Borrelia burgdorferi RevA antigen is a surface-exposed outer membrane protein whose expression is regulated in response to environmental temperature and pH.

Borrelia burgdorferi, the causative agent of Lyme disease, produces RevA protein during the early stages of mammalian infection. B. burgdorferi apparently uses temperature as a cue to its location, producing proteins required for infection of warm-blooded animals at temperatures corresponding to host body temperature, but does not produce such virulence factors at cooler, ambient temperatures. We have observed that B. burgdorferi regulates expression of RevA in response to temperature, with the protein being synthesized by bacteria cultivated at 34 degrees C but not by those grown at 23 degrees C. Tissues encountered by B. burgdorferi during its infectious cycle vary in their pH values, and the level of RevA expression was also found to be dependent upon pH of the culture medium. The cellular localization of RevA was also analyzed. Borrelial inner and outer membranes were purified by isopycnic centrifugation, and membrane fractions were conclusively identified by immunoblot analysis using antibodies raised against the integral inner membrane protein MotB and outer membrane-associated Erp lipoproteins. Immunoblot analyses indicated that RevA is located in the B. burgdorferi outer membrane. These analyses also demonstrated that an earlier report (H. A. Bledsoe et al., Infect. Immun. 176:7447-7455, 1994) had misidentified such B. burgdorferi membrane fractions. RevA was further demonstrated to be exposed to the external environment, where it could facilitate interactions with host tissues.

Distinct regulatory pathways control expression of Borrelia burgdorferi infection-associated OspC and Erp surface proteins.

Deciphering the mechanisms by which Borrelia burgdorferi controls the synthesis of proteins associated with mammalian infection will be an important step toward understanding the pathogenic properties of Lyme disease-causing bacteria. We present results of studies indicating that B. burgdorferi senses a wide variety of environmental stimuli, including soluble chemicals, which enables it to independently control synthesis of the Erp and OspC proteins. Regulation of OspC and Erp expression appears to occur at the level of transcription. In this regard, we observed that one or more DNA-binding proteins interact specifically with erp promoter DNA but not with the ospC promoter.

Evidence for a functional monomeric form of the bacteriophage T4 DdA helicase. Dda does not form stable oligomeric structures.

The active form of many helicases is oligomeric, possibly because oligomerization provides multiple DNA binding sites needed for unwinding of DNA. In order to understand the mechanism of the bacteriophage T4 Dda helicase, the potential requirement for oligomerization was investigated. Chemical cross-linking and high pressure gel filtration chromatography provided little evidence for the formation of an oligomeric species. The specific activity for ssDNA stimulated ATPase activity was independent of Dda concentration. Dda was mutated to produce an ATPase-deficient protein (K38A Dda) by altering a residue within a conserved, nucleotide binding loop. The helicase activity of K38A Dda was inactivated, although DNA binding properties were similar to Dda. In the presence of limiting DNA substrate, the rate of unwinding by Dda was not changed; however, the amplitude of product formation was reduced in the presence of increasing concentrations of K38A Dda. The reduction was between that expected for a monomeric or dimeric helicase based on simple competition for substrate binding. When unwinding of DNA was measured in the presence of excess DNA substrate, addition of K38A Dda caused no reduction in the observed rate for strand separation. Taken together, these results indicate that oligomerization of Dda is not required for DNA unwinding.

A second allele of eppA in Borrelia burgdorferi strain B31 is located on the previously undetected circular plasmid cp9-2.

Although sequence analysis of Borrelia burgdorferi isolate B31 was recently declared "complete," we found that cultures of this strain can contain a novel 9-kb circular plasmid, cp9-2. The newly described plasmid contains both sequence similarities with and differences from the previously identified B31 plasmid cp9-1 (formerly cp9). cp9-1 and cp9-2 each encode a unique allele of EppA, a putative membrane protein synthesized by B. burgdorferi during mammalian infection.

Attention deficit/hyperactivity disorder children with a 7-repeat allele of the dopamine receptor D4 gene have extreme behavior but normal performance on critical neuropsychological tests of attention.

An association of the dopamine receptor D4 (DRD4) gene located on chromosome 11p15.5 and attention deficit/hyperactivity disorder (ADHD) has been demonstrated and replicated by multiple investigators. A specific allele [the 7-repeat of a 48-bp variable number of tandem repeats (VNTR) in exon 3] has been proposed as an etiological factor in attentional deficits manifested in some children diagnosed with this disorder. In the current study, we evaluated ADHD subgroups defined by the presence or absence of the 7-repeat allele of the DRD4 gene, using neuropsychological tests with reaction time measures designed to probe attentional networks with neuroanatomical foci in D4-rich brain regions. Despite the same severity of symptoms on parent and teacher ratings for the ADHD subgroups, the average reaction times of the 7-present subgroup showed normal speed and variability of response whereas the average reaction times of the 7-absent subgroup showed the expected abnormalities (slow and variable responses). This was opposite the primary prediction of the study. The 7-present subgroup seemed to be free of some of the neuropsychological abnormalities thought to characterize ADHD.

Borrelia burgdorferi B31 Erp proteins that are dominant immunoblot antigens of animals infected with isolate B31 are recognized by only a subset of human lyme disease patient sera.

Sera from animals infected with Borrelia burgdorferi isolates yield intense immunoblot signals from the B31 ErpA/I/N and ErpB/J/O proteins, which have apparent molecular masses of 19 and 60 kDa, respectively. Since B. burgdorferi proteins with those molecular masses are of immunodiagnostic importance, Lyme disease patient sera were used in studies of B31 lysates and recombinant B31 ErpA/I/N and ErpB/J/O proteins. Immunoblot analyses indicated that only a minority of the patients produced antibodies that recognized the tested B31 Erp proteins. Southern blot analyses of Lyme disease spirochetes cultured from 16 of the patients indicated that all these bacteria contain genes related to the B31 erpA/I/N and erpB/J/O genes, although signal strengths indicated only weak similarities in many cases, suggestive of genetic variability of erp genes among these bacteria. These data indicate that Erp proteins are generally not the 19- and 60-kDa antigens observed on serodiagnostic immunoblots.

Copper/zinc-Superoxide dismutase is required for oxytetracycline resistance of Saccharomyces cerevisiae.

Saccharomyces cerevisiae, along with other eukaryotes, is resistant to tetracyclines. We found that deletion of SOD1 (encoding Cu/Zn superoxide dismutase) rendered S. cerevisiae hypersensitive to oxytetracycline (OTC): a sod1Delta mutant exhibited a >95% reduction in colony-forming ability at an OTC concentration of 20 microg ml(-1), whereas concentrations of up to 1,000 microg ml(-1) had no effect on the growth of the wild type. OTC resistance was restored in the sod1Delta mutant by complementation with wild-type SOD1. The effect of OTC appeared to be cytotoxic and was not evident in a ctt1Delta (cytosolic catalase) mutant or in the presence of tetracycline. SOD1 transcription was not induced by OTC, suggesting that constitutive SOD1 expression is sufficient for wild-type OTC resistance. OTC uptake levels in wild-type and sod1Delta strains were similar. However, lipid peroxidation and protein oxidation were both enhanced during exposure of the sod1Delta mutant, but not the wild type, to OTC. We propose that Sod1p protects S. cerevisiae against a mode of OTC action that is dependent on oxidative damage.

Impediments to writing do-not-resuscitate orders.

BACKGROUND: Physicians are frequently unaware of their patients' desires regarding end-of-life care. Consequently, opportunities to implement do-not-resuscitate (DNR) orders are often missed. OBJECTIVE: To determine the reasons attending physicians do not write DNR orders when patients face increased mortality. METHODS: Over 4 months, the medical records of all inpatients on the General Medicine Service were reviewed at the time of discharge to identify patients with conditions predicting increased mortality. These cases were presented to a 5-member panel who decided if a DNR order was indicated. Reasons for missing DNR orders were discussed with the attending physicians. RESULTS: Of 613 consecutive admissions, the panel identified 149 patients (24%) for whom DNR orders were indicated. In 88 (59%) of these, DNR orders were absent. The lack of a DNR order did not correlate with age (P = .95), sex (P = .61), or race (P = .80). The attending physicians' explanations for not writing DNR orders in these 88 cases included the belief that the patient was not in imminent danger of death (n = 49 [56%]), the belief that the primary physician should discuss DNR issues (n = 43 [49%]), and the lack of an appropriate opportunity to discuss end-of-life issues (n = 38 [43%]). In 11 (12%) of the 88 cases, patients or their families did not accept the recommendation for a DNR order. No physicians expressed concerns regarding the morality of DNR orders, discomfort discussing end-of-life issues, or the threat of litigation as reasons for not writing a DNR order. CONCLUSIONS: Limitations in the extent and depth of the physician-patient relationship appear to be the most frequent impediments to writing DNR orders in our institution.

Genetic Regulation of Development in Sorghum bicolor: V. The ma(3) Allele Results in Gibberellin Enrichment.

Sorghum bicolor genotypes, near isogenic with different alleles at the third maturity locus, were compared for development, for responsiveness to GA(3) and a GA synthesis inhibitor, and occurrence and concentrations of endogenous GAs, IAA, and ABA. At 14 days the genotype 58M (ma(3) (R)ma(3) (R)) exhibited 2.5-fold greater culm height, 1.75-fold greater total height, and 1.38-fold greater dry weight than 90M (ma(3)ma(3)) or 100M (Ma(3)Ma(3)). All three genotypes exhibited similar shoot elongation in response to GA(3), and 58M showed GA(3)-mediated hastening of floral initiation when harvested at day 18 or 21. Both 90M and 100M had exhibited hastening of floral initiation by GA(3) previously, at later application dates. Tetcyclacis reduced height, promoted tillering, and delayed flowering of 58M resulting in plants which were near phenocopies of 90M and 100M. Based on bioassay activity, HPLC retention times, cochromatography with (2)H(2)-labeled standards on capillary column GC and matching mass spectrometer fragmentation patterns (ions [m/z] and relative abundances), GA(1), GA(19), GA(20), GA(53), and GA(3) were identified in extracts of all three genotypes. In addition, based on published Kovats retention index values and correspondence in ion masses and relative abundances, GA(44) and GA(17) were detected. Quantitation was based on recovery of coinjected, (2)H(2)-labeled standards. In 14 day-old-plants, total GA-like bioactivity and GA(1) concentrations (nanograms GA/gram dry weight) were two- to six-fold higher in 58M than 90M and 100M in leaf blades, apex samples, and whole plants while concentrations in culms were similar. Similar trends occurred if data were expressed on a per plant basis. GA(1) concentrations for whole plants were about two-fold higher in 58M than 90M and 100M from day 7 to day 14. Concentrations of ABA and IAA did not vary between the genotypes. The results indicate the mutant allele ma(3) (R) causes a two- to six-fold increase in GA(1) concentrations, does not result in a GA-receptor or transduction mutation and is associated with phenotypic characteristics that can be enhanced by GA(3) and reduced by GA synthesis inhibitor. These observations support the hypothesis that the allele ma(3) (R) causes an overproduction of GAs which results in altered leaf morphology, reduced tillering, earlier flowering, and other phenotypic differences between 58M and 90M or 100M.