Inhibition of Rho-kinase induces alphaB-crystallin expression in lens epithelial cells

The small heat shock protein, alphaB-crystallin, has been shown to interact with actin and intermediate filament proteins. However, little is known regarding the cellular mechanisms regulating such interactions. In this study, we explored the role of the Rho/Rho-kinase pathway in alphaB-crystallin distribution and expression in porcine lens epithelial cells. alphaB-crystallin was distributed uniformly throughout the cytoplasm and did not exhibit any unique redistribution in response to actin depolymerization induced by Rho/Rho-kinase inhibitors (C3-exoenzyme or Y-27632) or by overexpression of the dominant negative mutant of Rho-kinase (DNRK) in porcine lens epithelial cells. Interestingly, alphaB-crystallin levels markedly increased in lens epithelial cells treated with the inhibitors of Rho/Rho-kinase proteins (lovastatin, Y-27632 or DNRK) while a protein kinase C inhibitor (GF109203x) was found to have no effect. Further, Y-27632 showed a dose (2-50 microM) response effect on alphaB-crystallin induction. Nocodazole, a microtubule-depolymerizing agent, elicited an increase in alphaB-crystallin levels but latrunculin, an actin depolymerizing agent, did not show any significant effect. Pretreatment with cycloheximide or genistein blocked the Rho-kinase inhibitor-induced increase in alphaB-crystallin protein levels. Rho-kinase inhibitor-induced increases in alphaB-crystallin levels were found to be associated with activation of P38 mitogen-activated protein kinase (MAPK). These results suggest that Rho/Rho-kinase negatively regulates alphaB-crystallin expression, and this response appears to be dependent on tyrosine-protein kinase and P38 MAPK function. Finally, alphaB-crystallin induction appears to be better correlated with the direct inhibition of Rho/Rho-kinase than with actin depolymerization per se.     

Effect of host species on recG phenotypes in Helicobacter pylori and Escherichia coli

Recombination is a fundamental mechanism for the generation of genetic variation. Helicobacter pylori strains have different frequencies of intragenomic recombination, arising from deletions and duplications between DNA repeat sequences, as well as intergenomic recombination, facilitated by their natural competence. We identified a gene, hp1523, that influences recombination frequencies in this highly diverse bacterium and demonstrate its importance in maintaining genomic integrity by limiting recombination events. HP1523 shows homology to RecG, an ATP-dependent helicase that in Escherichia coli allows repair of damaged replication forks to proceed without recourse to potentially mutagenic recombination. Cross-species studies done show that hp1523 can complement E. coli recG mutants in trans to the same extent as E. coli recG can, indicating that hp1523 has recG function. The E. coli recG gene only partially complements the hp1523 mutation in H. pylori. Unlike other recG homologs, hp1523 is not involved in DNA repair in H. pylori, although it has the ability to repair DNA when expressed in E. coli. Therefore, host context appears critical in defining the function of recG. The fact that in E. coli recG phenotypes are not constant in other species indicates the diverse roles for conserved recombination genes in prokaryotic evolution.     

Biochemical investigations of control of replication initiation of plasmid R6K

The mechanistic basis of control of replication initiation of plasmid R6K was investigated by addressing the following questions. What are the biochemical attributes of mutations in the pi initiator protein that caused loss of negative control of initiation? Did the primary control involve only initiator protein-ori DNA interaction or did it also involve protein-protein interactions between pi and several host-encoded proteins? Mutations at two different regions of the pi-encoding sequence individually caused some loss of negative control as indicated by a relatively modest increase in copy number. However, combinations of the mutation P42L, which caused loss of DNA looping, with those located in the region between the residues 106 and 113 induced a robust enhancement of copy number. These mutant forms promoted higher levels of replication in vitro in a reconstituted system consisting of 22 purified proteins. The mutant forms of pi were susceptible to pronounced iteron-induced monomerization in comparison with the WT protein. As contrasted with the changes in DNA-protein interaction, we found no detectable differences in protein-protein interaction between wild type pi with DnaA, DnaB helicase, and DnaG primase on one hand and between the high copy mutant forms and the same host proteins on the other. The DnaG-pi interaction reported here is novel. Taken together, the results suggest that both loss of negative control due to iteron-induced monomerization of the initiator and enhanced iteron-initiator interaction appear to be the principal causes of enhanced copy number.     

On-line oxygen uptake rate and culture viability measurement of animal cell culture using microplates with integrated oxygen sensors

O2 uptake rates of animal cells (Chinese hamster ovary-CHO) were measured in 96-well microtiter plates by integrating with fluorescent sensors thereby measuring fluorescence intensity ratios of an O2-sensitive and an insensitive fluorophor. O2 consumption rate was estimated from measured dissolved O2 and from O2 mass transfer coefficient determined in advance. Specific uptake decreased with time from 3.2 x 10(-13) mol O2 cell(-1) h(-1) at 15 h cultivation to 1.8 x 10(-13) mol O2 cell(-1) h(-1) at 48 h. Specific O2 uptake was also determined by sampling from a spinner-flask culture giving identical values. A cell viability assay for cultures based on O2 measurements is described in which cells are incubated outside the fluorescence reader and then the dissolved O2 is measured only once at a fixed time after the start of incubation. This protocol can be directly applied for high-throughput measurements.     

Structural bases for CRMP function in plexin-dependent semaphorin3A signaling

Collapsin response mediator proteins (CRMPs) are cytosolic phosphoproteins involved in neuronal differentiation and axonal guidance. CRMP2 was previously shown to mediate the repulsive effect of Sema3A on axons and to participate in axonal specification. The X-ray crystal structure of murine CRMP1 was determined at 2.1 A resolution and demonstrates that CRMP1 is a bilobed 'lung-shaped' protein forming a tetrameric assembly. Structure-based mutagenesis of surface-exposed residues was employed to map functional domains. As a rapid assay for CRMP, we exploited a reconstituted Sema3A signaling system in COS-7 cells expressing the receptor components Neuropilin1 and PlexinA1 (NP1/PlexA1). In these cells, CRMP and PlexA1 form a physical complex that is reduced in amount by NP1 but enhanced by Sema3A/NP1. Furthermore, CRMP accelerates Sema3A-induced cell contraction. Alanine substitutions in one domain of CRMP1 produce a constitutively active protein that causes Sema3A-independent COS-7 contraction. This mutant CRMP mimics the DRG neurite outgrowth-inhibiting effects of Sema3A and reduces Sema3A-induced axonal repulsion. These data provide a structural view of CRMP function in Plex-dependent Sema3A signaling.     

Synthesis and blood-schizontocidal antimalarial activities of 2-substituted/2,5-disubstituted-8-quinolinamines and some of their amino acid conjugates

Thirteen new analogues (32-40, 45-48) of recently discovered potent blood-schizontocidal antimalarial agent, 2-tert-butylprimaquine (2) are synthesized and evaluated for in vivo antimalarial activities against drug-sensitive P. berghei strain and multi-drug resistant P. yoelii nigeriensis strain. Two of the amino acid conjugates (47-48) have exhibited potent antimalarial activities similar to that of 2 against both drug-sensitive and multi-drug resistant strains.     

Synthesis and antimycobacterial activities of ring-substituted quinolinecarboxylic acid/ester analogues. Part 1

Structural optimization of recently discovered new chemical entity, 2,8-dicyclopentyl-4-methylquinoline (DCMQ; MIC= 6.25 microg/mL, M. tuberculosis H37Rv) resulted in the synthesis of four new series of ring-substituted quinolinecarboxylic acids/esters constituting 45 analogues. All new derivatives were evaluated for in vitro antimycobacterial activities against M. tuberculosis H37Rv. Certain ring-substituted-2-quinolinecarboxylic acid ester and ring-substituted-2-quinoline acetic acid ester analogues described herein showed moderate to good inhibitory activity. In particular, three analogues methyl 4,5-dicyclopentyl-2-quinolinecarboxylate (3b), methyl 4,8-dicyclopentyl-2-quinolinecarboxylate (3c) and ethyl 2-(2,8-dicyclopentyl-4-quinolyl)acetate (14g) exhibited excellent MIC values of 1.00, 2.00 and 4.00microg/mL, respectively. Results obtained indicate that substitution of the quinoline ring with dicyclopentyl substituent presumably enhances the antimycobacterial activities in the quinoline analogues described herein.     

Mitochondrial DNA of Hydra attenuata (Cnidaria): A Sequence That Includes an End of One Linear Molecule and the Genes for l-rRNA, tRNAf-Met, tRNATrp, COII, and ATPase8

The 3231-nucleotide-pair (ntp) sequence of one end of one of the two linear mitochondrial (mt) DNA molecules of Hydra attenuata (phylum Cnidaria, class Hydrozoa, order Anthomedusae) has been determined. This segment contains complete genes for tRNA^f-Met, l-rRNA, tRNA^Trp, subunit 2 of cytochrome c oxidase (COII), subunit 8 of ATP synthetase (ATPase8), and the 5′ 136 ntp of ATPase6. These genes are arranged in the order given and are transcribed from the same strand of the molecule. As in two other cnidarians, the hexacorallian anthozoan Metridium senile and the octocorallian anthozoan Sarcophyton glaucum, the mt-genetic code of H. attenuata is near standard. The only modification appears to be that TGA specifies tryptophan rather than termination. Also as in M. senile and S. glaucum, the encoded H. attenuata mt-tRNA^f-Met has primary and secondary structural features resembling those of Escherichia coli initiator tRNA^t-Met. As the encoded mt-tRNA^Trp cannot be folded into a totally orthodox secondary structure, two alternative forms are suggested. The encoded H. attenuata mt-l-rRNA is 1738 nt, which is 451 nt shorter than the M. senile mt-l-rRNA. Comparisons of secondary structure models of these two mt-l-rRNAs indicate that most of the size difference results from loss of nucleotides in the H. attenuata molecule at a minimum of 46 locations, which includes elimination of six distinct helical elements. Received: 9 March 2000 / Accepted: 24 July 2000     

Butyrated ManNAc analog improves protein expression in Chinese hamster ovary cells

The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4‐O‐Bu₃ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4‐O‐Bu₃ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation. In contrast, sodium butyrate treatment resulted in a ～20% decrease in maximal viable cell density and ～30% decrease in cell viability at the end of cell cultures compared to untreated or 1,3,4‐O‐Bu₃ManNAc treated CHO cell lines for both products. While NaBu treatment enhanced product yields more than the 1,3,4‐O‐Bu₃ManNAc treatment, the NaBu treated cells also exhibited higher levels of caspase 3 positive cells using microscopy analysis. Furthermore, the mRNA levels of four cell apoptosis genes (Cul2, BAK, BAX, and BCL2L11) were up‐regulated more in sodium butyrate treated wild‐type, erythropoietin, or IgG expressing CHO‐K1 cell lines while most of the mRNA levels of apoptosis genes in 1,3,4‐O‐Bu₃ManNAc treated cell lines remained equal or increased only slightly compared to the levels in untreated CHO cell lines. Finally, lectin blot analysis revealed that the 1,3,4‐O‐Bu₃ManNAc‐treated cells displayed higher relative sialylation levels on recombinant EPO, consistent with the effect of the ManNAc component of this additive, compared to control while NaBu treatment led to lower sialylation levels than control, or 1,3,4‐O‐Bu₃ManNAc‐treatment. These findings demonstrate that 1,3,4‐O‐Bu₃ManNAc has fewer negative effects on cell cytotoxicity and apoptosis, perhaps as a result of a more deliberate uptake and release of the butyrate compounds, while simultaneously increasing the expression of multiple recombinant proteins, and improving the glycosylation characteristics when applied at comparable molarity levels to NaBu. Thus, 1,3,4‐O‐Bu₃ManNAc represents a highly promising media additive alternative in cell culture for improving protein yields without sacrificing cell mass and product quality in future bioproduction processes.