Alpha-D-galactosidases from Thermotoga species.
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Base Sequence↗
Biomedical subjects
E S Miller
Publications and source records attributed to E S Miller.
Subtilisins of Bacillus spp. hydrolyze keratin and allow growth on feathers.
Keratinase is a serine protease produced by Bacillus licheniformis PWD-1 that effectively degrades keratin and confers the ability to grow on feathers to a protease-deficient B. subtilis strain. Studies presented herein demonstrate that B. licheniformis Carlsberg strain NCIMB 6816, which produces the well-characterized serine protease subtilisin Carlsberg, also degrades and grows on feathers. The PWD-1 and Carlsberg strains showed a similar time-course of enzyme production, and the purified serine proteases have similar enzymatic properties on insoluble azokeratin and soluble FITC-casein. Kinetic analysis of both enzymes demonstrated that they have high specificity for aromatic and hydrophobic amino acids in the P1 substrate position, although keratinase discriminates more than subtilisin Carlsberg against charged residues at this site. Nucleotide sequence analysis of the serine protease genes from B. licheniformis strains PWD-1, Carlsberg NCIMB 6816, ATCC 12759, and NCIMB 10689 showed that the kerA-encoded protease of PWD-1 differs from the others only by having V222, rather than A222, near the active site serine S220. Further, high-level expression of subE-encoded subtilisin from B. subtilis (78% similar to subtilisin Carlsberg) also confers growth on feathers on a protease-deficient B. subtilis strain. While strain PWD-1 and the kerA protease efficiently degrade keratin, keratin hydrolysis and growth on feathers is a property that can be conferred by appropriate expression of the major subtilisins, including the industrially produced enzymes.
Animals↗
RNA-binding properties of in vitro expressed histidine-tagged RB69 RegA translational repressor protein.
To facilitate RNA-binding studies of the phage RB69 RegA translational repressor protein, regA was configured to add six histidines to the carboxyl end of the protein. In vitro transcription-translation from the T7 promoter on plasmid pSA1 yielded a RegA69-His6 protein that binds nickel-Sepharose and elutes with 0.5 M imidazole. The system was further modified to avoid cloning and the toxic effects of RegA on Escherichia coli by the polymerase chain reaction (PCR), producing linear templates with the configuration T7 promoter-TIR-regA-His6. A translation initiation region was used that conforms to consensus E. coli and eukaryotic initiation sites and eliminates the target for RegA autogenous repression. RegA69-His6 synthesized in E. coli S30 or wheat germ extracts displayed RNA-binding properties similar to wild-type RB69 RegA. Specificity of RNA binding was demonstrated by in vitro repression of T4 gp44 and gp45 but not beta-lactamase, by differential binding to poly(U)- and poly(C)-agarose, and by site-specific binding to a 23-base gene 44 target RNA but not to mutant 44 RNA. Therefore, addition of the His6 tag to the C-terminus of RB69 RegA does not dramatically alter RNA binding, indicating that this region is not directly involved in site recognition. With access to several T4-like phage genomes and regA mutant sequences, in vitro synthesis of His-tagged proteins directly from linear PCR products provides a convenient and efficient system to study RegA and other interesting RNA-binding proteins.
Bacteriophage T4↗
Effects of spaceflight and PEG-IL-2 on rat physiological and immunological responses.
Sprague-Dawley rats were subjected to two 8-day spaceflights on the space shuttle. Rats housed in the National Aeronautics and Space Administration's animal enclosure were injected (iv or sc) with pegylated interleukin-2 (PEG-IL-2) or a placebo. We tested the hypothesis that PEG-IL-2 would ameliorate some of the effects of spaceflight. We measured body and organ weights; blood cell differentials; plasma corticosterone; colony-forming units (macrophage and granulocyte macrophage); lymphocyte mitogenic, superantigenic, and interferon-gamma responses; bone marrow cell and peritoneal macrophage cytokine secretion; and bone strength and mass. Few immunological parameters were affected by spaceflight. However, some spaceflight effects were observed in each flight. Specifically, peritoneal macrophage spontaneous secretion of tumor necrosis factor-alpha occurred in the first but not in the second flight. A significant monocytopenia and lymphocytopenia were detected in the second but not in the first flight. The second mission produced bone changes more consistent with past spaceflight investigations. PEG-IL-2 did not appear to be beneficial; however, this was mostly due to the lack of spaceflight effects. These studies reflect the difficulty in reproducing experimental models by using current space shuttle conditions.
Animals↗
2-deoxy-D-glucose-induced metabolic stress enhances resistance to Listeria monocytogenes infection in mice.
Exposure to different forms of psychological and physiological stress can elicit a host stress response, which alters normal parameters of neuroendocrine homeostasis. The present study evaluated the influence of the metabolic stressor 2-deoxy-D-glucose (2-DG; a glucose analog, which when administered to rodents, induces acute periods of metabolic stress) on the capacity of mice to resist infection with the facultative intracellular bacterial pathogen Listeria monocytogenes. Female BDF1 mice were injected with 2-DG (500 mg/kg b. wt.) once every 48 h prior to, concurrent with, or after the onset of a sublethal dose of virulent L. monocytogenes. Kinetics of bacterial growth in mice were not altered if 2-DG was applied concurrently or after the start of the infection. In contrast, mice exposed to 2-DG prior to infection demonstrated an enhanced resistance to the listeria challenge. The enhanced bacterial clearance in vivo could not be explained by 2-DG exerting a toxic effect on the listeria, based on the results of two experiments. First, 2-DG did not inhibit listeria replication in trypticase soy broth. Second, replication of L. monocytogenes was not inhibited in bone marrow-derived macrophage cultures exposed to 2-DG. Production of neopterin and lysozyme, indicators of macrophage activation, were enhanced following exposure to 2-DG, which correlated with the increased resistance to L. monocytogenes. These results support the contention that the host response to 2-DG-induced metabolic stress can influence the capacity of the immune system to resist infection by certain classes of microbial pathogens.
Adjuvants, Immunologic↗
Spaceflight and development of immune responses.
The NIH.R1 Space Shuttle experiment was designed to study the effects of spaceflight on rodent development. Pregnant rats were flown on the Space Shuttle for 11 days, and pregnant control rats were maintained in animal enclosure modules in a ground-based chamber under conditions approximating those in flight. Additional controls were in standard housing. The effects of the flight on immunological parameters of dams, fetuses, and pups were determined. Blastogenesis of spleen cells in response to mitogen was inhibited in flown dams but was not inhibited in cells from their pups. Interferon-gamma production by spleen cells showed a trend toward inhibition in flown dams but not in their pups. The response of bone marrow cells to colony-stimulating factor showed a trend toward inhibition after spaceflight in dams, but the response of fetus and pup liver cells was not inhibited. Total serum IgG was not affected by spaceflight. None of the examined immune parameters that were altered in rat dams after spaceflight was found to be altered in their offspring.
Animals↗
An E. coli B mutation, rpoB5081, that prevents growth of phage T4 strains defective in host DNA degradation.
An E. coli B Tab strain, EM121, was isolated that restricts T4 denA (DNA endonuclease II) mutants at 37 degrees C and above, but is permissive for wild-type T4 at all temperatures examined. At 42 degrees C, other mutants affected in nucleic acid metabolism (T4 dexA, regA and uvsW strains) are also restricted. Genetic analysis revealed that one mutation (rpoB5081) in the RNA polymerase beta subunit gene is sufficient for restricting all denA mutants. rpoB5081, together with a second linked mutation, is also required for restricting the other T4 mutants, rpoB5081 (P806S), previously shown to increase transcription termination in E. coli K-12, causes delayed synthesis of T4 late proteins and reduced DNA synthesis in denA infections. Thus, T4 DNA synthesis and gene expression are impaired by the rpoB5081 beta subunit when degradation of host DNA is reduced. Because the restricted T4 mutants are not readily distinguished from wild-type phage under typical plating conditions, EM121 is an important host for screening and mapping T4 denA mutations.
Alleles↗
Expression of the Bacillus licheniformis PWD-1 keratinase gene in B. subtilis.
The kerA gene which encodes the enzyme keratinase was isolated from the feather-degrading bacterium Bacillus licheniformis PWD-1. The entire gene, including pre-, pro- and mature protein regions, was cloned with Pker, its own promoter, P43, the vegetative growth promoter, or the combination of P43-Pker into plasmid pUB18. Transformation of the protease-deficient strain B. subtilis DB104 with these plasmids generated transformant strains FDB-3, FDB-108 and FDB-29 respectively. All transformants expressed active keratinase in both feather and LB media, in contrast to PWD-1, in which kerA was repressed when grown in LB medium. With P43-Pker upstream of kerA, FDB-29 displayed the highest activity in feather medium. Production of keratinase in PWD-1 and transformants was further characterized when glucose or casamino acids were supplemented into the feather medium. These studies help understand the regulation of kerA expression and, in the long run, can help strain development and medium conditioning for the production of this industrially important keratinase.
Bacillus↗
Vulvar basal cell carcinoma.
BACKGROUND: Vulvar basal cell carcinoma is a rare neoplasm that often displays aggressive biologic behavior. Recurrence after simple excision is common, and metastases have been reported. Early diagnosis is essential and rests upon histopathologic examination of biopsy specimens, since the clinical appearance of these neoplasms may be deceptively innocent. CASE: A 65-year-old woman was seen with a nonhealing skin lesion on the right vulva. Physical examination revealed a 0.3 X 0.3 cm crusted papule on the right labium majus. A saucerization biopsy was performed. Histopathologic examination of the biopsy specimen revealed basal cell carcinoma. The patient underwent Mohs micrographic excision of the tumor, the total extent of which measured 0.9 X 1.8 cm. CONCLUSION: Vulvar basal cell carcinoma is a rare but important consideration in the differential diagnosis of cutaneous vulvar lesions. Accurate diagnosis depends upon a high index of suspicion, biopsy, and histopathologic examination. Mohs micrographic excision is the treatment of choice.
Aged↗
Progressive osseous heteroplasia.
BACKGROUND: Primary heterotopic ossification beginning in childhood is quite rare but occurs in several well-described conditions, such as Albright hereditary osteodystrophy, fibrodysplasia ossificans progressiva, and platelike osteoma cutis. Recently, a new disorder called progressive osseous heteroplasia (POH) has been described in the orthopedic literature. Primary cutaneous calcification and ossification beginning in infancy are presenting signs of this progressive and potentially debilitating disorder. OBSERVATIONS: We describe 2 children with POH who were recently seen at Children's Hospital of Wisconsin. Both children were female and developed cutaneous calcification and ossification within the first 6 months of life. Both girls had progression of the lesions to involve ossification of the deeper tissues. No abnormalities in calcium, phosphorus, vitamin D, or parathyroid hormone levels were identified in these patients. No associated anomalies were detected. Biopsy results from the lesions showed calcinosis cutis superficially, with both endochondral and intramembranous bone formation in the deeper tissues. CONCLUSIONS: Progressive osseous heteroplasia must now be included in the differential diagnosis of primary cutaneous ossification beginning in childhood. Because the first clinical manifestations of POH appear in the skin. It is important for dermatologists to be aware of this newly described condition.
Calcinosis↗
Treating mental health problems of health care workers.
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England↗
Nucleotide sequence and expression of kerA, the gene encoding a keratinolytic protease of Bacillus licheniformis PWD-1.
Bacillus licheniformis PWD-1 (ATCC 53757) secretes keratinase, a proteolytic enzyme which is active on whole feathers. By amino acid sequence similarity and phenylmethylsulfonyl fluoride inhibition, the keratinase was demonstrated to be a serine protease. The entire nucleotide sequence of the coding and flanking regions of the keratinase structure gene, kerA, was determined. A fixed oligonucleotide primer derived from the N-terminal sequence of the purified enzyme and a second random oligonucleotide primer were used in a procedure called PCR walking, which was developed to amplify and sequence the upstream and downstream regions of kerA. Another method, PCR screening, was conducted with a lambda phage vector with inserted PWD-1 genomic DNA fragments as templates and with the known sequences of the vector arms and the N-terminal sequence of the enzyme as primers. PCR amplification and sequence analysis of the lambda library completed the entire kerA sequence and established a set of gene deletions. The kerA gene shares a 97% sequence identity with the gene encoding subtilisin Carlsberg from B. licheniformis NCIMB 6816. The putative promoters, ribosome binding sites, and transcriptional terminators are also similar in these two bacteria. The deduced amino acid sequences indicate only three amino acid differences between the two mature proteases. Northern (RNA) analysis demonstrates that transcriptional regulation controls kerA expression on different growth media.
Amino Acid Sequence↗
Influence of spaceflight on the production of interleukin-3 and interleukin-6 by rat spleen and thymus cells.
Six adult male Sprague-Dawley rats were flown on the 7-day US space shuttle mission STS-54. After flight, the spleen and thymus from each animal were assayed for the capacity to secrete the cytokines interleukin-3 (IL-3) and IL-6. Spleen and thymus cells (5 x 10(6)/ml) were incubated for 48 h in the presence of 5 micrograms/ml of concanavalin A or 2 micrograms/ml of bacterial lipopolysaccharide to stimulate the production of IL-3 and IL-6. IL-3 activity was measured using the IL-3/colony-stimulating-factor-dependent cell line 32D. IL-6 activity was measured using the IL-6-dependent cell line 7TD1. Spleen and thymus cells harvested from flown rats secreted significantly higher titers of biologically active IL-3 compared with ground control rats. Spaceflight significantly enhanced IL-6 production by thymus, but not spleen, cells. The results of this study demonstrate that spaceflight can enhance the production of certain cytokines by cells of the immune system.
Animals↗
RNA-protein interactions of the bacteriophage RB69 RegA translational repressor protein.
We report the RNA-protein interactions of phage RB69 RegA and RegA691 proteins. In RNase protection assays, RB69 RegA protects from 10 (44 mRNA) to >70 (rpbA) nucleotides; for rpbA in particular, the length of the protected region is RegA-concentration dependent. Quantitative fluorescence quenching measurements with RB69 wildtype RegA and RegA691 show that both proteins bind poly(U) similarly, but RegA691 binds with lower affinity to both 44 and rpbA RNA. The results indicate that both RB69 RegA RNA-protein and protein-protein interactions contribute to a translational repression hierarchy.
Bacteriophage T4↗
Influence of antiorthostatic suspension on resistance to murine Listeria monocytogenes infection.
The present study was designed to evaluate the influence of antiorthostatic suspension, a ground-based modeling system employed to simulate certain aspects of weightlessness that occur during space flight, on the capacity of mice to resist infection with the facultative intracellular bacterial pathogen Listeria monocytogenes. Female BDF1 mice were suspended by the tail in the orthostatic or antiorthostatic position and were infected with a sublethal dose of virulent L. monocytogenes at various times during the suspension. It was found that suspension did not influence the kinetics of bacterial growth in vivo if the infection was started concurrently with the suspension. However, mice that were antiorthostatically suspended 2, 4, or 7 days before the onset of infection exhibited an enhanced capacity to eliminate the challenge infection. Suspending mice on day 2 of the infection did not alter the kinetics of bacterial growth. Finally, the enhancement of resistance to the primary Listeria infection was accompanied by failure of the mice to generate long-term protective immunological memory to the challenge organism. Collectively, these results indicate that the stress of antiorthostatic suspension can influence the capacity of mice to resist bacterial infection.
Animals↗
Inhibition of murine splenic T lymphocyte proliferation by 2-deoxy-D-glucose-induced metabolic stress.
Female Swiss-Webster mice were injected with the glucose analogue 2-deoxy-D-glucose (2-DG), which when administered to rodents induces acute periods of metabolic stress. A single or multiple injections of 2-DG invoked a stress response, as evidenced by increases in serum corticosterone levels. The influence of this metabolic stressor on the blastogenic potential of splenic T lymphocytes was then examined. It was found that one, two, or three injections of 2-DG resulted in depressed T cell proliferative responses, with an attenuation of the effect occurring by the fifth injection. The 2-DG-induced inhibition of T cell proliferation was not attributable to 2-DG-induced cytolysis, as in vitro incubation of naive T cells with varying concentrations of 2-DG did not result in a reduction in cell number or viability, and flow cytometric analysis demonstrated that percentages of CD3, CD4, and CD8 splenic T cells were not altered as a result of 2-DG-induced stress. Incubating naive T cells in varying concentrations of 2-DG resulted in a dose-dependent inhibition of T cell blastogenic potential. Following in vivo exposure to 2-DG, T cell proliferation did not return to normal levels until 3 days after the cessation of 2-DG injections. Administering the beta-adrenergic receptor antagonist propranolol did not reverse the inhibited lymphoproliferation in 2-DG-treated mice. The inhibition in T cell proliferation was not observed, however, in mice that had been adrenalectomized or hypophysectomized and injected with 2-DG.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenalectomy↗
The Rhesus monkey as a model for testing the immunological effects of space flight.
The Rhesus monkey has been proposed as a model for the effects of space flight on immunity. In order to determine the feasibility of the use of the Rhesus monkey as a model, we studied the use of Rhesus monkey cells for immunological procedures that have been shown to be affected by space flight in both rodents and humans. We have shown that both lymph node cells and peripheral blood leukocytes can be stained with monoclonal antibodies to detect the following surface markers: CD4, CD-8, Ia and surface immunoglobulin. Also, the level of Ia antigen expression was increased by treatment of the cells with human interferon-gamma. In addition, cells were induced to produce interferons and interleukins. Isolated neutrophils also demonstrated increased oxidative burst. These data indicate that the Rhesus monkey will be a useful model for space flight studies of immunity.
Animals↗
Influence of suspension on the oxidative burst by rat neutrophils.
The influence of spaceflight on the oxidative burst of neutrophils is not known. The present study was designed to evaluate the influence of antiorthostatic suspension, a ground-based modeling system designed to simulate certain aspects of weightlessness that occur after spaceflight, on the capacity of rat neutrophils to express the oxidative burst, an important host defense mechanism against microbial pathogens. Rats were suspended in whole body harnesses in the antiorthostatic orientation for a 3- or 7-day period. Control rats were suspended orthostatically or allowed to remain in vivarium cages without the attachment of any suspension materials. After suspension, peripheral blood was harvested and neutrophils were isolated by density gradient centrifugation. The enriched neutrophil preparations were stimulated with N-formyl-methionyl-leucine-phenylalanine and phorbol myristic acid to induce the oxidative burst. It was found that neutrophils isolated from suspended animals released the same levels of superoxide anion as did vivarium control animals that were not suspended, indicating that whole body suspension did not alter this aspect of rat neutrophil function.
Animals↗