Modifications in the surgical correction of trigonocephaly

A modified technique for the surgical correction of trigonocephaly is presented. The technical modifications are designed both to increase the stability of fixation of the supraorbital bar and lateral canthal advancements and to increase interorbital distance and anterior cranial fossa volume when utilized in those patients who exhibit the full expression of trigonocephaly, including midline ridging, bifrontal recession, hypotelorbitism, shortened anterior cranial fossa, deficient projection of lateral orbit, and bitemporal narrowing. It is a modification of the supraorbital bar remodeling/advancement procedure as originally described by Marchac with the introduction of a nasofrontal osteotomy and superior osteotomy bone graft and midline miniplate fixation of the supraorbital bar to the nasofrontal junction. Its use in 20 patients has been favorable.     

Evidence for a plasmid conferring salt-tolerance in the plant-root associated,diazotrophKlebsiella sp. NIAB-I

Summary Using analytical and preparative methods, we demonstrated the presence of an indigenous plasmid (pNIAB-I) in a diazotroph,Klebsiella sp. NIAB-I isolated, from the roots of Kallar grass, growing on saline lands in Pakistan. The plasmid is approximately 50 kilobase (kb) in size. Transformation experiments indicated that non-halophilic bacteria such asE. coli K12 strain (MV10) andK. pneumoniae M5AI on acquiring this plasmid become tolerant to high salt (NaCl) and alkaline pH.     

An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region.

Translation of the human hepatitis C virus (HCV) RNA genome occurs by a mechanism known as "internal ribosome entry." This unusual strategy of translation is employed by naturally uncapped picornaviral genomic RNAs and several cellular mRNAs. A common feature of these RNAs is a relatively long 5' noncoding region (NCR) that folds into a complex secondary structure harboring an internal ribosome entry site (IRES). Evidence derived from the use of dicistronic expression systems, combined with an extensive mutational analysis, demonstrated the presence of an IRES within the HCV 5'NCR. The results of our continued mutational analysis to map the critical structural elements of the HCV IRES has led to the identification of a pseudoknot structure upstream of the initiator AUG. The evidence presented in this study is based upon the mutational analysis of the putative pseudoknot structure. This is further substantiated by biochemical and enzymatic probing of the wild-type and mutant 5'NCR. Further, the thermodynamic calculations, based upon a modified RNAKNOT program, are consistent with the presence of a pseudoknot structure located upstream of the initiator AUG. Maintenance of this structural element is critical for internal initiation of translation. The pseudoknot structure in the 5'NCR represents a highly conserved feature of all HCV subtypes and members of the pestivirus family, including hog cholera virus and bovine viral diarrhea virus.     

Identification and characterization of a novel repressor site in the human tumor necrosis factor alpha gene.

In human monocytic cell lines, tumor necrosis factor alpha (TNF alpha) expression is induced by phorbol myristate acetate (PMA). We have identified positive and negative cis-acting elements in the TNF alpha promoter by deletion analysis. Here we present the initial characterization of the repressor element. The repressor element was shown to function in either orientation and at various distances upstream from the positive element of the TNF alpha promoter. The TNF alpha repressor site (TRS) has been localized to a 25 bp region between base pairs -254 and -230 in the promoter. This region contains a 10 bp sequence with homology to the binding site of the activator protein AP-2. Mutation of the 6 C's of this 10 bp AP-2-like site abolish TRS repressor function. However, this AP-2-like site is not a binding site for AP-2 protein based on gel retardation analysis. In addition, a well-characterized AP-2-binding site placed upstream of the positive element of the TNF alpha gene did not cause repression. Therefore, this repression is very likely mediated by a novel protein(s) which interacts with the AP-2 consensus site in the TRS.     

A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA.

Translation of hepatitis C virus (HCV) RNA is initiated by cap-independent internal ribosome binding to the 5' noncoding region (NCR). To identify the sequences and structural elements within the 5' NCR of HCV RNA that contribute to the initiation of translation, a series of point mutations was introduced within this sequence. Since the pyrimidine-rich tract is considered a characteristic feature of picornavirus internal ribosome entry site (IRES) elements, our mutational analysis focused on two putative pyrimidine tracts (Py-I and Py-II) within the HCV 5' NCR. Translational efficiency of these mutant RNAs was examined by in vitro translation and after RNA transfection into liver-derived cells. Mutational analysis of Py-I (nucleotides 120 to 130), supported by compensatory mutants, demonstrates that the primary sequence of this motif is not important but that a helical structural element associated with this region is critical for HCV IRES function. Mutations in Py-II (nucleotides 191 to 199) show that this motif is dispensable for IRES function as well. Thus, the pyrimidine-rich tract motif, which is considered as an essential element of the picornavirus IRES elements, does not appear to be a functional component of the HCV IRES. Further, the insertional mutagenesis study suggests a requirement for proper spacing between the initiator AUG and the upstream structures of the HCV IRES element for internal initiation of translation.     

Regulation of N-Methyl-d-aspartate receptor-mediated calcium transport and norepinephrine release in rat hippocampus synaptosomes by polyamines

The role of polyamines (PA) synthesis in NMDA receptor-mediated⁴⁵Ca²⁺ fluxes and norepinephrine release was studied in rat hippocampal synaptosomes. NMDA (50M) caused a sharp (>2-fold) transient increase in PA synthesis regulating enzyme, ornithine decarboxylase (ODC) activity with concomitant elevation in PA levels in the order putrescine>spermidine>spermine. ODC inhibitor, -difluoromethylornithine (DFMO), and NMDA antagonist, 2-amino-5-phosphonovaleric acid (D-AP5), both blocked increases in ODC activity and PA levels. Activation of NMDA receptors induced a sharp (3 to 4-fold) and quick (15 seconds) increase in⁴⁵Ca²⁺ uptake by synaptosomes within 15 seconds of exposure at 37°C. The efflux of⁴⁵Ca²⁺ and³H-norepinephrine (NE) release at 22°C from pre-loaded synaptosomes was also significantly (2 to 4-fold) enhanced by NMDA within 15 seconds. These NMDA receptor-mediated effects on calcium fluxes and NE release were blocked by NMDA receptor-antagonists (DAP-5 and MK-801) and PA synthesis inhibitor, DFMO and the DFMO inhibition nullified by exogenous putrescine. These observations establish that ODC/PA cascade play an important role in transduction of excitatory amino acid mediated signals at NMDA receptors.Special issue dedicated to Dr. Sidney Ochs.     

Inhibition of seed germination by Macrophomina phaseolina is related to phaseolinone production

The production of phaseolinone, a phytotoxic metabolite of Macrophomina phaseolina in infected Phaseolus mungo seeds grown on soil, was estimated by enzyme-linked immunosorbent assay and HPLC. The degree of inhibition of seed germination correlated well with the amount of toxin produced; 50% inhibition was observed at a toxin level of 2.1 μg g^-1 of wet tissue. A comparison of the toxin-producing ability of nine isolates of the fungus obtained from different hosts and localities showed that the strain MPK'83 produced a significantly larger amount of the toxin, both in liquid culture and in infected seeds. The virulence of the isolates was related to their ability to produce phaseolinone.     

Age-mediated gut microbiota dysbiosis promotes the loss of dendritic cells tolerance

The old age-related loss of immune tolerance inflicts a person with a wide range of autoimmune and inflammatory diseases. Dendritic cells (DCs) are the sentinels of the immune system that maintain immune tolerance through cytokines and regulatory T-cells generation. Aging disturbs the microbial composition of the gut, causing immune system dysregulation. However, the vis-à-vis role of gut dysbiosis on DCs tolerance remains highly elusive. Consequently, we studied the influence of aging on gut dysbiosis and its impact on the loss of DC tolerance. We show that DCs generated from either the aged (DC^Old) or gut-dysbiotic young (DC^Dysbiotic) but not young (DC^Young) mice exhibited loss of tolerance, as evidenced by their failure to optimally induce the generation of Tregs and control the overactivation of CD4⁺ T cells. The mechanism deciphered for the loss of DC^Old and DC^Dysbiotic tolerance was chiefly through the overactivation of NF-κB, impaired frequency of Tregs, upregulation in the level of pro-inflammatory molecules (IL-6, IL-1β, TNF-α, IL-12, IFN-γ), and decline in the anti-inflammatory moieties (IL-10, TGF-β, IL-4, IDO, arginase, NO, IRF-4, IRF-8, PDL1, BTLA4, ALDH2). Importantly, a significant decline in the frequency of the Lactobacillus genus was noticed in the gut. Replenishing the gut of old mice with the Lactobacillus plantarum reinvigorated the tolerogenic function of DCs through the rewiring of inflammatory and metabolic pathways. Thus, for the first time, we demonstrate the impact of age-related gut dysbiosis on the loss of DC tolerance. This finding may open avenues for therapeutic intervention for treating age-associated disorders with the Lactobacillus plantarum.     

Cryopreservation of immature spring wheat zygotic embryos using an abscisic acid pretreatment

Spring wheat (Triticum aestivum L.) zygotic embryos were successfully cryopreserved, without the addition of exogenous cryoprotectants, using only an abscisic acid (ABA) pretreatment. Optimum survival was obtained when embryos were cultured in vitro for 10 days on semisolid Murashige and Skoog (MS) nutrient medium supplemented with 0.5 mg/L (±) ABA prior to cryopreservation. The embryos resumed growth within three days when returned to MS medium devoid of ABA but containing 2mg/L 2,4-dichlorophenoxyacetic acid. The embryogenic calli produced from these embryos exhibited normal plant regeneration on auxin-free media. Changes in dw/fw ratio, as well as the esterified fatty acid and sucrose concentrations correlated positively with the development of tolerance to cryopreservation.NRCC Publication No. 33519     

Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.