Isolation of three novel cholinergic neuron-specific gangliosides from bovine brain and theirin vitro syntheses

In the present study, three extremely minor but novel Chol-1 antigens, termed X1, X2, and X3 have been isolated from bovine brain gangliosides. Based on the results of sialidase degradation, TLC-immunostaining with anti-Chol-1 antibody and fast atom bombardment mass spectrometry, their chemical structures were identified as: $$\begin{gathered} III^6 NeuAc--GgOse4Cer (X1:GM1\alpha ) \hfill \\ III^6 NeuAc,II^3 NeuAc--GgOse4Cer (X2:GT1a\alpha ) \hfill \\ III^6 NeuAc,II^3 NeuAc--NeuGc--GgOse4Cer (X3:GT1b\alpha ) \hfill \\ \end{gathered} $$ The yields of GM1α, GD1aα, and GT1bα, were approximately 150, 20, and 10 µg, respectively, from 10 g of the bovine brain ganglioside mixture. In conjunction with our previous observations, all gangliosides with anti-Chol-1 reactivity were found to contain a common sialyl α2–6N-acetylgalactosamine residue, indicating that this unique sialyl linkage is the specific antigenic determinant. We subsequently examined the biosyntheses of the three novel Chol-1 gangliosides using rat liver Golgi fraction as an enzyme source. The results showed that GM1α, GD1aα, and GT1bα were synthesized from asialo-GM1, GM1a, and GD1b, respectively, by the action of a GalNAc α2-6sialyltransferase.     

Effects of long-term treatment with eicosapentaenoic acid on the heart subjected to ischemia/reperfusion and hypoxia/reoxygenation in rats

The effects of eicosapentaenoic acid (EPA) and long-term treatment with EPA-ethylester (EPA-E) were examined in perfused rat hearts subjected to ischemia/reperfusion and adult rat cardiomyocytes subjected to hypoxia/reoxygenation. EPA (0.1 M) improved postischmic contractile dysfunction of the ischemic/reperfused heart. EPA (10 M) attenuated hypoxia/reoxygenation-induced morphological deterioration of cardiomyocytes. The results suggest the presence of direct cardioprotective effects of EPA. Rats were orally treated for 4 weeks with 1 g/kg/day of EPA-E to elucidate ex vivo effects of EPA, and the fatty acid composition of cardiac phospholipids was determined. The percent ratio of EPA in total fatty acids of cardiac phospholipids increased whereas that of arachidonic acid decreased. The percent ratio of n-3/n-6 fatty acid did not increase. Treatment with EPA-E did not improve the post-ischemic contractile function, but attenuated the ischemia/reperfusion-induced release of prostaglandins during reperfusion. Treatment with EPA-E preserved a better morphological appearance of the cardiomyocytes subjected to hypoxia/reoxygenation. The results suggest that the mechanisms responsible for cytoprotective effects of hypoxic/reoxygeanted cardiomyocytes or inhibition of metabolic alterations of the ischemic/reperfused heart by long-term EPA-E treatment did not contribute substantially to recovery of post-ischemic contractile dysfunction. The direct in vitro effects of EPA may play a role in the protection of the heart from ischemia/reperfusion or hypoxia/reoxygenation injury.     

Correlation between cell-associated mannose-sensitive hemagglutination by Vibrio parahaemolyticus and adherence to a human colonic cell line Caco-2

Abstract Cell-associated hemagglutination (cHA) activity with human erythrocytes was examined for 468 clinical and 71 environmental strains of Vibrio parahaemolyticus . Approximately 95% of the strains tested were cHA positive irrespective of source or Kanagawa phenomenon. 75% of clinical strains showed relatively strong mannose-sensitive hemagglutination (MSHA), whereas 88% of the environmental strains showed relatively weak mannose-resistant hemagglutination (MRHA). Adherence of V. parahaemolyticus to Caco-2 cells was also determined. A clear positive correlation between cell-associated MSHA and adherence to Caco-2 cells was observed.     

A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) mainly stimulates proliferation and maturation of myeloid progenitor cells. Although the signal transduction pathways triggered by GM-CSF receptor (GMR) have been extensively characterized, the roles of GMR signals in differentiation have remained to be elucidated. To examine the relationship between receptor expression and differentiation of hemopoietic cells, we used transgenic mice (Tg-mice) that constitutively express human (h) GMR at almost all stages of hemopoietic cell development. Proliferation and differentiation of hemopoietic progenitors in bone marrow cells from these Tg-mice were analyzed by methylcellulose colony formation assay. High affinity GMR interacts with GM-CSF in a species-specific manner, therefore one can analyze the effects of hGMR signals on differentiation of mouse hemopoietic progenitors using hGM-CSF. Although mouse (m) GM-CSF yielded only GM colonies, hGM-CSF supported various types of colonies including GM, eosinophil, mast cell, erythrocyte, megakaryocyte, blast cell, and mixed hemopoietic colonies. Thus, the effects of hGM-CSF on colony formation more closely resembled mIL-3 than those of mGM-CSF. In addition, hGM-CSF generated a much larger number of blast cell colonies and mixed cell colonies than did mIL-3. hGM-CSF also generated erythrocyte colonies in the absence of erythropoietin. Therefore, GM-CSF apparently has the capacity to promote growth of cells of almost all hemopoietic cell lineages, if functional hGMR is present.     

Deficiency in fertilization by morphologically abnormal sperm produced by Azh mutant mice

Male mice homozygous for the azh mutation produce spermatozoa with abnormal head shapes and have significantly reduced fecundity, to between 5% and 10% that of wild-type or heterozygous mice. Several possible causes of this infertility were investigated. No gross endocrine disorders in azh/azh male mice were observed, and they exhibited apparently normal mating behavior. In addition, their sperm were motile, were capable of hyperactivated motility, and did not show premature acrosome reactions. However, quantitative analysis revealed slight but significant reductions in several motility parameters. Analysis of embryos following mating of azh/azh males with superovulated females indicated a reduction in the number of fertilized eggs compared to control matings. In vitro, spermatozoa from azh/azh mice failed to fertilize cumulus-intact/zona-intact and cumulus-free/zona-intact ova, although they successfully fertilized zonafree ova. These results indicate that the primary defect in fertility of azh/azh male mice is a result of sperm quality, likely, in sperm morphology, and is manifest at the level of interaction with the zona pellucida. © 1994 Wiley-Liss, Inc.     

Effect of neuromuscular electrical stimulation on motor cortex excitability upon release of tonic muscle contraction

The aim of the present study was to investigate the neurophysiological triggers underlying muscle relaxation from the contracted state, and to examine the mechanisms involved in this process and their subsequent modification by neuromuscular electrical stimulation (NMES). Single-pulse transcranial magnetic stimulation (TMS) was used to produce motor-evoked potentials (MEPs) and short-interval intracortical inhibition (SICI) in 23 healthy participants, wherein motor cortex excitability was examined at the onset of voluntary muscle relaxation following a period of voluntary tonic muscle contraction. In addition, the effects of afferent input on motor cortex excitability, as produced by NMES during muscle contraction, were examined. In particular, two NMES intensities were used for analysis: 1.2 times the sensory threshold and 1.2 times the motor threshold (MT). Participants were directed to execute constant wrist extensions and to release muscle contraction in response to an auditory “GO” signal. MEPs were recorded from the flexor carpi radialis (FCR) and extensor carpi radialis (ECR) muscles, and TMS was applied at three different time intervals (30, 60, and 90?ms) after the “GO” signal. Motor cortex excitability was greater during voluntary ECR and FCR relaxation using high-intensity NMES, and relaxation time was decreased. Each parameter differed significantly between 30 and 60?ms. Moreover, in both muscles, SICI was larger in the presence than in the absence of NMES. Therefore, the present findings suggest that terminating a muscle contraction triggers transient neurophysiological mechanisms that facilitate the NMES-induced modulation of cortical motor excitability in the period prior to muscle relaxation. High-intensity NMES might facilitate motor cortical excitability as a function of increased inhibitory intracortical activity, and therefore serve as a transient trigger for the relaxation of prime mover muscles in a therapeutic context.     

The preventive effect of fish oil on abdominal aortic aneurysm development

Abdominal aortic aneurysm (AAA) is a vascular disease involving gradual dilation of the abdominal aorta and high rupture‐related mortality rates. AAA is histologically characterized by oxidative stress, chronic inflammation, and extracellular matrix degradation in the vascular wall. We previously demonstrated that aortic hypoperfusion could cause the vascular inflammation and AAA formation. However, the preventive method for hypoperfusion‐induced AAA remains unknown. In this study, we evaluated the effect of fish oil on AAA development using a hypoperfusion‐induced AAA animal model. Dilation of the abdominal aorta in the fish oil administration group was smaller than in the control group. Collagen destruction and oxidative stress were suppressed in the fish oil administration group than in the control group. These results suggested that fish oil could prevent the development of AAA induced by hypoperfusion.     

The p.R92W variant of <Emphasis Type="Italic">NR5A1/Nr5a1</Emphasis> induces testicular development of 46,XX gonads in humans,but not in mice: phenotypic comparison of human patients and mutation-induced mice

NR5A1 is the key regulator of adrenal and gonadal development in both humans and mice. Recently, a missense substitution in human NR5A1, p.R92W, was shown to underlie gonadal dysgenesis in genetic males and testicular formation in genetic females. Here, we investigated the phenotypic effects of the p.R92W mutation on murine development. Mice carrying the p.R92W mutation manifested a similar but milder phenotype than that of the previously described Nr5a1 knockout mice. Importantly, mutation-positive XX mice showed no signs of masculinization. These results, together with prior observations, indicate that the p.R92W mutation in NR5A1/Nr5a1 encodes unique molecules that disrupt male gonadal development in both humans and mice and induces testicular formation specifically in human females. Our findings provide novel insights into the conservation and divergence in the molecular networks underlying mammalian sexual development.     

Developmental changes of C-series polysialogangliosides in chick brains revealed by mouse monoclonal antibodies M6704 and M7103 with different epitope specificities

We have established many mouse monoclonal antibodies detecting developmentally regulated antigens in chicken embryonic neural tissues of the otic vesicles and neural tube by immunizing mice with the membrane fraction of the neural tube and somite prepared from 3-d chick embryos. Among them, three monoclonal antibodies (MAbs) M6703, M6704, and M7103 were shown to react with the gangliosides isolated from chicken embryonic brains. The precise specificity of the antibodies was determined mainly by enzyme-immunostaining on thin layer plates. MAbs M6703 and M6704 bound to C-series polysialogangliosides including GT3, GT2, GT1c, GQ1c, and GP1c isolated from cod fish brains, but never to A-series, B-series, or X-series gangliosides. On the other hand, MAb M7103 antibody has a rather narrow specificity, reacting with GT1c, GQ1c, and GP1c, but not with ganglioside GT3. This indicates that the epitopes defined by these two MAbs are not with ganglioside GT3. This indicates that the epitopes defined by these two MAbs are different from each other. MAbs M6703 and M6704 recognize a trisialosyl residue, NeuAc alpha 2-8NeuAc alpha 2-8NeuAc alpha 2-3, while M7103 requires both a trisialosyl residue and the gangliotetraosy backbone structure for binding. As compared to similar MAbs which have been reported in the literature. M6703 and M6704 are unique in that they react equally with all of the C-series gangliosides and belong to the IgG3 subclass.(ABSTRACT TRUNCATED AT 250 WORDS)