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Biomedical subjects

G Kramer

Publications and source records attributed to G Kramer.

At least 73 records · Page 4Linked to original sources
PubMed1999

N-terminal and C-terminal modifications affect folding, release from the ribosomes and stability of in vitro synthesized proteins.

Important aspects of translation are release and folding of the synthesized protein into its three-dimensional structure. Studies from our group indicated that during in vitro protein synthesis a large portion of full-length polypeptides apparently accumulated as peptidyl-tRNA on ribosomes. We have also shown that some proteins though released in biologically active form may be inactivated without being degraded. These experiments were carried out by coupled transcription/translation using an Escherichia coli extract in which eukaryotic or prokaryotic test proteins were synthesized from their coding sequence inserted into specific plasmids. Experiments described here were designed to analyze the effects of N-terminal and C-terminal modifications of the coding sequence on the ribosomal release/termination process and on the stability of the newly synthesized protein. Elimination of the leader sequence in two proteins tested, mitichondrial rhodanese and bacterial beta-lactamase, caused an increase in the percentage of polypeptides released from the ribosomes relative to total synthesis. Conversely, an N-terminal extension such as a histidine-lag impaired the ribosomal release process. Also, a hydrophobic N-terminal modification of the synthesized protein reduced release of newly formed protein from the ribosomes. A C-terminal extension of the coding sequence for rhodanese by one amino acid decreased the percentage released polypeptide and furthermore affected the stability of the in vitro formed protein. We propose that a regulatory mechanism exists by which N-terminal and C-terminal sequences of a newly synthesized protein have feed-back effects on the termination factor-mediated release and on the stability of the native three-dimensional structure.

Cell-Free System↗
PubMed1999

Evaluation of the state-of-the-art of butyl- and phenyltin compound determinations in freshwater sediment prior to certification of a reference material.

In order to control the quality of butyl- and phenyltin compound determinations in sediment, the Standards, Measurements and Testing Programme (formerly BCR) of the European Commission has started a project, the final aim of which is to certify a freshwater sediment for its contents of a range of organotin compounds (mono-, di- and tributyltin, and mono-, di- and triphenyltin). The first part of this project involved an interlaboratory study which aimed to test the feasibility of preparation of candidate freshwater sediment reference material and to detect and remove most of the pitfalls observed in organotin determinations. This paper presents the main results of this interlaboratory study carried out prior to the certification campaign. The agreement obtained among laboratories for the six compounds determined was considered to reflect the state-of-the-art and was encouraging enough to decide upon the organisation of a certification campaign, which will be concluded in June 1999.

Environmental Monitoring↗
PubMed1998

Laterally Resolved Measurement of Interaction Forces between Surfaces That Are Partly Covered with Polyelectrolytes.

The adsorption of polycation poly(diallyldimethylammonium chloride), PDADMAC for short, on oxidized silicon wafers is investigated by direct force measurements using an atomic force microscope. It is shown that the electrostatic forces as well as the adhesive properties are significantly influenced by adsorbed PDADMAC. By using force mapping and monitoring the increased adhesion between the surfaces the adsorbed polymer can be two-dimensionally monitored on the surface. Different coverages of the polycation were investigated, which qualitatively agree well with results of XPS and electrokinetic measurements. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Regulation of heat shock protein 27 expression of prostatic cells in response to heat treatment.

BACKGROUND: The clinical value of heat-induced coagulation of prostatic tissue is evaluated as a minimally invasive treatment for patients with benign prostatic hyperplasia (BPH) and, more recently, localized prostate cancer (PC). To obtain a more detailed insight on the effect of heat on prostatic cells, heat shock protein (HSP) 27 expression of normal and malignant prostatic cells was studied. METHODS: In vitro, HSP27 expression of prostatic stromal cells and the human prostate cancer cell line LNCaP was studied by Western blotting when cultured at 37 degrees C. Subsequently, the effect of a sublethal heat shock from 43-49 degrees C for 60 min on HSP27 expression of LNCaP was determined. In vivo, HSP27 expression pattern of nine human prostates, which were treated in vivo by thermoablation with transrectal high-intensity focused ultrasound (HIFU) 3 hr-8 days prior to surgical removal, was analyzed by immunohistochemistry. Untreated BPH (n = 10) and PC (n = 7) specimens served as controls. RESULTS: Under physiologic conditions (37 degrees C), LNCaP and prostatic stromal cells expressed a 27-kD and 56-kD anti-HSP27 reactive molecule. Following sublethal cell heating, HSP27 (27 kD) expression of LNCaP increased by 3-4-fold in a temperature-dependent manner. In untreated BPH specimens (n = 10), muscle cells stained HSP27-positive in all samples, while epithelial cells (EC) were negative in 6 out of 10 specimens. At the border of the high-intensity focused ultrasound (HIFU) necrosis, increased HSP27 expression was consistently demonstrable (n = 9). HSP27 upregulation was strongest 2-3 hr after HIFU but still demonstrable after 5-8 days. In this border zone, basal and secretory EC as well as muscle cells stained strongly for HSP27. CONCLUSIONS: Benign and malignant human prostatic cells respond to heat by increased expression of HSP27 in vitro and in vivo. Transrectal HIFU therapy induces intraprostatic thermonecrosis surrounded by a zone characterized by a massive upregulation of HSP27 expression.

Cells, Cultured↗
PubMed1998

Truncations at the NH2 terminus of rhodanese destabilize the enzyme and decrease its heterologous expression.

Rhodanese mutants containing sequential NH2-terminal deletions were constructed to test the distinct contributions of this region of the protein to expression, folding, and stability. The results indicate that the first 11 residues are nonessential for folding to the active conformation, but they are necessary for attaining an active, stable structure when expressed in Escherichia coli. Rhodanese species with up to 9 residues deleted were expressed and purified. Kinetic parameters for the mutants were similar to those of the full-length enzyme. Compared with shorter truncations, mutants missing 7 or 9 residues were (a) increasingly inactivated by urea denaturation, (b) more susceptible to inactivation by dithiothreitol, (c) less able to be reactivated, and (d) less rapidly inactivated by incubation at 37 degreesC. Immunoprecipitation showed that mutants lacking 10-23 NH2-terminal amino acids were expressed as inactive species of the expected size but were rapidly eliminated. Cell-free transcription/translation at 37 degreesC showed mutants deleted through residue 9 were enzymatically active, but they were inactive when deleted further, just as in vivo. However, at 30 degreesC in vitro, both Delta1-10 and Delta1-11 showed considerable activity. Truncations in the NH2 terminus affect the chemical stability of the distantly located active site. Residues Ser-11 through Gly-22, which form the NH2-proximal alpha-helix, contribute to folding to an active conformation, to resisting degradation during heterologous expression, and to chemical stability in vitro.

Amino Acid Sequence↗
PubMed1998

Expression of the aryl hydrocarbon receptor (AhR) and the aryl hydrocarbon receptor nuclear translocator (ARNT) in fetal, benign hyperplastic, and malignant prostate.

BACKGROUND: Androgen-dependent tissue has been reported to be affected by chemical ligands of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, which heterodimerizes with the aryl hydrocarbon receptor nuclear translocator protein (ARNT). METHODS: Fetal (n = 3), benign hyperplastic (BPH) (n = 10), and carcinomatous (CaP) (n = 19) prostate tissues were analyzed using immunohistochemistry. Western blot analysis was used to confirm the identity of the recognized proteins. RESULTS: Immunoblotting of enriched prostatic epithelial cells (EC) and stromal cells revealed constitutive expression of bands at around 110 kDa and 90 kDa, using anti-AhR and anti-ARNT, respectively. Immunohistology of the fetal specimens revealed heterogeneous cytoplasmic and nuclear AhR expression of immature EC and mesenchymal cells. Constitutive expression of AhR (primarily cytoplasmic) and ARNT (nuclear and cytoplasmic) by the majority of adult basal and secretory EC, CaP, and smooth muscle cells was confirmed in situ. The most intense anti-AhR/-ARNT reactivity was found on smooth muscle cells, followed by EC and fibrocytes. Secretory BPH-EC revealed significantly decreased AhR expression when compared to normal tissue segments. By contrast, anti-AhR reactivity was frequently increased in the more dedifferentiated tumor areas. CONCLUSIONS: These findings suggest that an undefined physiologic AhR ligand(s) as well as environmental factors may exert effects on EC and smooth muscle cells in the prostate through binding to these receptors.

Adult↗
PubMed1998

Different conformations of nascent peptides on ribosomes.

The length at which the N terminus of nascent proteins becomes available to antibodies during their synthesis on ribosomes was determined. Three different proteins, bovine rhodanese, bacterial chloramphenicol acetyltransferase and MS2 coat protein, were synthesized with coumarin at their N terminus in a cell-free system derived from Escherichia coli. A derivative of coumarin was cotranslationally incorporated as N-coumarin-methionine at the N terminus of polypeptides. The interaction of specific anti-coumarin antibodies with this N-terminal coumarin of ribosome-bound nascent peptides was examined. The results indicate that short nascent peptides of each of the three proteins are unreactive, that the length at which they become accessible to the antibodies is different for the three proteins, and that longer peptides differ in their reactivity. It is suggested that these differences are due to differences in the conformation acquired by the peptides as they are synthesized on the ribosomes.

Capsid↗
PubMed1998

A cross-sectional study of vitamin intake in postoperative non-small cell lung cancer patients.

BACKGROUND AND OBJECTIVES: This cross-sectional study of postoperative non-small cell lung cancer (NSCLC) patients examined possible effects of vitamin intake and folate status on disease-free survival. METHODS: Supplemental vitamin usage, dietary vitamin intake (Willett Food Frequency Questionnaire), red blood cell (RBC) folate, and serum folate concentrations were assessed in patients with a history of NSCLC. Exclusion criteria included factors that alter folate status or that are associated with altered nutritional habits: (1) evidence of cancer on history, physical, or chest radiograph; (2) tobacco, alcohol ingestion (>2 drinks/ day), or cancer treatment within 3 months; (3) use of folate antagonists; and (4) age <60 years. RESULTS: 36 subjects were evaluated. The median disease-free censored survival was 24 months (range 4-41). Nineteen of 36 patients (53%) reported vitamin supplementation. Vitamin users had a longer median censored survival compared with nonusers (41 months versus 11 months; P = 0.002). With adjustment for cancer stage, the association between RBC folate and censored survival (r = 0.35; P = 0.055) and between serum folate and censored survival (r = 0.32; P = 0.083) approached statistical significance. CONCLUSIONS: NSCLC patients who took vitamin supplements were more likely to be long-term survivors in the patients studied; a similar trend toward long-term survival was seen among patients with higher circulating folate concentrations.

Aged↗
PubMed1998

Reactivation of thermally inactivated pre-beta-lactamase by DnaK, DnaJ, and GrpE.

To understand the role of the 23-amino acid signal sequence in the folding and stability of beta-lactamase, the precursor and a mutant beta-lactamase with a 19-amino acid signal sequence deletion were synthesized in vitro using an Escherichia coli cell-free coupled transcription/translation system. Approximately 30% of the newly synthesized full-length precursor and 60% of the deletion mutant polypeptides were terminated and released from the ribosomes as active enzyme. Activity of the pre-beta-lactamase, but not the mutant, was unstable at 37 degrees C, suggesting that the signal sequence causes the enzyme to unfold. This inactivation was independent of ATP. Pre-beta-lactamase activity was stabilized by lowering the temperature to 30 degrees C. Furthermore, addition of the molecular chaperones DnaK/J and GrpE, in the presence of ATP and Mg2+, restored the activity of the temperature-inactivated precursor. The precursor formed a stable complex with DnaK and GrpE. Both ATP and DnaJ were required for recovery of enzymatic activity, indicating that DnaJ may bind transiently to the complex. These results suggest that the signal sequence of the pre-beta-lactamase causes a temperature-dependent unfolding of the synthesized enzyme and that DnaK/J and GrpE interact with unfolded pre-beta-lactamase to promote refolding of the protein into its native, enzymatically active conformation.

Adenosine Triphosphate↗
PubMed1997

Renaturation of rhodanese by translational elongation factor (EF) Tu. Protein refolding by EF-Tu flexing.

The translation elongation factor (EF) Tu has chaperone-like capacity to promote renaturation of denatured rhodanese. This renaturation activity is greatly increased under conditions in which the factor can oscillate between the open and closed conformations that are induced by GDP and GTP, respectively. Oscillation occurs during GTP hydrolysis and subsequent replacement of GDP by EF-Ts which is then displaced by GTP. Renaturation of rhodanese and GTP hydrolysis by EF-Tu are greatly enhanced by the guanine nucleotide exchange factor EF-Ts. However, renaturation is reduced under conditions that stabilize EF-Tu in either the open or closed conformation. Both GDP and the nonhydrolyzable analog of GTP, GMP-PCP, inhibit renaturation. Kirromycin and pulvomycin, antibiotics that specifically bind to EF-Tu and inhibit its activity in peptide elongation, also strongly inhibit EF-Tu-mediated renaturation of denatured rhodanese to levels near those observed for spontaneous, unassisted refolding. Kirromycin locks EF-Tu in the open conformation in the presence of either GTP or GDP, whereas pulvomycin locks the factor in the closed conformation. The results lead to the conclusion that flexing of EF-Tu, especially as occurs between its open and closed conformations, is a major factor in its chaperone-like refolding activity.

Aminoglycosides↗
PubMed1997

Ribosomes and ribosomal RNA as chaperones for folding of proteins.

BACKGROUND: Provocative recent reports indicate that the large subunits of either prokaryotic or eukaryotic ribosomes have the capacity to promote refolding of denatured enzymes. RESULTS: Salt-washed Escherichia coli ribosomes are shown to promote refolding of denatured rhodanese. The ability of the ribosomes to carry out renaturation is a property of the 50S ribosomal subunit, specifically the 23S rRNA. Refolding and release of enzymatically active rhodanese leaves the ribosomes in an inactive state or conformation for subsequent rounds refolding. Inactive ribosomes can be activated by elongation factor G (EF-G) plus GTP or by cleavage of their 23S rRNA by alpha-sarcin. Activation by either mechanism is strongly inhibited by the EF-G.GDP.fusidic acid complex. CONCLUSIONS: Large subunits of E. coli ribosomes, specifically 23S rRNA, have the capacity to mediate refolding of denatured rhodanese. Refolding activity is related to the state or conformation of ribosomes that is promoted by EF-G. Activation by either mechanism is strongly inhibited by the EF-G.GDP.fusidic acid complex.

Endoribonucleases↗
PubMed1997

Conservative and surgical semen retrieval in patients with spinal cord injury.

Assisted semen retrieval was applied in 219 men with spinal cord injury (212 supranuclear, seven infranuclear lesions). Vibrostimulation in supranuclear lesions was successful in 133, and in five more after physostigmine injection. Electroejaculation was successful in all seven infranuclear lesions and in four supranuclear patients failing with vibrostimulation. Eight more supranuclear patients responded to electroejaculation and physostigmine. Surgical retrieval was applied in 27 patients. Nine Wagenknecht spermatoceles showed only one success, from all 18 implanted Brindley reservoirs semen could be collected. Three patients, in whom no pregnancy could be induced after Brindley reservoir implantation, had testicular sperm aspirated. In 109 patients who wanted to have offspring, 73 pregnancies could be induced in 46 couples, leading to 54 births (four twins), 16 abortions and three pregnancies. Conservative semen retrieval was possible in 82 of these men (63 pregnancies in 37 couples) and surgical methods were used in 27 (10 pregnancies in nine couples).

Adult↗
PubMed1997

Bladder autoaugmentation in adult patients with neurogenic voiding dysfunction.

Bladder autoaugmentation offers an alternative to enterocystoplasties for patients with low capacity high pressure bladders caused by non-malignant pathology. In particular for patients with neuropathic hyperreflexive bladders, refractory to less invasive therapy, bladder autoaugmentation will attain comparable results with less impact on life quality compared to other bladder augmentation procedures. The procedure does not preclude later enterocystoplasty or deafferentations. Over the last 7 years, 50 patients were treated by this method, some of them for other than neurogenic voiding dysfunction. Substantial increase of bladder capacity and compliance resulted, at the cost of increased residual urine and the need for intermittent catheterisation in most patients. Some patients are able to perform complete voiding at will. The time lapse between surgery and functional rehabilitation of the bladder (substantial increase of capacity and detrusor compliance) cannot be predicted yet. In most patients this amelioration was observed 1-6 months after surgery, but in some the effect was equivocal for a year or longer. Low-dose anticholinergics appear to speed up the therapeutic effect. One patient had enterocystoplasty and one deafferentation later, one had rupture of the bladder, probably from the use of an artificial sphincter, and two are rated as failures because of psychogenic bladder-centred problems. Bladder autoaugmentation is a favourable treatment for patients who are motivated to wait possibly longer for functional changes to occur and are willing to perform intermittent catheterisation afterwards.

Adolescent↗