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K Saxena

Publications and source records attributed to K Saxena.

At least 19 recordsLinked to original sources
PubMed2000

Unusual hydration properties of C16:0 sulfatide bilayer membranes.

After deacylation of bovine brain sulfatide under mild alkaline conditions and reacylation using palmitoyl chloride (, Chem. Phys. Lipids. 34:41-53), the anionic glycosphingolipid N-palmitoyl galactosulfatide (C16:0-GalSulf) has been synthesized. By differential scanning calorimetry (DSC), anhydrous C16:0-GalSulf exhibits an endothermic transition, T(M) = 93 degrees C (DeltaH = 5. 5 kcal/mol C16:0-GalSulf) on heating. With increasing hydration (50 mM sodium phosphate buffer, pH 7.0; 50 mM NaCl), T(M) decreases, reaching a limiting value of 49 degrees C (DeltaH = 8.2 kcal/mol C16:0-GalSulf) at 20 wt% buffer. X-ray diffraction data have been recorded over the hydration range 0-62% at temperatures below (20 degrees C) and above (60 degrees C) T(M). At 20 degrees C, sharp wide-angle reflections at approximately 1/4.4 A(-1), approximately 1/4.1 A(-1), and approximately 1/3.8 A(-1) indicate the presence of an ordered-chain gel phase, whereas at 60 degrees C a broad reflection at 1/4.5 A(-1) characteristic of a melted-chain phase is observed. Lamellar diffraction patterns consistent with the presence of bilayer phases are observed at both temperatures. At 60 degrees C, in the liquid-crystalline L(alpha) phase, the bilayer periodicity increases with hydration, in both water and 100 mM Na(+) buffer. Interestingly, in the gel phase at 20 degrees C, the bilayer periodicity (d = 64 A) is insensitive to hydration (over the range 30-60 wt%) with either water or buffer. The continuous swelling behavior exhibited by the L(alpha) bilayer phase of C16:0-GalSulf is typical of lipids bearing a net negative charge and confirms that the presence of 100 mM Na(+) is insufficient to shield the charge contributed by the sulfate group. In contrast, the lack of continuous swelling behavior of the bilayer gel phase of C16:0-GalSulf is unusual and resembles that of Na(+) soaps. Thus, presumably, alterations in the surface charge characteristics of the C16:0-GalSulf bilayer occur on hydrocarbon chain melting and lead to major changes in lipid hydration.

Calorimetry, Differential Scanning↗
PubMed2000

Bilayer properties of totally synthetic C16:0-lactosyl-ceramide.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the structural and thermal properties of totally synthetic D-erythro-N-palmitoyl-lactosyl-C(18)-sphingosine (C16:0-LacCer). Over the temperature range 0-90 degrees C, fully hydrated C16:0-LacCer shows complex thermal transitions characteristic of polymorphic behavior of exclusively bilayer phases. On heating at 5 degrees C/min, hydrated C16:0-LacCer undergoes a complex two-peak endothermic transition with maxima at 69 degrees C and 74 degrees C and a total enthalpy of 14.6 kcal/mol C16:0-LacCer. At a slower heating rate (1.5 degrees C/min), two endothermic transitions are observed at 66 degrees C and 78 degrees C. After cooling to 0 degrees C, the subsequent heating run shows three overlapping endothermic transitions at 66 degrees C, 69 degrees C, and 71.5 degrees C, followed by a chain-melting endothermic transition at 78 degrees C. Two thermal protocols were used to completely convert C16:0-LacCer to its stable, high melting temperature (78 degrees C) form. As revealed by x-ray diffraction, over the temperature range 20-78 degrees C this stable phase exhibits a bilayer structure, periodicity d approximately 65 A with an ordered chain packing mode. At the phase transition (78 degrees C) chain melting occurs, and C16:0-LacCer converts to a liquid crystalline bilayer (L(alpha)) phase of reduced periodicity d approximately 59 A. On cooling from the L(alpha) phase, C16:0-LacCer converts to metastable bilayer phases undergoing transitions at 66-72 degrees C. These studies allow comparisons to be made with the behavior of the corresponding C16:0-Cer (. J. Lipid Res. 36:1936-1944) and C16:0-GluCer and C16:0-GalCer (. J. Lipid Res. 40:839-849). Our systematic studies are aimed at understanding the role of oligosaccharide complexity in regulating glycosphingolipid structure and properties.

Antigens, CD↗
PubMed1999

Ion selectivity reversal and induction of voltage-gating by site-directed mutations in the Paracoccus denitrificans porin.

The porin from Paracoccus denitrificans, a slightly anion specific outer membrane pore protein, was expressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of urea and detergents. The purified recombinant protein was reconstituted into black lipid bilayer membranes and showed no difference in its functional properties in comparison to the native porin isolated from P.denitrificans membranes. To investigate the molecular basis of its ion selectivity and voltage-gating, a series of site-directed mutants was constructed, comprising acidic residues located on the third extracellular loop (L3), which forms the constriction zone of the channel, and basic residues along the opposing barrel wall. Measurements using zero-current membrane potentials indicated that the selectivity changed drastically from a slight anion to a distinct cation selectivity with the exchange of residues R29 and R31 by glutamate, whereas replacements on the L3 loop went largely unaffected. However, when assaying the voltage-dependent closure of channels, only mutations located on the L3 loop showed an effect, in contrast to the voltage-independent recombinant and native Paracoccus porin.

Electric Conductivity↗
PubMed1999

The WD repeat: a common architecture for diverse functions.

Our knowledge of the large family of proteins that contain the WD repeat continues to accumulate. The WD-repeat proteins are found in all eukaryotes and are implicated in a wide variety of crucial functions. The solution of the three-dimensional structure of one WD-repeat protein and the assumption that the structure will be common to all members of this family has allowed subfamilies of WD-repeat proteins to be defined on the basis of probable surface similarity. Proteins that have very similar surfaces are likely to have common binding partners and similar functions.

Animals↗
PubMed1999

Crystal structures of the XLP protein SAP reveal a class of SH2 domains with extended, phosphotyrosine-independent sequence recognition.

SAP, the product of the gene mutated in X-linked lymphoproliferative syndrome (XLP), consists of a single SH2 domain that has been shown to bind the cytoplasmic tail of the lymphocyte coreceptor SLAM. Here we describe structures that show that SAP binds phosphorylated and nonphosphorylated SLAM peptides in a similar mode, with the tyrosine or phosphotyrosine residue inserted into the phosphotyrosine-binding pocket. We find that specific interactions with residues N-terminal to the tyrosine, in addition to more characteristic C-terminal interactions, stabilize the complexes. A phosphopeptide library screen and analysis of mutations identified in XLP patients confirm that these extended interactions are required for SAP function. Further, we show that SAP and the similar protein EAT-2 recognize the sequence motif TIpYXX(V/I).

Amino Acid Sequence↗
PubMed1999

Structure and properties of totally synthetic galacto- and gluco-cerebrosides.

The structural and thermal properties of aqueous dispersions of the totally synthetic cerebrosides, D-erythro-N-palmitoyl galactosyl- and glucosyl-C18-sphingosine (C16:0-GalCer and C16:0-GluCer, respectively) have been studied using differential scanning calorimetry (DSC) and X-ray diffraction. Over the temperature range 0-100 degrees C, both C16:0-GalCer and C16:0-GluCer show complex thermal transitions characteristic of polymorphic behavior of exclusively bilayer phases. On heating, hydrated C16:0-GalCer undergoes an exothermic bilayer-bilayer transition at 59 degrees C to produce a stable bilayer crystal form. X-ray diffraction at 70 degrees C reveals a bilayer structure with an ordered hydrocarbon chain-packing arrangement. This ordered bilayer phase undergoes an endothermic chain-melting transition at 85 degrees C to the bilayer liquid crystalline state. Similar behavior is exhibited by hydrated C16:0-GluCer which undergoes the exothermic transition at 49 degrees C and a chain-melting transition at 87 degrees C. The exothermic transitions observed on heating hydrated C16:0-GalCer and C16:0-GluCer are irreversible and dependent upon previous chain melting, prior cooling rate, and time of incubation at low temperatures. Thus, the structure and properties of totally synthetic C16:0-GalCer and C16:0-GluCer with identical sphingosine (C18:1) and fatty acid (C16:0) chains are quite similar, suggesting that the precise isomeric structure of the linked sugar plays only a minor role in regulating the properties of hydrated cerebrosides. Further, these studies indicate that the complex thermal behavior and bilayer phase formation exhibited by these single-sugar cerebrosides are intrinsic properties and not due to the heterogeneity of the sphingosine base found in natural and partially synthetic cerebrosides.

Calorimetry, Differential Scanning↗
PubMed1998

Sites important for PLCbeta2 activation by the G protein betagamma subunit map to the sides of the beta propeller structure.

The betagamma subunits of the heterotrimeric GTP-binding proteins (G proteins) that couple heptahelical, plasma membrane-bound receptors to intracellular effector enzymes or ion channels directly regulate several types of effectors, including phospholipase Cbeta and adenylyl cyclase. The beta subunit is made up of two structurally different regions: an N-terminal alpha helix followed by a toroidal structure made up of 7 blades, each of which is a twisted beta sheet composed of four anti-parallel beta strands (Wall, M. A., Coleman, D. E., Lee, E., Iñiguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319). We have previously shown that sites for activation of PLCbeta2, PLCbeta3, and adenylyl cyclase II overlap on the "top" surface of the propeller, where Galpha also binds (Li, Y., Sternweis, P. M., Charnecki, S., Smith, T. F., Gilman, A. G., Neer, E. J., and Kozasa, T. (1998) J. Biol. Chem. 273, 16265-16272). The present study was undertaken to identify the regions on the side of the torus that might be important for effector interactions. We made mutations in each of the outer beta strands of the G protein beta1 propeller, as well as mutations in the loops that connect the outer strands to the adjacent beta strands. Our results suggest that activation of PLCbeta2 involves residues in the outer strands of blades 2, 6, and 7 of the propeller. We tested three of the mutations that most severely affected PLCbeta2 activity against two forms of adenylyl cyclase (ACI and ACII). Both inhibition of ACI and activation of ACII were unaffected by these mutations, suggesting that if ACI and ACII contact the outer strands, the sites of contact are different from those for PLCbeta2. We propose that distinct sets of contacts along the sides of the propeller will define the specificity of the interaction of betagamma with effectors.

Adenylyl Cyclases↗
PubMed1997

Structural studies of detergent-solubilized and vesicle-reconstituted low-density lipoprotein (LDL) receptor.

The low-density lipoprotein (LDL) receptor plays a key role in maintaining circulating and cellular cholesterol homeostasis. The LDL receptor is a transmembrane glycoprotein whose biochemical and genetic properties have been extensively studied notably by Brown, Goldstein and colleagues [Brown, M. S., & Goldstein, J. L., (1986) Science 232, 34-47]. However, few if any structural studies of the LDL receptor have been reported, and details of its secondary and tertiary structure are lacking. In an attempt to determine the low-resolution structure of the LDL receptor, we have purified the receptor from bovine adrenal cortices using modifications of the method of Schneider et al. [Schneider, W. J., Goldstein, J. L., & Brown, M. S. (1985) Methods in Enzymol.109, 405-417]. Using circular dichroism, the secondary structure of the detergent-solubilized bovine LDL receptor at 25 degrees C was shown to be 19% alpha-helix, 42% beta-sheet, and 39% random coil. Interestingly, the detergent-solubilized receptor appeared to be quite resistant to changes in secondary structure over the temperature range 10-90 degrees C, with only minor but reversible changes being observed. In contrast, a more pronounced unfolding of the detergent-solubilized receptor was observed in the presence of guanidinium hydrochloride. Using the complete sequence of the human LDL receptor, sequence analysis by the Chou-Fasman prediction algorithm showed quite good agreement with the experimentally determined secondary structure of the bovine LDL receptor at 25 degrees C. Finally, the purified, bovine LDL receptor was reconstituted into large unilamellar vesicles of egg yolk phosphatidylcholine using a procedure exploiting preformed vesicles and detergent dialysis. We showed previously using negative stain electron microscopy that reconstituted vesicles bind LDL. Now, using cryoelectron microscopy of frozen hydrated reconstituted vesicles evidence of an extended, stick-like morphology (length approximately 120 A) for the extracellular domain of the LDL receptor has been obtained. Successful purification of the receptor, its incorporation into single bilayer vesicles, and its direct visualization by cryoelectron microscopy pave the way for more detailed structural studies of the LDL receptor and the receptor-LDL complex.

Adrenal Cortex↗
PubMed1997

Molecular cloning and functional characterization of the Paracoccus denitrificans porin.

Bacterial porins facilitate the passive uptake of small solutes across the outer membrane of the cell. The channel properties and the primary structure of the porin from Paracoccus denitrificans were investigated. As judged from single-channel conductance experiments, this porin forms trimeric pores that show no ion selectivity in potassium chloride solution, which indicates that the charges within or near the channel are balanced. Based on peptide fragment sequence, the gene porG, which codes for this general pore protein, was cloned and analyzed. Its primary translation product contains a 20-residue signal sequence, followed by the 295 amino acids of the mature protein with a molecular mass of 31.9 kDa. Sequence alignments with porins from Rhodopseudomonas blastica and Rhodobacter capsulatus and secondary structure predictions suggest a typical rigid barrel structure consisting of 16 antiparallel beta-strands.

Amino Acid Sequence↗
PubMed1997

The structure of porin from Paracoccus denitrificans at 3.1 A resolution.

The crystal structure of a non-specific porin from Paracoccus denitrificans at 3.1 A resolution has been solved by molecular replacement using the porin from Rhodopseudomonas blastica as the search model. Paracoccus porin is very similar to other non-specific porins of known structure: a trimer of 16 stranded beta-barrels each with a central pore constricted by a long extracellular loop folding back against the barrel wall. The distinctive distribution of charged residues of this non-specific porin contributes to understanding the relation between structure and ion selectivity.

Amino Acid Sequence↗
PubMed1997

Term newborns who are at risk for sepsis: are lumbar punctures necessary?

OBJECTIVES: To determine: (1) whether a lumbar puncture (LP) is indicated in asymptomatic full-term newborns delivered by mothers at risk of intrapartum sepsis; and (2) whether gentamicin improves bacterial coverage for such newborns when used with ampicillin. DESIGN: A retrospective chart review from 1987 through 1993 of all newborns with positive blood and/or cerebrospinal fluid cultures in the first 7 days of life. METHODS: Pregnant women were screened in the second trimester for group B streptococci and given ampicillin during labor if two or more risk factors were present: group B streptococci colonization, maternal fever or leukocytosis, rupture of membranes at more than 18 hours, foul-smelling amniotic fluid, and fetal tachycardia. After sepsis evaluation (LP, blood culture, white blood cell count, and differential), asymptomatic infants received ampicillin and gentamicin for 48 to 72 hours unless cultures grew pathogens. RESULTS: Of approximately 24 452 full-term births in 7 years, 7% (1712) had evaluations for symptoms of sepsis, and 14% (3423) were asymptomatic but had evaluations for maternal risk factors. There were 11 cases of meningitis, all involving symptomatic newborns; 10 of these 11 had positive blood cultures for the same organism. In asymptomatic infants, none of the 3423 had meningitis (95% confidence interval, 0 to 0.0008), although 35 grew contaminants. Of 73 pathogens isolated from blood or cerebrospinal fluid, 7 (9.5%) were resistant to ampicillin. Addition of gentamicin provided coverage for only 2 of these 7 pathogens. Of 5135 infants who received ampicillin and gentamicin, only 2 required gentamicin for improved coverage. CONCLUSIONS: (1) LP is unnecessary in asymptomatic full-term newborns. (2) Empiric coverage for asymptomatic newborns with maternal risk factors need not include gentamicin at all hospitals, because it only improved the coverage of ampicillin alone from 90% to 93% of pathogens, but it exposed more than 5000 infants to the side effects of gentamicin. (3) The presence of leukopenia (<5000 white blood cells/mm) is highly predictive of bacteremia.

Ampicillin↗
PubMed1996

Analysis of the physical properties and molecular modeling of Sec13: A WD repeat protein involved in vesicular traffic.

WD repeat proteins are a family of proteins that contain a series of highly conserved internal repeat motifs, usually ending with WD (Trp-Asp). The G beta subunit of heterotrimeric guanine nucleotide binding protein is a member of this family, and its crystal structure has been recently solved at high resolution (Wall et al. (1995) Cell 83, 1047-1058; Sondek et al. (1996) Nature 379, 369-374). Based on the coordinates of G beta, we have constructed a model for the structure of Sec13, a 33 kDa WD repeat protein from Saccharomyces cerevesiae essential for vesicular traffic. The model has been tested using a combination of biophysical and biochemical methods. Sec13 was expressed in Escherichia coli as a hexa-His-tagged protein (H6Sec13) and purified to homogeneity. In contrast to some other WD repeat proteins that are unable to fold into monomeric structures when expressed in E. coli, H6Sec13 was soluble and monomeric in the absence of detergent. The far-UV circular dichroism (CD) spectra of H6Sec13 indicated less than 10% alpha-helix consistent with the model which predicts primarily beta-sheets. H6Sec13 shows a cooperative and irreversible thermal denaturation curve consistent with a tightly packed structure. The CD spectrum shows an unusual positive ellipticity at 229 nm that was attributed to interactions of surface tryptophans since the 229 nm maximum could be abolished by modification of 6.3 +/- 0.3 (n = 3) tryptophans (out of 15 total in the molecule) with N-bromosuccinimide. Our model predicts that three sets of tryptophans are clustered near the surface. As predicted by the model, purified H6Sec13 was completely resistant to trypsin digestion. The concordance of the model of Sec13 presented in this paper with the biochemical and biophysical studies suggests that this model can be useful as a guide to further experiments designed to elucidate the function of Sec13 in vesicular traffic.

Amino Acid Sequence↗
PubMed1995

Enhanced induction of the mitochondrial permeability transition following acute menadione administration.

Induction of the mitochondrial permeability transition in vitro is well-characterized and widely implicated in the mechanism of oxidant-induced cell death. Despite an abundance of in vitro evidence, implication of mitochondrial dysfunction in the mechanism of chemical toxicity in vivo awaits demonstration of the induction of the mitochondrial permeability transition in tissues from intoxicated animals. Menadione (2-methyl-1,4-naphthoquinone), an agent known to induce the permeability transition in isolated liver mitochondrial in vitro, was administered as a single bolus to adult male rats, and hepatic mitochondria were isolated 24 h later. Mitochondria from menadione-treated rats exhibited an increased sensitivity to calcium-induced inhibition of state 3 respiration and loss of respiratory control, as well as a greater sensitivity to calcium-induced calcium release that was inhibited by cyclosporine A. Associated with this was the depolarization of membrane potential and swelling of mitochondria from menadione-treated animals, but not control animals. Both the calcium-dependent depolarization and swelling of mitochondria from menadione-treated rats were inhibited by adding either cyclosporine A or ruthenium red. The results are consistent with the induction of the mitochondrial permeability transition and provide the first evidence for the manifestation of an increased sensitivity to this response as a result of chemical exposure in vivo.

Animals↗
PubMed1994

Evaluation of PGE2 gel for cervical ripening and induction of labour.

In a prospective study a single dose PGE2 gel 0.5 mg was given in 97 Low Bishop Score subjects, 12 hrs prior to indicated oxytocin or PGE2 tab induction for cervical ripening. Analysis of 97 subjects shows that it is very highly effective. 56 primi and 21 multi delivered spontaneously with gel administration only while 4 primi and 2 multi delivered vaginally following oxytocin and 3 more cases delivered vaginally following PGE2 tab administration and fewer caesarian section (11) were performed. The endo cervical administration of PGE2 was well tolerated and systemic PGE2 effects were minimal.

Administration, Oral↗
PubMed1994

Surface epithelial tumours of the ovary.

Surface epithelial tumours (SET) constituted 65.7% of all the ovarian tumours. Benign tumours were 182 (71.9%), of low malignant potential (LMP) 11 (4.4%) and frank malignant 60 (23.7%). Maximum number of cases, 102 (40.3%) belonged to 3rd decade. Mean age for serous cystoma was 31.5% years as compared to 30.8 years for mucinous cystoma. The commonest presenting feature was the abdominal lump observed in 182 cases (71.9%) and pain in abdomen in 120 (47.4%). Serous cystomas were t he most frequent tumours and comprised of 32.21% of all the ovarian tumours or 46.01% of all the SET or 65.56% of all the cystic SET. Seventeen (11.7%) of serous tumours were bilateral. Mucinous cystomas constituted 14.55% of all the ovarian tumours or 30.8% of all the SET. These tumours were bulky (78.6%; 15 cm diameter) and multilocular (83.9%). Mucinous cystadenocarcinoma was the commonest malignant epithelial tum our (36.6%). Endometroid carcinoma comprised 3.65% of all the SET or 8.4% of all the ovarian malignancy. Squamous metaplasia was seen in one case whereas 2 cases were of mesodermal mixed tumour with heterologous element as rhabdomyosarcoma. Clear cell carcinoma, Brenner tumour and unclassified group constituted 0.79%, 1.18% and 1.58% of all SET respectively.

Adenocarcinoma, Clear Cell↗
PubMed1993

Solid tumours of the ovary.

One hundred seventeen solid ovarian tumours, diagnosed histologically during the period between 1971 and 1990, were studied. The overall incidence of solid ovarian tumours was 23.4% of all the ovarian tumours (500 cases) studied. Of 117 solid ovarian tumours 19 (16.2%) were benign and the rest 98 (83.8%) were malignant. Epithelial tumours were the commonest (28.2%) followed by germ cell tumours (22.2%), sex cord stromal tumours (21.4%), metastatic tumours (19.7%) and non-specific tumours (8.5%). The average age (26.8 years) was comparatively low for germ cell tumours otherwise in other groups it ranged between 42 and 45 years. Below 15 years immature teratoma was diagnosed in 4 cases and granulosa cell tumour in 2 cases. Bilateral tumours were seen in 22 cases (23.2%) out of 95 cases observed. Pain and fullness in the pelvic region were observed in 94.9% cases. Varied morphological picture was seen in 6 cases of endometrioid carcinoma. Dysgerminoma constituted 6.8% of solid ovarian tumours. Gliomatosis peritonei was noticed in one case of immature teratoma. Granulosa cell tumour accounted for 11.1% of solid ovarian tumours. Of 23 cases of metastatic tumours, Krukenberg tumour was noticed in 88.9% cases. Diagnostic problems as well as prognostic factors of solid ovarian tumours have been discussed.

Adolescent↗
PubMed1990

Scopolamine withdrawal syndrome.

As travel by air and ship becomes increasingly popular, more and more travelers are using transdermal scopolamine to avoid motion sickness. In fact, it has become almost fashionable for ocean travelers to sit on the sun deck with a patch behind the ear. This article describes withdrawal symptoms in a patient who used transdermal scopolamine beyond the recommended 3 days.

Administration, Cutaneous↗