Creation of salt-insensitive 3'(2'),5'-bisphosphate nucleotidase by modeling and mutagenesis approach

3′(2′),5′-Bisphosphate nucleotidase, (EC 3.1.3.7) (BPntase) is a ubiquitous enzyme. Recently, these enzymes have drawn considerable attention as in vivo targets of salt toxicity as well as therapeutic targets of lithium that is used for the treatment of manic-depressive disorders. They belong to the Mg²⁺-dependent Li⁺-sensitive phosphomonoesterase super-family and are highly sensitive to lithium and sodium ions. However, the molecular mechanism of inhibition of this group of enzymes by monovalent cations has not been completely understood. Previously we have identified a BPntase (Dhal2p) from a highly halotolerant yeast Debaryomyces hansenii. Molecular characterization revealed a number of unique features in Dhal2p, indicating this is an extraordinary member of the family. In this study, we have carried out the structure-function analysis of Dhal2p through the combination of molecular modeling and in vitro mutagenesis approach. We have not only provided the explanation for the role played by the functionally important elements that are conserved among the members of this family but also identified important, novel structural elements in this enzyme. Our study for the first time unraveled the role of a flap as well as a loop region in the functioning of this enzyme. Most importantly, mutations in the loop region resulted in the creation of a BPntase that was insensitive to salt.     

Genetic deletion of the tumor necrosis factor receptor p60 or p80 abrogates ligand-mediated activation of nuclear factor-kappa B and of mitogen-activated protein kinases in macrophages.

Tumor necrosis factor (TNF) is a pleiotropic cytokine known to regulate cell growth, viral replication, inflammation, immune system functioning, angiogenesis, and tumorigenesis. These effects are mediated through two different receptors, TNFR1 and TNFR2 (also called p60 and p80, respectively), with p60 receptor being expressed on all cell types and p80 receptor only on cells of the immune system and on endothelial cells. Although the role of p60 receptor in TNF signaling is well established, the role of p80 is less clear. In this report, by using macrophages derived from wild-type mice (having both receptors) and mice in which the gene for either p60 (p60(-/-)), or p80 (p80(-/-)), or both (p60(-/-) p80(-/-)) receptor have been deleted, we have redefined the role of these receptors in TNF-induced activation of nuclear factor (NF)-kappa B and of mitogen-activated protein kinases. TNF activated NF-kappa B in a dose- and time-dependent manner in wild-type macrophages but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. These results correlated with the I kappa B alpha degradation needed for NF-kappa B activation. We also found that TNF activated c-Jun N-terminal protein kinase in a dose- and time-dependent manner in wild-type macrophages but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. TNF activated p38 MAPK and p44/p42 MAPK in wild-type but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. TNF induced the proliferation of wild-type macrophages, but for p60(-/-) and p80(-/-) macrophages proliferation was lower, and in p60(-/-) p80(-/-) it was absent. Overall, our studies suggest that both types of TNF receptors are needed in macrophages for optimum TNF cell signaling.     

Hydraulic permeability and activation energy of human keratinocytes at subzero temperatures

Studies on isolated human keratinocytes provide a model for design of optimal freeze-thaw protocols for skin cryopreservation and banking. Nucleated keratinocytes from the basal layer of split thickness human cadaveric skin were separated by a combined trypsin and DNAse digestion and suspended in Dulbecco's minimal essential medium with fetal calf serum. A small volume of suspension was frozen on a microprocessor controlled cryostage. Extracellular ice was nucleated at predetermined subzero temperatures, and the temperature was held constant for the duration of the experiment. The osmotic response of the cells to the formation of extracellular ice was recorded on 35-mm photographic film. Selected serial frames were digitized for automated computer evaluation of metric parameters of specific cells. Changes in the apparent cell volume were quantified over a period of several minutes to obtain dehydration curves associated with exposure to concentrated extracellular electrolytes. The Kedem-Katchalsky coupled flow transport model was statistically fit to the data using a personal computer. Values for the permeability coefficients were adjusted to optimize the correlation between the theory and the data. An activation energy of 44.8 kJ/mol and a water permeability of 0.035 micron (atm.min) at 0 degrees C were derived from the data measured over a temperature range from -2 to -9 degrees C.     

Sensory Neuron-Derived Eph Regulates Glomerular Arbors and Modulatory Function of a Central Serotonergic Neuron

Olfactory sensory neurons connect to the antennal lobe of the fly to create the primary units for processing odor cues, the glomeruli. Unique amongst antennal-lobe neurons is an identified wide-field serotonergic neuron, the contralaterally-projecting, serotonin-immunoreactive deutocerebral neuron (CSDn). The CSDn spreads its termini all over the contralateral antennal lobe, suggesting a diffuse neuromodulatory role. A closer examination, however, reveals a restricted pattern of the CSDn arborization in some glomeruli. We show that sensory neuron-derived Eph interacts with Ephrin in the CSDn, to regulate these arborizations. Behavioural analysis of animals with altered Eph-ephrin signaling and with consequent arborization defects suggests that neuromodulation requires local glomerular-specific patterning of the CSDn termini. Our results show the importance of developmental regulation of terminal arborization of even the diffuse modulatory neurons to allow them to route sensory-inputs according to the behavioural contexts.     

Molecular cloning, characterization, and engineering of xylitol dehydrogenase from Debaryomyces hansenii

Because of its natural ability to utilize both xylose and arabinose, the halotolerant and osmotolerant yeast Debaryomyces hansenii is considered as a potential microbial platform for exploiting lignocellulosic biomass. To gain better understanding of the xylose metabolism in D. hansenii, we have cloned and characterized a xylitol dehydrogenase gene (DhXDH). The cloned gene appeared to be essential for xylose metabolism in D. hansenii as the deletion of this gene abolished the growth of the cells on xylose. The expression of DhXDH was strongly upregulated in the presence of xylose. Recombinant DhXdhp was expressed and purified from Escherichia coli. DhXdhp was highly active against xylitol and sorbitol as substrate. Our results showed that DhXdhp was thermo-sensitive, and except this, its biochemical properties were quite comparable with XDH from other yeast species. Furthermore, to make this enzyme suitable for metabolic engineering of D. hansenii, we have improved its thermotolerance and modified cofactor requirement through modelling and mutagenesis approach.     

Effect of the mi allele on mast cells, basophils, natural killer cells, and osteoclasts in C57Bl/6J mice

The osteopetrotic, microphthalmic (mi/mi) mouse lacks functional osteoclasts and has also been reported to be deficient in mast cells and natural-killer (NK) cells. The later deficiencies could be secondary to the osteopetrotic marrow, or a direct result of the mi allele. Therefore, heterozygotes were examined for these cell types, since these mice do not exhibit osteopetrosis. Adult +/mi animals have approximately 50%, and mi/mi animals examined by histologic techniques or tissue histamine levels have 0-10%, of the peritoneal, dermal, and intestinal mast cells compared with that of +/+ animals. Leukocyte histamine, indicative of the number of basophils, demonstrates the same pattern. Histamine content per mast cell in +/+ and +/mi animals is identical. The number of large granular lymphocytes (LGL) in splenic leukocyte preparations from +/mi animals is 50% that of +/+ animals, and these cells are undetectable in preparations from mi/mi mice. NK activity against YAC-1 cells paralleled the number of LGL present. The resorptive response of neonatal calvaria to parathyroid hormone was delayed in the case of cultured +/mi bone compared with that of +/+ bone, but the final rate of calcium release was identical. These data indicate that 1) the presence of one mi allele can affect the development of four distinct cell types, and 2) osteopetrosis alone does not account for the lack of mast cells, basophils, and NK cells in mi/mi mice.     

Cell density-dependent regulation of cell surface expression of two types of human tumor necrosis factor receptors and its effect on cellular response

Tumor necrosis factor (TNF) is a multipotential cytokine known to regulate the growth of a wide variety of normal and tumor cells. It has been shown that the density of cells in culture can modulate the growth regulatory activities of TNF, the mechanism of which, however, is not understood. In this report, we investigated the effect of cell density on the expression of TNF receptors. The receptors were examined on epithelial cells (e.g., HeLa), which primarily express the p60 form, and on myeloid cells (e.g., HL-60) known to express mainly the p80 form. We observed that binding of TNF to both cell lines decreased with increase in cell density. Scatchard analysis of binding on HeLa and HL-60 cells revealed a 4- to 5-fold reduction in the number of TNF receptors without any significant change in receptor affinity in both cell types at high density. The decrease in TNF receptor numbers at high cell density was also observed in several other epithelial and myeloid cell lines. The downmodulation at high cell density was unique to TNF receptors, since minimum change in other cell surface proteins was observed as revealed by fluorescent activated cell sorter analysis. Neutralization of binding with antibodies specific to each type of the receptors revealed that both the p60 and p80 forms of the TNF receptor were equally downmodulated. A decrease in leucine incorporation into proteins was observed with increase in cell density, suggesting a reduction in protein synthesis. Since inhibition of protein synthesis by cycloheximide also leads to a decrease in TNF receptors, it is possible that the density-dependent reduction in TNF receptor number is due to an overall decrease in protein synthesis. The density-dependent decrease in TNF receptors was accompanied by a decrease in intracellular reduced glutathione levels. A reduction in the number of receptors on TNF sensitive tumor cells induced by cell-density correlated with increase in resistance to the cytokine.     

Biology of <Emphasis Type="Italic">B. sorokiniana</Emphasis> (syn. <Emphasis Type="Italic">Cochliobolus sativus</Emphasis>) in genomics era

Bipolaris sorokiniana (Sacc.) Shoemaker is a hemi-biotrophic fungal pathogen, which is an anamorph (teleomorph Cochlibolus sativus). It causes spot blotch, root rot and leaf spot diseases in a number of cereals including wheat, barley and other small grain cereals. In the genomics era, the fungus has been subjected to a variety of studies using molecular approaches. Correct chromosome number was determined and molecular karyotypes were prepared using contour-clamped homogeneous electric field. Molecular maps were prepared using markers like RFLPs, SSRs, RAPDs and SNPs. For this purpose, segregating progenies derived from crosses between diverse isolates of the pathogen were used. Whole genome sequencing (WGS) data was collected not only for B. sorokiniana isolates, but also for several species of Cochliobolus. Genes involved in secondary metabolism and virulence were identified from genome sequences. The WGS data has also been utilized for comparative genomics giving useful information about evolutionary trends. A brief account of this information is presented in this review.     

"To Be or Not to Be" Protonated: Atomic Details of Human Carbonic Anhydrase-Clinical Drug Complexes by Neutron Crystallography and Simulation

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