Putting the heat on sex determination

Sex determination and differentiation are inherently fascinating to both layperson and geneticist. Major advances have accelerated interest in the molecular genetic events mediating these processes in nematodes, flies, mice and humans. Far less attention has been paid to those organisms, particularly reptiles, where sex is determined by environmental cues. However, recent experimental evidence suggests that the two modes of sex determination may not only share common genetic elements, but may also be regulated by similar mechanisms. We argue that the ability to manipulate sex by temperature provides a particularly suitable model for exploring the molecular basis of this fundamental biological process.     

Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.     

Molecular basis for the species selectivity of the neurokinin-1 receptor antagonists CP-96,345 and RP67580.

Two non-peptide substance P antagonists exhibit opposite rank orders of potency for the human and rat neurokinin-1 receptors. CP-96,345 shows selectivity for the human receptor, whereas RP67580 shows selectivity for the rat receptor. Amino acid sequence comparison of the two receptors reveals 22 divergent residues. To elucidate the molecular basis for the species selectivity of these antagonists, divergent residues in the human neurokinin-1 receptor were substituted by the rat homologs. Analysis of mutant receptors revealed that substitution of 2 residues (V116L and I290S) in the transmembrane domain of the human neurokinin-1 receptor is both necessary and sufficient to reproduce the antagonist binding affinities of the rat receptor. The nature of these substitutions and the magnitude of the changes in binding affinity suggest that residues 116 and 290 do not interact directly with the antagonist molecules. The present results support a model in which phylogenetically conserved residues interact directly with the antagonists, while phylogenetically divergent residues affect the local helical packing of the receptor. Such a change in local structure would lead to increased binding affinity for one class of antagonists and decreased affinity for another.     

A gymnosperm extensin contains the serine-tetrahydroxyproline motif

The extensin family is a diverse group of hydroxyproline-rich glycoproteins located in the cell wall and characterized by repetitive peptide motifs glycosylated to various degrees. The origin of this diversity and its relationship to function led us earlier to compare extensins of the two major groups of angiosperms from which we concluded that the highly glycosylated Ser-Hyp₄ motif was characteristic of advanced herbaceous dicots, occurring rarely or not at all in a representative graminaceous monocot (Zea mays) and a chenopod (Beta vulgaris) representative of primitive dicots. Because these results could arise either from loss or acquisition of a characteristic feature, we chose a typical gymnosperm representing seed-bearing plants more primitive than the angiosperms. Thus, salt eluates of Douglas fir (Pseudotsuga menziesii) cell suspension cultures yielded two monomeric extensins differing in size and composition. The larger extensin reported earlier lacked the Ser-Hyp₄ motif, was rich in proline and hydroxyproline, and contained peptide motifs similar to the dicot repetitive proline-rich proteins. The smaller extensin monomer reported here (Superose-6 peak 2 [SP2]) was compositionally similar to typical dicot extensins such as tomato P1, mainly consisting of Hyp, Thr, Ser, Pro, Val, Tyr, Lys, His, abundant arabinose, and a small but significant galactose content. A chymotryptic peptide map (on Hamilton PRP-1) of anhydrous hydrogen fluoride-deglycosylated SP2 yielded eight peptides sequenced after further purification on a high-resolution fast-sizing column (polyhydroxyethyl aspartamide; Poly LC). Significantly, two of the eight peptides contained the Ser-Hyp₄ motif, consistent both with the SP2 amino acid composition as well as the presence of hydroxyproline tetraarabinoside as a small (4% of total Hyp) component of the hydroxyproline arabinoside profile; thus, hydroxyproline tetraarabinoside corroborates the presence of Ser-Hyp₄, in agreement with our earlier observation that Hyp contiguity and Hyp glycosylation are positively correlated. Interestingly, other peptide sequences indicate that SP2 contains motifs such as Ser-Hyp₃-Thr-Hyp-Tyr, Ser-Hyp₄-Lys, and (Ala-Hyp)_n repeats that are related to and typify dicot extensins P1, P3, and arabinogalactan proteins, respectively. Overall, these peptide sequences confirm our previous prediction that Ser-Hyp₄ is indeed an ancient motif and also strongly support our suggestion that the extensins comprise an extraordinarily diverse, but nevertheless phylogenetically related, family of cell wall hydroxyproline-rich glycoproteins.     

Wild-type and mutant bacterioopsins D85N, D96N, and R82Q: high-level expression in Escherichia coli

The integral membrane protein bacterioopsin, found in the extremely halophilic archaebacterium Halobacterium halobium, was expressed in Escherichia coli as a fusion protein containing 13 heterologous amino acids at the amino terminus. The expressed protein was localized primarily to the E. coli cytoplasmic membrane (greater than 80%) and had an in vivo half-life of 26 min. The amount of bacterioopsin in E. coli crude lysates was quantitated immunologically from Western blots and was expressed at 10-20-fold higher levels than seen previously (i.e., 17 mg/L; 5.6% of the total protein). Three distinct forms of the protein were detected immunologically: two of the forms were generated by the removal of either one or four amino acid residues at the amino terminus; the third form remained unaltered.     

Activation and inhibition of human alcohol dehydrogenase by monoclonal antibodies

The human class I alcohol dehydrogenase isozyme beta 2 beta 2 was used as the antigen to raise monoclonal antibodies. Altogether seven lines of hybridomas secreting monoclonal antibodies were obtained. None of the antibodies was isozyme specific and all of them exhibited a similar affinity against all isozymes of the human class I ADH. Five out of the seven monoclonal antibodies had no effect on beta 2 beta 2 activity. Antibody G3 acted as a non-competitive inhibitor with a KI of 3 micrograms/ml at pH 7.5. Increasing pH was effective in reducing the level of inhibition. On the other hand, antibody 1D4 exhibited a pH-dependent activation of ADH activity. In the presence of this antibody, the pH optimum of beta 2 beta 2 was shifted from 9 to 8.5 and total activity was increased by 70% at this optimal pH. Kinetic analysis indicates that 1D4 probably acts as a non-competitive activator and may exert its action by interacting with the coenzyme binding site.     

Territorialit?t und Brutbiologie der FeldlercheAlauda arvensis in einer intensiv genutzten Agrarlandschaft

Zusammenfassung In einer intensiv landwirtschaftlich genutzten Fläche des schweizerischen Mittellandes wurden 1983–1987 vier Feldlerchenaggregationen mit maximal 39 Brutpaaren untersucht. Die Tradition (Ortstreue) bestimmte in erster Linie die Revierwahl und das Verteilungsmuster. Die Reviere waren mit ha sehr groß; ihre Größe korrelierte negativ mit der Anzahl Parzellen pro Fläche und der Kulturendiversität. Die Nutzung des Ressourcenangebots (Nistplatz, Nahrung, Schutz) war abhängig von den Fortbewegungsmöglichkeiten. In großparzellig strukturierten Flächen mit geringer Kulturendiversität kam es häufig zu Revierverschiebungen und Reviervergrößerungen. Die Feldlerche versucht, im Laufe der Brutperiode den Getreideanteil im Revier zu verkleinern und Kulturen wie Rüben, Kartoffeln und auch Mais zu aquirieren. Die Nistplatzwahl zeigte eine deutliche Präferenz für alle nicht zu dicht stehenden, grasartigen Kulturen (Weizen, Hafer, Fettwiese). Eine Vegetationshöhe von 15–25 cm und eine Bodenbedeckung von 20–50 % bieten optimale Bedingungen für den Nestbau. 51 % aller Nester wurden in der Fettwiese angelegt, 20 % im Winterweizen und 18 % im Mais. Je nach Bewirtschaftungsart brüteten die im Mittel 2,4–2,8mal pro Brutsaison. Die Schlüpfrate von 220 Gelegen betrug 0,58. 44 % der geschlüpften Jungvögel verließen das Nest. Der Bruterfolg (nestverlassende Junge/gelegte Eier) lag bei max. 25 %. Dem Fütterungsverhalten der Altvögel nach zu schließen, wurden maximal 50 % der nestverlassenden Jungen flügge. Durchschnittlich war mit 0,9 flüggen Jungen pro Brutpaar und Jahr zu rechnen. Brutverluste waren kulturspezifisch und daher in erster Linie vom Neststandort abhängig.

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Territoriality and breeding biology of Skylark (Alauda arvensis) in an intensively farmed area in Switzerland

Summary In 1983–1987 four plots (total surface 307 ha) with up to 39 breeding pairs were studied in an area of intensive cultivation in the Swiss Lowlands (390 m asl). Distribution pattern and selection of territories are determined by a strong site tenacity of the birds. The size of territories averaged 3,3±0,9 hectares. It correlates negatively with the number of different crops per unit area and the diversity of cultures.The mobility within the vegetation determined which types of farmland ressources may be used for nesting, feeding and cover. Shifts of territories and attempts to enlarge them were frequent in areas of large plots with low crop diversity. During the season, Skylarks tried to reduce the proportion of cereal fields within their territory and to gain plots planted with sugar-beet, potatoes and corn.Their choice of nesting sites showed a strong preference for less dense grass crops such as wheat, oat or meadows. Optimal conditions for nest sites were in fields with a vegetation height of 15–25 cm and a ground coverage of 20–50 %. 51 % of 220 nests were constructed in heavily manured meadows, 20 % in winter wheat fields and 18 % in corn fields. Females bred on average 2,4–2,8 times per breeding season, depending on the type of cultivation (agriculture, husbandry). Hatching rate was 58 % out of 220 clutches, and 44 % of the hatched young left the nest. Maximum breeding success (i. e. young birds leaving the nest/number of eggs laid) was thus maximal 25 %. Observations of parental feeding behaviour suggest that a maximum of 50 % of the young larks which leave the nests survive to the age of fledging. It is estimated that 0,9 fledglings are raised per breeding pairs and year. Losses of broods vary according to farming practises in the different crops and therefore are largely depending on nesting sites.

     

The effect of chronic fatty acid treatment on lipolysis in 3T3-L1 adipocytes.

Various saturated and unsaturated fatty acids were included in the culture medium to test their effects on lipolysis in 3T3-L1 adipocytes. Following prolonged incubation, only oleate was found to exert enhancing effect on basal and isoproterenol-stimulated lipolysis. The effect of oleate was concentration-dependent and was accompanied with increased intracellular cAMP content. Furthermore, the lipolytic response induced by isobutyl-methylxanthine, forskolin or dibutyryl cAMP was also increased in adipocytes treated with oleate. Thus, it appears that in addition to an increased cAMP accumulation, a step distal to cAMP production in the cells may be involved in inducing enhanced lipolysis in 3T3-L1 adipocytes by prolonged exposure to oleate.