SOS and UVM pathways have lesion-specific additive and competing effects on mutation fixation at replication-blocking DNA lesions

Escherichia coli cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli. Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response. In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3,N4-ethenocytosine (epsilonC) and abasic (AP) sites. Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective E. coli strains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD' and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous lac-derived promoter. M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway. Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure. Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at epsilonC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion. Although mutagenesis at epsilonC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for epsilonC under SOS and UVM pathways are distinct. Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at epsilonC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced. These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (epsilonC) but also at classical SOS-dependent (class 1) lesions such as AP sites. Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network.     

Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion

We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin's action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation.     

Possible evidence for shift work schedules in the media workers of the ant species Camponotus compressus

The locomotor activity rhythm of the media workers of the ant species Camponotus compressus was monitored under constant conditions of the laboratory to understand the role of circadian clocks in social organization. The locomotor activity rhythm of most ants entrained to a 24 h light/dark (12:12 h; LD) cycle and free-ran under constant darkness (DD) with circadian periodicities. Under entrained conditions about 75% of media workers displayed nocturnal activity patterns, and the rest showed diurnal activity patterns. In free-running conditions these ants displayed three types of activity patterns (turn-around). The free-running period (τ) of the locomotor activity rhythm of some ants (10 out of 21) showed period lengthening, and those of a few (6 out of 21) showed period shortening, whereas the locomotor activity rhythm of the rest of the ants (5 out of 21) underwent large phase shifts. Interestingly, the pre-turn-around τ of those ants that showed nocturnal activity patterns during earlier LD entrainment was shorter than 24 h, which became greater than 24 h after 6-9 days of free-run in DD. On the other hand, the pre-turn-around τ of those ants, which exhibited diurnal patterns during earlier LD entrainment, was greater than 24 h, which became shorter than 24 h after 6-9 days of free-run in DD. The patterns of activity under LD cycles and the turn-around of activity patterns in DD regime suggest that these ants are shift workers in their respective colonies, and they probably use their circadian clocks for this purpose. Circadian plasticity thus appears to be a general strategy of the media workers of the ant species C. compressus to cope with the challenges arising due to their roles in the colony constantly exposed to a fluctuating environment.     

Local Perfusion of Nicotine Differentially Modulates Somatodendritic Dopamine Release in the Rat Ventral Tegmental Area After Nicotine Preexposure

We examined the effects of nicotine perfusion into the ventral tegmental area (VTA) on extracellular dopamine (DA) levels in rats using in vivo microdialysis. Local perfusion with nicotine for 80 min (10–100 M) modestly increased (105–131% of basal) the extracellular DA levels in the VTA of rats that had been pretreated with saline for 5 days. In animals that had been pretreated with nicotine for 5 days (0.3 mg/kg, s.c.), perfusion with nicotine for 80 min (10–100 M) dose-dependently increased the extracellular DA levels in the VTA of rats and did so to a greater extent than in saline-pretreated animals (125–171% of basal). Co-perfusion through the dialysis probe with 100 M mecamylamine, a nonselective nicotinic acetylcholine receptor (nAChR) antagonist, or 100 M dihydro--erythroidine, a high affinity and competitive nAChR antagonist, attenuated the enhancement of extracellular DA levels produced by 100 M nicotine alone. These results suggest that local nicotine challenge potentiated the somatodendritic DA release after nicotine preexposure by stimulation of high-affinity nAChRs in the VTA.     

Structure-function analysis of the presumptive Arabidopsis auxin permease AUX1

We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain.     

A repressor element in the 5'-untranslated region of human Pax5 exon 1A

Five members of the RecQ helicase family, RECQL, WRN, BLM, RTS and RECQL5, have been found in human and three of them (WRN, BLM and RTS) were disclosed to be the genes responsible for Werner, Bloom and Rothmund–Thomson syndromes, respectively. RECQL5 (RecQ helicase protein-like 5) was isolated as the fifth member of the family in humans through a search of homologous expressed sequence tags. The gene is expressed with at least three alternative splicing products, , β and γ. Here, we isolated mouse RECQL5β and determined the DNA sequence of full-length cDNA as well as the genome organization and chromosome locus. The mouse RECQL5β gene consists of 2949 bp coding 982 amino acid residues. Comparison of amino acid sequence among human (Homo sapiens), mouse (Mus musculus), Drosophila melanogaster and Caenorhabditis elegans RECQL5β homologs revealed three portions of highly conserved regions in addition to the helicase domain. Nineteen exons are dispersed over 40 kbp in the genome and all of the acceptor and donor sites for the splicing of each exon conform to the GT/AG rule. The gene is localized to the mouse chromosome 11E2, which has a syntenic relation to human 17q25.2-q25.3 where human RECQL5β exists. Our genetic characterizations of the mouse RECQL5β gene will contribute to functional studies on the RECQL5β products.     

The immunomodulatory effects of gentamicin, imipenem, piperacillin and amphotericin B on LAK effector function in vitro

An understanding of the immunomodulating effects of anti-microbial regimens on recombinant interleukin-2 (rIL-2) induced peripheral leukocyte function, i.e. lymphokine-activated killer (LAK)-cell efficacy, would be clinically useful in the selection of commonly employed bone marrow transplantation (BMT) antibiotics to avoid post-transplant complications and optimize anti-microbial, anti-viral, anti-tumor therapies. In this report we evaluated the modulatory effects of a number of antibiotics used in BMT on LAK-cell cytotoxicities, in vitro. Our data showed that, even at serum trough titer, amphotericin B was significantly (P < or =0.05) immunostimulatory, whereas gentamicin, imipenem, and piperacillin, individually, were significantly (P < or =0.05) immunosuppressive. Statistical analysis detected no modulation due to aztreonam, amikacin, cotrimoxazole, or ceftazidime, or any of the six cephalosporins tested at molar equivalent concentration. We conclude that certain antibiotics may be more suitable for infection prone BMT hosts.     

Acute and sub-acute effects of 2-butenoic acid-3-(diethoxy phosphinothioyl) methyl ester (RPR-II) on testis of albino rat

Acute and sub-acute toxic effects of a novel phosphorothionate coded as RPR-II on testis of albino rats were studied. In acute study rats received a single dose of 12.3 mg/kg of RPR-II and sacrificed after 24 hr. For sub-acute study 0.58 mg/kg/day was administered orally to rats for 10 and 21 days. Acute exposure of rats to RPR-II brought no change either in the gonadosomatic index (GSI) or in the structure of testis or in the serum levels of testosterone. Testis glutathione (GSH) level and glutathione S-transferase (GST) activity was significantly decreased whereas, acid phosphatase (AcP) levels increased significantly at 24 hr post-treatment. On 7th day (withdrawal period) after the cessation of the treatment the GSH, GST, AcP, and AkP levels reached to near control. The sub-acute study revealed a significant decrease in GSI on 10th and 21st day of the treatment. In contrast, a time-dependent and significant increased in GSH level and GST activity was observed on 100th and 21st day of post-treatment, except GSH level on 10th day, which was declined. Due to RPR-II treatment the testis AcP and alkaline phosphatase (AkP) levels were significant at both 10th and 21st day of medication but AcP levels were increased whereas AkP levels decreased. The histopathological studies on day 10th showed considerable loss of spermatozoids in testis and at 21st day complete derangement of cellular organization was observed. Testosterone levels decreased significantly on 10th day and remained significantly low at 21st day. However, withdrawal studies showed a recovery in testis of rat treated with RPR-II. GST, GSH, GSI, AcP and AkP values recovered, testosterone levels were also well recovered but recovery in testis structure remained at a low profile. The present study suggests that RPR-II may cause testicular toxicity in rats affecting the normal functioning of testis and it also gave some new information in withdrawal studies.     

The gene for juvenile hyaline fibromatosis maps to chromosome 4q21

Juvenile hyaline fibromatosis (JHF) is an autosomal recessive condition characterized by multiple subcutaneous nodular tumors, gingival fibromatosis, flexion contractures of the joints, and an accumulation of hyaline in the dermis. We performed a genomewide linkage search in two families with JHF from the same region of the Indian state of Gujarat and identified a region of homozygosity on chromosome 4q21. Dense microsatellite analyses within this interval in five families with JHF who were from diverse origins demonstrate that all are compatible with linkage to chromosome 4q21 (multipoint LOD score 5.5). Meiotic recombinants place the gene for JHF within a 7-cM interval bounded by D4S2393 and D4S395.