Genetics and Regulation of Chitobiose Utilization in Borrelia burgdorferi

Borrelia burgdorferi spends a significant proportion of its life cycle within an ixodid tick, which has a cuticle containing chitin, a polymer of N-acetylglucosamine (GlcNAc). The B. burgdorferi celA, celB, and celC genes encode products homologous to transporters for cellobiose and chitobiose (the dimer subunit of chitin) in other bacteria, which could be useful for bacterial nutrient acquisition during growth within ticks. We found that chitobiose efficiently substituted for GlcNAc during bacterial growth in culture medium. We inactivated the celB gene, which encodes the putative membrane-spanning component of the transporter, and compared growth of the mutant in various media to that of its isogenic parent. The mutant was no longer able to utilize chitobiose, while neither the mutant nor the wild type can utilize cellobiose. We propose renaming the three genes chbA, chbB, and chbC, since they probably encode a chitobiose transporter. We also found that the chbC gene was regulated in response to growth temperature and during growth in medium lacking GlcNAc.     

Mitochondrial targeting of vitamin E succinate enhances its pro-apoptotic and anti-cancer activity via mitochondrial complex II

Mitochondrial complex II (CII) has been recently identified as a novel target for anti-cancer drugs. Mitochondrially targeted vitamin E succinate (MitoVES) is modified so that it is preferentially localized to mitochondria, greatly enhancing its pro-apoptotic and anti-cancer activity. Using genetically manipulated cells, MitoVES caused apoptosis and generation of reactive oxygen species (ROS) in CII-proficient malignant cells but not their CII-dysfunctional counterparts. MitoVES inhibited the succinate dehydrogenase (SDH) activity of CII with IC(50) of 80 μM, whereas the electron transfer from CII to CIII was inhibited with IC(50) of 1.5 μM. The agent had no effect either on the enzymatic activity of CI or on electron transfer from CI to CIII. Over 24 h, MitoVES caused stabilization of the oxygen-dependent destruction domain of HIF1α fused to GFP, indicating promotion of the state of pseudohypoxia. Molecular modeling predicted the succinyl group anchored into the proximal CII ubiquinone (UbQ)-binding site and successively reduced interaction energies for serially shorter phytyl chain homologs of MitoVES correlated with their lower effects on apoptosis induction, ROS generation, and SDH activity. Mutation of the UbQ-binding Ser(68) within the proximal site of the CII SDHC subunit (S68A or S68L) suppressed both ROS generation and apoptosis induction by MitoVES. In vivo studies indicated that MitoVES also acts by causing pseudohypoxia in the context of tumor suppression. We propose that mitochondrial targeting of VES with an 11-carbon chain localizes the agent into an ideal position across the interface of the mitochondrial inner membrane and matrix, optimizing its biological effects as an anti-cancer drug.     

Benzomorphan alkaloids: Natural peptidomimetics of opioid peptide pharmacophores

Summary The synthesis of hybrid analogues of alkaloid nalindol acid and C-terminal fragments of opioid peptides aims to provide the final proof of the proposed topographical relation between opioid peptides and benzomorphan skeleton-containing alkaloids during interaction with receptors. The biological data indicate that the topographical requirements for Tyr¹ and Phe⁴ in opioid peptides are similar for interactions with both and receptor types.     

Independence of bacteriophage N15 lytic and linear plasmid replication from the heat shock proteins DnaJ, DnaK, and GrpE.

The chromosome of the temperate bacteriophage N15 replicates as a linear plasmid with covalently closed ends (or hairpins) when it forms a lysogen. I found that, in contrast to the cases for lambda and the low-copy-number plasmids F and P1, both phage and plasmid replication of N15 are independent of the heat shock proteins DnaJ, DnaK, and GrpE.     

Regulation of phosphoinositide hydrolysis induced by histamine and guanine nucleotides in human HeLa carcinoma cells. Calcium and pH dependence and inhibitory role of protein kinase C

The regulation of phospholipase C has been investigated in both intact and streptolysin-O permeabilized human HeLa carcinoma cells. Stimulation of phospholipase C by histamine and guanosine-5'-O-thiotriphosphate (GTP[S]) requires the presence of at least 10 nM free Ca2+, but is not significantly further increased by raising [Ca2+]i to greater than 10(-6) M. The pH optimum of the inositol phosphate response is at pH 6.8, while small changes in intracellular pH, as occur during hormonal stimulation (0.2-0.4 unit) attenuate the histamine/GTP[S]-induced stimulation of phospholipase C. Increasing cellular cAMP levels, either through addition of cell permeable cAMP analogues to intact cells or by stimulation with isoproterenol, does not affect histamine responsiveness, arguing against cross-talk between both signalling pathways. In contrast, we found that the response to histamine and/or GTP[S] is largely inhibited after brief pretreatment of the cells with phorbol esters or synthetic diacylglycerol prior to permeabilization, suggesting that protein kinase C exerts feedback inhibition at the level of, or downstream from, the putative GTP-binding protein.     

Prognostic value of response after three MOPP cycles in Hodgkin's disease--stage III and IV

Sixty-eight patients with Hodgkin's disease stage III and IV were evaluated after three out of six MOPP cycles. At that time, 46 (68%) were classified as early responders and 22 as slow responders. The criteria of response were: disappearance of B symptoms, decrease in the size of the largest lymph nodes (by more than 50%) and significant reduction (more than 20%) of mediastinal enlargement. Out of 43 early responders, 38 were in complete remission after six MOPP cycles and only five out of 22 slow responders. Such an early response is only related to the absence of B symptoms at the time of diagnosis (p less than 0.05). The survival curves of early responders and slow responders were significantly different (p less than 0.02). A rapid erythrocyte sedimentation rate (ESR) (greater than 50 mm) was the most frequently abnormal sign found in the group not responding after three MOPP cycles (p less than 0.0001). Such a significant prognostic value of early response is observed for stage III but not for stage IV patients. We conclude that early clinical response after three MOPP cycles is a good prognostic factor which must be kept in mind in the formulation of the therapeutic regimen for Hodgkin's disease stage III and IV.     

Rb fluxes in Chinese hamster ovary cells as a function of membrane cholesterol content

Steady-state fluxes of ⁸⁶Rb⁺ (as a tracer for K⁺) were measured in Chinese hamster ovary cells (CHO-K1) and a mutant (CR1) defective in the regulation of cholesterol biosynthesis; the membrane cholesterol content of this mutant was varied by growing it on a range of cholesterol supplements to lipid-free medium (Sinensky, M. (1978) Proc. Natl. Acad. Sci. U.S. 75, 1247–1249).Analogous to previous findings in ascites tumor cells, ⁸⁶Rb⁺ influx in the parent strain was differentiated into a ouabain-inhibitable ‘pump’ flux, furosemide-sensitive, chloride-dependent exchange diffusion, and a residual ‘leak’ flux.On the basis of this flux characterization, ⁸⁶Rb⁺ pump and leak fluxes were measured in the mutant as a function of membrane cholesterol content. Pump and leak fluxes, when expressed per ml cell water, were independent of the cholesterol content of the mutant. Moreover, ⁸⁶Rb⁺ fluxes in the mutant were equal to those in the parent strain. Our data imply that the flux behavior of K⁺ in the steady state is independent of the ordering of membrane lipid acyl chains.     

Current concepts in Bcl-2 family member regulation of female germ cell development and survival

Since the cloning of the bcl-2 gene in 1985, considerable progress has been made in elucidating the function of Bcl-2 and related proteins in controlling apoptosis. Although much of this work initially relied on the ectopic expression of bcl-2 gene family members in cell lines in vitro, a number of genetically manipulated mice have been generated to better understand the in vivo significance of specific family members to organ development and homeostasis. Of the many tissues that exhibit apoptosis at some point during fetal or postnatal life, the female gonads arguably possess one of the highest and most protracted incidences of apoptosis, associated with development and maturation of the germ line. Moreover, female germ cells (oocytes) are, for as-yet poorly understood reasons, extremely vulnerable to a host of pathological insults, such as anti-cancer therapies, that ultimately cause premature ovarian failure and infertility due to accelerated oocyte death. Accordingly, efforts to understand the occurrence and regulation of apoptosis in the ovary are of considerable importance from both biological and clinical perspectives. This review will highlight what is known of apoptosis in the female gonads, and the role that Bcl-2 family members play in regulating this process.