Loss of thick filaments from fast-twitch glucolytic muscle fibers of the pigeon pectoralis after chronic administration of dantrolene sodium.

Adult pigeons received dantrolene sodium, a skeletal muscle relaxant which blocks the release of calcium during excitation-contraction coupling, for 12 to 16 weeks. The pectoralis muscles of these birds were analyzed for changes occurring in the various fiber types of the muscle. Both histochemistry (ATPase and SDH activity) and electron microscopy (mitochondrial and lipid volume percentages) differentiated two fiber types. The two fiber-types consisted of fast-twitch glycolytic fibers (FG) and fast-twitch oxidative-glycolytic (FOG) fibers. After dantrolene treatment some FG fibers showed little or no ATPase activity. Dantrolene treatment also produced a disappearance of thick filaments in some FG fibers. We infer that the fibers without thick filaments are the ones lacking ATPase activity. The FOG fibers were nearly normal. Since drug-fed birds lose weight, a few birds were starved to determine whether the filament loss was related solely to the bird's loss in weight. No fibers in starved birds showed reduced ATPase activity or loss of thick filaments. In fibers that showed thick filament disappearance, the I-bands remained organized and intact, suggesting that the I-band maintains its integrity without interaction with the thick filaments. Changes in activity patterns may cause loss of thick filaments by inhibiting either their synthesis or assembly.     

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants.

A single-step most-probable-number method for enumerating fecal coliforms in sewage treatment plant effluents is described. The method requires the use of only one lactose-based medium and a single incubation temperature of 44.5 degrees C, and it can be completed in 18 h or less, as compared with up to 72 h for the standard most-probable-number method. The appearance of growth is the sole criterion used for designating positives, which can be determined either by increases in the electrical impedance ratio of inoculated medium, as compared to an uninoculated control using a Bactometer model 32, or by visual examination of inoculated medium for turbidity. In trials with 53 samples of unchlorinated sewage treatment plant effluent, fecal coliform counts by the single-step most-probable-number method, throughout a range of less than 10 to almost 10(7) fecal coliforms per 100 ml of effluent, were in excellent agreement with counts abtained by the standard most-probable-number procedure. Similar agreement was obtained in comparative trials with 31 chlorinated effluent samples from two sewage treatment plants. Overall, 87% of 452 positives were confirmed as containing fecal coliforms. The applicability of the single-step most-probable-number method to automated sewage treatment plant operations is discussed.     

Creatine kinase elevations in marathon runners: relationship to training and competition.

Elevation of creatine kinase (CK) in serum after exertion is a reliable marker of skeletal muscle injury. Limited data exist on CK levels in conditioned athletes after endurance training and competition. Serum CK was measured by a kinetic UV method (normal < 100 U/L) in 15 long distance runners before (pre-race), 24 hours after (post-race) and four weeks following (post-race) the 1979 Boston Marathon. CK levels were elevated throughout the study. Mean values for all runners and for those finishing before and after three hours and 30 minutes are as follows: Post-race CK was significantly elevated among the ten faster as compared to the five slower runners (p = 0.025). Elevations of creatine kinase drawn 24 hours post-marathon are inversely related to finishing times among the runners tested.     

Phase variation: genetic analysis of switching mutants

Site-specific inversion of a controlling element is responsible for flagellar phase transition in Salmonella. When a 900 bp DNA sequence is in one configuration, it allows the expression of the H2 gene, a structural gene which codes for the flagellar antigen. When it is in the opposite configuration, the H2 gene is not expressed. A hybrid λ phage containing the invertible control region and the adjacent H2 gene was constructed, and expression of the H2 gene was shown to be regulated by the orientation of the inversion region. Transposon Tn5 insertion derivatives of this hybrid phage were isolated and λH2::Tn5 mutants defective for inversion (H2 switching) were selected and characterized. Two classes of switching phenotypes were observed among the mutants—those which had slightly reduced frequencies of transition from expression of the H2 gene (H2 on) to nonexpression (H2 off) (intermediate class) and those in which the frequency of transition was reduced at least three orders of magnitude (null class). Physical mapping of the Tn5 insertion sites revealed that in all mutants the insertion was located inside the inversion region. Tn5 insertion sites in the null class of mutants defined a region of DNA including approximately 500 bp which was necessary for inversion. Genetic complementation tests showed that these λH2::Tn5 mutants could invert the H2 gene control element if the 500 bp region was introduced in the trans configuration. It is concluded that a gene is located inside the inversion segment and codes for a protein which is required for the inversion event. Furthermore, the two sites at which the crossover event occurred functioned in a cis configuration and were required for inversion. The presence of a gene which is involved in controlling site-specific recombination events may be a general feature of transposon-like elements.     

A rapid method for preparation of calf spleen exonuclease.

Chromatography on Concanavalin A sepharose is a simple procedure for partial purification of spleen phosphodiesterase. This procedure removes much, but not all, of the ribonuclease activity found as contaminant in most spleen phosphdisterase preparations.     

Sexual Therapy of Patients with Cardiovascular Disease

Physical illness or disability inevitably has a damaging effect on sexual relationships. Physicians are usually unaware of the sexual consequences of illness on their patients, and lack experience in treating sexual dysfunctions.The report of treatment of a couple with serious cardiovascular disease illustrates the potential efficacy of brief sex therapy for improving the quality of a patient''s life. If a primary physician lacks the skills to conduct sex therapy, he may collaborate with nonphysician therapists. The physician''s knowledge of the physiological and psychological effects of a specific illness on his patient is essential to successful therapy. Often, simple education, encouragement or reassurance by the physician is enough to overcome the damaging effects of illness on a patient''s sex life.     

Model for DNA packaging into bacteriophage T4 heads.

The mechanism of DNA packaging into bacteriophage T4 heads in vivo was investigated by glucosylation of hydroxymethylcytosine residues in a conditionally glucose-deficient host. Cytoplasmic DNA associated with partially packaged ts49 heads can be fully glucosylated, whereas DNA already packaged into these heads is shown to be resistant to glucosylation. After temperature shift and completion of arrested packaging into the reversible temperature-sensitive ts49 head, the structure of the DNA in the mature ts49 phage was investigated by restriction enzyme digestion, autoradiography, and other techniques. Such mature DNA appears to be fully glucosylated along part of its length and nonglucosylated on the remainder. Its structure suggests that the DNA is run into the head linearly and unidirectionally from one mature end and that there is little sequence specificity in that portion of the T4 DNA which first enters the capsid. This technique should be useful in investigation of the three-dimensional structure of first- and last-packaged DNA within the head; preliminary studies including autoradiography of osmotically shocked phage suggest that the DNA which first enters the head is deposited toward the center of the capsid and that the end of the DNA which first enters the head exits first upon injection. In conjunction with studies of the structure of condensed DNA, the positions and functions of T4 capsid proteins in DNA packaging, and the order of T4 packaging functions [Earnshaw and Harrison, Nature (London) 268:598-602, 1977; Hsiao and Black, Proc. Natl. Acad. Sci. U.S.A. 74:3652-3656, 1977; Müller-Salamin et al., J. Virol. 24:121-134, 1977; Richards et al., J. Mol. Biol. 78:255-259, 1973], the features described above suggest the following model: the first DNA end is fixed to the proximal apex of the head at p20 and the DNA is then pumped into the head enzymatically by proteins (p20 + p17) which induce torsion in the DNA molecule.     

Transfer of immunity to Plasmodium berghei by spleen and lymph node immune RNA.

The RNA from spleens and lymph nodes of Lewis rats immune to Plasmodium berghei protected A/J mice against a lethal challenge of the blood stages of P. berghei, NK65. The RNA was extracted by the hot phenol procedure from freshly removed spleens and lymph nodes. Protection was measured by survival and level of parasitemia as compared to controls. The levels of RNA administered were 10, 50, and 100 μg of RNA. There was observed 100% survival with 50 and 100 μg of immune spleen RNA. The maximum percentage of parasitemia was not reduced below that of the controls in the groups given immune RNA from lymph nodes, but was significantly reduced below that of the controls in the groups given immune RNA from spleens.     

Affinity labelling and characterization of the ppp(A2'p)nA-dependent endoribonuclease from different mammalian sources

The ppp(A2'p)nA-dependent endoribonucleases from a number of different mammalian sources have been investigated. The enzyme from reticulocyte lysates shows optimal activity of 50-150 mM KCl and requires the presence of Mg2+. Whilst the enzyme is inactivated after passage of reticulocyte lysates through Sephadex columns in the absence of ATP, it retains full activity provided ATP is included in the column buffer. The activity of the partially purified nuclease was unaffected by the addition of reticulocyte RNase inhibitor, which, in contrast, effectively inhibited other endogenous endonucleases. The ppp(A2'p)nA-dependent Rnase co-purified with a ppp(A2'p)nA-binding protein and with a protein which could be specifically covalently labelled with an oxidised radioactive analogue of ppp(A2'p)nA. This covalent labelling could be carried out either with the partially purified RNase or in crude extracts from rabbit reticulocytes, mouse Krebs and Ehrlich ascites tumour cells and human lymphoblastoid (Daudi) or HeLa cells. In each case the affinity labelled protein migrated to a position corresponding to a apparent molecular weight of about 85 000 on electrophoresis on dodecylsulphate/polyacrylamide gels. In all cases labelling could be prevented by the addition of an excess of unlabelled ppp(A2'p)nA but not, for example, by a similar excess of the biologically inactive dimer ppp(A2'p)'A. It is concluded that the RNase and ppp(A2'p)nA binding activities are likely to reside in the same molecule.     

Structure and expression of a cluster of human hematopoietic serine protease genes found on chromosome 14q11.2

We previously identified a cluster of hematopoietic serine protease genes on chromosome 14 at band q11.2. This cluster contains the cathepsin G gene and the two related cathepsin G-like genes CGL-1 and CGL-2. The CGL-1 gene is identical with the cytotoxic T cell serine protease CSP-B (also called SECT, and in mice, CCP1, granzyme B, or CTLA-1). In this report, we determined that CGL-2 is identical with a recently described gene called h-CCPX. The coding sequences of CG, CGL-1, and CGL-2 are 65-75% identical at the DNA level. The intervening sequences are much less conserved, except for introns 3 of the CGL-1 and CGL-2 genes, which are 93% identical. Each of the genes has the same overall organization, with 5 exons and 4 introns, very short 5' untranslated regions, and identical splice phases for all of the introns. Cathepsin G is expressed at high levels in promyelocytes/promonocytes, and CGL-1/CSP-B is expressed at high levels in activated cytolytic T cells, lymphokine-activated killer (LAK), and natural killer (NK) cells. CGL-2/h-CCPX is expressed at much lower levels in activated peripheral blood lymphocytes, LAK and NK cells. To begin to define the regulatory elements that target expression of each of these genes to their specific lineages at specific times, the 5' flanking region of each gene was sequenced. The 5' flanking regions are minimally related and have few conserved consensus elements. Further experiments will be required to determine the critical cis-acting regulatory sequences required for tissue- and development-specific expression of each of these genes.