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R Kraft

Publications and source records attributed to R Kraft.

At least 145 records · Page 8Linked to original sources
PubMed1988

Electrostatic interactions between cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase.

At pH 8.2 and a 100-fold molar excess of the amino group specific label 2-methoxy-5-nitrotropone (MNT) over protein 2 mol MNT/mol P-450 LM2 were bound, which caused a 50% decrease in the overall activity due to a decreased electron transfer rate from reductase to the hemoprotein. However, different from FITC modification, which produces the same effects, the label is not selectively bound to the alpha-amino group and to lysine 384, but reacts with lysines in positions 49, 100, 139, 144, 251, 384 and 433. The decrease in the overall activity and reduction rate thereby correlates with a relative increase in the modification of lysines 139, 144, 251 and 384. Thus, besides lysine 384 the epsilon-amino groups of lysines 139, 144 and 251 are further candidates for participation in the interaction with reductase. This finding supports our model of charge-pair contacts between P-450 and reductase, where amino groups of P-450 LM2 form salt bridges to carboxylic groups of reductase. The decrease of reductase supported P-450 reduction velocity in microsomes at high salt concentration (I greater than 222 mM) indicates the dominant electrostatic character of P-450/reductase interaction. Based on these results and data from the literature a model of membrane topography of P-450 LM2 has been proposed. Extension of the charge pair interaction model to interaction mechanisms of other P-450 isoenzymes and forms with their respective electron donors is discussed.

Amino Acid Sequence↗
PubMed1988

A simple determination of the sideness of the NH2-terminus in the membrane bound cytochrome P-450 LM2.

A simple and fast procedure for determining the sideness of the NH2-terminus in membrane proteins using FITC as label has been developed and applied to microsomal cytochrome P-450 LM2. The main advantages of FITC, which was shown not to penetrate lipid membranes, as compared to other labels are that it is fluorescent and that it can be used for manual microsequencing of proteins. By use of FITC it was directly shown that the NH2-terminus of P-450 LM2 is localized at the cytoplasmic side of the endoplasmic reticulum membrane. Based on this result the validity of recently published models of the membrane topology and the membrane insertion of P-450 are discussed.

Animals↗
PubMed1988

Purification of the growth-related protein p25 of the Ehrlich ascites tumor and analysis of its isoforms.

1. Two of the three isoforms of the growth-related protein p25 of the Ehrlich ascites tumor have been purified to homogeneity by giant two-dimensional polyacrylamide gel electrophoresis. 2. Antibodies raised against the isoform p25/1 react also with isoforms p25/2 and p25/3. 3. Limited tryptic digestion of p25/1 and p25/2 resulted in similar oligopeptide patterns. Corresponding oligopeptides of both isoforms have identical amino acid sequences. 4. The isoforms p25/2 and p25/3 are phosphorylated derivatives of unphosphorylated p25/1. The phosphorus is bound to serine and a further unknown phosphorylation site.

Amino Acid Sequence↗
PubMed1988

Comparative studies on the accessibility and functional importance of tyrosine residues in cytochrome P-450 isozymes.

Cytochromes P-450 LM2 and P-450 LM4 from rabbit liver microsomes were chemically modified with tetranitromethane. Nitration of two tyrosine residues of both isozymes inhibits the benzphetamine N-demethylase activity of P-450 LM2 as well as the p-nitrophenetole O-deethylase activity of P-450 LM4 by about 80%. For identification of the modified tyrosine residues the inactivated enzymes were digested with trypsin, and the labeled peptides were separated by HPLC. Sequencing of the 3-nitrotyrosine-containing peptides from cytochrome P-450 LM2 showed that the tyrosine residues at positions 235 and 380 were nearly fully nitrated, while Tyr-348, Tyr-484 and Tyr-111 were only partially labeled (about 40-50%). In the presence of the heme-binding inhibitor metyrapone, Tyr-380 is partially protected against modification, and the extent of inactivation is diminished as well. These results suggest that Tyr-380 of cytochrome P-450 LM2 presents a catalytically essential amino acid residue at its active center. Sequence analyses of the 3-nitrotyrosine-containing peptides from cytochrome P-450 LM4 revealed that mainly Tyr-243 and Tyr-271 were labeled, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent (about 30-45%). Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are suggested to be functionally involved in the interaction with NADPH-cytochrome P-450-reductase.

Amino Acid Sequence↗
PubMed1987

Chemical modification of cytochrome P-450 LM4. Identification of functionally linked tyrosine residues.

Cytochrome P-450 LM4 (RH, reduced flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes was chemically modified with tetranitromethane. Nitration of two tyrosine residues inhibits the p-nitrophenetole O-deethylase activity of the enzyme by about 80%. Sequencing the 3-nitrotyrosine-containing peptides after HPLC tryptic peptide mapping reveals that mainly Tyr-243 and Tyr-271 are nitrated, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent. Nitration of tyrosine residues affects the complex formation with p-nitrophenetole, alpha-naphthoflavone and metyrapone as indicated by an increased affinity towards p-nitrophenetole and by a decreased affinity for the latter compounds. Furthermore, nitration interferes with the electron transfer from NADPH-cytochrome P-450-reductase to cytochrome P-450 LM4 resulting in a slowed down reduction reaction. The results suggest that Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are functionally involved in the interaction with NADPH-cytochrome P-450 reductase.

Amino Acid Sequence↗
PubMed1987

Identification of a polypeptide growth inhibitor from bovine mammary gland. Sequence homology to fatty acid- and retinoid-binding proteins.

A polypeptide growth inhibitor purified from bovine mammary gland (mammary-derived growth inhibitor) has been shown to reversibly inhibit proliferation of mammary carcinoma cells at concentrations of about 10(-10) M. The carrier of inhibitory activity has been identified biochemically as an about 13-kDa polypeptide and chemically by elucidating the amino acid sequence. No homology to any of the hitherto structurally investigated growth inhibitors (transforming growth factor beta, interferons) has been observed. The data revealed extensive sequence homology of mammary-derived growth inhibitor to a family of low molecular mass hydrophobic ligand-binding proteins, among them a fatty acid-binding protein from rat heart, myelin P2, a differentiation associated protein in adipocytes (p422) and the cellular retinoic acid-binding protein. Interaction with as yet unknown hydrophobic ligands might play a functional role in the mechanism of growth inhibition excerted by mammary-derived growth inhibitor.

Animals↗
PubMed1987

Accumulation of anthracotic particles along lymphatics of the human lung: relevance to "hot spot" formation after inhalation of poorly soluble radionuclides.

Large lung sections of humans of advanced adult age revealed a markedly nonuniform retention pattern of dense anthracotic particle aggregates, with an impressive accumulation of this material along pulmonary lymphatics, i.e. the deep (peribronchial), septal (perivenous) and superficial (pleural) networks. Conversely, the alveolar parenchyme contained only occasional, small aggregates of macrophages heavily loaded with carbon, representing little more than 2% of this material in lung tissue. Although translocation kinetics of anthracotic particles cannot readily be compared to those of highly toxic alpha-emitting, poorly soluble radionuclides such as 239PuO2, lymphatic drainage of the latter over the years may also be expected to lead to a concentration of radioactive material along lymph vessels. Since human data on the effects of inhaled 239PuO2 are virtually lacking, the above distribution pattern is apt to help in identifying cells and other tissue components most heavily at risk. Findings are also relevant to the problem of "hot spot" formation in vivo and its possible sequelae. The latter are briefly discussed with regard to both stochastic and non-stochastic effects.

Cell Aggregation↗
PubMed1987

Biochemical and immunological characterization of structural proteins from retrovirus-D/New England and comparison to Mason-Pfizer monkey virus and permanent human fibroblast virus.

Biochemical and immunological properties of retrovirus-D/New England (here referred as R-D/NE) recently isolated at the New England Regional Primate Research Center, Southborough, Ma, from a rhesus monkey with acquired immune deficiency syndrome were investigated and compared to the prototype type D retroviruses Mason-Pfizer monkey virus (MPMV) and permanent human fibroblast virus (PMFV) isolated from a breast carcinoma of a rhesus monkey and a continuous human cell line, respectively. The polypeptide composition of R-D/NE propagated in a human lymphoid B cell line (Raji cells) has been investigated using SDS-polyacrylamide gel electrophoresis. Staining with Coomassie blue and labelling with 14C amino acids revealed seven viral polypeptides with molecular weights of 4,000, 10,000, 12,000, 15,000, 18,000, 27,000, and 80,000 Da which were also shared by MPMV and PMFV. The 80,000 Da protein was shown to be a glycoprotein by incorporation of 3H glucosamine. The 18,000 Da protein was identified as a phosphoprotein of R-D/NE. p18 structural proteins of MPMV and PMFV represent phosphoproteins of their respective viruses as well. All three phosphorylated proteins contain O-phosphoserine as major phosphoamino acid. The comparison of tryptic peptide maps of the major internal structural proteins of R-D/NE, MPMV, and PMFV revealed a striking similarity among p 10/p 12 and p 15. proteins. A minor difference was detected among the tryptic peptide digests of p 4 and p 27 proteins. Antiserum against p 15 of MPMV showed a significantly weaker binding to R-D/NE than to MPMV and PMFV at high dilutions.

Amino Acid Sequence↗
PubMed1987

Satisfactory primary tetanus antitoxin responses but markedly reduced germinal centre formation in first draining lymph nodes of ageing mice.

The present report deals with primary antibody responses to tetanus toxoid in 50-54-week-old ('ageing') as compared to 8-9-week-old ('young adult') mice. Antitoxin in the serum appeared 6 days earlier in the older than in the young animals, but in the latter reached 5 times higher titres on day 20. The magnitude of the proliferative response in the paracortex and the medulla of popliteal lymph nodes, as estimated by combined 3H-thymidine autoradiography and planimetry, was 3-7 times greater in the younger than in the older age group, thus approximately reflecting the difference in antibody titres on day 20. In contrast, germinal centre formation in response to the stimulus proved to be about 14 times less in ageing than in young adult mice. The findings demonstrate that, in the model system used, the age-related slopes of decline in humoral antibody responsiveness and proliferative reactivity in paracortex and medulla of first regional lymph nodes tend to be in parallel, while the ability of the immune apparatus to form germinal centres at this site deteriorates at a considerably faster pace. Results are also in line with the notion that centroblasts/centrocytes contribute little, if anything, to the ongoing antibody production elicited by the same stimulus which had triggered germinal centre formation. Finally, the observations made disprove the general validity of the suggestion that immune reactivity is maintained on the same level throughout life if tested with a novel antigen.

Aging↗
PubMed1986

[Results after radical prostatectomy for prostate carcinoma].

35 of 54 patients undergoing radical prostatectomy for localized cancer were asymptomatic and the tumor had been found during a routine check-up by the family physician. In 53 of 54 patients the indication for surgery was based on a positive cytological finding in fine-needle aspiration biopsy of the suspected tumor. In all patients the carcinoma was confirmed by histological work-up of the radical prostatectomy specimen. Cytological and histological grading was identical in three quarters of all patients. However, the preoperative tumor stage (T) was confirmed histologically (pT) only in half of all the patients. The extension of the carcinoma was often underestimated at the preoperative clinical staging. After a mean observation time of close to 4 years, the survival rate in patients undergoing radical prostatectomy for localized cancer is comparable to an age and sex matched control group. Our data suggest that fine-needle aspiration biopsy of the prostate is a very reliable procedure in the diagnosis of prostatic carcinoma, provided the aspiration technique is correct and the smears are interpreted by a well-trained cytologist. Moreover, patients with localized prostate cancer seem to have an excellent chance of survival with low morbidity, provided the indication for surgery is restricted to carefully selected patients.

Aged↗
PubMed1986

Health care provider and family acceptance of a rural community-based nursing service for chronically ill children.

Health care providers' and families' overall acceptance of a community-based nursing program was assessed in relationship to their perceptions of the program nurses' accessibility, professional competence, and personal qualities. Perceptions were measured by questionnaires completed by 139 health care providers and 140 families. Overall, providers indicated that the program has been of benefit to both the children it serves and the medical community. The majority of families felt that the nurses were helpful to them despite a few complaints related to poor availability.

Adult↗
PubMed1986

Structural components of bovine leukemia virus: further biochemical and immunological characterization of major structural proteins and glycoproteins.

Proteins from bovine leukemia virus (BLV) were investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the presence and absence of 14C amino acids. Virus grown either in fetal lamb kidney (FLK) or bat lung (BL) cells revealed seven major proteins designated p10, p12, p15(1), p15(2), p24, gp30, and gp64. By tryptic peptide analysis, N-terminal amino acid analysis and radioimmunoassay it could be shown that p10, a basic protein with hydrophobic properties, is a cleavage product of p15(2), the acidic and slightly hydrophobic phosphoprotein of BLV. The structural protein p12 was shown to be a phosphorylated protein identifying it as a second major viral protein to contain phosphorous. Investigation of [3H]glucosamine incorporation, N-terminal amino acid analysis and hydrophobic properties by chloroform-methanol extraction confirmed properties of gp30 and gp64 predicted by nucleotide sequence data. The two p15 proteins have been characterized in more detail.

Amino Acid Sequence↗
PubMed1986

Morphometric distinction of granulomas in tuberculosis and sarcoidosis. Difference in nuclear profiles.

Morphometric measurements were carried out on epithelioid-cell nuclei of noncaseating granulomas in paraffin-embedded sections of lymph node biopsy specimens originating from 15 patients with sarcoidosis and from 18 patients with tuberculosis. The results, which were obtained with the help of a computer-assisted tracing device, established highly significant differences in shape and size of epithelioid-cell nuclear profiles in these two diseases. Direct measurements and computations on these results showed that epithelioid-cell nuclear profiles in sarcoidosis have a smaller mean and median perimeter (boundary length), area and long diameter (maximum length of the nuclear profile) and exhibit a more plump, elliptical-like shape than do those in tuberculous granulomas. Epithelioid-cell nuclear profiles of the latter, as a whole, show more irregular contours, resembling those, e.g., of the sole of a shoe or other elongated patterns. Differences in shape of nuclear profiles were best demonstrated by a size-independent form factor (4 pi *area/perimeter2).

Adolescent↗