Mutational Analysis of the Drosophila Miniature-Dusky (M-Dy) Locus: Effects on Cell Size and Circadian Rhythms

A mutational analysis has been performed to explore the function of the Drosophila melanogaster miniature-dusky (m-dy) locus. Mutations at this locus affect wing development, fertility and behavior. The genetic characterization of 13 different mutations suggests that m and dy variants are alleles of a single complex gene. All of these mutations alter wing size, apparently by reducing the volume of individual epidermal cells of the developing wing. In m mutants, epidermal cell boundaries persist in the mature wing, whereas they normally degenerate 1-2 hr after eclosion in wild-type or dy flies. This has permitted the direct visualization of cell size differences among several m mutants. Mutations at the m-dy locus also affect behavioral processes. Three out of nine dy alleles (dyn1, dyn3 and dyn4) lengthen the circadian period of the activity and eclosion rhythms by approximately 1.5 hr. In contrast, m mutants have normal circadian periods, but an abnormally large percentage of individuals express aperiodic bouts of activity. These behavior genetic studies also indicate that an existing "rhythm" mutation known as Andante is an allele of the m-dy locus. The differential effects of certain m-dy mutations on wing and behavioral phenotypes suggest that separable domains of function exist within this locus.     

Endocrine, estrous and pregnancy response to varying dosages of Luprostiol in beef cows

Multiparous lactating beef cows were observed for estrus and randomly assigned to one of four Luprostiol (13, thia-PG-F(2)alpha analog) treatment groups receiving 3.8 (LI), 7.5 (LII), 15 (LIII) or 30 (LIV) mg Luprostiol, respectively, or to an untreated control group (C), or to a positive control group (E) receiving 500 mcg Estrumate. Cows received their respective treatments in a single dosage on Day 7, 8 or 9 of the estrous cycle (estrus = Day 0) and were artificially inseminated 12 h following the subsequent estrus. Blood samples were collected from all groups immediately prior to treatment and at 12-h intervals to 48 h post treatment and analyzed for progesterone (P(4)). Blood samples were collected at 3-h intervals from 24 to 72 h post treatment for animals in Group LIII and for 48 h (or observed estrus) starting on Day 19 of the estrous cycle for animals in Group C. These samples were analyzed for estradiol-17beta(E(2)), follicle stimulating hormone (FSH) and luteinizing hormone (LH). Treatment with Luprostiol at doses >/= 7.5 mg resulted in a synchronous estrous response during the first 5 d post treatment in 75 to 95% of cows treated. Luteal function, as evaluated by systemic P(4) concentration, paralleled results observed for estrous response. Treatment with a 15 or 30 mg dose of Luprostiol resulted in greater overall pregnancy rate at synchronized estrus. No biologically significant differences were found in blood levels of E(2), FSH or LH around the time of estrus between cows in Groups C and LIII. Results from these studies indicate treatment with Luprostiol at doses >/= 7.5 mg resulted in a synchronous estrus during the first 5 d after treatment. Pregnancy rates and endocrine changes were similar to those observed in control and Estrumate-treated cows.     

Solubilization and purification of rat liver 5''-nucleotidase by use of a zwitterionic detergent and a monoclonal-antibody immunoadsorbent.

1. A variety of detergents were used to solubilize 5'-nucleotidase from rat liver plasma membranes. 2. The zwitterionic detergent Sulphobetaine 14 gave optimal solubilization by the criteria of release into a high-speed-centrifugation supernatant and the formation of the smallest and least polydisperse active enzyme observed on polyacrylamide-gel electrophoresis. 3. The Sulphobetaine 14-solubilized enzyme from rat liver was purified by using the conventional techniques of ion-exchange chromatography and gel filtration, or by an immunoaffinity step with a monoclonal antibody immunoadsorbent. 4. 5'-Nucleotidase was purified at least 12 000-fold relative to liver homogenate by the immunoaffinity purification scheme and had a specific activity in the range 285-340 mumol/min per mg of protein. The yield was in the range 9-16%. 5. The purified enzyme shows a major polypeptide band of apparent Mr 70 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and a minor band of apparent Mr 38 000. 6. A rational approach to the general problem of the purification of minor intrinsic membrane proteins is discussed, with the use of polyacrylamide-gel electrophoresis to determine the most appropriate detergent and monoclonal antibodies in subsequent immunoaffinity purification.     

Role of melanin in fungal biosorption of tributyltin chloride

Summary Intact biomass of an albino and a melanic strain of Aureobacidium pullulans, as well as purified melanin from the latter strain, was capable of tributyltin chloride (TBTC) removal from solution. Melanized biomass had a greater biosorptive capacity than albino biomass, this difference being attributable to the presence of melanin. Purified melanin had a large capacity for TBTC biosorption, the calculated maximum uptake capacity, q _e, being approximately 35 mmol (g dry wt)^–1. TBTC biosorption by intact biomass and melanin obeyed the Langmuir adsorption isotherm over the concentration range used, and was relatively unaffected by external pH between pH 3.5 and 6.5: an approximate 20% decrease in TBTC biosorption resulted at external pH 2.5. A TBTC concentration of 0.3 M in growth medium resulted in a lag period which was longer with the albino strain (approximately 50 h) than with the pigmented strain (approximately 25 h). The addition of melanin to TBTC-containing growth media resulted in a reduction in toxicity and attainment of higher cell yields. The applied and environmental significance of these interactions are discussed. Offprint requests to: G. M. Gadd     

Partial purification and properties of an AMP-specific soluble 5''-nucleotidase from pigeon heart.

A soluble 5'-nucleotidase was purified 200-fold from pigeon heart. The enzyme (1) had an apparent molecular mass close to 150 kDa, (2) had a neutral pH optimum and hydrolysed a wide range of nucleoside 5'-monophosphates with a 15-fold preference for AMP over IMP, (3) at near-physiological concentrations of AMP was activated by ADP but not by ATP, (4) was inhibited by high Mg2+ concentration and high ionic strength, (5) was weakly inhibited by p-nitrophenol phosphate and Pi, and (6) was non-competitively inhibited more potently by 5'-deoxy-5'-isobutylthioinosine than by 5'-deoxy-5'-isobutylthioadenosine, but not by [alpha,beta-methylene]ADP. The data show that the enzyme is distinct from the ecto-5'-nucleotidase and from the previously purified IMP-specific 5'-nucleotidase. They also predict that the enzyme is activated during ATP catabolism and hence will generate a more-than-linear increase in the adenosine-formation rate in response to an increase in cytosolic AMP concentration.     

Macronuclear Genetics of Tetrahymena. II. Macronuclear Location of Somatic Mutations to Cycloheximide Resistance

Somatic cycloheximide-resistant mutants of syngen 1 of Tetrahymena pyriformis were isolated and genetically characterized. Two properties of the mutants were independently examined: (a) The transmission of the mutant phenotype during conjugation and (b) the kinetics of phenotypic assortment during vegetative propagation. The results of both studies strongly support the idea that these somatic mutations have a macronuclear location. The kinetics of assortment are consistent with the idea that the syngen 1 macronucleus contains about 45 assorting genetic units. The sib-selection method employed here, used in conjunction with the analysis of assortment kinetics and a previously described test for randomness of distribution, provides a probe of macronuclear genetics applicable to many ciliates, including those in which conjugation is not known to occur or is not under experimental control.     

Role of adenosine deaminase, ecto-(5''-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.     

Regulation of vascular smooth muscle cell proliferation, migration and death by heparan sulfate 6-O-endosulfatase1

Migration, proliferation and death of vascular smooth muscle cells (VSMC) are important events in vascular pathology regulated by heparan sulfate proteoglycans and hence potentially by cell surface HS 6-O-endosulfatase1 (sulf1). Sulf1 mRNA expression was increased in cultured VSMC compared to rat aorta. Furthermore, adenovirus mediated overexpression of quail sulf1 decreased adhesion, and increased proliferation and apoptosis of VSMC. Overexpression of a dominant negative variant also decreased adhesion of VSMC and increased proliferation, apoptosis, migration and chemotaxis of VSMC. Our results imply that only normal levels of 6-O-sulfation maintained by sulf1 are optimal for several functions of VSMC.