Immunodominant CD4+ T-cell epitope within nonstructural protein 3 in acute hepatitis C virus infection.

In acute hepatitis C virus infection, 50 to 70% of patients develop chronic disease. Considering the low rate of spontaneous viral clearance during chronic hepatitis C infection, the first few months of interaction between the patient's immune system and the viral population seem to be crucial in determining the outcome of infection. We previously reported the association between a strong and sustained CD4+ T-cell response to nonstructural protein 3 (NS3) of the hepatitis C virus and a self-limited course of acute hepatitis C infection. In this study, we identify an immunodominant CD4+ T-cell epitope (amino acids 1248 to 1261) that was recognized by the majority (14 of 23) of NS3-specific CD4+ T-cell clones from four of five patients with acute hepatitis C infection. This epitope can be presented to CD4+ T cells by HLA-DR4, -DR11, -DR12, -DR13, and -DR16. HLA-binding studies revealed a high binding affinity for 10 of 13 common HLA-DR alleles. Two additional CD4+ T-cell epitopes, amino acids 1388 to 1407 and amino acids 1450 to 1469, showed a very narrow pattern of binding to individual HLA-DR alleles. Our data suggest that the NS3-specific CD4+ T-cell response in acute hepatitis C infection is dominated by a single, promiscuous peptide epitope which could become a promising candidate for the development of a CD4+ T-cell vaccine.     

Changes in organic solutes,volume, energy state,and metabolism associated with osmotic stress in a glial cell line: A multinuclear NMR study

Diffusion-weighted in vivo¹H-NMR spectroscopy of F98 glioma cells embedded in basement membrane gel threads showed that the initial cell swelling to about 180% of the original volume induced under hypotonic stress was followed by a regulatory volume decrease to nearly 100% of the control volume in Dulbecco's modified Eagle's medium (DMEM) but only to 130% in Krebs-Henseleit buffer (KHB, containing only glucose as a substrate) after 7 h. The initial cell shrinkage to approx. 70% induced by the hypertonic stress was compensated by a regulatory volume increase which after 7 h reached almost 100% of the control value in KHB and 75% in DMEM.¹H-,¹³C-and³¹P-NMR spectroscopy of perchloric acid extracts showed that these volume regulatory processes were accompanied by pronounced changes in the content of organic osmolytes. Adaptation of intra- to extracellular osmolarity was preferentially mediated by a decrease in the cytosolic taurine level under hypotonic stress and by an intracellular accumulation of amino acids under hypertonic stress. If these solutes were not available in sufficient quantities (as in KHB), the osmolarity of the cytosol was increasingly modified by biosynthesis of products and intermediates of essential metabolic pathways, such as alanine, glutamate and glycerophosphocholine in addition to ethanolamine. The cellular nucleoside triphosphate level measured by in vivo³¹P-NMR spectroscopy indicated that the energy state of the cells was more easily sustained under hypotonic than hypertonic conditions.To whom to address reprint requests.     

The eukaryotic cofactor for the human immunodeficiency virus type 1 (HIV-1) rev protein, eIF-5A, maps to chromosome 17p12–p13: three eIF-5A pseudogenes map to 10q23.3, 17q25, and 19q13.2

The eukaryotic initiation factor 5A (eIF-5A) has been identified as an essential cofactor for the HIV-1 trans-activator protein Rev. Rev plays a key role in the complex regulation of HIV-1 gene expression and thereby in the generation of infectious virus particles. Expression of eIF-5A is vital for Rev function, and inhibition of this interaction leads to a block of the viral replication cycle. In humans, four different eIF-5A genes have been identified. One codes for the eIF-5A protein and the other three are pseudogenes. Using a panel of somatic rodent—human cell hybrids in combination with fluorescence in situ hybridization analysis, we show that the four genes map to threedifferent chromosomes. The coding eIF-5A gene (EIF5A) maps to 17p12–p13, and the three pseudogenes EIF5AP1, EIF5AP2, and EIF5AP3 map to 10q23.3, 17q25, and 19q13.2, respectively. This is the first localization report for a eukaryotic cofactor for a regulatory HIV-1 protein.     

Modifications of S100-protein immunoreactivity in rat brain induced by tissue preparation

Immunocytochemistry using antibodies against various molecular forms of the Ca⁺⁺ and Zn⁺⁺-binding S100 proteins predominantly labelled astrocytes. However, especially in the neocortex the staining pattern is variable. Methods of tissue preparation have been evaluated with the aim to preserve as much S 100 immunoreactivity as possible. Optimal results were obtained after perfusion fixation with 4–5% aldehydes, 0.1M sodium cacodylate, 0.1% CaCl₂, pH 7.3. In such preparations, astrocytes were completely labelled including their lamellar compartments in large parts of the central nervous system. Ca⁺⁺-withdrawal had adverse affects on S100 immunoreactivity. Cryostat sections treated with EDTA-containing solutions before fixation showed that Ca⁺⁺-free S100 can apparently not be fixed to the tissue. Perfusion fixatives containing EDTA resulted in inhomogeneous loss of S100 staining, indicating a differential susceptibility of astrocytic subpopulations. A different type of reduction in S100 immunoreactivity occurred around large neocortical blood vessels. Perivascular defects in immunostaining occasionally appeared even after optimal fixation, but could be regularly provoked by mildly acidic fixation (pH 6.6) or prolonged barbiturate anaesthesia. These defects might be based on S100 release into the cerebrospinal fluid. Presumably under none of the conditions studied can the immunoreactivity of all S100-forms and-fractions be completely preserved in the tissue. However, recommendations are presented for optimizing tissue preparation, to the extent that premortal modifications affecting the stainability of astrocytes may be detected by S100 immunohistochemistry in fixed brain tissue.     

Determinants of the high-methionine trait in wild and exotic germplasm may have escaped selection during early cultivation of maize

The 18 kDa high-methionine δ-class zein gene from maize has been cloned, and its regulation, structure, and map position studied. These studies have shown that (i) zein genes may also contain tryptophan and lysine codons, (ii) the 18 kDa and the related 10 kDa zein gene are coordinately regulated, but their products accumulate to different levels in a genotype-dependent manner, (iii) the duplication of δ-zein genes probably involved unequal crossing over, (iv) no copy correction in either direction has occurred from teosinte to modern corn, and (v) the duplication of of the 18 kDa zein gene probably occurred before the tetraploidization of a progenitor chromosome. The work shows that important nutritional quality determinants like the high-methionine seed proteins are abundant in several exotic and wild corn varieties and low in most of the inbreds screened. The lack of a selectable phenotype for such quality traits during initial domestication and breeding of corn would have eliminated cis and trans regulatory determinants from the germplasm used in modern corn breeding. Examples of the high-methionine δ-class zeins shown here may be generally applicable in explaining the low nutritional quality of most present-day corn grown.     

C4 polymorphism in the dog: Molecular heterogeneity of the C4α and C4γ subunit chains

Using an immunoblotting technique and goat antihuman C4, we observed five distinct electrophoretic variants of C4 in a panel of 60 random dogs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated C4 showed that dog C4 is composed of three polypeptide subunit chains (, , and ) and that structural variability occurs within the - and -chain regions. Two distinct molecular weight forms of both the C4- ( _A and _B) and C4-( _A and _B) chain were detected. The variant forms of C4 and C4 were found in association with particular C4 allotypes.     

Biophoton emission

The phenomenon of ultraweak photon emission from living systems was further investigated in order to elucidate the physical properties of this radiation and its possible source. We obtained evidence that the light has a high degree of coherence because of (1) its photon count statistics, (2) its spectral distribution, (3) its decay behavior after exposure to light illumination, and (4) its transparency through optically thick materials. Moroever, DNA is apparently at least an important source, since conformational changes induced with ethidium bromide in vivo are clearly reflected by changes of the photon emission of cells. The physical properties of the radiation are described, taking DNA as an exciplex laser system, where a stable state can be reached far from thermal equilibrium at threshold.     

Influence of fatty acids on energy metabolism. 2. Kinetics of changes in metabolic rates and changes in subcellular adenine nucleotide contents and pH gradients following addition of octanoate and oleate in perfused rat liver

The uncoupling-like effect of fatty acids [ Scholz , R., Schwabe , U., and Soboll , S. (1984) Eur. J. Biochem. 141, 223-230] was further substantiated in experiments with perfused rat livers by two ways: firstly the kinetics of changes in metabolic rates (oxygen consumption, ketogenesis, fatty acid oxidation) were analysed; secondly subcellular contents of adenine nucleotides and pH gradients across the mitochondrial membrane were determined following fractionation of freeze-fixed and dried tissues in non-aqueous solvents. The following results were obtained. The relaxation kinetics of the increase in oxygen consumption following fatty acid infusion revealed two components, a rapid one with a half-time around 10 s and a slow one with a half-time of more than 100 s. The rapid component was similar to the kinetics of fatty acid oxidation (ketogenesis and 14CO2 production from labelled fatty acids) whereas the half-time of the slow component was in the range of half-times observed with the increase in oxygen consumption following addition of carbonylcyanide p-trifluoromethoxyphenylhydrazone. In the presence of fatty acids, the cytosolic ATP concentrations and ATP/ADP ratios decreased, whereas the corresponding parameters for the mitochondrial space were either increased (oleate) or decreased (octanoate). The effects of oleate were dependent on the albumin concentrations in the perfusate. The normally large difference between cytosolic and mitochondrial ATP/ADP ratios became smaller. Similar observations were obtained with uncoupling agents. The pH gradient across the mitochondrial membrane as calculated from the subcellular distribution of 5,5 dimethyl[2-14C]oxazolidine-2,4-dione was inversed following the addition of both carbonylcyanide p-trifluoromethoxyphenylhydrazone and fatty acids, i.e. the mitochondrial matrix became more acidic than the cytosol. The pH gradient was not affected when oleate was added in the presence of high albumin concentrations. The data support the hypothesis that the increase in hepatic oxygen consumption due to octanoate or oleate is, in part, caused by a mechanism similar to uncoupling of oxidative phosphorylation. This mechanism seems not to be an artifact of isolated systems; it may be of physiological importance for processes in which reducing equivalents are removed independently of the ATP demand of the hepatocyte.     

Amino acid sequence of the phosphorylation site of isocitrate dehydrogenase fromEscherichia coli

InEscherichia coli, NADP⁺-specific isocitrate dehydrogenase (EC 1.1.1.42) may undergo a phosphorylation catalyzed by a cAMP-independent protein kinase, with a concomitant decrease in catalytic activity. In this report, we describe the purification and amino acid sequence of a³²P-labeled peptide obtained from in vivo³²P-labeled isocitrate dehydrogenase. The³²P-labeled peptide was isolated from a tryptic digest and found to contain seven amino acids, including a single serine residue. Following automated Edman degradation and reversephase high-pressure liquid chromatography of the phenylthiohydantoin-amino acids, the sequence of this peptide was established to be-Ser(P)-Leu-Asn-Val-Ala-Leu-Arg.     

Can Nocodazole,an Inhibitor of Microtubule Formation,Be Used to Synchronize Mammalian Cells?

Abstract Nocodazole, a temporary inhibitor of microtubule formation, has been used to partly synchronize Ehrlich ascites tumour cells growing in suspension. the gradual entry of cells into mitosis and into the next cell cycle without division during drug treatment has been studied by flow cytometric determination of mitotic cells, analysing red and green fluorescence after low pH treatment and acridine orange staining. Determination of the mitotic index (MI) by this method has been combined with DNA distribution analysis to measure cell-cycle phase durations in asynchronous populations growing in the presence of the drug. With synchronized cells, it was shown that in the concentration range 0.4–4.0 μg/l, cells could only be arrested in mitosis for about 7 hr and at 0.04 μg/ml, for about 5 hr. After these time intervals, the DNA content in nocodazole-blocked cells was found to be increased, and, in parallel, the ratio of red and green fluorescence was found to have changed, showing entry of cells into a next cell cycle without division (polyploidization). It was therefore only possible to partially synchronize an asynchronous population by nocodazole. However, a presynchronized population, e.g. selected G₁ cells or metabolically blocked G₁/S cells, were readily and without harmful effect resynchronized in M phase by a short treatment (0.4 μg/ml, 3–4 hr) with nocodazole; after removal of the drug, cells divided and progressed in a highly synchronized fashion through the next cell cycle.