Localization of adhesion molecules on human spermatozoa by fluorescence microscopy

Summary.  The expression of adhesion molecules on human spermatozoa of healthy probands was analysed. The localization patterns of adhesion molecules (AM) on the spermatozoal surface were documented by fluorescence microscopy. Spermatozoa were incubated with antibodies against α1 (CD49a), α2 (CD49b), α3 (CD49c), α4 (CD49d), α5 (CD49e), α6 (CD49f) chains of β1 integrins, β1 (CD29), β2 (CD18), αV (CD51), β3 (CD61) and β4 integrin chains, the LFA-3 (Lymphocyte function antigen, CD58) from the immunoglobulin superfamily and the extracellular matrix proteins laminin, fibronectin and collagen IV. For collagen IV, α1 and α2 chains no expression could be noticed. Laminin was detected at the acrosomal membrane, fibronectin and β4 chain mainly at the equatorial membrane. The fibronectin receptors α3, α4 and α5 chains of the β1 integrins were mainly located on acrosomal and equatorial membrane areas. Laminin receptor α6 chain was located postacrosomal and less frequently acrosomal. β2 chain and vitronectin receptors αV and β3 chains had a mainly postacrosomal localization pattern. LFA-3 was found constantly on postacrosomal membrane areas. Double staining technique was used to prove the simultaneous occurrence of fibronectin and its integrin receptors α3, α4 and α5 chains and of αV and β2 chains on spermatozoa. The localization patterns of integrins on double stained spermatozoa were similar to the patterns described for single stained spermatozoa. The localization of fibronectin appeared to be influenced by the presence of integrins: the typical equatorial fibronectin band disappeared in case of an equatorial localization of integrins.     

Expression of human T-cell lymphotropic virus type-1 gene products in the short-term cultured skin tissues of an adult T-cell leukemia/lymphoma patient with cutaneous manifestations.

Adult T-cell leukemia/lymphoma (ATLL) is recognized as a disease etiologically associated with human T lymphotropic virus type-1 (HTLV-1) infection, but, neither viral replication nor specific virus antigen expression have been detected on ATLL cells distributed in organs, including skin. To examine the latent expression of HTLV-1 in the cutaneous lesions of ATLL patients, we cultured the lesional skin tissues in vitro and applied immunofluorescence staining with mouse monoclonal antibodies Lt-4, GIN-14, and F10, which react with p40tax, p19 and gp21, respectively. We recognized HTLV-1 specific antigens on clustered ATLL cells only in the deeper dermis of the skin after 24 hrs cultivation of the lesional skin tissue from an ATLL patient in RPMI-1640 medium supplemented with 20% fetal calf serum. In the electron microscope, we observed HTLV-1 like particles, 80-140 nm in diameter with envelope and core structures, in the same tissue specimen. These findings suggest that HTLV-1 gene products may be expressed in the skin lesions of ATLL patients and involved in the pathogenesis of skin eruptions in cutaneous type ATLLs. To our knowledge, this is the first report that envisages the potency of intracutaneous HTLV-1 expression in vivo.     

High level production of soluble single chain antibodies in small-scale Escherichia coli cultures

We have investigated the effect of growth and induction conditions on the production of soluble single-chain Fv antibody fragments in Escherichia coli under the control of wt lac promoter. The scFv was directed into the periplasmic space by a pelB leader sequence. Addition of sucrose to the medium gave a 15–25-fold increase in the yield of soluble scFv-phOx (3.0 mg/l) for bacterial shake-tube cultures and an increase of 80–150-fold (16.5 mg/l) for shake-flask cultures. Using flask culture in the presence of 0.4 M sucrose, a significant amount of scFv was released into the medium. We found that the scFv could be made to accumulate in the periplasm or be secreted into the medium by simply changing the incubation conditions and the concentration of the inducer. The ratio between soluble antibody fragments and insoluble scFv aggregates proved to be dependent on the strength of the promoter. Lowering the incubation temperature below 20°C had no effect on the yield of soluble antibody fragments in the periplasm, but they were no longer secreted into the medium. An example of high level production in shake-flask cultures and one-step purification by immobilized metal affinity chromatography (IMAC) is described for a soluble scFv specific for the T cell surface antigen CD3. The biological activity of the purified anti-CD3 scFv was demonstrated by flow cytometry. This method should be especially useful for the functional screening of a large number of clones in small-scale cultures.     

Analysis of gene expression in MOG-induced experimental autoimmune encephalomyelitis after treatment with a novel brain-penetrating antioxidant

Accumulating data from experimental studies indicate that oxidative stress has a major role in the pathogenesis of multiple sclerosis (MS). It has been suggested that local production of reactive oxygen species, probably by macrophages, mediates axonal damage in both MS patients and the mouse model experimental autoimmune encephalomyelitis (EAE). We have shown previously that our novel brain-penetrating antioxidant, N-acetylcysteine amide (AD4), reduces the clinical and pathological symptoms, including inflammation and axonal damage in myelin oligodendrocyte glycoprotein (MOG)-induced chronic EAE in mice. The aim of this study was to examine the molecular mechanism by which AD4 exerts protection in MOG-induced EAE mice. Therefore, we analyzed gene-expression profile in the spinal cords of MOG-induced chronic EAE mice and compared them with MOG-induced mice treated with AD4, using a cDNA microarray. We found that MOG treatment up-regulated genes encoding growth factors, cytokines, death receptors, proteases, and myelin structure proteins, whereas MOG- and AD4-treated mice demonstrated gene expression profiles similar to that seen in na?ve healthy mice. In conclusion, our study shows that chronic AD4 administration suppresses the induction of various pathological pathways that play a role in EAE and probably in MS.     

Psychological,situational, and gender predictors of cardiovascular reactivity to stress: A multivariate approach

This study examined whether relationships between anger expression, hostility, social evaluative anxiety, and a presumed mechanism for coronary heart disease development, cardiovascular reactivity (CVR) to stress, are moderated by stress situation and gender and whether such relationships are attenuated by inadequate assessments. Subjects (47 men, 47 women) were assigned randomly to either a Harassment or a Social Evaluation condition, under which they performed a reaction time task. SBP, DBP, and HR measures were recorded during baseline and task. Multiple regression analyses indicated that expressed anger was related to CVR only among men in the Harassment condition; that hostile men who express anger showed the most CVR across situations, and that the traits assessed here did not predict CVR among women. Results suggest that assessments of coronary-risk and interventions to reduce risk may need to take into account attitudes, styles of emotional expression, environmental factors, and gender.This research was supported in part by an NIMH predoctoral fellowship (F31MH09836) awarded to John W. Burns and by a grant from the American Heart Association (89-01-3G) awarded to Edward S. Katkin.     

Anorexia Induced by the Parasitic Nematode, Nippostrongylus brasiliensis: Effects on NPY and CRF Gene Expression in the Rat Hypothalamus

Infections of the gastrointestinal nematode, Nippostrongylus brasiliensis, in the laboratory rat result in a characteristic biphasic anorexia which is followed by hyperphagia once the worm burden has been cleared. Despite the importance of parasite-induced anorexia, relatively little is known of the underlying mechanisms. We have investigated the involvement of the central appetite drive in this anorexia by studying the gene expression of two neuropeptides with opposing actions on energy balance, NPY and CRF. Gene expression was assessed by in situ hybridization at 2, 8 and 16 days post-infection (p.i.) in infected rats, in uninfected controls, and in a group with food intake restricted to match that taken voluntarily by the parasitized animals. The sampling intervals corresponded to each of the two phases of maximum anorexia and the period of compensatory hyperphagia. Surprisingly, we found that increases in NPY gene expression in the hypothalamic arcuate nucleus (ARC) accompany anorexia in rats infected with N. brasiliensis; there was a significant relationship between degree of anorexia and induction of NPY mRNA after 8 days of infection. Furthermore, ARC NPY mRNA levels in parasitized animals were similar to those in pair-fed individuals with food intake restricted to match the infected rats. The number of larvae used to establish the infection affected both the degree of anorexia and the level of NPY mRNA at 8 days p.i. in a dose-dependent manner. NPY gene expression remained elevated in infected rats during at least the initial stages of compensatory hyperphagia. This suggests that animals detect a state of energy deficit during the early stages of the infection, yet do not feed, but become hyperphagic coincident with worm loss. The failure of anorectic parasitized animals to feed in response to activation of the NPYergic system makes this a novel system in which to study the regulation of hypothalamic NPY by physiological challenge. There were no significant differences in CRF gene expression between the groups at any of the sampling intervals.     

Tyrosine hydroxylase and serotonin containing cells in embryonic rat rhombencephalon: a whole-mount immunocytochemical study

Rhombencephala from rat embryos were processed as whole-mounts for immunocytochemical detection of monoaminergic cell populations, using antibodies to tyrosine hydroxylase (TH) and serotonin (5-HT). Specific advantages of the whole-mount technique over the classical serial-section method were that even isolated immunoreactive (IR) cells could be detected easily, and three-dimensional relationships could be ascertained without the need for serial reconstruction. Embryos between embryonic days (E) 12 and 16 (the day following nocturnal mating being considered as E1) were used in this study. Both TH and 5-HT immunoreactivities were already detectable at E12, even in the smallest embryos (crown-rump length: 6 mm), but there was a striking difference in the number and regional distribution of these two types of IR cells. TH was expressed in several cell groups located in the rostral rhombencephalon (the presumed anlage of the A4-7 complex) as well as in the caudal rhombencephalon (the presumed anlagen of groups A1-2 and C1-3), whereas 5-HT was expressed in very few cells located near the rostral border of the rhombencephalon (presumed anlage of the B4-9 complex). Although the three-dimensional distribution of the TH-IR cell groups underwent some modifications during the period studied, its general pattern remained relatively stable after E12. This contrasted with the sequential appearance of the 5-HT-IR cell groups and their spatial transformations during this period. Using the rhombencephalic isthmus as a landmark, we found that conspicuous 5-HT-IR fibre bundles penetrated into the mesencephalon from E13 onwards, but that the 5-HT IR cell bodies were exclusively located caudal to the borderline between the mesencephalon and the rhombencephalon (the rhombencephalic isthmus). We therefore suggest the term "rostral rhombencephalic raphe nuclei" for the rostral 5-HT cell groups instead of "mesencephalic raphe nuclei," which is a misnomer. Close spatial association between TH and 5-HT-IR elements was observed mainly in the caudal rhombencephalon, where 5-HT-IR fibres coursed through an area containing numerous TH-IR cell bodies (the presumed anlagen of groups A1-2 and C1-3).     

Polysialic acid, a unique glycan that is developmentally regulated by two polysialyltransferases, PST and STX, in the central nervous system: From biosynthesis to function

Polysialic acid is a developmentally regulated carbohydrate composed of a linear homopolymer of a-2,a-linked sialic acid residues. This unique glycan is mainly attached to the neural cell adhesion molecule (N-CAM) and implicated in many morphogenic events of the neural cells by modulating the adhesive property of N-CAM. Recently, the cDNA that encodes polysialyltransferase, which is responsible for the polysialylation of N-CAM, was successfully cloned from three mammalian species. This review focuses on the molecular cloning of human polysialyltransferase, designated PST. it then describes the number of enzymes actually required for the polysialylation of N-CAM using an in vitro polysialyltransferase assay. Comparisons between PST and another polysialyltransferase, sialyltransferase X (STX), are made and it Is demonstrated that both enzymes can independently form polysiatic acid In vitro , but that during neural development they coordinately but distinctly synthesize polysialic acid on N-CAM. The role of polysialic acid in the central nervous system is also discussed. Finally, evidence that the two polysialyltransferases, PST and STX, apparently have distinct roles in the development of neural cells is provided by using a neurite outgrowth assay.