scholarly journals Role of maltase in the utilization of sucrose by Candida albicans

1993 ◽  
Vol 291 (3) ◽  
pp. 765-771 ◽  
Author(s):  
P R Williamson ◽  
M A Huber ◽  
J E Bennett
Keyword(s):  
Candida Albicans ◽  
Intracellular Localization ◽  
Cross Reactivity ◽  
Neutral Sugar ◽  
Sequence Specificity ◽  
Western Blots ◽  
Sds Page ◽  
Molecular Masses ◽  
Kinetics Of

Two isoenzymes of maltase (EC 3.2.1.20) were purified to homogeneity from Candida albicans. Isoenzymes I and II were found to have apparent molecular masses of 63 and 66 kDa on SDS/PAGE with isoelectric points of 5.0 and 4.6 respectively. Both isoenzymes resembled each other in similar N-terminal sequence, specificity for the alpha(1-−>4) glycosidic linkage and immune cross-reactivity on Western blots using a maltase II antigen-purified rabbit antibody. Maltase was induced by growth on sucrose whereas beta-fructofuranosidase activity could not be detected under similar conditions. Maltase I and II were shown to be unglycosylated enzymes by neutral sugar assay, and more than 90% of alpha-glucosidase activity was recoverable from spheroplasts. These data, in combination with other results from this laboratory [Geber, Williamson, Rex, Sweeney and Bennett (1992) J. Bacteriol. 174, 6992-6996] showing lack of a plausible leader sequence in genomic or mRNA transcripts, suggest an intracellular localization of the enzyme. To establish further the mechanism of sucrose assimilation by maltase, the existence of a sucrose-inducible H+/sucrose syn-transporter was demonstrated by (1) the kinetics of sucrose-induced [14C]sucrose uptake, (2) recovery of intact [14C]sucrose from ground cells by t.l.c. and (3) transport of 0.83 mol of H+/mol of [14C]sucrose. In total, the above is consistent with a mechanism whereby sucrose is transported into C. albicans to be hydrolysed by an intracellular maltase.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

Role of phosphoprotein phosphatases in the corpus luteum: I Identification and characterisation of serine/threonine phosphoprotein phosphatases in isolated rat luteal cells

1996 ◽  
Vol 150 (2) ◽  
pp. 205-211 ◽  
Author(s):  
S L Ford ◽  
D R E Abayasekara ◽  
S J Persaud ◽  
P M Jones
Keyword(s):  
Polyclonal Antibodies ◽  
Luteal Cell ◽  
Luteal Cells ◽  
Cell Functions ◽  
Phosphoprotein Phosphatases ◽  
Sds Page ◽  
Molecular Masses ◽  
Dose Dependent Decrease

Abstract Although the role of protein kinases and phosphorylation in steroidogenesis has received much attention, very little is known about the activities of phosphoprotein phosphatases (PP) and dephosphorylation in steroidogenic tissues. The aims of the present study were therefore to identify which of those serine/threonine PPs more commonly involved in intracellular signalling are expressed in rat luteal cells; to quantify, in vitro, the effects of inhibitors on PP activity extracted from purified rat luteal cells; and to measure the effects of PP inhibitors on the phosphorylation of endogenous luteal cell proteins. Polyclonal antibodies raised against the catalytic subunits of PP types 1 and 2A, and a monoclonal antibody raised against the Ca2+-binding subunit of PP2B, were used to identify immunoreactive proteins that migrated on SDS-PAGE with approximate molecular masses of 37, 34 and 16 kDa, corresponding well with the reported molecular mass of PP1, PP2A and PP2B respectively. Five selective inhibitors of PP1/PP2A: okadaic acid, calyculin A, cantharidin, tautomycin and microcystin-RR, each caused a dose-dependent decrease in the activity of PPs in luteal cell homogenates, and also enhanced 32P incorporation into numerous luteal cell proteins; most notably, proteins with approximate molecular masses of 20 and 22 kDa. The results of this study suggest that PPs may play an important role in the regulation of rat luteal cell functions. Journal of Endocrinology (1996) 150, 205–211

Genetic and immunological characterization of the microsporidianSeptata intestinalisCali, Kotler and Orenstein, 1993: reclassification toEncephalitozoon intestinalis

Parasitology ◽  
1995 ◽  
Vol 110 (3) ◽  
pp. 277-285 ◽  
Author(s):  
R. A. Hartskeerl ◽  
T. Van Gool ◽  
A. R. J. Schuitema ◽  
E. S. Didier ◽  
W. J. Terpstra
Keyword(s):  
Protein Composition ◽  
Rflp Analysis ◽  
Small Subunit ◽  
Cross Reactivity ◽  
Antigenic Structure ◽  
Phenotypic Characterization ◽  
Western Blots ◽  
Microsporidian Species ◽  
Sequence Identity ◽  
Sds Page

SUMMARYTh relationships between theEncephalitozoon-likeSeptata intestinalisand other microsporidia that occur in humans, notablyEncephalitozoon cuniculiandEncephalitozoon hellem, is insufficiently documented using morphological descriptions alone. To assess mutual relationships, we have examined other phenotypic as well as genetic aspects ofS. intestinalis, obtained both from tissue culture and clinical specimens, in comparison with a number of other microsporidia. Phenotypic characterization was performed by analysis of the protein composition and antigenic structure of various microsporidian spores by SDS-PAGE and Western blotting. The genetic characterization consisted of the determination of the sequence of theS. intestinalis rrsgene encoding the small subunit ribosomal RNA (srRNA), restriction fragment length polymorphism (RFLP) analysis of amplifiedrrsgenes and establishment of the degree of sequence identity betweenrrsgenes of various microsporidian species. The unique sequence ofrrsofS. intestinalisas well as the distinct RFLP and SDS-PAGE profiles indicate thatS. intestinalisis clearly different from other human microsporidian species. However, itsrrsgene shared about 90% sequence identity withrrsof bothEncephalitozoonspp.,E. cuniculiandE. hellem. This is remarkably higher than the about 70% identity observed betweenrrsof microsporidian species which belong to different genera and thus suggests that S.intestinalisshould be regarded as a species of the genusEncephalitozoon. Western blots revealed a marked cross-reactivity betweenS. intestinalisand both species ofEncephalitozoonwhich also stresses the close relationship between these organisms. It is concluded thatS. intestinalisis so closely related toE. cuniculi, the type species ofEncephalitozoon, that it should be reclassified asEncephalitozoon intestinalis.

Physicochemical, molecular and immunological characterization of camel calf rennet: a comparison with buffalo rennet

2000 ◽  
Vol 67 (1) ◽  
pp. 73-81 ◽  
Author(s):  
ELSAYED I. ELAGAMY
Keyword(s):  
Marked Decrease ◽  
Cross Reactivity ◽  
Buffalo Calf ◽  
Immunological Characterization ◽  
Milk Clotting ◽  
Electrophoretic Patterns ◽  
Sds Page ◽  
Molecular Masses ◽  
Milk Clotting Activity

Camel calf rennet (CCR) and buffalo calf rennet (BCR) were prepared from dried abomasa to study their physicochemical properties and electrophoretic behaviour and to carry out an immunological characterization of the rennet proteins. CCR was more thermostable than BCR. The milk clotting activity of both rennets increased as pH decreased. The optimum temperatures for CCR and BCR were 50 and 45 °C respectively. CCR was more sensitive to increased CaCl2 in milk than BCR. Addition of NaCl to milk in the range 0–100 g/l resulted in a marked decrease in the clotting activity of both rennets. When the rennets were treated with acetone, the activity of BCR was completely destroyed, but that of CCR was unaffected. The proteolytic activity of CCR was higher than that of BCR and pepsin towards both camel and cows' milk caseins at pH 6·0. SDS-PAGE electrophoretic patterns of CCR and BCR proteins gave two major bands with molecular masses estimated as 52 and 39 kDa for CCR and 50 and 35 kDa for BCR. Immunodiffusion and immunoelectrophoresis using anti-CCR serum demonstrated immunological cross reactivity between CCR and BCR.

scholarly journals Changes in protein kinase C ∊ phosphorylation status and intracellular localization as 3T3 and 3T6 fibroblasts grow to confluency and quiescence: a role for phosphorylation at Ser-729?

2000 ◽  
Vol 352 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Karen ENGLAND ◽  
Martin G. RUMSBY
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Localization ◽  
C6 Glioma Cells ◽  
Nuclear Fraction ◽  
Cell Fractionation ◽  
Cytosol Fraction ◽  
Kinase C ◽  
Sds Page ◽  
Molecular Masses

Protein kinase C (PKC) ε in 3T3 and 3T6 fibroblasts and in C6 glioma cells migrated on SDS/PAGE predominantly as a doublet with molecular masses of 87 and 95kDa (PKC ε87 and PKC ε95 respectively). PKC ε95 predominates when cells reach confluency but PKC ε87 was the main form detected within 15min when confluent cells were passaged at low cell density into fresh medium containing serum and allowed to adhere. Matrix-assisted laser-desorption ionization–time-of-flight MS analysis and experiments with phosphospecific antibodies revealed that PKC ε87 is phosphorylated at Thr-566 and Ser-703, and PKC ε95 is additionally phosphorylated at Ser-729. Cell fractionation studies revealed that PKC ε95 is associated with the nuclear fraction, whereas PKC ε87 was found in the 100000g cytosol fraction. Immunofluorescence studies confirmed these findings and showed that PKC ε95 had a perinuclear, probably Golgi, localization and PKC ε87 was distributed in the cytosol. It is proposed that phosphorylation at Ser-729 may be important for determining the intracellular localization of PKC ε, and that a specific Ser-729 phosphatase may be activated on cell passage to convert PKC ε95 to PKC ε87.

Abstract 30: ApoE and ApoCIII Interact to Modulate the Metabolism of HDL ApoA-I in Humans

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Allyson Morton ◽  
Carlos O Mendivil ◽  
Liyun Wang ◽  
Jeremy D Furtado ◽  
Frank M Sacks
Keyword(s):  
No Reduction ◽  
Multicompartmental Model ◽  
Preliminary Model ◽  
Sds Page ◽  
Modeling Software ◽  
Low Hdl ◽  
Vldl Metabolism ◽  
Hdl Metabolism ◽  
Kinetics Of

ApoE has potential roles in HDL metabolism by promoting enlargement and clearance, and apoCIII could delay apoE-mediated clearance by the liver as it does for VLDL metabolism. To determine whether apoE and apoCIII modulate the kinetics of apoA-I HDL, we compared the metabolism of apoA-I in HDL subspecies that have apoE, apoCIII, both, or neither. We recruited 10 participants (4M, 6F) with low HDL-C (range 24-54 mg/dl) and BMI between 25-35 kg/m 2 . They were given an IV bolus of d3-leucine and blood collected up to 46hr. HDL was isolated from plasma by anti-apoA-I immunoaffinity chromatography, separated by sequential anti-apoE and anti-apoCIII chromatography, and size-separated using NDPAGE into alpha-1, alpha-2, alpha-3, and prebeta-1 HDL. ApoA-I was purified from HDL subspecies on SDS-PAGE, and pool size of apoA-I was determined from the protein bands, adjusted to plasma total apoA-I. D3-leucine enrichment was measured by GC-MS. We used SAAM-II modeling software to compute apoA-I fractional catabolic rates (FCR) and fluxes for each HDL subspecies using a published multicompartmental model. The main findings from our preliminary model investigation are: - The liver secretes a range of HDL sizes for each of these HDL subspecies. About 2-6% of plasma HDL apoA-I is associated with apoE and/or apoCIII. Regardless of size, apoE- and apoCIII-containing HDL are detectable in the circulation slightly earlier after tracer administration than HDL containing neither apoE nor apoCIII. - HDL that contains apoE but not apoCIII is especially active in size conversions, such as generating prebeta-1 HDL. Prebeta-1 HDL types are not a universal precursor of larger size HDL. - HDL that contains apoE but not apoCIII has about a 4-fold increased FCR (range 1.3-8.8) across all sizes of HDL compared to other HDL subspecies, consistent with the role of apoE as a liver receptor ligand. When coexisting with apoE, apoCIII abolished the apoE-accelerated clearance, making the FCR similar to that of HDL that does not have apoE. But, when apoCIII is present on HDL that does not have apoE, there is no reduction in clearance compared to HDL containing neither apolipoprotein. In conclusion, these results suggest that apoE accelerates the metabolism of HDL apoA-I, whereas apoCIII impedes this process.

scholarly journals Characterization of quail intestinal mucin as a ligand for endogenous quail lectin

1993 ◽  
Vol 293 (3) ◽  
pp. 867-872 ◽  
Author(s):  
R Fang ◽  
M Mantle ◽  
H Ceri
Keyword(s):  
Goblet Cells ◽  
Cross Reactivity ◽  
Mucosal Surface ◽  
Immunoperoxidase Technique ◽  
Western Blots ◽  
Intestinal Mucin ◽  
Sds Page ◽  
Endogenous Lectins ◽  
Antibody Preparations ◽  
Intestinal Mucins

The S-type lectins have been shown to be components of mucosal scrapings, and in avian systems these lectins have been localized immunohistochemically to the mucosal surface and goblet cells of the intestine. The interaction of lectin specifically with purified mucin has not, however, been established. Quail intestinal mucin was purified by two subsequent isopycnic density-gradient centrifugations in CsCl and chromatography on Sepharose Cl-2B. Purified mucin, obtained from the void volume of the Sepharose column, was characterized by SDS/PAGE, amino acid and carbohydrate analyses, sensitivity to thiol reduction, and cross-reactivity with antibody preparations to rat and human intestinal mucins on Western blots. Antibody raised against purified quail mucin partially cross-reacts with purified rat, rabbit and human intestinal mucins, and specifically labels the mucosal surface and goblet cells of quail intestine by the immunoperoxidase technique. Protein eluted by lactose from an affinity matrix composed of quail intestinal mucin possessed the same molecular mass on SDS/PAGE as intestinal lectin and reacted on Western blots with a lectin-specific antibody. The data clearly demonstrate the co-localization of lectin and mucin in the quail intestine and also the ability of the lectin to specifically interact with the purified mucin, raising the question of the role of endogenous lectins in secretions.

scholarly journals Identification and Purification of SpecificPenicillium marneffei Antigens and Their Recognition by Human Immune Sera

1998 ◽  
Vol 36 (4) ◽  
pp. 949-954 ◽  
Author(s):  
L. Jeavons ◽  
A. J. Hamilton ◽  
N. Vanittanakom ◽  
R. Ungpakorn ◽  
E. G. V. Evans ◽  
...  
Keyword(s):  
Western Blotting ◽  
Gel Electrophoresis ◽  
Pathogenic Fungus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Sds Page ◽  
Molecular Masses

Disseminated infection with the dimorphic pathogenic fungusPenicillium marneffei is increasingly seen among patients with AIDS in southeast Asian countries. Previous studies have demonstrated the presence of humoral immune responses to this fungus in patient sera; we have confirmed this work using sera from P. marneffei-infected patients (n = 21) to develop Western blots of P. marneffei cytoplasmic yeast antigen (CYA). P. marneffei CYA was then partially purified by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Immunoenzyme development of the Western blots with pooled sera from patients with P. marneffei infection and with pooled sera from patients with aspergillosis (n = 20), candidiasis (n = 10), cryptococcosis (n = 9), and histoplasmosis (n = 11) revealed three antigens with relative molecular masses of 61, 54, and 50 kDa. These antigens were specifically recognized by the pooled sera from the P. marneffei-infected patients. The 61- and 54-kDa antigens were subsequently purified to homogeneity by preparative gel electrophoresis, and the 50-kDa antigen was partially purified by the same technique. N-terminal amino acid sequencing revealed that the 61-kDa antigen had a strong homology (87% identity) with the antioxidant enzyme catalase. The three antigens were then subjected to SDS-PAGE and Western blotting and to immunoenzyme development with individual patient sera; sera from 86% of P. marneffei-infected patients recognized the 61-kDa antigen, sera from 71% recognized the 54-kDa antigen, and sera from 48% recognized the 50-kDa antigen. These specifically recognized antigens are the first to be purified from P. marneffei and can be used either singly or in combination to detect antibody responses in a large percentage of individuals infected with P. marneffei.

scholarly journals Studies on the incorporation of a covalently bound disubstituted phosphate residue into Azotobacter vinelandii flavodoxin in vivo

1990 ◽  
Vol 268 (3) ◽  
pp. 745-749 ◽  
Author(s):  
M H Boylan ◽  
D E Edmondson
Keyword(s):  
Azotobacter Vinelandii ◽  
Conclusive Evidence ◽  
Cross Link ◽  
Western Blots ◽  
Cell Extracts ◽  
Sds Page ◽  
Nif Gene ◽  
Covalently Bound

Previous studies have shown the flavodoxin from Azotobacter vinelandii (strain OP, Berkeley) to contain a covalently bound disubstituted phosphate residue [Edmondson & James (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3786-3789]. Phosphorylation of the protein in vivo was investigated by the addition of [32P]phosphate to cells grown under N2-fixing conditions, under conditions of nif-gene repression and under conditions of nif-gene de-repression. Rocket immunoelectrophoresis of cell extracts showed an approx. 5-fold decrease in the concentration of flavodoxin expressed in cells grown in the presence of NH4+ as compared with those grown under N2-fixing conditions. A similar increase in flavodoxin concentration was observed on nif-gene de-repression. Incorporation of [32P]phosphate occurs only into newly synthesized flavodoxin, as observed on SDS/PAGE of immunoprecipitates of cell extracts. Western blots demonstrated no observable precursor forms of flavodoxin. These data provide conclusive evidence for the phosphorylation of Azotobacter strain OP flavodoxin in vivo and suggest that the covalently bound phosphate residue does not exchange with cellular phosphate pools. Thus the role of this phosphodiester cross-link is proposed to be structural rather than regulatory.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

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