Akt Activation Emulates Chk1 Inhibition and Bcl2 Overexpression and Abrogates G2 Cell Cycle Checkpoint by Inhibiting BRCA1 Foci

Akt is perhaps the most frequently activated oncoprotein in human cancers. Overriding cell cycle checkpoint in combination with the inhibition of apoptosis are two principal requirements for predisposition to cancer. Here we show that the activation of Akt is sufficient to promote these two principal processes, by inhibiting Chk1 activation with concomitant inhibition of apoptosis. These activities of Akt cannot be recapitulated by the knockdown of Chk1 alone or by overexpression of Bcl2. Rather the combination of Chk1 knockdown and Bcl2 overexpression is required to recapitulate Akt activities. Akt was shown to directly phosphorylate Chk1. However, we found that Chk1 mutants in the Akt phosphorylation sites behave like wild-type Chk1 in mediating G2 arrest, suggesting that the phosphorylation of Chk1 by Akt is either dispensable for Chk1 activity or insufficient by itself to exert an effect on Chk1 activity. Here we report a new mechanism by which Akt affects G2 cell cycle arrest. We show that Akt inhibits BRCA1 function that induces G2 cell cycle arrest. Akt prevents the translocation of BRCA1 to DNA damage foci and, thereby, inhibiting the activation of Chk1 following DNA damage.     

Assignment of the prealbumin (PALB) gene (familial amyloidotic polyneuropathy) to human chromosome region 18q11.2–q12.1

Summary The assignment of the human prealbumin (PALB) gene to chromosome region 18q11–q12.1 has been achieved using a human genomic probe in the study of human-mouse somatic cell hybrids and by in situ hybridization. Because familial amyloidotic polyneuropathy was reported previously to be due to a mutation in prealbumin, it can be inferred that the gene for this disorder also maps to 18q11.2–q12.1.     

Regulation of branched-chain amino acid biosynthesis in Streptomyces fradiae,a producer of tylosin

Synthesis of threonine dehydratase in Streptomyces fradiae was positively influenced by valine and negatively by isoleucine. However, these two amino acids had no effect on the activity of this enzyme. Synthesis of threonine dehydratase in -aminobutyrate resistant mutants of S. fradiae was pronouncedly less sensitive to the positive effect of valine and this change in regulation led to valine overproduction. Synthesis of acetohydroxy acid synthase is regulated in a similar manner to that of threonine dehydratase, however a lower level of expression was detected in -aminobutyrate resistant mutants. And again, no effect of branched-chain amino acids on acetohydroxy acid synthase activity was observed. It follows that in S. fradiae synthesis of threonine dehydratase is the main regulatory mechanism governing production and the mutual ratio of synthesized valine and isoleucine.Abbreviations -AB -aminobutyrate - AHAS acetohydroxy acid synthase - -KB -ketobutyrate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - TD threonine dehydratase - Trans. B. transaminase of branched-chain amino acids - VDH valine dehydrogenase     

Functional and ultrastructural effects of nontypeable haemophilus influenzae in a hamster trachea organ culture system

Summary A hamster trachea organ culture system was utilized to evaluate quantitatively the effects of a strain of nontypeableHaemophilus influenzae (NTHI) and culture supernatants of the same strain on ciliary activity. Tracheal explants were maintained in organ culture for 96 to 144 h and ciliary activity was observed daily with an inverted microscope. Explants continuously exposed to a strain of NTHI had a progressive decline in ciliary activity which was significantly lower than uninfected controls evaluated concomitantly by 48 h of exposure and thereafter. Histologic studies revealed a progressive degeneration of mucosal cells and exfoliation of ciliated cells. Scanning electron microscopy showed little adherence of NTHI to the mucosal surface. Sterile broth cultures of NTHI and supernatants of organ cultures infected with the same NTHI strain had no adverse effect on ciliary activity. Infected tracheal explants treated with ampicillin 24, 48, or 72 h after continuous bacterial challenge had no significant decline in ciliary activity compared to controls. The lack of adherence and the histologic changes observed when hamster trachea cultures were infected with NTHI suggested a toxin might mediate the damage observed. Broth and organ culture supernatants, however, produced no damage. Therefore, further studies are needed to determine the role, if any, of a toxin in the production of damage to hamster tracheal explants by NTHI. This work was supported by a Merit Review grant from the Veterans Administration and by Grant AI-19641 from the National Institute of Allergy and Infectious Diseases.     

Assignment of the αB-crystallin gene to human chromosome 11

Using a human αB-crystallin genomic probe and human-mouse somatic cell hybrids, the human αB-gene was assigned to chromosome 11 and further corroborated by in situ hybridization to normal metaphase chromosomes. This assignment confirmed and regionally mapped the locus to q22.3–23.1.     

Respiratory Rate as a Regulatory Factor in the Biosynthesis of the Denitrification Pathway of the Bacterium Paracoccus denitrificans

The decrease in the electron flow of the aerobic respiratory chain of the bacterium Paracoccus denitrificans, owing to either the drop in the saturation of terminal oxidases by oxygen or to the inhibition of the rate of respiration by azide or nitrite, resulted in the synthesis of dissimilatory nitrate reductase and nitrite reductase. The dependence of the resulting activities of the two enzymes (after a three-hour adaptation) on the initial value of the parameter V_max/k_La (oxidase activity of the volume unit of the culture divided by the volumetric oxygen transfer coefficient) or on the concentrations of the inhibitors had a similar form, characterized by the appearance of a maximum. The increasing parts of the obtained curves reflect the synthesis of enzymes, probably initiated by the increase in the intracellular degree of reduction, the subsequent drop being evidently in connection with the lack of metabolic energy for biosynthesis. The possible mechanisms of the effect of nitrogenous terminal acceptors (NO^-₃ and NO^-₂) on the formation of the denitrification pathway are discussed.     

Lipooligosaccharide biosynthesis in Neiseria gonorrhoeae: cloning, identification and characterization of the α1,5 heptosyltransferase I gene (rfaC)

The Escherichia coli arginine repressor (ArgR) is an l -arginine-dependent DNA-binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site-specific recombination at cer and related recombination sites in plasmids. Site-directed mutagenesis was used to isolate two mutants of E. coli ArgR that were defective in arginine binding. Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine-independent manner. Both mutants support Xer site-specific recombination at cer. One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild-type ArgR.