Clues from a halophilic methanogen about aromatic amino acid biosynthesis in archaebacteria

Extensive diversity in features of aromatic amino acid biosynthesis and regulation has become recognized in eubacteria, but almost nothing is known about the extent to which such diversity exists within the archaebacteria. Methanohalophilus mahii, a methylotrophic halophilic methanogen, was found to synthesize l-phenylalanine and l-tyrosine via phenylpyruvate and 4-hydroxyphenylpyruvate, respectively. Enzymes capable of using l-arogenate as substrate were not found. Prephenate dehydrogenase was highly sensitive to feedback inhibition by l-tyrosine and could utilize either NADP⁺ (preferred) or NAD⁺ as cosubstrate. Tyrosine-pathway dehydrogenases having the combination of narrow specificity for a cyclohexadienyl substrate but broad specificity for pyridine nucleotide cofactor have not been described before. The chorismate mutase enzyme found is a member of a class which is insensitive to allosteric control. The most noteworthy character state was prephenate dehydratase which proved to be subject to multimetabolite control by feedback inhibitor (l-phenylalanine) and allosteric activators (l-tyrosine, l-tryptophan, l-leucine, l-methionine and l-isoleucine). This interlock type of prephenate dehydratase, also known to be broadly distributed among the gram-positive lineage of the eubacteria, was previously shown to exist in the extreme halophile, Halobacterium vallismortis. The results are consistent with the conclusion based upon 16S rRNA analyses that Methanomicrobiales and the extreme halophiles cluster together.Abbreviation DAHP 3-deoxy-d-arabino-heptulosonate-7-phosphate     

Metastatic sweat gland adenocarcinoma: A clinico-pathological dilemma

Background  

Sweat gland adenocarcinoma is a rare malignancy with high metastatic potential seen more commonly in later years of life. Scalp is the most common site of occurrence and it usually spreads to lymph nodes. Liver, lung and bones are the distant sites of metastasis with fatal results. The differentiation between apocrine and eccrine metastatic sweat gland carcinoma is often difficult. The criteria's are inadequate to be of any practical utility.     

Role of glycosylation on the secretion and biological activity of erythropoietin.

The erythropoietin (EPO) molecule contains four carbohydrate chains. Three contain N-linkages to asparagines at positions 24, 38, and 83, and one contains an O-linkage to a serine at position 126. We constructed human EPO variants that eliminated the three N-glycosylation sites by replacing the asparagines with glutamines singly or in combination. The O-linked carbohydrate chain was removed by replacing the serine with glutamine, valine, histidine, or alanine. A variant with a double mutation (Gln38,83) and another with a triple mutation (Gln24,38,83) were secreted poorly from COS1 and CHO cells even though RNA encoding these variants was present. All other variants with mutations in N-linked glycosylation sites were secreted normally. Removal of any of the N-glycosylation sites reduced the in vivo but not the in vitro biological activity of the EPO molecule. All the mutations at Ser126, the O-glycosylation site, were secreted normally. In vitro activity was also unaffected except for Ala126 which had a 50-fold decrease. The Val126 variant was tested in vivo, and its specific activity was only slightly less than that of the native EPO, which indicates that the O-linked carbohydrate is not essential for activity.     

Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile

We present a strategy for generating and analyzing comprehensive genetic-interaction maps, termed E-MAPs (epistatic miniarray profiles), comprising quantitative measures of aggravating or alleviating interactions between gene pairs. Crucial to the interpretation of E-MAPs is their high-density nature made possible by focusing on logically connected gene subsets and including essential genes. Described here is the analysis of an E-MAP of genes acting in the yeast early secretory pathway. Hierarchical clustering, together with novel analytical strategies and experimental verification, revealed or clarified the role of many proteins involved in extensively studied processes such as sphingolipid metabolism and retention of HDEL proteins. At a broader level, analysis of the E-MAP delineated pathway organization and components of physical complexes and illustrated the interconnection between the various secretory processes. Extension of this strategy to other logically connected gene subsets in yeast and higher eukaryotes should provide critical insights into the functional/organizational principles of biological systems.     

The phosphatidylinositol 4,5-biphosphate and TORC2 binding proteins Slm1 and Slm2 function in sphingolipid regulation

The Stt4 phosphatidylinositol 4-kinase has been shown to generate a pool of phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, critical for actin cytoskeleton organization and cell viability. To further understand the essential role of Stt4-mediated PI4P production, we performed a genetic screen using the stt4(ts) mutation to identify candidate regulators and effectors of PI4P. From this analysis, we identified several genes that have been previously implicated in lipid metabolism. In particular, we observed synthetic lethality when both sphingolipid and PI4P synthesis were modestly diminished. Consistent with these data, we show that the previously characterized phosphoinositide effectors, Slm1 and Slm2, which regulate actin organization, are also necessary for normal sphingolipid metabolism, at least in part through regulation of the calcium/calmodulin-dependent phosphatase calcineurin, which binds directly to both proteins. Additionally, we identify Isc1, an inositol phosphosphingolipid phospholipase C, as an additional target of Slm1 and Slm2 negative regulation. Together, our data suggest that Slm1 and Slm2 define a molecular link between phosphoinositide and sphingolipid signaling and thereby regulate actin cytoskeleton organization.     

Selective growth of malignant cells by in vitro incubation on Teflon

The selective growth of malignant cells on non-adhesive Teflon substrates is reported. Neoplastically transformed cell lines of human and murine origin proliferated on Teflon whereas similarly seeded normal non-tumorigenic cells were unable to undergo cell division and subsequent growth. This substrate should be especially useful in establishing primary cultures from human tumor material.     

Sexual dimorphism in oxytocin responses to health perception and disgust,with implications for theories on pathogen detection

In response to a recent hypothesis that the neuropeptide oxytocin might be involved in human pathogen avoidance mechanisms, we report the results of a study in which we investigate the effect of intranasal oxytocin on two behaviors serving as proxies for pathogen detection. Participants received either oxytocin or a placebo and were asked to evaluate (1) the health of Caucasian male computer-generated pictures that varied in facial redness (an indicator of hemoglobin perfusion) and (2) a series of pictures depicting disgusting scenarios. Men, but not women, evaluated all faces, regardless of color, as less healthy when given oxytocin compared to a placebo. Women, on the other hand, expressed decreased disgust when given oxytocin compared to a placebo. These results suggest that intranasal oxytocin administration does not facilitate pathogen detection based on visual cues, but instead reveal clear sex differences in the perception of health and sickness cues.     

The DNA damage response pathway contributes to the stability of chromosome III derivatives lacking efficient replicators

In eukaryotic chromosomes, DNA replication initiates at multiple origins. Large inter-origin gaps arise when several adjacent origins fail to fire. Little is known about how cells cope with this situation. We created a derivative of Saccharomyces cerevisiae chromosome III lacking all efficient origins, the 5ORIΔ-ΔR fragment, as a model for chromosomes with large inter-origin gaps. We used this construct in a modified synthetic genetic array screen to identify genes whose products facilitate replication of long inter-origin gaps. Genes identified are enriched in components of the DNA damage and replication stress signaling pathways. Mrc1p is activated by replication stress and mediates transduction of the replication stress signal to downstream proteins; however, the response-defective mrc1(AQ) allele did not affect 5ORIΔ-ΔR fragment maintenance, indicating that this pathway does not contribute to its stability. Deletions of genes encoding the DNA-damage-specific mediator, Rad9p, and several components shared between the two signaling pathways preferentially destabilized the 5ORIΔ-ΔR fragment, implicating the DNA damage response pathway in its maintenance. We found unexpected differences between contributions of components of the DNA damage response pathway to maintenance of ORIΔ chromosome derivatives and their contributions to DNA repair. Of the effector kinases encoded by RAD53 and CHK1, Chk1p appears to be more important in wild-type cells for reducing chromosomal instability caused by origin depletion, while Rad53p becomes important in the absence of Chk1p. In contrast, RAD53 plays a more important role than CHK1 in cell survival and replication fork stability following treatment with DNA damaging agents and hydroxyurea. Maintenance of ORIΔ chromosomes does not depend on homologous recombination. These observations suggest that a DNA-damage-independent mechanism enhances ORIΔ chromosome stability. Thus, components of the DNA damage response pathway contribute to genome stability, not simply by detecting and responding to DNA template damage, but also by facilitating replication of large inter-origin gaps.     

Production and response to interleukin 2 in vitro and in vivo after bone marrow transplantation in mice

Lymphoid cells from bone marrow radiation chimeras do not produce normal levels of IL 2 but are capable of responding to IL 2 in mitogenic and cytotoxic assays in vitro. The administration of recombinant human IL 2 into host mice that have received allogeneic, H-2-compatible marrow did not enhance mortality.