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The primary insect steroid hormone ecdysone requires a membrane transporter to enter its target cells. Although an organic anion-transporting polypeptide (OATP) named Ecdysone Importer (EcI) serves this role in the fruit fly Drosophila melanogaster and most likely in other arthropod species, this highly conserved transporter is apparently missing in mosquitoes. Here we report three additional OATPs that facilitate cellular incorporation of ecdysone in Drosophila and the yellow fever mosquito Aedes aegypti. These additional ecdysone importers (EcI-2, 3, and 4) are dispensable for development and reproduction in Drosophila, consistent with the predominant role of EcI. In contrast, in Aedes, EcI-2 is indispensable for ecdysone-mediated development, whereas EcI-4 is critical for vitellogenesis induced by ecdysone in adult females. Altogether, our results indicate unique and essential functions of these additional ecdysone importers in mosquito development and reproduction, making them attractive molecular targets for species- and stage-specific control of ecdysone signaling in mosquitoes.

In vertebrates and invertebrates, the insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) cascade is highly conserved and plays a vital role in many different physiological processes . Among the many tissues that respond to IIS in mosquitoes, the fat body has a central role in metabolism, lifespan, reproduction, and innate immunity. We previously demonstrated that fat body specific expression of active Akt, a key IIS signaling molecule, in adult Anopheles stephensi and Aedes aegypti activated the IIS cascade and extended lifespan. Additionally, we found that transgenic females produced more vitellogenin (Vg) protein than non-transgenic mosquitoes, although this did not translate into increased fecundity. These results prompted us to further examine how IIS impacts immunity, metabolism, growth and development of these transgenic mosquitoes. We observed significant changes in glycogen, trehalose , triglycerides , glucose, and protein in young (3–5 d) transgenic mosquitoes relative to non-transgenic sibling controls, while only triglycerides were significantly changed in older (18 d) transgenic mosquitoes. More importantly, we demonstrated that enhanced fat body IIS decreased both the prevalence and intensity of Plasmodium falciparum infection in transgenic An. stephensi. Additionally, challenging transgenic An. stephensi with Gram-positive and Gram-negative bacteria altered the expression of several antimicrobial peptides ( AMPs) and two anti-Plasmodium genes, nitric oxide synthase (NOS) and thioester complement-like protein (TEP1), relative to non-transgenic controls. Increased IIS in the fat body of adult female An. stephensi had little to no impact on body size, growth or development of progeny from transgenic mosquitoes relative to non-transgenic controls. This study both confirms and expands our understanding of the critical roles insulin signaling plays in regulating the diverse functions of the mosquito fat body.

CodY is a global transcriptional regulator that is activated by branched-chain amino acids. A palindromic 15-bp sequence motif, AATTTTCNGAAAATT, is associated with CodY DNA binding. A gel mobility shift assay was used to examine the effect of pH on the binding of Bacillus subtilis CodY to the hutPp and ureAp 3 promoters. CodY at pH 6.0 has higher affinity for DNA, more enhanced activation by isoleucine, and a lower propensity for nonspecific DNA binding than CodY at pH 8.0. DNase I footprinting was used to identify the CodY-protected regions in the hutPp and ureAp 3 promoters. The CodY-protected sequences for both promoters were found to contain multiple copies of the 15-bp motif with 6-bp overlaps. Mutational analysis of the hutPp regulatory region revealed that two overlapping sequence motifs were required for CodY-mediated regulation. The presence of overlapping sequence motifs in the regulatory regions of many B. subtilis CodY-regulated genes suggests that CodY binds to native operators that contain overlapping binding sites.

The Bacillus subtilis GlnR transcription factor regulates gene expression in response to changes in nitrogen availability. Glutamine synthetase transmits the nitrogen regulatory signal to GlnR. The DNA-binding activity of GlnR is activated by a transient protein-protein interaction with feedback-inhibited glutamine synthetase that stabilizes GlnR-DNA complexes. This signal transduction mechanism was analysed by creating mutant GlnR proteins with partial or complete truncations of their C-terminal domains. The truncated GlnR proteins were found to constitutively repress gene expression in vivo. This constitutive repression did not require glutamine synthetase. Purified mutant GlnR proteins bound DNA in vitro more tightly than wild-type GlnR protein and this binding was not activated by feedback-inhibited glutamine synthetase. While full-length GlnR is monomeric, the truncated GlnR proteins contained significant levels of dimers. These results indicate that the C-terminal region of GlnR acts as an autoinhibitory domain that prevents GlnR dimerization and thus impedes DNA binding. The GlnR C-terminal domain is also required for the interaction between GlnR and feedback-inhibited glutamine synthetase. Compared with the full-length GlnR protein, the truncated GlnR proteins were defective in their interaction with feedback-inhibited glutamine synthetase in cross-linking experiments.

Staphylococcus epidermidis has emerged as a major nosocomial pathogen that is often associated with infections of indwelling medical devices. Microbial adhesion to implanted foreign materials is a prerequisite for establishing infection. We studied the time-dependent anti-adhesion effects of linezolid and vancomycin on three S. epidermidis clinical isolates. Minimum inhibitory concentration (MIC) values were identical for both agents for all three isolates (2 mg/l). Bacterial suspensions were added to polystyrene wells and treated with 0.5–4 times the MIC of linezolid or vancomycin at 0, 2, 4 or 6 h post-inoculation. Supra-inhibitory (2 and 4× MIC) and inhibitory (MIC) concentrations of linezolid demonstrated potent anti-adhesion activity following 2 and 4 h deferred treatments. Even at sub-inhibitory concentrations (0.5× MIC), suppression of staphylococcal adherence to polystyrene was still evident in most cultures. Linezolid at two and four times the MIC also exerted significant inhibitory effects in cultures that had been treated with a 6-h delay. Supra-inhibitory and inhibitory concentrations of vancomycin administered 2 h post-infection appeared equally effective as linezolid. However, sub-inhibitory concentrations of vancomycin showed minimal or no activity against bacterial adhesion. When vancomycin treatments were delayed by 4 h, only concentrations above the MIC prevented adherence. Linezolid has promising in vitro anti-adhesion activity that merits further studies to determine its role in the management of foreign-body infections.

The efficacy of linezolid, alone or in combination with rifampin, against methicillin-susceptible Staphylococcus aureus in rabbits with experimental endocarditis was investigated. Linezolid (50 or 75 mg/kg of body weight), rifampin, and linezolid (25, 50, or 75 mg/kg) plus rifampin produced statistically significant reductions in bacterial counts compared with those in untreated controls. Plasma or valvular vegetation levels of linezolid in the groups treated with the linezolid-rifampin combination were similar to those in the respective linezolid-only treatment groups. At therapeutic levels of linezolid, rifampin resistance was not observed. The results from this experimental model of endocarditis suggest that while rifampin did not provide synergy to the linezolid dosing, it did not antagonize the efficacy of linezolid.

Author Institution: Department of Chemistry, Oberlin College; Department of Chemistry, Kent, State University; Department of Partment of Chemistry, University of Michigan

Bacillus subtilis TnrA, a global regulator of transcription, responds to nitrogen availability, but the specific signal to which it responds has been elusive. Genetic studies indicate that glutamine synthetase is required for the regulation of TnrA activity in vivo. We report here that the feedback-inhibited form of glutamine synthetase directly interacts with TnrA and blocks the DNA binding activity of TnrA. Mutations in the tnrA gene ( tnrA C ) that allow constitutive high level expression of tnrA -activated genes were isolated and characterized. Feedback-inhibited glutamine synthetase had a significantly reduced ability to block the in vitro DNA binding by three of the TnrA C proteins. Thus, glutamine synthetase, an enzyme of central metabolism, directly interacts with and regulates the DNA binding activity of TnrA.

The present study determined tumorigenicity, tumor classification and DNA damage induced in infant mice by benzo[a]pyrene (B[a]P) or Manufactured Gas Plant (MGP) residues after a single exposure. Male and female B 6 C 3 F 1 mice were exposed to B[a]P or MGP residue from a single environmental site (MGP-4) and males were also exposed to MGP residue composite from seven different sites (MGP-M7). At 26, 39 and 52 weeks after exposure tumorigenesis was assessed in lung, forestomach and liver. Formation and persistence of DNA adducts were quantified by 32 P-postlabeling. Exposure of males to B[a]P induced liver tumors in a dose and time dependent manner. MGP induced more advanced tumors than B[a]P. Only a single liver tumor was found in MGP-4 treated females. No forestomach and few pulmonary adenomas were induced in males or females. MGP-4, MGP-M7 or B[a]P induced DNA adducts in males and females. Adducts in liver, lung and forestomach peaked on different days and decreased at different rates. At 24 h post-exposure, no significant differences in initial DNA adduct levels occurred in males and females exposed to MGP-4 or B[a]P. Lack of DNA damage (adducted DNA) did not account for non-responsiveness of lung and forestomach in B 6 C 3 F 1 genders as well as in liver in females. MGP tumorigenicity could not be accounted for solely by B[a]P content nor did it reflect additivity of B[a]P and other carcinogenic polycyclic aromatic hydrocarbons (PAHs) in MGP. Synergy among MGP-PAHs, presence of unidentified carcinogens and/or promoters in MGP may account for MGP potency. The B 6 C 3 F 1 infant male model is a convenient and rapid assay for assessing MGP liver tumorigenicity and potency.

Hazard and risk assessment of individual toxic chemicals utilizes data obtained from chronic toxicity and carcinogenicity studies in laboratory animals for regulating emission, cleanup or intake levels for individual chemicals. This approach can be utilized for problems associated with a single chemical emitted from a point source; however, in most situations, toxic chemicals are formed and emitted into the environment as complex mixtures of different structural classes of toxic/carcinogenic chemicals. Methodologies for risk assessment of complex mixtures have been developed for polychlorinated dibenzo‐p‐dioxins (PCDDs) and dibenzofurans (PCDFs) which are found as industrial and combustion by‐products. This methodology is based on the common mechanism of action for these chemicals and utilizes the most toxic PCDD/PCDF congener, 2, 3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), as a reference compound. The relative potencies or toxic equivalency factors (TEFs) are assigned to the other relevant PCDD/PCDF congen...

Fifteen day old B6C3F1 male mice were exposed by intraperitoneal (IP) injection to a Maximally Tolerated Dose of Manufactured Gas Plant (MGP) residue (1:32 dilution in DMSO or 1:8 in corn oil), 375 μg total of benzo[a]pyrene in DMSO or corn oil (CO) or DMSO and CO vehicle alone. On days 2, 4 and 7 post-treatment mice were injected IP with bromodeoxyuridine (BRdU) at a concentration of 50 μg/g of weight 1 hour prior to euthanasia. Immunoperoxidase staining for BRdU was performed and mitotic index (MI) determined for target tissues: bronchial mucosa, forestomach and liver. In MGP-exposed mice, MI in bronchial mucosa was increased 842% with DMSO vehicle and 555% in CO vehicle at day 2, MI was increased in forestomach mucosa by 40% at day 4 and MI was increased in liver by 50% at day 7 compared to controls. Therefore, complex mixtures such as MGP residue are capable of inducing transient cell proliferation. (Supported by EPRI RP2963-04)

The relative potencies of benzo[a]pyrene and a complex mixture of polycyclic aromatic hydrocarbons (PAH) produced as by-products of manufactured gas plant (MGP) residues as inducers of hepatic microsomal ethoxyresorufin O-deethylase (EROD) were determined in the B6C3F1 mouse. The ED50 values for the induction response were 78 and 65 mg/kg for benzo[a]pyrene and the MGP-PAH mixture, respectively, although benzo[a]pyrene and other compounds containing four or more rings were only trace components of this mixture. A comparison of the EROD induction potencies of benzo[a]pyrene and the MGP-PAH mixture showed that the mixture was approximately 706 times more active than expected based on its benzo[a]pyrene content (0.17%). The enhanced activity of the MGP-PAH mixture was also observed for several other aryl hydrocarbon (Ah) receptor-mediated responses, including inhibition of the splenic plaque-forming cell (PFC) response to both T-cell-dependent and independent antigens in B6C3F1 mice. The nature of t...

Class III agents have been associated with development of a polymorphic ventricular tachycardia (PVT) known as torsades de pointes. We compared the class III agent ibutilide, which prolongs repolarization through enhancement of an inward sodium current, with the potassium channel blockers E-4031, UK-68,798, clofilium, and D-sotalol for proarrhythmic effects in an anesthetized rabbit model. In these animals, prolongation of repolarization during alpha 1 stimulation with methoxamine produces early after depolarizations (EADs) and a pause-dependent torsades de pointes-like PVT. Agents were compared over dosage ranges that produced maximal increases in QTc interval and monophasic action potential duration (MAPD). PVT typically developed after atrioventricular (A-V) conduction block and slowing of heart rate (HR), and was preceded by development of repolarization arrhythmias characterized by EADs and triggered activity producing extrasystolic beats. Ibutilide administration resulted in significantly lower EAD amplitudes and a lower incidence of repolarization arrhythmias and PVT as compared with administration of other class III agents. The percentage of rabbits developing PVT for each agent was ibutilide 12%, D-sotalol 70%, E-4031 56%, UK-68,798 69%, and clofilium 80%. Rabbits receiving saline vehicle instead of a class III agent never developed conduction or repolarization abnormalities or PVT. Under the conditions of this study at doses that generate maximal class III effects, ibutilide produces lesser increases in QTc interval and MAPD, and EADs of lower amplitude, resulting in a lower incidence of repolarization arrhythmias and PVT as compared with other class III agents.

During growth of Bacillus subtilis in nutrient sporulation medium containing histidine (DSM-His medium), the expression of histidase, the first enzyme in the histidine-degradative pathway (hut), is derepressed 40- to 200-fold at the onset of stationary phase. To identify the gene products responsible for this regulation, histidase expression was examined in various hut regulatory mutants as well as in mutants defective in stationary-phase gene regulation. Histidase expression during growth in DSM-His medium was significantly altered only in a strain containing the hutC1 mutation. The hutC1 mutation allows the hut operon to be expressed in the absence of its inducer, histidine. During logarithmic growth in DSM-His medium, histidase levels were 25-fold higher in the HutC mutant than in wild-type cells. Moreover, histidase expression in the HutC mutant increased only four- to eightfold after the end of exponential growth in DSM-His medium. This suggests that histidine transport is reduced in wild-type cells during exponential growth in DSM-His medium and that this reduction is largely responsible for the repression of hut expression in cells growing logarithmically in this medium. Indeed, the rate of histidine uptake in DSM-His medium was fourfold lower in exponentially growing cells than in stationary-phase cells. The observation that the degradation of histidine is inhibited when B. subtilis is growing rapidly in medium containing a mixture of amino acids suggests that a hierarchy of amino acid utilization may be present in this bacterium.

The first enzymes of the histidine (hut) and proline degradative pathways, histidase and proline oxidase, could not be induced in Bacillus subtilis cells growing in glucose minimal medium containing a mixture of 16 amino acids. Addition of the 16-amino-acid mixture to induced wild-type cells growing in citrate minimal medium repressed histidase synthesis 25- to 250-fold and proline oxidase synthesis 16-fold. A strain containing a transcriptional fusion of the hut promoter to the beta-galactosidase gene was isolated from a library of Tn917-lacZ transpositions. Examination of histidase and beta-galactosidase expression in extracts of a hut-lacZ fusion strain grown in various media showed that induction, catabolite repression, and amino acid repression of the hut operon were mediated at the level of transcription. This result was confirmed by measurement of the steady-state level of hut RNA in cells grown in various media. Since amino acid repression was not defective in B. subtilis mutants deficient in nitrogen regulation of glutamine synthetase and catabolite repression, amino acid repression appears to be mediated by a system that functions independently of these regulatory systems.

U74006F, a novel 21-amino steroid is a potent inhibitor of iron-mediated lipid peroxidation and has been shown to be of therapeutic benefit in central nervous system ischemia. As oxygen radicals have been implicated in the development of postischemic myocardial dysfunction, we examined the efficacy of U74006F to enhance the recovery of function in a canine model of stunned, reperfused myocardium. Twenty-six dogs were randomized to either a vehicle (n = 11), U74006F (n = 10), or U74006F-paced group (n = 5). U74006F (6 mg/kg i.v.) was administered 15 min prior to coronary artery occlusion. Myocardial blood flows were measured by the microsphere technique, and function data were obtained by sonomicrometry. Both U74006F-treated groups demonstrated a significant increase in posterior wall thickening as compared to the vehicle treatment (U74006F-paced, 27.0 +/- 12.8%; U74006F, 22.4 +/- 11%; vehicle, -13.5 +/- 9.9%, p less than 0.001 following 3 h of reperfusion). Enhanced function recovery was accompanied by lower heart rates in the U74006F-treated group following reperfusion (treated versus vehicle, 109 +/- 6.7 versus 131 +/- 8.8 beats/min, p = 0.004). The U74006F-paced group was maintained at the same rate as the vehicle group, with no diminution in function recovery compared to the unpaced group. No effects in systemic hemodynamics or nutrient blood flow were evident as a function of drug treatment. We conclude that pretreatment with U74006F enhances the recovery of function in stunned canine myocardium via the inhibition of oxygen radicals and lipid peroxidation products. This activity suggests that this compound represents a new therapeutic adjunct in reperfusion and recanalization therapies.

In view of the reports that polymorpho- nuclear leukocytes (PMN) of patients with localized aggressive periodontitis (LAP) exhibit hyper-re- sponsiveness to stimulation, it has been suggested that such abnormalities could lead to PMN-medi- ated tissue damage during inflammation. To deter- mine whether these abnormalities include signal transduction, we compared cytoplasmic calcium concentration ((Ca 2 )i) and cytoplasmic pH (pH i ) changes, early stimulus responses to chemo- tactic agents, of LAP versus control (C)-PMN and explored whether these could be modulated by sensitizing cytokines or calcium channel-blocking agents. PMN responses of LAP patients were compared with age- and gender-matched con- trols. (Ca 2 )i and pHi were measured fluori- metrically using 1H-indole-6-carboxylic acid, 2-(4-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)- amino)-3-(2-(2-(bis(2-((acetyloxy)methoxy)-2- oxoethyl)amino)-5-methylphenoxy)ethoxy)phenyl)-1 and 2,7-bis-(carboxyethyl)-5(6)-carboxyfluores- cein as respective probes. Not only was the maxi- mal calcium response to chemoattractants higher in LAP-PMN, but also their subsequent intracellu- lar calcium redistribution was significantly slower. The slower calcium redistribution of LAP-PMN, but not their higher maximal calcium response, was successfully mimicked in C-PMN treated with Nifedipine™ or 1-(b-(3-(4-methoxyphenyl)propoxy)- 4-methoxyphenethyl)-1H-imidazole-HCl, both known to be inhibitors of membrane-associated calcium influx, but this redistribution was not af- fected when inhibitors of other calcium influx mechanisms, Diltiazem™ or Verapamil™, were used. Taken together, our findings indicate that certain early stimulus responses are aberrant in LAP-PMN, that internal redistribution of cytoplas- mic-free calcium is compromised, and, addition- ally, that a membrane-associated Ca 2 transport defect may be present. J. Leukoc. Biol. 78: 612-619; 2005.

were >100 in the present study, although statistical comparisons did not confirm a correlation with significant antibacterial effects. Studies in standard animal models of lung and thigh infection have shown that the efficacy of β-lactams and macrolides does not correlate with AUC/MIC, but does correlate with the percentage of time that the drug levels were maintained above the MIC for the pathogen used. Preliminary evidence shows a positive correlation between antibacterial effects and maintenance of linezolid plasma levels above the MIC for ∼40% of the dosing interval in a neutropenic mouse model of thigh infection. 5 In the present endocarditis study, linezolid trough plasma levels were maintained above the MIC for this MRSA strain for the entire dosing interval on each day except day 1. Other rabbit endocarditis studies have stressed the importance of maintaining plasma levels above the MIC to achieve efficacy. 6 It is important to consider that the pharmacokinetic requirements for efficacy in experimental endocarditis are more stringent compared with those for lung or thigh infections. Bacterial endocarditis is a deep-seated infection that is difficult to cure for several reasons. The vegetation is avascular, and is comprised of layers of platelets and fibrin, which reduces both neutrophil host-defence mechanisms and penetration of antimicrobial agents. In addition, the metabolic state of the bacteria within the vegetation may be reduced, lowering the effectiveness of drugs that target actively growing bacteria. In this setting, antibacterial agents such as β-lactams that have slow killing rates and no post-antibiotic effect must be administered at short intervals in order to maintain drug levels above the MIC throughout the entire dosing interval. The present study with linezolid demonstrates that maintaining plasma and valve vegetation drug levels above the MIC for the entire dosing interval over multiple days results in progressive reduction in bacterial counts in infected valves. While regional linezolid concentrations within the valve vegetation were not determined, the antibacterial effects and substantial drug concentration in valve homogenates suggest sufficient penetration of the drug.

The efficacies of orally (p.o.) dosed linezolid and intravenously (i.v.) dosed vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) in rabbits with experimental aortic-valve endocarditis were investigated. After endocarditis was established with a recent clinical MRSA isolate, rabbits were dosed for 5 days with linezolid (p.o., three times a day) at either 25, 50, or 75 mg/kg of body weight or vancomycin (i.v., twice a day) at 25 mg/kg. The 25-mg/kg linezolid group had a high mortality rate and bacterial counts in the valve vegetations that were not different from those of the controls. Linezolid dosed p.o. at 50 and 75 mg/kg and i.v. vancomycin produced statistically significant reductions in bacterial counts compared to those of the untreated controls. The reduced bacterial counts and culture-negative valve rates for the animals treated with linezolid at 75 mg/kg were similar to those for the vancomycin-treated animals. Concentrations of linezolid in plasma were determined at several points in the dosing regimen. These results suggest that the efficacy of linezolid in this infection model is related to trough levels in plasma that remain above the MIC for this microorganism. At the ineffective dose of linezolid (25 mg/kg) the concentration at sacrifice was 0.045 times the MIC, whereas the concentrations of linezolid in plasma in the 50- and 75-mg/kg groups were 2 and 5 times the MIC at sacrifice, respectively. The results from this experimental model suggest that the oxazolidinone linezolid may be effective for the treatment of serious staphylococcal infections when resistance to other antimicrobials is present.

A rabbit endocarditis model was used to test the efficacy of oral linezolid and iv vancomycin. Twenty-four hours after catheter placement across the aortic valve, rabbits were infected with 3.5 x 10 6 cfu of Staphylococcus aureus (UC-9258). Two days after infection, control rabbits were killed, and treated rabbits were given 5 days of therapy with linezolid at 8 h intervals (tds) using either 25, 50 or 75 mg/kg/dose, or vancomycin at 12 h intervals (bd) using 25 mg/kg/dose. Linezolid at 75 and 50 mg/kg, and vancomycin significantly reduced S. aureus in aortic valve vegetations compared with the control. Linezolid at 25 mg/kg was ineffective. The efficacy of 75 and 50 mg/kg linezolid was related to maintenance of plasma drug levels near or above the linezolid MIC for UC-9258 (2 mg/L).

In Bacillus subtilis , CcpA-dependent carbon catabolite repression (CCR) mediated at several cis -acting carbon repression elements ( cre ) requires the seryl-phosphorylated form of both the HPr ( ptsH ) and Crh ( crh ) proteins. During growth in minimal medium, the ptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization ( hut ) enzymes in cells grown in minimal medium showed that neither the ptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, the ptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase ( glnA ). In addition, these glnA mutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.

In Bacillus subtilis , carbon catabolite repression (CCR) of many genes is mediated at cis -acting carbon repression elements ( cre ) by the catabolite repressor protein CcpA. Mutations in transcription-repair coupling factor ( mfd ) partially relieve CCR at cre sites located downstream of transcriptional start sites by abolishing the Mfd-mediated displacement of RNA polymerase stalled at cre sites which act as transcriptional roadblocks. Although the acsA cre is centered 44.5 bp downstream of the acsA transcriptional start site, CCR of acsA expression is not affected by an mfd mutation. When the acsA cre is centered 161.5 bp downstream of the transcriptional start site for the unregulated tms promoter, CCR is partially relieved by the mfd mutation. Since CCR mediated at an acsA cre centered 44.5 bp downstream of the tms start site is not affected by the mfd mutation, the inability of Mfd to modulate CCR of acsA expression most likely results from the location of the acsA cre . Higher levels of CCR were found to occur at cre sites flanked by A+T-rich sequences than at cre sites bordered by G and C nucleotides. This suggests that nucleotides adjacent to the proposed 14-bp cre consensus sequence participate in the formation of the CcpA catabolite repression complex at cre sites. Examination of CCR of acsA expression revealed that this regulation required the Crh and seryl-phosphorylated form of the HPr proteins but not glucose kinase.

A Bacillus subtilis mutant that partially relieves carbon catabolite repression (CCR) of the hut operon was isolated by transposon mutagenesis. Characterization of this mutant revealed that the transposon had inserted into the gene, mfd, that encodes transcription-repair coupling factor. The Mfd protein is known to promote strand-specific DNA repair by displacing RNA polymerase stalled at a nucleotide lesion and directing the (A)BC excinuclease to the DNA damage site. A set of transcriptional lacZ fusions was used to demonstrate that the mfd mutation relieves CCR of hut and gnt expression at the cis-acting cre sequences located downstream of the transcriptional start site but does not affect CCR at sites located at the promoters. CCR of the amyE and bglPH genes, which contain cre sequences that overlap their promoters, is not altered by the mfd mutation. These results support a model in which the Mfd protein displaces RNA polymerase stalled at downstream cre sites that function as transcriptional roadblocks and reveal a new role for Mfd in cellular physiology.

Expression of urease, which is encoded by the ureABC operon, is regulated in response to nitrogen availability in Bacillus subtilis. Three ureABC promoters were identified in primer extension experiments and by examination of beta-galactosidase expression from ure-lacZ fusions. P1, a low-level constitutive promoter, lies immediately upstream of ureA. The P2 promoter is transcribed by the E sigmaH form of RNA polymerase and initiates transcription 270 bp upstream of the ureA start codon. The transcriptional start site for the sigmaA-dependent P3 promoter is located 839 bp upstream of the ureA start codon. To identify transcription factors that control ureABC expression, regulation of the P2 and P3 promoters was examined in wild-type and mutant strains. During rapid growth in minimal medium containing glucose and amino acids, CodY represses expression of the P2 and P3 promoters 30- and 60-fold, respectively. TnrA activates expression of the P3 promoter 10-fold in nitrogen-limited cells, while GlnR represses transcription from the P3 promoter 55-fold during growth on excess nitrogen. Expression of the ureABC operon increases 10-fold at the end of exponential growth in nutrient sporulation medium. This elevation in expression results from the relief of CodY-mediated repression during exponential growth and increased sigmaH-dependent transcription during stationary phase.

Expression from the Bacillus subtilis nrg-21 locus Increases 26-fold during nitrogen-limited growth. The DNA corresponding to this locus was cloned and sequenced. The nucleotide sequence revealed a gene that could encode a protein with sequence similarity to the Escherichia coll gamma-aminobutyric acid (GABA) permease. A transposon insertion in this locus eliminated the uptake of GABA and severely inhibited the utilization of GABA as a nitrogen source. Primer extension analysis revealed that the B. subtilis gabP gene was transcribed from two overlapping promoters. Transcription from the P1 promoter was repressed during growth in the presence of amino acids. The product of the codY gene proved to be required for this repression. Transcription from the P2 promoter increased during nitrogen-limited growth and was dependent upon the product of the tnrA gene. Deletion analysis revealed that activation of the P2 promoter during nitrogen-limited growth requires a nucleotide sequence located upstream of its -35 region. Regulation of gabP expression by the CodY and TnrA regulatory systems, which respond to different physiological signals, allows for a wide range of gabP expression during growth on various nitrogen sources.

The tumorigenic activity of manufactured gas plant residue (MGP) was evaluated in female A/J mice using a F0927 basal gel diet system. Adulterated diets containing MGP (0.10% or 0.25%) or benzo[a]pyrene (B[alpha]P; 16 or 98 ppm) were fed for 260 days. A negative control group was maintained on a nonadulterated basal gel diet. Mice dosed with a single ip injection of 1.79 mg of B[a]P in a tricaprylin vehicle and maintained on a NIH-07 pellet diet were positive controls. In addition, a nontreated group of mice and a group dosed with vehicle only were maintained on a NIH-07 pellet diet and used as negative controls. Animal body weight and consumption of MGP and B[a]P were monitored throughout the study. Ingestion of a 0.10 or 0.25% MGP adulterated diet resulted in 70 and 100% of the mice developing lung tumors with a multiplicity of 1.19 and 12.17 tumors/mouse, respectively. Mice maintained on a 0.10% MGP diet consumed 0.7 g of MGP containing 1.8 mg of B[a]P while those fed a 0.25% MGP diet ingested 1.5 g of MGP containing 4.2 mg of B[a]P. The incidence of lung tumors in mice fed only B[a]P was considerably lower than that observed for animals fed a MGP diet. A diet containing 98 ppm B[a]P produced a significant incidence of tumor-bearing mice with 52% developing lung tumors. The multiplicity observed in these animals, however, was not significant at 0.59 tumors/mouse. A diet containing 16 ppm B[a]P did not produce a significant tumorigenic response in lung. Animals fed a 16 or 98 ppm B[a]P diet consumed a total of 11 and 67 mg of B[a]P, respectively. A single ip dose of B[alpha]P (1.79 mg in 0.25 mL of tricaprylin) resulted in 100% lung tumorigenesis with a multiplicity of 15.79 tumors/mouse. In contrast to observed induction of lung tumors, no forestomach tumors were detected in any animal fed a 0.10 or 0.25% MGP adulterated diet. However, ingestion of a diet containing only 16 or 98 ppm of B[a]P resulted in 20 and 100% of the mice developing forestomach tumors, respectively. The multiplicity for forestomach tumors was 0.24 and 4.22 tumors/mouse, respectively. The incidence of forestomach carcinomas in tumor bearing mice was 8 and 52%, respectively. The ip administration of 1.79 mg of B[a]P resulted in an 83% forestomach tumor incidence having a multiplicity of 1.83 tumors/mouse. Forestomach carcinomas were induced in 34% of the mice exhibiting forestomach tumors. These data indicate that chronic ingestion of MGP- or B[a]P-adulterated diets produces significant differences in the tumorigenic response of female A/J mouse forestomach and lung tissues.

The potential non-additive interactions of polynuclear aromatic hydrocarbon (PAH) mixtures as inducers of Cyp1a-1 and Cyp1a-2 gene expression were investigated in B6C3F1 mice using a reconstituted PAH mixture. The chemical composition (% by weight) of the reconstituted PAH mixture was: 2-ring PAHs--indan (0.22), naphthalene (23.8), 2-methylnaphthalene (23.2) and 1-methylnaphthalene (13.3); 3-ring PAHs--acenaphthylene (7.7), acenaphthene (0.6), dibenzofuran (0.7), fluorene (4.3), phenanthrene (10.5) and anthracene (3.4); > or = 4-ring PAHs--fluoranthene (2.4), pyrene (4.3), benz[a]anthracene (1.4), chrysene (1.5), benzo[b]fluoranthene (0.8), benzo[k]fluoranthene (0.9) and benzo[a]pyrene (0.9). The composition of the 2-, 3- and > or = 4-ring PAH fractions were based on the relative concentration of individual PAHs as noted above. The > or = 4-ring PAH fractions were based on the relative concentration of individual PAHs as noted above. The > or = 4-ring PAH fraction and reconstituted mixture induced hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity and Cyp1a-1 mRNA levels, whereas the 2- and 3-ring PAHs were only weakly active. Direct comparison of the potencies of the reconstituted mixture and > or = 4-ring PAHs showed that the Cyp1a-1 induction activity of the reconstituted mixture was due to the > or = 4-ring PAHs. The reconstituted PAH mixture and > or = 4-ring PAHs also induced Cyp1a-2 hepatic mRNA levels and microsomal methoxyresorufin O-deethylase (MROD) activity; however, their dose-response curves indicated that the reconstituted PAH mixture was more potent as a Cyp1a-2 inducer than the > or = 4 ring PAHs. The differences in potency were due to 3-ring PAHs which were found to be strong inducers of hepatic Cyp1a-2 mRNA levels and microsomal MROD activity at the lowest dose administered (37 mg/kg). The 3-ring mixture caused a maximal 29-fold increase in hepatic MROD activity at a dose of 292 mg/kg, but only 28% of maximal induction of EROD activity. Northern analysis of liver mRNA from mice treated with 3-ring PAHs showed that there was minimal induction of Cyp1a-1 mRNA levels. The 3-ring PAHs did not competitively bind to the mouse hepatic cytosolic aryl hydrocarbon (Ah) receptor suggesting that 3-ring PAHs are a new class of Cyp1a-2 inducers which do not act through the Ah receptor.

Toward the ultimate goal of deriving quantitative protein data from silver-stained proteins resolved in complex two-dimensional electrophoresis gels we have performed computerized real-time analysis during pattern protein "silver stain" development. Three points emerged from this study: (i) Development time is a far more important variable than originally envisioned. (ii) Previous studies which, on the basis of a single development time, sought to relate silver stain slope and threshold properties to amino acid composition are non-informative since it will be shown that slope values change markedly with development time. (iii) The combination of real-time analysis and total protein load consideration allows, for the first time, protein "spots" to be grouped into categories, which in turn aid in quantification.

Conformationally distinct chromatin populations were utilized as substrates to quantitate the relative amount of and accessibility of internal 5'-phosphomonoester breaks in DNA-chromatin. In these studies, a constant amount of chromatin as well as deproteinized DNA derived from the respective chromatin sample was titrated with increasing quantities of adenylated polynucleotide ligase intermediate. This enzyme intermediate releases its AMP moiety while repairing a DNA single strand interruption, release of AMP being directly proportional to the number of internal 5'-phosphomonoester breaks repaired. Results of this study indicate that the ability of polynucleotide ligase to repair DNA breaks within chromatin was affected by the conformational state of the chromatin. The degree of conformational constraint present in a given chromatin, therefore, determined the capacity of the enzyme to repair internal DNA 5'-phosphomonoester breaks.

A mathematical model describes the carburization kinetics of tungsten powders mixed with carbon and heated in hydrogen. It is based on diffusion of carbon through a shell of WC growing into particles which are modeled as spheres. The activation energy is 58 kcal/mole in the temperature range 1056 to 1833 °C. Hydrogen gas is important to transport carbon as methane or acetylene, but increased hydrogen pressure increases the rate of carburization so little that an adsorbed species such as CH probably controls the carbon concentration at the particle surface.

Deposition of silver or silver compounds in proteins separated in linear gradient polyacrylamide electrophoretic gels is influenced by protein concentration and location, polyacrylamide concentration, extent of washing after incubation in ammoniacal silver hydroxide solution, and development time in a citric acid: formaldehyde solution. Our modification of existing silver staining methods provided routine resolution of polypeptides separated in 1.0 to 1.5-mm-thick gels without high background staining or surface staining and with picogram detection sensitivity. Removing electrophoretic components, such as sodium dodecyl sulfate (SDS), sulfhydryl reagents (2-mercaptoethanol, dithiothreitol and dithioerythreitol), glycerol, tris-glycine, carrier ampholytes, urea, and acetic acid, was essential in order to attain silver staining of proteins with a negligible background. Ammoniacal silver ions or silver ions, associated and unassociated with proteins separated in polyacrylamide gels, were removed during the washing step following incubation in ammoniacal silver hydroxide solution. Loss of stain intensity at discrete protein clusters was shown to correlate with polyacrylamide concentration in gradient gels. Standardizing the washing step enhanced reproducibility of staining. Using this silver staining method and computer-assisted image processing, it was possible to detect quantitatively as little as 27 picograms of protein per mm in 10 %-20 % linear gradient polyacrylamide gels (1.2 mm thick). Based on the lowest detection limits for Coomassie Brilliant Blue R-250 staining of protein (10 nanograms), this method for silver staining is at least 370-fold more sensitive.

The concentration of protein actually solubilized in sample buffer in preparation for analysis by two-dimensional polyacrylamide gel electrophoresis cannot be directly determined by the Lowry, Biuret, or Bradford protein methods due to interference by the combinational effect of presence of urea, detergents, carrier ampholytes, and thiol compounds in sample solubilization buffers. Determinations of the actual amount of protein solubilized and applied to gels is required to accurately quantitatively and qualitatively evaluate second-dimension polypeptide maps. It was found that when sample buffer consisting of 9 M urea, 4 % Nonidet P-40, 2 % Ampholines, and 2 % 2-mercaptoethanol containing solubilized sample(s) was acidified prior to dilution, protein concentrations over a range of 0.5 to 50 μg could be reproducibly determined utilizing a modified Bradford assay. The modified assay generates two near-linear segments, one over the range < 0.5 to 5 μg total protein that permits the application of Beer's law and a second linear response encompassing 5 to 50 μg total protein. The assay did not tolerate presence of greater than 0.1 % sodium dodecyl sulfate but addition of sodium chloride and protamine sulfate did not adversely affect protein quantitation. The modified assay allows direct quantitation of protein solubilized in sample buffers containing urea, carrier ampholytes, nonionic detergents, and thiol compounds.

Development of techniques for silver staining of proteins separated by polyacrylamide gel electrophoresis has increased experimental detection limits. However, the precise basis for the reaction between silver and polypeptides is still unclear and, depending upon the choice of silver reagent, may even differ. We compared protein stain intensity with silver diamine (ammoniacal silver) and silver nitrate reagents based on amino acid composition by determining the protein amount required to generate an arbitrary staining intensity and then calculating moles of each amino acid present in some representative proteins. We compared these values for each amino acid, taken singly, as well as for combinations of two and more. In no case could staining be attributed entirely to specific or classes of amino acids, thus supporting the argument that an inherent difference exists between primary reactive centers for the two staining reagents.

The development of methotrexate (MTX) resistance in cultured cells results in increased levels of the drug's target enzyme dihydrofolate reductase (DHFR). Stepwise-selected MTX-resistant sublines originating from an MTX-sensitive human-mouse hybrid expressed elevated DHFR levels and human-DHFR specific gene sequence amplification. By high resolution two-dimensional polyacrylamide gradient electrophoresis, human DHFR was shown to be selectively overproduced in VB2a-100 MTX-resistant cells whereas mouse DHFR protein “spots” present in MTX-sensitive parental hybrid were absent in these cells exhibiting 100 μM MTX resistance. These findings and those in a parallel study indicate that concurrent with overproduction of human DHFR and amplification DHFR sequences in VB2a-100, a loss of mouse-specific DHFR gene sequences occurred.

Purified, normal rat liver chromatin was sheared under controlled conditions in a Virtis “60” homogenizer and then separated into template-active (euchromatin) and template-inactive (heterochromatin) fractions by glycerol gradient centrifugation. The euchromatin portions of the glycerol gradients possessed greater than 90% of the in vitro template activity when estimated with Escherichia coli RNA polymerase and contained more than 90% of the nascent RNA formed in vivo . Inhibitor studies performed in vivo indicated that the leading edge of the heterochromatin portion of the glycerol gradients contains the genes coding for ribosomal RNA. Recentrifugation of putative eu- and heterochromatin fractions demonstrated that the isolation procedure produces fractions which are distinct and are characterized by little or no cross contamination. The data suggest that controlled shearing in conjunction with gradient centrifugation provides a means of fractionating with great fidelity the transcribable portions of the rat liver genome.

Normal and leukemic lymphocytes were subjected to lysis with a non-ionic detergent and 2.0 M NaCl. The intact nuclear DNA and the associated molecules resulting from these lyses (the nucleoids) were then separated from DNA-dissociated molecules and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Most of the proteins undissociable by 2.0 M NaCl from normal lymphocyte nuclear DNA or from DNA-associated molecules appear to be intermolecularly S-S linked. A distinct set of these proteins was either absent or only present in greatly decreased amounts in association with the leukemic lymphocyte nuclear DNA exposed to 2.0 M NaCl. On the other hand, the opposite relationship was found for a an MW 71,000 protein with leukemic lymphocytes of every patient studied regardless of the subtype of leukemia. Conversion of normal quiescent lymphocytes to cycling cells by lectin stimulation rendered them similar to leukemic cells in some respects but not in all. Our results show an altered nuclo(cyto)skeletal structure in leukemic cells; in general the proteins involved appear to be sulfhydryl proteins whose redox states and/or DNA association properties are modified by the neoplastic transformation of lymphocytes.

Recently, total chemical synthesis of thromboxane was achieved. The in vitro activity of synthetic thromboxane A2 is indistinguishable from biologically generated material. The present study describes the in vivo characterization of synthetic thromboxane A2 on the regional blood flow distribution of the canine heart. Local injections of synthetic thromboxane A2 into the coronary vasculature caused marked reductions in coronary blood flow, measured by both radiolabeled microsphere injection and an electromagnetic flow device. The threshold concentration required to bring about this effect varied greatly between dogs and ranged from 0.125 microgram/kg to 2.0 micrograms/kg. Similarly, the dose of thromboxane A2 required to aggregate dog platelets in vitro varied from 30 ng/ml to 1,000 ng/ml. Bolus injections of 2 micrograms/ml thromboxane A2 into the circumflex or left anterior coronary artery resulted in a simultaneous reduction in platelet count in coronary sinus blood of 83 +/- 5.2% (mean +/- SEM, n = 4, p = .0005). Both flow reduction and platelet effects were transient and localized. The time taken from onset to recovery of the response to control levels was 77 +/- 6.0 seconds (mean +/- SEM) for flow and 70-80 seconds for platelet count. Injections of thromboxane A2 caused a small but significant increase in heart rate with no change in systemic blood pressure. In conclusion, the in vivo actions of synthetic thromboxane A2 are consistent with the vasoconstrictor and platelet aggregatory effects seen in vitro, but dogs vary considerably in their sensitivity.

Methods for standardization of silver-“stained” protein position in two-dimensional polyacrylamide gel electrophoresis are presented for their application to simultaneous multiple gel systems. Four methods are discussed: the use of a single gel per simultaneous run for standardization with either (1) known quantities of commercially prepared high-or low-molecular-weight standards or (2) a known biological preparation such as T4 phage coat proteins; the use of each gel per simultaneous run to standardize itself by (3) including an internal carbamylation train such as creatine phosphokinase and using a one-dimensional gel pattern of proteins such as rat heart whole homogenate along the margin(s) or (4) identifying “marker” protein spots in test samples that occur in all gels in a given study and standardizing to relative position. Method four is discussed in detail and examples given of its use in a comparative study of proteins in spontaneous primary hepatocellular carcinomas occurring in mice, and its use compared to the other methods.

or sulfur compounds have found rather wide application in water and sewage treatment. One of the more recent uses has been as an aid in the prevention of corrosion in industrial water systems. A modern method of deoxygenation for corrosion prevention is the use of reducing agents to combine with the dissolved oxygen. The reaction between sodium sulfite, for example, and dissolved oxygen to form sodium sulfate is quite rapid at the temperatures found in boilers so it is extensively utilized in the deoxygenation of boiler feed water.

1. Dilute solutions of mercuric chloride in sea-water cause typical membrane elevation in the sea-urchin egg in spite of the fact that they presumably do not lower surface tension.2. The action of the mercuric chloride is favored by aging the eggs. Eggs fresh from the ovary are not acted upon, and the percentage of membrane elevation on treatment with the sublimate solution increases in proportion to the time the eggs have stood in sea-water before being subjected to the reagent.3. The favorable effect of aging is apparently due to the removal of carbon dioxide. The addition of carbon dioxide prevents membrane elevation by mercuric chloride.4. If eggs are centrifuged one or two minutes after the treatment with mercuric chloride is begun, membrane elevation is generally prevented.5. Solutions of mercuric chloride in contact with eggs lose their power of provoking membrane elevation.6. The facts cited in 4 and 5 are regarded as evidence in favor of the view that mercuric chloride reacts with the jelly or co...

We have previously examined the genetic organization and regulation of the Tn10 tetracycline-resistance determinant in Escherichia coli K-12. The structural genes for tetA, the Tn10 tetracycline-resistance function, and for tetR, the Tn10 tet repressor, are transcribed in opposite directions from promoters in a regulatory region located between the two structural genes. Expression of both tetA and tetR is induced by tetracycline. Here we report the DNA sequence of the Tn10 tet regulatory region. The locations of the tetA and tetR promoters within this region were defined by S1 nuclease mapping of the 5' ends of in vivo tet RNA. The tetA and tetR promoters overlap; the transcription start points are separated by 36 bp. We propose that two similar regions of dyad symmetry within the Tn10 tet regulatory region are operator sites at which tet repressor binds to tet DNA, thereby inhibiting transcription initiation at the tetA and tetR promoters. The Tn10 tet regulatory region and the pBR322 tet regulatory region show significant DNA sequence homology (53%).

The regulatory region of the tetracycline resistance determinant from transposon Tn10 has been used to construct plasmid vectors for gene expression regulated by tetracycline. Plasmids pRS tetBam-8 and pRS tetBam-16 include the tet regulatory region, the segment coding for the first four amino acids of the tetracycline resistance protein (tetA protein), and a linker region with SalI, HpaII, and BamHI restriction sites for gene fusions. Plasmid pTB-1, a derivative of pRS tetBam-8 and of the beta-galactosidase gene-containing plasmid pMC1403, constitutively expresses a tetA fragment-beta-galactosidase fusion protein. If a multicopy runaway replication plasmid, pMOBglII-16 that includes a 2.7-kb BglII DNA fragment from Tnl10 that provides tetR protein is present along with pTB-1, the expression of beta-galactosidase is reduced eightfold. Tetracycline acts as an inducer of the system and restores the level of beta-galactosidase activity measured in transformants containing pTB-1 alone. Plasmid mutants unable to produce active tetR protein are ineffective in reducing expression. Escherichia coli carrying plasmids that express both tetA protein and tetR protein show an increase in the tetracycline resistance level after incubation with the drug. The observations are consistent with the previously proposed mechanism of regulation of tetracycline resistance in Tn10.

Chromatin isolated from adult rat liver was fractionated into template active and inactive components by controlled shearing and glycerol gradient centrifugation. The fractionated chromatin was assayed for DNA-dependent DNA polymerase (DNA mucleotidyl transferase EC 2.7.7.7) activity with and without exogenous activated DNA serving as template. With endogenous chromatin as template, it was found that 90% of the endogenous chromatin bound DNA polymerase activity was located in the transcriptionally active fraction of chromatin, while the distribution of DNA polymerase assayed with exogenous activated DNA was found to be 65% in the transcriptionally active and 35% in the inactive fractions. However, when DNA polymerase was solubilized from these fractions by salt extraction, enzyme activity was found to be equally distributed, suggesting that the difference in endogenous DNA polymerase activity observed between eu- and heterochromatin is due to the restricted template found in repressed fractions.

The effects of VirTis shearing on chromatin subunit structure were investigated by enzymatic digestion, thermal denaturation, and electron microscopy. While initial rates of micrococcal nuclease and DNase I digestion were greater postshearing, limit digest values were similar to those for unsheared chromatin. Fractionated chromatin digestion kinetics varied with sedimentation. Digestion of all chromatins produced monomer and dimer DNA fragment lengths, but only unsheared chromatins exhibited higher order nucleosome oligomer lengths. Mononucleosomes and core particles were resolved in digests of sheared and gradient fractions analyzed by electrophoresis. All chromatins exposed to DNase I showed discrete 10-base pair nicking patterns. The presence of nucleosomes was confirmed by electron microscopy. Electron microscopy and histone content of gradient fractions showed that nucleosome density along the chromatin axis increased in rapidly sedimenting fractions. Thermal denaturation detected no appreciable generation of protein-free DNA fragments as a result of shearing. The results indicate that VirTis blending conserves subunit structure with loss of less than 12–15% of nucleosome structure.