Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Ovine fetal adaptations to chronically reduced urine flow: preservation of amniotic fluid volume

Mann, Stephanie E., Mark J. M. Nijland, and Michael G. Ross.Ovine fetal adaptations to chronically reduced urine flow: preservation of amniotic fluid volume. J. Appl.Physiol. 81(6): 2588-2594, 1996.Adequateamniotic fluid (AF) volume is maintained by a balance of fetal fluidproduction (lung liquid and urine) and resorption (swallowing andintramembranous flow). Because fetal urine is the principle source ofAF, alterations in urine flow and composition directly impact AFdynamics. Intra-amniotic 1-desamino-8-D-argininevasopressin (DDAVP) is rapidly absorbed into fetal plasma and induces amarked fetal urinary antidiuresis. To examine the effect ofintra-amniotic- DDAVP-induced fetal urinary responses on AF volume andcomposition, six chronically prepared ewes with singleton fetuses(gestation 128 ± 2 days) were studied for 72 h after a singleintra-amniotic DDAVP (50-µg) injection. After DDAVP, fetal urineosmolality significantly increased at 2 h (157 ± 13 to 253 ± 21 mosmol/kg) and remained elevated at 72 h (400 ± 13 mosmol/kg). Urinary sodium (33.0 ± 4.5 to 117.2 ± 9.7 meq/l)and chloride (26.0 ± 2.8 to 92.4 ± 8.1 meq/l) concentrations similarly increased. AF osmolality increased (285 ± 3 to 299 ± 4 mosmol/kgH₂O), although there was no change in fetalplasma osmolality (294 ± 2 mosmol/kg). Despite a 50% reductionin fetal urine flow (0.12 ± 0.03 to 0.05 ± 0.02 ml ^· kg^{1 ·} min¹at 2 h and 0.06 ± 0.01 ml ^· kg^{1 ·} min¹after 72 h), AF volume did not change (693 ± 226 to 679 ± 214 ml). There were no changes in fetal arterial blood pressures, pH,PCO₂, orPO₂ after DDAVP. We conclude the following. 1)Intra-amniotic DDAVP injection induces a prolonged decrease in fetalurine flow and increases in urine and AF osmolalities. 2) Despite decreased urine flow, AFvolume does not change. We speculate that, in response to DDAVP-inducedfetal oliguria, reversed intramembranous flow (from isotonic fetalplasma to hypertonic AF) preserves AF volume.

     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

A Branched Biosynthetic Pathway Is Involved in Production of Roquefortine and Related Compounds in Penicillium chrysogenum

Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.     

Two minimal Tat translocases in Bacillus

Activity of the Tat machinery for protein transport across the inner membrane of Escherichia coli and the chloroplast thylakoidal membrane requires the presence of three membrane proteins: TatA, TatB and TatC. Here, we show that the Tat machinery of the Gram-positive bacterium Bacillus subtilis is very different because it contains at least two minimal Tat translocases, each composed of one specific TatA and one specific TatC component. A third, TatB-like component is apparently not required. This implies that TatA proteins of B. subtilis perform the functions of both TatA and TatB of E. coli and thylakoids. Notably, the two B. subtilis translocases named TatAdCd and TatAyCy both function as individual, substrate-specific translocases for the twin-arginine preproteins PhoD and YwbN, respectively. Importantly, these minimal TatAC translocases of B. subtilis are representative for the Tat machinery of the vast majority of Gram-positive bacteria, Streptomycetes being the only known exception with TatABC translocases.     

Pre- and postjunctional alpha(2)-adrenergic receptors in fetal and adult ovine cerebral arteries

In ovine cerebral arteries, adrenergic-mediated vasoconstrictor responses differ significantly with developmental age. We tested the hypothesis that, in part, these differences are a consequence of altered alpha(2)-adrenergic receptor (alpha(2)-AR) density and/or affinity. In fetal (approximately 140 days) and adult sheep, we measured alpha(2)-AR density and affinity with the antagonist [(3)H]idazoxan in main branch cerebral arteries and other vessels. We also quantified contractile responses in middle cerebral artery (MCA) to norepinephrine (NE) or phenylephrine in the presence of the alpha(2)-AR antagonists yohimbine and idazoxan and contractile responses to the alpha(2)-AR agonists clonidine and UK-14304. In fetal and adult cerebral artery homogenates, alpha(2)-AR density was 201 +/- 18 and 52 +/- 6 fmol/mg protein, respectively (P < 0.01); however, antagonist affinity values did not differ. In fetal, but not adult, MCA, 10(-7) M yohimbine significantly decreased the pD(2) for NE-induced tension in the presence of 3 x 10(-5) M cocaine, 10(-5) M deoxycorticosterone, and 10(-6) M tetrodotoxin. In fetal, but not adult, MCA, UK-14304 induced a significant decrease in pD(2) for the phenylephrine dose-response relation. In addition, stimulation-evoked fractional NE release was significantly greater in fetal than in adult cerebral arteries. In the presence of 10(-6) M idazoxan to block alpha(2)-AR-mediated inhibition of prejunctional NE release, the fractional NE release was significantly increased in both age groups. We conclude that in fetal and adult ovine cerebral arteries, alpha(2)-AR appear to be chiefly prejunctional. Nonetheless, the fetal cerebral arteries appear to have a significant component of postjunctional alpha(2)-AR.     

Evidence for microvascular dysfunction after prenatal dexamethasone at 0.7, 0.75, and 0.8 gestation in sheep

Dexamethasone (DM) was administered to pregnant ewes as three weekly courses of four injections of 2 mg at 12-h intervals. DM (n = 7) or saline (n = 7) was given starting at 103 days of gestation (dGA; term approximately 149 days). Fetal femoral arteries (approximately 300-microm internal diameter) were evaluated using wire myography at 119 dGA. DM-exposed fetuses were significantly smaller than saline-exposed fetuses. DM exposure increased maximal contraction to 125 mM KCl, and maximum tension developed along with sensitivity to endothelin-1 and relaxation to bradykinin. Preincubation with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester shifted the dose-response curves to endothelin-1 and acetylcholine to the right in controls but not in the DM-exposed group. Relaxation to acetylcholine and to the nitric oxide donor sodium nitroprusside was similar in both groups. The combination of enhanced endothelin-induced vasoconstriction, abnormal endothelium-dependent relaxation, and normal endothelium-independent relaxation indicates microvessel dysfunction following antenatal DM administration. Because such dysfunction is associated with several forms of adult hypertension, our results indicate the potential for consequences of antenatal glucocorticoid exposure on adult cardiovascular health.     

Central neuropeptide Y stimulates ingestive behavior and increases urine output in the ovine fetus

We hypothesized that central neuropeptide Y (NPY) increases swallowing activity and alters renal function in the near-term ovine fetus. Six ewes with singleton fetuses (130 +/- 2 days of gestation; 148 days = term) were chronically prepared with arterial and venous catheters, a fetal lateral cerebroventricular cannula, and fetal bladder and amniotic fluid catheters. For determination of fetal swallowing, electromyogram wires were placed in the fetal thyrohyoid muscle and the upper and lower nuchal esophagus. Electrodes were implanted on the parietal dura for determination of fetal electrocorticogram (ECoG). After 5 days of recovery, fetal swallowing, ECoG, blood pressure, and heart rate were monitored during a 3-h basal period. At t = 3 h, ovine NPY (0.05 mg/kg) was administered into the lateral ventricle, and fetuses were monitored for an additional 8 h. A control study of central administration of artificial cerebral spinal fluid was performed on an alternate day. Central NPY significantly increased swallowing activity during low-voltage ECoG from basal activity (1.26 +/- 0.15 swallows/min) at 4 h (1.93 +/- 0.37 swallows/min), 6 h (1.69 +/- 0.27 swallows/min), and 8 h (2.38 +/- 0.31 swallows/min). NPY significantly increased fetal urine flow (basal: 0.13 +/- 0.02; 4 h: 0.21 +/- 0.04; 6 h: 0. 19 +/- 0.03 ml.kg(-1).min(-1)). These results demonstrate that central NPY stimulates fetal swallowing activity and increases urine output, which may contribute to the in utero development of ingestive behavior.     

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.