Poly(ADP-ribose) polymerase null mouse cells synthesize ADP-ribose polymers

Poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30), the only enzyme known to synthesize ADP-ribose polymers from NAD+, is activated in response to DNA strand breaks and functions in the maintenance of genomic integrity. Mice homozygous for a disrupted gene encoding PARP are viable but have severe sensitivity to gamma-radiation and alkylating agents. We demonstrate here that both 3T3 and primary embryo cells derived from PARP-/- mice synthesized ADP-ribose polymers following treatment with the DNA-damaging agent, N-methyl-N'-nitro-N-nitrosoguanidine, despite the fact that no PARP protein was detected in these cells. ADP-ribose polymers isolated from PARP-/- cells were indistinguishable from that of PARP+/+ cells by several criteria. First, they bound to a boronate resin selective for ADP-ribose polymers. Second, treatment of polymers with snake venom phosphodiesterase and alkaline phosphatase yielded ribosyladenosine, a nucleoside diagnostic for the unique ribosyl-ribosyl linkages of ADP-ribose polymers. Third, they were digested by treatment with recombinant poly(ADP-ribose) glycohydrolase, an enzyme highly specific for ADP-ribose polymers. Collectively, these data demonstrate that ADP-ribose polymers are formed in PARP-/- cells in a DNA damage-dependent manner. Because the PARP gene has been disrupted, these results suggest the presence of a previously unreported activity capable of synthesizing ADP-ribose polymers in PARP-/- cells.     

Rescue of thymocytes from glucocorticoid-induced cell death mediated by CD28/CTLA-4 costimulatory interactions with B7-1/B7-2

During the differentiation of thymocytes to mature T cells the processes of positive and negative selection result in signals that either protect thymocytes from cell death, or delete, through apoptosis, thymocytes with self-reactive T cell receptors (TCR). Glucocorticoids have been shown to induce thymocyte apoptosis and are produced within the thymic microenvironment. Furthermore, steroid-induced apoptosis of thymocytes has been suggested as a potential mechanism for removal of nonselected thymocytes. In this report, we demonstrate that thymocytes can be rescued from glucocorticoid-induced apoptosis by incubation with cells that express high levels of B7-1 or B7-2. In addition, the ability to be rescued by B7-1 and/or B7-2 can precede expression of the TCR. We demonstrate that CD3(+)-depleted or CD3+/ TCR-beta(+)-doubly depleted thymocytes can be rescued from glucocorticoid-induced apoptosis through the interaction of CD28 or CTLA-4 on thymocytes with cells bearing high levels of B7-1 or B7-2. Furthermore, these transfected cells are major histocompatibility complex (MHC) class II negative and, while they may express MHC class I, there is no preferential rescue of CD8+ thymocytes in the presence of glucocorticoids. Together, these data suggest that the rescue of thymocytes from glucocorticoids can be independent of the TCR. We also demonstrate that, in addition to CD28, CTLA-4 is expressed on thymocytes, suggesting that rescue from glucocorticoid-induced cell death can be mediated by both CD28 and CTLA-4. A CTLA-4Ig fusion protein which binds to both B7-1 and B7-2 was shown to completely block the rescue of thymocytes from glucocorticoid-induced cell death. Therefore, we conclude that interactions between B7-1/B7-2 and CD28/CTLA-4 are sufficient and necessary for rescue of thymocytes from glucocorticoid-induced cell death.     

Role of opioids in chronic non-cancer pain

A familial investigation was made in three families with deafness patients caused by aminoglycoside (AMI). The results showed that there was cross susceptibility among a few AMI antibiotics. The people with familial history of deafness caused by AMI were easier to be toxic than those without familial history, even though little dose of AMI for the former, especially for children. The cross susceptibility is dominated by inheritance of matriarchal heredity and by general chromosome. The data suggest that medical history should be inquired before treatment with AMI, and patients with matriarchal heredity must be prohibited from using AMI.     

An improved analysis for chlorinated pesticides and polychlorinated biphenyls (PCBs) in human and bovine sera using solid-phase extraction

Chlorinated pesticides and polychlorinated biphenyls (PCBs) remain public health concerns because of their unresolved health impact and their persistence in humans. Current epidemiological studies of cancer, non-Hodgkins lymphoma, and endocrine disruption in National Center for Environmental Health (NCEH) laboratories require exposure assessment of many analytes in thousands of people. Previous methods of analyzing pesticides and PCBs in serum have proven inadequate for timely processing of the number of samples required for epidemiological studies. A new method that involves solid-phase extraction (SPE) and cleanup followed by dual-column gas chromatographic separation and electron capture detection has been developed. Nine surrogate compounds were added to the serum prior to sample workup to provide quality assurance for the SPE steps. These surrogates mimic the chemistry of the analytes in the extraction, cleanup, and gas chromatographic analysis steps. To increase selectivity, extracts were injected onto two gas chromatographs with different capillary columns, a DB-1701 and a DB-5. Recoveries of 17 pesticides, 28 PCB congeners, and one polybrominated biphenyl congener ranged from 40 to 80%. Recoveries from this procedure were found to be similar to those from the previously used liquid-liquid extraction method. Correlation of analyte and surrogate recoveries were compared to examine the ruggedness of the technique. The SPE method was found to provide improved sample throughput by a factor of 15.     

katG mutations in isoniazid-resistant Mycobacterium tuberculosis isolates recovered from Finnish patients

katG and inhA genes from isoniazid-resistant Mycobacterium tuberculosis strains isolated in Finland were examined by PCR or sequencing. By PCR, katG was not detected in 3 of 54 strains. Sequencing of katG from 13 strains showed small point mutations or insertions; a previously described mutation causing a Ser-to-Thr change at position 315 was found in 4 strains, and there were nine new missense mutations of katG. A 209-bp segment of inhA from 17 strains was sequenced, but no mutations were observed. This result indicates that different mutations prevail in different geographical areas.     

Adjunctive use of a subgingival controlled-release chlorhexidine chip reduces probing depth and improves attachment level compared with scaling and root planing alone

The present studies evaluated the efficacy of a controlled-release biodegradable chlorhexidine (CHX) (2.5 mg) chip when used as an adjunct to scaling and root planing on reducing probing depth (PD) and improving clinical attachment level (CAL) in adult periodontitis. Two double-blind, randomized, placebo-controlled multi-center clinical trials (5 centers each) were conducted; pooled data are reported from all 10 centers (447 patients). At baseline, following 1 hour of scaling and root planing (SRP) in patients free of supragingival calculus, the chip was placed in target sites with PD 5 to 8 mm which bled on probing. Chip placement was repeated at 3 and/or 6 months if PD remained > or = 5 mm. Study sites in active chip subjects received either CHX chip plus SRP or SRP alone (to maintain study blind). Sites in placebo chip subjects received either placebo chip plus SRP or SRP alone. Examinations were performed at baseline; 7 days; 6 weeks; and 3, 6, and 9 months. At 9 months significant reductions from baseline favoring the chlorhexidine chip compared with both control treatments were observed with respect to PD (chlorhexidine chip plus SRP, 0.95 +/- 0.05 mm; SRP alone, 0.65 +/- 0.05 mm, P < 0.001; placebo chip plus SRP, 0.69 +/- 0.05 mm, P < 0.001) and CAL (chlorhexidine chip plus SRP, 0.75 +/- 0.06 mm; SRP alone, 0.58 +/- 0.06 mm, P < 0.05; placebo chip plus SRP, 0.55 +/- 0.06 mm, P < 0.05). The proportion of patients who evidenced a PD reduction from baseline of 2 mm or more at 9 months was significantly greater in the chlorhexidine chip group (19%) compared with SRP controls (8%) (P < 0.05). Adverse effects were minor and transient toothache, including pain, tenderness, aching, throbbing, soreness, discomfort, or sensitivity was the only adverse effect that was higher in the chlorhexidine group as compared to placebo (P = 0.042). These data demonstrate that the adjunctive use of the chlorhexidine chip results in a significant reduction of PD when compared with both SRP alone or the adjunctive use of a placebo chip. These multi-center randomized control trials suggest that the chlorhexidine chip is a safe and effective adjunctive chemotherapy for the treatment of adult periodontitis.     

Troglitazone improves defects in insulin action, insulin secretion, ovarian steroidogenesis, and fibrinolysis in women with polycystic ovary syndrome

Women with polycystic ovary syndrome (PCOS) are characterized by defects in insulin action, insulin secretion, ovarian steroidogenesis, and fibrinolysis. We administered the insulin-sensitizing agent troglitazone to 13 obese women with PCOS and impaired glucose tolerance to determine whether attenuation of hyperinsulinemia ameliorates these defects. All subjects had oligomenorrhea, hirsutism, polycystic ovaries, and hyperandrogenemia. Before and after treatment with troglitazone (400 mg daily for 12 weeks), all had 1) a GnRH agonist (leuprolide) test, 2) a 75-g oral glucose tolerance test, 3) a frequently sampled iv glucose tolerance test to determine the insulin sensitivity index and the acute insulin response to glucose, 4) an oscillatory glucose infusion to assess the ability of the beta-cell to entrain to glucose as quantitated by the normalized spectral power for the insulin secretion rate, and 5) measures of fibrinolytic capacity [plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator]. There was no change in body mass index (39.9 +/- 1.4 vs. 40.2 +/- 1.4 kg/m2) or body fat distribution after treatment. Both the fasting (91 +/- 3 vs. 103 +/- 3 mg/dL; P < 0.001) and 2 h (146 +/- 8 vs. 171 +/- 6 mg/dL; P < 0.02) plasma glucose concentrations during the oral glucose tolerance test declined significantly. There was a concordant reduction in glycosylated hemoglobin to 5.7 +/- 0.1 from a pretreatment level of 6.1 +/- 0.1% (P < 0.03). Insulin sensitivity increased from 0.58 +/- 0.14 to 0.95 +/- 0.26 10(-5) min-1/pmol.L (P < 0.01) after treatment as did the disposition index (745 +/- 135 vs. 381 +/- 96; P < 0.05). The ability of the beta-cell to appropriately detect and respond to an oscillatory glucose infusion improved significantly after troglitazone treatment; the normalized spectral power for the insulin secretion rate increased to 5.9 +/- 1.1 from 4.3 +/- 0.8 (P < 0.05). Basal levels of total testosterone (109.3 +/- 15.2 vs. 79.4 +/- 9.8 ng/dL; P < 0.05) and free testosterone (33.3 +/- 4.0 vs. 21.2 +/- 2.6 pg/mL; P < 0.01) declined significantly after troglitazone treatment. Leuprolide-stimulated levels of 17-hydroxyprogesterone, androstenedione, and total testosterone were significantly lower posttreatment compared to pretreatment. The reduction in androgen levels occurred independently of any changes in gonadotropin levels. A decreased functional activity of PAI-1 in blood (from 12.7 +/- 2.8 to 6.3 +/- 1.4 AU/mL P < 0.05) was associated with a decreased concentration of PAI-1 protein (from 64.9 +/- 9.1 to 44.8 +/- 6.1 ng/mL; P < 0.05). No change in the functional activity of tissue plasminogen activator (from 5.3 +/- 0.4 to 5.1 +/- 0.5 IU/mL) was observed despite a decrease in its concentration (from 9.6 +/- 0.9 to 8.2 +/- 0.7 ng/mL; P < 0.05). The marked reduction in PAI-1 could be expected to improve the fibrinolytic response to thrombosis in these subjects. We conclude that administration of troglitazone to women with PCOS and impaired glucose tolerance ameliorates the metabolic and hormonal derangements characteristic of the syndrome. Troglitazone holds potential as a useful primary or adjunctive treatment for women with PCOS.     

Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila

The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein (Suhw) that binds to gypsy retrovirus insertions between enhancers and promoters. Suhw bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer-promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. This implicates Chip in enhancer-promoter communication. We cloned Chip and find that it encodes a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins that bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is present in all nuclei examined and at numerous sites along the salivary gland polytene chromosomes. Embryos without Chip activity lack segments and show abnormal gap and pair-rule gene expression, although no LIM domain proteins are known to regulate segmentation. We conclude that Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. We suggest that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer-promoter interactions.     

Constitutive intra-articular expression of human IL-1 beta following gene transfer to rabbit synovium produces all major pathologies of human rheumatoid arthritis

To investigate the pathophysiologic effects of chronically elevated intra-articular levels of IL-1 beta, we used an ex vivo gene transfer method to deliver and express human IL-1 beta (hIL-1 beta) in the knee joints of rabbits. Expression of hIL-1 beta resulted in a severe, highly aggressive form of arthritis analogous to chronic rheumatoid arthritis in humans. Intra-articular manifestations included intense inflammation, leukocytosis, synovial hypertrophy and hyperplasia, and highly aggressive pannus formation with erosion of the articular cartilage and periarticular bone. Systemic effects were also observed, including diarrhea, fever, weight loss, and an increased erythrocyte sedimentation rate. In addition, the hIL-1 beta was found to induce elevated levels of both rabbit IL-1 beta and TNF-alpha in synovial fluid. Following the loss of hIL-1 beta transgene expression between 14 and 28 days post-transplantation, many of these changes began to normalize. These results suggest that chronically elevated intra-articular levels of IL-1 beta alone are sufficient to produce virtually all the pathologies found in rheumatoid arthritis, and furthermore, demonstrate that gene transfer can be used to investigate the roles of specific gene products in the pathogenesis of arthritis.