Search PubMed⌕ Search

Biomedical subjects

L A Davidson

Publications and source records attributed to L A Davidson.

At least 37 records · Page 2Linked to original sources
PubMed1997

Butyrate alters activity of specific cAMP-receptor proteins in a transgenic mouse colonic cell line.

There is great interest in utilizing butyrate as a chemotherapeutic agent. To elucidate its mechanism of action, the effect of butyrate on cAMP receptor protein kinase (PKA) activity in young adult mouse colon (YAMC) cells isolated from transgenic mice bearing a temperature sensitive mutation of the SV40 large T antigen gene was investigated. Conditionally immortalized cultures were plated at the permissive temperature (33 degrees C) or growth arrested by incubation at the nonpermissive temperature (39 degrees C). In addition, cells were incubated at 33 degrees C with or without 1 mmol/L butyrate for 24 h. Butyrate treatment reduced cell proliferation by 28% and enhanced apoptosis by 350% compared with cultures not exposed to butyrate. The PKA type I/II isozyme activity ratio was lower (P < 0.05) in cells incubated with butyrate. The relative level of PKA I isozyme was higher in proliferating cells at 33 degrees C (63% of total PKA), while the relative level of PKA II was higher in nonproliferating cells undergoing apoptosis at 39 degrees C (59% of total PKA). Neither incubation conditions (33 vs. 39 degrees C) nor butyrate treatment altered total PKA activity. When YAMC cells were incubated with 8-CI-cAMP, an activator of PKA II, growth was markedly inhibited in cells at both temperatures. Consistent with in vitro data, increased PKA I isozyme levels were associated with dysregulated growth in vivo. Specifically, the relative level of PKA I isozyme was three- to fivefold higher in rat colonic tumors compared with normal nontransformed colonic mucosa. These data indicate that the biological effects of butyrate on colonocyte proliferation and apoptosis are associated with changes in PKA isozyme-dependent signal transduction, and the YAMC cell line is a relevant model to examine the molecular mechanisms by which dietary-derived factors affect relative cancer risk.

Animals↗
PubMed1997

Intrahepatic splenic tissue.

Intrahepatic splenic tissue is uncommon being reported to date in three humans and one pig. This report is of a 54 year old man with chronic asthma who died from acute bronchial asthma. Twenty years previously he had undergone a splenectomy (the spleen was histologically normal). Necropsy revealed a well defined, smooth bordered, bilobed red mass on the left hepatic lobe; one lobe projected outwards the other was embedded in the liver. Histologically the mass was splenic tissue. This case of intrahepatic splenic tissue differing from the three human cases reported previously in that there was a common capsule beneath which splenic pulp directly abuts on hepatic tissue. This suggests that this case is more probably one of hyperplasia of congenitally ectopic splenic tissue following splenectomy rather than limited splenosis after implantation onto the serosal surface of splenic tissue released by trauma. Splenic ectopia should be considered in the differential diagnosis of hepatic lesions detected post-splenectomy and the liver should be considered as a possible site of residual splenic tissue if splenic function returns following splenectomy.

Choristoma↗
PubMed1996

Modulation of protein kinase C-related signal transduction by 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibits cell cycle dependence.

The modulation of protein kinase C (PKC)-mediated protein phosphorylation in quiescent vascular smooth muscle cells (SMCs) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exhibits a discrete temporal pattern in which early reductions of kinase activity are followed by marked increases in activity. This profile may be accounted for by transcriptional- and/or cell cycle-related effects of TCDD. To test this hypothesis, experiments were conducted to examine the influence of TCDD on PKC activity during different phases of the cell cycle in vascular (aortic) SMCs. Increased PKC activity was observed in the cytosolic and particulate fractions of randomly cycling SMC cultures derived from female rats treated in vivo with 10 microgram/kg TCDD relative to corn oil. Treatment of cycling naive SMC cultures with TCDD (0.1 to 1000 nM) for 0.5 h caused a concentration-dependent increase of particulate PKC activity and no changes in cytosolic counterparts. Extended challenge of SMCs with TCDD for 24 h increased PKC activity in both cellular fractions. Incubation of SMCs with various concentrations of fetal bovine serum for 72 h to differentially regulate cell cycling followed by challenge with 10 nM TCDD for 24 h reduced cytosolic and particulate PKC activity in quiescent cells, but enhanced activity in cycling cells. To determine if this serum-related profile was strictly dependent upon cell cycle-related events, SMCs were synchronized in the G0 phase and then pulsed with 10 nM TCDD during different phases of the cell cycle. Differential profiles were observed where reduced C-kinase activity occurred during the G0/G1 transition followed by increases during G1/S and no changes during S. Western blot analysis confirmed the patterns of PKC activity observed during the G0/G1 and G1/S transitions. PKCalpha, beta II, and delta isoforms were reduced during G0/G1, while only PKCbetaII and delta were increased during G1/S. These data show that modulation of PKC by TCDD in vascular SMCs exhibits cell cycle dependence and isoform specificity.

Animals↗
PubMed1996

Lung tumours immunoreactive for parathyroid hormone related peptide: analysis of serum calcium levels and tumour type.

Secretion by tumours of parathyroid hormone-related peptide (PTHrP) in quantities sufficient to raise circulating levels results in the syndrome of humoral hypercalcaemia of malignancy (HHM). Since HHM is commonly associated with squamous carcinoma of lung and rarely with adenocarcinoma or lung neuroendocrine tumours, immunopositivity was related to tumour type, to assess whether this difference was due to a low general incidence of PTHrP expression in the latter two groups. Seventy-six of 82 tumours were immunopositive: 22 of 22 squamous carcinomas, 21 of 25 small cell lung carcinomas, 14 of 15 carcinoids, and 19 of 20 adenocarcinomas. These data confirm and extend previous observations on squamous and neuroendocrine tumours but are in contrast with previous findings in adenocarcinoma, which have suggested that only a small proportion of cases express the peptide. They suggest that the differences in incidence of HHM in the various tumour types are due to patterns of secretion, rather than differences in expression of PTHrP. The second aim of this study was therefore to assess whether tumours immunopositive for PTHrP, but not associated with HHM, might secrete PTHrP at levels which might result in more subtle changes in calcium metabolism. Preoperative calcium levels were analysed in a series of 56 patients with immunopositive lung tumours of all types. One small cell carcinoma was associated with hypercalcaemia, but there was no evidence of any other alteration in serum calcium. These data indicate that the majority of tumours expressing PTHrP do not secrete it in amounts sufficient to alter calcium metabolism.

Adenocarcinoma↗
PubMed1996

Rapid competitive PCR determination of relative gene expression in limiting tissue samples.

Reverse transcriptase (RT)-PCR is widely used to study gene transcription in many biological systems. Despite the development of a variety of procedures, quantification of RT-PCR products remains difficult, particularly when processing a large number of samples. Therefore, we developed a novel alternative PCR technique that we term "rapid competitive PCR" (RC-PCR), designed to study the relative expression of specific genes in a large number of small tissue biopsies. RC-PCR is characterized by measuring relative gene expression at the mRNA level of two or more samples with a nonradioactive assay based on competitive PCR amplification between identical sequences of internal standard and target cDNA. Only a single reaction tube per sample is used in this technique, and it was validated by comparing RC-PCR of protein kinase C zeta and alpha expression in rat colonic mucosa samples with competitive RT-PCR analysis (requiring 6-8 reaction tubes per sample). We conclude that RC-PCR is a simple, rapid, highly sensitive technique that is capable of detecting less than twofold differences in mRNA expression.

Animals↗
PubMed1996

Early post-operative mortality following primary total hip replacement.

A study was undertaken to determine the excess risk of death following primary total hip replacement (THR). All patients who underwent THR or were placed on the waiting list for THR during an 18-month period were studied as to their mortality. The dates of all deaths among both of these populations, and age- and sex-specific mortality rates for the Scottish population, were obtained from the Registrar General for Scotland. It was possible to determine a mortality rate for those patients operated upon and those patients who waited 1 year for their operation. The crude mortality rate, standardized mortality ratio (SMR) and excess risk of death were calculated for the patients operated upon. The crude mortality rate was 1.8% and the SMR was 45.5 for the operated-upon group. The excess risk of death associated with THR within the first 3 post-operative months was calculated in two ways. The relative mortality ratio was determined to be 2.37, and the comparative mortality ratio was calculated to be 1.6 for the operated-upon population. Patients selected for THR are in general fitter than average, and the excess risk of death in the first 3 post-operative months after THR is of the order of 1.6.

Case-Control Studies↗
PubMed1995

Dietary fat and fiber alter rat colonic protein kinase C isozyme expression.

To better understand the biochemical mechanisms by which select fats and fibers modulate colonic cell proliferation, we determined the profile of protein kinase C (PKC) isozymes and cell proliferation in rat proximal and distal colonic mucosa following diet manipulation, because enhanced cell proliferation has been correlated with colon cancer incidence. Rats were assigned to one of four diets (each with 15 g fat + 6 g fiber/100 g diet) for 3 wk: fiber-free fish oil (FF), fiber-free corn oil (FC), cellulose + corn oil (CC), or pectin + corn oil (PC). Stead-state levels of colonic mucosal cytosolic and membrane PKC isozymes were determined. In vivo cell proliferation was determined by bromodeoxyuridine incorporation into DNA. In addition, viable exfoliated colonic epithelial cells were isolated from feces using Percoll-bovine serum albumin gradients. We found that 1) proximal and distal colonic mucosa possessed different steady-state levels and relative proportions of PKC isozymes; 2) PKC alpha and delta expression were significantly greater in distal membrane of the PC-fed group compared with the other dietary groups; 3) the number of exfoliated cells per 4-h fecal collection generally was proportional to the diet-induced changes in cell proliferation (PC > FC > CC > FF). These data demonstrate that dietary treatment altered colonic PKC isozyme expression, with animals fed the fiber-containing diets generally expressing higher steady-state levels of PKC alpha and delta.

Animals↗
PubMed1995

How do sea urchins invaginate? Using biomechanics to distinguish between mechanisms of primary invagination.

The forces that drive sea urchin primary invagination remain mysterious. To solve this mystery we have developed a set of finite element simulations that test five hypothesized mechanisms. Our models show that each of these mechanisms can generate an invagination; however, the mechanical properties of an epithelial sheet required for proper invagination are different for each mechanism. For example, we find that the gel swelling hypothesis of Lane et al. (Lane, M. C., Koehl, M. A. R., Wilt, F. and Keller, R. (1993) Development 117, 1049-1060) requires the embryo to possess a mechanically stiff apical extracellular matrix and highly deformable cells, whereas a hypothesis based on apical constriction of the epithelial cells requires a more compliant extracellular matrix. For each mechanism, we have mapped out a range of embryo designs that work. Additionally, the simulations predict specific cell shape changes accompanying each mechanism. This allows us to design experiments that can distinguish between different mechanisms, all of which can, in principle, drive primary invagination.

Animals↗
PubMed1995

Noninvasive detection of putative biomarkers for colon cancer using fecal messenger RNA.

Deaths from colon cancer number over 60,000 each year in the United States. Because early detection results in a high cure rate, development of noninvasive techniques for detection of colon cancer has received much interest. The ability to detect early changes in colonocyte genes and gene expression would provide valuable information. We have shown previously that alterations in protein kinase C (PKC) isoform expression are associated with changes in colonic cell proliferation, a key intermediate marker for the prediction of tumorigenesis. Here, we describe a method for the quantitative detection of mRNAs for select PKC isoforms isolated from rat feces containing exfoliated colonocytes. After total RNA extraction from fresh fecal material, polyadenylated RNA was selectively purified and quantitated with slot blotting and hybridization to oligodeoxythymidylic acid. Fecal polyadenylated RNA was used for semiquantitative (mimic) RT-PCR to quantitate PKC isoform mRNA expression. We detected mRNA for PKC-alpha, PKC-delta, PKC-epsilon, and PKC-sigma, but not for PKC-beta or PKC-gamma, which is consistent with the profile of isoforms detected previously in scraped colonic mucosa using immunoblot analysis. This noninvasive method, utilizing feces containing exfoliated colonocytes, is a sensitive noninvasive technique for quantitating luminal mRNAs. This provides a means to monitor gene expression of colonic epithelial cells, which may have predictive value in monitoring the neoplastic process.

Animals↗
PubMed1995

Localization of protein kinase C isozymes in rat colon.

We have demonstrated previously the presence of classical (alpha), novel (delta and epsilon), and atypical (zeta) protein kinase C (PKC) isozymes in human and rat colonic mucosa (L. A. Davidson et al., Arch. Biochem. Biophys., 312:547-553, 1994). To gain insight into the functions of individual PKC isozymes in colonic epithelium in situ, we determined the localization of the major PKC isozymes expressed in normal rat colonic epithelial cells using in situ reverse transcription (RT)-PCR and immunohistochemistry (IH). Cytokeratin, a positive biological control known to be expressed in epithelial cells, was shown by in situ RT-PCR and IH to be expressed only in epithelial cells within the colonic crypt. PKC gamma, a negative control for the colon since it is expressed only in the central nervous system, was not detectable in colon sections by either methodology. In situ RT-PCR analysis revealed that PKC alpha, delta, epsilon, and zeta mRNAs are expressed in epithelial cells along the entire colonic crypt. In addition, PKC delta and zeta mRNA are expressed in the stromal layer. All four PKC isozymes in the colonic epithelial cells were also detected by IH. However, in general, isozyme protein expression was greater at the top of the crypt axis, associated primarily with cells having acquired a differentiated phenotype. These results suggest that PKC isozyme protein expression may be localized to mature differentiated cells at the top of the colonic crypt. Therefore, PKC isozyme-dependent signal transduction may play a role in colonic epithelial cell ontogeny along the colonic crypt axis.

Animals↗
PubMed1994

Dietary modulation of rat colonic cAMP-dependent protein kinase activity.

Malignant transformation of cells is associated with enhanced proliferation and alterations in cAMP-dependent protein kinase (PKA) activity. To investigate the role of PKA in normal colonic cell proliferation, PKA was characterized in rat colonic mucosa. In addition, rats were fed diets containing different fats (corn oil, fish oil) and fibers (pectin, cellulose, fiber free) to elicit varying levels of colonic cell proliferation in order to study this signaling system under normal physiologic conditions. Overall, PKA activities were higher in cytosolic compared to membrane fractions. PKA type II (PKA II) isozyme contributed 89 +/- 1% and 96 +/- 1% of total PKA activity in cytosolic and membrane fractions, respectively. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of mRNA for both the alpha and beta isoforms of the regulatory subunits of PKA II. PKA activities were 21-33% higher in distal membrane and total distal fractions in rats fed a cellulose/corn oil diet compared to animals consuming the other fiber/fat diets. These effects were seen only in the distal colon, where the number of cells per crypt column was elevated only in animals fed the cellulose/corn oil diet relative to other diets. Diet-induced mitogenic responses did not involve significant changes in the relative activity of PKA I and II isozymes. These data demonstrate that dietary effects on PKA activity in the distal colon may be related to changes in cell differentiation as indicated by the number of cells per crypt column.

Animals↗
PubMed1994

Protein kinase C isoforms in human and rat colonic mucosa.

The protein kinase C (PKC) family of enzymes plays a key role in the regulation of cellular events, including cell proliferation and differentiation. Work from our laboratory has shown that the effects of dietary fat and fiber on colonic cell proliferation were positively correlated with membrane/cytosol PKC activity ratios (Chapkin et al., 1993, J. Nutr. 123, 649-655). The presence and subcellular distribution of specific PKC isoforms in rat and human colon were therefore determined in cytosolic and membrane extracts. Tissue extracts were probed with antibodies to individual PKC isoforms. PKC alpha, beta, delta, epsilon, and zeta were detected in both rat and human colonic mucosa, while PKC eta was detected in human colonic mucosa only. PKC alpha, beta, and zeta were predominantly localized in the cytosolic fraction, whereas the majority of PKC delta, epsilon, and eta were found in the membrane-associated fraction. Presence of mRNA for individual PKC isoforms was determined by reverse transcriptase PCR (RT-PCR). Using rat colonic mucosa, mRNA for PKC alpha, beta, delta, epsilon, eta, and zeta were detected by RT-PCR with identity confirmed by sequencing. The relative steady-state levels of PKC isoforms in human colon adenocarcinoma as compared with normal colonic mucosa were determined, with adenocarcinomas having higher amounts of cytosolic PKC beta, delta, epsilon, eta, and zeta. PKC isoforms were also detected in viable, exfoliated colonic cells isolated from human feces, demonstrating that this noninvasive method can be utilized to examine PKC expression in colonic cells. These results demonstrate that colonic mucosa expresses both calcium-dependent (classical) and calcium-independent (novel and atypical) PKC isoforms with distinct subcellular distributions for each. The dynamics of these PKC isoforms may have implications in the development of colon carcinogenesis.

Adenocarcinoma↗
PubMed1994

Characterization of a particulate pathway for copper in K562 cells.

More than half of the 67Cu recovered from K562 cells following a brief incubation with 67Cu-ceruloplasmin was recovered in particulate fractions of the cell. The fractions in Percoll had densities that ranged between 1.040 and 1.060 g/dl. In as early as 5 min, two fractions, densities of 1.051 and 1.056, respectively, were discernible. Components in the 1.051 fraction tested positive for clathrin and catalase. Those in the 1.056 fraction sedimented near the marker for lysosomes. The 67Cu in both fractions was stable to treatment by EDTA, nitrilotriacetate, alpha,alpha'-dipyridyl, heparinase, and ascorbate, but dissociated when treated with pronase, trypsin, or sodium dodecylsulfate. Continuous incubation with 67Cu-ceruloplasmin intensified the 67Cu activity in the 1.051 and 1.056 fractions. Cells incubated with 125I-transferrin displayed the label primarily in the 1.051 fraction. Continuous incubation intensified the label but unlike 67Cu, it did not shift to lighter or heavier fractions. Electron micrographs of the 1.051 fraction showed fields dominated by membranous structures some of which were enclosed. Micrographs of whole cells showed numerous invaginations resembling coated pits with sealed structures along and beneath the membrane surface suggesting the membrane was engaged in a rather extensive endocytosis. These data provide evidence that a large fraction of Cu from ceruloplasmin enters the K562 cell bound to membranous-like vesicles, part of which are sealed and coated with clathrin. This particulate pathway accounts for most of the copper entering the cell.

2,2'-Dipyridyl↗
PubMed1992

Phospholipid molecular species composition of mouse liver nuclei. Influence of dietary n-3 fatty acid ethyl esters.

The effect of dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ethyl esters on the individual molecular species composition of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was determined in mouse liver nuclei. After a 10 day feeding period, there was a depletion of the sn-2 position of n-6 polyunsaturated fatty acids (PUFA) and substitution with n-3 PUFA. EPA feeding significantly increased (P less than 0.05) diacyl PC and PE 16:0-20:5, n-3, 16:0-22:6,n-3, 18:0-20:5,n-3 and 18:0-22:6,n-3 relative to control (safflower oil ethyl ester fed) animals. In comparison, DHA feeding significantly increased (P less than 0.05) 22:6 n-3-containing species, specifically 18:1-22:6,n-3, 16:0-22:6,n-3 and 18:0-22:6,n-3 in PC, and 18:1-22:6,n-3, 16:0-22:6,n-3 and 18:0-22:6,n-3 in PE. In addition, the presence of 18:0-20:5,n-3 PC in the nuclei of DHA-fed rats and of 18:2-20:5,n-3, 18:1-20:5,n-3 and 18:0-20:5,n-3 in nuclear PE indicate that incorporation of DHA retroconversion (22:6,n-3-->20:5,n-3) products. These results indicate both EPA and DHA are extensively incorporated into nuclear phospholipids, and therefore could potentially influence gene function.

Animals↗
PubMed1990

Fecal alpha 1-antitrypsin in breast-fed infants is derived from human milk and is not indicative of enteric protein loss.

Alpha 1-antitrypsin is a serum protein commonly used as a marker of enteric protein loss. In this study, we have quantified alpha 1-antitrypsin concentration in human milk and its excretion by healthy term breast-fed infants. We found high concentrations of alpha 1-antitrypsin in early milk (0.3-0.6 mg/ml during the first week of lactation) while the concentration fell during subsequent weeks, being detectable through at least 3-4 months of lactation. Significant quantities of intact alpha 1-antitrypsin were found to be excreted by the breast-fed infants studied. The amount excreted was typically higher in early weeks (as much as 200 mg/24 h) and decreased with infant age, possibly due to both decreased intake from the milk as well as increased digestion of the protein by the maturing infant. In vitro studies demonstrated that the trypsin-alpha 1-antitrypsin complex resisted proteolysis by pepsin and pancreatic enzymes; thus, alpha 1-antitrypsin in milk can escape gastrointestinal degradation. We conclude that alpha 1-antitrypsin is not a suitable marker for intestinal protein loss by term breast-fed infants.

Breast Feeding↗