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PubMed1994

Strongyloides stercoralis: characterization of immunodiagnostic larval antigens.

Forty-one-, 31-, and 28-kDa proteins of Strongyloides stercoralis filariform larvae have previously been demonstrated to be sensitively and specifically recognized by serum IgG in individuals with strongyloidiasis. Characteristics of these proteins, their immunodominant epitopes, and reactive antibodies are described here. The proteins are soluble in aqueous as well as detergent extracts. The immunodominant epitopes are present in S. stercoralis but not in S. cebus or S. ratti. Epitopes on the three proteins are not shared, as determined by cross-adsorption of serum with each of the size components on nitrocellulose. In most sera from strongyloidiasis patients there was reactivity to each of the proteins by IgG1 and IgG4, but reactivity by IgG2 or IgG3 was detectable only in a minority. A rabbit antiserum raised to a 41-kDa size fraction of S. stercoralis larvae reacted against a doublet of 41-kDa which was distinct from the immunodiagnostic 41-kDa protein.

Animals↗
PubMed1986

Steroid hormone-induced changes in secreted proteins in the filamentous fungus Achlya.

In the fungus Achlya, sexual development in the male strain E87 is mediated by the steroid hormone antheridiol. Treatment of vegetatively growing cells of E87 with antheridiol resulted in changes in the [35S]methionine labeling of several secreted proteins. The most heavily labeled group of proteins observed in the secreted fraction from control cells appeared on one-dimensional gels as a prominent wide band which could be resolved into three closely spaced components with relative molecular weights (MWs) of 57, 54, and 50 kD, respectively. After hormone treatment the two lower MW proteins of the group were barely detectable. Concomitant with the observed reductions in the labeling of the 54 and 50 kD proteins was the increased labeling of a doublet of very prominent proteins with relative MWs of 44.4 and 43 kD, respectively. The results of experiments with Endoglycosidase H suggested that the 44.4 and 43 kD proteins seen in hormone-treated cultures, might result from the removal or reduced synthesis of high mannose oligosaccharide groups found on the 54 and 50 kD proteins normally seen in control cultures. Additional support for this suggestion was provided by the observation that the 54 and 50 kD proteins from control cultures appeared to bind to conA columns and to be eluted with alpha-methylmannoside, while there was little or no binding of the 44.4 and 43 kD proteins from hormone-treated cells. Although other possibilities are not excluded, the results are suggestive of a steroid hormone-induced alteration in glycoprotein processing. The functions of the observed hormonally-responsive secreted proteins as well as those of the secreted proteins in non-hormone-treated cultures, are not known at this time.

Acetylglucosaminidase↗
PubMed1989

Protein composition of human aqueous humor: SDS-PAGE analysis of surgical and post-mortem samples.

We analyzed the protein composition of human aqueous humor. Samples were obtained by paracentesis from 25 human eyes (age range 64-92 years) at elective cataract surgery, and from 20 age-matched post-mortem eyes within 1.5 to 18 hr after death. Individual samples were assayed for total protein, and the polypeptides were separated by qualitative SDS-PAGE into high-, medium- and low-molecular-weight ranges and then silver-stained. The clinical samples showed a remarkable consistency in the total protein values (mean +/- SEM: 12.4 +/- 2.0 mg per 100 ml) and no detectable variations in the profiles of the silver-stained proteins. Twelve major protein fractions, with apparent molecular weights of 140, 80 (doublet), 67, 60 (doublet), 35, 27, 25, 17, 14.6 and 9 kDa, were present. A preliminary analysis showed that the 17 kDa band contained a molecule resembling basic fibroblast growth factor. Two additional samples of aqueous humor from patients whose blood/aqueous barrier was compromised during paracentesis showed a quantitative and qualitative increase in the polypeptides that were present. Compared with the samples of aqueous humor obtained at surgery, the post-mortem samples exhibited a greater variability in total protein content (56.1 +/- 11.6 mg per 100 ml) and an increased number of high- and low-molecular-weight protein fractions. In view of wide differences in the clinical parameters, including ocular and systemic medications, systemic illness, surgical premedications, anesthesia and total serum protein values, the similarity in the protein profiles of the carefully drawn surgical samples is most remarkable. Our results indicate that, in patients who underwent elective cataract surgery, the levels of major proteins in human aqueous humor are not affected by wide individual variations in the clinical parameters. We attribute this finding to the care taken in the collection of aqueous humor samples.

Aged↗
PubMed1991

Expression of angiotensin converting enzyme mRNA in rat brain.

Using molecular genetic approaches, two forms of angiotensin-converting enzyme (ACE) have so far been identified; a pulmonary form and a testicular form. This study utilized both northern blot analysis and in-situ hybridization to examine the expression of ACE mRNA in the rat brain. Northern blot analysis using oligonucleotide probes specific to the testicular or pulmonary forms of ACE demonstrated expression of a 4-5kb mRNA doublet in the rat brain, identical in size to pulmonary ACE mRNA. No expression of the testicular form of ACE mRNA was detected in the brain. In situ hybridization demonstrated that ACE mRNA was expressed in the choroid plexus, caudate putamen and cerebellum. A weak signal was also observed in the hippocampus. This distribution correlated well with the expression of ACE mapped by radioligand binding and autoradiography.

Amino Acid Sequence↗
PubMed1996

Characterization of insulin-like growth factor binding proteins and regulation of IGFBP3 in human mesangial cells.

IGF-I regulates renal growth and development. Insulin-like growth factor binding proteins (IGFBPs) are synthesized by the kidney and may modulate the local autocrine and/or paracrine actions of IGF-I. We have previously demonstrated that mesangial cells (MC) release IGF-I and IGF-binding activity; however, the specific IGFBPs produced by these cells and the factors involved in their regulation are unknown. We examined MC for expression of IGFBP-1 to -6 mRNAs and proteins. RNase protection assays using total RNA demonstrated that MC express all of the IGFBPs. [125I]IGF-I Western ligand blot of conditioned medium demonstrated that MC release IGFBPs of 24, 29, 32 kDa, and a doublet at 46 kDa, consistent with IGFBP-4, -5, -2 and -3, respectively. IGFBP species of 28 and 34 kDa were also detected. Since IGF-I and TGF-beta are implicated in glomerular hypertrophy and matrix expansion, we tested their effect on IGFBPs released by MC. IGF-I (100 ng/ml), TGF-beta (2 ng/ml) and forskolin (10(-5) M) differentially regulated the abundance of IGFBPs released in the conditioned medium in a time-dependent manner. IGF-I and TGF-beta were potent inducers of the release of IGFBP3 protein; however, TGF-beta, but not IGF-I, increased IGFBP3 mRNA levels. Recombinant IGFBP3 was tested for its effect on IGF-I-induced mitogenesis. IGFBP3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner with a peak effect observed at 50 nM IGFBP3. Although TGF-beta is a potent inhibitor of IGF-I-stimulated DNA synthesis, this effect is not mediated via IGFBPs. Expression of IGFBP-1 to -6 by MC suggests that these proteins may modulate IGF-I bioavailability in the glomerulus. IGF-I itself, TGF-beta and cAMP agonists may indirectly modulate the effects of IGF-I via the release of IGFBPs by MC.

Colforsin↗
PubMed2004

A non-DM1, non-DM2 multisystem myotonic disorder with frontotemporal dementia: phenotype and suggestive mapping of the DM3 locus to chromosome 15q21-24.

The majority of proximal myotonic myopathy syndromes reported so far have been related to the myotonic dystrophy (DM) type 2 (DM2) mutation, an expanded (CCTG)n repeat in the ZNF9 gene. Here, we describe the phenotype and the histological features in muscle and brain of the first large pedigree with a non-myotonic dystrophy type 1 (DM1) non-DM2 multisystem myotonic disorder associated with severe frontotemporal dementia. Thirty individuals from three generations underwent detailed neurological, neuropsychological, electrophysiological, brain imaging and molecular analyses. Ten of them had proximal muscle weakness at onset, clinical/electrical myotonia and DM-type cataracts. The mean age at onset was 46.7 +/- 12.6 years (range: 32-69). Dementia was observed later in the course of the disease. On muscle biopsies, rare nuclear clumps, rimmed vacuoles and small angulated type 1 and type 2 fibres were seen early in the disease. They were replaced by fibrous adipose tissue at later stages. Immunohistochemical analysis of myosin heavy chain isoforms showed no selective fibre type atrophy-both type 1 and type 2 fibres being affected. Cortical atrophy without white matter lesions was seen on brain MRI. A brain single photon emission computed tomography (SPECT) study revealed marked frontotemporal hypoperfusion. Post-mortem examination of the brains of two patients showing prominent frontotemporal spongiosis, neuronal loss and rare neuronal and glial tau inclusions suggested frontotemporal dementia. Western blot analyses of the tau protein showed a triplet of isoforms (60, 64 and 69 kDa) in neocortical areas, and a doublet (64 and 69 kDa) in subcortical areas that distinguish our myotonic disorder from other's myotonic dystrophies. Molecular analyses failed to detect a repeat expansion in the DMPK and ZNF9 genes excluding both DM1 and DM2, whereas a genome-wide linkage analysis strongly suggested a linkage to chromosome 15q21-24. This previously unreported multisystem myotonic disorder including findings resembling DM1, DM2 and frontotemporal dementia provides further evidence of the clinical and genetic heterogeneity of the myotonic dystrophies. We propose to designate this disease myotonic dystrophy type 3, DM3.

Adult↗
PubMed1988

Antigens recognised by the human immune response to infection with Leptospira interrogans serovar hardjo.

Serum samples from patients infected with Leptospira interrogans serovar hardjo were tested by the microscopic agglutination test (MAT), enzyme immunoassay (EIA) and immunoblotting. There was no apparent correlation between MAT titre and EIA optical density (OD) for individual serum samples, but sequential serum samples produced similar profiles in both tests during the course of an infection. Immunoblotting of hardjo sonicate with patients' sera revealed reactions with a number of bands, in the mol. wt (10(3] range 14.4-95. However, all serum samples reacted with the major 28 x 10(3)-mol. wt sub-unit of hardjo lipopolysaccharide (LPS) and most reacted with a (34.5-35) x 10(3)-mol. wt flagella doublet. Examination of sequential serum samples obtained over a period of about 3 months after infection revealed little change in the antigens detected after the second to third week of infection. Absorption of patients' sera with whole viable leptospires revealed that antibodies to several exposed antigens, including LPS, were produced. Sera which reacted with hardjo flagella also reacted with bands of similar mol. wts in preparations from other serovars.

Agglutination Tests↗
PubMed1994

Proton magnetic resonance spectroscopy of cerebrospinal fluid as an aid in neurological diagnosis.

The proton magnetic resonance (1H-NMR) spectra were obtained from 38 cerebrospinal fluid (CSF) samples from 34 patients with various central nervous system (CNS) diseases and the H2O signals were reduced by the presaturation method. A doublet signal for CH3 protons of lactate, as well as a singlet one for alpha-CH proton of glucose, were clearly detected in all the spectra obtained. The relative lactate concentration values were calculated on the basis of the glucose concentrations which had been measured by a routine laboratory method in the hospital and the ratios of the peak heights (lactate CH3/glucose alpha-CH). In the CSF samples examined, the relative lactate values semi-quantitatively determined in this way were clearly elevated in cerebral infarction and bacterial meningitis, but not in other disorders including viral meningitis. On the other hand, one CSF component whose enhancement was found only in a sample from a hepatic encephalitis patient was identified as glutamine from the double quantum filtered-shift correlation spectrum. These results suggest that 1H-NMR spectroscopy of CSF can become a powerful aid in biochemical diagnosis of CNS diseases.

Adult↗
PubMed1995

Different molecular and messenger ribonucleic acid forms of insulin-like growth factor-binding protein-3 in the pregnant baboon (Papio anubis).

The ratio of the serum concentrations of insulin-like growth factors (IGF) to IGF-binding protein (IGFBP)-3 is highly correlated (Baxter & Martin 1986). During pregnancy in the baboon, this ratio is perturbed; serum IGFBP-3 concentrations increase 10-fold, yet IGF-I levels are unaltered and IGF-II is increased only 2-fold (Giudice et al. 1993). The aims of this study were to determine the molecular distribution of IGFBP-3 and to identify the tissue source and form(s) of IGFBP-3 during pregnancy in the baboon. Serum of non-pregnant and pregnant baboons, and conditioned media of decidua and placental explant cultures were characterized using neutral size-exclusion chromatography in combination with Western ligand blot, Western immunoblot, an IGFBP-3 radioimmunoassay (RIA) and an IGFBP-3 protease assay. Localization of immunoreactive IGFBP-3 was determined by immunocytochemistry, and expression of IGFBP-3 mRNA in the placental and decidual explants was examined by Northern blot analysis. RIA confirmed that immunoreactive IGFBP-3 is increased 10-fold in pregnancy serum compared with non-pregnancy serum. Size-exclusion chromatography combined with an IGFBP-3 RIA revealed that, unlike non-pregnancy serum where 70% of the immunoreactive IGFBP-3 elutes in the 150 kDa ternary complex, equal amounts of immunoreactive IGFBP-3 were measured in pregnancy serum in the < or = 150 and 60 kDa IGFBP regions. Western analysis revealed that non-pregnancy serum contained predominantly a 45-40 kDa IGFBP-3 doublet and a 28 kDa immunoreactive form of IGFBP-3, while in pregnancy serum IGFBP-3 existed as a 45-40 kDa doublet, as well as 26-28 kDa and 18 kDa immunoreactive forms. These alternative forms of IGFBP-3 were not attributable to detectable IGFBP-3 protease activity. To identify the source of the increased serum levels of IGFBP-3 during pregnancy, we examined explant culture media of baboon decidua and placenta. Size-exclusion chromatography combined with RIA and Western analysis revealed that: (1) more immunoreactive IGFBP-3 was produced by decidual cultures than by placental explants, but less 45-40 kDa IGFBP-3 was present in decidua; (2) the immunoreactive forms of IGFBP-3 detected in decidua were similar to those found in maternal serum; (3) placental explants secreted only 45-40 kDa IGFBP-3 in culture. IGFBP-3 was immunohistochemically localized to the cells of placental villi, and to the perinuclear region of the decidual cells and staining for IGFBP-3 was more intense in the decidua than in the placenta. Northern analysis of the explant cultures revealed two IGFBP-3 mRNA transcripts of 2.4 and 1.7 kb in both decidua and placenta which may account for the different immunoreactive forms of IGFBP-3 detected in the baboon. However analysis of non-pregnancy liver also revealed two IGFBP-3 transcripts of 2.4 and 1.7 kb. These data suggest that the two transcripts are not solely pregnancy-associated and levels of protein may be the reason for detection of multiple immunoreactive IGFBP-3 fragments in pregnancy.

Animals↗
PubMed1990

Bone acidic glycoprotein-75 is a major synthetic product of osteoblastic cells and localized as 75- and/or 50-kDa forms in mineralized phases of bone and growth plate and in serum.

Anti-peptide and anti-protein antisera were produced which both recognize bone acidic glycoprotein-75 (Mr = 75,000) and an apparent fragment or biosynthetic intermediate (Mr = 50,000) in calcified tissues and/or serum. A fragment-precursor relationship is suggested from the fact that closely spaced doublet polypeptides of Mr = 50,000 could be produced by proteolysis of the purified protein upon long term storage. No reactivity was detected with osteopontin, bone sialoprotein, or small bone proteoglycans. Bone acidic glycoprotein-75 represents 0.5-1% of the total radiolabeled proteins synthesized by explant cultures of neonatal calvaria or growth plate, by calvarial outgrowth cultures, and by rat osteosarcoma cells. Amounts produced by explant cultures and calvarial outgrowth cultures were similar to that for osteopontin, a major product of osteoblasts. In osteosarcoma cultures, 80% of labeled antigens were associated with the cell layer fraction wherein specific immunoprecipitation pelleted Mr = 50,000 and 75,000 sized antigens. Bone acidic glycoprotein-75 (Mr = 75,000) is enriched in 4 M guanidine HCl/0.5 EDTA extracts of neonatal rat bone and growth plate tissues, whereas largely absent from heart, lung, spleen, liver, brain, and kidney. Explant cultures of these noncalcifying tissues also synthesized bone acidic glycoprotein-75 antigen, but the quantities produced were only 5% or less that obtained with calvaria. By immunohistochemistry, antigenicity is associated with the bony shaft and calcified cartilage of long bones, but is absent from associated soft tissues. These finding demonstrate that bone acidic glycoprotein-75 is antigenically distinct, predominantly localized to calcified tissues, represents a major product of normal osteoblastic cells and may undergo a characteristic fragmentation in vivo and in vitro.

Animals↗
PubMed1978

Detection of genetic variation with radioactive ligands. III. genetic polymorphism of transcobalamin II in human plasma.

We detected genetically determined, electrophoretic variants of vitamin B12 binding proteins, most probably transcobalamin II, in human plasma. Polymorphic variants were observed in all populations tested; the two most common alleles (of at least four detected to date) attain frequencies of greater than 40% in Caucasians and Orientals. The variants are autosomally inherited and are seen as doublets in homozygotes, and four-banded patterns, the sum of two dissimilar homozygote patterns, in heterozygotes. The technique used in this survey, polyacrylamide gel electrophoresis (PAGE) autoradiography of plasma and serum labeled in vitro with 57Co-vitamin B12 is particularly applicable to the study of trace proteins such as the transcobalamins (10(-9)M). Possible functional variation in the TC II allele products is described, and the selective significance of this worldwide polymorphism is considered.

Autoradiography↗
PubMed1995

Microdomain formation in phosphatidylethanolamine bilayers detected by 2H-NMR.

In deuterium NMR spectra of phosphatidylethanolamine bilayers with an extremely high content of saturated fatty acids, each C1 deuteron of the glycerol backbone gave rise to a doublet [Yoshikawa et al., (1988) Biochim. Biophys. Acta 944, 321-328]. This suggests the presence of two backbone conformations, the exchange between which is slow on an NMR time-scale. The origin of the two conformations has been investigated in this work using saturated 1,2-diacyl-sn-glycero-3-phosphoethanolamine specifically deuterated in the glycerol backbone. The results showed that the two conformations originate from different domains, which have different fatty acid compositions. The differential scanning calorimetry of the bilayers suggested that the size of the domain is not large enough to show an independent phase transition. Thus, the formation of microdomains in the phosphatidylethanolamine bilayers has been concluded. Conformational difference in different domains was shown to be restricted to the C1 position of the glycerol backbone. The microdomains of phosphatidylethanolamine were retained even in the presence of other phospholipids.

Calorimetry, Differential Scanning↗
PubMed2000

Evidence that proximal multiple mutations in Big Blue transgenic mice are dependent events.

To characterize the nature of multiple mutations in the tissues of an intact animal, the Big Blue transgenic mouse mutation detection system was used to examine 1459 mutants from eight normal tissues and 507 mutants from 11 tumors. Multiple mutations occurred and predominantly doublet mutants were identified (i.e. two mutations within one mutant lacI gene), but multiplets of up to five mutations were observed. The frequency of doublets in normal tissues and spontaneous tumors from p53-deficient mice was enhanced to the same degree (660 and 667 fold, respectively) over that expected for two independent mutational events. Doublets, multiplets and singlets have similar patterns of mutation. The distance between mutations in doublets fits an exponential distribution, not that expected for randomly spaced events, suggesting that many doublets occur in rapid succession within the same cell cycle.

Animals↗
PubMed2002

Novel rectangular [Fe4(mu4-OHO)(mu-OH)(2)](7+) versus "butterfly" [Fe4(mu3-O2](8+) core topology in the Fe(III)/RCO2(-)/phen reaction systems (R = Me, Ph; phen = 1,10-phenanthroline): preparation and properties of [Fe4(OHO)(OH)(2)(O2CMe)(4)(phen)(4)](ClO4)(3), [Fe4O2(O2CPh)(7)(phen)(2)](ClO4), and [Fe4O2(O2CPh)(8)(phen)(2)].

The preparations, X-ray structures, and detailed physical characterizations are presented for three new tetranuclear Fe(III)/RCO(2)(-)/phen complexes, where phen = 1,10-phenanthroline: [Fe(4)(OHO)(OH)(2)(O(2)CMe)(4)(phen)(4)](ClO(4))(3).4.4MeCN.H(2)O (1.4.4MeCN.H(2)O); [Fe(4)O(2)(O(2)CPh)(7)(phen)(2)](ClO(4)).2MeCN (2.2MeCN); [Fe(4)O(2)(O(2)CPh)(8)(phen)(2)].2H(2)O (3.2H(2)O). Complex 1.4.4MeCN.H(2)O crystallizes in space group P2(1)/n, with a = 18.162(9) A, b = 39.016(19) A, c = 13.054(7) A, beta = 104.29(2) degrees, Z = 4, and V = 8963.7 A(3). Complex 2.2MeCN crystallizes in space group P2(1)/n, with a = 18.532(2) A, b = 35.908(3) A, c = 11.591(1) A, beta = 96.42(1) degrees, Z = 4, and V = 7665(1) A(3). Complex 3.2H(2)O crystallizes in space group I2/a, with a = 18.79(1) A, b = 22.80(1) A, c = 20.74(1) A, beta = 113.21(2) degrees, Z = 4, and V = 8166(1) A(3). The cation of 1 contains the novel [Fe(4)(mu(4)-OHO)(mu-OH)(2)](7+) core. The core structure of 2 and 3 consists of a tetranuclear bis(mu(3)-O) cluster disposed in a "butterfly" arrangement. Magnetic susceptibility data were collected on 1-3 in the 2-300 K range. For the rectangular complex 1, fitting the data to the appropriate theoretical chi(M) vs T expression gave J(1) = -75.4 cm(-1), J(2) = -21.4 cm(-1), and g = 2.0(1), where J(1) and J(2) refer to the Fe(III)O(O(2)CMe)(2)Fe(III) and Fe(III)(OH)Fe(III) pairwise exchange interactions, respectively. The S = 0 ground state of 1 was confirmed by 2 K magnetization data. The data for 2 and 3 reveal a diamagnetic ground state with antiferromagnetic exchange interactions among the four high-spin Fe(III) ions. The exchange coupling constant J(bb) ("body-body" interaction) is indeterminate due to prevailing spin frustration, but the "wing-body" antiferromagnetic interaction (J(wb)) was evaluated to be -77.6 and -65.7 cm(-1) for 2 and 3, respectively, using the appropriate spin Hamiltonian approach. Mössbauer spectra of 1-3 are consistent with high-spin Fe(III) ions. The data indicated asymmetry of the Fe(4) core of 1 at 80 K, which is not detected at room temperature due to thermal motion of the core. The spectra of 2 and 3 analyze as two quadrupole-split doublets which were assigned to the body and wing-tip pairs of metal ions. (1)H NMR spectra are reported for 1-3 with assignment of the main resonances.

Journal Article↗
PubMed2002

Characterization of an antagonist interleukin-6 dimer by stable isotope labeling, cross-linking, and mass spectrometry.

The homodimeric form of a recombinant cytokine interleukin-6 (IL-6(D)) is known to antagonize IL-6 signaling. In this study, spatially proximal residues between IL-6 chains in IL-6(D) were identified using a method for specific recognition of intermolecular cross-linked peptides. Our strategy involved mixing 1:1 (15)N-labeled and unlabeled ((14)N) protein to form a mixture of isotopically labeled and unlabeled homodimers, which was chemically cross-linked. This cross-linked IL-6(D) was subjected to proteolysis by trypsin and the generated peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (MS). Molecular ions from cross-linked peptides of intermolecular origin are labeled with [(15)N/(15)N] + [(15)N/(14)N] + [(14)N/(15)N] + [(14)N/(14)N] yielding readily identified triplet/quadruplet MS peaks. All other peptide species are labeled with [(15)N] + [(14)N] yielding doublet peaks. Intermolecular cross-linked peptides were identified by MS, and cross-linked residues were identified. This intermolecular cross-link detection method, which we have designated "mixed isotope cross-linking" MIX may have more general application to protein-protein interaction studies. The pattern of proximal residues found was consistent with IL-6(D) having a domain-swapped fold similar to IL-10 and interferon-gamma. This fold implies that IL-6(D)-mediated antagonism of IL-6 signaling is caused by obstruction of cooperative gp130 binding on IL-6(D), rather than direct blocking of gp-130-binding sites on IL-6(D).

Dimerization↗
PubMed1989

Identification of subunits of gonococcal RNA polymerase by immunoblot analysis: evidence for multiple sigma factors.

Heparin-agarose and single-stranded DNA-cellulose chromatography were used to purify RNA polymerase 25-fold from Neisseria gonorrhoeae, and the activity of the polymerase was characterized in altered assay systems. The core subunits (beta, beta', and alpha) were tentatively identified as major proteins copurifying with polymerase activity. The identification of the core subunits was confirmed by Western (immunoblot) analysis with polyclonal antisera to Escherichia coli core RNA polymerase. Gonococcal sigma factor heterogeneity was examined by Western blot analysis with polyclonal antiserum to the major E. coli sigma factor, sigma 70, to the E. coli heat shock sigma factor, sigma 32, and with a monoclonal antiserum to Salmonella typhimurium NtrA (sigma 54). Purified RNA polymerase and whole-cell extracts from type 1, type 4, heat-shocked, and anaerobically grown gonococci were examined. Four putative gonococcal sigma factors were detected in purified RNA polymerase preparations and in whole-cell extracts from all cell types. Two of these bands appeared as a doublet, which had an estimated Mr of 80,000. A single lower-Mr band, estimated to be 40,000, was also present. All three of these bands reacted with antisera to E. coli sigma 70 and to E. coli sigma 32. A fourth gonococcal protein reacted solely with a highly specific monoclonal antibody to sigma 54 and had an Mr of 90,000. We conclude that N. gonorrhoeae may contain multiple sigma factors, which it may use to regulate gene expression.

Animals↗
PubMed1991

Proteins of the mammalian mitotic spindle: phosphorylation/dephosphorylation of MAP-4 during mitosis.

The phosphoprotein composition of isolated CHO spindles was analyzed using the MPM-1 and MPM-2 antibodies, which are reactive with a phosphorylated epitope enriched in mitotic cells and present on the centrosome, kinetochores, midbody and fibers of the mitotic spindle. Several high molecular weight phosphorylated spindle proteins were detected on immunoblots, including species of 410 x 10(3) Mr, 350 x 10(3) Mr, a 230-240 X 10(3) Mr doublet, 210 x 10(3) Mr and 120 x 10(3) Mr. The temporal and spatial distribution of the MPM-reactive phosphoproteins was determined by examining spindle structures isolated from cells at various stages of mitosis. The susceptibility of the staining pattern to extraction with salt, a procedure known to remove most microtubule-associated proteins (MAPs), was also examined. The phosphorylated 210 x 10(3) Mr species was identified as MAP-4 and localized to the spindle fibers using (1) a polyclonal antibody raised against this species, that reacted with known MAPs, and (2) established MAP-4 antibodies that reacted with the spindle 210 x 10(3) Mr MPM-reactive proteins. The comparative immunoblot and immunofluorescence analysis establishes a cycle of phosphorylation/dephosphorylation of MAP-4 upon entry and exit from mitosis. Regarding the other MPM-reactive proteins, comparative immunofluorescence staining and immunoblot analysis of isolated spindle samples before and after salt extraction indicate that they may be constituents of the centrosome, kinetochores or midbody, but their definitive identification awaits the production of monospecific antibodies.

Animals↗
PubMed1990

Study of human Entamoeba histolytica infection by zymodeme, indirect hemagglutination and electroimmunotransfer blot assays.

Human amebiasis is a parasitic infection which involves asymptomatic as well as intestinal and extraintestinal symptomatic stages in individuals harbouring Entamoeba histolytica. Several factors have been proposed to explain the variability in the outcome of the disease. Among these are differences in virulence of E. histolytica strains and methods such as zymodeme analysis have been used to differentiate invasive from non invasive strains of this parasite (Sargeaunt and Williams, 1978). In order to establish a possible correlation between zymodeme analysis and serological data in human cases of amebiasis, a cross-sectional epidemiological study was carried out in the community area of Cadereyta, State of Queretaro, Mexico. In this study, fecal and serum samples from individuals were also tested by Indirect Hemagglutination assay (IHA) to determine antibody titre and by a standardized Electroimmunotransfer blot assay (EITB) for recognition of E. histolytica specific antigens. Zymodemes were determined in a total of 81 samples. Of these, 27 had a pathogen zymodeme (PZ) while 54 had a non pathogen zymodeme (NPZ). Values obtained by IHA varied from negative to titres up to 1:256 in serum samples tested. Reactivity to E. histolytica antigens determined by EITB was observed in all sera. Patterns of antigen recognition were complex and showed reactivity to several parasite molecules. Among these, components with M. Wt. of 165, 119, and doublets of 98-100 and 50-52 Kd were more frequently recognized by antibodies present in the sera tested. In general, antibody titres detected by IHA did not showed a direct correlation with zymodeme analysis. Samples with PZ or NPZ had similar variable levels of reactivity to E. histolytica antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗