Cloning and expression of a plasmid pBS195 gene, determining oxygenase activity, in Escherichia coli cells

Plasmid pBS195, detected in a strain of Lactobacillus sp. isolated from long-living persons, has a broad host range, including Gram-positive and Gram-negative microorganisms [1]. Plasmid-harboring colonies of the strain Escherichia coli HB101 give a color reaction with catechol. This indicates that genes mediating the activity of oxygenase are present in this plasmid. The high activity level of this enzyme, mediated by pBS195, and substrate specificity, which has not bee detected in any known metapyrocatechases, were found in cells of E. coli. Hybridization with a 32P-labeled fragment containing the NahC gene revealed a region of homology with a 1.6-kb EcoR I- BamH I fragment of plasmid pBS195. Deletion variants of this plasmid that lost oxygenase activity confirmed the location of the oxygenase gene in this region. The gene responsible for oxygenase activity in the plasmid was cloned on the pUC19 vector in E. coli cells. The expression of the cloned gene is controlled by the lac promoter of this vector. Physical, hybridization, and deletion analyses as well as analysis of polypeptides, which are synthesized in E. coli mini-cells, showed that this activity requires the participation of a polypeptide with molecular mass of 34 kDa.     

Estimation of the magnitude and spatial distribution of combustible materials in urban areas. A case study of the San Jose area,California

This paper describes a methodology for estimating the magnitude and spatial distribution of urban fuel loadings using San Jose, California, as a case study. The study was undertaken to provide basic inputs into the modeling of large urban fires ignited by nuclear explosions, which has important implications for studies of the global climatic effects of nuclear war, or the so-called ‘nuclear winter’ phenomenon. The methodology is based on the measurement of the numbers and sizes of different building occupancy types from aerial photographs for approximately 6000 1000-ft (304.8-m) grid cells covering the study area. These measurements were combined with literature-derived average fuel loadings for each building type to yield the total fuel loading per cell. The total fuel loading thus calculated is approximately 4558.56 × 10⁶ kg, yielding an average of 8.4 kg m⁻² (1.7 lb ft⁻²) for the built-up area of the city and 9.4 kg m⁻² (1.9 lb ft⁻²) if unoccupied cells (i.e. cells containing no fuel) within the built-up area are excluded. These figures are lower than previously published average values, due to the low-density character of San Jose and the exclusion of certain components of total fuel loading by the methodology.     

Evidence for the lumenal location of the 53 kDa glycoprotein of sarcoplasmic reticulum

The 53 kDa glycoprotein from sarcoplasmic reticulum was shown to be protected from proteolysis by trypsin, V8 proteinase and proteinase K in intact vesicles yet readily digested in the presence of the non-denaturing detergent C12E8. Competitive ELISAs with a library of seven monoclonal antibodies raised against the 53 kDa glycoprotein showed that the epitopes for these antibodies were only accessible in C12E8 solubilised and not intact sarcoplasmic reticulum. When the monoclonal antibodies against the 53 kDa glycoprotein were assessed for their effect on the uptake of Ca2+ by sarcoplasmic reticulum no effect was detected; neither were these antibodies able to augment the inhibitory influences of anti-(Ca(2+)-Mg2+)-ATPase monoclonal antibodies on Ca2+ uptake. These data indicate that the 53 kDa glycoprotein is located in the lumen of the sarcoplasmic reticulum.     

Comparison of chronic neutral endopeptidase inhibition and furosemide in an ovine model of heart failure

OBJECTIVE: Due to the elevated levels of hematopoietically active cytokines such as tumor necrosis factor (TNF) and granulocyte macrophage colony stimulating factor (GMCSF) in rheumatoid arthritis (RA) serum and synovium, the increased bone marrow activity in RA, and the effectiveness of GMCSF in mobilizing progenitor cell release from the bone marrow into the periphery, we hypothesized that hematopoietic progenitors are altered in the peripheral blood (PB) of patients with RA. METHODS: Flow cytometry assisted cell surface analysis was employed to compare the distribution of myeloid (CD34+CD33+), B lymphoid (CD34+CD10+), and erythroid (CD34+CD71+) committed progenitor cell subsets in the PB of healthy controls and patients with RA. Since RA and Sjogren's syndrome (SS) are related autoimmune disorders, primary SS PB was also investigated. RESULTS: Only those patients with RA exhibiting clinically active disease (RA-A) demonstrated increases in myeloid and B lymphoid progenitor cell subsets. Growth of RA-A progenitors in cytokines promoting myelopoiesis (GMCSF, TNF, stem cell factor) produced increased monocyte and dendritic cell progeny, in support of the flow cytometry data. Lineage committed (CD34+CD38+) progenitors were increased in SS PB (p <0.03). However, these did not correlate with either the myeloid, erythroid, or B lymphoid lineages. CONCLUSION: Distinct alterations in the distribution of PB progenitors are present in RA and primary SS. Since progenitor cells retain a proliferative capacity, their infiltration into the synovial/glandular environment may contribute to the accumulation of inflammatory cells within these sites. We propose that PB progenitors enter the diseased microenvironment through similar mechanisms as mature hematopoietic elements.     

Precipitated immune complexes of IgM induce the generation of reactive oxygen species by rabbit polymorphonuclear leucocytes

We report that immune complexes of IgM (ICIgM) antibodies and ovalbumin in the form of a precipitate from the equivalence zone induce the generation of reactive oxygen species by rabbit blood polymorphonuclear leucocytes (PMN), as measured by the chemiluminescence (CL) production in the presence of luminol. The kinetics of CL generation induced by ICIgM is quite different from that induced by precipitated immune complexes of IgG (ICIgG): the maximum rate of CL production for ICIgM occurs around 14 min, whereas for ICIgG it occurs about 5 min after incubation with the cells. Also the triggering of the process requires a higher concentration of ICIgM than of ICIgG. Evidence is presented that these effects are not mediated by interaction of the antigen (ovalbumin) with the cell, since immune precipitates of ovalbumin and the F(ab')2 fragment had no effect. Our observations that precipitated ICIgM can also be an effective stimulus for CL generation and thus for O2- production reveal a new functional capability of PMN. These results may have implications for the understanding of the participation of ICIgM (as well as of ICIgG) in inflammatory reactions mediated by PMN in immune complex diseases, and in the mechanisms of defense against microbes and other non-self agents.     

The role of glutathione and cysteine conjugates in the nephrotoxicity of o-xylene in rats

One-hundred-eighty cylindrical monocortical titanium implants, 4mm diameter and 12mm long, with three different coatings: fluorohydroxyapatite (group A), hydroxyapatite (group B), and titanium oxide (group C), all applied by vacuum plasma spray were bilaterally, randomly implanted into the femurs and tibiae of twelve adult mongrel sheep. The sheep were divided into four groups (1, 2, 3 and 4) numbering three sheep each. Sheep of groups 1, 2, 3 and 4 were euthanized at two weeks, one month, three and nine months after implantation, respectively Biomechanical and histomorphological analysis were performed. Extraction torque increased over time in all groups until the nine months period. At all the studied periods, the bone-implant contact was higher in Groups A and B compared to Group C. However, only at nine months did this difference reach statistical significance (p<0.005 comparing Groups A and B to C). The results of this study show that all the three coatings could be recommended for clinical applications.     

The genetics of psoriasis: a complex disorder of the skin and immune system

In the last few years, molecular genetics analyses have permitted novel insights into psoriasis, a disease characterized by uncontrolled proliferation of keratinocytes and recruitment of T cells into the skin. The disease affects approximately 1-2% of the Caucasian population and can occur in association with other inflammatory diseases such as Crohn's disease and in association with human immunodeficiency virus (HIV) infection. Given that psoriasis has characteristics of an autoimmune disease, it is not surprising that HLA studies revealed an association with certain alleles, notably HLA-Cw6. Despite this HLA component, psoriasis in some families is inherited as an autosomal dominant trait with high penetrance. Loci at chromosome 17q25 and 4q have been identified following genome-wide linkage scans of large, multiply affected families. In the case of at least the susceptibility locus at 17q25, the development of psoriasis does not require the presence of HLA-Cw6. Sib-pair analyses have confirmed the association with HLA-Cw6, confirmed the existence of a locus at 17q25 and identified other possible susceptibility loci. Two independent groups have reported a third region on chromosome 20p. Despite these findings, the extent of genetic heterogeneity and the role of environmental triggers and modifier genes is still not clear. The precise role of HLA also still needs to be defined. The isolation of novel susceptibility genes will provide insights into the precise biochemical pathways that control this disease. Such pathways will also reveal additional candidate genes that can be tested for molecular alterations resulting in disease susceptibility.     

Clinical assessment of spasticity in spinal cord injury: a multidimensional problem

OBJECTIVE: To determine the relation between various components of spasticity evaluated clinically in persons with spinal cord injury (SCI). DESIGN: Case series evaluating spasticity using clinical scales commonly referenced in contemporary literature, including the Penn Spasm Frequency Scale, the Ashworth Scale, and standard scales of tendon taps, clonus, and plantar stimulation. SETTING. A Veterans Affairs Medical Center Spinal Cord Injury Center. PATIENTS. Eighty-five spinal cord injured individuals with varying degrees of spasticity. RESULTS: Correlations demonstrated weak relationships between Spasm Frequency Scale and self-report scales of interference with function (.407) and painful spasms (.312). No clinical examination score correlated with self-report scores greater than 0.4. Three clinical examination scores correlated modestly (> 0.5)-Ashworth score with patellar tendon taps (.553), ankle clonus with Achilles tendon tap (.663), and patellar tendon tap with adductor tendon tap (.512). Two other clinical scales correlated weakly (> 0.4)-Achilles tendon tap with patellar tendon tap (.417) and plantar reflex with adductor tendon taps (.423). CONCLUSIONS: Clinical scales currently used to evaluate spasticity in SCI correlate poorly with each other, suggesting that they each assess different aspects of spasticity. The use of any single scale is likely to underrepresent the magnitude and severity of spasticity in the SCI population. In the absence of agreement among these various scales and with the absence of an appropriate criterion standard for evaluation of spasticity, assessments of spasticity, whether clinical or neurophysiological in nature, should be comprehensive in scope.