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Biomedical subjects

G Krause

Publications and source records attributed to G Krause.

At least 91 records · Page 5Linked to original sources
PubMed1997

Atherogenesis and the homocysteine-folate-cobalamin triad: do we need standardized analyses?

BACKGROUND: Bioscientists, physicians and nutritionists are newly interested in the homocysteine-folate-cobalamin triad, in part because homocysteine may be important both in atherogenesis and thrombogenesis. Homocysteine imbalance may be an early marker for cobalamin disorders because cobalamin is a cofactor in remethylation of homocysteine to methionine. METHODS: In 139 men and 32 women of similar mean age of 65 years, we measured markers which have been cited as risk for atherosclerosis: serum homocysteine, folate, total cobalamin, holotranscobalamin I and II, (TCI and TCII), total serum cholesterol (SCHOL), high density lipoprotein cholesterol (HDLC), triglycerides (STG) as well as red blood cell (RBC) folate, food records and body composition by whole body counting of potassium-forty (40K). RESULTS: Statistical relationships among the data showed healthy women had lower mean serum homocysteine and their mean RBC folate and TCI and TCII were higher than men. Eighty-three subjects had TCII much lower than 60 pg/ml (subnormal), yet only 11 of these men and two women had total cobalamin < 200 pg/ml (abnormal). Fifty-two subjects with serum homocysteine greater than 17.5 nmol/ml had TCII less than 60 pg/ml, suggesting serum homocysteine may be a marker for early cobalamin negative balance. None of the subjects in the study had serum folate below abnormal values, i.e., less than 1.6 mg/ml. All subjects had RBC folate within normal range. Serum homocysteine showed inverse relationship with RBC folate and serum total cobalamin, TCI and TCII. CONCLUSIONS: 1) importance of using serum holotranscobalamin TCI and TCII as markers of cobalamin deficiency, 2) necessity to use documented quantitative components of dietary intake if strong comparisons are to be made among quantitative values of serum or plasma homocysteine, folate, cobalamin, and nutrients in food intake.

Adult↗
PubMed1997

Complementary anchor PCR of rearranged variable T-cell receptor beta-chain cDNA regions.

Sequencible amplificates comprising the variable cDNA sequences of the rearranged T-cell receptor (TCR) beta-chain were obtained from the T-leukemia cell line Jurkat using a single-sided PCR approach based on five synthetic oligonucleotides derived from the flanking constant sequence. Double-stranded cDNA was cleaved by a restriction enzyme creating cohesive ends, to which an anchor oligonucleotide was ligated. Since this anchor was complementary to the antisense strand of the known constant region, exclusively the desired ligation product folded into a stem-loop-structure that was enzymatically extended to yield a PCR template, now flanked at both ends by primer binding sites appropriate for nested PCR.

Binding Sites↗
PubMed1996

Effect of adenosine 3',5'-cyclic monophosphate (cAMP) on human non-tumorigenic and tumorigenic parotid acinar cells in culture.

Adenosine 3',5'-cyclic monophosphate (cAMP) regulates growth and/or differentiation of some tumor cells in culture. The effects of cAMP on human parotid acinar cells have never been studied. Results showed that 2HPC8 (non-tumorigenic cells derived from human pleomorphic parotid adenoma) were more sensitive to dibutyryl cAMP, an analog of cAMP, and RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, than 2HP1G cells (derived from spontaneous transformation of 2HPC8 cells), suggesting that human parotid adenoma cells maintain a higher degree of cAMP-responsiveness than parotid carcinoma cells. Prostaglandin E1 (PGE1), a stimulator of adenylate cyclase, which increased cAMP levels in 2HPC8 cells, but not in 2HP1G cells, caused a greater degree of growth inhibition in 2HP1G cells than in 2HPC8 cells, suggesting that this effect of PGE1 on tumorigenic cells is not related to cAMP. In the normal parotid and parotid adenoma cell cultures, about 5-10% of cells were small and round, whereas in tumorigenic cell cultures, all cells were small and round. Some indirect evidence suggests that these round cells may be the primary target for transformation. These tumor cells formed cytoplasmic processes of variable length after treatment with cAMP stimulating agents and prostaglandin E1.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Epidemiologic investigation of serum levels of the soluble forms of CD25, CD54 and CD58, and T cell responsiveness after stimulation via the CD2-dependent pathway in a random sample of the general population.

A sero-epidemiologic correlation study on immune parameters which would correlate with the frequency of common colds (FCC) had been conducted in 1992. There, an inverse relationship between circulating adhesion molecules CD54 and CD58 and FCC was found. Eighteen months later we performed an analogous assessment in order to verify the previous findings and to carry out additional experiments including in vitro proliferative responses of T cells and their production of various cytokines (IFN-gamma, IL-2, IL-6 and IL-10). The additional examinations showed that individuals with frequent common colds exert a higher T cell proliferation and higher production of cytokines than persons which experience never or few common cold infections. These findings could be confirmed statistically. Taken together, the results suggest consistently in individuals with frequent common colds an association with low serum levels of the immunosuppressive soluble adhesion molecules sCD54 and sCD58, high proliferation of unstimulated and stimulated T cells and secretion of higher concentrations of cytokines (IFN-gamma, IL-2, IL-6 and IL-10) into the cell culture supernatants.

Age Factors↗
PubMed1995

Influence of alpha-helicity, amphipathicity and D-amino acid incorporation on the peptide-induced mast cell activation.

Mast cell activation by polycationic substances is believed to result from a direct activation of G protein alpha subunits and it was suggested that the adaption of amphipathic, alpha-helical conformations would allow the peptide to reach the cytosolic compartment to interact with G proteins (Mousli et al., 194, Immunopharmacology 27, 1, for review). We investigated the histamine-releasing activity of model peptides as well as analogues of magainin 2 amide and neuropeptide Y with different amphipathicities and alpha-helix content on rat peritoneal mast cells. Amphipathic helicity is not a prerequisite for mast cell activation. Moreover, non-helical magainin peptides with high histamine-releasing activity were less active in the liberation of carboxyfluoresceine from negatively charged liposomes, indicating that peptide-induced mast cell activation and peptide-induced membrane perturbation do not correlate. In contrast to the negligible influence of the secondary structure, amino acid configuration may exert a striking influence on peptide-induced mast cell activation. Thus histamine-release by substance P was markedly impaired when the L-amino acids in the positively charged N-terminal region were replaced by D-amino acids, with [D-Arg1)substance P being the most inactive substance P diastreoisomer.

Amino Acid Sequence↗
PubMed1995

Structure effects of double D-amino acid replacements: a nuclear magnetic resonance and circular dichroism study using amphipathic model helices.

D-Amino acid replacements and the determination of resulting structural changes are a useful tool to recognize amphipathic helices in biologically active peptides such as neuropeptide Y and corticotropin-releasing factor. In this paper the secondary structures of one amphipathic alpha-helical peptide and its double D-amino acid analog have been determined by means of 1H NMR and CD spectroscopies under equivalent conditions. The chemical shifts (NH and C alpha H) and the analysis of nuclear Overhauser effects show a split of the continuous helix for the all-L peptide into two helices at the position of double D-amino acid replacement. Hydrogen exchange rates correlate with water accessibilities in the hydrophobic/hydrophilic face and confirm the amphipathic helical structure in the all-L peptide as well as in its double D-amino acid analog. A significantly accelerated hydrogen isotope exchange rate is observed for the D-Ala9 backbone proton, implying an increased flexibility at that position. These results show that the incorporation of an adjacent pair of D-amino acids only causes a local change in structure and flexibility, which makes the double D replacement interesting as a tool for specific helix-disturbing modifications to search for helical conformations in biologically active peptides.

Amino Acid Sequence↗
PubMed1995

Comparison of body composition measured by total body potassium and infrared interactance.

OBJECTIVE: The purpose of this study was to find how closely a near infrared instrument (IRI), Futrex 5000, would compare to whole body counting (WBC) of potassium-40 (40K) in assessing body composition. DESIGN: We measured lean body mass (LBM) and percent total body fat (PCF) in 177 males and 27 females by total body potassium (TBK) and by infrared interactance (IRI). TBK was assessed by 2 pi whole body counter (WBC) and IRI by Futrex (F) instrument. These subjects were measured for TBK and for IRI within 1 week of each other. RESULT: A large mean difference (p < or = 0.001) was found between the results using the two instruments: IRI data for LBM were higher and PCF lower compared to TBK. CONCLUSION: As shown by repeated TBK measurements over a 22-year period in these same volunteers, this technique is reliable. The IRI assessments did not come to a close fit with TBK.

Adult↗
PubMed1994

Distinction of weakly homologous cDNA amplificates by single-strand conformation polymorphism analysis: application to guanylyl cyclase isozymes.

By use of the polymerase chain reaction (PCR), uniform amplification products of 225 to 240 bp length were obtained from five cDNA clones representing different types of guanylyl cyclases. These short DNA double strands were differentiated by single-strand conformation polymorphism (SSCP), using polyacrylamide gel electrophoresis with the Pharmacia Phast-System. Following heat denaturation, the samples were separated on native polyacrylamide gels at different running temperatures. Nucleic acids on the gel were detected by an automated silver stain procedure. Using 7.5% homogeneous or 4-15% gradient polyacrylamide gels at a temperature of 12 degrees C, single-strand conformations of amplificates, representing three different particulate guanylyl cyclases and the two subunits of soluble guanylyl cyclase, were differentiated. The characteristic banding patterns resulting from dissimilar migration of the single-strand conformations were assigned to different guanylyl cyclase types. For the enzyme family of guanylyl cyclases, the feasibility of a combined PCR and electrophoresis approach for analyzing the expression of related genes was demonstrated. This application of the PCR-SSCP technique provided a rapid and sensitive tool for the characterization of PCR products obtained with a common primer pair and suggested its use for investigating the tissue distribution of gene expression within a class of homologous proteins.

Animals↗
PubMed1994

The effect of folate and cobalamin on osteoarthritic hands.

OBJECTIVE: Historically diet and arthritis have been cause/effect associated but the idea is controversial with little evidence that specific diet components are effective treatment. This controlled, double-blinded, crossover study reports the effect of folate and cobalamin supplements in 26 humans diagnosed for an average 5.7 years with idiopathic osteoarthritis of the hands who had been medicated by prescribed nonsteroidal anti-inflammatory drugs (NSAID). METHODS: Subjects entered the study after a 10-day washout period from use of all anti-arthritis drugs, vitamins, and minerals. They were randomly allocated to consume daily 6400 micrograms folate or 6400 micrograms folate plus 20 micrograms cobalamin or lactose placebo each for 2 months within self-selected diets. Pain was to be medicated by acetaminophen as needed, and at the end of each phase they returned for assessment and dispensing of the next treatment. Serum folate and cobalamin, red blood cell folate, blood smears, diet records, standard rheumatology assessment and hand grip measurements were reviewed and statistically analyzed. RESULTS: For all subjects mean right and left hand grip values were higher with combined cobalamin-folate ingestion than with other "vitamin" supplements and were equivalent to NSAID use. Number of tender hand joints were greater with use of NSAID than with use of cobalamin-folate. Side effects with the vitamin combination were none; side effects of NSAID are many, and the cost of vitamins and acetaminophen also is lower. CONCLUSION: The limited number of subjects in this study demonstrates that ingestion of a prescribed cobalamin-folate supplement and acetaminophen as needed resulted in positive outcomes.

Acetaminophen↗
PubMed1994

Distribution of membrane bound guanylyl cyclases in human intestine.

The quantification and distinction of particulate guanylyl cyclases in the human intestine were considered by an enzymatic approach, which comprised the signal transduction from receptor binding to cGMP formation, and, in addition, by showing the expression of an intracellular portion of these transmembrane proteins. Basal guanylyl cyclase (GC) activities were 50 to 80 pmol cGMP formation/min/mg protein and were stimulated up to twofold by heat stable enterotoxin, but were not significantly influenced by atrial natriuretic factor. Enzymatic analysis of colonoscopic specimens pointed to the prevalence of guanylyl cyclase C in the terminal ileum and in the large bowel including colon ascendens, colon descendens, sigmoid, and rectum. The availability of sequence information on human guanylyl cyclases permitted the development of a polymerase chain reaction approach for distinguishing the expression of GC-A and GC-C in human tissue samples. The expression levels of particulate guanylyl cyclases found by polymerase chain reaction in surgical biopsy specimens confirmed the enzymatic data, in that substantial expression of GC-C was found not only in the small intestine but also in the large bowel. According to the restriction mapping of amplificates, GC-C prevailed over GC-A throughout the human intestine, particularly in the mucosal layers.

Adult↗
PubMed1992

A Pro to His mutation in active site of thioredoxin increases its disulfide-isomerase activity 10-fold. New refolding systems for reduced or randomly oxidized ribonuclease.

Thioredoxin (Trx) from Escherichia coli was compared with bovine protein disulfide-isomerase (PDI) for its ability to catalyze native disulfide formation in either reduced or randomly oxidized (scrambled) ribonuclease A (RNase). On a molar basis, a 100-fold higher concentration of Trx than of PDI was required to give the same rate of native disulfide formation measured as recovery of RNase activity. A Pro-34 to His (P34H Trx) mutation in the active site of E. coli Trx (WCGPC), mimicking the two suggested active sites in PDI (WCGHC), increased the catalytic activity in disulfide formation about 10-fold. The mutant P34H Trx displayed a 35-mV higher redox potential (E'0) of the active site disulfide/dithiol relative to wild type Trx, making it more similar to the redox potential observed for PDI. This higher redox potential correlates well with the enhanced activity and suggests a role for the histidine side chain. Enzymatic isomerization of disulfides in scrambled, oxidized RNase requires the presence of a catalytic thiol such as GSH to initiate the thiol-disulfide interchange. Bovine thioredoxin reductase, together with NADPH, could replace GSH. For oxidative folding of reduced RNase in air with Trx, P34H Trx, or PDI, catalytic amounts of sodium selenite (1 microM) resulted in rapid disulfide formation and high yields of ribonuclease activity equivalent to previously known redox buffers of GSH and GSSG. These results demonstrate no obligatory role for glutathione in disulfide formation. A possible mechanism for the unknown thiol oxidative process accompanying folding and protein disulfide formation in vivo is discussed.

Amino Acid Sequence↗
PubMed1992

Influence of Corynebacterium parvum-PER on disease progression in the NZB/W model of systemic lupus erythematosus.

Murine models of systemic lupus erythematosus (SLE) exhibit some, but not all, of the characteristics of human disease. Disease progression in the animal models is regulated by autoantibodies, genetics and inflammatory processes. In the present study, the influence of the pyridine extract residue of Corynebacterium parvum Type I (CP-PER) on disease progression in the NZB/W model of SLE was investigated. CP-PER is known to activate the reticuloendothelial (RE) system of mice and can alter a number of host responses. Injection of NZB/W females with CP-PER at 10 weeks of age, prior to the onset of overt disease, led to a transient activation of the RE system which then appeared to return to levels observed in untreated animals. Sera from treated and untreated animals were analyzed for anti-nuclear antibodies, and anti-ssDNA antibodies. The most prominant change in the treated animals was an increase in the titer of anti-ssDNA. On average, animals in the early treatment group also lived somewhat longer than those in the untreated control group. Treatment of animals with CP-PER at 6 months of age, when the disease was evident, again led to transient RES activation and an altered autoantibody profile. Animals treated with CP-PER at 6 months of age exhibited a slightly accelerated death rate when compared with the untreated controls. These results indicate that bacterial response modifiers such as C. parvum-PER, which contains primarily the RES activating activity of C. parvum, can alter disease progression in the NZB/W model.

Animals↗
PubMed1992

Aging in humans: a continuous 20-year study of physiologic and dietary parameters.

The old adage, "You are what you eat," is not always reliable, as demonstrated in this mixed-longitudinal study of men that began in 1969. Mean values of percent body fat, total body potassium (TBK), and total serum cholesterol (SCHOL) did not show changes that correlated with any studied nutrient from repeated 4-day diet records. Mean blood pressure increased with increased body weight as age increased. High-density lipoprotein cholesterol decreased when polyunsaturated fat intake increased. The men had decreased mean height, TBK and increase in percent body fat as age increased. Food energy intake decreased equally from carbohydrates, protein and fat as age increased. Large standard deviations of mean values of measured parameters document heterogeneity of these subjects. A subset of 144 male cohorts was studied serially for 20 years in time-age, cross-sectional and longitudinal series. As they aged, height and TBK decreased (p < or = 0.05), percent body fat and blood pressure increased (p < or = 0.04) and SCHOL had no significant change. Intake of all nutrients decreased significantly (p < or = 0.03) longitudinally and with time, showing a time effect.

Adipose Tissue↗
PubMed1991

Motion of aromatic side chains, picosecond fluorescence, and internal energy transfer in Escherichia coli thioredoxin studied by site-directed mutagenesis, time-resolved fluorescence spectroscopy, and molecular dynamics simulations.

We have determined the picosecond fluorescence of the four aromatic amino acid residues (W28, W31, Y49, and Y70) in wild-type Escherichia coli thioredoxin (wt Trx) and a mutant Trx with W31 replaced by phenylalanine, Trx-W28-W31F. The internal motions of the four aromatic side chains were also analyzed. We examined the possibility of using internal energy transfer from tyrosine to tryptophan as a measure of long-range distances. The major features of the lifetime distribution of tryptophan fluorescence were unchanged in the W31F mutation, indicating that the environment of W28 is similar in both wt Trx and Trx-W28-W31F. However, the mutation of W31F changed the mobility of W28, situated close to the active-site disulfide/dithiol, but not the mobility of two tyrosines, Y49 and Y70, situated on the other side of the molecule. The mobility of the two tyrosine residues increased upon reduction of the active-site disulfide, indicating a looser structure with reduction. This increased motion could also be seen from molecular dynamics simulations. The change in energy transfer rates, as judged by tyrosine fluorescence lifetimes, was in agreement with energy transfer rates calculated from the molecular dynamics simulations. The anisotropy of tryptophan and tyrosine fluorescence could be separated in three parts: (I) overall rotation of the protein (10(-9)s), (II) internal mobility of side chains (10(-10)s), and (III) a very fast relaxation (10(-12)s). We can only experimentally detect this very fast relaxation when the internal motion is not present.

Amino Acids↗
PubMed1991

Assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin.

As a necessary first step in the use of heteronuclear correlated spectra to obtain high resolution solution structures of the protein, assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin (Mr 12,000) uniformly labeled with 15N has been performed. The 15N chemical shifts of backbone amide nitrogen atoms have been determined for both oxidation states of thioredoxin using 15N-1H correlated and two-dimensional heteronuclear single-quantum coherence (HSQC) TOCSY and NOESY spectra. The backbone assignments are complete, except for the proline imide nitrogen resonances and include Gly33, whose amide proton resonance is difficult to observe in homonuclear 1H spectra. The differences in the 15N chemical shift between oxidized and reduced thioredoxin, which occur mainly in the vicinity of the two active site cysteines, including residues distant in the amino acid sequence which form a hydrophobic surface close to the active site, are consistent with the differences observed for proton chemical shifts in earlier work on thioredoxin.

Amino Acid Sequence↗
PubMed1991

Mimicking the active site of protein disulfide-isomerase by substitution of proline 34 in Escherichia coli thioredoxin.

To mimic the active sites (Trp-Cys-Gly-His-Cys) contained in two thioredoxin-like domains of the eukaryotic enzyme protein disulfide-isomerase (PDI, EC 5.3.4.1), the Pro-34 residue of Escherichia coli thioredoxin (Trx) was replaced by His using site-directed mutagenesis. The mutant P34H Trx was isolated in high yield and was stable. The equilibrium between Trx and NADPH in the thioredoxin reductase (TR)-catalyzed reaction revealed that the redox potential (E'o) or P34H Trx at pH 7.0 was -235 mV as compared with -270 mV for wild type (wt) Trx. The higher E'o value made P34H Trx more similar to PDI and contributed to prominent changes in Trx functions, e.g. improved activity with TR and slower reduction of protein disulfides. Compared to wt Trx, the P34H oxidized Trx was about twice as good a substrate for TR from E. coli and four times as efficient with calf thymus TR. A novel fluorimetric assay permitted direct recording of the reaction between insulin disulfide(s) and reduced Trx. At pH 8 and 15 degrees C, second-order rate constants for wt Trx of 2 x 10(4) M-1 s-1 and for P34H Trx of 3 x 10(3) M-1 s-1 were obtained, and a different equilibrium was observed consistent with differences in E'o values. Also when the reduction mechanism of insulin was examined using NADPH and TR, P34H Trx behaved differently from wt Trx or PDI. P34H Trx may be useful as an analogue of PDI for disulfide formation in vivo and in vitro.

Amino Acid Sequence↗
PubMed1991

Substitution of the conserved tryptophan 31 in Escherichia coli thioredoxin by site-directed mutagenesis and structure-function analysis.

All prokaryotic and eukaryotic thioredoxins contain a conserved tryptophan residue, exposed at the active site disulfide/dithiol. The role of this W31 in Escherichia coli thioredoxin (Trx) was studied by site-directed mutagenesis. Four mutant Trx with W31Y, W31F, W31H, and W31A replacements were characterized. Very low tryptophan fluorescence emission from the remaining W28 was observed in all mutant Trx; reduction resulted in large, but variable increases (up to 11-fold) of fluorescence, to levels higher than in native or denatured wild-type Trx, demonstrating a previously postulated change involving W28. All W31 mutant Trx were good substrates for E. coli thioredoxin reductase. Compared with wild type, the apparent Km values were increased less than 2-fold for the W31A, W31H, and W31F Trx and the W31Y Trx showed even slightly higher catalytic efficiency (kcat/Km value). Functions of reduced Trx with ribonucleotide reductase and in reduction of insulin disulfides were more strongly influenced by the W31 replacements, in particular at low pH for A and H residues. T7 DNA polymerase activity generated by T7 gene 5 protein and reduced Trx was lowered by large factors for W31Y, W31A, or W31H compared with W31F or the wild-type protein. The in vivo function of Trx was studied by using pUC118-trxA expression in an E. coli trxA- background. The trxA genes with W31Y and W31F substitutions restored, fully and partly, the methionine sulfoxide utilization of a trxA- metE- test strain; W31A and W31H mutations resulted in no growth. Propagation of M13 was moderately impeded by W31Y and W31F or severely by W31A and W31H replacements. Growth of a phage T3/7 hybrid was possible only with the W31Y and W31F substitutions reflecting the in vitro results for T7 DNA polymerase.

Amino Acid Sequence↗