Biomedical subjects

M A Wells

Publications and source records attributed to M A Wells.

At least 19 recordsLinked to original sources

PubMed1997

Purification and properties of a lipid transfer particle from Bombyx mori: comparison to the lipid transfer particle from Manduca sexta.

A lipid transfer particle (LTP) was purified from the hemolymph of the silkworm Bombyx mori. Like other insect LTPs, the B. mori LTP is a very high density lipoprotein containing 21% lipid and three apoproteins of mass approximately 350 kDa, approximately 85 kDa, and approximately 60 kDa. B. mori LTP catalyzes the exchange of lipids between different density class lipoproteins found in adult hemolymph and between adult lipoproteins and vitellogenin. However, in no case was net lipid transfer observed. Manduca sexta LTP also catalyzed exchange of lipids, but not net transfer of lipids, between different density class lipoproteins found in adult hemolymph.

Animals↗

PubMed1997

Juvenile hormone controls early trypsin gene transcription in the midgut of Aedes aegypti.

Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first few hours after ingestion of a blood meal. The enzymatic activity of early trypsin plays an essential role in the transcriptional activation of the late trypsin gene, which encodes the major midgut endoprotease involved in blood meal protein digestion. Transcription of the early trypsin gene is part of the normal post-emergence maturation of the midgut in the adult female. Abdominal ligation within 1 h of emergence completely prevented the transcription of the early trypsin gene. Topically applied JH III or methoprene induced transcription of the early trypsin gene in ligated abdomens to levels similar to those observed in non-ligated females. The induction of early trypsin transcription by JH is dose-dependent and 'head-independent', suggesting that factors coming from the neuro-secretory axis are not required.

Aedes↗

PubMed1997

Adipokinetic hormone-induced lipolysis in the fat body of an insect, Manduca sexta: synthesis of sn-1,2-diacylglycerols.

The pathway for the adipokinetic hormone-stimulated synthesis of sn-1,2-diacylglycerols in the adult Manduca sexta fat body was studied. Adult fat body lipids were labeled by feeding 5th instar larvae either with [9,10(n)-3H]oleic acid or [1(3)-3H] glycerol and after 32 days insects at the adult stage were used. This long-term prelabeling led to labeled fat body acylglycerols in which triacylglycerols comprised the main radioactive lipid component (95.5%), regardless of the radiolabeled compound used. Because the distribution of radioactivity among the lipid classes was very close to the mass distribution of the fat body lipid subspecies, it was concluded that homogeneous labeling of fat body lipids was obtained. After adipokinetic hormone treatment, an accumulation of radioactivity in the sn-1,2-diacylglycerol fraction was the only significant change found in the distribution of radioactivity among fat body lipids. The size of diacylglycerol pool increased 280% 60 min after adipokinetic hormone stimulation, whereas the fatty acid, monoacylglycerol and phosphatidic acid pool sizes remained constant. These results support the hypothesis that adipokinetic hormone-stimulated synthesis of sn-1,2-diacylglycerol in the fat body involves stereospecific hydrolysis of the triacylglycerol stores.

Animals↗

PubMed1996

Purification and partial characterization of a lutein-binding protein from the midgut of the silkworm Bombyx mori.

A lutein-binding protein was purified from fifth instar larval midgut of Bombyx mori by a combination of ammonium sulfate fractionation and three chromatographic procedures, gel filtration, chromatofocusing, and anion exchange chromatography. The protein has a pI of 5.4 and an apparent molecular mass of 35,000 Da, as determined by a linear gradient SDS-polyacrylamide gel electrophoresis. The lutein-protein complex is water-soluble and more stable than the carotenoid or protein alone. The carotenoid moiety was identified by thin layer chromatography, light absorption spectroscopy, and high performance liquid chromatography as all-trans-lutein. Lutein is specifically and stoichiometrically bound to the protein, with a ratio of 3 mol of lutein per mol of protein. Binding of lutein (absorption maximum in hexane at 454 nm) to the apoprotein results in a marked red spectral shift of about 38 nm, giving rise to absorption maxima at 432, 462, and 492 nm in 20 mM Tris-HCl, pH 7.0. The lutein-protein complex is characterized by fine spectral structure indicating that lutein is in a relatively rigid environment. This protein is distributed in equal amounts throughout the midgut and in all developmental stages of the larval B. mori.

Ammonium Sulfate↗

PubMed1996

Role of diacylglycerol and apolipophorin-III in regulation of physiochemical properties of the lipophorin surface: metabolic implications.

Manduca Sexta adults insects have two defined lipophorin species of densities 1.09 g/mL, [high-density lipophorin (HDLp)] and 1.02 g/mL [low-density lipophorin (LDLp)], respectively, and a continuous broad range of lipophorin particles of intermediate size and density, intermediate-density lipophorin (IDLp). The transformation of HDLp into IDLp and LDLp is the result of the progressive loading of HDLp with diacylglycerol (DG) and an exchangeable apolipoprotein, apolipophorin-III (apoLp-III). In this paper, we describe the physiochemical changes which occur in the lipophorin surface as a result of the transformation of HDLp into LDLp. (1) The increase in apoLp-III content, from 0 to 16 molecules per particle, is accompanied by a gradual increase in the zeta-potential which, at pH 8.6 ranges from /1.02 mV for lipophorins without apoLp-III to -7.76 mV for lipophorins containing 16 molecules of apoLp-III. (2) As judged by the changes in the partition constant for trimethylammonium diphenylhexatriene and oleic acid, an average 2-fold increase in the size of the lipophorin lipid surface takes place when HDLp is loaded with Dg and transformed into LDLp. (3) These data, as well as the results obtained by end point lipolysis with a triacylglycerol (TG) lipase, indicated that the accessible DG content increases 4-7 times when HDLp is converted in LDLp. (4) Fluorescence polarization of the cationic and anionic lipid probes, trimethylammonium diphenylhexatriene and cis-parinaric acid, embedded in eight different subspecies of lipophorin, containing from 12 to 50% DG, showed a small decrease in the surface lipid order when going from HDLp (25% DG) to LDLp (50% DG). (5) Porcine pancreatic phospholipase A2 was used as a probe of the lipoprotein surface. As the DG content of the lipoprotein increased, a higher enzyme activity against the lipoprotein-phospholipids was observed, with a maximum activity 5-fold higher against LDLp than against HDLp. Overall, the changes observed as the lipoprotein particles are loaded with DG and apoLp-III provide a link between the structure and properties of the lipophorin surface and the physiological roles of HDLp and LDLp particles.

Animals↗

PubMed1996

Synthesis of sn-1,2-diacylglycerols by monoacylglycerol acyltransferase from Manduca sexta fat body.

The pathway for the synthesis of sn-1,2-diacylglycerol stimulated by the action of adipokinetic hormone (AKH) in the insect fat body is unknown. Previous results from this laboratory suggested that the hydrolysis of stored triacylglycerol to sn-2-monoacylglycerol followed by the stereospecific acylation of sn-2-monoacylglycerol catalyzed by a monoacylglycerol-acyltransferase (MGAT) could be the major route of AKH-stimulated sn-1,2-diacylglycerol synthesis. Thus, MGAT might represent a key enzyme of this pathway. In this study we characterized the MGAT activity from the Manduca sexta fat body. The activity, which was assayed by acylation of 2-monoolein using radioactive labeled palmitoyl-CoA, was found to be primarily a microsomal enzyme. The products of the acylation of 2-monoolein were 1,2-diacylglycerol (40-50%), 1,3-diacylglycerol (20-30%), and triacylglycerol (30-40%). The presence of triacylglycerol as a product revealed the presence of diacylglycerol-acyltransferase activity in the fat body microsomes. The pH optimum of MGAT activity was 7.0, and the dependence of the activity on the concentration of 2-monoolein showed saturation kinetics. An endogenous MGAT activity, which represented 20% of the maximal activity observed with added substrate, was detected. Optimal concentrations of palmitoyl-CoA ranged between 0.10-0.20 mM. The specific activity of MGAT, measured under optimal conditions, was about 0.6 nmol DG formed/min-mg protein. MGAT activity was greatest with 2-monoolein, and lower activity was observed when a saturated 2-monoacylglycerol was employed. The activity observed with sn-1-monoacylglycerol was lower than that observed with sn-2-monoacylglycerol. AKH did not stimulate MGAT activity, suggesting that either the enzyme is not under hormonal regulation or the monoacylglycerol pathway is not involved in the AKH-stimulated production of sn-1,2-diacylglycerol in the M. sexta fat body.

Acyltransferases↗

PubMed1996

Mitochondrial cytochrome C oxidase subunit I of Manduca sexta and a comparison with other invertebrate genes.

A cDNA encoding mitochondrial cytochrome c oxidase subunit I (mt COI) from Manduca sexta (Lepidoptera: Sphingidae) was cloned and sequenced. AT (adenine-thymine) content is high and codon usage is biased and likely reflects the role of mt COI in electron transport. The encoded protein is 514 amino acids long, contains seven invariant His residues observed in COIs in all organisms and would be predicted to be composed of 12 transmembrane regions.

Amino Acid Sequence↗

PubMed1996

Early trypsin, a female-specific midgut protease in Aedes aegypti: isolation, aminoterminal sequence determination, and cloning and sequencing of the gene.

Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first hours after ingestion of a blood meal. It plays an essential role in the transcriptional activation of the late trypsin form, the major midgut endoprotease involved in the blood meal digestion. Early trypsin is the most abundant midgut polypeptide isolated by benzamidine-sepharose affinity chromatography 3 h after feeding. The amino-terminal sequence of the early trypsin protein matches that of the 3a1 cDNA for a putative trypsinogen described by Kalhok et al. (Insect. Molec. Biol., 2, 71-79, 1993). The early trypsin cDNA was over expressed in Escherichia coli. Polyclonal antibodies generated against this recombinant protein were used to show that the enzyme was present in the midgut during the first 4 h after feeding. A 2.5 kb genomic clone of the early trypsin was isolated, mapped and subcloned. A 1.56 kb subclone, corresponding to 1303 bp of the upstream regulatory region and 265 bp of the coding region, was sequenced. The gene contains a 64 nucleotide intron which interrupts the codon for Val at position 18 of the protein. This Val is located toward the end of the putative signal sequence of the protein.

Aedes↗

PubMed1996

Aedes aegypti midgut early trypsin is post-transcriptionally regulated by blood feeding.

Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first hours after ingestion of a blood meal. Early trypsin gene expression was studied by Northern blot analysis. The early trypsin mRNA, absent in larvae, pupae and newly emerged females, reaches detectable levels at 24 h post-emergence and attains a maximum level at an adult age of 4-7 days. After the first week there is a decrease in the steady-state level of the transcript, but it remains readily detectable for up to a month after emergence. Despite the high levels of early trypsin mRNA present in the midgut of the unfed female, translation of the early trypsin mRNA occurs only after a blood or a protein meal. Early trypsin mRNA levels rapidly decrease during the first 24 h after feeding, but the steady-state level of the transcript rises again at the end of the blood digestion cycle (60 h), as the mosquito prepares for a second blood meal.

Aedes↗

PubMed1995

Low concentrations of diacylglycerol promote the binding of apolipophorin III to a phospholipid bilayer: a surface plasmon resonance spectroscopy study.

The binding of the exchangeable apolipoprotein apolipophorin III (apoLp-III) to an egg phosphatidylcholine bilayer as a function of the concentration of diacylglycerol (DG) in the bilayer was studied by surface plasmon resonance spectroscopy. At a DG concentration of 2 mol % in the bilayer, the binding of apoLp-III reached saturation. Under saturating conditions, apoLp-III forms a closely packed monolayer approximately 55 A thick, in which each molecule of protein occupies approximately 500 A2 at the membrane surface. These dimensions are consistent with the molecular size of the apoLp-III molecule determined by x-ray crystallography, if apoLp-III binds to the bilayer with the long axis of the apoLp-III normal to the membrane surface. In the absence of protein, the overall structure of the lipid bilayer was not significantly changed up to 2.5 mol% DG. However, at 4 and 6 mol % DG, the presence of nonbilayer structures was observed. The addition of apoLp-III to a membrane containing 6 mol % DG promoted the formation of large lipid-protein complexes. These data support a two-step sequential binding mechanism for binding of apoLp-III to a lipid surface. The first step is a recognition process, consisting of the adsorption of apoLp-III to a nascent hydrophobic defect in the phospholipid bilayer caused by the presence of DG. This recognition process might depend on the presence of a hydrophobic sensor located at one of the ends of the long axis of the apoLp-III molecule but would be consolidated through H-bond and electrostatic interactions. Once primary binding is achieved, subsequent enlargement of the hydrophobic defect in the lipid surface would trigger the unfolding of the apolipoprotein and binding via the amphipathic alpha-helices. This two-step sequential binding mechanism could be a general mechanism for all exchangeable apolipoproteins. A possible physiological role of the ability of apoLp-III to bind to lipid structures in two orientations is also proposed.

Animals↗

PubMed1995

Early trypsin activity is part of the signal transduction system that activates transcription of the late trypsin gene in the midgut of the mosquito, Aedes aegypti.

Trypsin activity during the first hours after feeding is essential to induce late trypsin gene expression. These results are consistent with the idea that free amino acids or other products released during digestion might be the initial signal for transcriptional activation of late trypsin. Besides early trypsin, some other factor(s) have to be translated for induction of late trypsin. This is the first case in which the proteolytic activity of a digestive enzyme is part of the signal transduction system which regulates expression of a second gene. The presence of two trypsins allows the mosquito to assess the quality of the meal and adjust the levels of late trypsin for a particular meal with remarkable flexibility.

Aedes↗

PubMed1995

Isolation and characterization of apolipophorin-III from the giant water bug (Lethocerus medius).

Upon injection of synthetic adipokinetic hormone, lipophorin from Lethocerus medius decreased in density and became associated with apolipophorin-III (apoLp-III). ApoLp-III isolated from hemolymph of Lethocerus medius had a M(r) = 19,000 and an amino acid composition high in methionine, in comparison with other apoLp-IIIs. Its circular dichroism spectrum was consistent with a protein with secondary structure of predominantly alpha-helix. NH2-terminal sequence alignment with apoLp-III sequences from other species showed a conservation of the hydrophobic or hydrophilic properties of residues at each position rather than of specific amino acids. ApoLp-III from Lethocerus medius has the potential to form amphipathic alpha-helices, similar to those found in the three-dimensional structure of Locusta migratoria apoLp-III. A portion of the apoLp-III molecules that are not associated with lipophorin contained the blue chromophore, biliverdin.

Amino Acid Sequence↗

PubMed1995

Purification, characterization and cDNA sequence of an alkaline chymotrypsin from the midgut of Manduca sexta.

The chymotrypsin in the midgut of Manduca sexta has been purified, characterized and the cDNA encoding the protein has been cloned. The enzyme exists as a monomer of approx. 24 kDa and shows maximal activity between pH 10.5 and 11.0. Kinetic studies reveal that the Michaelis constant (Km) for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide varies only slightly between pH 7.5 and 11.5 and the Dixon plot shows a kinetically significant pKa at 9.2. The specificity of the purified enzyme was determined to be the peptide bond on the carboxyl side of tyrosine, phenylalanine, tryptophan, histidine, leucine, threonine and glycine. The protease is inhibited by TPCK, PMSF, chymostatin and DFP. A 1 kilobase chymotrypsin cDNA clone was isolated and sequenced. The cDNA sequence encodes a preproenzyme with a putative 17 amino acid signal sequence, a 41 amino acid activation peptide and a mature enzyme of 235 amino acids. The isolated clone encodes the highly conserved active site residues (His, Asp, Ser) and specificity pocket residues present in bovine chymotrypsinogen B. Northern analysis localizes the mRNA for the chymotrypsin to the anterior and middle third of the midgut.

Amino Acid Sequence↗

PubMed1995

Purification and properties of glycogen phosphorylase from the fat body of larval Manduca sexta.

Glycogen phosphorylase b has been purified to homogeneity from the fat body of larval Manduca sexta. The purification procedure involved ammonium sulfate precipitation, and chromatography of DEAE-cellulose, 5'-AMP-Sepharose and Q-Sepharose. The final product, which showed a single band on SDS-PAGE with a M(r) = 92,500, was purified 50-fold from the original homogenate in a yield of about 3%. The molecular mass of the native purified phosphorylase b was estimated to be 186,000 Da from gel filtration, suggesting that the native enzyme is a dimer. The apparent Km values for glycogen, phosphate and 5'-AMP were 1.4 mM, 82 mM and 1.1 mM, respectively. The enzyme had a pH optimum of 7.05, and was inhibited by ATP, ADP and glucose, but not by trehalose, even at high concentration. Conversion of phosphorylase b into the a form was achieved by incubation with rabbit phosphorylase kinase and Mg(2+)-ATP. The molecular mass of phosphorylase a was estimated to be 250,000 Da by gel filtration chromatography. The specific activity of the a form in the presence of 5'-AMP was 1.6-1.7-fold higher than the specific activity of the b form under the same conditions. Thus, 5'-AMP activates the a form by about 20%, whereas ATP has no effect on the phosphorylase a activity.

Animals↗

PubMed1994

Hydration and localization of diacylglycerol in the insect lipoprotein lipophorin. A 13C-NMR study.

In order to probe the organization of diacylglycerol (DG) in lipophorin, 13C-enriched lipophorin was prepared for NMR investigations. We obtained 13C-enriched lipophorin labeled exclusively in DG by feeding insects tobacco leaves coated with [1-13C]palmitic acid or [1-13C]oleic acid. Lipophorins enriched up to 5% with a [13C]fatty acid were obtained by this procedure. NMR studies of the isolated lipophorin DG showed that palmitic acid accumulates almost entirely (> 90%) in the sn-1 position. Oleic acid was found equally distributed between the sn-1 and sn-2 positions, yielding a DG enriched equally at both positions. The 13C-NMR spectra of both [13C]palmitate- and [13C]oleate-enriched lipophorins showed that DG had one narrow carbonyl resonance indicative of rapid motion. A comparative analysis of the 13C carbonyl chemical shift data for DG in organic solvents, aqueous solutions, and dispersions with the DG carbonyl chemical shift of native lipophorin enriched in [13C]palmitate or [13C]oleate shows a high degree of water exclusion from the DG carbonyls in lipophorin. This result is consistent with the existence of a lipophorin lipid core containing most of the lipophorin DG. This study represents the first attempt to elucidate the organization of DG in lipophorin. The possibility of obtaining [13C]DG-enriched lipophorins, selectively enriched in one or both acyl chains of DG, should provide a powerful tool for further analysis of the organization and the dynamic properties of DG in native lipoproteins.

Animals↗

PubMed1994

Effect of diacylglycerol content on some physicochemical properties of the insect lipoprotein, lipophorin. Correlation with the binding of apolipophorin-III.

Diacylglycerol (DG) is the main lipid component of the insect lipoprotein lipophorin. In order to study the effect of DG content on the structure and properties of lipophorin and to analyze the role of DG in the binding of apolipophorin-III (apoLp-III), an exchangeable apolipoprotein, we developed a method that allows the modification of the DG content of lipoproteins. This method employs sn-1,2-dioctanoyl glycerol (diC8-DG). The degree of incorporation of diC8-DG was determined by including [14C]-diC8DG in the incubation and subsequent purification of the diC8-DG-loaded lipophorins in a KBr gradient. The efficiency of diC8-DG loading of lipophorin is time dependent, but high levels of loading are obtained in relatively short periods of time (100% in 3-4 h). For DG loading up to about 15% (w/w), the efficiency and rate of diC8-DG loading are independent of the presence of apoLp-III. DG loading above 15% (w/w) in the absence of apoLp-III resulted in aggregation of the particles. Lipophorin particles enriched up to about 30% with diC8-DG were obtained by this procedure. When lipophorin particles were loaded with diC8-DG in the presence of apoLp-III, it was observed that binding of apoLp-III was proportional to the amount of diC8-DG incorporated into the lipophorin particle, indicating that the only requirement for apoLp-III binding to lipophorin is an increased DG content. A decrease in the degree of order of the lipid phase of the lipoproteins was observed by anisotropy of fluorescence of diphenylhexatriene as the content of diC8-DG was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Cation-promoted cyclic voltammetry of recombinant rat outer mitochondrial membrane cytochrome b5 at a gold electrode modified with beta-mercaptopropionic acid.

Reversible cyclic voltammetry of recombinant rat outer membrane (OM) cytochrome b5 was observed at a gold electrode modified with beta-mercaptopropionic acid. Electron transfer between the negative electrode surface and the negatively charged OM cytochrome b5 was promoted by the addition of divalent metal ions such as Mg2+ or Ca2+ and by the positively charged species poly-L-lysine. The titration of OM cytochrome b5 (0.1 mM) with poly-L-lysine resulted in a gradual positive shift of the E1/2 value which leveled off at +8 mV vs NHE when the poly-L-lysine:cytochrome b5 ratio reached a value of 2:1. Since the further addition of poly-L-lysine had no effect on the E1/2 value of the protein, it was concluded that a complex is formed in which two molecules of poly-L-lysine bind to each molecule of OM cytochrome b5. When the OM cytochrome b5-poly-L-lysine complex (0.1 mM) was titrated with Mg2+ or Ca2+ ions, the E1/2 value shifted gradually in the negative direction and leveled off at -40 mV vs NHE when the concentration of divalent ions reached 85 mM. When the voltammetric response of 0.1 mM cytochrome b5 was promoted by Mg2+ or Ca2+ ions, the minimum concentration of divalent cation necessary to produce a reversible voltammogram was 40 mM and the observed E1/2 was -46 mV vs NHE. On the other hand, only 0.2 mM [Cr(NH3)6]3+ was necessary to promote the reversible electrochemistry of 0.1 mM cytochrome b5. The half-wave potential observed under these conditions was -78 mV vs NHE. This indicates that there is a large dependence of the reduction potential of cytochromes b5 on the kind and concentration of multivalent ions in solution. A reduction potential of -102 mV vs NHE was obtained for OM cytochrome b5 (0.60 mM) by spectroelectrochemical titration in the presence of 0.4 mM [Ru(NH3)6]3+ and 1 mM methyl viologen, pH 7.0, mu = 0.1 M. This value is approximately 100 mV more negative than the reduction potentials reported for microsomal cytochromes b5 obtained from other sources under the same conditions. The binding interactions between OM cytochrome b5 and poly-L-lysine or Mg2+ ions were probed by investigating the isotropically shifted 1H NMR resonances arising from the heme in the OM cytochrome b5.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Mercaptopropionic Acid↗

PubMed1994

Codon usage patterns among genes for lepidopteran hemolymph proteins.

Patterns in codon usage were examined for the coding regions of the 23 known lepidopteran hemolymph proteins. Coding triplets are GC rich at the third position and a significant linear relationship between GC content of silent and nonsilent (replacement) sites was demonstrated. Intron GC content was significantly lower than in coding regions and no relationship between intron GC content and the same at silent and nonsilent sites was found. Though hemolymph proteins are all produced by the same tissue--fat body--significantly less bias was observed when all moth sequences were pooled than when sequences of the two major species were analyzed separately, as predicted by the genome hypothesis. In cases where no statistically significant bias was observed, polar or acidic/basic amino acids were almost exclusively involved. Calculation of codon adaptation indices (CAI) was of limited value in quantifying the degree of codon bias and probably reflects the complexity of multicellular-organism life cycles and the changing patterns of gene expression over different developmental stages.

Animals↗