Three distinct H-2Ks molecules differing at the carboxy terminus are expressed on a tumor from SJL/J mice

A dimethylbenzanthracene-induced leukemia of H-2s origin expressed at least two class I molecules on the cell surface that were precipitated by anti-H-2.19, an alloantiserum prepared against the private H-2Ks specificity. Mapping studies in recombinant inbred strains along with comparisons of tryptic peptide maps and N-terminal sequences indicated that the proteins were virtually identical and probably encoded by the same class I gene. When cells were labeled in the presence of tunicamycin, the proteins precipitated by anti-H-2.19 were further resolved into three distinct peptides. Experiments were performed to determine which of these various proteins were phosphorylated and which were recognized by an anti-synthetic peptide serum directed against the ultimate C-terminus of H-2K class I molecules. The results indicate that a single class I gene from the H-2Ks region may encode three class I molecules that differ only at the C-terminus due to alternative splicing of pre-mRNA.     

Transforming growth factor beta is an important immunomodulatory protein for human B lymphocytes

The growth and differentiation of B cells to immunoglobulin (Ig)-secreting cells is regulated by a variety of soluble factors. This study presents data that support a role for transforming growth factor (TGF)-beta in this regulatory process. B lymphocytes were shown to have high-affinity receptors for TGF-beta that were increased fivefold to sixfold after in vitro activation. The addition of picogram quantities of TGF-beta to B cell cultures suppressed factor-dependent, interleukin 2 (IL 2) B cell proliferation and markedly suppressed factor-dependent (IL 2 or B cell differentiation factor) B cell Ig secretion. In contrast, the constitutive IgG production by an Epstein Barr virus-transformed B cell line was not modified by the presence of TGF-beta in culture. This cell line was found to lack high-affinity TGF-beta receptors. The degree of inhibition of B cell proliferation observed in in vitro cultures was found to be dependent not only on the concentration of TGF-beta added but also on the concentration of the growth stimulatory substance (IL 2) present. By increasing the IL 2 concentrations in culture, the inhibition of proliferation induced by TGF-beta could be partially overcome. In contrast, the inhibition of Ig secretion induced by TGF-beta could not be overcome by a higher concentration of stimulatory factor, demonstrating that the suppression of B cell differentiation by TGF-beta is not due solely to its effects on proliferation. Furthermore, it was demonstrated that B lymphocytes secrete TGF-beta. Unactivated tonsillar B cells had detectable amounts of TGF-beta mRNA on Northern blot analysis, and B cell activation with Staphylococcus aureus Cowan (SAC) resulted in a twofold to threefold increase in TGF-beta mRNA. Supernatants conditioned by unactivated B cells had small amounts of TGF-beta, SAC activation of the B cells resulted in a sixfold to sevenfold increase in the amount of TGF-beta present in the supernatants. Thus, B lymphocytes synthesize and secrete TGF-beta and express receptors for TGF-beta. The addition of exogenous TGF-beta to cultures of stimulated B cells inhibits subsequent proliferation and Ig secretion. TGF-beta may function as an autocrine growth inhibitor that limits B lymphocyte proliferation and ultimate differentiation.     

Synchronized synthesis and intracellular transport of serum albumin and apolipoprotein B in cultured rat hepatocytes as studied by double immunofluorescence

Synthesis and intracellular transport of two secretory proteins, serum albumin (SA) and apolipoprotein B (apo B) have been synchronized in primary cultures of normal rat hepatocytes to make possible immunocytochemical study of the transport pathway. Under appropriate conditions of cycloheximide treatment, synthesis of new protein was inhibited and, by double immunofluorescent labeling, the cells were found to be largely depleted of the SA and apo B previously synthesized. Re-initiation of protein synthesis led to sequential appearance of SA and apo B, first in the endoplasmic reticulum, then in the Golgi complex, and finally at the cell surface. These results indicate that it should be feasible to use this cell system for high-resolution investigation of the sequence of structures involved in intracellular transport of SA and apo B by corresponding immunolabeling experiments as observed by electron microscopy.     

The immune response towards beta-adrenergic ligands and their receptors. VI. Idiotypy of monoclonal anti-alprenolol antibodies

Four murine monoclonal antibodies specific for alprenolol, a synthetic beta-adrenergic ligand, with different binding properties towards alprenolol and other beta-adrenergic antagonists and agonists (as described in a previous report) were used to induce anti-idiotypic responses in rabbits and mice. Three of the rabbit anti-idiotypes inhibited, and one increased the binding of tritiated dihydroalprenolol to the Ab1 against which they were induced. The syngeneic mouse anti-idiotypes all had an inhibitory effect on the ligand binding to their corresponding Ab1. Cross-reactivity tests of the xenogeneic and syngeneic anti-idiotypes were positive only for two monoclonal anti-alprenolol antibodies. Cross-reaction could be shown neither on a panel of 15 other monoclonals, nor on polyclonal anti-alprenolol antibodies of the BALB/c and the C57BL/10 mice. These results suggest that the immune response against alprenolol results in antibodies with mostly private idiotypic determinants. Moreover, the properties of the anti-idiotypic response against the same monoclonal antibody seem to be different according to the species used for anti-idiotypic induction.     

Arbovirological survey in Silica plateau area, Roznava District, Czechoslovakia

The serosurveys conducted in the Silica plateau area of the Slovak karst region revealed the presence of specific neutralizing antibody against tick-borne encephalitis (TBE) virus in 18% of local inhabitants (33 examined, mostly goats and sheep farmers), 54% of goats (26 examined), 18% of sheep (120 examined) and 13% of cattle (60 examined), against Lipovník (LIP) virus in 30% of inhabitants, 88% of goats, 55% of sheep and 45% of cattle, and against Bhanja (BHA) virus in 27% of inhabitants, 46% of goats, 29% of sheep and 23% of cattle. The results of hemagglutination-inhibition tests with TBE and BHA antigens were analogous. A detailed analysis of these serologic data points to a recent enhancement of the circulation of LIP and BHA viruses and to a very low TBE virus activity in this natural focus of arboviral infections. The immunological surveys of the 32 former "Roznava disease" patients, conducted 25 years after an extensive epidemic of a TBE virus infection that originated in Roznava in 1951, revealed the presence of neutralizing (and also hemagglutination-inhibiting) antibodies against TBE virus in as many as 78% of cases. Antibodies against LIP and BHA viruses were also detectable in the sera of 16% and 9%, respectively, of these individuals. Populations of the ectoparasites examined for the presence of arbovirus comprised 231 Ixodes ricinus, 806 Dermacentor marginatus and 204 Haemaphysalis punctata ticks and 117 specimens of the louse-flies Melophagus ovinus. Two strains of arbivirus that were antigenically related to Lipovník and Tribec viruses belonging to a group of Kemerovo viruses were isolated from male and female I. ricinus ticks collected from cattle.     

Effect of gamma radiation on resting B lymphocytes. I. Oxygen-dependent damage to the plasma membrane results in increased permeability and cell enlargement

Although the susceptibility of resting B lymphocytes to radiation-induced interphase death is well known, the mechanism by which this occurs is not understood. In this report, we use three measures of plasma membrane integrity (increase in cell volume, uptake of trypan blue, and release of 51Cr) to assess the effect of radiation on the resting B cell plasma membrane. The delivery of 500 to 1000 rad caused the majority of resting B cells to enlarge slightly, whereas 3000 rad caused virtually all of the cells to approximately double in size within 3 to 4 hr. Measurement of the release of 51Cr from resting B cells revealed a similar relationship between the dose of radiation and the loss of radioactive label. Trypan blue exclusion was also found to diminish as a function of radiation dose. An analysis of a variety of lymphoid cells suggested that sensitivity to the membrane damaging effects of gamma radiation was in the order of resting B cells greater than resting T cells greater than a long-term L3T4+ T cell clone greater than a B cell lymphoma. LPS-induced B cell blasts treated with 3000 rad were equivalent to 1000 rad-treated resting B cells. The effects of the gamma radiation could be ameliorated by excluding oxygen (a diradical molecule that can potentially enhance the generation and propagation of highly reactive free radicals) at the time of irradiation, or by adding the free radical scavenging agent cysteamine. These data are compatible with the hypothesis that gamma radiation results in damage to the plasma membrane of resting lymphocytes via the generation of highly reactive free radical species. This damage is reflected in a rapid increase in plasma membrane permeability and swelling of the cells, and may play a major role in causing interphase death.     

Isolation and characterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-gamma

By using a preparation of inactivated rabies virus, the blood mononuclear cells from five rabies vaccine recipients were stimulated in vitro in the presence of interleukin 2. T cell lines that displayed significant proliferative responses to whole rabies virus and to preparations of rabies glycoprotein and nucleocapsid were obtained from all the individuals. Other antigens, such as diphtheria and tetanus toxoids, influenza A virus, hepatitis B surface antigen, and serum albumin, failed to induce the proliferation of the T cell lines. One of these rabies-specific T cell lines was found to proliferate in response to rabies antigens only when the antigen-presenting cells expressed homologous HLA-DR antigens. The use of mouse monoclonal antibodies specific for human T cell surface markers revealed that most of the cells of these rabies-reactive lines were of the helper/inducer class of T lymphocytes. Stimulation of the T cell lines with the rabies antigens induced the production of interferon-gamma, a lymphokine with potent antiviral activity. Several T cell clones were isolated from two of these cell lines, and most of them appeared to be specific for the antigenic components of the viral nucleocapsid. Two T cell clones specific for the rabies glycoprotein were also isolated from one of these lymphocyte interleukin 2-dependent lines. Further in vitro studies with rabies-specific T cells could help us to understand in more depth the role of regulatory T cells in the human immune response to rabies virus.     

Effect of serotonin on murine macrophages: suppression of Ia expression by serotonin and its reversal by 5-HT2 serotonergic receptor antagonists

Serotonin (5-HT), a mediator released from platelets at sites of inflammation, suppressed IFN-gamma-induced Ia expression in mouse bone marrow macrophages maintained in vitro. (Mean percent suppression = 63.9% +/- 9.2, n = 40.) This suppression was not toxic or endotoxin-related, was concentration-dependent, and occurred at the physiologic concentrations of 5-HT present at inflammatory sites. The concentration of 5-HT producing the half-maximal effect was 2.5 to 5.5 X 10(-8) M. Related compounds, dopamine, histamine, and tryptamine, were much less potent in suppressing IFN-gamma-induced Ia, with maximally suppressing concentrations more than 100-fold higher than the maximally suppressing 5-HT concentration. L-5-hydroxytryptophan (5-HTP), the most potent analog tested, was 10-fold less potent than 5-HT in suppressing Ia expression. The concentration of 5-HTP producing the half-maximal effect = 4 X 10(-7) M. 5-HT suppression of IFN-gamma-induced Ia expression was antagonized by the 5-HT2 type receptor antagonists spiperone, ketanserin, and LY53857. Concentrations of these agents resulting in 50% inhibition of the serotonin effect were 1.5 X 10(-8) M, 7.5 X 10(-8) M, and 4.5 X 10(-12) M, respectively. 5-HT was most effective in suppressing IFN-gamma-induced Ia when added early in culture simultaneously with IFN-gamma. These data provide functional evidence that 5-HT suppression of IFN-gamma-induced Ia expression is mediated through a 5-HT receptor with some characteristics of the 5-HT2 type. 5-HT may play a physiologic role at sites of inflammation as a modulator of the effects of IFN-gamma on macrophage function.     

Investigation of the tertiary folding of Escherichia coli ribosomal RNA by intra-RNA cross-linking in vivo

"In vivo" cross-links were introduced into ribosomal RNA by direct ultraviolet irradiation of intact Escherichia coli cells, during growth in a 32P-labelled medium. Ribosomes were isolated from the irradiated cultures, dissociated into subunits and subjected to partial digestion with cobra venom nuclease. The intra-RNA cross-linked fragments were separated by two-dimensional gel electrophoresis and the sites of cross-linking determined, using our published methodology. A comparison with the data previously obtained by this procedure, after irradiation of isolated 30 S and 50 S subunits, showed that in the case of the 50 S subunit nine out of the ten previous cross-links in the 23 S RNA could be identified in the "in vivo" experiments, and correspondingly in the 30 S subunit five out of the six previous cross-links in the 16 S RNA were identified. Some new cross-links were found, as well as two cross-links in the 16 S RNA, which had hitherto only been observed after partial digestion of irradiated 30 S subunits with ribonuclease T1. The relevance of these data to the tertiary folding of the rRNA in situ is discussed, with particular reference to the work of other authors, in which "naked" RNA was used as the substrate for cross-linking and model-building studies.     

Crystal structure determination, refinement and the molecular model of the alpha-amylase inhibitor Hoe-467A

The crystal and molecular structure of the alpha-amylase inhibitor Hoe-467A has been determined and refined at high resolution. The polypeptide chain is folded in two triple-stranded sheets, which form a barrel. The topology of folding is as found in the immunoglobulin domains. The amino acid triplet Trp18-Arg19-Tyr20 has an exceptional conformation and position in the molecule and is possibly involved in inhibitory activity.