Donor- and ligand-dependent differences in C-C chemokine receptor 5 reexpression

N-terminal modifications of the chemokine RANTES bind to C-C chemokine receptor 5 (CCR5) and block human immunodeficiency virus type 1 (HIV-1) infection with greater efficacy than native RANTES. Modified RANTES compounds induce rapid CCR5 internalization and much slower receptor reexpression than native RANTES, suggesting that receptor sequestration is one mode of anti-HIV activity. The rates of CCR5 internalization and reexpression were compared using the potent n-nonanoyl (NNY)-RANTES derivative and CD4(+) T cells derived from donors with different CCR5 gene polymorphisms. NNY-RANTES caused even more rapid receptor internalization and slower reexpression than aminooxypentane (AOP)-RANTES. Polymorphisms in the promoter and coding regions of CCR5 significantly affected the receptor reexpression rate after exposure of cells to NNY-RANTES. These observations may be relevant for understanding the protective effects of different CCR5 genotypes against HIV-1 disease progression.     

The Amino Acid Sequence of Ribitol Dehydrogenase-F, a Mutant Enzyme with Improved Xylitol Dehydrogenase Activity

A mutant ribitol dehydrogenase (RDH-F) was purified from Klebsiella aerogenes strain F which evolved from the wild-type strain A under selective pressure to improve growth on xylitol, a poor substrate used as sole carbon source. The ratio of activities on xylitol (500 mM) and ribitol (50 mM) was 0.154 for RDH-F compared to 0.033 for the wild-type (RDH-A) enzyme. The complete amino acid sequence of RDH-F showed the mutations. Q60 for E60 and V215 for L215 in the single polypeptide chain of 249 amino acid residues. Structural modeling based on homologies with two other microbial dehydrogenases suggests that E60 Q60 is a neutral mutation, since it lies in a region far from the catalytic site and should not cause structural perturbations. In contrast, L215 V215 lies in variable region II and would shift a loop that interacts with the NADH cofactor. Another improved ribitol dehydrogenase, RDH-D, contains an A196 P196 mutation that would disrupt a surface -helix in region II. Hence conformational changes in this region appear to be responsible for the improved xylitol specificity.     

Noninvasive ultrasonic measurement of arterial wall motion in mice

Despite the extensive use of genetically altered mice to study cardiovascular physiology and pathology, it remains difficult to quantify arterial function noninvasively in vivo. We have developed a noninvasive Doppler method for quantifying vessel wall motion in anesthetized mice. A 20-MHz probe was held by an alligator clip and positioned over the carotid arteries of 16 mice, including six 3- to 5-mo-old wild-type (WT), four 30-mo-old senescent (old), two apolipoprotein E null (ApoE), and four alpha-smooth muscle actin null (alpha-SMA) mice. Doppler signals were obtained simultaneously from both vessel walls and from blood flow. The calculated displacement signals from the near and far walls were subtracted to generate a diameter signal from which the excursion and an augmentation index were calculated. The excursion ranged between 13 microm (in ApoE) and 95 microm (in alpha-SMA). The augmentation index was lowest in the WT mice (0.06) and highest in the old mice (0.29). We conclude that Doppler signal processing may be used to measure vessel wall motion in mice with high spatial and temporal resolution and that diameter signals can replace pressure signals for calculating the augmentation index. This noninvasive method is able to identify and confirm characteristic changes in arterial properties previously associated with age, atherosclerosis, and the absence of vascular tone.     

The "Goldilocks Effect" in Cystic Fibrosis: identification of a lung phenotype in the <Emphasis Type="Italic">cftr</Emphasis> knockout and heterozygous mouse

Background  

Cystic Fibrosis is a pleiotropic disease in humans with primary morbidity and mortality associated with a lung disease phenotype. However, knockout in the mouse of cftr, the gene whose mutant alleles are responsible for cystic fibrosis, has previously failed to produce a readily, quantifiable lung phenotype.     

Saccharomyces cerevisiae RAD5 influences the excision repair of DNA minor groove adducts

Nucleotide excision repair (NER) is the primary pathway for the removal of DNA adducts that distort the double helix. In the yeast Saccharomyces cerevisiae the RAD6 epistasis group defines a more poorly characterized set of DNA damage response pathways, believed to be distinct from NER. Here we show that the elimination of the DNA minor groove adducts formed by an important class of anticancer antibiotic (CC-1065 family) requires NER factors in S. cerevisiae. We also demonstrate that the elimination of this class of minor groove adduct from the active MFA2 gene depends upon functional Rad18 and Rad6. This is most clear for the repair of adducts on the transcribed strand, where an absolute requirement for Rad6 and Rad18 was seen. Further experiments revealed that a specific RAD6-RAD18-controlled subpathway, the RAD5 branch, mediates these events. Cells disrupted for rad5 are highly sensitive to this minor groove binding agent, and rad5 cells exhibit an in vivo adduct elimination defect indistinguishable from that seen in rad6 and rad18 cells as well as in NER-defective cells. Our results indicate that the RAD5 subpathway may interact with NER factors during the repair of certain DNA adducts.     

Defects in interstrand cross-link uncoupling do not account for the extreme sensitivity of ERCC1 and XPF cells to cisplatin

The anticancer drug cisplatin reacts with DNA leading to the formation of interstrand and intrastrand cross-links that are the critical cytotoxic lesions. In contrast to cells bearing mutations in other components of the nucleotide excision repair apparatus (XPB, XPD, XPG and CSB), cells defective for the ERCC1-XPF structure-specific nuclease are highly sensitive to cisplatin. To determine if the extreme sensitivity of XPF and ERCC1 cells to cisplatin results from specific defects in the repair of either intrastrand or interstrand cross-links we measured the elimination of both lesions in a range of nucleotide excision repair Chinese hamster mutant cell lines, including XPF- and ERCC1-defective cells. Compared to the parental, repair-proficient cell line all the mutants tested were defective in the elimination of both classes of adduct despite their very different levels of increased sensitivity. Consequently, there is no clear relationship between initial incisions at interstrand cross-links or removal of intrastrand adducts and cellular sensitivity. These results demonstrate that the high cisplatin sensitivity of ERCC1 and XPF cells likely results from a defect other than in excision repair. In contrast to other conventional DNA cross-linking agents, we found that the repair of cisplatin adducts does not involve the formation of DNA double-strand breaks. Surprisingly, XRCC2 and XRCC3 cells are defective in the uncoupling step of cisplatin interstrand cross-link repair, suggesting that homologous recombination might be initiated prior to excision of this type of cross-link.