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Biomedical subjects

J Weber

Publications and source records attributed to J Weber.

At least 235 records · Page 13Linked to original sources
PubMed1996

Genetic heterogeneity of HIV type 1 in Russia: identification of H variants and relationship with epidemiological data.

HIV-genetic subtypes were analyzed in 130 subjects from the Russian Federation, by the HMA technique. Six subtypes were identified in heterosexuals, including A, B, C, D, G, and H; however, homosexual men were infected predominantly with the B subtype (33 of 35). The subtype A isolates were found in population of intravenous drug users. HMA successfully identifies 128/130 DNA samples; the phylogenetic analysis of the V1/V5 gp120 encoding region derived from another two samples demonstrated that these isolate belong to subtype H.

Amino Acid Sequence↗
PubMed1996

Therapeutic effect of m-[131I]- and m-[125I]iodobenzylguanidine on neuroblastoma multicellular tumor spheroids of different sizes.

m-[l25I]iodobenzylguanidine (m-[125I]MIBG) has been suggested as an alternative to m-[131I]MIBG for the treatment of metastatic neuroblastoma to achieve a higher radiation dose in micrometastases. To compare these two radiopharmaceuticals, a mathematical model was developed in the present study that allows for the calculation of radiation dose rates within small spherical tumors for different distributions of 131I and 125I. Furthermore, the relationship between tumor size and the therapeutic effects of m-[131I]- and m-[125I]MIBG was studied in vitro using multicellular tumor spheroids of the neuroblastoma cell line SK-N-SH. According to the calculations, higher mean dose rates can be achieved by m-[125I]MIBG than by m-[131I]MIBG up to a tumor diameter of 100 microm when both substances are homogeneously distributed within the tumor. In larger tumors, however, mean dose rates achieved by 131I are up to 8-fold higher. Evaluation of various activity distributions demonstrated that even in tumors of less than 100 microm in diameter, marked heterogeneities of the dose rate can occur when m-[125I]MIBG is not distributed homogeneously. By treatment with m-[131I]MIBG, the growth of tumor spheroids ranging from 100 to 250 microm in diameter was inhibited more effectively in the larger than in the smaller spheroids. The growth inhibition of spheroids treated with m-[125I]MIBG was independent of the spheroid size. In consistency with the calculations, the therapeutic effect of m-[125I]- and m-[131I]MIBG was equal in spheroids with diameters of about 100 microm. In larger spheroids, m-[131I]MIBG induced a more pronounced delay in spheroid growth than m-[125I]MIBG. According to these calculations and in vitro data, m-[125I]MIBG as a single agent does not seem to be a promising alternative to m-[131I]MIBG for treatment of metastatic neuroblastoma. However, the combined use of m-[131I]- and m-[125I]MIBG may be more effective than treatment with m-[131I]MIBG alone.

3-Iodobenzylguanidine↗
PubMed1996

Specific tryptophan substitution in catalytic sites of Escherichia coli F1-ATPase allows differentiation between bound substrate ATP and product ADP in steady-state catalysis.

Tryptophan was specifically inserted as the residue immediately preceding the P-loop sequence in F1-ATPase catalytic sites. The mutant enzyme (betaF148W) showed normal enzymatic characteristics. The fluorescence responses of beta-tryptophan 148 enabled us to differentiate between nucleoside di- and triphosphate bound in catalytic sites; MgADP quenched at 350 nm, whereas MgAMPPNP and MgADP.BeFx complex enhanced the fluorescence at 325 nm. With MgATP, both effects were seen simultaneously. This allowed analysis of bound catalytic site nucleotides directly under steady-state MgATP hydrolysis conditions. At mM concentration of MgATP (Vmax conditions) one of the three catalytic sites was filled with substrate MgATP and the other two sites were filled with product MgADP. A model for F1-ATPase steady-state turnover is presented that encompasses these findings. Given the structural similarity of the P-loop in nucleotide-binding proteins, this approach may prove widely useful.

Adenosine Diphosphate↗
PubMed1996

F1F0-ATP synthase: development of direct optical probes of the catalytic mechanism.

Using strategically-placed tryptophan (Trp) residues as optical probes to monitor nucleotide binding and hydrolysis, we demonstrate that all three catalytic nucleotide binding sites in F1-ATPase must be filled to obtain physiological (Vmax) MgATP hydrolysis rates. At Vmax hydrolysis rates, the predominant enzyme species has one of the three catalytic sites filled with unhydrolyzed substrate MgATP, the other two sites are filled with product MgADP. A specifically-inserted Trp probe was also developed to characterize nucleotide binding to the noncatalytic sites, and a model to explain the specificity of these sites is shown. These sites appear to play no role in ATP hydrolysis.

Adenosine Triphosphate↗
PubMed1996

The role of the adenovirus protease on virus entry into cells.

Adenovirus uncoating is a stepwise process which culminates in the release of the viral DNA into the nucleus through the nuclear pore complexes and dissociation of the capsid. Using quantitative biochemical, immunochemical and morphological methods, we demonstrate that inhibitors of the cystine protease, L3/p23, located inside the capsid block the degradation of the capsid-stabilizing protein VI, and prevent virus uncoating at the nuclear membrane. There was no effect on virus internalization, fiber shedding and virus binding to the nuclear envelope. The viral enzyme (dormant in the extracellular virus) was activated by two separate signals, neither of which was sufficient alone; virus interaction with the integrin receptor (inhibited with RGD peptides) and re-entry of the virus particle into a reducing environment in the endosome or the cytosol. Incorrectly assembled mutant viruses that lack the functional protease (ts1) failed at releasing fibers and penetrating into the cytosol. The results indicated that L3/p23 is needed not only to assemble an entry-competent virus but also to disassemble the incoming virus.

Adenoviruses, Human↗
PubMed1996

High activated and memory cytotoxic T-cell responses to HTLV-1 in healthy carriers and patients with tropical spastic paraparesis.

The cytotoxic T-lymphocyte (CTL) response to HTLV-1 is directed mainly against the Tax protein. Circulating, activated Tax-specific CTL can be found in a majority of healthy carriers and patients with the HTLV-1-associated disease tropical spastic paraparesis (HAM/TSP). In this study we present data on the Tax-specific CTL response of 26 HTLV-1 carriers, including 10 newly recruited subjects. Rex-specific CTL were not found in any subjects investigated. Activated and memory CTL responses were determined separately in 4 healthy carriers, 3 HAM/TSP patients, and 1 "seronegative HAM/TSP." In all subjects, the mean frequency of peptide-specific memory cells per epitope (1/1307) was high. There was no significant difference in mean memory CTL frequency per epitope or in the proportion of subjects with activated CTL between healthy carriers and HAM/TSP patients. One individual with HAM/TSP had an unusually high frequency response to two peptides, suggesting immunodominance of epitope recognition in this individual. We conclude that the magnitude and components of the HLTV-1-specific CTL response do not differ between healthy carriers and HAM/TSP patients. These data do not support a specific CTL-mediated component in the pathogenesis of HAM/TSP.

Amino Acid Sequence↗
PubMed1996

Binding and hydrolysis of TNP-ATP by Escherichia coli F1-ATPase.

It had previously been suggested that Vmax hydrolysis rate of 2', 3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) by F1-ATPase required filling of only two catalytic sites on the enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727), whereas recently it was shown that Vmax rate of ATP hydrolysis requires that all three catalytic sites are filled (Weber, J., Wilke-Mounts, S., Lee, R. S. F., Grell, E., and Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133). To resolve this apparent discrepancy, we measured equilibrium binding and hydrolysis of MgTNP-ATP under identical conditions, using betaY331W mutant Escherichia coli F1-ATPase, in which the genetically engineered tryptophan provides a direct fluorescent probe of catalytic site occupancy. We found that MgTNP-ATP hydrolysis at Vmax rate did require filling of all three catalytic sites, but in contrast to the situation with MgATP, "bisite hydrolysis" of MgTNP-ATP amounted to a substantial fraction (approximately 40%) of Vmax. Binding of MgTNP-ATP to the three catalytic sites showed strong binding cooperativity (Kd1 < 1 nm, Kd2 = 23 nm, Kd3 = 1.4 microM). Free TNP-ATP (i.e. in presence of EDTA) bound to all three catalytic sites with lower affinity but was not hydrolyzed. These data emphasize that the presence of Mg2+ is critical for cooperativity of substrate binding, formation of the very high affinity first catalytic site, and hydrolytic activity in F1-ATPases and that these three properties are strongly correlated.

Adenosine Triphosphate↗
PubMed1996

Visualization of dedicated catheters using fast scanning techniques with potential for MR-guided vascular interventions.

This paper reports the development of dedicated catheters and real-time MR scan techniques for guiding vascular interventional procedures. By way of phantom experiments, it is shown that proper modification of the magnetic properties of catheters allows their conspicuous and consistent visualization in subsecond 2D gradient echo images and phase contrast angiograms. Dynamic scan times as low as 0.5 s could be achieved by exploiting the keyhole technique and purposeful postprocessing. The temporal resolution and spatial resolution of the resultant scan protocol shows promise for adequate tracking of catheter manipulation.

Blood Flow Velocity↗
PubMed1996

The effects of prolonged treatment with citicoline in temporary experimental focal ischemia.

Potential therapeutic effects of cytidine 5-diphosphocholine (citicoline), a key intermediary in the biosynthesis of the membrane phospholipid, phosphatidylcholine, are presumably related to enhanced phospholipid synthesis in the ischemic brain. We evaluated prolonged citicoline treatment in a temporary focal ischemia model. Using the suture occlusion model, we induced 2 hours of temporary ischemia in 30 Sprague-Dawley rats. The rats were randomly and blindly assigned to receive intraperitoneally 500 mg/kg citicoline (HD), 100 mg/kg citicoline (LD) or physiologic saline as the control group once daily for 7 days (n = 10 per group) beginning at the time of reperfusion. Neurological scoring (0-5 scale) was performed daily. After elective sacrifice on day 7, or earlier if death occurred prematurely, the brains underwent 2,3,5-triphenyltetrazolium chloride (TTC) staining for calculation of corrected infarct and edema volume. The mean corrected infarct volume in the HD group was 125 +/- 45.2 mm3 (mean +/- SD), significantly smaller than controls, 243.5 +/- 88.6 mm3 (p < 0.01, Scheffe's-test). The LD group infarct volume was 200.2 +/- 62.8 mm3 (N.S.). The mean amount of brain edema in the HD group was 46.4 +/- 45.6 mm3 was smaller than the controls, 92.3 +/- 54.4 mm3 and the LD group, 84.9 +/- 71.7 mm3 (N.S.). Mortality before day 7 in the HD was 30% while it was 50% in the two other groups. The neurologic score on day 7 was 2.5 +/- 1.8 in the HD group, 3.3 +/- 1.8 in the LD group and 3.4 +/- 1.7 in controls (N.S.). These results demonstrate that extended high dose citicoline treatment significantly reduced infarct volume in this temporary focal ischemia model and that there was a trend toward reducing brain edema and mortality. These effects may be related to membrane stabilization and inhibition of free fatty acid release.

Animals↗
PubMed1996

Transplanting two unique beta-glucanase catalytic activities into one multienzyme, which forms glucose.

Endo cellulases of plant pathogenic erwinias degrade cellulose as well as the cellulosic domains of barley (1-3,1-4)-beta-glucan. Depolymerization of the latter substrate is mainly caused by (1-3,1-4)-beta-glucanases, which hydrolyze (1-4)-beta glycosidic linkages adjacent to (1-3)-beta linkages. To construct an enzyme for efficient degradation of barley (1-3,1-4)-beta-glucan, the sequence encoding the catalytic domain and interdomain linker of the cellulase from Erwinia carotovora subspecies atroseptica was fused to that for the heat stable Bacillus hybrid, H(A12-M) delta Y13 (1-3,1-4)-beta glucanase. The chimeric enzyme secreted from Escherichia coli cells did not remain covalently assembled as judged by SDS-PAGE. However, the glycosylated and intact enzyme (denoted CELGLU) is secreted from the yeast Pichia pastoris. CELGLU exhibits both cellulase and (1-3,1-4)-beta-glucanase catalytic activities, and was accordingly classified a true multienzyme. HPLC and NMR analyses revealed that among the products from CELGLU, di- and trimeric oligosaccharides were identical to those produced by the parental cellulase. Tetrameric oligosaccharides, derived from the (1-3,1-4)-beta-glucanase activity of CELGLU, were further degraded by the cellulase moiety to yield glucose and trimers. Compared with the parental enzymes, CELGLU exhibits substantially higher Vmax for degradation of both soluble cellulose and barley (1-3,1-4)-beta-glucan. These findings point to construction of multienzymes as an effective approach for engineering enzymes with novel characteristics.

Artificial Gene Fusion↗
PubMed1996

Evidence that a locus for familial psoriasis maps to chromosome 4q.

Psoriasis is an inflammatory skin disease that affects 2% of the population. It is characterised by red, scaly skin patches which are usually found on the scalp, elbows and knees, and may be associated with severe arthropathy. The lesions are caused by abnormal keratinocyte proliferation, and infiltration of inflammatory cells into the dermis and epidermis. The usual age of onset of psoriasis is between 15 and 30 years, although it can present at any age. Psoriasis is recognised to have a large genetic component. Twin studies show the concordance in monozygotic twins to be between 65-70%, compared to between 15-20% in dizygotic twins. Family studies estimate the risk to first degree relatives at between 8-23%. However, there are also several environmental factors, including streptococcal infection and stress, that affect the onset and presentation of the disease. The mode of inheritance of psoriasis is unclear. We conducted a genome-wide scan to search for psoriasis susceptibility loci in a single large multiplex family. Parametric linkage analysis indicated that a susceptibility locus for familial psoriasis was located on chromosome 4q. Investigation of this locus in five further multiplex families using both parametric and non-parametric methods gave significant localisation to chromosome 4q. The maximum total pairwise lod score obtained was 3.03 with the microsatellite marker D4S1535 at theta = 0.08. Non-parametric multipoint analysis with GENEHUNTER- demonstrated significant excess allele sharing, with a P value of 0.0026, at the same locus.

Chromosome Mapping↗