An investigation of the molecular properties and stability of intermediates of proenkephalin in isolated bovine adrenal medullary chromaffin granules

An antiserum to a synthetic peptide corresponding to residues 95-117 of bovine proenkephalin recognizes all the major intermediates of this prohormone in bovine adrenal medulla (Birch, N. P. and Christie, D. L. (1986) J. Biol. Chem. 261, 12213-12221). This antiserum enabled an investigation of the stability and molecular properties of intermediates in the processing of proenkephalin in bovine adrenal medullary chromaffin granules. Intact and hypotonic lysates of chromaffin granules were incubated at 37 degrees C and the stability of intermediates assessed by gel filtration followed by radioimmunoassay and gel electrophoresis in combination with immunoblotting. Processing was slow in intact granules compared with incubations of hypotonic lysates which resulted in the selective cleavage of an Mr 27,000 intermediate and increases in the amounts of immunoreactivity of lower molecular weight. Protease inhibitors increased the stability of the 27-kilodalton intermediate, the most effective being p-chloromercuribenzoate. Preliminary evidence was obtained for the regulation of the processing of this intermediate by soluble factors present in chromaffin granules. It appears that membrane-associated intermediates of proenkephalin are relatively stable, although analysis of soluble immunoreactivity released during the incubation of chromaffin granule membranes showed a decrease in the 27-kilodalton intermediate and increased amounts of lower molecular weight intermediates. Analysis of hypotonic lysates by two-dimensional gel-electrophoresis showed that proenkephalin intermediates exhibit significant microheterogeneity. It will be important to compare the products of proenkephalin generated by purified proteases with a putative role in the processing of this prohormone with the properties of endogenous intermediates as revealed in this study.     

Beta-endorphin. Biological activity of analogs containing dermorphin and dynorphin sequences: ileum and vas deferens assays

Biological activity of synthetic beta-endorphin (beta-EP) analogs containing dermorphin or dynorphin-A-(1-13) structure has been investigated using the guinea pig ileum and the vas deferens of the mouse, rat and rabbit. Replacement of NH2-terminal 1-7 segment of camel beta-EP [beta c-EP-(1-7)] with dermorphin caused a great increase in opiate potency of the analog. [Dermorphin (1-7)]-beta c-EP was 120 times more potent than beta c-EP in the guinea pig ileum assay, 49 times more potent in the mouse vas deferens assay; and only 4 times more potent in the rat vas deferens assay. Replacement of NH2-terminal 1-13 segment of human beta-EP [beta h-EP-(1-13)] with dynorphin-A-(1-13) caused an increase in opiate potency in both the guinea pig ileum and rabbit vas deferens assays, a complete loss of potency in the rat vas deferens assay, and no change in the mouse vas deferens assay. In comparison with dynorphin-A-(1-13), the hybrid peptide was less potent in the guinea pig ileum assay as well as in mouse and rabbit vas deferens assay. It is suggested that beta c-EP-(8-31) facilitates the dermorphin moiety to act on opiate mu and delta receptors but not on the epsilon receptor, while beta h-(14-31) reduces the action of dynorphin on mu, delta and kappa receptors.     

Bone marrow-derived macrophage expression of endogenous and transfected class II MHC genes during differentiation in vitro

C57BL/6 (H-2b) mice fail to express I-E molecules on the surface of their cells and thus are unable to respond to I-E-restricted antigens such as GL phi and cytochrome c. Previous experiments in our laboratory have involved developing a system for studying differentiation of bone marrow cells into mature macrophage to gain a better understanding of class II MHC gene expression and function. In this study, we have used this system to transfect the E alpha d gene (cosmid 17.2) into C57BL/6 bone marrow cells and subsequently observed I-E expression on bone marrow-derived macrophages (BMDM) after differentiation in vitro. By using a modified calcium phosphate protocol, we found that the optimal period for transfection of the bone marrow cells was after 2 days of culture in vitro. By using the anti-I-E monoclonal antibody (Ia.7) derived from hybridoma 14-4-4, we detected the I-E molecule on the surface of transfected macrophages by a radiobinding assay and immunoprecipitation. BMDM expressed the I-E product maximally at 5 days of differentiation, and expression then declined. Furthermore, we have found that the expression of the I-E molecule on transfected macrophage was dependent upon exposure to interferon-gamma. Expression of I-E molecules was also detected by the generation of an allogeneic response. Transfected BMDM were compared with (CB6)F1 BMDM for their ability to stimulate C57BL/6 T cells and they were found to be equally effective. By using these initial findings, we hope to further optimize the conditions for insertion and expression of class II MHC genes in bone marrow cells.     

Correlation of titers of antibodies to the measles virus by an immunoenzyme method and in the hemagglutination inhibition reaction

The authors analyze the results of comparative studies on 15 paired sera from children with suspected measles, of 32 sera from children and adolescents aged 1.5 to 16 immunized against measles, and of 21 sera from adults aged 19 to 86 with a history of the disease. EIA proved to be more sensitive than HAIT: the detection rate of positive sera was higher, as were the titers of antibodies detected by it, in examinations of the sera from vaccinated children and the adults. Analysis of the distribution of sera with different titers of antibody to measles virus in EIA and HAIT has revealed a correlation between the titers in the sera with high antibody levels. In the cases with low antihemagglutinin titers, no correlation between the titers determined in the two tests has been observed.     

Immune system function in candidiasis patients

To compare the clinical picture and the immunological characteristics, 58 candidiasis patients differing by the severity and dissemination of the disease were examined. Chronic candidiasis of the skin and mucous membranes, the most severe and disseminated form of the disease, is associated with a decrease in the number of T-lymphocytes and changes in their subpopulations, as well as high titers of Candida albicans antigen and antibodies to it in blood sera. The immune system of patients with visceral candidiasis and chronic vulvovaginal candidiasis was similar to that of healthy persons in the characteristics under study. Immediate and mixed hypersensitivity occurred in candidiasis patients more frequently than in healthy persons. In extremely severe forms of Candida infection immediate hypersensitivity prevailed.     

Studies on purine turnover in the camel (Camelus dromedarius) and zebu (Bos indicus)

Significantly higher hypoxanthine over uric acid ratios were found in camel plasma and urine, with respect to those of zebu. Enzyme levels of purine catabolism were markedly lower in camel than in zebu liver. Oxidation of hypoxanthine appears to be the limiting step of purine metabolism in camel liver. Any hepatic hypoxanthine appears to be actively converted into IMP in camel liver, rather than oxidized to uric acid.     

Olfactory rhythm in the electrical activity of the human amygdaloid nucleus

Summated electrical activity of the human amygdaloid nucleus was investigated in the neurosurgical clinic by chronically implanted electrodes. It was found that odoriferous stimulation of this structure produced bursts of rapid rhythm (20–30 cps, 30–50 µV). The quasisinusoidal waves of olfactory rhythm consist of sinusoidal components which are more pronounced within the 20–30-Hz frequency range. Spindling of 1–3 sec duration occurs at the end of inhalation and the beginning of exhalation in time with breathing. During monorhinal breathing this activity, whose amplitude depends on degree of olfactory stimulation, can only be recorded ipsilaterally. Room air also activates the amygdaloid nucleus, but less strongly than odoriferous substances: No characteristic odor-dependent differences were discovered in the frequency range of the olfactory rhythm within a 20–30-Hz band.Institute of Physiology, Kiev State University, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 1, pp. 61–69, January–February, 1986.     

Bioluminescent immunosorbent for rapid immunoassays

We describe a bioluminescent immunoassay procedure which does not require a separation step to remove excess free label. A luminescent immunosorbent constituted of bacterial luciferase, FMN oxidoreductase, and an antibody coimmobilized on Sepharose is used to determine specifically the label enzyme (glucose-6-phosphate dehydrogenase, coupled to an antigen) bound by a specific antibody. The immunosorbent confines the bioluminescent reaction in a small volume, and the bound label produces NADH, which is directly used by the nearby luciferase FMN oxidoreductase enzyme system. On the contrary NADH produced by dehydrogenases in solution is directly oxidized without emitting light. Dehydrogenases contained in the biological sample do not interfere with the assay, which can be performed directly on 25 microliter of serum. In this paper we describe the general procedure and we analyze the different parameters that must be optimized.     

Etiology of acute bronchitis in adults

A total of 126 adult patients with acute bronchitis were subjected to microbiological, virological and immunological examination. Influenza and other acute respiratory viral infections were detected in 104 (82.5%) cases of acute bronchitis. The results of microbiological examination and the study of local and systemic immunity showed that in 94.4 +/- 2.6% of the patients the development of acute bronchitis was linked with the appearance of acute pneumococcal infection irrespective of the presence, or absence, of virologically and serologically confirmed acute viral or mycoplasmal infections.