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Biomedical subjects

C Smith

Publications and source records attributed to C Smith.

At least 235 records · Page 13Linked to original sources
PubMed2000

Complexed prostate specific antigen provides significant enhancement of specificity compared with total prostate specific antigen for detecting prostate cancer.

PURPOSE: Determining serum total prostate specific antigen (PSA) has proved to be a valuable diagnostic aid for detecting prostatic carcinoma, although the lack of specificity has limited its usefulness. Studies indicate that the use of percent free PSA would improve specificity while maintaining sensitivity. Since complexed PSA represents the major proportion of measurable PSA in serum, we determined whether it represents a single test alternative to the use of percent free PSA for the early detection of prostate cancer. MATERIALS AND METHODS: Archival serum was obtained from 385 men with no evidence of malignancy on biopsy and 272 with biopsy confirmed prostate cancer. We determined the concentration and proportion of total, complexed and free PSA. RESULTS: Receiver operating characteristics analysis using total PSA results from all samples (range 0.32 to 117 ng./ml.) indicated that the areas under the curve for complexed PSA alone as well as the free-to-total and complexed-to-total PSA ratios were similar and significantly greater than those for total PSA alone. Within the range of 85% to 95% sensitivity receiver operating characteristics analysis revealed that the specificity of complexed PSA was higher than that of total PSA and equivalent to that of the free-to-total PSA ratio. We noted a similar improvement in specificity in the 4 to 10 ng./ml. total PSA range. Using published cutoff values for complexed, total and percent free PSA when total PSA was in the 4 to 10 ng./ml. range the sensitivity and specificity of complexed and percent free PSA were similar. Within the 4 to 10 ng./ml. total PSA range the population of patients with no evidence of malignancy and complexed PSA below the upper limit was different with respect to total PSA from that with no evidence of malignancy and free PSA greater than 25%. CONCLUSIONS: The measurement of complexed PSA represents an alternative to the use of percent free PSA, although the patient populations identified by the 2 tests are different.

Humans↗
PubMed2000

Inhibition of cholesteryl ester transfer protein by substituted dithiobisnicotinic acid dimethyl ester: involvement Of a critical cysteine.

SC-71952, a substituted analog of dithiobisnicotinic acid dimethyl ester, was identified as a potent inhibitor of cholesteryl ester transfer protein (CETP). When tested in an in vitro assay, the concentration of SC-71952 required for half-maximal inhibition was 1 microm. The potency of SC-71952 was enhanced 200-fold by preincubation of the inhibitor with CETP, and was decreased 50-fold by treatment with dithiothreitol. Analogs of SC-71952 that did not contain a disulfide linkage were less potent, did not display time dependency, and were not affected by dithiothreitol treatment. Kinetic and biochemical characterization of the inhibitory process of CETP by SC-71952 suggested that the inhibitor initially binds rapidly and reversibly to a hydrophobic site on CETP. With time, the bound inhibitor irreversibly inactivates CETP, presumably by reacting with one of the free cysteines of CETP. Liquid chromatography/mass spectroscopy (LC/MS) analyses of tryptic digests of untreated or SC-71952-inactivated CETP was used to identify which cysteine(s) were potentially involved in the time-dependent, irreversible component of inactivation by the inhibitor. One disulfide bond, Cys143-Cys184, was unaffected by treatment with the inhibitor. Inactivation of CETP by SC-71952 correlated with a progressive decrease in the abundance of free Cys-13 and Cys-333. Conversion of Cys-13 to alanine had no effect on the rapid reversible component of inactivation by SC-71952. However, it abolished the time-dependent enhancement in potency seen with the inhibitor when using wild-type CETP. These data indicate that Cys-13 is critical for the irreversible inactivation of CETP by SC-71952 and provides support for the structural model that places Cys-13 near the neutral lipid-binding site of CETP.

Amino Acid Substitution↗
PubMed1999

Streptavidin-based containment systems for genetically engineered microorganisms.

The use of genetically modified microorganisms for environmental remediation continues to be debated. Conditional lethal systems with tightly regulated gene expression can be used to contain released microorganisms and ameliorate some of the concerns about horizontal gene transfer. We have described streptavidin-based suicide systems to address these concerns and evaluated their function in Pseudomonas putida containing the TOL plasmid for aromatic hydrocarbon metabolism. Tight regulation of expression of a truncated streptavidin gene was required to avoid premature production of the toxic protein. Streptavidin expression was induced by the absence of 3-methyl benzoate (hydrocarbon substrate) which resulted in the elimination of 99.9% of the bacterial culture within eight hours. Low mutant escape rates at 10(-7) per cell per generation were also realized.

Bacteria↗
PubMed1999

APOE epsilon4 allele and amyloid beta-protein deposition in long term survivors of head injury.

Head injury and APOE epsilon4 are risk factors for Alzheimer's disease (AD). We previously found that deposits of amyloid beta-protein (Abeta) occur in fatal head injury, more frequently in patients with APOE epsilon4. We postulated that Abeta deposits triggered by injury could, in survivors, lead to AD-like pathology later in life. Here, we compared Abeta deposits in 21 long term survivors of head injury (up to 20 years) with age and APOE genotype matched controls. In both groups Abeta deposits were more common among patients with APOE epsilon4. However, Abeta deposits were not more common among survivors of head injury than controls. The findings support previous studies associating APOE epsilon4 with deposition of Abeta. However pathogenetic mechanisms other than Abeta deposition may explain the association of head injury with AD.

Adult↗
PubMed1999

CD45-induced tumor necrosis factor alpha production in monocytes is phosphatidylinositol 3-kinase-dependent and nuclear factor-kappaB-independent.

The pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha plays a pivotal role in the pathogenesis of rheumatoid arthritis. The mechanisms involved in regulating monocyte/macrophage TNFalpha production are not yet fully understood but are thought to involve both soluble factors and cell/cell contact with other cell types. Ligation of certain cell surface receptors, namely CD45, CD44, and CD58, can induce the production of TNFalpha in monocytes. In this paper, we investigate further the signaling pathways utilized by cell surface receptors (specifically CD45) to induce monocyte TNFalpha and compare the common/unique pathways involved with that of lipopolysaccharide. The results indicate that monocyte TNFalpha induced upon CD45 ligation or lipopolysaccharide stimulation is differentially modulated by phosphatidylinositol 3-kinase and nuclear factor-kappaB but similarly regulated by p38 mitogen-activated protein kinase. These results demonstrate that both common and unique signaling pathways are utilized by different stimuli for the induction of TNFalpha. These observations may have a major bearing on approaches to inhibiting TNFalpha production in disease where the cytokine has a pathogenic role.

Cells, Cultured↗
PubMed1999

Increased expression of bioactive chemokines in human cerebromicrovascular endothelial cells and astrocytes subjected to simulated ischemia in vitro.

Leukocyte infiltration into the brain has been implicated in the development of ischemic brain damage. In this study, simulated in vitro ischemia/reperfusion and IL-1beta were found to up-regulate both the expression of intercellular adhesion molecule- (ICAM-1) in cultured human cerebromicrovascular endothelial cells (HCEC) and the adhesion of allogenic neutrophils to HCEC. Both HCEC and human fetal astrocytes (FHAS) also responded to IL-1beta and to in vitro ischemia/reperfusion by a pronounced up-regulation of IL-8 and MCP-1 mRNA and by increased release of IL-8 and MCP-1 in cell culture media. FHAS were found to release 30-times higher levels of MCP-1 than HCEC under both basal and ischemic conditions. However, 100 u/ml IL-1beta induced greater stimulation of both IL-8 and MCP-1 secretion in HCEC (50 and 20 times above controls, respectively) than in FHAS (three and two times above controls, respectively). IL-8 was the principal neutrophil chemoattractant released from IL-1beta-treated HCEC, since IL-8 antibody completely inhibited neutrophil chemotaxis enticed by HCEC media. However, the IL-8 antibody neutralized only 50% of IL-1beta-stimulated neutrophil chemoattractants released from FHAS, and 40%-60% of ischemia-stimulated chemotactic activity released by either HCEC or FHAS. These results suggest that simulated in vitro ischemia, in addition to IL-8 and MCP-1, stimulates secretion of other bioactive chemokines from HCEC and FHAS.

Astrocytes↗
PubMed1999

Cell surface localization of tissue transglutaminase is dependent on a fibronectin-binding site in its N-terminal beta-sandwich domain.

Increasing evidence indicates that tissue transglutaminase (tTG) plays a role in the assembly and remodeling of extracellular matrices and promotes cell adhesion. Using an inducible system we have previously shown that tTG associates with the extracellular matrix deposited by stably transfected 3T3 fibroblasts overexpressing the enzyme. We now show by confocal microscopy that tTG colocalizes with pericellular fibronectin in these cells, and by immunogold electron microscopy that the two proteins are found in clusters at the cell surface. Expression vectors encoding the full-length tTG or a N-terminal truncated tTG lacking the proposed fibronectin-binding site (fused to the bacterial reporter enzyme beta-galactosidase) were generated to characterize the role of fibronectin in sequestration of tTG in the pericellular matrix. Enzyme-linked immunosorbent assay style procedures using extracts of transiently transfected COS-7 cells and immobilized fibronectin showed that the truncation abolished fibronectin binding. Similarly, the association of tTG with the pericellular matrix of cells in suspension or with the extracellular matrix deposited by cell monolayers was prevented by the truncation. These results demonstrate that tTG binds to the pericellular fibronectin coat of cells via its N-terminal beta-sandwich domain and that this interaction is crucial for cell surface association of tTG.

3T3 Cells↗
PubMed1999

Urinary kallikrein excretion in normotensive and hypertensive pregnancies: 8 years later.

In a previous longitudinal study of urinary kallikrein (UK) excretion in pregnancy we reported that women with low inactive UK (IUK) to creatinine ratio (IUK:Cr), measured at 16-20 weeks gestation, were at increased risk of developing pregnancy induced hypertension. In this study, 8 years later, we have recalled 14 women who had an IUK:Cr < or = 170 (Gp1) and became hypertensive in late pregnancy and 14 women who had IUK:Cr > 170 and remained normotensive (Gp2). Resting blood pressure (BP) and BP response following application of cutaneous cold to assess vasoconstrictor reactivity were measured. A timed urine sample was also collected for measurements of components of the kallikrein kinin system (KKS): IUK, active UK (AUK), kininogen (UKg), urinary kinins (UKi). Urinary creatinine (UCr) was also measured. The correlation between IUK:Cr during the 1989-1990 study and current measurement was r2 = 0.52, p < 0.0001. Although resting BP was higher in Gp1 than Gp2 this was not significantly different. However, there was a greater change in diastolic BP in Gp1 than in Gp2 in response to the cold pressor test (p < 0.04). Excretion rates of IUK, UKg and UKi were significantly lower in Gp1, p < 0.006, p < 0.003, p < 0.03, respectively. AUK was also reduced in Gp1 but did not reach statistical significance. Women with reduced activity of the renal KKS combined with increased sympathetic drive may be at increased risk of developing hypertension.

Adult↗
PubMed1999

Tissue transglutaminase is an important player at the surface of human endothelial cells: evidence for its externalization and its colocalization with the beta(1) integrin.

We recently reported [J. Cell Sci. 110, 2461-2472 (1997)] that reduced expression of tissue transglutaminase (tTgase, type II) in human endothelial cell line ECV304 led to impaired cell spreading and adhesion; however, there is no immunocytochemical evidence for its presence and specific location at the surface of these cells. In this report we have stably transfected the same cell line with the cDNA for human tTgase which has been tagged at the C-terminus of the encoded protein with a 12-amino-acid peptide from protein kinase C epsilon. Using antibodies directed against this epitope tag peptide we show for the first time using immunogold electron microscopy and fluorescent immunocytochemistry the presence of cell surface-related tTgase. In cells undergoing attachment and cell spreading the enzyme appears to be concentrated at cell adhesion points which are rich in beta(1) integrin, suggesting that these areas may be the initial focal points for enzyme externalization. In more spread and confluent cells the enzyme appears more diffusely distributed along the basal membrane, with increased concentrations found at areas of cell-cell and cell-substratum contact. These findings strengthen the argument for the enzyme's role in cell-matrix interactions.

Base Sequence↗
PubMed1999

Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity.

Because hematopoietic stem cells are rich in aldehyde dehydrogenase (ALDH) activity, we developed a fluorescent substrate for ALDH, termed BODIPY aminoacetaldehyde (BAAA), and tested its potential for isolating primitive human hematopoietic cells. A population of cells with low orthogonal light scattering and bright fluorescence intensity (SSC(lo)ALDH(br) cells) could be readily fractionated from human umbilical cord blood cells costained with BAAA and the multidrug-resistance inhibitor verapamil. The SSC(lo)ALDH(br) population was depleted of lineage-committed cells, 40-90% pure for CD34(+)CD38(lo/-) cells, and enriched 50- to 100-fold for primitive hematopoietic progenitors detected in short- and long-term culture analyses. Together, these observations indicate that fractionating human hematopoietic stem cells on the basis of ALDH activity using BAAA is an effective method for isolating primitive human hematopoietic progenitors. This technique may be useful for isolating stem cells from other tissues as well.

Aldehyde Dehydrogenase↗
PubMed1999

An automated movement assessment panel for upper limb motor functions in rhesus monkeys and humans.

As part of our studies of age-associated changes in motor functions, we have designed an automated movement assessment panel (MAP) to evaluate upper limb and hand movements. Here we describe two versions of the MAP, one for human testing and one for nonhuman primates, and methods for conducting parallel tests in rhesus monkeys and human volunteers. The results are reported from a battery of tests on young adult rhesus monkeys (n = 10, 5-8 years old), young adult human subjects (n = 10, 18-22 years old) and ten aged human subjects (n = 10, 66-68 years old) to demonstrate the capability of the MAP in quantifying arm and hand movement times. The performance times on the two simplest tasks tested were consistent from trial to trial, demonstrating that a stable behavioral baseline could be established for evaluating changes in motor functions over time and assessing treatments for improving motor functions. Motor learning was seen in the more complex movement tasks tested, indicating their usefulness in analyzing this behavior. Finally, age-associated changes in performance times were robustly delineated by the four tasks evaluated in the human subjects.

Adolescent↗
PubMed1999

Interfraction interval does not affect survival of patients with non-small cell lung cancer treated with chemotherapy and/or hyperfractionated radiotherapy: a multivariate analysis of 1076 RTOG patients.

PURPOSE: It was observed by Jeremic et al. that a shorter interfraction interval (IFI) was associated with an improved survival in patients (pts) with locally advanced non-small cell lung cancer (NSCLC) treated with hyperfractionated radiation therapy (HFX-RT), with or without chemotherapy (CT). Our analysis was undertaken to verify this hypothesis. METHODS AND MATERIALS: Records of patients treated on 5 Radiation Therapy Oncology Group (RTOG) studies were reviewed, and an actual IFI, defined as a mean of all daily IFIs, was calculated. RT dose was 1.2 Gy BID to 69.6 Gy. The relationship between the length of IFI and the median survival time and incidence of esophagitis was investigated. RESULTS: In 682 pts eligible for this analysis, a full dose of RT was delivered and at least 90% of all daily IFIs were available. The actual mean IFI was as follows: 4-4.9 h in 51% of pts; 5-5.9 h in 17%; 6-6.9 h in 28% and 7-8 h in 4%. In multivariate analysis, only lack of weight loss, use of CT, low nodal stage and good KPS, but not IFI (4-6 h vs. 6-8 h) were associated with an improved survival for all pts (p values: <0.0001; <0.0001; 0.006; 0.006, and 0.73, respectively), as well as for HFX-RT only pts. For the CT-HFX-RT pts, not enough data points are available for a meaningful analysis. Length of IFI did not influence the incidence of Grade 3 or higher esophagitis (p = 0.82), but use of CT was associated with a 12-fold greater risk of developing severe esophagitis (p < 0.0001). CONCLUSION: Length of IFI (4-6 h vs. 6-8 h) did not influence survival and acute complications incidence in pts with NSCLC treated in RTOG studies with HFX-RT to 69.6 Gy. Previously identified factors, such as use of CT, minimal weight loss, good KPS and low nodal stage, were confirmed again to be associated with a favorable prognosis in a multivariate analysis. Use of CT was associated with a 12-fold greater risk of developing severe esophagitis than HFX-RT alone. It appears that an IFI of 4-8 hr is acceptable in clinical practice for pts with NSCLC, treated with HFX-RT.

Adenocarcinoma↗
PubMed1999

Phosphatidylserine receptors: role of CD36 in binding of anionic phospholipid vesicles to monocytic cells.

Exposure of phosphatidylserine (PtdSer) has been implicated in the recognition and phagocytosis of senescent and apoptotic cells, and CD36 has been proposed as one receptor protein that recognizes PtdSer and other anionic phospholipids. We investigated the binding of phospholipid vesicles to the monocytic leukemia cell lines THP-1 and J774A.1 with a flow cytometric assay; vesicles contained 50 mol% PtdSer, phosphatidylinositol (PtdIns), or phosphatidylglycerol (PtdGro), with the balance being phosphatidylcholine. Specific, high affinity binding was observed for vesicles containing PtdSer, PtdIns, or PtdGro. Specificity of the assay was confirmed by control experiments with erythrocytes, which showed minimal vesicle binding, and with annexin V, which blocked the binding of PtdSer, PtdGro, and PtdIns vesicles to the THP-1 cells. However, O-phospho-L-serine (to 1 mM) had no effect on the binding of PtdSer vesicles, indicating that high affinity binding requires a surface containing multiple phosphoserine groups rather than a single molecule. A monoclonal antibody to CD36 blocked up to 60% of the specific binding of PtdSer vesicles but had minimal to no effect on the binding of PtdGro or PtdIns vesicles. This antibody also selectively inhibited the phagocytosis of PtdSer-containing vesicles as measured by fluorescence microscopy, indicating that CD36 is functionally significant for phagocytosis of this vesicle type. In addition, collagen and thrombospondin, two other putative ligands of CD36, were unable to inhibit the binding of PtdSer vesicles. We conclude that CD36 is the primary protein responsible for the high affinity binding of PtdSer vesicles to these monocyte-like cells. In addition, CD36 appears to be specific for PtdSer among anionic phospholipids, and non-phospholipid ligands of CD36 do not share binding sites with PtdSer on CD36.

Annexin A5↗
PubMed1999

A persistent activity-dependent facilitation in chromaffin cells is caused by Ca2+ activation of protein kinase C.

Activity-dependent facilitation was studied in bovine adrenal chromaffin cells. Stimulation with a train of depolarizations caused subsequent triggered exocytotic activity to be significantly enhanced. After the facilitating stimulus train, the readily releasable vesicle pool (RRP) size was estimated from capacitance jumps in response to paired depolarizations and found to be elevated for a period of at least 10 min. The time dependency of onset and degree of facilitation could be well fitted assuming protein kinase C (PKC)-dependent and independent Ca2+-mediated processes. Both processes increase the recruitment of vesicles from the reserve pool to the RRP, resulting in an greater number of releasable vesicles. The data suggest that cell activity can act as a trigger to increase cytosolic Ca2+ to a level sufficient to cause an increase in the number of readily releasable secretory vesicles, with the more persistent component of the evoked facilitation being mediated through activity-dependent activation of PKC.

Adrenal Glands↗