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Spherical splines are used to define approximate solutions to strongly elliptic pseudodifferential equations on the unit sphere. These equations arise from geodesy. The approximate solutions are found by using Galerkin method. We prove optimal convergence (in Sobolev norms) of the approximate solution by spherical splines to the exact solution. Our numerical results underlie the theoretical result. [ABSTRACT FROM AUTHOR]

Spherical radial basis functions are used to define approximate solutions to pseudodifferential equations of negative order on the unit sphere. These equations arise from geodesy. The approximate solutions are found by the collocation method. A salient feature of our approach in this paper is a simple error analysis for the collocation method using the same argument as that for the Galerkin method. [ABSTRACT FROM AUTHOR]

The existence of enteric dopaminergic neurons has been suspected; however, the innervation of the gut by sympathetic nerves, in which dopamine (DA) is the norepinephrine precursor, complicates analyses of enteric DA. We now report that transcripts encoding tyrosine hydroxylase (TH) and the DA transporter (DAT) are present in the murine bowel (small intestine > stomach or colon; proximal colon > distal colon). Because sympathetic neurons are extrinsic, transcripts encoding TH and DAT in the bowel are probably derived from intrinsic neurons. TH protein was demonstrated immunocytochemically in neuronal perikarya (submucosal » myenteric plexus; small intestine > stomach or colon). TH, DA, and DAT immunoreactivities were coincident in subsets of neurons (submucosal > myenteric) in guinea pig and mouse intestines in situ and in cultured guinea pig enteric ganglia. Surgical ablation of sympathetic nerves by extrinsic denervation of loops of the bowel did not affect DAT immunoreactivity but actually increased numbers of TH-immunoreactive neurons, expression of mRNA encoding TH and DAY, and enteric DOPAC (the specific dopamine metabolite). The fetal gut contains transiently catecholaminergic (TC) cells. TC cells are the proliferating crest-derived precursors of mature neurons that are not catecholaminergic and, thus, disappear after embryonic day (E) 14 (mouse) or E15 (rat). TC cells appear early in ontogeny, and their development/survival is dependent on mash-1 gene expression. In contrast, the intrinsic TH-expressing neurons of the murine bowel appear late (perinatally) and are mash-1 independent. We conclude that the enteric nervous system contains intrinsic dopaminergic neurons that arise from a mash-1-independent lineage of noncatecholaminergic precursors. [ABSTRACT FROM AUTHOR]

This paper deals with the histological and ultrastructural findings in a case of the Sézary syndrome. The striking nuclear and cytoplasmic features of the Sézary cell are illustrated, and the similarities of this cell to the mycosis fungoides cell are once more stressed. [ABSTRACT FROM PUBLISHER]

We evaluated the benefits of heat-stable carotenoid-producing Bacillus marisflavi SH8 spores individually and in combination with non-pigmented Bacillus subtilis SH23 spores on growth, colour change, nutritional content, innate immunity, and gut microbiota of white-leg shrimp. White-leg shrimp (Litopenaeus vannamei; n = 30 per tank; 2 tanks per group) were provided feed without (control group) or with SH8, SH23, or mixed spores (total, 1 × 106 cfu/g pellet) for 28 d. The SH8 and SH8-23 combination groups had significantly higher specific growth rates (9.6 and 11.0%), improved red-colour score (4 scores), astaxanthin concentration (1.8- and 2.3-fold), lipid contents (30 and 50%), and superoxidase dismutase activity (8.5 and 12.3%) than that of the control group. Analysis of shrimp's gut microbiome using 16S rRNA metagenome sequencing revealed increased abundance of four useful species and reduced abundance of four harmful species in the combination group than in the control group. Heat-stable Bacillus spore combination improved growth parameters, nutrient content, red-colour score, live counts, and abundance of useful bacteria in the gut of L. vannamei. This is the first study to show the benefits of combining highly heat-stable pigmented and non-pigmented Bacillus spores and their possible mechanisms in a shrimp model.

Southeast Asia is particularly susceptible to the negative impacts of increasing coastal pollution as coastal populations and cities grow at unprecedented rates. Although water chemistry can be monitored, there are greater advantages in using bioindicators as reflectors of the combined effect of multiple pollution types on coastal ecosystem health and for early detection of the negative impacts of pollutants on biotic systems. This study explores the utility and application of ostracods as pollution bioindicators and examines the response of ostracod assemblages to variable pollution in Lap An Lagoon, central Vietnam. From 14 sites within the lagoon, 79 species of 46 genera were identified and sediment grain size, total organic carbon, organic matter and heavy metal concentration were measured. Cluster analysis, detrended correspondence analysis and canonical correspondence analysis identified four distinct ostracod biofacies that were highly correlated to the physical environmental variables (salinity, depth, sediment type, heavy metal concentrations, total organic carbon and organic matter) and are shown to be the main factors controlling ostracod biofacies. Low ostracod diversities were found in silty sediments with heavy metal concentrations likely toxic. Sinocytheridea impressa was indicative of a marginally polluted environment within the lagoon. This study provides evidence for the potential for Southeast Asian ostracods to be used in water quality assessments and the data collected can be used as a baseline for future pollution monitoring., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In studies of germ cell transplantation, counting cells and measuring tubule diameters from different populations using labelled antibodies are important measurement processes. However, it is slow and sanity grinding to do these tasks manually. This paper proposes a way to accelerate these processes using a new image analysis framework based on several novel algorithms: centre points detection of tubules, tubule shape classification, skeleton-based polar-transformation, boundary weighting of polar-transformed image, and circular shortest path smoothing. The framework has been tested on a dataset consisting of 27 images which contain a total of 989 tubules. Experiments show that the detection results of our algorithm are very close to the results obtained manually and the novel approach can achieve a better performance than two existing methods., (© 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.)

This paper presents a segmentation methodology of abdominal axial CT images. The aim of the study is to determine the location of mesenteric area from the axial images so the organs enclosed within can be localized precisely for diagnostic purposes. The challenge confronted here is that there is no a certain deterministic shape of abdominal organs. The methodology implemented here utilizes a curvelets stage followed by morphological image processing to achieve a contour emphasized segmentation from the gestalts of surrounding organs. This paper gives a detailed analysis of approach taken with the problems faced and a brief comparison wrt to other wavelet approaches.

Neurotrophin-3 (NT-3) promotes enteric neuronal development in vitro; nevertheless, an enteric nervous system (ENS) is present in mice lacking NT-3 or TrkC. We thus analyzed the physiological significance of NT-3 in ENS development. Subsets of neurons developing in vitro in response to NT-3 became NT-3 dependent; NT-3 withdrawal led to apoptosis, selectively in TrkC-expressing neurons. Antibodies to NT-3, which blocked the developmental response of enteric crest-derived cells to exogenous NT-3, did not inhibit neuronal development in cultures of isolated crest-derived cells but did so in mixed cultures of crest- and non-neural crest-derived cells; therefore, the endogenous NT-3 that supports enteric neuronal development is probably obtained from noncrest-derived mesenchymal cells. In mature animals, retrograde transport of (125)I-NT-3, injected into the mucosa, labeled neurons in ganglia of the submucosal but not myenteric plexus; injections of (125)I-NT-3 into myenteric ganglia, the tertiary plexus, and muscle, labeled neurons in underlying submucosal and distant myenteric ganglia. The labeling pattern suggests that NT-3-dependent submucosal neurons may be intrinsic primary afferent and/or secretomotor, whereas NT-3-dependent myenteric neurons innervate other myenteric ganglia and/or the longitudinal muscle. Myenteric neurons were increased in number and size in transgenic mice that overexpress NT-3 directed to myenteric ganglia by the promoter for dopamine beta-hydroxylase. The numbers of neurons were regionally reduced in both plexuses in mice lacking NT-3 or TrkC. A neuropoietic cytokine (CNTF) interacted with NT-3 in vitro, and if applied sequentially, compensated for NT-3 withdrawal. These observations indicate that NT-3 is required for the normal development of the ENS.

The 3-morpholinosydnonimine (SIN-1) generates both nitric oxide (NO) and superoxide anion (O2-). It elicits dose-dependent vasodilation in vivo, in spite of the opposite effects of its breakdown products on vascular tone and platelet aggregation. This study was designed to investigate the influence of intravenous SIN-1 injection on platelet Ca2+ handling in patients undergoing coronary angiography. SIN-1 administration reduced cytosolic [Ca2+] in unstimulated platelets by decreasing Ca2+ influx. It attenuated Ca2+ mobilization from internal stores evoked by thrombin or thapsigargin. In vitro studies were used as an approach to investigate how simultaneous productions of NO and O2- from SIN-1 modify thrombin- or thapsigargin-induced platelet Ca2+ mobilization. Superoxide dismutase, the O2- scavenger, enhanced the capacity of SIN-1 to inhibit Ca2+ mobilization but catalase had no effect. This suggests that the effects of SIN-1 on platelet Ca2+ handling resemble those of NO, but are modulated by simultaneous O2- release, independently of H2O2 formation.

Avian leukosis retroviruses (ALV) cause lymphomas and other cancers in chickens. Previous studies have used enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) to detect ALV p27 group-specific antigens (GSA) in commercial chicken eggs. In the poultry industry eradication programme against exogenous ALV, ELISA assays are used to identify chickens infected with the virus. The inability of ELISA and IFA assays to discriminate between ALV GSA of endogenous or exogenous origin, and actual virus, have limited rigorous assessments of viral transmission dynamics. Here, we report the use of a newly developed reverse transcriptase-polymerase chain reaction (RT-PCR) assay, with direct sequencing of the RT-PCR product, to show endogenous and exogenous ALV in albumen from unfertilized chicken eggs. We found that 95% of 20 eggs from ALV-exposed commercial chickens and 14.2% of 240 egg samples from 20 randomly chosen New Orleans retail stores were ALV-positive by RT-PCR. In comparison, only 2.5% of the same egg samples from the retail stores were positive by ELISA. Corresponding direct sequencing of randomly chosen RT-PCR products showed that four of six egg samples contained endogenous ALV, while two of the six samples were positive for exogenous subgroup A ALV. The finding of endogenous subgroup E ALV in unfertilized chicken eggs emphasizes that the transmission of endogenous ALV is common and should be considered in the implementation of ALV eradication programmes by the poultry industry.

In response to stimuli, endothelial cells release arachidonic acid, a lipid precursor of various vasoactive substances. We have investigated the relationships between cytosolic Ca2+ movements and arachidonic acid release in human umbilical vein endothelial cells. Histamine, a receptor-dependent agonist, and thapsigargin, a specific inhibitor of sarco-/endoplasmic Ca2+ pumps, time- and dose-dependently increased the release of [1-14C]-arachidonic acid. This release was inhibited by AACOCF3, a selective inhibitor of cytosolic phospholipase A2 (PLA2). In the absence of Ca2+ influx, arachidonic acid release was suppressed in both histamine- and thapsigargin-stimulated cells, despite marked elevations of cytosolic Ca2+ concentration ([Ca2+]i). In the presence of Ca2+ influx, arachidonic acid release was reduced in cells treated with BAPTA, an intracellular Ca2+ buffer, or with SK&F 96365, a receptor-operated Ca2+ channel blocker. Arachidonic acid release was analyzed as a function of the two successive phases of Ca2+ response to stimulation: Ca2+ peak and plateau phase, reflecting Ca2+ mobilization from internal stores and Ca2+ influx, respectively. The amount of arachidonic acid released was directly related to [Ca2+]i values measured at the influx phase with a 80 nM [Ca2+]i threshold, similar to that reported for PLA2 translocation. This suggests that Ca2+ entry from the extracellular space is essential for activating cytosolic PLA2 in human endothelial cells.

A reverse transcriptase polymerase chain (RT-PCR) assay was developed to detect avian leukosis retrovirus (ALV) in egg albumen. Eggs of Single Comb White Leghorns were from a commercial breeder (stock F) and from a pathogen-free flock (stock N). RT-PCR was undertaken on isolated RNA from 20 unfertilized egg samples using seven sets of primers that correspond to the ALV gp85 envelope glycoprotein which determines the ALV subgroup classification. An ELISA assay for ALV gs antigen of egg albumen was positive for all stock F birds tested and negative for all stock N birds. Virus isolation was undertaken by inoculating egg albumen, feather pulp, or blood from five stock F chickens onto cultures of chicken embryo fibroblasts (C/E). IFA analysis of the inoculated C/E cultures indicated that all stock F birds tested contained infectious ALV. For the virus-positive stock F chickens, RT-PCR analyses using primers designed to detect all ALV subgroups detected ALV in 15/15 (100%) egg albumen samples, while primers designed to detect subgroup A ALV were positive for 12/15 (80%) egg albumen samples. RT-PCR products were not detected from five egg albumen samples from five stock N chickens by any primer sets. Direct sequencing using primers specific for subgroup A ALV verified the viral subgroup in the RT-PCR amplification products. The combined use of RT-PCR and direct sequencing of the RT-PCR product provides a new approach for identifying ALV-infected poultry.

In tropical areas, where unsupervised use of antimalarial drugs is common, patients with an illness consistent clinically with severe malaria but with negative blood smears pose a management dilemma. Malaria pigment is evident in peripheral blood leukocytes in greater than 90% of patients with severe malaria. To characterize the clearance kinetics of parasitized erythrocytes and malaria pigment-containing leukocytes, sequential peripheral blood and intradermal smears were assessed in 27 adult Vietnamese patients with severe falciparum malaria. The clearance of parasitized erythrocytes and pigment-containing monocytes (PCMs) followed first order kinetics. The elimination of pigment-containing neutrophils (PCNs) was first order initially, but deviated from this when counts were low. Clearance of peripheral blood PCMs (median clearance time, 216 hours; range, 84 to 492 hours) was significantly slower than that of parasitized erythrocytes (median, 96 hours; range, 36 to 168 hours) or PCNs (median, 72 hours; range, 0 to 168 hours; P < .0001). Intradermal PCM clearance times were the longest of all (median, 12 days; range, 6 to 23 days; significantly longer than peripheral blood PCM clearance, P < .001). Twenty-one (88%) patients still had signs, symptoms, or laboratory features of severe malaria after parasite clearance but before phagocyte pigment clearance. Sixteen of the 23 surviving patients (70%; 95% confidence interval, 50% to 87%) still had intraleukocytic malaria pigment on peripheral blood films 72 hours after parasite clearance. Thus, by determining the distribution of malaria pigment in peripheral blood and intradermal phagocytes, the time since effective antimalarial treatment started can be estimated. Microscopy for intraleukocytic pigment is valuable in the differential diagnosis of severe febrile illnesses in malarious areas where uncontrolled use of antimalarial drugs is widespread.

Enteric and sympathetic neurons have previously been proposed to be lineally related. We present independent lines of evidence that suggest that enteric neurons arise from at least two lineages, only one of which expresses markers in common with sympathoadrenal cells. In the rat, sympathoadrenal markers are expressed, in the same order as in sympathetic neurons, by a subset of enteric neuronal precursors, which also transiently express tyrosine hydroxylase. If this precursor pool is eliminated in vitro by complement-mediated lysis, enteric neurons continue to develop; however, none of these are serotonergic. In the mouse, the Mash-1-/- mutation, which eliminates sympathetic neurons, also prevents the development of enteric serotonergic neurons. Other enteric neuronal populations, however, including those that contain calcitonin gene related peptide are present. Enteric tyrosine hydroxylase-containing cells co-express Mash-1 and are eliminated by the Mash-1-/- mutation, consistent with the idea that in the mouse, as in the rat, these precursors generate serotonergic neurons. Serotonergic neurons are generated early in development, while calcitonin gene related peptide-containing enteric neurons are generated much later. These data suggest that enteric neurons are derived from at least two progenitor lineages. One transiently expresses sympathoadrenal markers, is Mash-1-dependent, and generates early-born enteric neurons, some of which are serotonergic. The other is Mash-1-independent, does not express sympathoadrenal markers, and generates late-born enteric neurons, some of which contain calcitonin gene related peptide.

The hypothesis was tested that developing enteric neurons withdraw from the cell cycle in a sequence related to their phenotype. The birthdays of immunocytochemically identified myenteric and submucosal neurons were determined in the murine duodenum and jejunum. [3H]thymidine ([3H]TdR) was injected into timed pregnant mice or pups at 4-8 hour intervals over a 24 hour period. Pups were killed on postnatal day 30 (P30). [3H]TdR incorporation was detected by radioautography in enteric neurons, which were phenotypically identified by the simultaneous detection of the immunoreactivities of 5-hydroxytryptamine (5-HT), choline acetyl transferase (ChAT), neuropeptide Y (NPY), enkephalin (ENK), calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP). The dates of the earliest withdrawal from the cell cycle of neurons containing these markers were determined, as well as the length of time during which the identified neurons continued to be born, and the date on which their rate of birth was maximal. The birthdates of myenteric neurons that contained 5-HT (E8-E14, peak at E10) or ChAT (E8-E15, peak at E12) tended to be earlier than those that contained ENK (E10-E18, peak at E14), NPY (E10-E18, peak at E15), VIP (E10-P5, peak at E15), or CGRP (E10-P3, peak at E17). For any given immunocytochemically defined neuronal phenotype, submucosal neurons tended to be born later than their myenteric counterparts and submucosal neurons that contained neuropeptides were born later than those that contained only ChAT immunoreactivity. The day (E8) on which the first 5-HT- and ChAT-immunoreactive neurons became postmitotic is earlier than the day (E9) on which the colonization of the bowel by crest-derived cells has been detected. The population of neural precursors that colonizes the gut, therefore, is heterogeneous; many cells are proliferating, but a specific subset, which will ultimately give rise to serotoninergic or cholinergic neurons, is already postmitotic. Neurons continued to be born throughout fetal life and even after birth. Consequently, terminally differentiated neurons coexist in the developing enteric nervous system with dividing neural precursor cells. This observation is consistent with the idea that early developing neurons could affect the development of enteric neural precursors; moreover, they also demonstrate that it is possible to add neurons to the enteric plexuses even after the neural circuits on which the bowel depends have become functional.

Recent data from the literature have shown that cDNA clones for the carboxyterminal domain of the core protein of large proteoglycan monomers from human cartilage contain an EGF-like domain, which appears to undergo alternative splicing. In the present study we have found that articular proteoglycans from human and baboon separated on agarose flat-bed gels and blotted onto nitrocellulose react with a rabbit antiserum to mouse EGF. In addition both forms of the proteoglycans (band I and band II) seen on these gels are reactive. Reactivity is seen with proteoglycans extracted from human articular cartilage of various ages (fetal, newborn, young and aged) and with proteoglycans extracted from cartilage of thanatophoric dysplasia and homozygous achondroplasia. Reactivity is dependent on prior digestion of the nitrocellulose blot with Chase ABC, suggesting masking of epitope by chondroitin sulfate. Reactivity of the EGF antiserum with cartilage proteoglycan core protein was also demonstrated in an ELISA system with core protein as coating antigen. The reactivity appears to reside in a tryptic peptide generated from Chase/keratanase digested core protein. The immunoreactive species migrates as a 68 KDa species on gradient gels. Immunological detection and quantitative analysis of the EGF-like domain could be useful for analysis of various proteoglycan samples.

We studied with ultrastructure techniques portions of right atrium resected at operation from 12 patients with chronic rheumatic heart disease and 6 patients with non-rheumatic valvular heart disease. The right atrial pressures, duration of symptoms and age of the patients were comparable in both groups. Ten of the 12 rheumatic and 4 of the 6 non-rheumatic patients had atrial fibrillation. In the 12 rheumatic patients we found severe interstitial fibrosis, extensive cellular degeneration (17% of cells studied) and marked cellular hypertrophy (average cell diameter 16 micrometers). The six non-rheumatic patients showed evidence of cellular hypertrophy (average cell diameter 15 micrometers) but minimal interstitial fibrosis or cellular degeneration. The degenerative changes in the rheumatic group did not correlate with the degree of hypertrophy or the extent of the hemodynamic alterations. Atrial fibrillation, present in both rheumatic and non-rheumatic patients, did not correlate with the presence of cellular degeneration. We conclude that the structural changes in atria of patients with chronic rheumatic heart disease may be part of the rheumatic process and are not entirely secondary to altered hemodynamics.

The effects of left atrial enlargement on atrial cell electrophysiology and structure were studied in dogs with mitral valve fibrosis. Thirteen dogs (Groups I) had left atrial enlargement and intermittent atrial arrhythmias; 10 dogs (Group II) had left atrial enlargement and chronic atrial fibrillation. The resting and action potentials of cells in isolated preparations from the enlarged left atrium were found not to differ from those in the nonenlarged right atrium or in the atrium of control dogs. The resting and action potentials of cells in Group II atria did not differ significantly from those in Group I atria. Some cells (15 percent of the total studied) in the atria of dogs in Groups I and II were inexcitable, but either superfusion with acetylcholine or norepinephrine restored excitability. The structural studies showed that the left atrium of the dogs in Groups I and II had a reduced number of muscle cell layers spanning the wall with an unusually large amount of connective tissue between greatly hypertrophied cells. Very few degenerating cells were seen. Dramatic abnormalities of cell electrophysiology may not be involved in the genesis of arrhythmias in the enlarged canine atrium, and the altered morphologic features of the atrium in these dogs may be important in the genesis of persistent atrial arrhythmias.

Portions of operatively resected right atrium from 15 patients with atrial septal defect were studied ultrastructurally to determine whether the cell hypertrophy in the right atrium of patients with increased right atrial blood flow and increased right atrial pressure is caused by the increased blood flow. In 12 patients with normal right atrial mean pressure but increased right atrial blood flow the atrium was dilated but no atrial arrhythmias were noted clinically. Ultrastructurally, the atrial myocardial cells in these patients were normal, measuring 6 to 10 mu in diameter, and there was no evidence of cell hypertrophy or degeneration. The remaining three patients had elevated right atrial mean pressure and increased right atrial blood flow. Ultrastructurally, the atrial myocardial cells in all three patients were hypertrophied, and two patients had evidence of focal cell degeneration; the atrium was markedly dilated, but atrial arrhythmias were not noted. The lack of cell hypertrophy in the right atrium of the 12 patients with increased blood flow but normal mean pressure suggests that in congenital heart disease volume overload alone does not lead to cell hypertrophy of the right atrial myocardium.

Aganglionosis occurs in the terminal colon of the ls/ls mouse because an intrinsic defect of the presumptive aganglionic tissue prevents the entry and colonization of this portion of the bowel by migrating neural crest cells. The current study was undertaken to determine if abnormalities of the extracellular matrix could be identified in this segment that might account for migratory failure. Since basal laminae of the muscularis mucosa are overproduced in the aganglionic segment of adult ls/ls mice, we examined components of basal laminae in fetal gut from Day E 11 to Day E 16 of gestation. This period spans the time of enteric ganglion formation. Laminin and collagen type IV were studied by immunocytochemistry and proteoglycans by staining glycosaminoglycans with Alcian blue. Abnormalities of each of these components occur during development of the presumptive aganglionic bowel in the ls/ls mouse and could be detected as early as Day E 11. These defects consist mainly of an overabundance of these materials, both in defined basal laminae and throughout the extracellular space of the mesenchyme. Electron microscopic observations in the presumptive aganglionic ls/ls colon revealed a thickening of basal laminae and exceptionally wide intercellular spaces between smooth muscle myoblasts that contained an irregular fibrillar material, consisting of 4.5- to 6.0-nm filaments associated with 14- to 20-nm granules. Fibrillar and flocculant material was continuous with formed basal laminae, and was concentrated in the same areas found to have an overabundance of laminin immunoreactivity. These observations indicate that there is an accumulation of extracellular matrix material, including components of basal laminae, that (i) precedes the formation of enteric ganglia, (ii) is in the path through which enteric neural precursors from the crest would have to migrate, and (iii) is limited to the aganglionic and hypoganglionic ls/ls bowel. These data are consistent with the hypothesis that components of basal laminae contribute to the inability of crest cells to colonize the terminal bowel of ls/ls mice.

Analytical electrophoresis on polyacrylamide-agarose gels of aggregating proteoglycan monomers from baboon articular cartilage produces two distinct bands, corresponding to two different aggregating monomer populations. A preparative electrophoresis procedure is described for isolating the two monomers. Proteoglycans were extracted from young baboon articular cartilage in 4 M guanidinium chloride containing proteolysis inhibitors and aggregated after hyaluronic acid addition. The aggregates were separated from non-aggregated proteoglycans by isopycnic centrifugation, followed by gel chromatography on Sepharose CL-2B. The monomers of the aggregates were obtained by isopycnic centrifugation under dissociative conditions. Two monomers were separated by preparative electrophoresis on 0.8 % agarose submerged gels. Approximately 60 % of the proteoglycans were recovered from the gel using a freeze-squeeze procedure. Aliquots of the separated monomers gave single bands when submitted to analytical polyacrylamide-agarose gel electrophoresis. Their migration and appearance were similar to that of the two bands present in the non separated preparation of monomers.

Human metaphase chromosomes, fixed in methanol-acetic acid, were ultraviolet irradiated to produce single-stranded regions of chromosomal DNA and treated with anti-5-methylcytidine. Using an immunoperoxidase procedure, regions of antibody binding were readily visualized by light microscopy in the centromeric heterochromatin regions of chromosomes 1, 9, 16, the short arm of chromosome 15, and in the distal portion of the Y. Electron microscopic visualization of the same whole mount chromosome preparations transferred to formvarcoated grids revealed additional details of the distribution and arrangement of 5-methylcytosine. A helical arrangement of 5-methylcytosine residues was seen below the centromere of chromosome 1. The Y chromosome showed a concentration of 5-methylcytosine residues on the distal long arm, and in areas just below and slightly above the centromere. In all the above chromosomes, especially chromosome 15, additional 5-methylcytosine residues were detected as isolated foci along the arms. Our findings support the concept that clusters of similar purine or pyrimidine residues exist along the arms of condensed metaphase chromosomes, with the possibility that concentrations of 5-methylcytosine residues might have been enhanced at the surface of the chromosomes during the condensation process.

The terminal portion of the bowel of the lethal spotted mutant mouse (ls/ls) lacks an enteric nervous system due to the failure of neural crest precursors to colonize this region during embryonic life. As a result, the mouse develops congenital megacolon. We have postulated that the defect occurs because the microenvironment of the aganglionic segment is segmentally abnormal and does not permit the migration and/or survival of the enteric neural or glial precursors in the affected zone. We have examined the terminal segment of adult ls/ls and control mice by light and electron microscopy to determine if the defect is associated with identifiable structural abnormalities that persist to maturity. A striking abnormality is an overgrowth of the muscularis mucosa in the adult ls/ls mouse, particularly in the outer longitudinal layer. Electron microscopy also reveals an extensive thickening of the basal lamina around smooth muscle cells. In addition, nerves that are derived from fibers that are extrinsic to this area are abnormal. Large bundles of nerve fibers, some of which contain myelinated axons, large-caliber unmyelinated axons, and abundant collagen, are prominent in the intermuscular region of the aganglionic segments and often reach into the submucosa. The supporting cells of the unmyelinated and myelinated nerves in the aganglionic segment have voluminous perineural cytoplasm typical of immature Schwann cells. They also exhibit intermediate filaments in their cytoplasm. Otherwise they have the typical morphology of peripheral Schwann cells, rather than enteric glia, including individual ensheathment of axons and a surrounding basal lamina. We suggest that the extracellular matrix and/or cells of mesenchymal origin of the terminal bowel of the ls/ls mouse may prevent the ingrowth of the normal precursors of the glia as well as neurons of the enteric nervous system, but may permit or even encourage the ingrowth of abnormal numbers of extrinsic axons.

The lethal spotted mutant mouse (ls/ls) develops congenital megacolon because of the absence of ganglia in the terminal colon. This aganglionosis results from a failure of neural crest cells to colonize this area during fetal life. We have postulated that the microenvironment of the aganglionic segment of bowel is abnormal. Our hypothesis suggests that this abnormal enteric microenvironment fosters the sprouting of neuritic processes. We further propose that neural and glial precursors cease to migrate once they have extended their definitive processes. As a result, the area distal to the site where neurite extension is favored does not become colonized by neural or glial precursors. A prediction of this hypothesis is that the aganglionic tissue should be innervated by axons from neurons located both in the more proximal ganglionated bowel and in ganglia located outside the gut. Neurons and their processes in control and ls/ls terminal gut were located by the histochemical demonstration of acetylcholinesterase (AChE) activity and their structure was classified as intrinsic (enteric) or extrinsic in type by electron microscopy. In ls/ls mice the submucosal plexus was much more severely affected than the myenteric plexus. No submucosal ganglia were found within 30 mm of the anus. In contrast, myenteric ganglia extended to within 4 mm of the anus on the mesenteric side of the gut and to within 15 mm on the antimesenteric side. Rostral to the areas that were absolutely aganglionic, both plexuses were hypoganglionic, especially the submucosal plexus, which was hypoganglionic throughout the entire colon. Both the aganglionic and caudal hypoganglionic zones of the ls/ls bowel were penetrated by large nerve trunks that had the ultrastructural characteristics of extra-enteric peripheral nerve. Unusual ganglia, outside the enteric musculature in the adventitia of the colon, were connected to these trunks. The location of the cell bodies of origin of the nerve fibers in the terminal colon of control mice and in the aganglionic segment of the bowel in ls/ls mice was determined by following the retrograde transport of tracers injected as close as possible to the anus. An extrinsic innervation originating from the inferior mesenteric ganglion and dorsal root ganglia (L6-S1) was found in both types of animal. In control but not ls/ls mice retrograde labeling was also observed in the sacral parasympathetic nucleus of the spinal cord. In addition, neuritic processes were traced to neurons in myenteric ganglia. In control mice, these labeled neurons were present in ganglia within the injection site as well as in bowel rostral and caudal to it.(ABSTRACT TRUNCATED AT 400 WORDS)

Surgical resection of the endocardium and subendocardium often abolishes chronic recurrent sustained ventricular tachycardia in patients with healed myocardial infarcts or ventricular aneurysms, presumably by interrupting the reentrant pathway. To define the morphologic characteristics of cells in the reentrant pathway, we studied the histology and ultrastructure of the endocardial resections of 23 patients who underwent this procedure. Bundles of apparently viable myocardial fibers embedded in dense fibrous tissue were identified throughout the endocardial resections from all patients. These bundles of cells were separated from one another by fibrous tissue but extended uninterrupted to the margins of the surgical resection. In 14 patients Purkinje fibers were identified beneath the thickened endocardium whereas the remaining bundles were composed of ventricular muscle. The Purkinje fibers appeared to have normal ultrastructure and ventricular cells with both normal and abnormal ultrastructures were present. The abnormal muscle cells were characterized by loss of contractile elements, aggregates of dilated sarcoplasmic reticulum, and osmiophilic dense bodies. The sarcolemma was intact and the nuclear chromatin was evenly dispersed suggesting that these cells were still viable. The abnormal structure and arrangement of the surviving cardiac fibers in the endocardium may cause the abnormal electrophysiologic function that results in ventricular tachycardia.

Proteoglycan monomers from articular cartilage of young baboons and of a baboon foetus were submitted to zonal rate centrifugation in sucrose gradients. The peaks obtained were analyzed by gel electrophoresis on polyacrylamide-agarose gels. The proteoglycan monomers corresponding to the bands obtained by gel-electrophoresis of unfractionated proteoglycans were separated in the gradients and the sedimentation distance correlated with the electrophoretic mobility.

To determine the storage site of platelet fibrinogen and of platelet factor 4 (PF4) in human platelets by immunoelectron microscopic techniques, washed human platelets were briefly exposed to Karnovsky's fixative and embedded in water-soluble Durcupan. Thin sections of platelets were exposed to Fab fragments of rabbit anti-human fibrinogen or of goat anti-human PF4, followed by a peroxidase conjugate of Fab fragments of antibodies to rabbit immunoglobulin (Ig) G or to goat IgG. The technique enabled preservation of the antigenic determinants of the platelet proteins, accessibility of Fab fragments to the platelet proteins, and maintenance of the ultrastructural integrity of the platelets. Using this approach, it was directly demonstrated that platelet fibrinogen and PF4 are stored in the alpha-granules of human platelets.

Ultrastructural studies were performed on portions of the operatively resected right atrium from six patients with a ventricular septal defect and six patients with an endocardial cushion defect. The six patients with a ventricular septal defect had normal right atrial mean pressure and no evidence of right atrial volume overload. Ultrastructurally, the atrial muscle cells in these patients appeared normal and measured 6 to 12 mu in diameter. The six patients with an endocardial cushion defect had elevated right atrial mean pressure and evidence of right atrial volume overload. Ultrastructurally, the atrial muscle cells in these patients were generally larger than 12 mu in diameter. The cells were irregular and had multiple and occasionally widened intercalated discs. In addition, there were degenerative changes in two patients with markedly increased atrial pressure. These changes included extensive loss of contractile elements, aggregation of small irregular mitochondria and proliferation of tubules of the sarcoplasmic reticulum. The structural changes suggest that hypertrophy of the right atrium may be secondary to volume overload of the atrium, whereas degenerative changes may be secondary to increased right atrial pressure.