PubMed Health⌕ Search

Biomedical subjects

V K Mishra

Publications and source records attributed to V K Mishra.

At least 19 recordsLinked to original sources
PubMed2001

Effects of increasing hydrophobicity on the physical-chemical and biological properties of a class A amphipathic helical peptide.

We have recently shown that a class A amphipathic peptide 5F with increased amphipathicity protected mice from diet-induced atherosclerosis (Garber et al. J. Lipid Res. 2001. 42: 545-552). We have now examined the effects of increasing the hydrophobicity of a series of homologous class A amphipathic peptides, including 5F, on physical and functional properties related to atherosclerosis inhibition by systematically replacing existing nonpolar amino acids with phenylalanine. The peptides, based on the sequence Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH(2) (Ac-18A-NH(2) or 2F) were: 3F(3)(Ac-F(3)18A-NH(2)), 3F(14)(Ac-F(14)18A-NH(2)), 4F(Ac-F(3,14)18A-NH(2)), 5F(Ac-F(11,14,17) 18A-NH(2)), 6F(Ac-F(10,11,14,17)18A-NH(2)), and 7F(Ac-F(3,10,11,14,17) 18A-NH(2)). Measurements of aqueous solubility, HPLC retention time, exclusion pressure for penetration into an egg phosphatidylcholine (EPC) monolayer, and rates of EPC solubilization revealed an abrupt increase in the hydrophobicity between peptides 4F and 5F; this was accompanied by increased ability to associate with phospholipids. The peptides 6F and 7F were less effective, indicating a limit to increased hydrophobicity for promoting lipid interaction in these peptides. Despite this marked increase in lipid affinity, these peptides were less effective than apoA-I in activating the plasma enzyme, lecithin:cholesterol acyltransferase, with 5F activating LCAT the best (80% of apoA-I). Peptides 4F, 5F, and 6F were equally potent in inhibiting LDL-induced monocyte chemotactic activity. These studies suggest that an appropriate balance between peptide-peptide and peptide-lipid interactions is required for optimal biological activity of amphipathic peptides. These studies provide a rationale for the design of small apoA-I-mimetics with increased potency for atherosclerosis inhibition.

Animals↗
PubMed1999

An amphipathic alpha-helix at a membrane interface: a structural study using a novel X-ray diffraction method.

The amphipathic alpha-helix is a recurrent feature of membrane-active proteins, peptides, and toxins. Despite extensive biophysical studies, the structural details of its affinity for membrane interfaces remain rather vague. We report here the first results of an effort to obtain detailed structural information about alpha-helices in membranes by means of a novel X-ray diffraction method. Specifically, we determined the transbilayer position and orientation of an archetypal class A amphipathic helical peptide in oriented fluid-state dioleoylphosphatidylcholine (DOPC) bilayers. The peptide, Ac-18A-NH2(Ac-DWLKAFYDKVAEKLKEAF-NH2), is a model for class A amphipathic helices of apolipoprotein A-I and other exchangeable lipoproteins. The diffraction method relies upon experimental determinations of absolute scattering-length density profiles along the bilayer normal and the transbilayer distribution of the DOPC double bonds by means of specific bromination, and molecular modeling of the perturbed lipid bilayer (derived using the transbilayer distribution of the double bonds) and the peptide. The diffraction results showed that Ac-18A-NH2was located in the bilayer interface and that its transbilayer distribution could be described by a Gaussian function with a 1/e-halfwidth of 4.5(+/-0.3) A located 17.1(+/-0.3) A from the bilayer center, close to the glycerol moiety. Molecular modeling suggested that Ac-18A-NH2is helical and oriented generally parallel with the bilayer plane. The helicity and orientation were confirmed by oriented circular dichroism measurements. The width of the Gaussian distribution, a measure of the diameter of the helix, indicated that the Ac-18A-NH2helix penetrated the hydrocarbon core to about the level of the DOPC double bonds. Bilayer perturbations caused by Ac-18A-NH2were surprisingly modest, consisting of a slight decrease in bilayer thickness with a concomitant shift of the double-bond distribution toward the bilayer center, as expected from a small increase in lipid-specific area caused by the peptide.

Amino Acid Sequence↗
PubMed1999

Biomass cooking fuels and prevalence of tuberculosis in India.

OBJECTIVES: To examine the relation between use of biomass cooking fuels (wood or dung) and prevalence of active tuberculosis in India. METHODS: The analysis is based on 260,162 persons age 20 and over in India's 1992-93 National Family Health Survey. Logistic regression is used to estimate the effects of biomass fuel use on prevalence of active tuberculosis, as reported by household heads, after controlling for a number of potentially confounding variables. RESULTS: Persons living in households that primarily use biomass for cooking fuel have substantially higher prevalence of active tuberculosis than persons living in households that use cleaner fuels (odds ratio [OR] = 3.56; 95% confidence interval [CI] = 2.82-4. 50). This effect is reduced somewhat when availability of a separate kitchen, house type, indoor crowding, age, gender, urban or rural residence, education, religion, caste or tribe, and geographic region are statistically controlled (OR = 2.58; 95% CI = 1.98-3.37). Fuel type also has a large effect when the analysis is done separately for men (OR = 2.46; 95% CI = 1.79-3.39) and women (OR = 2. 74; 95% CI = 1.86-4.05) and separately for urban areas (OR = 2.29; 95% CI = 1.61-3.23) and rural areas (OR = 2.65; 95% CI = 1.74-4.03). The analysis also indicates that, among persons age 20 years and over, 51% of the prevalence of active tuberculosis is attributable to cooking smoke. CONCLUSIONS: Results strongly suggest that use of biomass fuels for cooking substantially increases the risk of tuberculosis in India.

Adult↗
PubMed1998

Studies of synthetic peptides of human apolipoprotein A-I containing tandem amphipathic alpha-helixes.

In mature human apolipoprotein A-I (apo A-I), the amino acid residues 1-43 are encoded by exon 3, whereas residues 44-243 are encoded by exon 4 of the apo A-I gene. The region encoded by exon 4 of the apo A-I gene contains 10 tandem amphipathic alpha-helixes; their location and the class to which they belong are as follows: helix 1 (44-65, class A1), helix 2 (66-87, class A1), helix 3 (88-98, class Y), helix 4 (99-120, class Y), helix 5 (121-142, class A1), helix 6 (143-164, class A1), helix 7 (165-186, class A1), helix 8 (187-208, class A1), helix 9 (209-219, class Y), and helix 10 (220-241, class Y). To examine the effects of multiple tandem amphipathic helixes compared to individual helixes of apo A-I on lipid association, we have studied lipid-associating properties of the following peptides: Ac-44-87-NH2 (peptide 1-2), Ac-66-98-NH2 (peptide 2-3), Ac-66-120-NH2 (peptide 2-3-4), Ac-88-120-NH2 (peptide 3-4), Ac-99-142-NH2 (peptide 4-5), Ac-121-164-NH2 (peptide 5-6), Ac-143-186-NH2 (peptide 6-7), Ac-165-208-NH2 (peptide 7-8), Ac-187-219-NH2 (peptide 8-9), and Ac-209-241-NH2 (peptide 9-10). To study lipid-associating properties of the region encoded by exon 3 of the apo A-I gene, 1-33-NH2 (peptide G) has also been studied. The results of the present study indicate that, among the peptides studied, peptides 1-2 and 9-10 possess significantly higher lipid affinity than the other peptides, with peptide 9-10 having higher lipid affinity than peptide 1-2, as evidenced by (i) higher helical content in the presence of 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), (ii) faster rate of association with DMPC multilamellar vesicles (MLV), (iii) greater reduction in the enthalpy of gel to liquid-crystalline phase transition of DMPC MLV, (iv) higher exclusion pressure from an egg yolk phosphatidylcholine monolayer, and (v) higher partitioning into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine MLV. A comparison of the free energies of lipid association (DeltaG) of the peptides studied here with those studied previously by us [Palgunachari, M. N. , et al. (1996) Arterioscler. Thromb. Vasc. Biol. 16, 328-338] indicates that, except for the peptides 4-5 and 5-6, other peptides possess higher lipid affinities compared to constituent helixes. However, the lipid affinities of the peptides studied here are neither higher than nor equal to the sum of the lipid affinities of the constituent helixes. This indicates the absence of cooperativity among the adjacent amphipathic helical domains of apo A-I for lipid association. As indicated by DeltaG, the lipid affinity of peptide 4-5 is higher than peptide 5 but lower than peptide 4; the lipid affinity of peptide 5-6 is lower than both peptides 5 and 6. Implications of these results for the structure and function of apo A-I are discussed.

Apolipoprotein A-I↗
PubMed1998

Crystal structure of the channel-forming polypeptide antiamoebin in a membrane-mimetic environment.

Crystals of an ion-channel-forming peptaibol peptide in a partial membrane environment have been obtained by cocrystallizing antiamoebin with n-octanol. The antiamoebin molecule has a bent helical conformation very similar to that established for Leu-zervamicin, despite a significantly different sequence for residues 1-8. The bent helices assemble to form a polar channel in the shape of an hour glass that is quite comparable to that of Leu-zervamicin. The molecules of cocrystallized octanol are found in two different areas with respect to the assembly of peptide molecules. One octanol molecule mimics a membrane segment along the hydrophobic exterior of the channel assembly. The other octanol molecules fill the channel in such a way that their OH termini satisfy the C==O moieties directed into the interior of the channel. Structure parameters for C82 H27 N17 O20(.3) C8H18O are space group P2(1) 2(1) 2(1), a = 9.143(2) A, b = 28.590(8) A, c = 44.289(8) A, Z = 4, agreement factor R1 = 11.95% for 4,113 observed reflections [>4sigma(F)], resolution approximately 1.0 A.

Anti-Bacterial Agents↗
PubMed1998

Studies of kinetics and equilibrium membrane binding of class A and class L model amphipathic peptides.

We studied the kinetics and equilibrium membrane binding of two amphipathic alpha-helical peptides: the 18L peptide, which belongs to the class L (lytic peptides), and the Ac-18A-NH2 peptide of the class A (apolipoprotein), according to classification of Segrest et al. ((1990) Proteins, 8, 103-117). Both for cationic 18L and zwitterionic Ac-18A-NH2, the presence of acidic lipids increased the membrane binding constants by two orders of magnitude. The free energy of peptide-membrane association was in the range of 8.5-12.8 kcal/mol. Binding isotherms corresponded to monomer partitioning with saturation at high peptide/lipid ratios. This was also supported by stopped flow studies of the kinetics of peptide-membrane association as measured by peptide tryptophan fluorescence or by energy transfer from the peptide to the lipid-anchored anthrylvinyl fluorophor. The apparent time required for peptide-membrane equilibration was in the millisecond range. At low peptide/lipid ratios it depended on lipid concentration and was independent of the peptide concentration. The rate of peptide-membrane association was found to be relatively close to the diffusion limit. Thus peptide-membrane affinity was mostly determined by the peptide dissociation rate, i.e. higher membrane affinity correlated with a decrease in dissociation rate and with a slower peptide exchange. We have shown that the dynamic character of the peptide membrane equilibrium can be used for selective peptide targeting and disruption of membranes with a specific lipid composition.

Amino Acid Sequence↗
PubMed1998

Apolipoprotein B-100: conservation of lipid-associating amphipathic secondary structural motifs in nine species of vertebrates.

Development of a computer program called LOCATE allowed us to show that human apolipoprotein B-100 is composed of five domains, NH2-alpha1-beta1-alpha2-beta2-alpha3-COOH, enriched, alternately, in amphipathic alpha helixes and amphipathic beta strands. Using updated versions of this program, here we compare the complete sequence of human apolipoprotein B-100 with partial sequences from eight additional species of vertebrates (chicken, frog, hamster, monkey, mouse, pig, rat, and rabbit). The lipid-associating amphipathic alpha helixes cluster in domains alpha2 (between residues 2075 +/- 25 and 2575 +/- 25) and alpha3 (between residues 4100 +/- 100 and 4550 +/- 50) in all species for which those regions have been sequenced but with little conservation of individual helixes. Lipid-associating amphipathic beta strands cluster in domains beta1 (approximately residues 827-2000) and beta2 (approximately residue 2571 to residue 4000 +/- 50) in all species for which these regions have been sequenced, with conservation of several individual amphipathic beta strands. Hydrophobic segments are present in apolipoprotein B-100 sequences of all nine species but the frequency of occurrence is no greater than generally found in beta sheet-containing proteins. We conclude that four alternating lipid-associating domains, -beta1-alpha2-beta2-alpha3-COOH, are common supramolecular features of apolipoprotein B-100 in nine vertebrate species.

Amino Acid Sequence↗
PubMed1998

Testicular toxicity in rat to repeated oral administration of tetramethylthiuram disulfide (Thiram).

Thiram was administered to male rats through gavage at doses 5, 10 and 25 mg/kg/day for 180 and 360 days. Thiram has caused marginal increase in the relative weight of testes and epididymis and decrease in the weight of seminal vesicle and prostate. Marked degenerative changes were observed in seminiferous tubules together with alterations in testicular enzyme profile. The activity of testicular enzymes such as ACP, SDH and ATPase (Na+ + K+ dependent) was decreased whereas activity of LDH, G-6-PDH and ALP increased. The levels of serum cholesterol and testicular free sialic acid were enhanced, while the level of testicular protein was lowered. It is evident from the present study that long term treatment of thiram at tested dose levels has resulted in dose and time dependent morphological and biochemical changes in testes of rat.

Administration, Oral↗
PubMed1997

Truncation of the amino terminus of human apolipoprotein A-I substantially alters only the lipid-free conformation.

An amino-terminal deletion mutant (residues 1-43) of human apolipoprotein A-I (apo hA-I) has been produced from a bacterial expression system to explore the structural and functional role of these amino acids, encoded by exon 3, in apo hA-I. Lipid binding of apo delta (1-43)A-I and lipid binding of apo hA-I are very similar as assessed by surface activity, lipid association with palmitoyloleoylphosphatidylcholine (POPC) vesicles, and lipid association with plasma lipoproteins. Preliminary kinetic measurements appear to show that the reactivity of lecithin:cholesterol acyltransferase (LCAT) with the mutant is slightly decreased compared to wild-type apo hA-I. Collectively, these results indicate that the N-terminal region is not necessary for lipid binding or activation of LCAT. In contrast, there are significant structural differences between lipid-free apo delta (1-43)A-I and apo hA-I, as judged by denaturant-induced unfolding, binding of the fluorescent probe 1-anilinonaphthalene-8-sulfonate, surface balance measurements, and far- and near-ultraviolet circular dichroic spectroscopy. All spectral and physical measurements indicate apo delta (1-43)A-I has a folded, tertiary structure, although it is significantly less stable than that of apo hA-I. It is concluded that the N-terminal 43 residues are an important structural element of the lipid-free conformational state of apo hA-I, the absence of which induces a fundamentally different fold for the remaining carboxy-terminal residues, compared to those in native apo hA-I.

Anilino Naphthalenesulfonates↗
PubMed1996

Interaction of model class A1, class A2, and class Y amphipathic helical peptides with membranes.

To test the hypothesis that differences in the lipid affinity of exchangeable apolipoproteins are due to the presence of different classes of amphipathic alpha-helical motifs which differ primarily in the distribution of charged amino acid residues, we designed and synthesized model peptides mimicking class A1, class A2, and class Y amphipathic helices present in these apolipoproteins. Both class A1 and class A2 helices have positive residues at the polar-nonpolar interface and negative residues at the center of the polar face. However, clustering of positive and negative residues is less exact in class A1 compared to class A2 helices. The class Y helices have two negative residue clusters on the polar face separating the two arms and the base of the Y motif formed by three positive residue clusters. The lipid affinities of three 18 residue model peptides representing these classes, Ac-18A1-NH2 (Ac-ELLEKWAEKLAALKEALK-NH2), Ac-18A2-NH2 (Ac-ELLEKWKEALAALAEKLK-NH2), and Ac-18Y-NH2 (Ac-ELLKAWKEALEALKEKLA-NH2), were determined by right-angle light scattering, circular dichroism spectroscopy, differential scanning calorimetry, and fluorescence spectroscopy. The observed rank order of lipid affinity of these three peptides is: Ac-18A2-NH2 > Ac-18Y-NH2 > Ac-18A1-NH2. This order is consistent with the known lipid affinity of exchangeable apolipoproteins containing class A1, class A2, and class Y helices (class A2 > class Y > class A1). Results of this study illustrate the important role of interfacial lysine residues in modulating the lipid affinity of amphipathic helices and suggest that the effect of interfacial lysine residues in increasing lipid affinity is additive. We propose that interfacial lysine residues, in addition to widening the hydrophobic face because of snorkeling, also help anchor the amphipathic helix in the lipid bilayer.

Acrylamide↗
PubMed1996

Only the two end helixes of eight tandem amphipathic helical domains of human apo A-I have significant lipid affinity. Implications for HDL assembly.

Human apolipoprotein A-I (apo A-I) possesses multiple tandem repeating 22-mer amphipathic alpha-helixes. Computer analysis and studies of model synthetic peptides and recombinant protein-lipid complexes of phospholipids have suggested that apo A-I interacts with HDL surface lipids through cooperation among its individual amphipathic helical domains. To delineate the overall lipid-associating properties of apo A-I, the first step is to understand the lipid-associating properties of individual amphipathic helical domains. To this end, we synthesized and studied each of the eight tandem repeating 22-mer domains of apo A-I: residues 44-65, 66-87, 99-120, 121-142, 143-164, 165-186, 187-208, and 220-241. Among the 22-mers, only the N- and C-terminal peptides (44-65 and 220-241) were effective in clarifying multilamellar vesicles (MLVs) of dimyristoylphosphatidylcholine (DMPC). These two peptides also exhibited the highest partition coefficient into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine liposomes, the highest exclusion pressure for penetration into an egg yolk phosphatidylcholine monolayer, and the greatest reduction in the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. These results suggest that the strong, lipid-associating properties of apo A-I are localized to the N- and C-terminal amphipathic domains. Although each of the eight peptides studied has an amphipathic structure, models based on changes in residual effective amino acid hydrophobicity resulting from differing depths of helix penetration into the lipid are best able to explain the high lipid affinity possessed by the two terminal domains. Differential scanning calorimetry (DSC) studies showed that on a molar basis, apo A-I is about 10 times more effective than the most effective peptide analyzed in reducing the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. Because previous proteolysis experiments coupled with the present DSC results suggest that the lipid-associating domains of apo A-I are distributed throughout the length of the 243 amino acid residues, we propose that the terminal amphipathic helical domains are involved in the initial binding of apo A-I to the lipid surface to form HDL particles, followed by cooperative binding of the middle six amphipathic helical domains, perhaps aided by salt-bridge formation between adjacent helixes arranged in an antiparallel orientation.

Apolipoprotein A-I↗
PubMed1996

Probing structure and function of VLDL by synthetic amphipathic helical peptides.

Class A amphipathic helical peptides have been shown to mimic many properties of exchangeable apolipoproteins. The three analogs of the class A amphipathic peptides were used to probe the structure and function of human very low density lipoproteins (VLDL): 1) 18 residue peptide possessing a single helical domain (18A) with the sequence Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe; 2) two domains of 18A separated by a Pro (37pA); and 3) and 18A analog with the end groups protected to increase helicity (Ac-18A-NH2). Upon incubation of the peptides with VLDL at a peptide to VLDL, (protein) ratio of 1:1, the 37pA and Ac-18A-NH2 were able to displace most of apolipoprotein (apo) Cs and E from VLDL without alteration in its lipid composition and morphology while 18A had minimal effect. The extent of displacement was a function of the peptide to VLDL ratio. The rank order of displaceability of apolipoproteins on VLDL was apoE > C-III > C-II. The displacement of apoE and/or Cs from VLDL by peptides variably affected the ability of VLDL to interact with purified bovine milk lipoprotein lipase (LpL) and cultured macrophages. Treatment of VLDL with Ac-18A-NH2 markedly lowered its reactivity to LpL and its ability to induce lipid accumulation in cultured macrophages: however, treatment of VLDL with 37pA or 18A only minimally lowered their abilities. Ac-18A-NH2 treatment of VLDL resulted in the increase of apparent K(m) and a decrease of Vmax for lipoprotein lipase (LpL)-catalyzed hydrolysis of VLDL triglycerides. When an artificial triglyceride emulsion was used as a substrate of LpL, 37pA, but not Ac-18A-NH2, activated LpL. The above data indicate that 1) amphipathic helical peptides can alter the metabolic and functional properties of VLDL by dissociating the functionally important exchangeable apolipoproteins from VLDL as well as by acting as a functional element of VLDL after their incorporation; and 2) the class A amphipathic peptides having different lipid-associating properties exert significantly different effect on VLDL function.

Amino Acid Sequence↗
PubMed1995

Microenvironments of basic amino acids in amphipathic alpha-helices bound to phospholipid: 13C NMR studies using selectively labeled peptides.

The lipid-binding properties of serum apolipoproteins are mediated by class A amphipathic alpha-helices in which basic and acidic amino acid residues tend to be localized at the helix polar-nonpolar boundary and the center of the polar face, respectively. To better understand the role of the interfacial basic residues, the microenvironments and titration characteristics of lysine (Lys) residues have been examined using two 18-residue peptides. The parent 18A molecule (AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe) binds phospholipid relatively well because of its class A amphipathic helix, whereas 18R, which has the positions of basic and acidic residues reversed, binds to phospholipid relatively weakly. Lys residues were 13C-dimethylated, and NMR spectra were obtained of the peptides complexed with dimyristoylphosphatidylcholine (1/1 (w/w) DMPC/peptide). The four [13C]-epsilon-dimethyl-Lys in 18A gave four resonances at chemical shifts (delta) of 42-43 ppm, whereas only a single resonance at delta 42.70 ppm was observed from the four Lys in 18R. Measurements of delta as a function of pH gave pKa values. The four Lys in 18R had a common pKa of 10.3, indicating that the Lys in the polar face of the amphipathic helix have the pKa expected for noninteracting, fully hydrated groups. In contrast, the four Lys in Ac-18A-NH2 had pKa's in the range 9.4-11.0, indicating that they exist in various microenvironments at the polar-nonpolar boundary of the 18A helix. To examine individual Lys, 18A was synthesized with various Lys selectively labeled. Lys-4, -9, -13, and -15 in Ac-18A-NH2 associated with DMPC had pKa values of 11.0, 9.4, 9.4, and 10.3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Molecular basis for prokaryotic specificity of magainin-induced lysis.

Magainins and mastoparans are examples of peptide antibiotics and peptide venoms, respectively. They have been grouped together as class L amphipathic helixes [Segrest, J.P., et al. (1990) Proteins 8, 103-117] because of similarities in the distribution of Lys residues along the polar face of the helix. Class L venoms lyse both eukaryotic and prokaryotic cells whereas class L antibiotics specifically lyse bacteria. The structural basis for the specificity of class L antibiotics is not well understood. Sequence analysis showed that class L antibiotics have a Glu residue on the nonpolar face of the amphipathic helix; this is absent from class L venoms. We synthesized three model class L peptides with or without Glu on the nonpolar face: 18LMG (LGSIWKFIKAFVGGIKKF), [E14]18LMG and [G5,E14]18LMG. Hemolysis, bacteriolysis, and bacteriostasis studies using these peptides showed that the specificity of lysis is due to both the presence of a Glu residue on the nonpolar face of the helix and the bulk of the nonpolar face. Studies using large unilamellar phospholipid vesicles showed that the inclusion of cholesterol greatly inhibited leakage by the two Glu-containing peptides. These results cannot be attributed to changes in the phase behavior of the lipids caused by the inclusion of cholesterol or to differences in the secondary structure of the peptides. These results suggest that eukaryotic cells are resistant to lysis by magainins because of peptide-cholesterol interactions in their membranes that inhibit the formation of peptide structures capable of lysis, perhaps by hydrogen bonding between Glu and cholesterol. Bacterial membranes, lacking cholesterol, are susceptible to lysis by magainins.

Amino Acid Sequence↗
PubMed1995

Effect of the arrangement of tandem repeating units of class A amphipathic alpha-helixes on lipid interaction.

Exchangeable apolipoproteins possess tandem repeating units of class A amphipathic helical segments and many of them are linked together by proline residues. To understand the optimal arrangement of the amphipathic helixes for lipid association, we have studied the interactions of three model class A amphipathic helical peptides with lipids. The three peptides are: 37pA, a dimer of 18A (DWLKAFYDKVAEKLKEAF) linked together by a Pro (18A-Pro-18A); 37aA, a dimer of 18A linked together by an Ala (18A-Ala-18A); and 36A, a dimer of 18A without any linker residue (18A-18A). Circular dichroism (CD) spectra showed that the peptides are predominantly alpha-helical in aqueous and lipid environments. Temperature dependent CD studies indicated that in buffer helix stability decreases in the order 36A > 37aA > 37pA; however, in the presence of dimyristoyl phosphatidylcholine (DMPC), the above order is reversed. The retention times of the peptides on a C18 reversed-phase high performance liquid chromatography column decreased in the order 36A > 37aA > 37pA, consistent with the lengths of the nonpolar faces of the alpha-helixes being in the same order; the retention time of the parent 18A was shorter than 37pA. While 37pA adsorbed to egg phosphatidylcholine monolayers most strongly, the degree and rate of association of 36A were significantly lower. Differential scanning calorimetry indicated that, while 37pA was most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of DMPC multilamellar vesicles, 36A was least effective; 36A was even less effective than 18A. Fluorescence quenching experiments with iodide and acrylamide indicated that, in the presence of DMPC, Trp residues in 36A are most exposed to the quenchers while in 37pA they are least exposed. In the presence of DMPC, shielding of Trp in 18A from the quenchers was more than that observed with Trp residues in 36A. The results of this study suggest that the arrangement of tandem repeating amphipathic helical units which results in the formation of a class A amphipathic helix with a nonpolar face longer than five or six turns reduces the ability of the helix to associate with phospholipid.

Amino Acid Sequence↗
PubMed1995

Functional bladder neck obstruction: a rare cause of renal failure.

We studied 7 men and 6 women (average age 33 years) who presented in renal failure with obstructive voiding symptoms or retention. Of these patients 11 had a dilated upper tract and 2 had shrunken kidneys. Mean serum creatinine at presentation was 7.0 mg./dl. No abnormality was noted on cysto-panendoscopy, retrograde urethrography and voiding cystourethrography. The patients were initially treated with clean intermittent self-catheterization following 7 to 10 days of indwelling catheterization. The majority of patients had low pressure and low flow rate at initial presentation but high end filling pressure (mean 35.3 cm. water), high voiding pressure (mean 118.9 cm. water), high opening pressure (mean 95.3 cm. water) and low peak flow (mean 5.7 ml. per second) on video pressure flow electromyography. The external sphincter was relaxed during voiding but the bladder neck opened intermittently or inadequately. No proper funneling of the bladder neck was seen. Thus, functional bladder neck obstruction was considered to be responsible for obstructive voiding in these patients. Of the patients 3 void to completion with the help of alpha blockers alone, 5 underwent bladder neck incision and are voiding well, and 5 were practicing clean intermittent self-catheterization at last followup. Serum creatinine returned to near normal in 10 patients. End stage renal failure persisted in 2 patients, 1 of whom underwent renal transplantation and is voiding well but the other died without having undergone renal replacement therapy. In the remaining patient serum creatinine was stable at 3.2 mg./dl. Mean serum creatinine at 6 months of followup was 2.33 mg.%. Bladder neck obstruction is a rare cause of renal failure which can be corrected if treated appropriately.

Adult↗