The role of an iron-sulfur cluster in an enzymatic methylation reaction. Methylation of CO dehydrogenase/acetyl-CoA synthase by the methylated corrinoid iron-sulfur protein

This paper focuses on how a methyl group is transferred from a methyl-cobalt(III) species on one protein (the corrinoid iron-sulfur protein (CFeSP)) to a nickel iron-sulfur cluster on another protein (carbon monoxide dehydrogenase/acetyl-CoA synthase). This is an essential step in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation. The results described here strongly indicate that transfer of methyl group to carbon monoxide dehydrogenase/acetyl-CoA synthase occurs by an SN2 pathway. They also provide convincing evidence that oxidative inactivation of Co(I) competes with methylation. Under the conditions of our anaerobic assay, Co(I) escapes from the catalytic cycle one in every 100 turnover cycles. Reductive activation of the CFeSP is required to regenerate Co(I) and recruit the protein back into the catalytic cycle. Our results strongly indicate that the [4Fe-4S] cluster of the CFeSP is required for reductive activation. They support the hypothesis that the [4Fe-4S] cluster of the CFeSP does not participate directly in the methyl transfer step but provides a conduit for electron flow from physiological reductants to the cobalt center.     

Systematic analysis of N-linked sugar chains from whole tissue employing partial automation

A partially automated technique for the isolation and characterization of N-linked sugar chains from glycoproteins of crude tissue samples is established. The N-linked sugar chains from the acetone-extracted tissues are made free by a process of hydrazinolysis and subsequently N-acetylated by GlycoPrep 1000 (Oxford Glycosystems). These free sugar chains are further converted to pyridylamino derivatives by GlycoTag (Takara). Characterization of these sugar chains is achieved by a combination of HPLC columns using a highly sensitive fluorescence detector at femtomole levels. Tissue sample can be successfully pyridylaminated and analyzed to give highly reproducible results with consistent yield, requiring fewer purification steps, minimum skills, and less time. Moreover, fixed tissues can also be analyzed employing this technique, giving a similar sugar chain pattern compared to normal tissue samples. Using this method we show that the pattern of N-linked sugar chains present in human sera or in one small region of brain is strikingly similar among the different individuals. However, the absence of a highlighted peak in one of the samples suggests this method can be extrapolated to identify changes, if any, associated with disorders such as inflammation or cancer. Furthermore, this two-dimensional display of sugar chains would discover the function-specific molecules as we see in proteins.     

The translational repressor Pumilio regulates presynaptic morphology and controls postsynaptic accumulation of translation factor eIF-4E

Translational repression by Drosophila Pumilio (Pum) protein controls posterior patterning during embryonic development. Here, we show that Pum is an important mediator of synaptic growth and plasticity at the neuromuscular junction (NMJ). Pum is localized to the postsynaptic side of the NMJ in third instar larvae and is also expressed in larval neurons. Neuronal Pum regulates synaptic growth. In its absence, NMJ boutons are larger and fewer in number, while Pum overexpression increases bouton number and decreases bouton size. Postsynaptic Pum negatively regulates expression of the translation factor eIF-4E at the NMJ, and Pum binds selectively to the 3'UTR of eIF-4E mRNA. The GluRIIa glutamate receptor is upregulated in pum mutants. These results, together with genetic epistasis studies, suggest that postsynaptic Pum modulates synaptic function via direct control of eIF-4E expression.     

Development of high oleic and low linoleic acid transgenics in a zero erucic acid Brassica juncea L. (Indian mustard) line by antisense suppression of the fad2 gene

A zero erucic acid (C22:1) line of Brassica juncea (VH486), adapted to the agronomic conditions of Northern India, has been modified for its fatty acid composition in the seed oil with antisense constructs using the sequence of fad2 gene of B. rapa. The full-length B. rapa fad2 cDNA sequence was determined by 5 and 3 RACE of a partial sequence available in the EST database. Construct pASfad2.1 contained 315 to 1251 bp and construct pASfad2.2 contained 1 to 1251 bp fragment of the fad2 gene, both in antisense orientation, driven by a truncated napin promoter. Analysis of the levels of linoleic acid (C18:2) in the BC1 seeds of single-copy transgenics showed that the construct pASfad2.2 gave better suppression of the fad2 gene as compared to the construct pASfad2.1. The BC1 transgenic seeds containing the pASfad2.2 construct segregated into two distinct classes of C18:2>20% (putative null homozygotes) and C18:2<20% (putative heterozygotes) in a 1:1 ratio, while the T1 seeds segregated into three classes, C18:2>20%, C18:2 between 12% and 20%) and C18:2<12% (putative homozygotes) in a 1:2:1 ratio. Putative homozygous T1 seeds (C18:2<12% analyzed by the half-seed method) of four of the transgenic lines were grown to establish T2 homozygous lines. These had ca. 73% C18:1 and 8 to 9% each of C18:2 and C18:3 (-linolenic acid) fractions in comparison to ca. 53% C18:1, 24% C18:2 and 16% C18:3 in the parental line VH486.     

A basal membrane-like structure surrounding tumour nodules may prevent intraepithelial leucocyte infiltration in colorectal cancer

Epithelial tumours consist of an epithelial compartment and a stromal compartment, which are sometimes separated by a basal membrane-like structure. We sought to determine whether these factors have prognostic value in 84 curatively resected stage II and III colorectal cancer by immunohistochemically staining tumours for leucocytes (CD45) and extracellular matrix, and to assess the presence of a basal membrane-like structure. Leucocyte infiltration was also assessed in hematoxylin-eosin (HE) stained sections. Most leucocytes were located in the tumour stroma. A relatively high intraepithelial leucocyte infiltration was significantly correlated with a lower level of tumour recurrence (P=0.03) and a longer disease-free survival (P=0.05), whereas leucocytes located in the tumour stroma (P=0.92) or at the advancing margin (p=0.06) were not. Intraepithelial leucocyte infiltration was also significantly correlated with leucocyte infiltration in the tumour stroma (P=0.02) and at the advancing tumour margin (P=0.005), and as assessed in HE-stained tumour sections (P=0.05), but each of these parameters on its own did not have a prognostic value in predicting disease-free survival. Moreover, the presence of a basal membrane-like structure surrounding the tumour epithelium was inversely correlated with the number of intraepithelial leucocytes (P=0.05), suggesting that this membrane-like structure functions as a barrier to intraepithelial leucocyte infiltration. We conclude that leucocytes must be in the direct vicinity of tumour cells to affect tumour growth. The presence of an extracellular matrix barrier seems to prevent this interaction.     

The myelin-axolemmal complex: biochemical dissection and the role of galactosphingolipids

Myelin-axolemmal interactions regulate many cellular and molecular events, including gene expression, oligodendrocyte survival and ion channel clustering. Here we report the biochemical fractionation and enrichment of distinct subcellular domains from myelinated nerve fibers. Using antibodies against proteins found in compact myelin, non-compact myelin and axolemma, we show that a rigorous procedure designed to purify myelin also results in the isolation of the myelin-axolemmal complex, a high-affinity protein complex consisting of axonal and oligodendroglial components. Further, the isolation of distinct subcellular domains from galactolipid-deficient mice with disrupted axoglial junctions is altered in a manner consistent with the delocalization of axolemmal proteins observed in these animals. These results suggest a paradigm for identification of proteins involved in neuroglial signaling.     

A protocol for shoot multiplication from foliar meristem of Vanda spathulata (L.) Spreng

Leaf explants collected from flowering plants of Vanda spathulata were cultured in Mitra medium with combinations of 6-benzyladenine (BA; 13.2-88.8 microM) and indole-3-acetic acid (IAA; 0.0 -85.6 microM). Combination of BA (66.6 microM) and IAA (28.5 microM) induced maximum shoots (17.33) from foliar meristems (leaf base). BA individually did not induce caulogenesis in leaf explants. For optimized multiplication, BA:IAA (2:1 microM) was essential at 22.2- 88.8 microM of BA. Re-cultured leaf explants produced lesser number of shoots compared to original explants and were nearly equal at combinations of 22.2-44.4 microM of BA and 5.7-28.5 microM of IAA. Rooting of shoots (> 95%) occurred in medium containing banana pulp (75 gl(-1)) and IAA (5.7 microM) within 3-9 weeks. Plantlets with 2-5 roots of 2-5 cm length established easily in community pots at 80-90% rates without hardening.     

The functional and clinical roles of osteopontin in cancer and metastasis

Osteopontin (OPN) is a secreted and integrin-binding protein that has been implicated in a number of pathologies. In this review we will focus on the functional and clinical roles of OPN in cancer and metastasis, with a particular emphasis on breast cancer. While much evidence has suggested that OPN is associated with cancer, its functional contribution to cancer remains poorly understood. Here we will review evidence for mechanisms by which OPN may act to enhance malignancy, including evidence that signaling pathways directly induced by OPN, as well as interactions with growth factor receptor pathways, can combine to activate expression of genes and functions that contribute to metastasis. OPN has been shown to be over-expressed in a variety of human tumors and is present in elevated levels in the blood of some patients with metastatic cancers. We also will discuss recent clinical evidence that suggests that OPN is not only associated with several tumor types, but that levels of OPN in cancer patients' blood or tumors may provide prognostic information.     

Interaction of microtubules with active principles of Xanthium strumarium

Indigenous variety of Xanthium strumarium (X. strumarium) was screened for its antimitotic activity using the microtubule-tubulin system isolated from mammalian tissue. A preliminary phytochemical screening of the whole extracts of the plant was carried out followed by partial purification of the whole extract of X.strumarium. The separated fractions obtained were identified and used for in vitro polymerization studies. The whole as well as partially separated chemical constituents of X. strumarium showed effective inhibition of tubulin polymerization. The results thus suggest that X. strumarium may possess antimitotic components.     

Evidence that helix 8 of rhodopsin acts as a membrane-dependent conformational switch

The crystal structure of rhodopsin revealed a cytoplasmic helical segment (H8) extending from transmembrane (TM) helix seven to a pair of vicinal palmitoylated cysteine residues. We studied the structure of model peptides corresponding to H8 under a variety of conditions using steady-state fluorescence, fluorescence anisotropy, and circular dichroism spectroscopy. We find that H8 acts as a membrane-surface recognition domain, which adopts a helical structure only in the presence of membranes or membrane mimetics. The secondary structural properties of H8 further depend on membrane lipid composition with phosphatidylserine inducing helical structure. Fluorescence quenching experiments using brominated acyl chain phospholipids and vesicle leakage assays suggest that H8 lies within the membrane interfacial region where amino acid side chains can interact with phospholipid headgroups. We conclude that H8 in rhodopsin, in addition to its role in binding the G protein transducin, acts as a membrane-dependent conformational switch domain.