Taxonomic evaluation of Streptomyces albus and related species using multilocus sequence analysis and proposals to emend the description of Streptomyces albus and describe Streptomyces pathocidini sp. nov.

In phylogenetic analyses of the genus Streptomyces using 16S rRNA gene sequences, Streptomyces albus subsp. albus NRRL B-1811(T) forms a cluster with five other species having identical or nearly identical 16S rRNA gene sequences. Moreover, the morphological and physiological characteristics of these other species, including Streptomyces almquistii NRRL B-1685(T), Streptomyces flocculus NRRL B-2465(T), Streptomyces gibsonii NRRL B-1335(T) and Streptomyces rangoonensis NRRL B-12378(T) are quite similar. This cluster is of particular taxonomic interest because Streptomyces albus is the type species of the genus Streptomyces. The related strains were subjected to multilocus sequence analysis (MLSA) utilizing partial sequences of the housekeeping genes atpD, gyrB, recA, rpoB and trpB and confirmation of previously reported phenotypic characteristics. The five strains formed a coherent cluster supported by a 100 % bootstrap value in phylogenetic trees generated from sequence alignments prepared by concatenating the sequences of the housekeeping genes, and identical tree topology was observed using various different tree-making algorithms. Moreover, all but one strain, S. flocculus NRRL B-2465(T), exhibited identical sequences for all of the five housekeeping gene loci sequenced, but NRRL B-2465(T) still exhibited an MLSA evolutionary distance of 0.005 from the other strains, a value that is lower than the 0.007 MLSA evolutionary distance threshold proposed for species-level relatedness. These data support a proposal to reclassify S. almquistii, S. flocculus, S. gibsonii and S. rangoonensis as later heterotypic synonyms of S. albus with NRRL B-1811(T) as the type strain. The MLSA sequence database also demonstrated utility for quickly and conclusively confirming that numerous strains within the ARS Culture Collection had been previously misidentified as subspecies of S. albus and that Streptomyces albus subsp. pathocidicus should be redescribed as a novel species, Streptomyces pathocidini sp. nov., with the type strain NRRL B-24287(T).

Event excess in the MiniBooNE search for ¯νµâ†’¯νe oscillations.

The MiniBooNE experiment at Fermilab reports results from a search for ¯ν_{µ}→¯ν_{e} oscillations, using a data sample corresponding to 5.66×10²° protons on target. An excess of 20.9±14.0 events is observed in the energy range 475

Population genetic screening efficiently identifies carriers of autosomal dominant diseases.

Three inherited autosomal dominant conditions-BRCA-related hereditary breast and ovarian cancer (HBOC), Lynch syndrome (LS) and familial hypercholesterolemia (FH)-have been termed the Centers for Disease Control and Prevention Tier 1 (CDCT1) genetic conditions, for which early identification and intervention have a meaningful potential for clinical actionability and a positive impact on public health1. In typical medical practice, genetic testing for these conditions is based on personal or family history, ethnic background or other demographic characteristics2. In this study of a cohort of 26,906 participants in the Healthy Nevada Project (HNP), we first evaluated whether population screening could efficiently identify carriers of these genetic conditions and, second, we evaluated the impact of genetic risk on health outcomes for these participants. We found a 1.33% combined carrier rate for pathogenic and likely pathogenic (P/LP) genetic variants for HBOC, LS and FH. Of these carriers, 21.9% of participants had clinically relevant disease, among whom 70% had been diagnosed with relevant disease before age 65. Moreover, 90% of the risk carriers had not been previously identified, and less than 19.8% of these had documentation in their medical records of inherited genetic disease risk, including family history. In a direct follow-up survey with all carriers, only 25.2% of individuals reported a family history of relevant disease. Our experience with the HNP suggests that genetic screening in patients could identify at-risk carriers, who would not be otherwise identified in routine care.

Evidence for a fourteen-gene, phnC to phnP locus for phosphonate metabolism in Escherichia coli.

The Escherichia coli phn (psiD) locus consists of a large gene cluster encoding proteins necessary for the use of phosphonates (Pn) as a sole phosphorus source. On the basis of nucleotide (nt) sequence analysis, the phn locus contains a 12.6-kb operon of seventeen genes named, in alphabetical order, phnA to phnQ [Chen et al., J. Biol. Chem. 265 (1990) 4461-4471]. New Pn+ plasmids were made which are suitable for mutational analysis of this gene cluster. These plasmids contain the R6K origin for DNA replication, can be conjugatively transferred, contain the tetAR genes, and therefore provide a way for allele replacement. The construction of these plasmids showed that phnA and phnB have no role in Pn metabolism. Also, these plasmids were employed to introduce nonpolar phnD::lacZ and phnD::uidA fusions into the chromosome, which allowed us to show that phnD probably has a role in transport. In addition, it was shown that phnP is the most distal gene required for Pn use. This was done by testing the effect of phn::uidA insertions in or near the 3' end of phnP on Pn use. Altogether, these results show that all genes required for Pn use are in the 10.9-kb, fourteen-gene, phnCDEFGHIJKLMNOP locus.

Construction of new beta-glucuronidase cassettes for making transcriptional fusions and their use with new methods for allele replacement.

Five cassettes carrying uidA, encoding beta-glucuronidase, were made for the construction of insertion mutants with transcriptional fusions to uidA. Three uidA cassettes contain antibiotic-resistance genes, for chloramphenicol (Cm), for kanamycin (Km) and neomycin (Nm), or for streptomycin (Sm) and spectinomycin (Sp). Some cause polar insertions while others provide a promoter for downstream gene expression. The expression of these uidA cassettes was compared to the expression of lacZ at the same site in phnD, a phosphate-regulated gene for phosphonate use. Several phn::uidA or phn::lacZ insertions were recombined onto the chromosome to test mutational effects and to measure gene expression in single copy. This was done using one of three methods for allele replacement. A new method involved recombination of mutations in M13 onto the chromosome by infection of an Escherichia coli rep mutant that fails to propagate single-stranded DNA phages. Merodiploid recombinants were selected using a resistance marker carried by the M13 phage; segregants lacking M13 sequences were then selected as deoxycholate-resistant (DocR) ones. An improved method for recombination of mutations in pir-dependent, oriR6K vectors involved the use of plasmids containing genes for tetracycline resistance (TcR). Merodiploid recombinants were selected by conjugative transfer of such plasmids into a recipient lacking pir (encoding the pi protein of the R6K plasmid); segregants lacking vector sequences were subsequently selected as Tc-sensitive ones. Both procedures are efficient and allow for recombining marked as well as unmarked mutations onto the chromosome. In addition, some insertions with an antibiotic-resistance marker were directly recombined onto the chromosome by transformation of a recD mutant with linear DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6K gamma origin plasmids at different copy numbers.

Escherichia coli hosts were constructed for maintenance of vectors containing the gamma replication origin of the R6K plasmid (oriRR6K gamma) at different copy numbers (15 or 250/cell). Such vectors require the trans-acting II protein (the pir gene product) for replication. New hosts carry pir+ or pir-116 on the chromosome within uidA, the E. coli gene encoding beta-glucuronidase. They were made using the rep technique for allele replacement and KmR M13 delta uid A::pir+ or M13 delta uidA::pir-116 phage. Because M13 cannot replicate in a rep mutant, KmR transductants arose by integration into the chromosomal uidA locus. Segregants lacking M13 sequences (which were selected as deoxycholate-resistant (DocR) ones) frequently contained delta uidA::pir+ or delta uidA::pir-116 on the chromosome. In principle, this procedure could be used for the introduction of any foreign gene into any nonessential gene on the E. coli chromosome. The delta uidA::pir+ and delta uidA::pir-116 loci were subsequently transferred to a variety of E. coli strains. One such strain is a suppressor-negative one that is especially useful for transposon (Tn) mutagenesis. This strain has an integrated RP4 derivative for conjugative transfer of oriRR6K gamma plasmids also containing oriT from RP4. In addition, new oriRR6K gamma, oriT+ vectors carrying the TcR-encoding genes tetAR from Tn10 are described. These can be used for allele replacement by conjugative transfer of an oriRR6K gamma, oriT+, tetAR plasmid containing a mutated gene into a non-pir recipient and by subsequent selection for Tc-sensitive exconjugants.

Lymphocyte fluorescent profiles (LFP): a possible screening technique in neoplasia.

A technique involving fluorescent protein staining and microfluorometry has been developed for measuring the lymphocyte fluorescent profile (LFP) of peripheral blood lymphocytes. In contrast to normal humans who display a regular bell-shaped curve, the profile from patients with cancer is irregular, showing a bimodal distribution of fluorescence, with a significant population of cells fluorescing at a higher relative intensity. It is suggested that this elevation in protein concentration is due to an immune response to the presence of a neoplasm, and thus this technique may prove to be a useful indicator of malignancy.

The kinetic differences between sodium nitrite, amyl nitrite and nitroglycerin oxidation of hemoglobin.

The effect of sodium nitrite, amyl nitrite and nitroglycerin (glyceryl trinitrate) on the hemoglobin of adult erythrocytes was examined in vitro. Both amyl nitrite and nitroglycerin reacted immediately with oxyhemoglobin to effect oxidation into methemoglobin while sodium nitrite required an inductionary period (lag phase) prior to the reaction. Kinetic studies of the biomolecular rate law for each of the preceding reaction's reactionary periods (log phases) allowed rate constant calculations to be made. The values are 1.14 x 10(4) M-1 min-1, 7.45 x 10(4) M-1 min-1, and 3.50 x 10(1) M-1 min-1 for sodium nitrite, amyl nitrite and nitroglycerin, respectively. A comparison of the amyl nitrite and nitroglycerin rate constants reveals that amyl nitrite is approximately 2000-fold more toxic to oxyhemoglobin than nitroglycerin. These oxidant's effect on in vitro hemoglobin solutions are comparable since both reactions approximate to rectangular hyperbolae. Sodium nitrite reacts about 300-fold faster with oxyhemoglobin than does nitroglycerin. However, the sodium nitrite reaction proceeds in a sigmoidal fashion which makes a strict comparison between these compounds relative toxicities less clear cut.

Molecular genetic studies of a 10.9-kb operon in Escherichia coli for phosphonate uptake and biodegradation.

Bacteria that use phosphonates as a phosphorus source must be able to break the stable carbon-phosphorus bond. In Escherichia coli phosphonates are broken down by a C-P lyase that has a broad substrate specificity. Evidence for a lyase is based on in vivo studies of product formation because it has been proven difficult to detect the activity in vitro. By using molecular genetic techniques, we have studied the genes for phosphonate uptake and degradation in E. coli, which are organized in an operon of 14 genes, named phnC to phnP. As expected for genes involved in P acquisition, the phnC-phnP operon is a member of the PHO regulon and is induced many hundred-fold during phosphate limitation. Three gene products (PhnC, PhnD and PhnE) comprise a binding protein-dependent phosphonate transporter, which also transports phosphate, phosphite, and certain phosphate esters such as phosphoserine; two gene products (PhnF and PhnO) may have a role in gene regulation; and nine gene products (PhnG, PhnH, PhnI, PhnJ, PhnK, PhnL, PhnM, PhnN, and PhnP) probably comprise a membrane-associated C-P lyase enzyme complex. Although E. coli can degrade many different phosphonates, the ability to use certain phosphonates appears to be limited by the specificity of the PhnCDE transporter and not by the specificity of the C-P lyase.

Kinetics of amyl nitrite-induced hemoglobin oxidation in cord and adult blood.

The effect of amyl nitrite on the erythrocytes of adult and cord hemoglobin was examined in vitro. This study revealed that amyl nitrite caused oxyhemoglobin to become oxidized to methemoglobin wherein a rectangular hyperbolic curve was generated as the reaction progressed. This curve consisted of a reactionary log phase, and a terminal asymptotic phase only, with no inductionary lag phase. A comparative study of human cord blood oxidation times and adult blood was undertaken. It was revealed that cord blood erythrocytes were oxidized by amyl nitrite at a 5-6-fold greater rate than adult blood erythrocytes. Based on an independent Student's t-test, the time taken for cord blood erythrocytes to undergo oxidation was significantly shorter (P less than 0.05) than adult controls. This greatly enhanced reactivity of cord blood erythrocytes parallels earlier findings when sodium nitrite was used instead of amyl nitrite. However, this difference defies a simple explanation and must be attributed to many factors which may include pH, structural differences, and solubility phenomenon.

Reliability of quantitative muscle testing in healthy children and in children with Duchenne muscular dystrophy using a hand-held dynamometer.

The purpose of this study was to examine intratester and test-retest reliability using a hand-held dynamometer for the measurement of isometric muscle strength in 28 healthy children and children with Duchenne muscular dystrophy. The Dystrophic Group consisted of 14 children diagnosed with Duchenne muscular dystrophy, and the Healthy Group consisted of 14 age-matched children with no history of orthopedic or neuromuscular disorders. One physical therapist tested hip and knee extension, elbow flexion, and shoulder abduction in each child bilaterally. A two-way analysis of variance for repeated measures was used to analyze differences between measurements taken within and across the testing sessions. Pearson product-moment correlation coefficients were determined on mean values across the testing sessions for each variable. No significant differences (p greater than .05) between measurements taken within or across testing sessions were found in either the Dystrophic Group or the Healthy Group. Correlation coefficients for the Dystrophic Group ranged from .83 to .99 for the variables tested. Correlation coefficients for the Healthy Group ranged from .74 to .99. The results suggest that the hand-held dynamometer can be used as a reliable instrument in measuring the isometric strength of selected muscles in children.

Changes in the lymphocyte cytoplasmic refractive index following typhoid vaccination.

The refractive index of the circulating blood lymphocytes (LCRI) of healthy young adults vaccinated with typhoid vaccine has been measured by immersion refractometry and phase contrast microscopy. A rise in the LCRI preceeds the rise in the antibody titre by two to three days. A more pronounced rise also occurred in one patient prior to a rejection crises following a kidney transplant.

Morphometric analysis of rabbit skeletal muscle and peripheral nerve tissue after electrical stimulation.

Transcutaneous medium frequency electrical stimulation is used extensively to improve muscle strength in people who encounter difficulty in improving strength voluntarily. The purpose of this study was to describe some morphometric effects of electrical stimulation applied to rabbit skeletal muscle and peripheral nerve tissue (N = 5 control and 7 experimental rabbits). Intermittent electrical current (4000 Hz pulse modulated at 50 Hz) was applied transcutaneously to adult female rabbit thigh muscle 3 times/week for 3 months. Muscle ATPase histochemical staining, followed by morphometric analysis, demonstrated that type IIB fibers in stimulated muscles (sartorius and vastus lateralis) had larger cross-sectional areas in comparison to nonstimulated muscle fibers. Type IIA fibers of the stimulated sartorius muscle also were hypertrophied in comparison to nonstimulated muscle fibers. The percentage distribution of muscle fiber types did not change significantly as a result of stimulation. The femoral nerves of these rabbits were fixed and stained. Morphometric analysis did not detect any significant change in myelin cross-sectional area or thickness. Also, nerve axoplasmic cross-sectional area in stimulated femoral nerves was not significantly different from controls. These data suggest that electrical stimulation can increase the size of skeletal muscle fibers if applied consistently for an extended period of time. Further research is needed to further characterize this effect and to determine whether the same effect can be observed in humans after prolonged stimulation. J Orthop Sports Phys Ther 1990;11(7):313-320.