Your career guide. Winding down, bowing out. Leaving practice, but not the profession.
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Career Mobility↗
Biomedical subjects
L M Walker
Publications and source records attributed to L M Walker.
Nicotinic regulation of c-fos and osteopontin expression in human-derived osteoblast-like cells and human trabecular bone organ culture.
Long-term in vivo studies have highlighted smoking as a risk factor in postmenopausal osteoporosis, bone fracture incidence, and increased nonunion rates. In contrast, there are few data postulating the effects of smoking at the cellular level in human skeletal tissue. In this study, we present novel evidence demonstrating that the nicotinic receptor alpha4 subunit is present in human primary bone cells by using reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, we demonstrate direct cellular effects of nicotine on primary human bone cells and blockage of these effects with a nicotinic receptor antagonist, D-tubocurarine. Nicotine effects on cell proliferation were biphasic with toxic, antiproliferative effects at high levels of nicotine (>1 mmol/L) and stimulatory effects at very low levels (0.01-10 micromol/L) after 72 h. This nicotine-induced increase in cell proliferation was inhibited in a dose-dependent manner by the addition of D-tubocurarine. In addition, proliferation effects from low-level treatment correlated with an upregulation of expression of the AP-1 transcription factor, c-fos, within 1 h, which was blocked by incubation with D-tubocurarine. To determine in situ bone cell responses within their trabecular matrix, cores of human bone isolated from biopsies were perfused with 0.1 micromol/L nicotine for 24 h. Western analysis of proteins isolated from the cores highlighted an increase in osteopontin, a bone matrix protein implicated in regulating resorption, which was partially inhibited by the addition of D-tubocurarine. To conclude, our results suggest that nicotine has a direct effect on human bone cells in modulating proliferation, upregulation of the c-fos transcription factor, and the synthesis of the bone matrix protein, osteopontin.
Base Sequence↗
Oxidative stress and reactive nitrogen species generation during renal ischemia.
Previous evidence suggests that both oxygen radicals and nitric oxide (NO) are important mediators of injury during renal ischemia-reperfusion (I-R) injury. However, the generation of reactive nitrogen species (RNS) has not been evaluated in this model at early time points. The purpose of these studies was to examine the development of oxidant stress and the formation of RNS during I-R injury. Male Sprague-Dawley rats were anesthetized and subjected to 40 min of bilateral renal ischemia followed by 0, 3, or 6 h of reperfusion. Control animals received a sham operation. Plasma urea nitrogen and creatinine levels were monitored as markers of renal injury. Glutathione (GSH) oxidation and 4-hydroxynonenal (4-HNE)-protein adducts were used as markers of oxidant stress. 3-Nitrotyrosine (3-NT) was used as a biomarker of RNS formation. Significant increases in plasma creatinine concentrations and urea nitrogen levels were found following both 3 and 6 h of reperfusion. Increases in GSH oxidation, 4-HNE-protein adduct levels, and 3-NT levels were observed following 40 min of ischemia with no reperfusion. Since these results suggested RNS generation during the 40 min of ischemia, a time course of RNS generation following 0, 5, 10, 20, and 40 min of ischemia was evaluated. Significant increases in 3-NT generation was detected as early as 10 min of ischemia and rose to values nearly 10-fold higher than Control at 40 min of ischemia. No additional increase was observed following reperfusion. The data clearly demonstrate that oxidative stress and RNS generation occur in the kidney during ischemia.
Aldehydes↗
Getting down to the numbers. Can the value of health care really be quantified?
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Absenteeism↗
Is there life after work? You bet!
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Adult↗
Where you go to get away from it all.
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Data Collection↗
Calcium-channel activation and matrix protein upregulation in bone cells in response to mechanical strain.
Femur-derived osteoblasts cultured from rat femora were loaded with Fluo-3 using the AM ester. A quantifiable stretch was applied and [Ca(2+)]i levels monitored by analysis of fluorescent images obtained using an inverted microscope and laser scanning confocal imaging system. Application of a single pulse of tensile strain via an expandable membrane resulted in immediate increase in [Ca(2+)]i in a proportion of the cells, followed by a slow and steady decrease to prestimulation levels. Application of parathyroid hormone (10(-6) M) prior to mechanical stimulation potentiated the load-induced elevation of [Ca(2+)]i. Mechanically stimulating osteoblasts in Ca(2+)-free media or in the presence of either nifedipine (10 microM; L-type Ca(2+)-channel blocker) or thapsigargin (1 microM; depletes intracellular Ca(2+) stores) reduced strain-induced increases in [Ca(2+) ]i. Furthermore, strain-induced increases in [Ca(2+)]i were enhanced in the presence of Bayer K 8644 (500 nm), an agonist of L-type calcium channels. The effects of mechanical strain with and without inhibitors and agonists are described on the total cell population and on single cell responses. Application of strain and strain in the presence of the calcium-channel agonist Bay K 8644 to periosteal-derived osteoblasts increased levels of the extracellular matrix proteins osteopontin and osteocalcin within 24 h postload. This mechanically induced increase in osteopontin and osteocalcin was inhibited by the addition of the calcium-channel antagonist, nifedipine. Our results suggest an important role for L-type calcium channels and a thapsigargin-sensitive component in early mechanical strain transduction pathways in osteoblasts.
Animals↗
Lack of a role for inducible nitric oxide synthase in an experimental model of nephrotic syndrome.
Puromycin aminonucleoside (PAN) administration in rats produces an experimental model of nephrotic syndrome characterized by glomerular epithelial cell injury and proteinuria. The purpose of this study was to examine the role of nitric oxide (NO) in this model of minimal change glomerular disease. Aminoguanidine (AG) was used to inhibit inducible nitric oxide synthase (iNOS). Sprague-Dawley rats were divided into Control (N = 9), PAN (N = 14), AG (N = 2), and PAN + AG (N = 12) treatment groups. Control animals received saline (i.v. ), PAN animals received PAN (75 mg/kg, i.v.), and PAN + AG animals received PAN plus AG (50 mg/kg, i.p., twice daily). AG animals received a saline injection (i.v.) on day 0 in the place of PAN and then AG on the same schedule as the PAN + AG group. Animals were kept in metabolic cages, and urinary protein excretion and nitrite (NO(2)(-)) excretion were measured daily. PAN administration increased urinary NO(2)(-) excretion by day 2, and levels remained elevated through day 7. AG prevented this PAN-induced increase in urinary NO(2)(-) excretion. Plasma nitrate (NO(3)(-)) and NO(2)(-) (NOx) concentrations were also increased in the PAN and PAN + AG groups. iNOS protein expression was not detected in either the glomeruli or the cortex at day 7. Proteinuria developed in PAN animals on day 4 and increased steadily through day 7. PAN + AG animals showed a pattern similar to that of the PAN group. These results indicated that in contrast to models of proliferative glomerulonephritis, NO formation during PAN-induced nephrotic syndrome is increased but does not participate in the development of glomerular injury as measured by proteinuria.
Animals↗
Role of nitric oxide in lipopolysaccharide-induced oxidant stress in the rat kidney.
Lipopolysaccharide (LPS)-induced renal oxidant injury and the role of nitric oxide (NO) were evaluated using the inducible nitric oxide synthase (iNOS) inhibitor L-iminoethyl-lysine (L-NIL). One group of male rats received LPS (Salmonella minnesota; 2 mg/kg, i.v.). A second group received LPS plus L-NIL (3 mg/kg, i.p.). A third group received saline i.v. At 6 hr, iNOS protein was induced in the kidney cortex, and plasma nitrate/nitrite levels were increased from 4 +/- 2 nmol/mL in the Saline group to 431 +/- 23 nmol/mL in the LPS group. The value for the LPS + L-NIL group was reduced significantly to 42 +/- 9 nmol/mL. LPS increased blood urea nitrogen levels from 13 +/- 1 to 47 +/- 3 mg/dL. LPS + L-NIL reduced these levels significantly to 29 +/- 2 mg/dL. Plasma creatinine levels were unchanged in all groups. Tissue lipid peroxidation products in the kidney were increased from 0.16 +/- 0.01 nmol/mg in the Saline group to 0.30 +/- 0.03 nmol/mg in the LPS group. LPS + L-NIL reduced the values significantly to 0.22 +/- 0.02 nmol/mg. Intracellular glutathione levels were decreased in the kidneys from 1.32 +/- 0.1 nmol/mg in the Saline group to 0.66 +/- 0.08 nmol/mg in the LPS group. LPS + L-NIL increased the levels significantly to 0.99 +/- 0.13 nmol/mg. LPS increased the 3-nitrotyrosine-protein adducts in renal tubules as detected by immunohistochemistry, indicating the generation of peroxynitrite. L-NIL decreased adduct formation. These data indicated that LPS-induced NO generation resulted in peroxynitrite formation and oxidant stress in the kidney and that inhibitors of iNOS may offer protection against LPS-induced renal toxicity.
Animals↗
Hormonal regulation of [Ca(2+)](i) in periosteal-derived osteoblasts: effects of parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandin E(2).
The effects of hormonal modulators of osteoblast function, parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandins on [Ca(2+)](i) in periosteal-derived osteoblasts from rat femurs have been investigated. Our results show that application of parathyroid hormone PTH (10(-5) M) and prostaglandin E(2) (PGE(2)) (4 microM) result in a rapid heterogeneous elevation in [Ca(2+)](i) that, in the case of PTH, is dependent on both extracellular and intracellular sources of calcium. Variable responses to treatments have been found within populations of cells. The PGE(2) response is dose dependent. Treatment with 1,25(OH)(2)D(3) (10(-8) M) induces a brief (60-90 sec) elevation in [Ca(2+)](i) that is almost totally abolished in EGTA-buffered Ca(2+)-free medium. Interactive effects of multiple hormone treatments have been observed. Pretreatment with 1,25(OH)(2)D(3) results in near-total inhibition of the PTH and PGE(2) responses. In conclusion, modulation of [Ca(2+)](i) appears to play a role not only in the direct effects of osteotropic hormones on osteoblasts but also in the synergistic and antagonistic effects between circulating hormones.
Aniline Compounds↗
AXR2 encodes a member of the Aux/IAA protein family.
The dominant gain-of-function axr2-1 mutation of Arabidopsis causes agravitropic root and shoot growth, a short hypocotyl and stem, and auxin-resistant root growth. We have cloned the AXR2 gene using a map-based approach, and find that it is the same as IAA7, a member of the IAA (indole-3-acetic acid) family of auxin-inducible genes. The axr2-1 mutation changes a single amino acid in conserved domain II of AXR2/IAA7. We isolated loss-of-function mutations in AXR2/IAA7 as intragenic suppressors of axr2-1 or in a screen for insertion mutations in IAA genes. A null mutant has a slightly longer hypocotyl than wild-type plants, indicating that AXR2/IAA7 controls development in light-grown seedlings, perhaps in concert with other gene products. Dark-grown axr2-1 mutant plants have short hypocotyls and make leaves, suggesting that activation of AXR2/IAA7 is sufficient to induce morphological responses normally elicited by light. Previously described semidominant mutations in two other Arabidopsis IAA genes cause some of the same phenotypes as axr2-1, but also cause distinct phenotypes. These results illustrate functional differences among members of the Arabidopsis IAA gene family.
Arabidopsis↗
Oxidant stress in rat liver after lipopolysaccharide administration: effect of inducible nitric-oxide synthase inhibition.
The role of inducible nitric-oxide synthase (iNOS) in lipopolysaccharide (LPS)-induced hepatic oxidant stress was evaluated using the iNOS inhibitor L-iminoethyl-lysine (L-NIL). Male rats were divided into three groups. One group received LPS (Salmonella minnesota) 2 mg/kg i.v. A second group received LPS plus L-NIL (3 mg/kg i.p.) at the time of LPS administration followed by a second dose 3 h later. A third group received saline i.v. At 6 h, blood and liver tissue were collected. Serum nitrate/nitrite (metabolic products of nitric oxide) levels were increased from 5.4 +/- 1.5 nmol/ml in the saline group to 360 +/- 48 nmol/ml in the LPS group (n = 5). Values for the LPS + L-NIL group were significantly reduced to 35 +/- 7 nmol/ml. Tissue malondialdehyde levels were increased from 0.20 +/- 0.02 nmol/mg (n = 4) in the saline group to 0.41 +/- 0.03 nmol/mg (n = 4) in the LPS group. L-NIL significantly reduced the values in the LPS group to 0.29 +/- 0.02 nmol/mg (n = 4). 4-Hydroxynonenal-protein adducts levels were increased 3.6-fold by LPS treatment as compared with saline. L-NIL significantly reversed the levels to 1.6-fold (n = 4). Intracellular GSH levels were decreased from 8.49 +/- 0.64 nmol/mg (n = 4) in the saline group to 5.63 +/- 0.51 nmol/mg in the LPS group (n = 7). L-NIL significantly increased the levels in the LPS group to 7.04 +/- 0.46 nmol/mg (n = 7). These data indicate that LPS-induced nitric oxide generation can result in oxidant stress in the liver, and that inhibitors of iNOS may offer some protection in LPS-induced hepatic toxicity.
Animals↗
Evidence for peroxynitrite formation in renal ischemia-reperfusion injury: studies with the inducible nitric oxide synthase inhibitor L-N(6)-(1-Iminoethyl)lysine.
Reactive oxygen species are suggested to participate in ischemia-reperfusion (I-R) injury. However, induction of inducible nitric oxide synthase (iNOS) and production of high levels of nitric oxide (NO) also contribute to this injury. NO can combine with superoxide to form the potent oxidant peroxynitrite (ONOO(-)). NO and ONOO(-) were investigated in a rat model of renal I-R injury using the selective iNOS inhibitor L-N(6)-(1-iminoethyl)lysine (L-NIL). Sprague-Dawley rats were subjected to 40 min of bilateral renal ischemia followed by 6 h of reperfusion with or without L-NIL administration. Control animals received a sham surgery and had plasma creatinine values of 0.4 +/- 0.1 mg/dl. I-R surgery significantly increased plasma creatinine levels to 1.9 +/- 0.3 mg/dl (P <.05) and caused renal cortical necrosis. L-NIL administration (3 mg/kg) in animals subjected to I-R significantly decreased plasma creatinine levels to 1.2 +/- 0.10 mg/dl (P <.05 compared with I-R) and reduced tubular damage. ONOO(-) formation was evaluated by detecting 3-nitrotyrosine-protein adducts, a stable biomarker of ONOO(-) formation. Immunohistochemistry and HPLC revealed that the kidneys from I-R animals had increased levels of 3-nitrotyrosine-protein adducts compared with control animals. L-NIL-treated rats (3 mg/kg) subjected to I-R showed decreased levels of 3-nitrotyrosine-protein adducts. These results support the hypothesis that iNOS-generated NO mediates damage in I-R injury possibly through ONOO(-) formation.
Animals↗
Smoke exposure in dogs: 27 cases (1988-1997).
OBJECTIVE: To evaluate clinical findings, prognostic variables, and clinical course of dogs exposed to smoke. DESIGN: Retrospective study. ANIMALS: 27 dogs exposed to smoke in residential fires. PROCEDURE: Medical records were reviewed for 1988-1997. Time of year, signalment, interval from fire to arrival at veterinary hospital, duration of smoke exposure, clinical signs at the fire scene, physical examination findings, changes in respiratory tract signs, radiographic findings, hematologic and arterial blood gas analyses, initial treatment, clinical course, and outcome were recorded. RESULTS: 27 dogs (16 uncomplicated and 11 complicated cases) were identified. In the complicated group, 4 dogs died, 4 were euthanatized, and 3 had a complicated clinical course. Stupor or coma (8 of 17 dogs), coughing or gagging (6), and respiratory difficulty (6) were commonly observed at the fire scene. Substantial improvement was evident within 5 to 30 minutes after dogs were given supplemental oxygen at the fire scene. More severely affected dogs had a higher median PCV (58%) than less severely affected dogs (50%). Most common thoracic radiographic findings were an alveolar (10 dogs) or an interstitial (3) pattern. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs exposed to smoke can develop respiratory or neurologic complications. Monitoring progression of respiratory problems on the day after the fire may provide clinicians with clues about the severity of each dog's condition.
Animals↗
Smoke exposure in cats: 22 cases (1986-1997).
OBJECTIVE: To review clinical findings and clinical course for cats exposed to smoke in residential fires and to determine clinical variables that may have prognostic importance. DESIGN: Retrospective study. ANIMALS: 22 cats admitted to our veterinary teaching hospital between 1986 and 1997 with a history of smoke exposure during a residential fire. PROCEDURE: Medical records were reviewed for history, clinical signs, physical examination findings, changes in respiratory tract signs, initial hematologic analysis, treatment, results of thoracic radiography, and outcome. RESULTS: Fifteen of 22 (68%) cats were categorized in the uncomplicated group, 5 (23%) in the complicated group, and 2 (9%) were discharged after a short period because of financial considerations. Twenty (91%) cats survived, but 2 (9%) were euthanatized because of severe respiratory compromise or neurologic changes. Predominant thoracic radiographic changes were diffuse interstitial pattern (6 cats) and focal alveolar pattern (5). The majority (8/13) of cats that were stable or had improved by the day after admission had an uncomplicated clinical course while hospitalized, whereas cats that were worse on the day after admission tended to have a complicated clinical course. CONCLUSIONS AND CLINICAL RELEVANCE: Cats that survive a residential fire and are admitted to a hospital have a good chance to be discharged. Cats that do not have signs of respiratory tract dysfunction at admission probably will not develop severe respiratory complications. For cats with signs of respiratory dysfunction at admission, better prognostic information will be determined by monitoring progression of the respiratory condition on the day after admission.
Animals↗
Mechanical manipulation of bone and cartilage cells with 'optical tweezers'.
The single beam optical gradient trap (optical tweezers) uses a single beam of laser light to non-invasively manipulate microscopic particles. Optical tweezers exerting a force of approximately 7 pN were applied to single bone and cartilage derived cells in culture and changes in intracellular calcium levels were observed using Fluo-3 labelling. Human derived osteoblasts responded to optical tweezers with an immediate increase in [Ca2+]i that was inhibited by the addition of a calcium channel blocker nifedipine. Force applied to different regions of cells resulted in a variable response. [Ca2+]i elevation in response to load was lower in rat femur derived osteoblasts, and not apparent in primary chondrocytes and the osteocytic cell line (MLO Y4).
Calcium↗
Radiographic, ultrasonographic, and endoscopic findings in cats with inflammatory bowel disease of the stomach and small intestine: 33 cases (1990-1997).
OBJECTIVE: To characterize imaging findings in cats with confirmed inflammatory bowel disease (IBD) of the upper gastrointestinal tract (i.e., stomach and small intestine) and relate these findings to clinical signs and histologic changes. DESIGN: Retrospective study. ANIMALS: 32 cats with clinical and histopathologic diagnoses of IBD. PROCEDURE: Medical records were reviewed for signalment, clinical signs, clinicopathologic findings, radiographic and ultrasonographic findings, and results of endoscopic examination. Histologic findings were reviewed and characterized by severity and type of inflammatory infiltrate. RESULTS: All cats had 1 or more clinical signs (e.g., vomiting, diarrhea, weight loss, and anorexia) consistent with IBD. Lymphocytic and plasmacytic infiltrates were observed in histologic sections of gastrointestinal tissue. Crypt distortion, villous blunting and fusion, and fibrosis were most commonly seen in cats with moderate or severe IBD. Clinicopathologic findings of some cats included anemia, leukocytosis or leukopenia, hypocholesterolemia, and hyper- or hypoproteinemia. Abnormalities were not found on abdominal radiographic views in 9 of 9 cats. However, contrast studies using barium revealed radiographic abnormalities in 1 of 3 cats. In 13 of 17 cats, abdominal ultrasonography revealed several intestinal abnormalities (e.g., poor intestinal wall layer definition, focal thickening) and large mesenteric lymph nodes with hypoechoic changes consistent with IBD. Endoscopic observation revealed findings (e.g., erythema, plaques, mucosal friability) consistent with inflammation in 9 of 18 cats. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with endoscopy of the gastrointestinal tract or abdominal radiography, clinical signs and ultrasonographic findings appear to have the best association with histologic grade of IBD in cats.
Anemia↗