Core bacterial community composition of a cryptoendolithic ecosystem in the Grand Staircase-Escalante National Monument, Utah.

Cryptoendolithic bacterial communities in the Jurassic Navajo Sandstones play an important ecological role in this ecosystem. Developing a better understanding of the role of these cryptoendolithic communities required a deeper knowledge of the microbial diversity present. We analyzed the bacterial diversity in eight sandstones samples from several microgeological features associated with a large sandstone dome. Cryptoendolithic bacterial diversity is clustered into three distinct groups which correlated with topography, suggesting the duration of water retention might be a factor. Comparisons of diversity between each cluster showed that a core bacterial community exists in this habitat. The overall bacterial community structure was dominated by Cyanobacteria, Proteobacteria, Bacteroidetes, and Actinobacteria. The most prevalent genera in cyanobacteria were Leptolyngbya, Chroococcidiopsis, and unclassified cyanobacteria accounting for the bulk of cyanobacterial sequences. Within the Proteobacteria, Alphaproteobacteria were the largest class detected, with members of the Acetobacteraceae, particularly the genus Acidiphilium, being the most abundant. Acidiphilium spp. are capable of aerobic ferric iron reduction under moderately acidic conditions, explaining the high levels of iron (II) in this system. This study highlights the extent of unexplored bacterial diversity in this habitat system and sets the premise for elaborating on the ecological function of cryptoendolithic communities.

In situ studies on the time-dependent degradation of recombinant corn DNA and protein in the bovine rumen.

An in situ technique was adopted to investigate the time-dependent ruminal degradation of chloroplast compared with recombinant DNA of Bt176 corn using conventional and quantitative PCR assays. In parallel, the Cry1Ab protein content and fragment sizes were determined by ELISA and immunoblotting techniques. Triplicate nylon bags filled with 5 g of each substrate (whole-plant isogenic, whole-plant transgenic, ensiled isogenic, and ensiled transgenic corn) were positioned within the rumen of 5 rumen-cannulated, nonlactating cows and incubated for 2, 4, 8, 16, 24, and 48 h. To investigate the DNA degradation process, PCR assays were developed to detect fragments of the endogenous highly abundant rubisco gene (173, 896, 1,197, and 1,753 bp) and of the recombinant cry1Ab gene (211, 420, 727, and 1,423 bp). Short fragments of rubisco (<431 bp) and cry1Ab DNA (211 bp) were amplifiable in whole-plant and ensiled corn samples incubated in the rumen for 48 h, whereas the traceability of larger fragments depended on previous processing of the sample (whole-plant or ensiled corn), the length of the target sequence, and concomitantly on the length of time incubated in the rumen. Quantification of rubisco and cry1Ab gene fragments applying real-time PCR assays revealed degradation to <20% of initial 0-h values within 2 h and <0.5% after 48 h of ruminal incubation. Analysis of Cry1Ab protein in whole-plant corn using the ELISA technique revealed a decrease to 28.0% of the initial value within 2 h and to 2.6% within 48 h. The concentration of Cry1Ab protein of ensiled corn was only 10% that of whole-plant corn. Ensiled corn Cry1Ab protein decreased to 10% of initial values after 48 h of ruminal incubation. Using an immunoblotting technique, the full-size Cry1Ab protein was only detectable up to 8 h; thereafter, only fragments of approximately 17 and 34 kDa size were found. In conclusion, ruminal digestion decreased the presence of functional cry1Ab gene fragments. It is unlikely that full-size, functional Cry1Ab protein will be present after 8 h of incubation in the rumen. Therefore, results based on ELISA measurements should be interpreted carefully and verified by another detection method that discriminates between the full-size and fragmented Cry1Ab protein.

Modeling intermolecular effects on nonlinear optical properties of transition-metal complexes. An effective core potential study.

An initial effort to study the nonlinear optical (NLO) properties of interacting transition-metal-oxo complexes is presented and studied by effective core potential approaches. Osmium tetroxide is used for this study. Favorable intermolecular interaction effects, even within this weak interaction regime, that yield enhancements in NLO properties have been found. Interaction effects increase alpha (polarizability) up to 6% and gamma (second hyperpolarizability) up to 100% relative to the isolated monomer result for OsO4. The magnitude of the interaction (hyper)polarizabilities, and indeed even the sign, is found to be quite sensitive to the relative orientation of the osmium tetroxide monomers.

Parenterally administered kainic acid induces a persistent hyperalgesia in the mouse and rat.

Nociceptive primary afferent C-fibers express a subset of glutamate receptors that are sensitive to kainic acid. Thus, we tested the possibility that activation of these receptors alters nociception. Intraperitoneal (i.p.) injection of kainic acid induced a persistent thermal hyperalgesia, when tested using the hot plate (mice) and tail flick (mice and rats) assays, and mechanical hyperalgesia when tested using von Frey monofilaments (rats), but had no effect on acetic acid-induced chemical nociception (mice). When administered i. p., 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an (R, S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid HBr/kainate (AMPA/KA) antagonist, completely blocked hyperalgesia. When injected intrathecally (i.t.), kainic acid itself failed to induce hyperalgesia and AMPA/KA antagonists given i.t. also failed to attenuate the hyperalgesic effect of kainic acid administered i.p. , indicating that the spinal cord is not the primary site of action. Kainic acid injected subcutaneously in the back of mice decreased response latencies in the hot plate and tail flick assays, indicating that hyperalgesia is achieved by a variety of parenteral routes of injection. Histological evaluation of rat spinal cord and dorsal root ganglia revealed no neurodegenerative changes 24 h after kainic acid. Together these data suggest that a persistent hyperalgesia results from the transient activation of AMPA/KA receptors that are located outside the spinal cord, perhaps on the distal projections of primary afferent fibers.

Use of diaminobenzidine to stain for cytochrome c oxidase activity in Caulobacter crescentus and Escherichia coli.

Caulobacter crescentus is an aerobic Gram negative bacterium found in bacterial biofilms. C. crescentus produces a sessile stalked cell during its life cycle that allows it to attach to surfaces. Due to the oxygen gradient found in the bacterial biofilm, we postulated that C. crescentus would localize the terminal cytochrome to the cell body that remains in the aerobic portion of the biofilm. The terminal electron acceptor in the electron transport system was determined using Kovac's oxidase test to be cytochrome c oxidase. We are able to use diaminobenzidine to locate the oxidase histochemically using transmission electron microscopy. Upon examination of sectioned bacteria, we determined that cytochrome c oxidase of C. crescentus is found only in the cell body.

Death of a wild wolf from canine parvoviral enteritis.

A 9-mo-old female wolf (Canis lupus) in the Superior National Forest of Minnesota (USA) died from a canine parvovirus (CPV) infection. This is the first direct evidence that this infection effects free-ranging wild wolves.

[Leukoencephalomalacia in two horses--moldy corn poisoning in Germany?]. / Leukoenzephalomalazie bei zwei Pferden--Moldy Corn Poisoning in Deutschland?

Moldy corn poisoning is a mycotoxicosis of Fusarium sp. causing a disease termed equine leukoencephalomalacia (ELEM). This article reviews the literature on ELEM and describes two cases with clinical signs and morphological findings comparable with fusariotoxicosis. Since in both cases neither a fungus nor a toxin proof were performed, the different causes of leukoencephalomalacia in horses are discussed.

Caffeine consumption is associated with decreased severe late toxicity after radiation to the pelvis.

PURPOSE: Recent studies have suggested that pentoxifylline, a methylxanthine, can prevent or ameliorate late radiation injury in animals and humans. Caffeine is a commonly consumed methylxanthine that provides a model for evaluating the impact of this category of drugs on radiation injury. A retrospective study was undertaken to determine if there is an association between caffeine consumption and a lower incidence of late radiation toxicity. METHODS AND MATERIALS: From 1984 through 1990, 82 patients with cervical cancer and 53 patients with endometrial cancer were treated with primary or adjuvant radiation therapy at the University of Washington. Patients were interviewed regarding ingestion of caffeine-containing beverages, and average daily caffeine consumption during the time of radiotherapy was estimated. The evaluable patients (42 cervical, 31 endometrial) were stratified by quantity of caffeine consumption for correlation with the incidence of radiation toxicity. RESULTS: Acute radiation toxicity was not associated with caffeine consumption for cervical or endometrial cancer. There was a nonstatistically significant trend toward a decrease in overall late radiation toxicity with increased caffeine intake for cervical cancer patients. Subgroup analysis revealed this trend to be attributable to a decreased incidence of severe late radiation injury in cervical cancer patients who consumed higher levels of caffeine at the time of their radiotherapy (p = 0.02). This relationship was not observable for late toxicity in the endometrial cancer patients due to the low incidence of severe late injury following radiation for that disease. CONCLUSIONS: This investigation is supportive of previous studies showing a radioprotective effect for pentoxifylline, and suggests that the mechanisms of radioprotection may be common to methylxanthines as a drug class.

The Caulobacter crescentus holdfast: identification of holdfast attachment complex genes.

Caulobacters are biofilm bacteria that attach to surfaces via a holdfast, an adhesive expressed at discrete cell surface sites. We have described a cluster of at least three genes involved in the adhesive attachment of the holdfast of Caulobacter crescentus CB2A to the cell, analyzing the sequence of two genes, hfaAB. Here we report hfaC and a fourth open reading frame, hfaD. hfaC predicts a protein of 41 kDa homologous to ATP-binding transport-related proteins, with ChvD of Agrobacterium tumefaciens as best match. HfaD is predicted to be 28 kDa with three membrane spanning regions. hfaA, hfaC, and hfaD were expressed in Escherichia coli; Western analysis with antisera against a holdfast-enriched preparation indicated HfaA was likely holdfast-associated. Cumulative findings predict HfaA and HfaB are developmentally regulated and one or both enhance hfaC transcription, HfaA is a mediator of adhesion, possibly between holdfast and a membrane-bound HfaD, and HfaC mediates export of an unidentified component required for holdfast attachment.

Possible role of the N-terminus of substance P in kainic acid-induced toxicity in rats.

Subcutaneously administered kainic acid (KA) in the rat results in brain damage accompanied by a behavioral response characterized by wet dog shakes (WDS), seizures and brain damage, an effect that is potentiated by opioids. Based on the potentiative effect of the N-terminus of substance P (SP) on the ability of KA to induce behavioral responses in mice, we tested the hypothesis that the N-terminus of SP also plays a role in KA-induced neurotoxicity in rats. Pretreatment i.p. with 1 or 10 nmol of SP(1-7), a major N-terminal metabolite of the undecapeptide SP, 15 min before administration of 12 mg/kg of KA potentiated the incidence of WDS. In contrast, after administration of 1 nmol of [D-Pro2, D-Phe7]SP(1-7) (D-SP(1-7)), the D-isomer of SP(1-7) and a substance P N-terminal antagonist, the intensity of KA-induced WDS was no different from those in either the KA- or saline-injected rats. However, pretreatment with D-SP(1-7) completely blocked the potentiative effect of SP(1-7) on the KA-induced WDS. While the severity of KA-induced lesions was not significantly altered by pretreatment with 1 nmol of SP(1-7), the effect of KA was not significantly different from that in control rats when administered with 1 nmol of D-SP(1-7). These results suggest a possible involvement of endogenous SP N-terminal activity in the effects following subcutaneous (s.c.) administration of KA.(ABSTRACT TRUNCATED AT 250 WORDS)

An "all-purpose" cellulase reporter for gene fusion studies and application to the paracrystalline surface (S)-layer protein of Caulobacter crescentus.

The secreted endoglucanase (CenA) from the Gram-positive bacterium Cellulomonas fimi and a deletion derivative (delta CenA) lacking the N-terminal leader peptide of native CenA were used to explore the potential of delta CenA as a reporter molecule in Caulobacter crescentus. Expression of cenA in C. crescentus yielded extracellular endoglucanase activity, suggesting that the N-terminal leader peptide of CenA could direct the enzyme to the periplasm where it subsequently leaked into the medium. In contrast, expression of delta cenA yielded only cell-associated endoglucanase activity; this suggested that the enzyme retained activity in the C. crescentus cytoplasm. Using the putative cytoplasmic and periplasmic forms of delta CenA as markers, a simple assay for periplasmic delta CenA hybrids was developed. This assay indicated that delta CenA activity was largely independent of cellular location. To facilitate the use of delta CenA as a reporter, a broad host range translational fusion vector (pEC215) incorporating delta cenA was constructed. This vector was used to investigate factors important to the expression of the gene (rsaA) encoding the paracrystalline surface protein (S-layer) of the bacterium. It was found that altering the 5' untranslated region of the rsaA mRNA reduced gene expression by 70%. One rsaA:delta cenA gene fusion resulting from these experiments that incorporated only rsaA translation initiation information was further modified to serve as a general reporter for creating transcriptional gene fusions with other promoters. Gene fusions between alkaline phosphatase (phoA) and either cenA or lacZ were used to supplement information about RsaA secretion derived from rsaA:phoA gene fusions. It was found that linkage of the N-terminal leader peptide of CenA to PhoA yielded 50-100 times more cell-associated PhoA activity in C. crescentus than linkage of the RsaA N terminus. Taken together, these experiments indicated that delta CenA was useful for tagging proteins localized to the cytoplasm, exported to the periplasm, or secreted from the cell, as well as for monitoring events in the cytoplasm such as examining factors important to the level of gene expression. Further, because delta CenA was active in all cell compartments, it could be used to estimate the efficiency of hybrid protein export-secretion from enzyme activity measurements alone. In short, delta CenA possessed many of the attributes of an "all-purpose" reporter.

Selective potentiation of NMDA-induced activity and release of excitatory amino acids by dynorphin: possible roles in paralysis and neurotoxicity.

Selective antagonists of N-methyl-D-aspartate (NMDA) excitatory amino acid (EAA) receptors have been shown to protect against dynorphin-A (DYN)-induced paralysis and neurotoxicity in the spinal cord. To test the hypothesis that either DYN-induced paralysis or neurotoxicity involves an enhanced release of EAAs, we used microdialysis to monitor aspartate (Asp) and glutamate (Glu) release in both the lumbar spinal cord extracellular fluid (ECF) and the spinal cord cerebral spinal fluid (CSF) of conscious rats in response to DYN (1-13). Injection of 5 nmol of DYN produced temporary paralysis in 8 of 10 animals, but did not significantly change Asp or Glu release in either the ECF or the CSF. Injection of 20 nmol of DYN caused permanent paralysis and neuronal cell loss in all animals tested as well as a significant increase of Asp and Glu in both the ECF and the CSF, and a decrease in glutamine (Gln) release only in the ECF. Pretreatment with 1 mg/kg of the NMDA antagonist MK-801 blocked both paralysis and amino acid changes in the ECF. Pretreatment of animals with 5 mg/kg naloxone inhibited glutamate release in the ECF, but did not block paralysis, Asp release or inhibition of Gln release. As MK-801 sensitive paralysis by DYN was not mediated through enhanced EAA release, we examined whether DYN could act through postsynaptic facilitation of NMDA receptors by testing the ability of DYN to alter the magnitude of a behavioral response produced by intrathecal injection of NMDA in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of a Caulobacter crescentus gene cluster involved in attachment of the holdfast to the cell.

Caulobacter crescentus firmly adheres to surfaces with a structure known as the holdfast, which is located at the flagellar pole of swarmer cells and at the stalk tip in stalked cells. A three-gene cluster (hfaAB and hfaC) is involved in attachment of the holdfast to the cell. Deletion and complementation analysis of the hfaAB locus revealed two genes in a single operon; both were required for holdfast attachment to the cell. Sequence analysis of the hfaAB locus showed two open reading frames with the potential to encode proteins of 15,000 and 26,000 Da, respectively. A protein migrating with an apparent size of 21 kDa in gel electrophoresis was encoded by the hfaA region when expressed in Escherichia coli under the control of the lac promoter, but no protein synthesis could be detected from the hfaB region. S1 nuclease analysis indicated that transcription of the hfaAB locus was initiated from a region containing a sequence nearly identical to the consensus for C. crescentus sigma 54-dependent promoters. In addition, a sequence with some similarity to ftr sequences (a consensus sequence associated with other Caulobacter sigma 54-dependent genes) was identified upstream of the hypothesized sigma 54 promoter. At least one of the hfaAB gene products was required for maximal transcription of hfaC. The sequence of hfaB showed some similarity to that of transcriptional activators of other bacteria. The C-terminal region of the putative gene product HfaA was found to be homologous to PapG and SmfG, which are adhesin molecules of enteropathogenic E. coli and Serratia marcescens, respectively. This information suggests that the protein encoded by the hfaA locus may have a direct role in the attachment of the holdfast to the cell, whereas hfaB may be involved in the positive regulation of hfaC.

Malignant hyperthermia susceptibility: cardiac histomorphometry of dogs and young and market-weight swine.

The defect causing malignant hyperthermia has been proposed to involve cardiac as well as skeletal muscle. We tested the hypothesis that histomorphometric parameters for ventricular wall from malignant hyperthermia-susceptible swine and dogs were abnormal. Hearts were obtained from: mature dogs, age- and weight-matched young swine (89 +/- 15 days, 30 +/- 3 kg); and market-weight swine (102 +/- 10 kg). Using light microscopy, estimates were made for muscle nuclear dimensions and the volume-fraction of nuclei, sarcoplasm, blood vessels, and interstitial space. Cardiac maturation in both MH and normal swine was accompanied by decreased myocyte volume-fraction due to decreased nuclear volume-fraction and increased interstitial space volume-fraction. Sarcoplasm and vasculature volume-fraction were unchanged after maturation. Nuclear volume-fraction was slightly greater (p less than 0.05) in the right ventricle than the left for malignant hyperthermia and normal swine. Myocyte nuclear dimensions were generally similar among animals. Dogs and the oldest group of swine were not significantly different. Myocytes of all swine contained multiple nuclei, closely spaced in rows of 2 to 12. In contrast, most myocytes of mature dogs apparently contained one or two nuclei. Histomorphometric values were not significantly different between normal and malignant hyperthermia young swine and dogs. However, within the market-weight swine, volume-fraction for malignant hyperthermia myocytes and myocyte nuclei was decreased and interstitial space was increased compared to normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of zearalenone on dairy cows.

Doses of 99% pure zearalenone (0.0, 31.25, 62.5, 125.0, 250.0, or 500.0 mg) in gelatin capsules were given once a day per os to 18 nonpregnant, nonlactating, multiparous dairy cows for 2 consecutive estrous cycles. There was no effect (P less than 0.10) on serum progesterone concentrations, RBC, WBC, PCV, hemoglobin, and estrous cycle length. Differential cell counts, clinical health, and sexual behavior were not affected by the zearalenone. One cow from each of the groups given zearalenone and a control were euthanatized at the end of the study. The zearalenone had no effect on the terminal bone marrow smears and did not induce any gross lesions discernible at necropsy or any microscopic lesions in representative samples of 30 tissues/cow. Rectal palpation of the reproductive tracts once a week indicated that the corpora lutea were small in cows given zearalenone. There was a general trend to increased hemoglobin concentrations in cows given the larger doses of zearalenone. Zearalenone of and by itself does not seem to be an important factor in dairy cow health.

Effect of zearalenone on the fertility of virgin dairy heifers.

Eighteen cycling, virgin, Holstein heifers daily were given 250 mg of 99% purified zearalenone in a gelatin capsule orally, and 18 controls were given an empty gelatin capsule once a day. The study lasted through 1 non-breeding estrous cycle and the next 2 consecutive estrous cycles during which the 36 heifers were bred, using artificial insemination. Serum concentrations of progesterone and complete blood cell counts were determined throughout the study. The treated and control heifers had conception rates of 62% and 87%, respectively. There was no effect (P less than 0.05) on the serum concentration of progesterone or the complete blood cell counts. Three heifers, bred but not pregnant by the end of the study, were euthanatized and necropsied. The treated heifer did not have any zearalenone-attributable lesions, and there was no effect seen in the bone marrow smears. The remaining 33 heifers were sold as a herd, and the 31 pregnant heifers calved normally. There was no effect (P less than 0.05) on the sex ratio of the offspring, which were all clinically healthy. Zearalenone did lower the conception rate of the treated heifers (P less than 0.065).

Porcine proliferative enteritis: serological, microbiological and pathological studies from three field epizootics.

Three outbreaks of porcine proliferative enteritis were evaluated clinically, pathologically, microbiologically and serologically. The disease was characterized by a chronic intermittent diarrhea. Pathological lesions included a thickened, turbid ileum with the microscopic appearance of proliferating ileal crypt epithelial cells. Comma shaped intracytoplasmic organisms were observed in the apical portions of the proliferating crypt epithelial cells with a Warthin-Starry silver stain. Microbiologically, both Campylobacter sputorum subspecies mucosalis and Campylobacter hyointestinalis, were cultured from ileal specimens of seven pigs with lesions of porcine proliferative enteritis. Microagglutination antibody titers were determined on sera from 12 of 14 pigs with porcine proliferative enteritis and on sera from 91 clinically normal swine. Pigs with porcine proliferative enteritis had a low antibody titer to subspecies mucosalis that ranged from 1-3 with a mean of 2.17. A varied C. hyointestinalis titer from 3-7 with mean of 4.83 was determined. Titers to either subspecies mucosalis and C. hyointestinalis were higher in non-porcine proliferative enteritis pigs. The results indicate that the presence of a positive titer to either C. hyointestinalis or subspecies mucosalis in swine is not indicative of clinical disease. The isolation of C. hyointestinalis from diseased ileal specimens (porcine proliferative enteritis) confirms previous reports implicating this agent in the disease.

"Campylobacter hyointestinalis" sp. nov.: a new species of Campylobacter found in the intestines of pigs and other animals.

The name "Campylobacter hyointestinalis" sp. nov. is proposed for a Campylobacter species that was isolated from the intestines of pigs with proliferative enteritis. "C. hyointestinalis" is also found in the feces of cattle and has been isolated from the intestine of a hamster. "C. hyointestinalis" is distinguished from previously described catalase-positive Campylobacter species by colony morphology, ability to produce H2S in triple sugar iron agar, ability to grow anaerobically in 0.1% trimethylamine N-oxide hydrochloride, resistance to nalidixic acid, susceptibility to cephalothin and metronidazole, and hydrogenase activity. Sixteen "C. hyointestinalis" strains were highly related (greater than or equal to 76%) by DNA-DNA hybridization (hydroxyapatite method, 50 and 65 degrees C). Other Campylobacter species were less than or equal to 30% related to "C. hyointestinalis." The type strain of "C. hyointestinalis" is designated 80-4577-4 (= ATCC 35217), and its DNA has a guanine-plus-cytosine content of 36 mol%.