Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes.

Recent evidence has shown that members of the Jak kinase family are activated after IL-6 binds to its receptor complex, leading to a tyrosine phosphorylation of gp130, the IL-6 signal-transducing subunit. The different members of the IL-6 cytokine subfamily induce distinct patterns of Jak-Tyk phosphorylation in different cell types. Using monospecific antibodies to gp130, Jak2 kinase, and phosphotyrosine, we investigated the kinetics of IL-6 stimulation of members of this pathway in primary hepatocytes. Our findings show that Jak 2 is maximally activated within 2 min of exposure to IL-6, followed by gp130 phosphorylation that reaches its peak in another 2 min then declines to basal level by 60 min. In vitro phosphorylation experiments show that activated Jak 2 is able to phosphorylate both native gp130 and a fusion peptide containing its cytoplasmic domain, demonstrating gp130 is a direct substrate of Jak 2 kinase. Experiments designed to explore the cell surface expression of gp130 show that > or = 2 h are required to get a second round of phosphorylation after the addition of more cytokines. This finding suggests that activated gp130 is internalized from the cell surface after IL-6 stimulation. Additional experiments using protein synthesis inhibitors reveal that new protein synthesis is required to get a second cycle of gp130 phosphorylation indicating gp130 must be synthesized de novo and inserted into the membrane. These findings provide strong evidence that down regulation of the IL-6 signal in hepatocytes involves the internalization and cytosol degradation of gp130.     

Characterization of Rhodobacter capsulatus genes encoding a molybdenum transport system and putative molybdenum-pterin-binding proteins.

The alternative, heterometal-free nitrogenase of Rhodobacter capsulatus is repressed by traces of molybdenum in the medium. Strains carrying mutations located downstream of nifB copy II were able to express the alternative nitrogenase even in the presence of high molybdate concentrations. DNA sequence analysis of a 5.5-kb fragment of this region revealed six open reading frames, designated modABCD, mopA, and mopB. The gene products of modB and modC are homologous to ChlJ and ChlD of Escherichia coli and represent an integral membrane protein and an ATP-binding protein typical of high-affinity transport systems, respectively. ModA and ModD exhibited no homology to known proteins, but a leader peptide characteristic of proteins cleaved during export to the periplasm is present in ModA, indicating that ModA might be a periplasmic molybdate-binding protein. The MopA and MopB proteins showed a high degree of amino acid sequence homology to each other. Both proteins contained a tandem repeat of a domain encompassing 70 amino acid residues, which had significant sequence similarity to low-molecular-weight molybdenum-pterin-binding proteins from Clostridium pasteurianum. Compared with that for the parental nifHDK deletion strain, the molybdenum concentrations necessary to repress the alternative nitrogenase were increased 4-fold in a modD mutant and 500-fold in modA, modB, and modC mutants. No significant inhibition of the heterometal-free nitrogenase by molybdate was observed for mopA mopB double mutants. The uptake of molybdenum by mod and mop mutants was estimated by measuring the activity of the conventional molybdenum-containing nitrogenase. Molybdenum transport was not affected in a mopA mopB double mutant, whereas strains carrying lesions in the binding-protein-dependent transport system were impaired in molybdenum uptake.     

Photolithoautotrophic growth and control of CO2 fixation in Rhodobacter sphaeroides and Rhodospirillum rubrum in the absence of ribulose bisphosphate carboxylase-oxygenase.

Rhodospirillum rubrum and Rhodobacter sphaeroides were shown to be capable of photolithoautotrophic growth in the absence of the reductive pentose phosphate (Calvin) cycle. Ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strains were incapable of photolithoautotrophic growth using hydrogen as an electron donor but were able to grow in the absence of organic carbon using less reduced inorganic electron donors, i.e., thiosulfate or sulfide. Wild-type R. rubrum grown in the presence of thiosulfate contained RubisCO levels that were 50-fold lower compared with those in cells growth with hydrogen as an electron donor without substantially influencing rates of photolithoautotrophic growth. These results suggest there are two independent CO2 fixation pathways that support photolithoautotrophic growth in purple nonsulfur photosynthetic bacteria, indicating that these organisms have developed sophisticated control mechanisms to regulate the flow of carbon from CO2 through these separate pathways.     

Regulation of fibronectin and laminin binding activity in cultured human lymphoblastic cell lines

The current study shows that a clonal derivative of the Jurkat cell line up-regulates both the avidity and density of the α 6/β1 receptor in response to phorbol 12-myristate 13-acetate (PMA). This derivative attaches to fibronectin and, to a lesser degree, laminin constitutively. Adhesion and spreading are dramatically up-regulated following treatment with PMA. The response on fibronectin peaks within 4 hours, is insensitive to cyclohexaminde, can be blocked by monoclonal antibodies (Mabs) to the β1 and α 5 subunits of the β1 family of integrins, and is not associated with increased expression of the α 5 or β1 epitopes at the cell surface. In contrast, the response on laminin is biphasic. The early phase parallels the response on fibronectin. The second phase peaks after 48–72 hours of treatment with PMA, is sensitive to cycloheximide, can be blocked by Mabs to the β1 and α 6 subunits, and is associated with increased expression of the α 6 epitope. Both the density independent and dependent responses to PMA in Jurkat cells are blocked by the protein kinase inhibitor staurosporine. The HSB-2, CEM, Molt-4, and HPB-ALL T-lymphoblastic cell lines also up-regulate attachment to fibronectin and laminin following treatment with PMA. All four lines constitutively attach to fibronectin and show rapid up-regulation of attachment following treatment with PMA. None of the lines attach to laminin prior to PMA treatment; however, specific adhesion developed after 4–120 hours of treatment. The most mature lines (Jurkat and HPB-ALL) up-regulated adhesion on laminin more rapidly than the less phenotypically mature lines (CEM, Molt-4, and HSB-2). In summary, clonal derivatives of the Jurkat cell line up-regulated attachment to laminin through protein kinase dependent increases in α /β1 receptor avidity and density. In addition, the expression of functional receptors for laminin is linked to developmental maturity in a series of T-lymphoblastic cell lines. © 1993 Wiley-Liss, Inc.     

The phylogenetic position of taxaceae based on 18S rRNA sequences

The evolutionary position of the yew family, Taxaceae, has been very controversial. Some plant taxonomists strongly advocate excluding Taxaceae from the conifer order and raising its taxonomic status to a new order or even class because of its absence of seed cones, contrary to the case in the majority of conifers. However, other authors believe that the Taxaceae are not fundamentally different from the rest of the conifers except in that they possess the most reduced solitary-ovule cones. To resolve the controversy, we have sequenced the 18S rRNA genes from representative gymnosperms: Taxus mairei (Taxaceae), Podocarpus nakaii (Podocarpaceae), Pinus luchuensis (Pinaceae), and Ginkgo biloba (Ginkgoales). Our phylogenetic analysis of the new sequence data with the published 18S rRNA sequence of Zamia pumila (a cycad) as an outgroup strongly indicates that Taxus, Pinus, and Podocarpus form a monophyletic group with the exclusion of Ginkgo and that Taxus is more closely related to Pinus than to Podocarpus. Therefore, Taxaceae should be classified as a family of Coniferales. Our finding that Taxaceae, Pinaceae, and Podocarpaceae form a clade contradicts both the view that the uniovulate seed of Taxaceae is a primitive character and the view that the Taxaceae are descendants of the Podocarpaceae. Rather, the uniovulate seed of Taxaceae and that of some species of Podocarpus appear to have different origins, probably all reduced from multiovulate cones. Correspondence to: W.-H. Li     

Cerebral Metabolic Compartmentation as Revealed by Nuclear Magnetic Resonance Analysis of D-[1-13C]Glucose Metabolism

Abstract: Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, γ-aminobutyrate (GABA), glutamine, and aspartate. d -[1^-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for ¹³C-NMR analysis and amino acid analysis. The absolute amount of ¹³C present within each carbon-atom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance ¹³C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates. Our results also are consistent with the concept that glutamine and/or a tricarboxylic acid cycle intermediate is supplied by astrocytes to neurons for replenishing the neurotransmitter pool of GABA. However, a similar role for astrocytes in replenishing the transmitter pool of glutamate was not substantiated, possibly due to difficulties in quantitating satellite peaks arising from ¹³C-¹³C coupling.     

Functional analysis of matrix proteins expressed from cloned genes of measles virus variants that cause subacute sclerosing panencephalitis reveals a common defect in nucleocapsid binding.

We have developed an in vitro nucleocapsid-binding assay for studying the function of the matrix (M) protein of measles virus (MV) (A. Hirano, A. H. Wang, A. F. Gombart, and T. C. Wong, Proc. Natl. Acad. Sci. USA, 89:8745-8749, 1992). In this communication we show that the M proteins of three MV strains that cause acute infection (Nagahata, Edmonston, and YN) bind efficiently to the viral nucleocapsids whereas the M proteins of four MV strains isolated from patients with subacute sclerosing panencephalitis (SSPE) (Biken, IP-3, Niigata, and Yamagata) fail to bind to the viral nucleocapsids. MV Biken (an SSPE-related virus) produces variant M sequences which encode two antigenically distinct forms of M protein. A serine-versus-leucine difference is responsible for the antigenic variation. MV IP-3 (an SSPE-related virus) also produces variant M sequences, some of which have been postulated to encode a functional M protein responsible for the production of an infectious revertant virus. However, the variant M proteins of Biken and IP-3 strains show no nucleocapsid-binding activity. These results demonstrate that the nucleocapsid-binding function is conserved in the M proteins of MV strains that cause acute infection and that the M proteins of MV strains that cause SSPE exhibit a common defect in this function. Analysis of chimeric M proteins indicates that mutations in the amino-terminal, carboxy-proximal, or carboxy-terminal region of the M protein all abrogate nucleocapsid binding, suggesting that the M protein conformation is important for interaction with the viral nucleocapsid.     

Distinctive effects of the carboxyl-terminal sequence of the insulin-like growth factor I receptor on its signaling functions.

We have shown previously that the extracellular sequences of the human insulin receptor (IR) and the insulin-like growth factor I receptor (IGFR) have an inhibitory effect on protein tyrosine kinase (PTK) activity and on the biological functions of their respective Gag-receptor fusion proteins. To study the role of IGFR carboxyl sequence in modulation of the Gag-IGFR PTK and biological activities, five mutants, CM1, CM2, CM3, CM4, and CM5, containing carboxyl deletions of 17, 27, 47, 67, and 88 amino acids (aa), respectively, were constructed from the parental virus UIGFR encoding the Gag-IGFR. Deletion of up to 27 aa had little effect on the cell-transforming and PTK activities of UIGFR. Deletions of 47 aa in CM3 abolished PTK and transforming activities. Surprisingly, a further deletion of 20 aa in CM4 beyond that in CM3 reactivated the kinase and transforming activities. CM5, containing a deletion of 20 aa beyond that in CM4, had only marginal transforming and PTK activities. We conclude that deletion of the carboxyl region of the Gag-IGFR inactivates, instead of activating as in the case with Gag-IR, its transforming activity and the amino acid sequence 1250 to 1310 is essential for PTK and transforming activities. Analysis of the ability of the full-length IGFR and its mutant receptors described above to associate with phosphatidylinositol 3 kinase indicated that the association required PTK activity and tyrosine phosphorylation of the receptors and correlated well with their transforming activities. The carboxyl 88 aa are not essential for the association.     

Eviostachya hoegii Stockmans在中国五通组的首次发现

首次描述了Eviostachya hoegii Stockmans的营养部分,通过大量标本的观察,修订了前人有关其生殖部分和解剖部分的描述,并对其生殖部分进行了复原.同意Emberger(1968)的观点,将其归于Eviostachyrales中.