Deduced sequence of the bovine coronavirus spike protein and identification of the internal proteolytic cleavage site

The sequence of the spike (also called peplomer or E2) protein gene of the Mebus strain of bovine coronavirus (BCV) was obtained from cDNA clones of genomic RNA. The gene sequence predicts a 150,825 mol wt apoprotein of 1363 amino acids having an N-terminal hydrophobic signal sequence of 17 amino acids, 19 potential N-linked glycosylation sites, a hydrophobic anchor sequence of approximately 17 amino acids near the C terminus, and a hydrophilic cysteine-rich C terminus of 35 amino acids. An internal Lys-Arg-Arg-Ser-Arg-Arg sequence predicts a protease cleavage site between amino acids 768 and 769 that would separate the S apoprotein into S1 and S2 segments of 85690 and 65153 mol wt, respectively. Amino terminal amino acid sequencing of the virion-derived gp 100 spike subunit confirmed the location of the predicted cleavage site, and established that gp 120 and gp 100 are the glycosylated virion forms of the S1 and S2 subunits, respectively. Sequence comparisons between BCV and the antigenically related mouse hepatitis coronavirus revealed more sequence divergence in the putative knob region of the spike protein (S1) than in the stem region (S2).     

A history of Combined-Integrated doctoral training in psychology

The history of doctoral training in psychology is reviewed, with special emphasis on the development of training models for applied psychology. Among applied psychology doctoral programs, most label themselves as being one of the three currently recognized practice areas: clinical, counseling, and school psychology; however, since the 1970s, APA also has accredited "combined" programs. The history of the development of the combined model is traced, including information from the programs themselves about the manner in which each evolved. Recently, new interest in the combined category -or Combined-Integrated (C-I) as these programs wish to become known-has emerged, and an organization representing these programs has formed. The C-I model is described, and the potential that the model may serve as a template for the future of professional training in psychology is discussed.     

Cytological diagnosis of nonulcerative penile neoplasms: report of two cases

Nonulcerative penile mass lesions are rare. Pathological diagnosis of these lesions would traditionally be a biopsy. We report two such primary penile lesions which were diagnosed by fine-needle aspiration cytology (FNAC). Both lesions were present in the shaft and were diagnosed as squamous cell carcinoma (SCC). The first patient had a recurrence on the penile stump of partial amputation without any ulceration. The second had a primary urethral carcinoma on the terminal penile shaft infiltrating the corpora cavernosa dorsally. Open biopsies were avoided in both cases. FNAC was associated with very little and tolerable discomfort. There were no complications. The aspirate yield was sufficient for cytological diagnosis. FNAC of nonulcerated penile lesions is safe, well tolerated, and capable of providing a cytological diagnosis. Hence, it is a very useful outpatient procedure and could be the procedure of choice for diagnosis.     

Polymorphic MHC ancestral haplotypes affect the activity of tumour necrosis factor-alpha.

It remains unclear which MHC loci are involved in susceptibility to autoimmune diseases and immune deficiencies. We have chosen to evaluate whether different alleles of tumour necrosis factor-alpha (TNF-alpha) are important, as TNF has been implicated in the etiology of many immunological disorders. We have shown previously that a restriction fragment length polymorphism in the TNF region correlates with MHC ancestral haplotypes. We therefore examined the effect of ancestral haplotype on the activity of TNF-alpha in culture supernatants of lymphoblastoid cell lines. The results demonstrate that TNF-alpha activity in supernatants of 8.1 (A1, B8, DR3) cell lines was higher than that present in the supernatants from cells homozygous for eight different MHC ancestral haplotypes, and indicate that polymorphisms in TNF-alpha, or in other MHC genes that regulate TNF, may be responsible for the 8.1 phenotype.     

Generation of functionally active suppressor cells by haemorrhage and haemorrhagic serum.

Mitogen (PHA)-induced proliferation of peripheral blood mononuclear cells (PBMC) was reduced by more than 70% 2 h after the haemorrhage of 30% of blood volume. Experiments using isolated macrophages and lymphocytes showed that post-haemorrhage macrophages were functionally normal and that lymphocytes were responsible for the observed haemorrhage-induced depression of proliferative response. Surface marker determinations showed that at least some, if not all, of the haemorrhage-induced suppressor cells are of the OX8+ phenotype. Exposure of PBMCs to serum from bled animals also brought about activation of OX8+ suppressor T cells. These results suggest that the depressed proliferative response of PBMCs induced by haemorrhage or by exposing the cells to haemorrhagic serum (serum from bled animals) is due to the activation of OX8+ suppressor T cells.     

Epitope definition by proteomic similarity analysis: identification of the linear determinant of the anti-Dsg3 MAb 5H10

Cell-based bioassays have been suggested for screening of hormones and drug bioactivities. They are a plausible alternative to animal based methods. The technique used is called receptor/reporter system. Receptor/reporter system was initially developed as a research technique to understand gene function. Often reporter constructs containing viral promoters were used because they could be expressed with very 'high' magnitude in a variety of cell types in the laboratory. On the other hand mammalian genes are expressed in a cell/tissue specific manner, which makes them (i.e. cells/tissues) specialized for specific function in vivo. Therefore, if the receptor/reporter system is to be used as a cell-based screen for testing of hormones and drugs for human therapy then the choice of cell line as well as the promoter in the reporter module is of prime importance so as to get a realistic measure of the bioactivities of 'test' compounds. We evaluated two conventionally used viral promoters and a natural mammalian promoter, regulated by steroid hormone progesterone, in a cell-based receptor/reporter system. The promoters were spliced into vectors expressing enzyme CAT (chloramphenicol acetyl transferase), which served as a reporter of their magnitudes and consistencies in controlling gene expressions. They were introduced into breast cell lines T47D and MCF-7, which served as a cell-based source of progesterone receptors. The yardstick of their reliability was highest magnitude as well as consistency in CAT expression on induction by sequential doses of progesterone. All the promoters responded to induction by progesterone doses ranging from 10^-12 to 10^-6 molar by expressing CAT enzyme, albeit with varying magnitudes and consistencies. The natural mammalian promoter showed the most coherence in magnitude as well as dose dependent expression profile in both the cell lines. Our study casts doubts on use of viral promoters in a cell-based bioassay for measuring bioactivities of drugs and hormones for human therapy and suggests caution regardingtranslation in toto, of a research technique as a cell-based bioassay for drug screening.     

Group I mGluRs increase excitability of hippocampal CA1 pyramidal neurons by a PLC-independent mechanism

Previous studies have implicated phospholipase C (PLC)-linked Group I metabotropic glutamate receptors (mGluRs) in regulating the excitability of hippocampal CA1 pyramidal neurons. We used intracellular recordings from rat hippocampal slices and specific antagonists to examine in more detail the mGluR receptor subtypes and signal transduction mechanisms underlying this effect. Application of the Group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) suppressed slow- and medium-duration afterhyperpolarizations (s- and mAHP) and caused a consequent increase in cell excitability as well as a depolarization of the membrane and an increase in input resistance. Interestingly, with the exception of the suppression of the mAHP, these effects were persistent, and in the case of the sAHP lasting for more than 1 h of drug washout. Preincubation with the specific mGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), reduced but did not completely prevent the effects of DHPG. However, preincubation with both MPEP and the mGluR1 antagonist LY367385 completely prevented the DHPG-induced changes. These results demonstrate that the DHPG-induced changes are mediated partly by mGluR5 and partly by mGluR1. Because Group I mGluRs are linked to PLC via G-protein activation, we also investigated pathways downstream of PLC activation, using chelerythrine and cyclopiazonic acid to block protein kinase C (PKC) and inositol 1,4,5-trisphosphate-(IP(3))-activated Ca(2+) stores, respectively. Neither inhibitor affected the DHPG-induced suppression of the sAHP or the increase in excitability nor did an inhibitor of PLC itself, U-73122. Taken together, these results argue that in CA1 pyramidal cells in the adult rat, DHPG activates mGluRs of both the mGluR5 and mGluR1 subtypes, causing a long-lasting suppression of the sAHP and a consequent persistent increase in excitability via a PLC-, PKC-, and IP(3)-independent transduction pathway.     

Adherence of streptococcus pyogenes, Escherichia coli, and Pseudomonas aeruginosa to fibronectin-coated and uncoated epithelial cells.

The relationship between the variability in the fibronectin (Fn) content on human buccal epithelial cells and the capacity of the cells to bind gram-positive (Streptococcus pyogenes) or gram-negative (Escherichia coli or Pseudomonas aeruginosa) bacteria was investigated. Adhesion experiments performed with mixtures of epithelial cells and mixed suspensions of either S. pyogenes and E. coli or S. pyogenes and P. aeruginosa exhibited three major populations of buccal cells: one of these was able to bind S. pyogenes (gram positive) but neither of the gram-negative bacteria; a second population was able to bind the gram-negative but not the gram-positive bacteria; and a third was able to bind various numbers of both types of organisms. Further adhesion experiments performed with a mixture of epithelial cells and a mixed suspension of S. pyrogens, E. coli, and fluoresceinconjugated methacrylate beads coated with immune immunoglobulin G directed against Fn revealed that the epithelial cells recognizing the gram-positive bacteria were rich in Fn, whereas those recognizing the gram-negative organisms were poor in Fn. Immunoelectron microscopy confirmed that cells of S. pyogenes bound to epithelial cells coated with Fn, whereas cells of E. coli bound to epithelial cells lacking Fn. These results suggest that Fn on the surfaces of epithelial cells may modulate the ecology of the human oropharyngeal cavity, especially with respect to the colonization of these surfaces by pathogenic gram-negative or gram-positive bacteria.     

Solubility properties in polymers and biological media. 2. The correlation and prediction of the solubilities of nonelectrolytes in biological tissues and fluids

Solubilities of a range of nonelectrolyte solutes in biological systems, such as blood, plasma, brain, lung, liver, kidney, muscle tissue, and human fat, are correlated and predicted through an equation that takes the form log Ltissue = c + w log Lwater + o log Loil, where L is the Ostwald solubility coefficient (or gas/liquid partition coefficient). The ratio of the constants o and w gives a measure of the "oiliness" of a given biological tissue or fluid. The strong possibility exists that, for many types of nonelectrolyte solutes, simple measurements of solubilities in water and oil (gas/liquid partition coefficients) will allow accurate predictions of solubilities in the above biological solvents, as well as tissue/blood partition coefficients. The solubility of rare gases and the inorganic gases H2, N2, CO, and O2 may be correlated through the simpler equation log Ltissue = l'RG + d', where l' and d' are constants that characterize the phase, and RG is a known parameter, obtained by normalizing and averaging solubilities over a range of solvent systems, that characterizes the solute. Both of the above equations allow prediction of L in biological solvents to within about 20%, which compares well with the precision of the experimental measurements.