicotyl cells in young flax plants. We applied the simultaneous use of a subtractive method and two imaging techniques (secondary ion mass spectrometry and electron microscopy after PATAg staining) to correlate the distribution of Ca2+ to pectic substances in cell walls of young flax plants. The calcium images were compared with the structural electron microscopy images. This suggests that the linkage of the pectic substances within the wall is mainly by calcium bridges in the intercellular junctions of most types of cells under study (epidermis, subepidermis, fiber layer, and endodermis) and in the outer part (close to the cuticle) of the wall of the epidermal cells. In the primary walls of the various types of cells under study and in the inner part (close to the cytoplasm) of the wall of the epidermal cells, the linkage of the pectic substances would be mainly by covalent bonds. In the middle lamellae of the various cells, and in the intercellular junctions within the cortical parenchyma, both types of linkages apparently coexist. The mechanism of "ionic condensation" may provide an interpretation for the chemical status of the Ca2+ ions which are associated with the pectic components solubilized in boiling water, and which do not seem to contribute to the linkage of these components within the wall.


Distribution of the extracellular matrix proteins tenascin, fibronectin, and vitronectin in fetal, infant, and adult human spleens. Using immunohistochemistry, we investigated the distribution of the extracellular matrix (ECM) glycoproteins tenascin, fibronectin, and vitronectin in fetal [16-24 gestational weeks (GW)], infant (40 GW), and adult human spleens to clarify the presence of these proteins during different phases of maturation. In the red and white pulp, tenascin and fibronectin were constant components of the reticular fibers from the age of 18 GW onwards, whereas vitronectin was seen only in adult spleens. The immunohistochemical staining patterns of tenascin and fibronectin remained unchanged at different fetal ages. Ring fibers, which are modified basement membranes around venous sinuses, became visible relatively late, and in adult spleens they contained both tenascin and vitronectin but lacked fibronectin. The composition of the ring fibers is therefore clearly different from that of ordinary basement membranes, which have not been reported to contain tenascin or vitronectin. The rapidly increasing number of reticular fibers in the spleen at the age of approximately 18 GW corresponds with the beginning of lymphatic colonization. Reticular fibers, rich in ECM glycoproteins, form a framework to which cells can migrate and attach. We suggest that the composition of these fibers might be important for lymphatic colonization and function of the spleen.


Novel method for performing carbonic anhydrase histochemistry and immunocytochemistry on cryosections. It is difficult to correlate structure with function in the kidney because of the extensive cell heterogeneity. Carbonic anhydrase (CA) is an enzyme that mediates renal acidification and is found predominantly in proximal tubule and collecting duct cells. We modified Hansson's method for histochemically identifying cellular CA activity on PLP-fixed rabbit kidney sections mounted on Millipore filters, and then removed the filters to perform peanut lectin and antibody labeling on the same sections. There was adequate preservation of morphology, and individual cells could be identified with CA activity in the cytosol and specific antibody or lectin labeling on the cell surfaces.


[Cellular characteristics of yolk sac tumor]. Morphological analysis of imprint cytology and histology was performed in 5 patients with yolk sac tumor (YST). The age of the patients ranged from 14 to 37. The level of AFP ranged from 460 ng/ml to 13,464 ng/ml. Five patients had a primary lesion in the ovary. These five cases of YST were histologically evaluated and classified into 4 patterns. As a result, the endodermal sinus pattern was the most frequently identified, followed by the polyvesicular vitelline pattern. As with imprint cytology findings in 4 YST cases, cuboidal cells were the most frequently observed; cuboidal cells and nuclei were both cuboidal in shape and 25-40 mu and 20-25 mu in diameter respectively. The chromatin had fine to coarse granular patterns. The nuclear.cytoplasmic (N/C) ratio was 50-70%, and each nucleus contained one to four nucleoli. Next to cuboidal cells, spindle-shaped cells were most frequently noticed; 20-40 mu in size with spindle-shaped nuclei 10-20 mu in diameter and had a fine to coarse granular chromatin pattern. The nuclei of the 30-50% N/C ratio cells contained one to two nucleoli. Naked cells and bizarred cells were also noticed.


Sulfated glycoconjugates demonstrated in combination with high iron diamine thiocarbohydrazide-silver proteinate and silver acetate physical development. Sulfated glycoconjugates in epithelial cells and mesenchymal cells were investigated after staining with high iron diamine-thiocarbohydrazide-silver proteinate. One purpose of the experiment was to apply a new physical developed to the staining. Instead of silver nitrate, silver lactate or silver bromide, we used silver acetate as an ion donor. This new method allowed physical development under normal lighting conditions, and resulted in the reduction of background staining even after amplification. As the developer did not contain gum arabic, troublesome treatment was not necessary. The time required for staining was very short and the electron density of the final reaction product was high and easily identifiable under the electron microscope. Fixing was not necessary. Very small amounts of reactive substance were detectable after physical development. This developmental procedure has been applied to both the preembedding staining and postembedding staining of sulfated glycoconjugates. The results obtained using this method are presented.


One-year follow-up on the safety and efficacy of isoprinosine for human immunodeficiency virus infection. Scandinavian Isoprinosine Study Group. The safety and clinical impact of isoprinosine in HIV-infected individuals were assessed in a multicentre, randomized, double-blind, 24-week study phase, followed by an optional 24-week open treatment phase. The results of the double-blind phase have been reported. Of 866 HIV-seropositive patients randomized, 832 subjects were eligible for efficacy analysis. On completion of the double-blind phase, 596 patients started open treatment. All patients were evaluated with regard to progression to AIDS and/or death. Within 48 weeks, 10/412 (2.4%) patients assigned isoprinosine and 27/420 (6.4%) patients assigned placebo progressed to AIDS (P = 0.005). Intention-to-treat analysis showed identical results. Viewing the open treatment phase in isolation revealed no difference in progression rates between those treated and those not receiving the drug, perhaps reflecting the higher proportion of patients receiving zidovudine or PCP prophylaxis in the latter group. No severe adverse reactions or toxicities were observed. We conclude that HIV-seropositive patients without AIDS may be safely and effectively treated with isoprinosine.


Hyperamylasemia following cardiopulmonary bypass. In order to study the occurrence of postbypass hyperamylasemia, 75 patients undergoing cardiopulmonary bypass (CPB) were studied from March 1989 to January 1990. There were 49 males and 26 females. Among them, 27 had congenital heart disease, 30 had valvular disease, and 18 had coronary artery disease. There were 27 patients with at least one elevated serum amylase sample after operation. Thus, the overall incidence of hyperamylasemia was 36%. As compared with the preoperative data (1.3%), there was a statistically significant difference in the occurrence of hyperamylasemia (p less than 0.05). Three patients had overt clinical pancreatitis postoperatively. There was no positive correlation between the serum amylase level and the occurrence of pancreatitis (p greater than 0.05). Forty-two cases had a significant elevation of the amylase creatinine clearance ratio (ACCR) after CPB. However, there was no significant difference between the groups with pulsatile and nonpulsatile CPB (p greater than 0.05). Three patients (4%) died in our series. The causes of death were heart failure in two and fulminant pancreatitis associated with low cardiac output in one. Although our experience in dealing with pancreatitis improved survival, mortality was still high (33.3%) in our series. Nevertheless, there was no apparent correlation between mortality and postbypass hyperamylasemia (p greater than 0.05). Logistic regression analysis was used to analyze the risk factors of the occurrence of hyperamylasemia, and the analysis revealed that patients with coronary artery disease were susceptible to postbypass hyperamylasemia. Our studies indicate that the use of total serum amylase or ACCR to monitor for the occurrence of pancreatitis in postbypass patients is inadequate.(ABSTRACT TRUNCATED AT 250 WORDS)


Taurine and serine supplementation modulates the metabolic response to tumor necrosis factor alpha in rats fed a low protein diet. Plasma taurine and serine decrease following trauma and in severe inflammatory disease. These changes may signify an increase in requirements for sulfur amino acids. We previously demonstrated that cysteine supplementation can restore the impaired ability of rats fed an 8% casein diet to increase hepatic zinc, glutathione (GSH) and protein concentrations in response to tumor necrosis factor alpha (TNF alpha). Here we examined whether serine or taurine produces a similar effect, because serine provides the carbon skeleton of cysteine and taurine is its major metabolite. After 7 d of receiving either a 20% casein diet supplemented with cysteine or an 8% casein diet supplemented with alanine, serine or taurine, rats received an intraperitoneal injection of human TNF alpha. Tumor necrosis factor caused no change in hepatic GSH but resulted in a lower GSH concentration in lung in rats fed the alanine-supplemented diet. Neither taurine nor serine increased liver GSH relative to that in rats fed alanine, but the depression in lung due to TNF injection was lessened. The absolute increase in ceruloplasmin in response to TNF was enhanced in rats fed the alanine-supplemented diet relative to those fed the 20% casein diet. Serine normalized this response. This observation--the effects of taurine and serine on lung GSH and a significant negative correlation between ceruloplasmin and liver and lung GSH concentration in rats fed TNF--suggests that supplemental serine and taurine may improve antioxidant defenses when dietary supplies of cysteine are low but do not influence cysteine availability for a normal response to TNF.


Raw soybeans stimulate human pancreatic proteinase secretion. Intraduodenal instillation of raw soybeans stimulated pancreatic proteinase secretion in humans. Raw soybeans almost abolished the activity of chymotrypsin and severely reduced (50%) the tryptic activity. Immunoreactive tryptic and chymotryptic material simultaneously appeared in amounts 2 to 4 times basal concentrations. This increase, demonstrated with rocket immunoelectrophoresis, was begun within the first 10 min of soybean instillation. The enhanced secretion also persisted throughout the succeeding saline instillation, and it is suggested that the presence of Kunitz trypsin inhibitor contributed to this postprandial stimulation. An amidase that hydrolyzes low-molecular-weight substrates (i.e., benzoyl-arginine p-nitroanilide) was found in raw soybeans. Its low activity was not assumed to substantially bias standard trypsin assays. The increased proteinase secretion was, as previously published, not preceded by an elevated plasma cholecystokinin concentration. The raw soybeans also caused a nonparallel secretion of amylase and proteinases. Nervous, perhaps cholinergic, regulation mediates the inhibitor-stimulated proteinase secretion in humans. This stimulation yields both a general increase of proteinases and also a specific inhibitor-resistant trypsin. This is consistent with the physiologic need for proenzyme-activation in the presence of inhibitors and for restoration of the proteolytic capacity of the duodenal juice.


Gastric and pancreatic enzyme activities and their relationship with some gut regulatory peptides during postnatal development and weaning in calves. Changes in the activities of three gastric and nine pancreatic enzymes plus colipase were determined during postnatal development and weaning in calves. In calves exclusively milk-fed for 2, 7, 28, 56, 70 and 119 d, the enzyme activities per kilogram of empty live weight increased with age for chymotrypsin, elastase, carboxypeptidases A and B, ribonuclease and alpha-amylase, decreased for chymosin, lysozyme and colipase but showed no change in the case of pepsin, trypsin, lipase and phospholipase A2 compared with animals at birth. The greatest increase was that in alpha-amylase activity (about 50-fold between d 2 and 119). In calves weaned between d 28 and 56, all the activities were higher than in milk-fed animals, except that of chymosin (which was slightly lower) and that of colipase (which did not change). At 119 d of age, chymotrypsin, carboxypeptidase A, alpha-amylase and lipase were 1.6- to fourfold higher in ruminants than in preruminants. Thus, most enzyme activities were modified first by colostrum and milk intake, and again upon weaning by development of the forestomachs and ingestion of solid food. These ontogenic patterns might be under the control of many gut regulatory peptides, the plasma concentrations of which changed simultaneously. Some gastric and pancreatic enzymes were correlated to plasma concentrations of these gut regulatory peptides.


Effect of sexual steroid hormones on spiramycin disposition in genital tract secretions of the ewe. The present work investigated the influence of sexual steroid compounds (estradiol 17 beta and fluorogestone) on antibiotic passage across the uterine barrier. Five healthy and mature ewes, with controlled hormonal impregnation, were given a single iv injection of spiramycin, a macrolide antibiotic, at a dose of 64,000 IU/kg. Plasma and uterine secretions were regularly sampled before the injection and for 30 h post-injection. Blood was collected from the jugular vein and uterine secretions were obtained by uterine flushing with a sterile saline solution containing 0.2% inulin. Spiramycin was concentrated in the uterine secretions, whatever the hormonal status; the secretions-to-plasma ratio was 4.68 +/- 1.88 under estrogen priming and 2.68 +/- 0.91 under progestagen priming. The area under the concentration-time curve (AUC) and the mean residence time (MRT) were significantly higher (p less than 0.001) in uterine secretions than in plasma. The AUC in uterine secretions was significantly higher (p less than 0.05) under estrogen priming (439.07 +/- 241.25 IU.h/mL) than under progestagen priming (141.41 +/- 89.37 IU.h/mL). The spiramycin MRTs in uterine secretions were 11.92 +/- 4.08 and 12.06 +/- 3.35 h for both estrogens and progestagen treatment, respectively. These experiments demonstrate that estrogens increase uterine bioavailability, but not the residence time, of spiramycin when administered by a systemic route.


Synthesis of 3-[(2,3-dihydro-1,1,3-trioxo-1,2-benzisothiazol-2-yl)alkyl] 1,4-dihydropyridine-3,5-dicarboxylate derivatives as calcium channel modulators. 1,4-Dihydropyridine (DHP) derivatives with a 1,2-benzisothiazol-3-one 1,1-dioxide group, linked through an alkylene bridge to the C-3 carboxylate of the DHP ring, with both vasoconstricting and vasorelaxant properties were obtained. In blocking Ca(2+)-evoked contractions of K(+)-depolarized rabbit aortic strips, compounds 12 and 41 were 10-fold more potent than nifedipine; 27 other compounds were 1-4-fold more potent. Their vascular versus cardiac selectivity was very pronounced; for instance, the selectivity index for compound 41 was 70-fold higher than that of nifedipine. This was also true for the vasoconstricting compound 22, which was as potent as Bay K 8644 in enhancing the Ca(2+)-evoked contractions of rabbit aorta strips, yet it had poor inotropic activity in rabbit left atria. Oral administration of compounds 38, 40, 43, and 53 (20 mg/kg) caused a 35-37% decrease in systolic blood pressure in spontaneously hypertensive rats (SHR); these effects were similar to those of nifedipine. However, iv administration of these compounds to anesthetized SHR caused a decrease in blood pressure which was more pronounced and long-lasting than that of nifedipine. When administered iv at 100 micrograms/kg, the vasoconstricting compound 22 caused a 40% increase in systolic and diastolic blood pressure. Compound 22 exhibited an unusually interesting feature over the other five Ca2+ DHP agonists: it had diester substitutions at the C-3 and C-5 positions of the DHP ring. Overall, compounds possessing these properties might be useful in treating clinical cardiovascular conditions in which DHP Ca2+ antagonists or agonists are indicated.


NMR studies of an FK-506 analog, [U-13C]ascomycin, bound to FK-506-binding protein. Multidimensional, heteronuclear NMR methods were used to determine the complete 1H and 13C resonance assignments for [U-13C]ascomycin bound to recombinant FKBP, including stereospecific assignment of all 22 methylene protons. The conformation of ascomycin was then determined from an analysis of NOEs observed in a 13C-edited 3D HMQC-NOESY spectrum of the [U-13C]ascomycin/FKBP. This structure is found to be quite different from the solution structure of the two forms of uncomplexed FK-506. However, it is very similar to the X-ray crystal structure of FK-506 bound to FKBP, rms deviation = 0.56 A. The methods used for resonance assignment and structure calculation are presented in detail. Furthermore, FKBP/ascomycin NOEs are reported which help define the structure of the ascomycin binding pocket. This structural information obtained in solution was compared to the recently described X-ray crystal structure of the FKBP/FK-506 complex.


Linkage of autosomal dominant dystrophic epidermolysis bullosa in three British families to the marker D3S2 close to the COL7A1 locus. Linkage of the anonymous marker D3S2 at 3p21 has been shown in three British families with dominant dystrophic epidermolysis bullosa with a combined lod score of 6.75 at theta = 0. This locus is close to the collagen type VII locus implying that abnormalities of this gene cause dominant dystrophic epidermolysis bullosa.


Preferential interaction of human immunodeficiency virus reverse transcriptase with two regions of primer tRNA(Lys) as evidenced by footprinting studies and inhibition with synthetic oligoribonucleotides. Primer tRNA regions involved in the interactions between human immunodeficiency virus reverse transcriptase (HIV RT) and tRNA(Lys) were studied by digestion of primer with pancreatic ribonuclease in the presence or absence of HIV RT. The acceptor stem of tRNA(Lys) is not noticeably protected against nuclease action in the presence of HIV RT, while this enzyme clearly protects part of the anticodon and dihydrouridine loops of tRNA(Lys). The acceptor stem of primer tRNA was digested by RNase A only in the presence of the retroviral enzyme, suggesting a partial destabilization of this region by the HIV RT. Synthetic oligoribonucleotides, corresponding to the anticodon and the dihydrouridine loops, inhibited strongly reverse transcription, confirming the strong interaction of these tRNA regions with the enzyme.


Impairment of helper T-cell function following severe head injury. Major infections, such as sepsis and pneumonia, occur in 50-75% of patients following isolated severe head injury. Previous studies have demonstrated that this high incidence of infection following severe head injury may be related to a decrease in helper T-cell activation and function. The present study was designed to investigate the effect of severe head injury on specific subgroups of helper T cells known to enhance or suppress cellular immune function. Specifically, peripheral blood lymphocytes (PBLs) from 10 head-injured patients and 10 matched controls were evaluated following in vitro stimulation with the T-cell mitogen, phytohemagglutinin (PHA). Subsets of helper T cells evaluated included activated helper (CD4+/CD25+) T cells; helper/inducer (CD4+/CDw29+) T cells, which enhance cellular immune activity; and suppressor/inducer (CD4+/CD45R+) T-cells, which induce suppressor (CD8+) T-cells. In addition, the effect of intraventricular fluid (IVF) on PHA-stimulated in vitro CD4 and CD25 expression was investigated to determine whether severe head injury results in the production of mediators within the central nervous system capable of affecting T-cell activation. The results of this study indicate that isolated severe head injury selectively reduces the ability of PHA-stimulated PBLs to express the helper/inducer (CD4+/CDw29+) T-cell (p = 0.023) and activated helper (CD4+/CD25+) T-cell (P = 0.041) phenotypes. There was no significant change in PHA-stimulated CD4 or CD25 expression following incubation of PBLs with intraventricular fluid (IVF) from head-injured patients. The relationship between these changes in specific helper T-cell subpopulations and the infectious complications of severe head injury are discussed.


Dextran-coupled deferoxamine improves outcome in a murine model of head injury. Tissue damage involving oxygen-derived free radicals may be greatly exacerbated by free, reactive iron, which acts as a catalyst in oxidative reactions. The effects of free iron can be attenuated by the administration of deferoxamine (DFO), an iron chelator. However, DFO has limited therapeutic utility because it has a short plasma half-life (approximately 5.5 min in mice) and produces profound hypotension upon intravenous infusion. These negative attributes have been circumvented by the covalent attachment of DFO to large polymers, such as dextran or hydroxyethyl starch. The ability of the dextran-conjugated DFO (DEX-DFO) to inhibit iron-catalyzed reactions with lipids was compared to that of the native molecule in an in vitro model of CNS lipid degradation in the presence of 200 microM ferrous iron. There was no difference between native DFO and the modified form. Modified and unmodified DFO were also compared for therapeutic efficacy in a murine model of head injury. Using a previously described "grip test" as a measure of neurologic impairment following injury, DEX-DFO, native DFO, and dextran were administered intravenously 3-5 min after injury. Dextran-DFO significantly decreased the incidence of severe neurologic impairment at dosage levels of 0.1 (n = 92), 1.0 (n = 76), and 10.0 (n = 80) mg/kg. Administration of native DFO or dextran had no effect at the same dosages and concentrations. These results suggest that the murine model of head injury contains a significant iron-dependent component that should be assessed in other models of neural injury.


The effects of normovolemic hemodilution with dextran-40 on acute myocardial ischemia/reperfusion injury in rabbits. The effects of decreasing blood viscosity by normovolemic hemodilution with dextran-40 or normal saline (NS) on myocardial lipid peroxides, superoxide dismutase, infarct size and left ventricular function during acute myocardial ischemia/reperfusion were studied in rabbits. It was found that normovolemic hemodilution with dextran-40 could decrease the content of ischemic myocardial malondialdehyde and preserve ischemic myocardial superoxide dismutase activity after 1 h of coronary occlusion followed by 1 h of reperfusion. However, after administration of NS only a tendency in this aspect exhibited without statistical significance. Besides, hemodilution with dextran-40 reduced infarct size and improved left ventricular systolic function after 1 h of ischemia followed by 23 h of reperfusion. These results suggest that normovolemic hemodilution with dextran-40 may have anti-injury effect on acute myocardial ischemia/reperfusion to a certain degree.


Simultaneous existence of galactose-containing glycoprotein and several neuropeptides in DRG of the rat. By use of light microscopic immunohistochemical and lectin histochemical methods, the interrelation of galactose-containing glycoprotein (GCGP), calcitonin gene-related peptide (CGRP)-like, leu-enkephalin (L-ENK)-like, and substance P (SP)-like peptides has been evaluated on consecutive sections of dorsal root ganglia from colchicine-treated rats. The results showed that GCGP, CGRP, L-ENK and SP exist simultaneously in individual neurons of the dorsal root ganglia in rats. Almost all small neurons in dorsal root ganglion contained both GCGP and CGRP. The stronger peanut lectin affinity with small neurons, the weaker CGRP immunoreactivity, and vice versa. Some neurons of medium size were of strong CGRP-like immunoreactivity; however, they lacked in affinity with peanut lectin. The large spinal ganglionic cells rarely showed CGRP immunoreactivity and affinity with peanut lectin. The results suggested that there was a negative interrelation between GCGP and CGRP in small primary sensory neurons. From the above it may be suggested that the GCGP plays an important role in recognizing and transmitting information in primary sensory neurons.


Analysis of antigenic polypeptides of Dane particles and antibody response ability of HBV infected subjects to PreS1 polypeptides. We demonstrated the constitutive polypeptides (PP) of Dane particles employing Western Blot and investigated the antibody response ability of HBV infected subjects to PreS1 PP in comparison with other serum markers from HBV infected individuals. The results indicated that 1) the major reason for discrepant results may be related to the detergents used in the sample solutions and the degree of denaturation the samples had undergone; 2) there are 12 bands in the PAGE-graph of Dane particles. By Western Blot it was confirmed that 5 PP (P24, P27, P36, P39, P42) are derived from S-open reading frame (S-ORF), P21 is associated with C-ORF, P24-25 possesses some epitopes of Pol protein, and P45 and P76 express similar epitopes to human IgG and IgM; and 3) the prevalence of anti-PreS1 PP was 17.24% in the group of healthy persons following latent HBV infection, much higher than that of HBV infected patients (1.21%). The above findings imply that antibody response ability of the host to PreS1 PP is attributing to the outcome of HBV infection. It may play an important role in the elimination of the virus.


Evaluation of efficacy of four pediculicides against head louse (Pediculus capitis) infestation. To evaluate the efficacy and to determine the minimum effective dosage of four pediculicides against head louse infestation, as well as to select a safe, effective, practical, and cheap agent, 1,657 infested school children in 25 primary schools in Szu-Hu, Kou-Hu, and Ku-Keng Districts of Yunlin County were treated and 1,611 of them were examined. The overall cure rate was 73% and the rate for boys (84%) was higher than that for girls (71%). The cure rate of Nix (permethrin 1%) cream rinse was 81%. The cure rate for single dosages of 1, 2, 3 and 4 cases/tube (56 gm/tube) was 87%, 83%, 81% and 71%, respectively. The cure rate of Para aerosol (bioallethrine 0.66%) was 78%. The cure rate for single dosages of 30 and 40 cases/tube (90 gm/tube) was 87% and 70%, respectively. The cure rate of Prioderm (malathion 1%) cream shampoo was 64%. The cure rate for single dosages of 10, 15 and 20 cases/tube (40 gm/tube) was 74%, 71% and 52%, respectively. The cure rate of Delice (1% gamma benzene hexachloride) was 71%. The cure rate for single dosages of 5 and 10 ml/case was 64% and 76%, respectively. Of the 226 infested girls, 181 (80%) were found to be infested with 1-10 head lice, 33 (15%) with 11-50 lice, 7(3%) with 51-100 lice and 5 (2%) with over 100 lice. Of the 2,160 head lice collected, 1,788 (83%) were nymphs, 284 (13%) females, and 88 (4%) males. The mean number of head lice in each infested girl was 10 (range 1-137). The low cure rates obtained in the present study may be due to the fact that many school girl & have long hair. In comparison, Nix had the highest cure rate (81%) but the highest price (NT$ 120/case). The cure rate and price of Para aerosol (78%, NT$ 7.2/case) and Delice (71%, NT$ 16.7/case) came next. Prioderm (64%, NT$ 10.0/case) had the lowest rate and a slight offensive smell. Preliminary trials show that based on the cost-effectiveness, Para aerosol is best in head louse infestation control.


Latent prostatic carcinomas found at autopsy in men over 90 years old. Step-sections of 29 whole prostate glands obtained at autopsy from elderly men aged 90 years or over have been investigated. Latent prostatic carcinomas were found in 17 cases (58.6%), all in the peripheral region. The majority of these latent prostatic carcinomas in very old individuals were considered to correspond to stage A1 tumors since, in 75% of cases, the diameter was less than 1 cm. Benign nodular hyperplasia and atypical hyperplasia were also observed in 26 (89.7%) and 14 cases (48.3%), respectively. There was a clear correlation between the appearance of atypical hyperplasia and the presence of latent prostatic carcinoma.


Use of nucleolar organizer regions in the histopathological diagnosis of colorectal epithelial neoplasia. The AgNOR technique, specific staining for nucleolar organizer regions, was applied to twenty-seven lesions (sixteen tubular adenomas with low grade atypia and eleven well-differentiated adenocarcinomas invading the submucosa) of colorectal epithelial neoplasia, to examine the possibility of its application to histopathological diagnosis. The mean AgNOR number per adenoma nucleus (2.62 +/- 0.16) was significantly higher (P less than 0.01) than that per carcinoma nucleus (2.28 +/- 0.25). The mean cross-sectional area and the maximum diameter of carcinoma AgNORs (1.91 +/- 0.20 micron2 and 1.89 +/- 0.09 micron, respectively) were significantly larger than those of adenoma (1.15 +/- 0.17 and 1.45 +/- 0.11, respectively). The result of the discriminant analysis using these two variants corresponded to the pathological diagnosis with an accuracy of 96.3% (26/27). We thus concluded the AgNOR technique to be one of use in the histopathological diagnosis of colorectal epithelial neoplasia.


[Effects of pH on the formation and dissociation of the complex of trypsin and PSTI]. The effects of pH on the formation and dissociation of the complex of trypsin and PSTI was investigated using pancreatic juice of two patients. Pancreatic juice was eluted from a Sephadex G100 column with the buffer in different pH values, and the trypsin inhibitory activity in the eluate was measured. Under alkaline and neutral conditions, the complex of PSTI and trypsin was observed, while in acidic condition, dissociation of the complex was occurred. Molar ratio of PSTI and trypsin in the complex was found to be one mole/one mole. Human serum, alpha 1-antitrypsin, and alpha 2-macroglobulin dissociated the complex of PSTI and trypsin.


[A case of primary anterior mediastinal embryonal carcinoma: report of a case]. A 15-year-old man was admitted to our hospital because of chest pain. The chest x-ray film and CT scan revealed a large anterior superior mediastinal mass. The serum alpha-fetoprotein (AFP) value was raised. Percutaneous biopsy of the tumor suggested embryonal carcinoma. The tumor was totally removed. Postoperatively combination chemotherapy including CDDP was performed. However the patient died of tumor recurrence 8 months after operation. AFP is very useful for its diagnosis and the follow-up of the clinical course.


[Efficacy of the use of a stabilized alpha2-macroglobulin concentrate in experimental burns]. The effectiveness of the use of a stabilized composition of alpha 2-macroglobulin concentrate obtained from fraction III of plasma proteins according to Cohn in burn disease in the experiment on guinea pigs was studied. Under the influence of intraabdominal administration for 4 days of alpha 2-macroglobulin concentrate with a stabilizer, healing of the burn wounds, normalization of a state of the animals occurred 2 times more rapidly than in control ones, activity of acid proteinases restored by day 5 (in control--by day 14) due to increase in activity of proteolytic enzyme inhibitors.


Basement membrane components enhance isolated enterocyte growth. Isolated enterocyte transplantation may have a potential role in increasing intestinal surface area. However, enterocytes are notoriously difficult to grow. Basement membrane components (BMC) promote adherence, migration, and differentiation of enterocytes. Our aim was to determine if BMC enhance enterocyte growth. Twenty-nine rabbits had 5-cm ileal segments resected and serosal pouches (n = 22) created from the serosal surface of the colon. Harvesting of enterocytes by warm trypsinization resulted in 92 +/- 6% cell viability and yielded 5.0 +/- 2.4 10(6) enterocytes/cm intestine. Enterocytes (10(5) were cultured in vitro (n = 7) in 10 ml growth media in plain flasks and flasks coated with laminin alone or Matrigel (an extract of mouse basement membrane containing laminin plus Type IV collagen and heparan sulfate). The cells were subcultured at 2 weeks and examined after Geimsa staining at 4 weeks. Epithelial growth was confirmed by light microscopy and staining for cytokeratin and quantitated by image analysis (JAVA). Epithelial coverage of the flasks was greater with Matrigel (80 +/- 15%) than laminin (66 +/- 15%) which was greater than control (44 +/- 20%) (P less than 0.01). For in vivo studies 10(5) harvested cells were infused into the serosal pouches either in growth media (n = 9) or media + Matrigel (n = 13). Epithelial growth in the pouch was evaluated by qualitative scoring of cytokeratin staining. Cytokeratin staining was similar on control colon serosa (n = 5) and serosa after infusion of cells in media alone. However, Matrigel significantly enhanced the area, depth, and intensity of staining.(ABSTRACT TRUNCATED AT 250 WORDS)


Determination of the resection line in early esophageal cancer using intraoperative endoscopic examination with Lugol staining. Determination of the resection line using intraoperative endoscopic examination with Lugol staining was performed when preoperative examinations such as an esophagogram could not be effectively carried out and the carcinoma was not palpable from the outer surface of the esophageal wall. During the past two years, we performed this technique on eight patients. The carcinoma was restricted within the epithelium in one, the mucosal layer in five, and the submucosal layer in two. Although intraepithelial carcinoma contiguous to the main lesion was seen in six and cancer multiplicity was evident in two, all of the resected stumps were free of any cancer tissue. There have been no cases of recurrence and a limited operation, such as distal esophagectomy, was able to be performed in six. Therefore, intraoperative endoscopic examination is useful for early esophageal cancer.


Enucleation of a secondary esophageal mural tumor. Metastatic tumors to the esophagus have been described as having the imaging characteristics of benign mural tumors such as leiomyomata, but application of enucleation in treating such tumors has not been described. Our report describes long esophageal myotomy and enucleation of tumor for palliation of dysphagia due to a mural tumor originating from a lung primary.


Immune response of peripheral blood mononuclear cells to antigenic determinants within hepatitis B core antigen in HB virus-infected man. Residues 72-146 within hepatitis B core Ag (HBcAg) represent T-cell recognition site in HB-virus-infected man. This study was undertaken to define critical residues involved in the immunogenicity of dominant T-cell determinants of HBcAg. For this purpose, p120-131 and its analog (p120-131 [A] containing alanine substitutions at residues 122 and 125, which were identified as epitopic residues in mice, were synthesized. These peptides and recombinant HBcAg were analyzed for their ability to stimulate peripheral blood mononuclear cells (PBMC) from 25 patients with chronic HBV infection and three patients with acute hepatitis B. PBMC from 18 out of 28 patients showed significantly increased IFN-gamma production and proliferative response in the presence of recombinant HBcAg. Eight patients responded to the two peptides, while 12 patients did not. Four patients responded only to p120-131, and four displayed a response only to p120-131 [A]. The responses to the two peptides were similar among HBeAg-positive and anti-HBe-positive patients, and did not depend on disease activity, except for HBeAg-positive asymptomatic carriers in whom there was no response to any additive. These results indicate that immune responses to p120-131 and its analog were similar in our patient groups. The dominant epitopic residues in this region of HBcAg may differ between man and mouse.


Increased steady-state levels of alpha-fetoprotein mRNA in hepatocellular carcinoma: an analysis by in situ hybridization. The molecular basis for the augmented production of alpha-fetoprotein is unknown. We have used in situ hybridization of alpha-fetoprotein cDNA to malignant hepatocytes to establish if increased serum alpha-fetoprotein concentrations are related to detectable steady-state levels of alpha-fetoprotein mRNA in hepatocytes. Tumor tissue from four patients with histologically confirmed hepatocellular carcinoma were examined, and the results compared to fetal liver. Northern blot hybridization for alpha-fetoprotein mRNA in tumor tissue was also analyzed. As expected a high number of grains was observed in fetal liver tissue, indicative of high levels of alpha-fetoprotein mRNA physiologically present during pre-natal development. Sections from all patients with high serum concentrations of alpha fetoprotein showed appreciable hybrid formation, which correlated semi-quantitatively with the serum concentrations. However, hybrids were not detected in a patient with a normal serum alpha-fetoprotein. The high alpha-fetoprotein mRNA levels in fetal and neoplastic liver suggest that gene transcription is the mechanism of alpha-fetoprotein production in malignancy, although the control of this mechanism remains speculative.