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R Kraft

Publications and source records attributed to R Kraft.

At least 73 records · Page 4Linked to original sources
PubMed1996

A third interferon-gamma-induced subunit exchange in the 20S proteasome.

The 20S proteasome is a protease complex of functional importance for antigen processing. Two of the 14 proteasome subunits, delta and MB1, can be replaced by the major histocompatibility complex (MHC)-encoded and interferon-gamma (IFN-gamma)-inducible subunits LMP2 and LMP7, respectively. LMP2 and LMP7 alter the cleavage site specificity of the 20S proteasome and are required for the efficient generation of T cell epitopes from a number of viral proteins and for optimal MHC class I cell surface expression. We compared the 20S proteasome subunit pattern from IFN-gamma-induced and non-induced mouse fibroblasts on two-dimensional gels and identified a third subunit exchange by microsequencing: the non-MHC-encoded subunit MECL-1 is induced by IFN-gamma and replaces a sofar barely characterized beta subunit designated 'MC14'. In analogy to LMP2 and LMP7, MECL-1 may be functional in MHC class I-restricted antigen presentation.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of the hydrogenase from Thiobacillus ferrooxidans.

Hydrogenase of Thiobacillus ferrooxidans ATCC 19859 was purified from cells grown lithoautotrophically with 80% hydrogen, 8.6% carbon dioxide, and 11.4% air. Hydrogenase was located in the 140,000 x g supernatant in cell-free extracts. The enzyme was purified 7.3-fold after chromatography on Procion Red and Q-Sepharose with a yield of 19%, resulting in a 85% pure preparation with a specific activity of 6.0 U (mg protein)-1. With native PAGE, a mol. mass of 100 and 200 kDa was determined. With SDS-PAGE, two subunits of 64 (HoxG) and of 34 kDa (HoxK) were observed. Hydrogenase reacted with methylene blue and other artificial electron acceptors, but not with NAD. The optimum of enzyme activity was at pH 9 and at 49 degrees C. Hydrogenase contained 0.72 mol nickel and 6.02 mol iron per mol enzyme. The relationship of the T. ferrooxidans hydrogenase to other proteins was examined. A 9.5-kb EcoRI fragment of T. ferrooxidans ATCC 19859 hybridized with a 2.2-kb XhoI fragment from Alcaligenes eutrophus encoding the membrane-bound hydrogenase. Antibodies against this enzyme did not react with the T. ferrooxidans hydrogenase in Western blot analysis. The N-terminal amino acid sequence (40 amino acids) of HoxK was 46% identical to that of the hydrogen sensor HupU of Bradyrhizobium japonicum and 39% identical to that of the HupS subunit of the Desulfovibrio baculatus hydrogenase. The N-terminal sequence of 20 amino acids of HoxG of T. ferrooxidans was 83.3% identical to that of the 60-kDa subunit. HupL, of the hydrogenase of Anabaena sp. Sequences of ten internal peptides of HoxG were 50-100% identical to the respective sequences of HupL of the Anabaena sp. hydrogenase.

Amino Acid Sequence↗
PubMed1996

Intravesical versus intravesical plus intradermal bacillus Calmette-Guerin: a prospective randomized study in patients with recurrent superficial bladder tumors.

PURPOSE: Intravesical instillation of bacillus Calmette-Guerin (BCG) induces various immunological reactions and decreases the recurrence rate of superficial bladder tumors. To determine whether additional immune stimulation with concomitant intradermal BCG applications could further lower the recurrence rate, 154 patients with superficial bladder tumors at high risk for recurrence were randomized to receive either 6 intravesical instillations of 120 mg. Pasteur strain BCG alone or combined with intradermal application. MATERIALS AND METHODS: A total of 76 patients received intravesical and intradermal BCG, while 78 received intravesical BCG only. Median followup was 41 months (range 2 to 89) and 36 months (range 2 to 86), respectively. Both treatment groups were comparable regarding patient age and number of previous transurethral bladder tumor resections, as well as tumor recurrence rate, stage and grade before BCG therapy. RESULTS: A highly significant decrease in the monthly tumor recurrence rate was observed in both arms after BCG compared to the pretreatment recurrence rates (p < 0.0001). Recurrence rate decreased from 0.73 +/- 1.07 (standard deviation) to 0.06 +/- 0.13 in the combined treatment group and from 0.71 +/- 0.90 to 0.074 +/- 0.17 in the intravesical treatment only group. However, we were unable to find any difference between the 2 groups regarding interval to initial recurrence or recurrence rates after BCG treatment. Changes in the purified protein derivative skin test performed before and after BCG therapy were not useful to predict response to treatment because 44% of our patients already had a positive test before treatment. Also, interpretation of the skin test was difficult and not always reliable. In the multivariate analysis, however, fever was an important prognostic factor. Patients with increased body temperature greater than 37.5C had a significantly lower recurrence rate than those without fever (37.5C or less) after BCG instillation (p = 0.009). Moreover, fever after BCG instillation was observed significantly more frequently in patients with a positive purified protein derivative skin test before treatment (p = 0.021). CONCLUSIONS: The therapeutic benefit from intravesical BCG apparently was not substantially improved by simultaneous intradermal BCG vaccination. Fever following intravesical BCG instillation is an important prognostic factor regarding superficial bladder tumor recurrence. Fever occurs predominantly in patients who were previously sensitized to mycobacteria (by BCG vaccination or infection) as shown by a positive pretreatment purified protein derivative skin test. This finding suggests that previously sensitized patients respond significantly better to a single course of intravesical BCG.

Adjuvants, Immunologic↗
PubMed1996

Abortive mitoses and nuclear DNA fragmentation in CD30+ large cells of Hodgkin's disease.

This study was undertaken to better comprehend the reasons for the scarcity of Hodgkin and Reed-Sternberg (H-RS) cells in Hodgkin's disease (HD) despite their expression of "proliferation-associated antigens". To this end, we assessed the relative frequency of mitotic phases and nuclear damage (detected by in situ end-labeling of DNA strand breaks) in CD30+ large cells of nodular sclerosis and mixed cellularity HD. Our results show that a) most CD30+ cells in HD exhibit abortive mitoses, with a highly significant arrest at the metaphase-ana/telophase transition, and b) many of these elements, i.e. mainly H-RS cells, show fragmentation of nuclear DNA, suggesting imminent or actual death. Percentages of CD30+ cells that entered mitosis and those with DNA strand breaks were of a similar order of magnitude and correlated significantly in a linear fashion. These findings are consistent with the concept that cell deletion is the major cause of the paucity of H-RS cells in HD.

Adolescent↗
PubMed1996

Extensive extramedullary hematopoiesis in a thyroid nodule.

We describe a case of extramedullary hematopoiesis in a hyperplastic thyroid nodule from a patient with primary myelofibrosis. The presence of extensive extramedullary hematopoiesis in the thyroid gland was suspected as a result of fine-needle aspiration cytologic examination and was confirmed by histo- and immunomorphologic examination of the resected nodule. The diagnosis of myelofibrosis was established on the basis of clinical findings, hematologic laboratory results, and bone marrow morphologic characteristics.

Aged↗
PubMed1995

Presence of the bcl-2 protein and apoptosis in non-Hodgkin lymphomas with diffuse growth pattern.

In an attempt to further clarify the role of the apoptosis-counteracting protein bcl-2, non-Hodgkin lymphomas (NHL, n = 170) were examined immunohistochemically, across the boundaries of histologic classification, for the presence of this oncoprotein, in comparison with apoptotic indices (AI) and percentages of Ki-67+ cells (growth fraction). The results of this retrospective study revealed a highly significant inverse relationship ("mirror image") between the proportion of bcl-2+ cells and the AI per case. Both these parameters, although variable, clearly distinguished low- from high-grade-malignancy lymphomas according to the Kiel classification. Cluster analysis detected 2 separate groups of high-grade NHL, one with rather high AI and low percentages of bcl-2+ cells, comprising most anaplastic large-cell lymphomas, the other group with reverse characteristics. We found no correlation between the percentage of bcl-2+ cells per case and overall survival.

Apoptosis↗
PubMed1995

Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope.

BACKGROUND: Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60kD importin protein (importin 60) as a single polypeptide. RESULTS: We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)-nitrolotriacetic acid-Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin-agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors. CONCLUSIONS: Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.

Amino Acid Sequence↗
PubMed1995

Incorporation of major histocompatibility complex--encoded subunits LMP2 and LMP7 changes the quality of the 20S proteasome polypeptide processing products independent of interferon-gamma.

The 20S proteasome is the enzyme complex responsible for the processing of antigens bound by major histocompatibility complex class I molecules. The role of the interferon-gamma (IFN-gamma)-inducible proteasome subunits LMP2 and LMP7 in this process is, however, still controversial. We have studied the effects of IFN-gamma-independent LMP incorporation on the quality of peptides processed from the murine cytomegalovirus IE pp89 25-mer polypeptide substrate through dual cleavages by 20S proteasomes. The incorporation of a single LMP subunit or both LMP2 and LMP7 induces changes in 20S proteasome subunit stoichiometry, alters its cleavage site preference and in consequence, the quality of the generated peptides. When the several hydrolytic activities are tested with short fluorogenic peptide substrates, the Vmax, S0.5 (Km), or both values of 20S proteasomes are altered, depending on the combination of LMP. There exists, however, no obvious correlation between the observed changes in hydrolytic activities against short fluorogenic peptides and the changes in dual cleavage site usage within the 25-mer polypeptide substrate. As judged from the calculated Hill coefficients, the presence of both LMP subunits induces a drastic increase in positive cooperativity between the proteasome subunits.

Amino Acid Sequence↗
PubMed1995

Low versus high cell turnover in diffusely growing non-Hodgkin's lymphomas.

Cell loss, perhaps as important as cell production in determining the size of an expanding cell population, has not usually been registered in quantitative cellular kinetic analyses of neoplastic disorders. The present retrospective study on various types and subtypes of non-Hodgkin's lymphomas (NHLs; n = 170) was designed to test the usefulness of a novel additional parameter, the 'turnover index' (TI), which is the sum per case of the mitotic index and the apoptotic index. Results document that TIs clearly distinguished between categories and subtypes of NHLs according to the Kiel classification. Cluster analysis of TIs plotted against the percentage of Ki-67-positive cells per case revealed that about one-third of the high-grade malignancy lymphomas actually belonged to the low-turnover lymphomas. Overall survival was longer in the low- than in the high-turnover group of lymphomas. Assessment of TIs can, for practical diagnostic purposes, be replaced by counting mitotic figures and apoptotic cells in several high-power fields. The TI concept may help to interpret the kinetics of NHLs in terms of accumulation vs. proliferation of cells.

Adolescent↗
PubMed1995

Analysis of early failures after intravesical instillation therapy with bacille Calmette-Guérin for carcinoma in situ of the bladder.

OBJECTIVE: To analyse the clinical and therapeutic consequences of early treatment failure after bacille Calmette-Guérin (BCG) instillation therapy for carcinoma in situ of the bladder. PATIENTS AND METHODS: A total of 115 patients with carcinoma in situ (Tis) of the bladder were treated by intravesical instillation of living BCG vaccine (Immun BCG Pasteur F). Twenty five patients had primary Tis and 90 had secondary Tis with synchronous or prior superficial papillary tumours. All papillary tumours were resected before instillation of BCG. All patients completed one series of 6 weekly instillations of 120 mg BCG. RESULTS: Twenty-two of 25 patients (88%) with primary Tis responded completely, with negative cytology and cystoscopy findings within a median follow-up period of 44 months. Three of the 25 (12%) had cytological evidence of disease within 9 months of therapy and were considered to be early treatment failures. One patient had muscle-invasive bladder cancer, one had Tis and invasive cancer of the prostatic urethra, and the last, in whom a second BCG course also failed, had Tis of both ureters. Seventy of 90 patients (78%) with secondary Tis had a complete response after treatment with BCG, with repeated negative cytology and cystoscopy examinations within a median follow-up time of 40 months. Twenty of the 90 (22%) with secondary Tis had positive cytology within 9 months after BCG therapy and were considered early treatment failures. Five of these 20 had a cystectomy, three for persistent Tis of the bladder and two for a solid urothelial carcinoma of the prostate. The remaining 15 early failures received a second course of BCG. Four of these 15 patients responded and the remaining 11 failed the second course. The 11 failures included two patients with multifocal T1 G3 bladder cancers. four with invasive bladder cancer, two with solid urothelial carcinomas of the prostatic urethra, and three with Tis of the upper urinary tract. CONCLUSIONS: According to these data, early treatment failure after 6 weekly instillations of 120 mg Immun BCG Pasteur F is an alarming signal which requires immediate re-assessment of the patient to exclude a muscle-invasive bladder cancer or an extravesical carcinoma in situ, either in the upper urinary tract or in the prostatic urethra.

Administration, Intravesical↗
PubMed1995

Adjuvant treatment with a vitamin A analogue (etretinate) after transurethral resection of superficial bladder tumors. Final analysis of a prospective, randomized multicenter trial in Switzerland.

Vitamin A and its derivatives, the retinoids, have antiproliferative effects and may induce cellular differentiation. Etretinate, a synthetic retinoid, has a more favorable therapeutic index experimentally than all-trans-retinoic acid or 13-cis-retinoic acid. Ninety patients with superficial papillary bladder tumors stages Ta and T1 entered a prospective randomized double-blind multicenter trial in Switzerland. Seventy-nine of the patients were eligible and received either 25 mg of etretinate or a placebo orally each day. The early withdrawal of a significantly greater number of patients in the placebo group for treatment failure during the first year of the study resulted in a secondary positive selection in this group. High-risk patients were removed and low-risk patients remained. In those patients who had tumor recurrences after randomization, the time to first recurrence was similar in both groups with 13.5 and 13.6 months in the placebo and etretinate groups, respectively. However, the mean interval to subsequent tumor recurrence was significantly longer in the etretinate group. The mean interval between recurrences in these subgroups was 12.7 months in the placebo arm and 20.3 months in the etretinate arm (p = 0.006). Consequently, the number of transurethral resections per patient-year was also reduced significantly in the etretinate group (p < 0.001). In patients with more than one transurethral resection of papillary tumors before randomization, the annual transurethral resection rate in the two treatment groups dropped from 1.7 to 1.3 in the 30 patients in the placebo group (NS, p = 0.1) and from 2.1 to 0.95 in the 25 patients in the etretinate group (p < 0.001). The side effects of etretinate (cheilitis, dryness of mucous membranes and skin) were acceptable to most patients. The relationship of the 3 myocardial infarcts observed in the etretinate group to the retinoid is not clear. Despite their significant effect on the recurrence rate of superficial papillary bladder tumors, retinoids should only be used in well-controlled prospective trials until more is known about their dosage-toxicity profiles.

Adult↗
PubMed1994

Spatial distribution of mitosis, apoptosis and small blood vessels in malignant diffuse follicular-center-cell lymphomas: a nearest-neighbor analysis.

To better comprehend the relationships between cell birth and cell death in neoplastic disorders, the topography of these events needs to be considered. We applied a computerized nearest-neighbor analysis to malignant, diffuse follicular-center-cell lymphomas in order to examine the spatial distribution of pyknotic (apoptotic) cells/bodies (A) and mitotic figures (M) in relation to capillaries (C) and venules (V). The results revealed a complex dispersion pattern, with significant aggregations of A and M, in addition to an even greater random distribution component. The greatest clustering displayed by A was around capillaries (A-C, 42% reduction of object frequency between the first and second distance class), followed by A-A (33%), A-V (24%) and A-M (12%). Additional values for mitotic figures were: M-M (45%), M-V (33%) and M-C (22%). These findings may reflect the relative importance of inherent properties of the neoplastic and host factors, respectively, in the regulation of cell birth and cell death rates.

Adult↗
PubMed1994

C-terminal region of adrenodoxin affects its structural integrity and determines differences in its electron transfer function to cytochrome P-450.

The role of the C-terminal region of adrenodoxin was studied by analyzing deletion mutants 4-114 and 4-108 lacking amino acids 1-3 and 115-128 or 109-128, respectively. Absorption spectra of these mutants were found to be identical to that of wild type adrenodoxin. However, EPR and CD studies indicated that the structure of deletion mutants 4-114 and 4-108 differs from that of wild type adrenodoxin. Mutant 4-107, which in addition to residues 109-128 lacks the unique proline 108, showed no EPR spectrum. This indicates that proline 108 plays an essential role for the formation of the iron-sulfur cluster. Deletion of residues 115-128 or 109-128 did not essentially affect adrenodoxin reductase binding as shown by nearly unchanged cytochrome c reduction activity. In a CYP11A1 assay, mutants 4-108 and 4-114 exhibited 3.2- and 5-fold decreased Km values, respectively, whilst the Kd values for CYP11A1 decreased 3- and 1.9-fold, respectively. Additionally, in a CYP11B1 assay, mutants 4-108 and 4-114 showed decreased Km values. Furthermore, the first step of electron transfer to CYP11B1, but not to CYP11A1, was accelerated up to 4.5-fold by the adrenodoxin mutants. The results suggest that the C-terminal peptide of adrenodoxin, especially proline 108, affects the structural integrity of the iron-sulfur cluster and that electron donation from adrenodoxin to CYP11A1 and CYP11B1 is determined at least in part by different features of the cytochromes.

Adrenal Glands↗
PubMed1994

Drosophila mode of metamerization in the embryogenesis of the lepidopteran insect Manduca sexta.

Insect embryos have been classified as intermediate- and short-germ embryos, in which posterior segments are thought to be generated sequentially from an uncommitted growth zone, or as long-term embryos, such as Drosophila melanogaster, which develop primordia for all segments simultaneously. In Drosophila the coordinated activities among a three-tiered cascade of zygotic segmentation genes subdivide the embryo into progressively smaller units along the anterior-posterior axis. The mode of pattern specification in lepidopteran embryos has not been determined, although on morphological grounds they have been characterized as intermediate-germ insects. We have cloned orthologues of Drosophila segmentation genes from the tobacco hawkmoth Manduca sexta and have found that the blastoderm expression patterns of these genes show a molecular prepatterning typical of Drosophila. Thus, successive segment formation in Manduca embryos may not be due to sequential addition but rather may be the consequence of a lateral compression of the embryo proceeding in an anterior-to-posterior progression. These data challenge the view that the classification of insect development according to morphological criteria can serve as a reliable indicator of the molecular mechanisms underlying segmentation.

Amino Acid Sequence↗
PubMed1994

Generation of the soluble and functional cytosolic domain of microsomal cytochrome P450 52A3.

The cytosolic domain of microsomal P450 52A3 (P450Cm1) was isolated as a soluble and functionally active protein. The NH2-terminal region that anchors the P450 protein to the endoplasmic reticulum was removed by sequence-specific proteolysis at a designed cleavage site. For that purpose, P450Cm1 was genetically engineered to establish at position 63-66 the sequence Ile-Glu-Gly-Arg, which is recognized by the restriction protease factor Xa. The modified P450 was produced in high yields as an integral membrane protein in Saccharomyces cerevisiae. In the microsomal fraction, it was accessible to factor Xa digestion, releasing a readily soluble, shortened P450 protein. For large scale preparation of the cytosolic domain, the modified P450Cm1 was first purified and then subjected to sequence-specific proteolysis. The highly purified delta(1-66)P450Cm1 exhibited unchanged spectral characteristics and catalyzed the hydroxylation of n-hexadecane with 85% of the activity determined for full-length wild-type P450Cm1. The method developed for the preparation of the cytosolic domain of P450Cm1 may be more generally applicable to facilitate structure-function studies on membrane-bound P450 forms.

Amino Acid Sequence↗