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At least 649 records · Page 36Linked to original sources
PubMed2001

A protein pre-organized to trap the nucleotide moiety of coenzyme B(12): refined solution structure of the B(12)-binding subunit of glutamate mutase from Clostridium tetanomorphum.

Uniformly (13)C,(15)N-labeled MutS, the coenzyme B(12)-binding subunit of glutamate mutase from Clostridium tetanomorphum, was prepared by overexpression from an Escherichia coli strain. Multidimensional heteronuclear NMR spectroscopic experiments with aqueous solutions of (13)C,(15)N-labeled MutS provided signal assignments for roughly 90% of the 1025 hydrogen, 651 carbon, and 173 nitrogen atoms and resulted in about 1800 experimental restraints. Based on the information from the NMR experiments, the structure of MutS was calculated, confirming the earlier, less detailed structure obtained with (15)N-labeled MutS. The refined analysis allowed a precise determination of the secondary and tertiary structure including several crucial side chain interactions. The structures of (the apoprotein) MutS in solution and of the B(12)-binding subunit in the crystal of the corresponding homologous holoenzyme from Clostridium cochlearium differ only in a section that forms the well-structured helix alpha1 in the crystal structure and that also comprises the cobalt-coordinating histidine residue. In the apoprotein MutS, this part of the B(12)-binding subunit is dynamic. The carboxy-terminal end of this section is conformationally flexible and has significant propensity for an alpha-helical structure ("nascent helix"). This dynamic section in MutS is a decisive element for the binding of the nucleotide moiety of coenzyme B(12) and appears to be stabilized as a helix (alpha1) upon trapping of the nucleotide of the B(12) cofactor.

Amino Acid Sequence↗
PubMed1985

Anomalous neutrophil granule distribution in a patient with lactoferrin deficiency: pertinence to the respiratory burst.

Neutrophils from a patient with lactoferrin deficiency were examined and the quantity and subcellular localization of protein markers were determined on Percoll density gradients. Distribution of azurophilic and specific granule markers was abnormal in that azurophilic granules were lighter than normal and appeared in the fraction of the gradient where normally the specific granules sediment. The specific granule membrane markers, cytochrome b-235 and its associated flavoprotein, were abnormally distributed in the gamma fraction, the site of the plasma membrane marker alkaline phosphatase. Thus, the b-cytochrome-flavoprotein complex had either been incorporated into the plasma membrane or was still present in the membranes of granules that were abnormally light and cosedimented with the plasma membranes. This is of particular interest in regard to the patient's respiratory burst oxidase function, since the b-cytochrome/flavoprotein complex normally translocates from the specific granules to the plasma membrane to constitute the active respiratory burst oxidase. The functional consequences of this abnormal distribution are discussed, as is the importance of characterizing both intragranular enzymatic markers and granule membrane proteins to define granular disorders.

Alkaline Phosphatase↗
PubMed1990

Low holotranscobalamin II is the earliest serum marker for subnormal vitamin B12 (cobalamin) absorption in patients with AIDS.

In AIDS, as previously found in pernicious anemia (PA), the earliest serum marker of subnormal vitamin B12 (cobalamin) absorption, and therefore of negative B12 balance, is low serum holotranscobalamin II (holo-TC II; B12-TC II) despite normal total serum B12 level, normal serum homocysteine, and normal classic (oral free radio-B12) Schilling test. This may be accompanied by subtle and insidious damage to hematopoietic, immunologic, neuropsychiatric, nutritional and alimentary systems, confirmed by correction on therapeutic trial with B12 therapy. Our studies suggest such selective B12 deficiency occurs in about half of the HIV-1 infected, in part due to frequent depression of B12 absorption by HIV-1 attack on the gastric mucosa and/or opportunistic infection attack on the small bowel, and in part due to a telescoping of the continuum of the stages of negative B12 balance in relation to damage to B12 delivery by the infective and/or systemic disease process. In AIDS, when total serum B12 is normal despite tissue depletion of B12, if the classic Schilling test does not reveal subnormal food B12 absorption, the food Schilling test does. We hypothesize that DNA-synthesizing cells of the hematopoietic, immunologic, neurologic and other systems which have surface receptors solely for holo-TC II, and which have low B12 stores, rapidly become dysfunctional due to B12 deficiency when holo-TC II is low, while cells (such as liver cells) which also have surface receptors for holohaptocorrin (B12-haptocorrin) remain B12-replete. We believe this to be another example of the concept of selective nutrient deficiency in one cell line but not another.

Absorption↗
PubMed1993

Measurement of red blood cell-vitamin B12: a study of the correlation between intracellular B12 content and concentrations of plasma holotranscobalamin II.

We have recently reported a new and rapid assay to measure plasma holotranscobalamin II (holo TC II) as a means of exploring vitamin B12 status. In order to further evaluate the significance of plasma holoTC II in determining tissue cobalamin, we have chosen the red blood cell-vitamin B12 (RBC-B12) assay as a measure of tissue vitamin B12 content and studied the relationship between RBC-B12 and plasma holoTC II levels. Plasma holoTC II and RBC-B12 concentrations were concomitantly assayed in 20 hematologically normal controls and cancer patients. In our groups of controls, the mean value of RBC-B12 was determined as 241 +/- 51 pg/ml of packed erythrocytes (PE) with a range varying from 180 to 355 pg/ml PE. Preliminary results obtained in 32 cancer patients revealed lower holoTC II and RBC-B12 levels than the control group and a required threshold value of 70 pg/ml of holoTC II in order to maintain a normal RBC-B12 greater than 180 pg/ml PE.

Erythrocytes↗
PubMed2001

Deficiency of the specific granule proteins, R-binder/transcobalamin I and lactoferrin, in plasma and saliva: a new disorder.

The mechanisms of hereditary deficiency of R binder, which originates in neutrophils and exocrine gland epithelium, are unknown and may be multiple. This led us to examine if defective R binder synthesis also involves proteins that colocalize with it in neutrophil-specific granules and exocrine epithelial cells and may be under common regulatory control. Stored plasma and saliva samples from five unrelated R binder-deficient patients and control subjects were assayed for R binder, lactoferrin, cationic antimicrobial protein-18, neutrophil gelatinase-associated lipocalin, gelatinase, lysozyme, and myeloperoxidase. One patient, patient A, had lactoferrin levels below the limits of detection in both plasma and saliva in addition to his R binder deficiency. Although his deficiency involved lactoferrin as well, he had no history of predisposition to infection. PCR amplification of his R binder gene promoter region and the beginning of the first exon revealed no DNA abnormalities. His son and the son of his equally deficient brother, both presumptive heterozygotes, had mild deficiency of both R binder and lactoferrin. The results show that R binder deficiency exists in at least two forms. One, presumably the less common of the two forms, is the new hereditary entity described here, which is characterized by deficiency of more than one specific granule protein in both plasma and saliva. Despite this more widely distributed absence of the proteins than is found in congenital specific granule deficiency, infection posed no clinical problem in the affected patient.

Female↗
PubMed1991

Induction of neutrophil-mediated cartilage degradation by interleukin-8.

Neutrophil influx into the inflamed joint is a characteristic feature of disease flares in patients with rheumatoid arthritis. Recently, a protein produced by monocytes and fibroblasts that has chemoattractive/activating properties for neutrophils has been identified and characterized. This protein has been called interleukin-8 (IL-8). In this study, we cocultured neutrophils with 35S-sulfate-labeled cartilage and found that the addition of recombinant human IL-8 (rHuIL-8) caused rapid, neutrophil-mediated cartilage degradation that was the result of induction of neutrophil degranulation by the cytokine. With 10(-7)M rHuIL-8, 23% of the radiolabel was released into the culture medium in 4 hours, compared with a 9% release without the factor. At concentrations of up to 10(-6)M, rHuIL-8 had no direct effect upon cartilage breakdown. These findings indicate that IL-8 may participate in the pathogenesis of rheumatoid arthritis through the induction of neutrophil-mediated cartilage damage.

Animals↗
PubMed1988

Osmotic forces are not critical for Ca2+-induced secretion from permeabilized human neutrophils.

In order to examine the role of osmotic forces in degranulation, the effects of solutes and osmolality on granule secretion were explored using both FMLP-stimulated, intact neutrophils and Ca2+-stimulated, permeabilized cells. We employed a HEPES-based buffer system which was supplemented with: a) permeant (KCl or NaCl) or impermeant (Na-isethionate or choline-Cl) ions, or b) permeant (urea) or impermeant (sucrose) uncharged solutes. Intact and permeabilized cells had significantly different solute requirements for degranulation. FMLP-stimulated release from intact cells was supported by NaCl or Na-isethionate greater than KCl greater than choline-Cl or sucrose greater than urea. In contrast, the rank order of Ca2+-stimulated release from permeabilized cells was choline-Cl greater than Na-isethionate, KCl, or NaCl greater than sucrose greater than urea. Hypo-osmotic conditions caused increased levels of background granule release from both intact and permeabilized neutrophils. However, hypo-osmolality inhibited both FMLP-stimulated degranulation from intact cells and Ca2+-induced release from permeabilized neutrophils. While hyperosmotic conditions inhibited stimulated release from intact cells, this inhibition was much less pronounced in permeabilized cells when the granules were directly exposed to these solutions. In fact, hyperosmotic sucrose greatly enhanced Ca2+-induced secretion. Although isolated specific and azurophil granules showed some lytic tendencies in hypo-osmotic buffers, the overall stability of the isolated granules did not indicate that swelling alone could effect degranulation. These results suggest that degranulation in permeabilized cells is neither due to nor driven by simple osmotic forces (under resting or stimulated conditions) and emphasize differences obtained by bathing both the granules and plasma membrane (as opposed to membranes alone) in various solutes.

Calcium↗
PubMed1988

Quantitation of intracellular Mac-1 (CD11b/CD18) pools in human neutrophils.

The adhesive glycoprotein Mac-1 (CD11b/CD18) of the CD11/CD18 complex contributes to multiple neutrophil inflammatory functions. Activation of neutrophils by chemotactic stimuli results in a rapid, protein synthesis-independent increase in surface Mac-1 derived from incompletely defined intracellular compartments. Therefore, we developed a novel quantitative lectin immunoblot technique to define intracellular pools of Mac-1 in subcellular neutrophil fractions resolved on discontinuous Percoll gradients. In cavitates of unstimulated neutrophils, 30% and 26% of total Mac-1 was identified in beta [1.10 gm/ml; vitamin B12 binding protein (vit B12 B.P.)-rich] or pre-gamma (1.07 gm/ml; vit B12 B.P.-poor) granular fractions, respectively, whereas 24% was associated with the plasma membrane-rich gamma (1.06 gm/ml) fractions. N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation (10(-8) M, 15 min, 37 degrees C) significantly diminished Mac-1 in pre-gamma (-18% of total, P less than 0.05) but not beta fractions (+6% of total). Under these conditions, the content of Mac-1 in gamma fractions increased 13% in association with four- to eightfold increase in surface Mac-1 expression (OKM-1 binding). These findings suggest that chemotactic stimuli increase plasma membrane and/or surface Mac-1 on human neutrophils by mobilizing a novel intracellular granule pool.

Adult↗
PubMed1991

Oxidative metabolism of polymorphonuclear leukocytes: modulation by adhesive stimuli.

Different agents such as phorbol myristate acetate (PMA), N-formyl-methionyl-leucylphenylalanine (fMet-Leu-Phe), or opsonized zymosan induced an oxidative burst in rat peritoneal polymorphonuclear leukocytes (PMNs) elicited by casein. Plastic adhesion of PMNs down-regulated superoxide (O2) release stimulated by PMA or fMet-Leu-Phe but had no effect on zymosan-induced O2 generation, indicating that the O2 forming enzyme, the NADPH oxidase, was not affected by modulation and that a common step of the transductional events induced by PMA or fMet-Leu-Phe might be involved in this regulation. We demonstrated that a differential translocation of protein kinase C (PKC) was not responsible for that modulation. PMA-induced secretion of granule content (vitamin B12-binding protein) was not susceptible to modulation, suggesting that the transductional pathways leading to O2 generation and granule secretion are partly separated. The adhesion of PMNs to different substrates (glass, plastic, albumin-, laminin-, fibronectin-, poly-lysine-, or concanavalin A-coated plastic) down-regulated to different extent superoxide release. Whether the nature of the biochemical signal induced by the diverse adhesive stimuli or a physical parameter such as binding strength was involved in this differential behavior remains to be elucidated. Since adhesiveness was dependent on the state of the cytoskeleton and O2 inducers were reported to stimulate actin polymerization, we studied the F-actin content and distribution of PMNs by using the specific fluorescent probe NBD-phallacidin and an original methodology allowing a quantitative analysis of fluorescence on both adherent and suspended cells. PMA induced a polarization of F-actin on suspended PMNs but had no effect on the intracellular distribution of F-actin in adherent PMNs. Thus, we suggest that the adhesion of PMNs induced an immobilization of F-actin, possibly correlated to the down-regulation of one of the transductional pathways involved in the NADPH oxidase activation.

Actins↗
PubMed1991

Actin assembly and regulation of neutrophil function: effects of cytochalasin B and tetracaine on chemotactic peptide-induced O2- production and degranulation.

Several studies indicate that the actin filament system is not only involved in cell motility, but also in the regulation of other neutrophil functions. The aim of the present investigation was to examine the mechanisms by which actin filament formation participates in the control of the respiratory burst and degranulation in human neutrophils. The approach taken was to use both an inhibitor (cytochalasin B) and a potentiator (tetracaine) of formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe)-induced actin polymerization. The total inhibition of fMet-Leu-Phe-induced actin polymerization in cytochalasin B-treated cells was accompanied by an impressive potentiation of both oxidase activity and degranulation (azurophilic and specific granules). However, preincubation with tetracaine, which causes an enhanced accumulation of F-actin in the periphery of fMet-Leu-Phe-stimulated cells, also augmented the rate and duration of peptide-induced superoxide anion production, but inhibited degranulation (specific granules). A likely explanation for the potentiating effects of cytochalasin B and tetracaine is provided by our observation that both of these substances reduced the acid-resistant binding of fMet-Leu-Phe (interpreted as a decrease in the internalization of fMet-Leu-Phe-receptor-complexes), resulting in an enhanced formation of second messengers (diacylglycerol). The findings that tetracaine potentiated the activity of the oxidase, whereas it inhibited degranulation (specific granules), suggest that actin polymerization per se plays a role in the latter process. Consequently, this study argues against the idea of a direct inhibitory effect of F-actin on chemotactic factor-induced oxidase activation, but supports an active role of actin filaments in the translocation and release of granule components.

Actins↗
PubMed1992

Assessment of neutrophil leukocyte secretory response to fMLP in whole blood in vitro.

A simple, precise method has been developed for assessing neutrophil secretory responses (release of vitamin B12 binding protein from specific granules) to challenge of aliquots of whole blood with the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Dose-response studies performed on blood from normal healthy volunteers showed higher maximal secretory responses in males than females (33.3 +/- SEM 2.2 vs. 27.4 +/- 2.5, P less than .005) a left shift in dose-response curves after feeding compared to fasting (P less than .005), spontaneous up-regulation of responses in blood incubated at 37 degrees C for 1 h, and marked upregulation in response to preincubation with endotoxin. This whole blood challenge method may be used to study neutrophil responses in groups of individuals or patients without the confounding effects of changes in cell responses resulting from cell isolation procedures. The method may also be used as a bioassay for neutrophil-activating factors.

Cytochalasin B↗
PubMed1993

Quartz selectively down-regulates CR1 on activated human granulocytes.

We have investigated the interaction between quartz and granulocytes with respect to complement receptor expression. When N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated leukocytes were exposed to quartz at 37 degrees C, CR1 was down-regulated but CR3 was not affected. This was a direct effect on granulocytes because it occurred in a similar fashion when mixed leukocyte suspensions and isolated granulocyte populations were used as targets for quartz. The observed down-regulation by quartz was not affected by the microfilament-disrupting agent cytochalasin B and the total detectable pool of CR1 was reduced after quartz exposure. When protease inhibitors, such as aprotinin or phenylmethanesulfonyl fluoride, were present during quartz exposure, the down-regulation of CR1 was less pronounced, but this was not the case not when protease inhibitors such as EDTA-Na2 and pepstatin were present. Exposure to quartz was not accompanied by a pronounced release of beta-glucuronidase (marker for the primary granules) or vitamin B12 binding protein (marker for the secondary granules). In contrast to quartz, exposure to alumina did not affect the expression of CR1 and CR3. The spontaneous mobilization of CR1 at 37 degrees C was reduced when quartz was present but the CR3 mobilization was unaffected. Our results indicate that quartz induces a granule protease-dependent selective shedding of CR1 but not CR3 despite a low degree of degranulation.

Aprotinin↗
PubMed1995

Improved method for silver staining of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels.

A method for detection of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels was developed by a combination of (i) initial periodic acid oxidation/Alcian blue staining and (ii) subsequent staining with silver nitrate. The procedure allowed detection of as little as 1.6 ng of alpha 1-acid glycoprotein and 8-40 ng of a polydisperse mucin sample, which is at least 10 times more sensitive than previously published methods. The method should be very useful for assessment of sample purity and detection of glycoproteins in dilute mixed samples.

Alcian Blue↗
PubMed1999

Vitamin B12 transporters.

The uptake of vitamin B12 from the intestine into the circulation is perhaps the most complex uptake mechanism of all the vitamins, involving no less than five separate VB12-binding molecules, receptors and transporters. Each molecule involved in uptake has a separate affinity and specificity for VB12 as well as a separate cell receptor. Thus VB12 is initially bound by haptocorrin in the stomach, then by IF in the small intestine. An IF receptor is then involved in uptake of the IF-VB12 complex by the intestinal epithelial cell, with the subsequent proteolytic release of VB12 and subsequent binding to TcII. The TcII receptor then transports the TcII-VB12 complex across the cell, whence it is released into the circulation. It is surprising, then, that despite its complexity, it has been possible to harness the vitamin VB12 uptake mechanism to enhance the oral uptake of peptides, proteins, and nanoparticles.

Amino Acid Sequence↗