A synthetic decapeptide of influenza virus hemagglutinin elicits helper T cells with the same fine recognition specificities as occur in response to whole virus

The immunogenicity of an isolated murine helper T cell determinant was studied. Mice were immunized with a synthetic peptide corresponding to amino acid residues 111-120 of the influenza PR8 hemagglutinin (HA) heavy chain, a region previously identified as a major target of the helper T cell response to the HA molecule in virus-primed BALB/c mice. Lymph node T cells from these mice were fused with BW 5147 cells to produce T hybrids for clonal analysis of their recognition specificities. Three T cell hybridoma clones, obtained from two different mice, responded to the immunizing peptide when presented by syngeneic antigen-presenting cells. All of these clones responded also to antigen provided as intact wild-type PR8 virus. The fine specificity of the peptide-induced T cell hybridomas, in response to a panel of mutant and variant influenza viruses, was indistinguishable from the fine specificities of T cells to the corresponding region of the HA1 chain of the HA molecule which had been generated by priming of mice with intact wild-type virus. These results suggest that an immunogenic determinant is contained within the 111-120 sequence that is able to elicit anti-influenza virus T cells with a similar repertoire to those elicited by immunization with whole virus.     

Murine TH response to influenza virus: recognition of hemagglutinin, neuraminidase, matrix, and nucleoproteins

BALB/c mice were primed with type A influenza virus by footpad injection or by aerosol infection with PR8 [A/PR/8/34-(H1N1)]. Isolated T cells from draining lymph nodes were then tested for their proliferation in the presence of purified viral proteins hemagglutinin, neuraminidase, matrix, and nucleoprotein. Significant responses [( 3H]thymidine incorporation) were seen against each of the four proteins after either priming scheme. When helper T (TH) cell clones were isolated by hybridoma formation from two different strains of mice, responsiveness (interleukin 2 production) towards each protein was against apparent. Of 12 virus-specific T cell hybridomas isolated, four responded to matrix, three to nucleoprotein, one to neuraminidase, three to hemagglutinin, and one cell was of undefined specificity. Each hybridoma was also tested for recognition of the HK virus [A/Hong Kong/1/68-(H3N2)], which differs in subtype from the priming strain. All matrix-specific cells, two nucleoprotein-specific cells, and the cell of undefined specificity were cross-reactive with HK virus. H1-subtype specificity was seen for all hemagglutinin and neuraminidase-specific cells and one of the three nucleoprotein-specific cells. Because many virus-immune TH cells recognize antigenically variable determinants, a significant fraction of TH cell function may be lost after virus evolution. When selecting priming schemes for long-term immunization against influenza, the isolated enhancement of TH cells recognizing conserved determinants on matrix and nucleoprotein may therefore be considered.     

Translation and functional expression of cell-cell channel mRNA inXenopus oocytes

Summary mRNA from estrogen-stimulated rat myometrium, a tissue known to upregulate cell-cell channels in response to this hormone, was microinjected intoXenopus laevis oocytes. The oocytes had been freed from covering layers of follicle cells and vitelline to allow direct cell membrane interactions when paired. About 4 hours after the mRNA injection, paired oocytes become electrically coupled. This coupling was due to the presence of typical cell-cell channels characterized by size-limited intercellular tracer flux, the presence of gap junctions at the oocyte-oocyte interface, and the reversible uncoupling that occurred in the presence of carbon dioxide. The induction of new cell-cell channels in the oocyte membrane was observed against a zero background or a low level of endogenous coupling, depending on the maturation stage of the oocytes. The time course of development of cell-cell coupling after the microinjection of mRNA was determined. The mRNA capable of inducing cell-cell coupling was confined to an intermediate size class when fractionated on a sucrose gradient.     

Improved Method for Isolating Synaptosomes from 11 Regions of One Rat Brain: Electron Microscopic and Biochemical Characterization and Use in the Study of Drug Effects on Nerve Terminal γ-Aminobutyric Acid in Vivo

A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3-phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll-sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional gamma-aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3-mercaptopropionic acid (3-MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GABA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium-free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA-synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3-MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3-MP-induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3-MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.     

Consequences of iron deficiency on photosynthetic and respiratory electron transport in blue-green algae

Growth of Aphanocapsa in low iron media resulted in a decrease of the endogenous iron pool. Below a critical concentration photosynthetic electron transfer was specifically depressed. This was caused by a strong inhibition of the synthesis of cytochromes b-559 of PSII, cytochromes b-563, f-557, and the Rieske Fe-S center of the cytochrome complex and especially the Fe-S centers of PSI. The influence of iron limitation on respiration and chlorphyll formation was negligible.Paper presented at the FESPP meeting (Strasbourg, 1984)     

Action of neurotensin on size, composition, and growth of pancreas and stomach in the rat

Since the gastrointestinal peptide neurotensin has a stimulatory effect on the secretion of the exocrine pancreas and an inhibitory effect on secretion and motility of the stomach, we investigated whether chronic parenteral administration of neurotensin would affect pancreatic and gastric growth. We therefore infused synthetic neurotensin subcutaneously (dose, 43 and 282 pmol X kg-1 X min-1) in 20 Wistar rats for 2 weeks using Alzet osmotic minipumps and compared pancreatic weight, DNA, RNA, protein, lipase, amylase, pancreatic polypeptide and insulin with these parameters in 10 control rats from the same litter with subcutaneously implanted plastic cylinders approximately the size of the minipumps. In another experiment, synthetic neurotensin (836 pmol X kg-1) was injected intraperitoneally three times a day for 3 days in 12 rats. Thereafter, we measured pancreatic DNA and in vitro incorporation of [3H]thymidine into pancreatic DNA. These effects were compared with the actions of caerulein and normal saline. Long term infusion of the high neurotensin dose induced an increase of pancreatic weight (control: 0.87 g, neurotensin: 1.02 g) and of DNA (control: 2.5 micrograms; neurotensin: 3.5 micrograms) and pancreatic polypeptide (control: 2.4 ng; neurotensin: 7.4 ng) contents, whereas pancreatic protein, RNA, amylase and lipase contents were not stimulated. In relation to DNA, these parameters even were significantly depressed. Insulin remained unchanged. Intraperitoneal injection of neurotensin induced an increase of pancreatic DNA content and stimulated [3H]thymidine incorporation into DNA (control: 11 000 dpm/g; neurotensin: 15 800 dpm/g pancreas). Moreover, long-term neurotensin infusion with the high dose led to a rise in protein concentration and an increase in the thickness of the gastric antrum; antral DNA concentration was insignificantly stimulated. Parenteral neurotensin in the doses and at the times administered, led therefore, to hyperplasia of the pancreas and induced growth of the gastric antrum. It is concluded that neurotensin can act as a trophic factor on pancreas and gastric antrum of the rat. It remains to be determined whether this represents a physiological effect of neurotensin.     

Combined immunostaining of neurofilaments, neuron specific enolase, GFAP and S-100. A possible means for assessing the morphological and functional status of the enteric nervous system

Neurofilaments, part of the cytoskeletal network, and neuron specific enolase, a major enzyme in glycolysis, are both present in central and peripheral neurons. Glial fibrillary acidic protein and S-100, on the other hand, are soluble proteins which are found exclusively in the supportive cells of the nervous system, i.e. the glial cells. Examination was made, using immunocytochemistry, of all main areas of the gastrointestinal tract of three mammalian species, rat, pig and man. By applying serial tissue sectioning, it was possible to study the relative occurrences of the two neuronal markers in the same cell bodies and to examine the relationships of the neurons with the glial cells as revealed by the antibodies to glial fibrillary acidic protein and S-100. Both neurofilaments and neuron specific enolase were localised to an extensive system of enteric nerves, with the level of neuron specific enolase-immunoreactivity showing greater variability than that observed using antibodies to neurofilaments. Comparison of the occurrence of neuron specific enolase and neurofilament immunoreactivity in serially sectioned neuronal cell bodies revealed that a minor population stained only with antibodies to neurofilaments. The equivocal or absent neuron specific enolase-immunoreactivity in some perikarya may reflect variations in functional status within the nervous system. Glial fibrillary acidic protein- and S-100-immunoreactivities were confined to glial cells which, in this normal tissue, were always in close association with the neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

The primary structure of hemoglobin from amur-leopard (Panthera pardus orientalis)

The complete amino acid sequence of the major component of hemoglobin from amur-leopard (Panthera pardus orientalis) is presented. The major component accounts for more than 90% of the total hemoglobin. Separation of the globin subunits was achieved by ion-exchange chromatography on CM-cellulose in urea. The sequence was studied by automatic Edman degradation of tryptic and hydrolytic peptides. Alignment was carried out with human hemoglobin sequence. The NH₂ terminus is blocked with Ac-serine. The data are compared with other mammalian hemoglobins and results are discussed with respect to sequence and physiology.85th communication on hemoglobin.     

Identification and isolation of the primary aggregation factor from the cell membrane of the sponge Geodia cydonium

Summary The primary aggregation factor (pAF) of sponge cells is a glycoprotein that is firmly associated with the cell membrane. Polyspecific antibodies (anti-GM) prepared from sera raised against membranes of cells from the siliceous sponge Geodia cydonium were found to inhibit initial aggregation of homologous cells. The inhibition of aggregation, caused by anti-GM was neutralized by pAF. The pAF had been successfully solubilized and enriched by affinity chromatography, gel filtration and density gradient centrifugation, if checked by polyacrylamide gel electrophoresis in the presence of urea. The M_r of the native pAF was approximately 40 000 as estimated by gel filtration; under denaturing conditions three protein species (M_r: 16 500, 15 500 and 13 500) were identified in the pAF preparation. The pAF was precipitable by Ca⁺⁺ and did not cross-react with antisera against homologous purified secondary aggregation factor and lectin. It is mainly composed of protein (48.0%) and carbohydrate (50.2%). The isolated pAF restored the aggregation potency not only of factor-depleted Geodia cells but also of cells from other Demospongiae. However, the pAF displayed no aggregation enhancing effect on urea-treated cells from species belonging to the Calcispongiae or Hexactinellida. We hypothesize that in contrast to the secondary aggregation, the initial aggregation of Geodia cells is mediated by the one-component system, the bivalent and bifunctional pAF.