Pig-n, a mammalian homologue of yeast Mcd4p, is involved in transferring phosphoethanolamine to the first mannose of the glycosylphosphatidylinositol.

Many cell surface proteins are anchored to the membrane via a glycosylphosphatidylinositol (GPI) moiety, which is attached to the C terminus of the proteins. The core of the GPI anchor is conserved in all eukaryotes but is modified by various side chains. We cloned a mouse phosphatidylinositol glycan-class N (Pig-n) gene that encodes a 931amino acid protein expressed in the endoplasmic reticulum, which is homologous to yeast Mcd4p. We disrupted the gene in F9 embryonal carcinoma cells. In the Pig-n knockout cells, the first mannose in the GPI precursors was not modified by phosphoethanolamine. Nevertheless, further biosynthetic steps continued with the addition of the third mannose and the terminal phosphoethanolamine. The surface expression of Thy-1 was only partially affected, indicating that modification of the first mannose by phosphoethanolamine is not essential for attachment of GPI anchors in mammalian cells. An inhibitor of GPI biosynthesis, YW3548/BE49385A, inhibited transfer of phosphoethanolamine to the first mannose in mammalian cells but only slightly affected the surface expression of GPI-anchored proteins. Biosynthesis of GPI in the Pig-n knockout cells was not affected by YW3548/BE49385A, and yeast overexpressing MCD4 was highly resistant to YW3548/BE49385A, suggesting that Pig-n and Mcd4p are targets of this drug.

Effectiveness of cholecystokinin-stimulated cholescintigraphy in the diagnosis and treatment of acalculous gallbladder disease.

We retrospectively reviewed the medical records of 107 patients in two community hospitals who had undergone cholecystokinin-stimulated cholescintigraphy with ejection fraction to determine whether this test is reliable in identifying patients whose symptoms will improve following cholecystectomy. Patients with cholelithiasis or incomplete medical records and patients who could not be interviewed were excluded from the study. Forty-two of 58 study patients (72%) had an abnormal ejection fraction (defined as 35% or less); 27 of 42 patients (64%) underwent cholecystectomy. Twenty-six of 27 (96%) reported lessening of or resolution of symptoms following cholecystectomy. Sixty-seven per cent of the surgical specimens from the 27 patients demonstrated chronic cholecystitis. Fifteen of 42 patients (36%) with abnormal ejection fractions did not undergo cholecystectomy; 12 of 15 (80%) also reported lessening or resolution of symptoms. Of the 16 of 58 patients with a normal ejection fraction, 2 underwent cholecystectomy and reported resolution of symptoms. Five of 14 (36%) with normal ejection fractions who did not undergo cholecystectomy reported improvement. In this series, most patients with an abnormal ejection fraction had lessening of symptoms regardless of whether they underwent cholecystectomy.

Bilateral renal artery occlusion: an unusual presentation of atrial fibrillation and hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy predisposes patients to atrial fibrillation and the development of systemic embolization. We describe a rare case of bilateral renal artery thrombosis which presented as acute renal failure requiring dialysis. The patient was successfully treated with a selective, continuous infusion of urokinase which resulted in the return of adequate renal function.

Men and their bodies: a comparison of homosexual and heterosexual men.

The study examined influences on body satisfaction, disordered eating, and exercise behavior of a male subculture that places a heightened emphasis on appearance: the homosexual male subculture. Subjects were 71 homosexual and 71 heterosexual men. Relative to heterosexual men, homosexual men showed more body dissatisfaction and considered appearance more central to their sense of self. Also, their exercise was more motivated by a desire to improve attractiveness. Among the homosexual but not the heterosexual group, men who desired to be thinner showed more attitudes and behaviors associated with disordered eating than men who were thinner than their desired size. In contrast, heterosexual but not homosexual men who wished to be heavier had lower self-esteem scores than men who were heavier than or equal to their desired size. The findings support a view that a male subculture that emphasizes appearance may heighten the vulnerability of its members to body dissatisfaction and disordered eating.

Alcohol and masculinity.

Alcohol use--and abuse--has always been more prevalent among males than among females. The sex role prescription for men to affirm their masculinity by drinking is a major determinant of this sex difference. This paper reviews the intricate interrelationship between masculinity and both social and alcoholic drinking. A large body of evidence indicates that social drinking is a primary cultural symbol of manliness; portrayals in the media strengthen this association. Less evidence exists to connect masculinity issues with alcoholic dependence, but there has been much speculation: Three psychodynamic theories of alcoholism--the repressed homosexuality, dependency, and power theories--hypothesized that men who drink addictively have the most fragile masculine identities. The 1980s have witnessed a widespread recognition of the dangers of equating drinking and manliness, and societal changes suggest that drinking may be gradually losing its masculine aura.

Recent developments in chloroplast protein transport.

Most proteins located in chloroplasts are encoded by nuclear genes, synthesized in the cytoplasm, and transported into the organelle. The study of protein uptake by chloroplasts has greatly expanded over the past few years. The increased activity in this field is due, in part, to the application of recombinant DNA methodology to the analysis of protein translocation. Added interest has also been gained by the realization that the transport mechanisms that mediate protein uptake by chloroplasts, mitochondria and the endoplasmic reticulum display certain characteristics in common. These include amino terminal sequences that target proteins to particular organelles, a transport process that is mechanistically independent from the events of translation, and an ATP-requiring transport step that is thought to involve partial unfolding of the protein to be translocated. In this review we examine recent studies on the binding of precursors to the chloroplast surface, the energy-dependent uptake of proteins into the stroma, and the targeting of proteins to the thylakoid lumen. These aspects of protein transport into chloroplasts are discussed in the context of recent studies on protein uptake by mitochondria.

Specific heat shock proteins are transported into chloroplasts.

We demonstrate that in three plant species-soybean, pea, and corn-certain nuclear-encoded heat shock proteins are transported into chloroplasts. In vitro translation products of poly(A)-RNA from control or heat-shocked plants were incubated with isolated intact pea chloroplasts and differences in the profile of imported proteins were analyzed. In all three species, abundant polypeptides between 21 and 27 kDa are present in the heat shock sample and absent in the controls. These polypeptides are protected from trypsin and chymotrypsin digestion after their import into chloroplasts and are recovered primarily with the soluble chloroplast protein fraction. Chloroplasts isolated from pea or corn leaves labeled in vivo at heat shock temperatures, but not at normal growth temperatures, contain the same polypeptides observed in vitro. Synthesis of the heat shock polypeptides can be inhibited in vivo by cycloheximide but not by chloramphenicol, further indicating they are products of cytoplasmic protein synthesis. The in vitro transport experiments demonstrate that synthesis of the chloroplast-localized heat shock proteins results from heat-induced accumulation of the corresponding poly(A)-RNAs. The same mRNAs are also produced in response to heat shock by a nonphotosynthetic tissue, the etiolated soybean hypocotyl.

Functional determinants in transit sequences: import and partial maturation by vascular plant chloroplasts of the ribulose-1,5-bisphosphate carboxylase small subunit of Chlamydomonas.

The precursor of the ribulose-1,5-bisphosphate carboxylase small subunit and other proteins from Chlamydomonas reinhardtii are efficiently transported into chloroplasts isolated from spinach and pea. Thus, similar determinants specify precursor-chloroplast interactions in the alga and vascular plants. Removal of all or part of its transit sequence was found to block import of the algal small subunit into isolated chloroplasts. Comparison of available sequences revealed a nine amino acid segment conserved in the transit sequences of all small subunit precursors. A protease in the vascular plant chloroplasts recognized this region in the Chlamydomonas precursor and produced an intermediate form of the small subunit. We propose that processing of the small subunit precursor involves at least two proteolytic events; only one of these has been evolutionarily conserved.

Characterization of a wheat germ agglutinin-like lectin from adult wheat plants.

Radioimmuno-and enzyme-linked immunosorbent assays show that a substantial amount of wheat germ agglutinin(WGA)-like protein is present at the base of the shoot and in the roots of adult wheat (Triticum aestivum L.) plants. The protein can be purified by hapten-and antibody-mediated affinity procedures. It forms an arc of identity with the embryo lectin upon Ouchterlony double-diffusion and is an active lectin that agglutinates trypsinized erythrocytes in an N-acetylglucosamine-and chitin-inhibitable manner. Reduced and carboxyamidated protein comigrates with the 18-kdalton subunits of embryo lectin on sodium dodecyl sulfate-polyacrylamide gels. Invivo labeling of 9-d-old, hydroponically grown plants with (35)S-labeled sulfate demonstrates that at least some of the WGA-like protein is synthesized de novo. Immunocytochemistry with rabbit anti-WGA and colloidal-gold-conjugated second antibody shows that cross-reactive protein is present at the tips of new adventitious roots. In reactive cells, the lectin is localized near the inner surface of the vacuole membrane. Wheat plants contain up to 100 ng of WGA-like protein after the first week of growth, but the level fluctuates thereafter. Since most of the lectin is present at the base of the shoot and much less is found in older roots, these fluctuations may be the consequence of changes in the initiation of new advantitious roots.

Posttranscriptional Regulation of Ribulose 1,5-bisphosphate Carboxylase Small Subunit Accumulation in Chlamydomonas reinhardtii.

The coordinated synthesis of the subunits of ribulose 1,5-bisphosphate carboxylase was examined by analysis of the chloroplast ribosome-deficient mutant of Chlamydomonas reinhardtii, ac20crl. The absence of the chloroplast-synthesized large subunit of this enzyme from cells of this strain is a direct consequence of the lack of chloroplast ribosomes. In contrast, the absence of the cytoplasmically-synthesized small subunit of ribulose 1,5-bisphosphate carboxylase from this mutant is not understood. To discern the cause of this absence, we have compared results of in vivo radioactive labeling experiments with those of cell-free translations of RNA from ac20crl. Protein products from these experiments were identified by one-and two-dimensional electrophoretic analyses. Neither subunit, revealed either as a stained band or by fluorography of proteins radioactively labeled in vivo for 2 hours, was detected in ac20crl. Cell-free translation of polyadenylated RNA obtained from ac20crl, however, revealed wild-type levels of mRNA for the precursor to the small subunit. This messenger was found to be associated with subribosomal RNP and polysomes. We conclude that the absence of the small subunit of ribulose 1,5-bisphosphate carboxylase from ac20crl is the result of a translational or posttranslational event.

Localization of wheat germ agglutinin--like lectins in various species of the gramineae.

Antigenically similar chitin-binding lectins are present in the embryos of wheat, barley, and rye, members of the Triticeae tribe of the grass family (Gramineae). However, the lectins display different localization patterns in these embryos. Lectin is absent from the coleoptile of barley but is present in the outer surface cells of this organ in wheat and in both inner and outer surface cells of rye coleoptiles. All three cereals contain lectin at the periphery of embryonic roots. Similar lectins were not detected in oats and pearl millet, members of other tribes of the Gramineae. Rice, a species only distantty related to wheat, contains a lectin that is antigenically similar to the other cereal lectins and located at the periphery of embryonic roots and throughut the coleoptile.

Rapid degradation of unassembled ribulose 1,5-bisphosphate carboxylase small subunits in chloroplasts.

We have detected a proteolytic mechanism in chloroplasts that selectively and rapidly degrades the imported small subunit of ribulose 1,5-bisphosphate carboxylase when pools of the chloroplast-synthesized large subunit are depleted. This degradation system is constitutively present and appears to be responsible for precise stoichiometric accumulation of the two subunits of the enzyme. We believe similar proteolytic mechanisms participate in regulating the accumulation of other photosynthetic proteins during chloroplast biogenesis.

Immunocytochemical localization of wheat germ agglutinin in wheat.

Immunocytological techniques were developed to localize the plant lectin, wheat germ agglutinin (WGA), in the tissues and cells of wheat plants. In a previous study we demonstrated with a radioimmunoassay that the lectin is present in wheat embryos and adult plants both in the roots and at the base of the stem. We have now found, using rhodamine, peroxidase, and ferritin-labeled secondary antibodies, that WGA is located in cells and tissues that establish direct contact with the soil during germination and growth of the plant In the embryo, WGA is found in the surface layer of the radicle, the first adventitious roots, the coleoptile, and the scutellum. Although found throughout the coleorhiza and epiblast, it is at its highest levels within the cells at the surface of these organs. In adult plants, WGA is located only in the caps and tips of adventitious roots. Reaction product for WGA was not visualized in embryonic or adult leaves or in other tissues of adult plants. At the subcellular level, WGA is located at the periphery of protein bodies, within electron-translucent regions of the cytoplasm, and at the cell wall-protoplast interface. Since WGA is found at potential infection sites and is known to have fungicidal properties, it may function in the defense against fungal pathogens.

Distribution of wheat germ agglutinin in young wheat plants.

A liquid phase, competition-binding radioimmunoassay for wheat germ agglutinin, with a detection limit of 10 nanograms, was developed in order to determine the distribution of this lectin in young wheat plants. Affinity columns for wheat germ agglutinin removed all antigenically detectable activity from crude extracts of wheat tissue; thus, the antigenic cross-reactivity detected by the assay possesses sugar-binding specificity similar to the wheat germ-derived lectin. The amount of lectin per dry grain is approximately 1 microgram, all associated with the embryo. At 34 days of growth, the level of lectin per plant was reduced by about 50%, with approximately one-third in the roots and two-thirds in the shoot. The data also indicate that actively growing regions of the plant (the bases of the leaves and rapidly growing adventitious roots) contain the highest levels of lectin. Half of the lectin associated with the roots could be solubilized by washing intact roots in buffer containing oligomers of N-acetylglucosamine, whereas the remainder is liberated only upon homogenization of the tissue.