Search PubMed⌕ Search

Biomedical subjects

S Smith

Publications and source records attributed to S Smith.

At least 505 records · Page 28Linked to original sources
PubMed1994

Effects of exogenous interleukin-10 in a murine model of graft-versus-host disease to minor histocompatibility antigens.

IL-10 is a regulatory cytokine of both T cells and monocytes. We have investigated the ability of IL-10 to regulate responses to alloantigens in vitro and in vivo. Addition of IL-10 to mixed lymphocyte cultures profoundly decreased the proliferation and IL-2 production by donor B10.BR cells stimulated with CBA cells expressing minor histocompatibility antigens. Administration of IL-10 for a period of 2 weeks after bone marrow transplantation decreased the expansion of CD4+ and CD8+ donor T cells. In addition, splenocytes from BMT mice treated with IL-10 secreted less IFN-gamma after stimulation with Con A in vitro. The suppression of the mitogen-driven proliferative response of lymphocytes from the IL-10-treated group could also be reversed with significantly less anti-IFN-gamma antibody than for saline-treated controls. However, treatment with IL-10 was not sufficient to alter significantly the clinical course of graft-versus-host disease in CBA recipient mice as assessed by survival, weight loss, and splenomegaly. The results suggest that exogenous IL-10 suppresses the afferent Th1 response in a graft-versus-host reaction but does not significantly diminish the effector stage of graft-versus-host disease.

Animals↗
PubMed1994

Evidence that the agent of equine grass sickness may reach neurons by retrograde axonal transport.

Sera from acute and chronic cases of natural grass sickness or normal horses were injected into the parotid salivary gland of ponies. This gland receives its sympathetic innervation from the ipsilateral cranial cervical ganglion. None of the ponies showed any local or systemic signs of illness. After one week the cranial cervical ganglia, stellate and coeliaco-mesenteric ganglia were removed for histological study. Pathological changes were found only in the cranial cervical ganglion ipsilateral to a parotid salivary gland which had received an injection of grass sickness serum. Four out of five batches of test sera from cases of acute natural grass sickness were associated with chromatolytic changes in neurons; the remaining batch of serum produced no abnormalities. The most severe chromatolytic changes were induced by two samples obtained from horses whose signs of grass sickness had been present for less than 12 hours. A serum sample from a chronic case of grass sickness of three weeks duration did not produce chromatolysis but was associated with a moderately severe inflammatory infiltrate and neuronophagia in the ipsilateral cranial cervical ganglion. One batch of serum was size fractionated to separate components with molecular weights above or below 30 kDa. Only the fraction containing components above 30 kDa induced chromatolytic changes.

Acute Disease↗
PubMed1994

Colocalization of vertebrate lamin B and lamin B receptor (LBR) in nuclear envelopes and in LBR-induced membrane stacks of the yeast Saccharomyces cerevisiae.

We have expressed human lamin B and the chicken lamin B receptor (LBR), either separately or together, in yeast and have monitored the subcellular location of the expressed proteins by immunofluorescence microscopy, immunoelectron microscopy, and cell fractionation. At the light microscopic level, the heterologous lamin B localized to the yeast nuclear rim and at electron microscopic resolution was found subjacent to the yeast inner nuclear membrane. These data indicate that vertebrate lamin B was correctly targeted in yeast. Expression of the heterologous LBR, either alone or together with the heterologous lamin B, resulted in the formation of membrane stacks primarily adjacent to the nuclear envelope, but also projecting from the nuclear envelope into the cytoplasm or under the plasma membrane. Double immunoelectron microscopy showed colocalization of the heterologous lamin B and LBR in the yeast nuclear envelope and in the LBR-induced membrane stacks. Cell fractionation showed the presence of the heterologous lamin B and LBR in a subnuclear fraction enriched in nuclear envelopes. The heterologous lamin B was extracted at 8 M urea, but not at 4 M urea, thus behaving as a peripheral membrane protein and indistinguishable from assembled lamins. The heterologous LBR was not extracted by 8 M urea, indicating that it was integrated into the membrane. The observed colocalization and cofractionation are consistent with previously reported in vitro binding data and suggest that heterologous lamin B and LBR interact with each other when coexpressed in yeast.

Animals↗
PubMed1994

Comparative toxicity of mitomycin C and 5-fluorouracil in vitro.

The adjunctive use of antimetabolites has revolutionized surgical procedures to correct glaucoma in the past decade. Although much is known about the in vitro and in vivo actions of 5-fluorouracil and mitomycin C, their use in the eye has raised new questions about cellular toxicity. For example, filtering blebs after a surgical procedure supplemented with mitomycin C are relatively avascular in comparison with those seen with 5-fluorouracil supplementation. We investigated the effect of 5-fluorouracil and mitomycin C on cultured capillary endothelial cells and fibroblasts. The viability and morphologic characteristics of 3T3 fibroblasts and capillary endothelial cells were studied in light of increasing doses of 5-fluorouracil and mitomycin. Both cell types showed similar sensitivity to mitomycin C. However, fibroblasts were far more sensitive to 5-fluorouracil than were the cultured microvascular endothelial cells. Thus 5-fluorouracil appears to be toxic to fibroblasts while sparing vascular endothelial cells, whereas mitomycin C is cytotoxic for both cell types.

3T3 Cells↗
PubMed1994

Extracardiac anomalies, aneuploidy and growth retardation in 100 consecutive fetal congenital heart defects.

The importance of extracardiac anomalies in 100 consecutive fetuses with congenital heart disease was evaluated. The most common cardiac diagnoses were hypoplastic left heart syndrome in 35% and atrioventricular canal defect in 15%. Extracardiac anomalies were present in 42%, abnormal karyotype in 19% and intrauterine growth retardation in 20%. There were 45 survivors (35 required neonatal surgery, ten did not require early surgery) and 80% (36/45) of the survivors had isolated congenital heart disease. Extracardiac anomalies and abnormal karyotype were more frequently present in non-survivors (p < 0.001). Also, intrauterine growth retardation was more frequent in non-survivors (p < 0.05).

Journal Article↗
PubMed1994

Structure and function of the 5' flanking sequences of the human alpha class glutathione S-transferase genes.

We have isolated the 5'flanking regions of two human Alpha class glutathione S-transferase genes, GSTA1 and GSTA2. The two genes share 95% sequence identity between nucleotide positions -1,300 and +500 from the transcriptional start site. Various DNA fragments from the 5' flanking region of the GSTA1 gene were fused to the chloramphenicol acetyltransferase reporter gene and transfected into HepG2 cells. The results indicated that negative regulatory and enhancer elements are located in the sequence upstream of the GSTA1 gene. Sequence analysis and functional assays have not found any evidence for xenobiotic- or antioxidant-responsive elements previously described in rodent Alpha class genes. Thus the transcriptional regulation of the human Alpha class glutathione S-transferase genes may be dramatically different from the regulation of Alpha class glutathione S-transferase genes in rodents.

Base Sequence↗
PubMed1994

Cloning and sequence analysis of the gene encoding the low potential cytochrome c of Synechocystis PCC 6803.

The gene for the low potential cytochrome c (petK) was isolated from a genomic library of Synechocystis PCC 6803. The nucleotide sequence of this gene contains two regions with sequence similarity to two regions in the gene for the high potential cytochrome c6 of the organism. The sixth iron ligand can be identified with a conserved histidine. Experiments demonstrate the reduction of the low potential cytochrome by reduced ferredoxin II. The heme of the cytochrome is flanked by lysines which may be involved in orienting the ferredoxin near the site of electron donation.

Amino Acid Sequence↗
PubMed1994

Crystallization of thioesterase II from Escherichia coli.

X-ray quality single crystals of the Escherichia coli thioesterase II have been obtained. The protein used for crystallization was overexpressed in parent organism. The crystals are orthorhombic, space group C222(1) with axial lengths a = 99.0 A, b = 121.1 A, c = 166.6 A. A complete homotetramer (120,000 Da) of four polypeptide chains, each 286 residues long, occupies the asymmetric unit. The diffraction pattern extends to approximately 2.4 A resolution using CuK alpha radiation from a rotating anode source. The elucidation of the three-dimensional structure of this unusual bacterial thioesterase will provide basis for the analysis of its unique catalytic mechanism and substrate specificity.

Crystallization↗
PubMed1994

Measurement of GABA in rat brain microdialysates using o-phthaldialdehyde-sulphite derivatization and high-performance liquid chromatography with electrochemical detection.

A number of recent studies have detected gamma-aminobutyric acid (GABA) in brain microdialysates by measuring an electroactive o-phthaldialdehyde (OPA)-alkylthiol derivative using HPLC with electrochemical detection. A particular problem of this approach is the stench of the thiol reagents involved, and the poor stability of the electroactive GABA derivative. Here we report that these practical disadvantages can be overcome by substitution of the OPA-alkylthiol reaction with an OPA-sulphite reaction. This provides a simple, sensitive and reliable means for determination of GABA in microdialysates of rat brain.

Animals↗
PubMed1994

Detection of increased amounts of the extracellular domain of the c-erbB-2 oncoprotein in serum during pulmonary carcinogenesis in humans.

Over-expression of the c-erbB-2 oncogene-encoded p185 protein product has been implicated in the pathogenesis of a wide variety of human malignancies, including lung cancer. Over-expression of p185 can be detected immunologically by quantification of the extracellular domain of p185 (c-erbB-2 oncopeptide) in extracellular fluid in vitro and in serum in vivo. An enzyme-linked immunosorbent assay (ELISA) for the c-erbB-2 oncopeptide was used to examine banked serum samples of 11 pneumoconiosis patients who subsequently developed lung cancer and serum samples from 11 hospital controls matched for age, sex, ethnic group and smoking as well as 55 unmatched general population controls. The mean serum level for the c-erbB-2 oncopeptide in human neu units/ml in the lung cancer cases (1,756 +/- 549 HNU/ml) was statistically significantly elevated (p < 0.001) in comparison to the mean level in the matched controls (976 +/- 488 HNU/ml) or the general population controls (888 +/- 655 HNU/ml). Defining a positive elevation of the serum c-erbB-2 oncopeptide as any value more than 2 standard deviations above the mean of the matched controls, 64% (7 of 11) of the lung cancer cases were positive compared to 0% (0 of 11) matched controls and 5% (3 of 55) of the unmatched controls. In addition, 4 of the 7 c-erbB-2 oncopeptide-positive cancer cases had positive serum samples prior to the time of disease diagnosis (average = 35 months). These results suggest that serum c-erbB-2 oncopeptide may be elevated at an early stage of pulmonary carcinogenesis and that further prospective study of the utility of this biomarker is warranted.

Aged↗
PubMed1994

Reengineering the specificity of a serine active-site enzyme. Two active-site mutations convert a hydrolase to a transferase.

Two residues are known to play important catalytic roles in fatty acyl-thioester hydrolase, thioesterase II: Ser-101, the site of a covalent acyl-enzyme intermediate, and His-237 which is within hydrogen bonding distance of Ser-101 and facilitates catalysis by increasing the nucleophilicity of this residue. In this study we have examined the effect of mutations at these two residues on the ability of the enzyme to function as a hydrolase and, in the presence of a thiol acceptor, as an acyltransferase. In the hydrolase reaction kcat values for the wild-type, H237R, S101C, and S101C, H237R thioesterase enzymes were 0.11, < 0.002, 0.10, and < 0.002 s-1, respectively, and at steady state, the proportion of each enzyme present as the covalent acyl-enzyme intermediate was 11, 91, 71, and 100%, respectively. In the acyltransferase reaction no activity could be detected for the wild-type or H237R enzymes but the specific activities of the S101C and S101C/H237R thioesterases were 170 and 1300 nmol/min/mg of protein, respectively. From this data we conclude the following: the wild-type enzyme functions exclusively as a hydrolase. The H237R mutant acts ineffectively as a hydrolase primarily because the deacylation reaction is drastically retarded. The S101C enzyme functions well as a hydrolase, even though the rate of deacylation is adversely affected, and this enzyme can also perform as an acyltransferase. Mutation of both catalytic residues leads to a complete loss of hydrolase activity and the S101C,H237R mutant functions as an effective acyltransferase exhibiting kcat values higher then those of the wild-type enzyme acting as a hydrolase. This study reveals that, with only two amino acid replacements, an enzyme capable of functioning exclusively as a hydrolase can be converted into an equally active enzyme performing solely as an acyltransferase.

Acylation↗
PubMed1994

Phenotypic analysis and characterization of CD34+ cells from normal human bone marrow, cord blood, peripheral blood, and mobilized peripheral blood from patients undergoing autologous stem cell transplantation.

Single- and multicolor flow cytometry were used to define progenitor subsets in normal human bone marrow and peripheral blood, cord blood, and blood following mobilization of CD34+ progenitor cells by cyclophosphamide or cyclophosphamide/etoposide/G-CSF treatment. CD34 cells were quantitated and subsets of CD34+ cells were defined by coexpression of CD33, CD13, CD10, CD19, CD45RA, and CD71. Myeloid and erythroid progenitors were quantitated by sorting single CD34+ cells into individual wells of 96-well plates containing methylcellulose, IL-3, GM-CSF, G-CSF, IL-6, and erythropoietin. Comparative studies of CD34 cells showed that the percentage of CD34+ mononuclear cells was greatest in blood samples from patients following mobilization treatment with cyclophosphamide/etoposide/G-CSF averaging 2%. By comparison, the remaining sample groups ranged from 1.68 to 0.15% CD34 cells in this order, bone marrow > cord blood > cyclophosphamide mobilized blood > peripheral blood. Comparison of CD34 cells per milliliter of bone marrow or blood showed a range of 22.4 x 10(4) to 0.65 x 10(4)/ml in the following order, bone marrow > chemotherapy/etoposide/G-CSF > cord blood > cyclophosphamide-mobilized blood. Comparative analysis of CD34 subsets from different sources showed significant differences, particularly bone marrow and blood samples. A distinct population of CD34+ CD19+ (Leu 12) CD10+ (CALLA) pre-B lymphocyte cells was defined in bone marrow with lower side and forward light scatter characteristics and was variable between donors (29.8 +/- 16.9%, mean +/- 1 SD; range, 3-54%; n = 8). This population was not found to a significant degree in blood and also expressed CD45RA (Leu 18). Coexpression studies of CD45RA and CD71 (transferrin receptor) expression on CD34+ cells defined a CD45RA- CD71+ population containing 89 +/- 6.3% (n = 4) BFU-E and a CD45RA+ CD71+ population that contained all CFU-GM (n = 4). LeuM7 (CD13) stained a larger percentage to a greater intensity than MY7 (CD13). Coexpression of CD45RA (Leu 18) and CD13 (LeuM7) defined a subset of CD13+ CD45RA+ cells enriched for CFU-GM and CFU-M with a cloning efficiency of 31%. Coexpression of CD33 (MY9) and CD13 (MY7) defined a population that was predominantly CFU-GM with a cloning efficiency of 38%. These studies define CD34+ phenotypes containing pure populations of B lymphocyte, granulocyte-macrophage, or erythroid progenitors and demonstrate the utility of multiparameter flow cytometry to define lineage-committed CD34+ cells.

Adult↗
PubMed1994

Isolation of a yeast artificial chromosome contig spanning the Greig cephalopolysyndactyly syndrome (GCPS) gene region.

Disruption of the zinc finger gene GLI3 has been shown to be the cause of Greig cephalopolysyndactyly syndrome (GCPS), at least in some GCPS translocation patients. To characterize this genomic region on human chromosome 7p13, we have isolated a YAC contig of more than 1000 kb including the GLI3 gene. In this contig the gene itself spans at least 200-250 kb. A CpG island is located in the vicinity of the 5' region of the known GLI3 cDNA, implying a potential promoter region.

Abnormalities, Multiple↗
PubMed1994

Potential diagnostic implications of breakpoints in the long arm of chromosome 8 in lipoblastoma.

We report a case of lipoblastoma in a 17-month-old male patient with a pseudodiploid karyotype of 46,XY,der(14)t(8;14)(q11.2;q24). The tumor cells show two normal chromosome 8 homologs in addition to the extra 8q11.2-->qter segment, making it trisomic for the region 8q11.2-->qter, and monosomic for 14q24-->qter. Reports of chromosome studies in lipoblastoma are rare; however, a breakpoint in the long arm of chromosome 8q11-13 appears to be emerging as a consistent finding.

Chromosome Aberrations↗