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Biomedical subjects

J Walter

Publications and source records attributed to J Walter.

At least 145 records · Page 8Linked to original sources
PubMed1996

M.BssHII, a multispecific cytosine-C5-DNA-methyltransferase with unusual target recognizing properties.

A new multispecific cytosine-C5-DNA-methyltransferase (C5-MTase), M.BssHII, was identified in Bacillus stearothermophilus H3. The M.BssHII gene was cloned and sequenced. The amino acid sequence deduced shows the characteristic building plan of a C5-MTase. By sequencing bisulfite-treated DNA methylated by M.BssHII and by restriction enzyme analysis, we defined the following methylation targets of M.BssHII: ACGCGT/CCGCGG (MluI/SacII), PuGCGCPy (HaeII), PuCCGGPy (Cfr10I) and GCGCGC (BssHII). The relative location of the specificity determinants in the C5-MTase was derived from the analysis of M.BssHII derivatives carrying deletions within the variable region "V" and chimeric C5-Mtases constructed between M.BssHII and the related monospecific enzyme M.phi3TII. Four of the M.BssHII specificities (MluI, SacII, Cfr10I and BssHII) could be associated with amino acid segments within the variable region "V". The determinant for HaeII activity had to be assigned to sequences defining the enzyme core, the first example of a C5-MTase in which a sequence-specific methylation potential is mediated by structures outside of the variable region. Another intriguing result came from the analysis of one particular chimera made between M.BssHII and M.phi3TII. This construct showed a relaxation of the methylation capacity, both with respect to the target recognized and the targeting of methylation within this sequence.

Amino Acid Sequence↗
PubMed1996

DNA binding specificity of two homeodomain proteins in vitro and in Drosophila embryos.

In previous experiments, the homeodomain proteins even-skipped and fushi-tarazu were found to UV cross-link to a surprisingly wide array of DNA sites in living Drosophila embryos. We now show that UV cross-linking gives a highly accurate measure of DNA binding by these proteins. In addition, the binding of even-skipped and fushi-tarazu proteins has been measured in vitro to the same DNA fragments that were examined in vivo. This analysis shows that these proteins have broad DNA recognition properties in vitro that are likely to be important determinants of their distribution on DNA in vivo, but it also shows that in vitro DNA binding specificity alone is not sufficient to explain the distribution of these proteins in embryos.

Animals↗
PubMed1996

Successful expression of human factor IX following repeat administration of adenoviral vector in mice.

Adenoviral vectors can direct high-level expression of a transgene, but, due to a host immune response to adenoviral antigens, expression is of limited duration, and repetitive administration has generally been unsuccessful. Exposure to foreign proteins beginning in the neonatal period may alter or ablate the immune response. We injected adult and neonatal (immunocompetent) CD-1 mice intravenously with an adenoviral vector expressing human blood coagulation factor IX. In both groups of mice, expression of human factor IX persisted for 12-16 weeks. However, in mice initially injected as adults, repeat administration of the vector resulted in no detectable expression of the transgene, whereas in mice initially injected in the neonatal period, repeat administration resulted in high-level expression of human factor IX. We show that animals that fail to express the transgene on repeat administration have developed high-titer neutralizing antibodies to adenovirus, whereas those that do express factor IX have not. This experimental model suggests that newborn mice can be tolerized to adenoviral vectors and demonstrates that at least one repeat injection of the adenoviral vector is possible; the model will be useful in elucidating the immunologic mechanisms underlying successful repeat administration of adenoviral vectors.

Adenoviridae↗
PubMed1996

Induced release of cell surface protein kinase yields CK1- and CK2-like enzymes in tandem.

Several types of cell exhibit cell surface protein kinase (ecto-PK) activities with Ser/Thr-specificity. Ecto-PK sharing certain characteristics of protein kinase CK2 can be detached from intact cells by interaction with exogenous substrates (Kübler, D., Pyerin, W., Burow, E., and Kinzel, V. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025). However, a detailed molecular analysis of this ecto-PK was hampered by the vanishingly small amounts of labile enzyme protein obtained by substrate-inducible enzyme release. We now describe the stabilization and enrichment of released ecto-PK by precipitation with polyethylene glycol followed by affinity chromatography on heparin-agarose. Ecto-PK is shown to consist of two separate forms released in tandem, ecto-PK I and ecto-PK II. Comparison with cell homogenates as well as cell surface biotinylation experiments excluded contamination with intracellular PK. Purified ecto-PK I and ecto-PK II exhibit respectively selective phosphorylation of CK1- and CK2-specific peptide substrates, a complementary sensitivity to inhibitory agents and a differential use of the cosubstrates ATP and GTP. Ecto-PK I consists of a 40-kDa moiety; the ecto-PK II is an ensemble of three components of 43- and 40-kDa (catalytic subunits) and a noncatalytic 28-kDa subunit. In addition, components of the ecto-PK II react with CK2-specific antibodies. Further, comparative peptide mapping and the results of mass spectrometry in combination with assignment of amino acid sequences confirmed that ecto-PK II is closely related if not identical to the protein kinase CK2. Assays with intact cells that result in the phosphorylation of a variety of endogenous membrane proteins showed that both ecto-PKs participate, and further, certain ecto-PK substrates become preferentially labeled by one or another of the enzymes, whereas others are phosphorylated by both ecto-PK activities.

Amino Acid Sequence↗
PubMed1996

In vitro studies and computer simulations to assess the use of a diode laser (850 nm) for laser-induced thermotherapy (LITT).

BACKGROUND AND OBJECTIVE: This study investigated the feasibility of 850 nm diode laser-induced thermotherapy (LITT), a new procedure providing local hyperthermia and photocoagulation, a minimally invasive treatment of tumors, and other disorders such as benign prostate hyperplasia. STUDY DESIGN/MATERIAL AND METHODS: An improved fiber optic diffuser inserted through a catheter was used to irradiate prostate, liver, and kidney tissue in-vitro. Experimental results were compared with predictions from mathematical simulations based on measured optical tissue parameters. RESULTS: After 10 minutes of irradiation with 2-4.6 W CW laser power, sizable coagulation diameters had been achieved. In prostate and kidney tissue, temperature rises and coagulation diameters after laser irradiation were found to be smaller than in liver tissue. Those tissues also had a higher threshold for carbonization, thus reducing the risk of damage to the delivery system. CONCLUSION: The data suggest that a low power diode laser can be recommended for use in LITT to treat prostate and kidney tissue.

Animals↗
PubMed1996

Cardiorespiratory responses of patients with rheumatoid arthritis during bicycle riding and running in water.

BACKGROUND AND PURPOSE: The purpose of this study was to compare exercise during dry-land bicycle ergometry and running in water with a flotation device in individuals with rheumatoid arthritis. SUBJECTS: Eight individuals with adult-onset rheumatoid arthritis, between the ages of 30 and 40 years (X = 35.88, SD = 2.85), participated. METHODS: Each subject did a graded maximal exercise test on a stationary bicycle and in the water wearing a flotation device, while oxygen uptake (VO2), heart rate (HR), pain, rating of perceived exertion (RPE), minute ventilation (VE), respiration rate, tidal volume (VT), and respiratory exchange ratio (R) were monitored. RESULTS: Higher maximum RPE and R were seen during water running, whereas higher VE and VT were seen during bicycle riding. Heart rate, R, and plateauing VO2 data indicated that a true physiological peak VO2 was reached during the bicycle test. Peak VO2 and HR were similar for either water or bicycle exercise. These findings show that with both forms of exercise, subjects were able to reach training levels as set by the American College of Sports Medicine. CONCLUSION AND DISCUSSION: The water exercise, therefore, provides a means of exercising for individuals with rheumatoid arthritis. It allows them to reach the needed training levels in a comfortable aquatic environment.

Adult↗
PubMed1996

The Alzheimer's disease-associated presenilins are differentially phosphorylated proteins located predominantly within the endoplasmic reticulum.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the deposition of extracellular senile plaques composed of amyloid beta-peptide (A beta). Whereas most cases of AD occur sporadically, about 10% of AD cases are inherited as a fully penetrant autosomal dominant trait. Mutations in the recently cloned Presenilin genes (PS-1 and PS-2) are by far the most common cause of early onset familial AD. MATERIALS AND METHODS: Cellular expression of endogenous and overexpressed PS proteins was analyzed by immunocytochemistry and metabolic labeling followed by immunoprecipitation. In vivo phosphorylation sites of PS proteins were analyzed by extensive mutagenesis. RESULTS: PS-1 as well as PS-2 proteins were localized predominantly within the endoplasmic reticulum (ER). However, small amounts of the PS proteins were detected within the Golgi compartment, where they colocalize with the beta-amyloid precursor protein (beta APP). The PS-2 protein was found to be highly phosphorylated, whereas very little phosphorylation was observed for PS-1. The selective phosphorylation of PS-2 occurs exclusively on serine residues. In vivo phosphorylation of PS-2 was mapped to serine residues 7, 9, and 19 within an acidic stretch at the N terminus, which is absent in PS-1. casein kinase (CK)-1 and CK-2 were shown to phosphorylate the N terminus of PS-2 in vitro. CONCLUSIONS: The majority of PS proteins were detected in the ER where little if any proteolytic processing of beta APP was reported. ER retention of PS proteins might occur by intramolecular aggregation. Small amounts of PS proteins were also detected in the Golgi where they colocalized with beta APP. This might suggest that potential interactions between PS proteins and beta APP could occur within the Golgi. Selective phosphorylation of PS-2 proteins within the acidic domain missing in PS-1 indicates differences in the biological functions and regulation of the two highly homologous proteins.

Alzheimer Disease↗
PubMed1995

Tumor-associated E6 protein of human papillomavirus type 16 contains an unusual H-2Kb-restricted cytotoxic T cell epitope.

We previously showed that CTL from H-2b mice immunized against the E6 protein of human papilloma virus (HPV) type 16 recognized a 10-mer peptide corresponding to amino acids 131 to 140. We show in this study that the minimal epitope, 130 to 137, is a 8-mer peptide presented by H-2Kb class I molecules. At position P8, the 130 to 137 peptide contains a hydrophobic methionine anchor residue, but P3 and P5 do not contain the typical anchor residues that are frequently found in Kb-bound peptides. Analysis with alanine-substituted peptides indicates that the tryptophan at P3 acts as an alternative anchor mediating Kb binding, while an arginine at P2 is a TCR contact residue. Synthetic 9-mer peptides corresponding to residues 130 to 138 are as efficiently recognized by CTL as 130 to 137 peptides. Analysis of extracts of E6-expressing cells suggests that Ag processing may produce multiple peptides containing the minimal 130 to 137 epitope. In vitro binding studies indicate that Kb binding of peptide 130 to 137 is approximately five orders of magnitude less efficient than Kb binding of previously identified CTL epitopes. In contrast, the E6 protein contains another potential CTL epitope in the region of amino acids 41 to 50. A synthetic peptide spanning this region binds very strongly to Kb and is capable of stimulating a strong peptide-specific CTL response. In the context of the whole protein, however, this epitope remains cryptic.

Amino Acid Sequence↗
PubMed1995

M.BssHII: a new multispecific C5-DNA-methyltransferase.

M.BssHII is a new multispecific C5-DNA-methyltransferase recognizing five different targets. As the enzyme has been isolated from a thermophilic Bacillus, the protein should show enhanced intrinsic thermostability and therefore be a promising candidate for crystallizing a multispecific MTase.

Bacillus↗
PubMed1995

Induction of CD44 expression by the Epstein-Barr virus latent membrane protein LMP1 is associated with lymphoma dissemination.

Epstein-Barr Virus (EBV) is implicated in the pathogenesis of endemic Burkitt's lymphoma (BL), B-cell lymphomas occurring under immunosuppression, nasopharyngeal carcinoma and Hodgkin's disease. Two distinct patterns of latent EBV gene expression occur in EBV-associated lymphomas. BLs typically display expression of the nuclear antigen EBNAI only, whereas EBV-associated, non-Burkitt B-cell lymphomas express at least 9 latent viral genes (6 EBNAs and 3 latent membrane proteins), reminiscent of in vitro EBV-immortalized lymphoblastoid cell lines (LCL). BLs are characterized by local, extra-nodal growth, whereas EBV-associated B-cell lymphomas often disseminate to peripheral lymphoid tissue. We show here that BL cells forming local tumors after xenotransplantation into SCID mice disseminate to lymphoid tissue following introduction of the latent membrane protein I (LMP 1) gene. Introduction of LMP 1 into BL cells induced expression of CD44 on the cell surface, a molecule implicated in enhanced lymphoid tumor growth and dissemination. Introduction of CD44 into LMP 1-/CD44-BL cells was observed to confer the disseminated tumor growth pattern associated with LMP 1 expression. Taken together our results show that expression of LMP 1 may regulate expression of CD44 and play an important role in the behavior of EBV-based lymphomas.

Animals↗
PubMed1995

Ligand mediated activation of ectopic EGF receptor promotes matrix protein adhesion and lung colonization of rat mammary adenocarcinoma cells.

Increased expression of EGF receptor (EGFR) in metastases of human mammary carcinoma as compared to cells of the primary cancer suggests a contribution of EGFR to mammary carcinoma metastasis. To test for a positive causative link, we investigated 13762NF rat mammary adenocarcinoma cloned tumor cell lines of high (MTLn3) or low (MTC) metastatic potential. While MTC cells expressed barely detectable amounts of EGFR, MTLn3 cells expressed readily detectable levels of functional receptors. A full length cDNA of the human EGFR (HER) was introduced by infection with a retroviral vector into MTC cells. Expression of HER was stable and receptors were functional with respect to surface expression, ligand binding and EGF-stimulated phosphorylation. Independent clones of the transfectants were isolated and characterized. Ligand stimulation of MTC HER cells and derived clones led to enhanced adhesion of cells to extracellular matrix proteins. Implantation of cells intravenously into female nu/nu mice revealed ligand-dependent enhancement of lung colonizing potential of EGFR-expressing cells.

Adenocarcinoma↗
PubMed1995

The beneficial effect of low initial dose and gradual increase of erythropoietin treatment in hemodialysis patients.

It is well known that the effects of human recombinant erythropoietin (rHuEPO) are dose-dependent. However, when higher doses of rHuEPO are used, the frequency of the side effects also increases. The aim of our study was to analyze the hematologic parameters and blood pressure response in hemodialysis patients treated with low initial and gradually increased rHuEPO. Sixteen regular hemodialysis patients were treated with 3 x 20 U/kg/week of rHuEPO subcutaneously during the first month. Every fourth week the dose was increased by 3 x 20 U/kg/week if the hematocrit did not rise by 2%. If the elevation was 2% or more, the dose of the rHuEPO was not changed. Blood count and blood pressure were checked every week. The antihypertensive treatment was also reviewed weekly. The hematocrit increased significantly from the second week, and 11 patients achieved the target level (30%) between Weeks 8 and 24. Two patients reached the 30% hematocrit level between Weeks 2 and 8, and another 2 patients reached the target level between Weeks 25 and 28. There was 1 nonresponder. The average rHuEPO dose needed to achieve the target hematocrit was 56.3 U/kg/week. We did not observe significant changes in the mean arterial or diastolic blood pressure. It was necessary to increase the doses of antihypertensive drugs, namely nifedipine and captopril, to control blood pressure. Encephalopathy occurred in none of the cases. The low initial dose and the gradual increase of rHuEPO treatment were beneficial to the hemodialysis patients. Although the target hematocrit took longer to achieve, high blood pressure and encephalopathy were prevented by close monitoring and administration of suitable antihypertensive treatment.

Adult↗
PubMed1995

Heliox improves pulsus paradoxus and peak expiratory flow in nonintubated patients with severe asthma.

Heliox is a blend of helium and oxygen with a gas density less than that of air that decreases airway resistance (Raw) in patients ventilated for status asthmaticus. We tested whether breathing an 80:20 mixture of helium:oxygen would reduce pulsus paradoxus (PP) and increase peak expiratory flow (PEF) in patients presenting to the emergency room with an exacerbation of asthma. After receiving 30 min of beta-agonist aerosols and intravenously administered methylprednisolone, 27 patients whose PP remained greater than 15 mm Hg and whose PEF remained less than 250 L/min consented to breathe heliox or room air for 15 min. PP decreased and PEF increased with time in control patients, indicating a time-related effect of routine bronchodilator therapy (p < 0.05). PP decreased in 15 of 16 patients during heliox, and the change with heliox was significantly greater than that during air breathing (p < 0.01). PEF measured with a Wright's peak flow meter calibrated for heliox increased in all patients breathing heliox. Again, the increase in PEF during heliox breathing was significantly greater than the corresponding change in control patients breathing air (p < 0.001). To the extent that PP reflects the inspiratory fall in pleural pressure, this reduction in PP indicates a substantial reduction in inspiratory Raw when the less dense gas is inspired through narrowed bronchi having turbulent flow regimes. The 35% increase in PEF while breathing heliox signals a similar reduction in expiratory Raw, which might diminish the hyperinflation often observed during an exacerbation of asthma.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗