Expression, characterization, and immunoreactivities of a soluble hepatitis E virus putative capsid protein species expressed in insect cells.

The hepatitis E virus (HEV) open reading frame-2 (ORF-2) is predicted to encode a 71-kDa putative capsid protein involved in virus particle formation. When insect Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus containing the entire ORF-2 sequence, two types of recombinant proteins were produced; an insoluble protein of 73 kDa and a soluble protein of 62 kDa. The 62-kDa species was shown to be a proteolytic cleavage product of the 73-kDa protein. N-terminal sequence analysis of the 62-kDa protein indicated that it lacked the first 111 amino acids that are present in the full-length 73-kDa protein. A soluble 62-kDa protein was produced without the proteolytic processing by inserting the coding sequence of amino acids 112 to 660 of ORF-2 in a baculovirus expression vector and using the corresponding virus to infect Sf9 cells. The two recombinant 62-kDa proteins made by different mechanisms displayed immunoreactivities very compatible to each other. The 62-kDa proteins obtained by both proteolytic processing and reengineering demonstrated much higher sensitivities in detecting anti-HEV antibodies in human sera than the antigens made from bacteria, as measured by enzyme-linked immunosorbent assay. The data suggest that the soluble 62-kDa protein made from insect cells contains additional epitopes not present in recombinant proteins made from bacteria. Therefore, the 62-kDa protein may be useful for HEV diagnostic improvement and vaccine development. The reengineered construct allows for the consistent large-scale production of the soluble 62-kDa protein without proteolytic processing.

Phospholipase C activity in NCB-20 cells is inhibited by protein kinase A-mediated phosphorylation of low molecular mass GTP-binding proteins.

We have previously shown that bradykinin-induced production of second messengers such as inositol trisphosphate and diacylglycerol in neurotumor cells is inhibited by raising cellular cyclic AMP levels, which in turn inhibit phospholipase C. A monoclonal antibody to phospholipase C-II immunoprecipitated the 140-kDa form of phospholipase C-II from [35S]methionine/[3H]eucine-labeled cells, but not [32P]orthophosphate-labeled phospholipase C-II, following treatment with either forskolin or dibutyryl cyclic AMP. This suggested that phospholipase C is not the target for cyclic AMP-dependent protein kinase-mediated phosphorylation. In vitro studies confirmed that phospholipase C activity was inhibited by raising cellular cAMP levels, and partial sensitivity to Bordetella pertussis toxin suggested the involvement of a GTP-binding protein which could be the target for protein kinase A. The involvement of a GTP-binding protein in coupling the bradykinin receptor to phospholipase C was further suggested by the ability of both guanosine 5'-O-(thio-triphosphate) and fluoride (NaF) to release inositol phosphates from NCB-20 cell membranes previously labeled with [3H]inositol. Both effects were blocked by pretreatment of the cells with protein kinase A activators, further suggesting a GTP-binding protein as the target for protein kinase A-mediated phosphorylation. When whole NCB-20 cell extracts were blotted onto nitrocellulose and incubated with [alpha- 32P]GTP, a major 24-kDa band plus minor bands at 22 and 20 kDa were revealed by autoradiography. A pH 3.0/6.0 soluble (basic protein) NCB-20 cell extract revealed the major 24-kDa band plus the 20-kDa band, and similar basic proteins were shown to be heavily phosphorylated following [32P]orthophosphate labeling and pretreatment with forskolin. The size and ability to bind GTP on Western blots are characteristic of the ras, rho, smg, etc. family of GTP-binding proteins recently suggested to be the much sought after GPLC (Lapetina, E.G., Lacal, J. C., Reep, B. R., and Molina y Vedia, L. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3131-3134; Wang, P., Nishihata, J., Takabori, E., Yamamoto, K., Toyoshima, S., and Osawa, T. (1989) J. Biochem. (Tokyo) 105, 461-466; Nagata, K.-I., Nagao, S., and Nozawa, Y. (1989) Biochem. Biophys. Res. Commun. 160, 235-242). We propose that GPLC is uniquely sensitive to protein kinase A-mediated phosphorylation and that phosphorylation inhibits stimulus-secretion coupling in these cells.

Rapid dephosphorylation of protein kinase C substrates by protein kinase A activators results from inhibition of diacylglycerol release.

The biochemical events encompassing the dephosphorylation of protein kinase C substrates by protein kinase A activators have been investigated in a neurotumor cell line, NCB-20. Treatment of [32P]orthophosphate-labeled cells with protein kinase A activators (e.g. forskolin, dibutyryl cAMP, prostaglandin E1) resulted in an inhibition of protein kinase C activity due to a failure of the protein kinase C complex to translocate into the membrane. Phospholipase C activity, as measured by the synchronous release of diacylglycerol and inositol phosphates (inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1-phosphate) in response to bradykinin, was inhibited up to 50% following exposure to protein kinase A activators. At the same time, phospholipase C-specific inositol phospholipid substrates (phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate) were found to accumulate in NCB-20 cells following treatment with protein kinase A activators. This suggests that phospholipase C may be altered through protein kinase A-mediated protein phosphorylation. Second messenger generation (inositol phosphates, diacylglycerol, and Ca2+) is therefore inhibited through cyclic AMP-mediated shutdown of the inositol lipid cycle at the level of phospholipase C.

Differential regulation of basic protein phosphorylation by calcium phospholipid and cyclic-AMP-dependent protein kinases.

Myelin basic protein, an 80-kilodalton (kDa) protein in rat oligodendrocytes, and an 80-kDa basic protein in neuroblastoma x neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl-acetylglycerol). TPA-stimulated phosphorylation was inhibited by pre-incubation with 50 microM psychosine (galactosyl-sphingosine), confirming that it is mediated through the phospholipid-dependent protein kinase C (PK-C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic-AMP-dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte-specific 2',3'-cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.: Proceedings of the National Academy of Sciences of the United States of America 85:939, 1988). The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol for PK-C activation is discussed.

Utilization of gyniatricians as primary health care providers for women.

The role, functions, activities, educational preparation and relationship with other health professionals of a new type of primary health care provider is described. The name gyniatrician has been applied to this kind of physician, who will be qualified to provide overall primary health care for women from the onset of puberty through adult life.

PIP: The role, functions, activities, educational preparation and relationship with other health professionals of a new type of primary health care provider for women -- a gyniatrician -- is described. The term gyniatrician is applied to this kind of physician who will be qualified to provide overall primary health care for women from the onset of puberty through adult life. Gyniatricians will have a wide range of functions and activities. They will assume responsibility for the provision, coordination, integration and management of the full range of women's health care and services. The care provided by gyniatricians will be based on a firm foundation of integrated knowledge from the medical, biologic, social, psychologic and behavioral sciences. The gyniatricians will be responsible for recognizing and evaluating their patients' total health needs and for teaching women how to enter the health care system. The gyniatrician's specialized education and training will be 5 years in duration, will begin during the 1st year in medical school, and will include a 1-year residence spent primarily in ambulatory settings. All 5 years of gyniatric training will include intensive study of all aspects of primary health care and services for women. Students desiring to become gyniatricians will be selected from those candidates for admission to medical school who have demonstrated special motivation and interest in primary health care for women. A table of the functions and activities of the gyniatrician is included along with a curriculum guide. Gyniatricians will work with obstetrician-gynecologists in giving the improved total health care that women are requesting. As a result of women's increasing awareness that the health care system is failing to provide what they need and want, women are requesting more pertinent, comprehensive, accessible and competent professional health care.

The school nurse practitioner: providing improved health care to children.

School nurse practitioners have the competence to participate in providing a broad range of direct primary health care and services to the school-age child. Greater utilization of these specially prepared and highly qualified nurse practitioners as providers of health care in the school is a practical and effective method of adding to the quality, availability, and continuity of health care for the school-age child.