A cooperative taxonomic study of mycobacteria isolated from armadillos infected with Mycobacterium leprae

Seventeen strains of mycobacteria, recovered from six armadillos experimentally infected with Mycobacterium leprae, were examined in ten different laboratories. This collaborative study included use of conventional bacteriological tests, lipid analyses, determination of mycobactins and peptidoglycans, characterization by Py-MS, and immunological, metabolic, pathological and DNA studies. These armadillo-derived mycobacteria (ADM) formed five homogeneous groups (numbered ADM 1 to 5) on the basis of phenetic analyses. However, DNA studies revealed only four homogeneous groups since group ADM 1 and one of the two strains in group ADM 3 showed a high level of DNA relatedness. The phenetic and DNA studies confirmed that the ADM strains differed from all other known mycobacteria. Cultural, biochemical, metabolic and pathogenic properties as well as DNA-DNA hybridizations clearly differentiated these ADM from M. leprae.     

Characterization of High-Affinity Dopamine D2 Receptors and Modulation of Affinity States by Guanine Nucleotides in Cholate-Solubilized Bovine Striatal Preparations

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnorapomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of guanylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5'-(gamma-thio)triphosphate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions.     

Immune responses to isolated human cytomegalovirus envelope proteins.

A group of envelope proteins of human cytomegalovirus, gA protein (L. Pereira, M. Hoffman, M. Tatsuno, and D. Dondero, Virology 139:73-86, 1984; L. Pereira, p. 383-404, in B. Roizman, ed., The herpesviruses, vol. 3, 1985), and two protein mixtures (58,000-molecular-weight [58K]-66K and 130K-66K), separated by serial columns prepared with anti-gA immunoglobulin G from sera of immunized guinea pigs, induced neutralizing antibodies and a cellular immune response in the animals. The gA is a disulfide-linked protein complex consisting of high-molecular-weight (greater than 200K), 130K-150K, and 55K-58K proteins.     

A rat oocyte differentiation antigen (OA-1) detected by a monoclonal antibody

Cell surface antigenic changes associated with differentiation of the rat oocyte and early embryo have been demonstrated with a monoclonal antibody (anti-OA-1). Antigen is first detectable coincident with initiation of oocyte growth, is a constant feature of all growing oocytes and displays a redistribution during meiotic maturation. Following fertilization, antigen is detectable on the surface of the embryo through the four-cell stage. This first monospecific marker for the rat oocyte and embryo should prove useful in probing structure/function relationships in oocyte growth, meiotic maturation fertilization, and/or early embryonic development.     

Pulmonary mechanics during rapid mechanical ventilation in rabbits with saline-lavaged lungs

Infants with respiratory failure are frequently mechanically ventilated at rates exceeding 60 breaths/min. We analyzed the effect of ventilatory rates of 30, 60, and 90 breaths/min (inspiratory times of 0.6, 0.3, and 0.2 s, respectively) on the pressure-flow relationships of the lungs of anesthetized paralyzed rabbits after saline lavage. Tidal volume and functional residual capacity were maintained constant. We computed effective inspiratory and expiratory resistance and compliance of the lungs by dividing changes in transpulmonary pressure into resistive and elastic components with a multiple linear regression. We found that mean pulmonary resistance was lower at higher ventilatory rates, while pulmonary compliance was independent of ventilatory rate. The transpulmonary pressure developed by the ventilator during inspiration approximated a linear ramp. Gas flow became constant and the pressure-volume relationship linear during the last portion of inspiration. Even at a ventilatory rate of 90 breaths/min, 28-56% of the tidal volume was delivered with a constant inspiratory flow. Our findings are consistent with the model of Bates et al. (J. Appl. Physiol. 58: 1840-1848, 1985), wherein the distribution of gas flow within the lungs depends predominantly on resistive factors while inspiratory flow is increasing, and on elastic factors while inspiratory flow is constant. This dynamic behavior of the surfactant-depleted lungs suggests that, even with very short inspiratory times, distribution of gas flow within the lungs is in large part determined by elastic factors. Unless the inspiratory time is further shortened, gas flow may be directed to areas of increased resistance, resulting in hyperinflation and barotrauma.     

Characterization of the O2-induced manganese-containing superoxide dismutase from Bacteroides fragilis

A manganese-containing superoxide dismutase (MnSOD) has been isolated from extracts of O2-induced Bacteroides fragilis. The enzyme, Mr 43,000, was a dimer composed of noncovalently associated subunits of equal size. A preparation whose specific activity was 1760 U/mg had 1.1 g-atoms Mn, 0.3 g-atoms Fe, and 0.2 g-atoms Zn per mol dimer. Exposing the enzyme to 5 M guanidinium chloride, 20 mM 8-hydroxyquinoline abolished enzymatic activity. Dialysis of the denatured apoprotein in buffer containing either Fe (NH4)2(SO4)2 or MnCl2 restored O2-. scavenging activity. The iron-reconstituted enzyme was inhibited 89% by 2 mM NaN3, similar to other Fe-containing superoxide dismutases. The Mn-reconstituted and native MnSOD were inhibited approximately 50% by 20 mM NaN3. Addition of ZnSO4 to dialysis buffer containing either the iron or manganese salt inhibited restoration of enzymatic activity to the denatured apoprotein. MnSOD migrated as a single protein band coincident with a single superoxide dismutase activity band in 7.5 or 10% acrylamide gels. Isoelectric focusing resulted in a major isozymic form with pI 5.3 and a minor form at pI 5.0. Mixtures of the MnSOD and the iron-containing superoxide (FeSOD), isolated from anaerobically maintained B. fragilis [E. M. Gregory and C. H. Dapper (1983) Arch. Biochem. Biophys. 220, 293-300], migrated as a single band on acrylamide gels and isoelectrically focused to a major protein band (pI 5.3) and a minor band at pI 5.0. The amino acid composition of MnSOD was virtually identical to that of the FeSOD. The data are consistent with synthesis of a single superoxide dismutase apoprotein capable of accepting either Mn or Fe to form the holoenzyme.     

Stylet penetration by the bayberry whitefly, as affected by leaf age in lemon, Citrus limon

Bayberry whitefly (Parabemisia myricae [Kuwana]) crawlers were placed on young and mature lemon leaves and were allowed 7–9 days to settle. Afterwards, the nymphs were fixed and sectioned in situ on the leaves and the area of leaf under each whitefly was examined at 1 000 x for stylet penetration. Both stylets and stylet tracks were readily visible in the sections. The path of penetration was mostly intercellular and the objective appeared to be the phloem. Passage of the stylets through the plant tissue did not cause detectable damage to most cells; however, damaged plant cells occasionally were noticed. Nymphs that had moulted during the 7–9 day settling period reached the phloem significantly more often than those that were still in their first instar. In each of the three replicates, penetration in the mature leaf occurred significantly less often than penetration in the young leaf (4% vs. 72%, p<0.01, ²). Penetration appears to be inhibited in the mature leaf either by the leaf cuticle or by factors detected by the nymphs after very shallow penetration into the leaf. The cuticle of mature leaves was much thicker than the cuticle of young leaves and may have been a barrier to stylet penetration.

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Résumé Des larves de premier stade (avant la fixation) de l'aleurode, Parabemisia myricae (Kuwana), ont été placées, pour qu'elles se fixent, pendánt 2 à 9 jours, sur des feuilles jeunes ou mûres de citronnier. Des morceaux de feuilles avec des larves fixées ont été trempés dans 2% d'agar; ce procédé a permis de maintenir les larves à leurs sites de fixation finale. Des pièces d'agar contenant les morceaux de feuilles avec les larves ont ensuite été trempées dans de la paraffine, et sectionnées en séries de 10 . Il était nécessaire de maintenir une basse température pendant l'opération afin d'obtenir de bonnes sections des feuilles mûres durcies. Les stylets et leurs traces étaient faciles à voir dans les sections teintées aux safranins, et fast green. Presque toutes les traces des stylets étaient intercellulaires et leur destination semblaient être de phloème. En général, la plupart des cellules ne semblaient pas endommagées, les larves qui avaient mué pendant la période de 7 à 9 jours de fixation avaient atteint significantivement plus le phloeme que les larves du premier stade. Dans trois essais, la pénétration des feuilles mûres était significativement moins fréquente que celle des feuilles jeunes (p<0.01, ²).La pénétration dans des feuilles mûres semble être empêchée par la cuticule ou par des facteurs perçus par les nymphes après une pénétration superficielle. La cuticule des feuilles mûres était beaucoup plus épaisse que la cuticule des jeunes feuilles et pourrait donc représenter une barrière à la pénétration des stylets.

     

Organogenesis in pepper tissue cultures

Knowledge concerning in vitro growth and developmental responses of bell and chile peppers (Capsicum annuum L.) has been limited. Shoot and root organogenesis in cultures of seedling explants was restricted to primary cultures or those less than three months old under 12-and 16-h photoperiod at 25°C. Shoot organogenesis was extended to 5 months under continuous light at 25°C, and to 8 months under continuous light at 28.5°C. Murashige and Skoog basal media containing 0.05mg/l each of IAA and BA promoted shoot elongation and rooting of some explant sources, while 0.05-4 mg/l IAA with 10–50 mg/l BA promoted adventitious shoot bud formation. Glucose was superior to sucrose as the carbon source. Leaf discs collected from greenhouse-grown plants regenerated shoots for at least 2 months. Incubation environment, carbon source, explant source, growth regulator treatment and passage number were not independent variables as demonstrated by statistical analysis. The plant regeneration techniques described here have useful but limited applications, not extending to unorganized callus or cell suspension cultures.Journal article no. 1151 of the New Mexico Agricultural Experiment Station.     

Alterations in the time of X chromosome replication induced by 5-azacytidine in a patient with 48,XXXY/47,XXY

It has recently been shown that 5-azacytidine (5-azaC) can induce altered replication patterns of the late-replicating X chromosome in normal female cells. This has been demonstrated by bromodeoxyuridine labelling of cells late in the S phase. In the present study the same method was applied to the lymphocytes of a Klinefelter patient (48,XXXY/47,XXY). Significant 5-azaC-induced changes in the replication of the entire inactive X chromosome, from late to early, were found in the lymphocytes of this patient. These results indicate that hypomethylating agents can not only alter the replication of individual bands, but also change the gross replication schedule of multiple inactive X chromosomes in the presence of a Y chromosome.     

Impact of nitrogen and phosphorus on [C]lignocellulose decomposition by stream wood microflora

Nutritional and physical factors affecting the decomposition of [C]lignocellulose prepared from Douglas fir (Pseudotsuga menziesii) were examined by incubating the labeled substrate with homogenized surface wood scrapings obtained from a Douglas fir log in a Pacific Northwest stream. Incubations were conducted in distilled water, in stream water collected from four different sources, or in a defined mineral salts solution with or without supplemental N (KNO(3)). Decomposition rates of [C]lignocellulose, as measured by CO(2) evolution, were greater in each of the four filter-sterilized sources of stream water than in distilled water alone. Decomposition experiments conducted in stream water media with the addition of defined mineral salts demonstrated that [C]cellulose decomposition was stimulated 50% by the addition of either KNO(3) or KH(2)PO(4)/K(2)HPO(4) and further enhanced (167%) by a combination of both. In contrast, [C]lignin decomposition was stimulated (65%) only by the addition of both N and P. Decomposition of [C]lignocellulose was greatest when supplemental KNO(3) was supplied in concentrations of at least 10.0 mg of N liter but not increased further by higher concentrations. The decomposition of [C]lignocellulose increased as the incubation temperature was raised and NO(3)-N supplementation further increased these rates between three-and sevenfold over the range of temperatures examined (5 to 22 degrees C). Accumulation of NH(4) (2 to 4 mg of N liter) was always observed in culture filtrates of incubations which had been supplemented with KNO(3), the quantity being independent of NO(3) concentrations >/= 10 mg of N liter. The role of supplemental NO(3) in the decomposition of [C]lignocellulose is discussed in relation to wood decomposition and the low concentrations of N found in stream ecosystems of the Pacific Northwest.