Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.     

Arrangement of MP26 in lens junctional membranes: Analysis with proteases and antibodies

Summary The major membrane protein of the bovine lens fiber cell is a 26-kilodalton (kD) protein (MP26), which appears to be a component of the extensive junctional specializations found in these cells. To examine the arrangement of MP26 within the junctional membranes, various proteases were incubated with fiber cell membranes that had been isolated with or without urea and/or detergents. These membranes were analyzed with electron microscopy and SDS-PAGE to determine whether the junctional specializations or the proteins were altered by proteolysis. Microscopy revealed no obvious structural changes. Electrophoresis showed that chymotrypsin, papain, and trypsin degraded MP26 to 21–22 kD species. A variety of protease treatments, including overnight digestions, failed to generate additional proteolysis. Regions on MP26 which were sensitive to these three proteases overlapped. Smaller peptides were cleaved from MP26 with V8 protease and carboxypptidases A and B. Protein domains cleaved by these proteases also overlapped with regions sensitive to chymotrypsin, papain, and trypsin. Specific inhibition of the carboxypeptidases suggested that cleavage obtained with these preparations was not likely due to contaminating endoproteases. Since antibodies are not thought to readily penetrate the 2–3 nm extracellular gap in the fiber cell junctions, antibodies to MP26 were used to analyze the location of the protease-sensitive domains. Antisera were applied to control (26 kD) and proteolyzed (22 kD) membranes, with binding being evaluated by means of ELISA reactions on intact membranes. Antibody labeling was also done following SDS-PAGE and transfer to derivatized paper. Both assays showed a significant decrease in binding following proteolysis, with the 22 kD product showing no reaction with the anti-MP26 sera. These investigations suggest that MP26 is arranged with approximately fourfifths of the primary sequence “protected” by the lipid bilayer and the narrow extracellular gap. One-fifth of the molecule, including the C-terminus, appears to be exposed on the cytoplasmic side of the membrane.     

Cholinomimetics Increase Glutamate Outflow via an Action on the Corticostriatal Pathway: Implications for Alzheimer's Disease

Abstract: Physostigmine, the acetylcholinesterase inhibitor (0.3 mg/kg, i.m.), increased extracellular glutamate but not aspartate concentrations in the striatum of anaesthetised rats, determined using microdialysis and HPLC. The rise was both tetrodotoxin and calcium dependent. In contrast, neither physostigmine (10 µ M ) added to the perfusion fluid nor vehicle (injected intramuscularly) affected amino acid concentrations. To obtain evidence that the action of acetylcholine was to modulate positively cortical pyramidal neurone activity via the M₁ receptor, the selective M₁ agonist PD 142505-0028 (10 µ M ) was topically applied to the frontal cortex. Like physostigmine, PD 142505-0028 rapidly increased glutamate but not aspartate concentrations in the striatum. Moreover, the effect of intramuscular physostigmine was blocked by a topically applied M₁ antagonist. These new data add to our hypothesis that cholinomimetics increase pyramidal neurone function.     

Final height data, body composition and glucose metabolism in growth hormone-treated short children born small for gestational age

Low birth weight has been associated with impaired insulin sensitivity, type 2 diabetes mellitus, hypertension and cardiovascular disease in later life. GH therapy is known to increase fasting and postprandial insulin levels. For this reason concern has been expressed regarding the possible detrimental effects of GH therapy in children born small for gestational age (SGA). To assess the effects of GH therapy on body composition, carbohydrate metabolism and final height in short SGA children, 165 prepubertal short children born SGA were enrolled in either a multicentre, double-blind, randomized, dose-response GH trial (n = 75) or in a GH controlled trial (n = 90). The inclusion criteria were: (1) birth length standard deviation score (SDS) below -2; (2) age 3-8 years; (3) height SDS below -2. The children's mean (SD) age was 7.3 (2.1) years (GH dose-response trial) and 6.0 (1.5) years (GH controlled trial), birth length SDS was -3.6 and height SDS was -3.0 (0.7). In the GH dose-response trial, children were randomly assigned to either 1 mg GH/m(2) per day (group A, n = 41) or 2 mg GH/m(2) per day (group B, n = 38) ( approximately 0.033 or 0.067 mg/kg per day, respectively). In the GH controlled trial, children were randomly assigned to 1 mg GH/m(2) per day (n = 60) or served as controls (n = 30). Subjects underwent standard oral glucose tolerance tests and measurement of body mass index, systolic and diastolic blood pressure and serum lipids at baseline and after 1 and 6 years of GH therapy and again 6 months after discontinuation of GH. Body composition was measured by dual energy x-ray absorptiometry at baseline and again after 3 years in the GH controlled trial. Mean (SD) final height SDS was not significantly different between the two GH dosage groups: -1.2 (0.7) in group A and -0.8 (0.7) in group B. At the start of GH therapy, 8% of children had impaired glucose tolerance (IGT). Systolic blood pressure was significantly higher in comparison with healthy peers. GH therapy induced considerably higher fasting and glucose-stimulated insulin levels after 1 and 6 years, regardless of GH dosage. After 6 years, 4% of children had IGT. Six months after discontinuation of GH, glucose levels remained normal, whereas fasting and glucose-stimulated insulin returned to levels comparable to those of healthy peers. None of the children developed diabetes. During 6 years of GH therapy both systolic and diastolic blood pressure decreased significantly and remained so after discontinuation of GH therapy. At baseline all children had reduced bone mineral content and lean body mass. Fat mass was not significantly lower than normal. Treatment with 1 mg GH/m(2) per day resulted in a significant increase in (and normalization of) bone mineral content and lean body mass in comparison with untreated short SGA controls. Fat mass decreased during the first year of GH but returned to values comparable to those at baseline in the following 2 years of GH therapy. We found that long-term, continuous GH therapy in short children born SGA leads to a normalization of height during childhood and to a normal final height in most children, regardless of GH dosage. Only very short or relatively older children may need a dosage of 2 mg GH/m(2) per day. Long-term GH therapy had no adverse effects on glucose levels and serum lipids and had a positive effect on blood pressure, even with GH dosages of up to 2 mg/m(2) per day. However, as has been reported in other patient groups, GH induced higher fasting and glucose-stimulated insulin levels, indicating insulin resistance. After discontinuation of GH serum insulin levels returned to normal age-reference levels. Short SGA children have a reduction in bone mineral content and lean body mass when compared with healthy controls, which significantly improved (normalized) with GH therapy at a dose of 1 mg/m(2) per day.     

Analysis of the Behavior of Mitochondria in the Ovaries of the Earthworm Dendrobaena veneta Rosa 1839

We examined six types of cells that form the ovary of the earthworm Dendrobena veneta ogonia, prooocytes, vitellogenic oocytes, trophocytes, fully grown postvitellogenic oocytes and somatic cells of the gonad. The quantitative stereological method revealed a much higher “volume density” of mitochondria in all of the types of germ-line cells except for the somatic cells. Fluorescent vital stain JC-1, however, showed a much higher oxidative activity of mitochondria in the somatic cells than in the germ-line cells. The distribution of active and inactive mitochondria within the studied cells was assessed using the computer program ImageJ. The analysis showed a higher luminosity of inactive mitochondria in all of the types of germ-line cells and a higher luminosity of active mitochondria in somatic cells. The OXPHOS activity was found in somatic cells mitochondria and in the peripheral mitochondria of the vitellogenic oocytes. The detection of reactive oxygen species (ROS) revealed a differentiated distribution of ROS in the different cell types. The amount of ROS substances was lower in somatic cells than in younger germ-line cells. The ROS level was also low in the cytoplasm of fully grown postwitellogenic oocytes. The distribution of the MnSOD enzyme that protects mitochondria against destructive role of ROS substances was high in the oogonia and in prooocytes and it was very high in vitellogenic and postvitellogenic oocytes. However, a much lower level of this protective enzyme was observed in the trophocytes and the lowest level was found in the cytoplasm of somatic cells. The lower mitochondrial activity and higher level of MnSOD activity in germ-line cells when compared to somatic cells testifies to the necessity of the organisms to protect the mitochondria of oocytes against the destructive role of the ROS that are produced during oxidative phosphorylation. The protection of the mitochondria in oocytes is essential for the transfer of healthy organelles to the next generation.     

Chronic infections and genetic factors in the development of ischemic stroke

The aim of this study was to examine whether chronic infections and genetic factors of the host play roles in the pathophysiology of acute noncardioembolic ischemic stroke. Blood samples from 59 subjects with ischemic stroke and 52 control patients were investigated by nested PCR for the presence of C. pneumoniae DNA, HCMV DNA and enterovirus RNA, by ELISA for the levels of antibodies to C. pneumoniae, HCMV, HSV, HHV-6, EBV and the inflammatory chemokine IL-8, and by PCR for promoter polymorphism of the IL-8 and CD14 host genes. Associations of stroke with the HCMV IgG and HSV-1 IgA antibody levels were observed. No association of stroke was detected with the presence of C. pneumoniae, HCMV or enterovirus nucleic acids in the peripheral blood, C. pneumoniae IgM, IgG and IgA, the HSV IgG, the EBV IgG, or HHV-6 IgG antibody levels, the pathogen burden, the IL-8 or CD14 promoter polymorphisms, or with the serum levels of IL-8 in the overall study population. These results are consistent with the hypothesis that certain pathogens are involved in the development of ischemic stroke.     

Synthesis and antiviral properties of aza-analogues of acyclovir

Aza-analogues of Acyclovir were obtained from N-(2-pivaloyloxyethyl)-N-(pivaloyloxymethyl)-p-toluenesulfonamide via a one-pot base silylation/nucleoside coupling procedure. The antiviral activities of all aza-nucleosides in vitro against a variety of viruses were evaluated. None of these compounds displayed any specific antiviral effects.     

The instantly released Drosophila immune proteome is infection-specific

In this study, we analyzed the hemolymph proteome of Drosophila third instar larvae, which were induced with a suspension of Gram-positive bacteria or yeast. Profiling of the hemolymph proteins of infected versus non-infected larvae was performed by two-dimensional difference gel electrophoresis. Infection with Micrococcus luteus or Saccharomyces cerevisiae induced, respectively, 20 and 19 differential protein spots. The majority of the spots are specifically regulated by one pathogen, whereas only a few spots correspond to proteins altered in all cases of challenging (including after challenge with lipopolysaccharides). All of the upregulated proteins can be assigned to specific aspects of the immune system, as they did not increase in the hemolymph of sterile pricked larvae. Next to known immune proteins, unannotated proteins were identified such as CG4306 protein, which has homologues with unknown function in all metazoan genome databases available today.