Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation.

In these studies, the Bordetella pertussis adenylate cyclase toxin-hemolysin homology to the Escherichia coli hemolysin is extended with the finding of cyaC, a homolog to the E. coli hlyC gene, which is required for the production of a functional hemolysin molecule in E. coli. Mutations produced in the chromosome of B. pertussis upstream from the structural gene for the adenylate cyclase toxin revealed a region which was necessary for toxin and hemolytic activities of the molecule. These mutants produced the 216-kDa adenylate cyclase toxin as determined by Western blot (immunoblot) analysis. The adenylate cyclase enzymatic activities of these mutants were equivalent to that of wild type, but toxin activities were less than 1% of that of wild type, and the mutants were nonhemolytic on blood agar plates and in in vitro assays. The upstream region restored hemolytic activity when returned in trans to the mutant strains. This genetic complementation defined a gene which acts in trans to activate the adenylate cyclase toxin posttranslationally. Sequence analysis of the upstream region defined an open reading frame with homology to the E. coli hlyC gene. In contrast to E. coli, this open reading frame is oriented oppositely from the adenylate cyclase toxin structural gene.     

Surrogate transformation of perennial ryegrass, Lolium perenne, using genetically modified Acremonium endophyte.

Summary Conditions have been developed for transforming protoplasts of the perennial ryegrass endophyteAcremonium strain 187BB. Unlike most other ryegrass endophytes, this strain does not produce the lolitrem B neurotoxin and is therefore suitable as a host for surrogate introduction of foreign genes into grasses. Transformation frequencies of 700–800 transformants/g DNA were obtained for both linear and circular forms of pAN7-1, a hygromycin (hph) resistant plasmid. Up to 80% of the linear transformants were stable on further culturing but only 25% of the circular transformants retained hygromycin resistance. Integration of pAN7-1 into the genome was confirmed by Southern blotting and probing of genomic digests of transformant DNA. Both single and tandemly repeated copies of the plasmid were found in the genome and both the number and sites of integration varied among the transformants. At least 13 chromosomes were identified in 187BB using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Probing of Southern blots of these gels confirmed that pAN7-1 had integrated into different chromosomes. The -glucuronidase (GUS) gene,uidA, was also introduced into 187BB by co-transformation of pNOM-2 with pAN7-1. GUS activity was detected by growing the transformants on plates containing 5-bromo-4-chloro-3-indolyl -D-glucuronic acid and by enzyme assays of mycelial extracts. Severalhph- anduidA-containing transformants were reintroduced into ryegrass seedlings and expression of GUS visualized in vivo, demonstrating that 187BB can be used as a surrogate host to introduce foreign genes into perennial ryegrass. Molecular analysis of fungal isolates from the leaf sheath confirmed that the pattern of pAN7-1 and pNOM-2 hybridizing fragments was identical to that observed in the fungus used as inoculum.     

Photoautotrophic growth of soybean cells in suspension culture. II. Optimization of culture medium and conditions

We have attempted to optimize the conditions under which a photoautotrophic soybean suspension culture line (SB-P; Horn et al. 1983) is grown. Magnesium, phosphate, and calcium concentrations were varied individually from one-tenth to five times the normal level found in the Murashige and Skoog (1962) recipe. After two subcultures, only phosphate at one-tenth the normal level caused the cells to show a substantial reduction in fresh and dry weight increase and chlorophyll level. Nitrate and ammonium levels were inversely varied in 20 millimolar increments of potassium nitrate and ammonium chloride. Neither N-source alone could support growth through two subcultures. A ratio of 40 millimolar potassium nitrate to 20 millimolar ammonium gave significantly better fresh and dry weight increases than did a ratio of 20:40 or 30:30 but the chlorophyll level was unchanged. The minor salts as a group resulted in a small improvement in growth when provided at twice the normal level.Indole-3-acetic acid at five milligrams per liter resulted in significantly better fresh and dry weight increases than did -naphthaleneacetic acid at any level but the final chlorophyll level was not changed. There was no correlation between growth and kinetin level and this resulted in the discovery that SB-P cells are cytokinin-autotrophic, as are heterotrophic SB cells, with regard to both growth and greening ability. Growing SB-P cells under a 14 h:10 h day:night photoperiod resulted in a slow but inevitable death. Increasing the carbon dioxide level to 10% for four weeks gave no increase in SB-P cell growth or chlorophyll level, but SB-P cells would not grow with carbon dioxide levels below 0.4%. The results clearly show that SB-P cells, despite their tenuous existence, are capable of adapting to a wide range of culture conditions. A simplified and improved culture medium for photoautotrophic cultures is given.Abbreviations SB-P photoautotrophic soybean cells - SB-M photomixotrophic soybean cells - SB-H heterotrophic soybean cells     

Erythropoietic progenitor cells from premature neonates studied using a validated serum-deprived medium

We have examined a serum-deprived culture system in order to verify that it is suitable for the study of burst forming unit erythroid (BFU-E) progenitor cells from premature neonates. Optimum growth of BFU-E from premature neonates was observed with each media constituent using the same concentration as that previously described for adult subjects. Growth of immature BFU-E from premature neonates were highly dependant upon a source of Burst Promoting Activity and mature BFU-E derived colonies emerged at day 12 compared to day 14 in adults. Our preliminary results with the validated medium suggest that premature infants have increased peripheral blood concentrations of BFU-E compared to healthy adult controls.Abbreviations Ad Adherent cells - BPA Burst promoting activity - BFU-E Burst forming unit erythroid - Epo Erythropoietin - IL3 Interleukin-3 - LDC Low density (<1.077 g ml¹) peripheral blood mononuclear cells     

Aminophylline increases submaximum power but not intrinsic velocity of shortening of frog muscle.

We hypothesized that methylxanthines, such as aminophylline, increase the power developed by submaximally activated frog skeletal muscles by increasing the force developed at any given velocity of shortening. Frog semitendinosus muscles were excised and tested at 20 degrees C in oxygenated control and aminophylline Ringer solutions. Force-velocity relationships were determined and power was calculated from muscles stimulated at frequencies of 80 and 300 Hz. The 300-Hz frequency of stimulation produced a maximum rate of force development. In 50 and 500 microM aminophylline, twitch force increased by 25 +/- 12 and 75 +/- 13%, respectively. Aminophylline did not affect maximum isometric force generation or the shortening velocity at any relative load. At 80-Hz stimulation and in the presence of 500 microM aminophylline, power increased by an average of 11% at 10 of 14 relative loads. At maximum frequencies of stimulation, aminophylline had no effect on any measured parameter. We conclude that aminophylline increases the power developed by submaximally activated frog muscles through an increase in the force generated particularly at the lower velocities of shortening.     

Structure-activity relationship of the neurotransmitter alpha-bag cell peptide on Aplysia LUQ neurons: Implications regarding its inactivation in the extracellular space

Alpha-bag cell peptide [α-BCP (Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu)] is a neurotransmitter that mediates bag cell-induced inhibition of left-upper-quadrant (LUQ) neurons L₂, L₃, L₄, and L₆ in the abdominal ganglion of Aplysia. Our recent biochemical studies have shown that α-BCP[1–9] is cleaved into α-BCP[1–2], [3–9], [1–5], [6–9], and [7–9] by a combination of three distinct peptidase activities located within the extracellular spaces of the CNS: A diaminopeptidase-IV (DAP-IV)-like enzyme cleaves α-BCP[1–9] at the 2–3 peptide bond; a neutral metalloendopeptidase (NEP)-like enzyme cleaves either α-BCP[1–9] or α-BCP[3–9] at the 5–6 bond; an aminopeptidase M-II (APM-II)-like enzyme cleaves α-BCP[6–9] at the 6–7 bond, but cleaves neither α-BCP[1–9], nor the other ganglionic peptidase products. To further understand the manner in which α-BCP is inactivated after release, that is loses its electro-physiological activity, we studied its structure-activity relationship by recording intracellularly from LUQ neurons in isolated abdominal ganglia that were arterially perfused with peptides dissolved in artificial sea water. The effects of α-BCP[1–9] and 15 of its fragments ([1–8], [1–7], [1–6], [1–5], [2–9], [3–9], [3–8], [6–9], [7–9], [8–9], [6–7], [6–8], [1–2], Phe, Tyr) indicated that the sequence Phe⁶-Tyr⁷ was both necessary and sufficient to produce LUQ inhibitory activity. The combined results of our electrophysiological and biochemical studies strongly suggest that α-BCP[1–9] is inactivated by the serial actions of the NEP-like and APM-II-like peptidases; that is, the NEP-like enzyme yields an electro-physiologically active product, α-BCP[6–9], that is cleaved by the APM-II-like enzyme to yield inactive α-BCP[7–9]. Furthermore, because α-BCP[6–9] is more active than α-BCP[1–9], cleavage by the NEP-like enzyme potentiates α-BCP's activity. © 1992 John Wiley & Sons, Inc.     

The shift from C-19 to C-21 steroid synthesis in spawning male common carp, Cyprinus carpio, is regulated by the inhibition of androgen production by progestogens produced by spermatozoa

There is a rapid shift in the steroidogenic pathway from androgen to progestogen production in spawning male common carp, Cyprinus carpio. Experiments were conducted to determine the mechanism regulating this shift using in vitro cultures of testicular fragments and isolated sperm of spermiating male carp. The levels of 11-ketotestosterone (11-KT) continually increased for 48 h with or without gonadotropin (GtH) stimulation, suggesting that 11-KT is the principal androgen produced by carp testes. Ovine prolactin (oPRL) enhanced GtH-stimulated 11-KT production, but by itself had no effect. Gonadotropin, carp pituitary extract, and pregnenolone all enhanced the production of 11-KT, testosterone (T), and 17 alpha-hydroxyprogesterone (17-P) in a dose-dependent manner. No 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) was detected in response to any of these agents; 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P) was not measured. Both 17,20 beta-P and 17,20 alpha-P inhibited 11-KT production in a dose-dependent manner in the presence of either GtH, 17-P, or T. Isolated sperm and testicular fragment preparations both produced 17,20 beta-P and approximately tenfold more 17,20 alpha-P when incubated with 17-P. Only testicular fragments, however, produced 11-KT. We conclude that androgen synthesis occurs only within somatic cells of common carp testes. GtH, and perhaps PRL, stimulates the production of steroid precursors that, under normal physiological conditions, are metabolized to androgens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nyssa talamancana (Cornaceae), an addition to the remanant Laurasian Tertiary flora of southern Central America

A new species,Nyssa talamancana, with fruits larger than those of any other, either living or fossil, is described from Costa Rica and Panama. In size, number of germination valves, and surface-sculpturing, its endocarps resemble those of the fossil assemblage more than those of the other living species. The occurrence of this distinctive new member of a definitely Laurasian family, in association with other endemic or nearly endemic Laurasian taxa, at wet mid-elevations lends credence to the idea that these forests harbor remnants of the really ancient flora of southern Central America.