PAK1 is involved in sensing the orientation of collagen stiffness gradients in mouse fibroblasts.

Migrating cells sense variations of stiffness in connective tissue matrices but how cells detect and respond to stiffness orientation is not defined. We examined cell extension formation on collagen with underlying support (vertical stiffness gradient) or on collagen laterally supported by nylon (lateral stiffness gradient). At 6 h after plating, cells plated on laterally-supported collagen exhibited >2-fold more abundant and ~2-fold longer cell extensions than cells plated on collagen with underlying support. We examined whether p21-activated kinase 1 (PAK1) influences extension formation that is dependent on the orientation of support. At 6 h after plating on collagen with underlying support, wild-type cell extensions were 40% shorter than PAK1 knockdown cells. In contrast, on laterally-supported collagen, wild-type cell extensions were 2-fold longer than PAK1 knockdown cells. In cells plated on laterally-supported collagen, there were ~2-fold reductions of collagen fiber alignment and compaction in PAK1 knockdown cells compared with wild-type cells. PAK1 knockdown did not affect collagen fiber alignment or compaction by cells plated on collagen with underlying support. Wild-type cells with lateral support of collagen exhibited 3-fold increases of phospho-myosin staining at 6h, which was 2-fold lower in PAK1 knockdown cells. In contrast, cells on collagen with underlying support showed no increase of phospho-myosin staining at any times. PAK1 knockdown did not affect α2 or ß1 integrin expression or function. We conclude that PAK1 is involved in the ability of cells to sense the orientation of stiffness in collagen substrates and generate contractile forces that affect cell extension formation.

Clinical significance of CDX2-positive circulating tumour cells in colorectal cancer patients.

BACKGROUND: Our recent work has shown the feasibility of using a refined immunomagnetic enrichment (IE) assay to detect cytokeratin 20-positive circulating tumour cells (CK20 pCTCs) in colorectal cancer (CRC) patients. We attempted to improve the sensitivity for CRC by detecting another intestinal-type differentiation marker, CDX2 pCTCs, using the same methodology. METHODS: CDX2 pCTCs were detected in patients with CRC, colorectal adenoma (CAD), benign colorectal diseases (BCD), other common cancers (OCC) and normal subjects (NS). Statistical analysis was used to correlate CDX2 pCTCs to the clinicohistopathological factors, recurrence, metastasis and survival after follow-up for 42 months in CRC patients. RESULTS: CDX2 pCTCs were detected in 81% CRC patients (73 out of 90, median number=21.5 CTCs), 7.5% CAD patients (3 out of 40), 0% patients with BCD (0 out of 90), 2.5% patients with OCC (2 out of 80) and 0% NS (0 out of 40). Furthermore, statistical analysis showed that CDX2 pCTC numbers were associated with tumour- node-metastasis stage and lymph node status. Using the median CDX2 pCTC numbers as the cutoff points, stratified groups of CRC patients had significant differences in their recurrence and survival. CONCLUSIONS: This study showed that the refined IE assay can detect CDX2 pCTCs with high sensitivity and that CDX2 pCTCs can generate clinically important information for CRC patients.

Dynamic formation of a hot field reversed configuration with improved confinement by supersonic merging of two colliding high-ß compact toroids.

A hot stable field-reversed configuration (FRC) has been produced in the C-2 experiment by colliding and merging two high-ß plasmoids preformed by the dynamic version of field-reversed θ-pinch technology. The merging process exhibits the highest poloidal flux amplification obtained in a magnetic confinement system (over tenfold increase). Most of the kinetic energy is converted into thermal energy with total temperature (T{i}+T{e}) exceeding 0.5 keV. The final FRC state exhibits a record FRC lifetime with flux confinement approaching classical values. These findings should have significant implications for fusion research and the physics of magnetic reconnection.

Polymorphisms of glutathione S-transferase genes and functional activity in smokers with or without COPD.

OBJECTIVE: To determine the role of polymorphisms of genes regulating glutathione S-transferase (GST) and its plasma GST activity in the pathogenesis of chronic obstructive pulmonary disease (COPD). DESIGN: Case-control study. METHODS: One hundred and sixty-three patients with stable COPD from several community or regional hospitals were matched for age and pack-years smoked with the same number of health controls from the general population. Each participant underwent an interview-based respiratory and smoking questionnaire, lung function testing and gave a blood sample. Genotyping was carried out using a polymerase chain reaction-based method for polymorphisms of glutathione S-transferase theta 1 (GSTT1), glutathione S-transferase mu 1 (GSTM1) and glutathione S-transferase P 1 (GSTP1) genes. Plasma GST activity was measured using the spectrofluorometric method. RESULTS: There were no significant differences in the distribution of various genotypes of polymorphisms of GSTT1, GSTM1 and GSTP1 between COPD patients and healthy controls. GST activity was significantly higher in patients compared with controls, irrespective of their different genotypes, and was not different between patients with different levels of airflow obstruction. CONCLUSION: Polymorphisms of GSTT1, GSTM1 and GSTP1 genes are unlikely to be involved in the pathogenesis of COPD in Chinese in Hong Kong and Southern China.

Determinants of chronic obstructive pulmonary disease in Chinese patients in Hong Kong.

BACKGROUND: The prevalence of chronic obstructive pulmonary disease (COPD) is rising in many parts of the world. This is a study of risk factors for COPD in Chinese patients in Hong Kong. DESIGN: Case-control study matched by sex and age (+/-5 years). METHODS: A total of 289 consecutive patients with COPD were recruited from out-patient clinics while healthy controls were recruited from two sources: random population and community centres for senior citizens. All patients and controls underwent a questionnaire-based interview and spirometry. RESULTS: The mean ages of COPD patients and controls were 71.1 +/- 9.4 and 67.5 +/- 9.3 years, respectively. The male to female ratio of COPD patients was 5 to 1. Smoking was found to be the most important determinant for COPD, followed by poor education and low body mass index adjusted for confounders. A dose-dependent relationship was found between the risk of COPD and pack-years smoked. Place of birth, exposure to environmental tobacco smoke and a history of asthma and tuberculosis were not associated with increased risk of COPD. CONCLUSION: Despite a progressive reduction in prevalence, smoking remains the most important predictor of COPD in Hong Kong. Greater anti-smoking efforts are warranted.

Prevalence and determinants of positive tuberculin reactions of residents in old age homes in Hong Kong.

OBJECTIVE: To determine the prevalence of tuberculous infection and the predictors of positive tuberculin reactivity in old age home residents in Hong Kong. DESIGN: Cross-sectional study. METHODS: A questionnaire-interview and review of medical records were carried out, together with measurement of weight and height/arm span and assessment of nutritional status. A one-stage tuberculin skin test (TST) was performed using two units of PPD RT23. An induration > or = 10 mm was considered as positive. RESULTS: Of 3682 residents (71.7% participation, mean age 82 years) who agreed to undergo a TST, 46.3% had a positive reaction. Factors associated with a significantly higher risk of a positive TST included being male, an ex- or current smoker and having a past history of tuberculosis (TB). Factors associated with reduced positive tuberculin reactivity included older age groups (> 70 years), a history of cancer and chronic obstructive pulmonary disease and low body mass index quartiles. CONCLUSION: In old age homes, the high prevalence of latent tuberculous infection is responsible for the high rate of active TB due to reactivation. Early diagnosis and treatment are necessary to prevent transmission of disease in these crowded environments with susceptible individuals.

Synthesis of lipoprotein lipase in cultured avian granulosa cells.

Avian granulosa cells cultured as a homogeneous parenchymal population contain lipolytic activity. This activity is stimulated 2--5-fold by serum, inhibited 90% by 1 M NaCl and inhibited 80% by specific anti-lipoprotein lipase immunoglobulins. 85% of the activity binds to heparin-Sepharose 4B, and 70% of bound activity is eluted with 1.5 M NaCl. Thus, the lipolytic activity of cultured granulosa cells is lipoprotein lipase. Granulosa cells were shown to synthesize lipoprotein lipase in culture by incorporating [3H]leucine into the enzyme protein, as measured with an immunoadsorption technique. Finally, colchicine was shown to increase intracellular lipolytic activity, suggesting an inhibition of secretion of this enzyme by cultured granulosa cells.

Human endothelin converting enzyme-2 (ECE2): characterization of mRNA species and chromosomal localization.

Generation of the functionally pleiotropic members of the endothelin vasoactive peptide family is critically catalyzed by unique type II metalloproteases, termed endothelin converting enzymes (ECE). Isolation of human ECE-2 (EC 3.4.24.71) cDNAs revealed deduced open reading frames of 787 and 765 amino acids with approximately 60% identity with human ECE-1. Characterization of mRNA variants revealed mRNA structural diversity at the 5'-terminus. Two mRNA species exist containing distinct first and second exons. Furthermore, in one of these species, an in-frame deletion of the intracytoplasmic domain removed 29 amino acids. Because of the previously reported human genetic diseases ascribed to germline mutations of member genes of the endothelin family, ECE2 was localized in human chromosomes with fluorescence in situ hybridization and radiation hybrid mapping to 3q28-q29 and SHGC-20171/D3S1571, respectively.

Species differences in prostatic steroid 5 alpha-reductases of rat, dog, and human.

The conversion of testosterone to 5 alpha-dihydrotestosterone by prostate particulates from rats, dogs, and humans was investigated, and significant species differences were found with their pH profiles, affinities for 4-azasteroidal inhibitors, and sensitivities to mercuric sulfhydryl reagents. The pH optima for the rat (pH 7), the dog (pH 6), and the human (pH 5) enzyme are significantly different. Mersalyl acid and p-hydroxymercuribenzoate inactivate only the rat 5 alpha-reductase, but not the human or dog enzyme. The rank orders of potencies of 24 3-oxo-4-azasteroids to inhibit 5 alpha-reductases of the rat, dog, and human prostate are different. The variation of the 17 beta-functional groups of the inhibitors demonstrates clearly the species differences. Those inhibitors with a 17 beta-diethylcarbamoyl, 17 beta-diisopropylcarbamoyl, 17 beta-t-butylcarbamoyl, or 17 beta-secbutylcarbonyl functional group are approximately equipotent as inhibitors of the rat and human enzymes, whereas they are only 0.1-15% as potent as inhibitors of the dog enzyme. On the other hand, those inhibitors with a 17 beta-spiroether functional group are most potent as inhibitors of the rat enzyme, are 15-50% as potent as inhibitors of the dog enzyme, and are 0.2-0.4% as potent as inhibitors of the human enzyme. Those inhibitors with a 17 beta-n-octylcarbamoyl, 17 beta-(1-carboxyethyl), or 17 beta-(1-carboxy-3-butyl) functional group are 2-3 orders of magnitude less potent as inhibitors of the dog and human enzymes than as inhibitors of the rat enzyme. These results suggest that prostatic 5 alpha-reductases of rats, dogs, and humans are significantly different. In spite of the significant species differences in inhibitor affinities, where determined, inhibition of the rat, dog and human enzymes by these compounds is competitive with testosterone. These 3-oxo-4-azasteroids have a similar rank order of potency as inhibitors of 5 alpha-reductase in human normal, benign hyperplastic, and cancerous prostates, indicating that the inhibitor-binding sites of 5 alpha-reductase in the prostate in different pathological states are similar. The affinities of the 3-oxo-4-azasteroids for rat prostatic cytosol receptor were determined. Five of these 5 alpha-reductase inhibitors have no significant affinity for the androgen receptor, whereas others do have an affinity for the receptor.

Specific activation of Gs by synthetic peptides corresponding to an intracellular loop of the beta-adrenergic receptor.

Peptides corresponding to the amino acid sequence of the hamster beta 2-adrenergic receptor (beta 2AR) were synthesized and their ability to activate purified G-proteins determined. Two peptides, comprising the N- and C-terminal 15 amino acids of the putative third intracellular loop region of the beta 2AR were found to activate the G-protein Gs but not to activate a preparation of Gi/Go. Other peptides corresponding to the internal portions of this loop and the C-terminal tail region failed to activate either G-protein. The presence of phospholipid vesicles was required for this activation. The observation that peptides with sequences corresponding to the ends of the third intracellular loop of the beta AR can specifically activate Gs confirms the results of previous mutagenesis studies on the receptor and demonstrates that the secondary structure conferred by the amino acid sequences in these regions is sufficient for the activation of G-proteins.

Azasteroids: structure-activity relationships for inhibition of 5 alpha-reductase and of androgen receptor binding.

A series of steroids, primarily 4-azasteroids, were prepared and tested in vitro as inhibitors of human and rat prostatic 5 alpha-reductase and of binding of dihydrotestosterone to the rat androgen receptor. The primary structural modifications were changes of the A ring and of moieties attached at the C-17 position of the steroid nucleus. New A-ring modifications included the 4-cyano-3-oxo-delta 4 system in the carbocyclic series and 1 alpha-CN, 1 alpha-CH3, 1 alpha,2 alpha-CH2, 2 beta-F, 2-aza, 2-oxa, and A-homo changes in the 3-oxo-4-aza series. In addition, 4-azasteroids with a D-homo ring or methyl substitution at C-7 (alpha and beta) or C-16 (alpha and beta) were prepared. The majority of the C-17 substituents were prepared from reactive intermediates derived from the 17 beta-COOH. Enhanced 5 alpha-reductase inhibition in both the human and rat enzyme assays is seen with 4-CN substitution on 3-oxo-delta 4 steroids and with a C-17 side chain incorporating a lipophilically substituted semipolar group on the 4-aza-3-oxo-5 alpha-androstane nucleus. Fewer highly active compounds were found in the human enzyme assay than in the rat assay. Structural requirements for inhibition of the rat androgen receptor are much different from those for inhibition of the enzyme. The 17 beta-OH moiety enhances potency more than any other feature while introduction of double bonds at C-1 or C-5 in the azasteroid gives a small improvement. Azasteroids unsubstituted at the 4-position show greatly diminished receptor activity.

Evaluation of a 20-MHz ultrasound transducer used in diagnosing porcine small bowl ischemia.

The authors have previously demonstrated the ability of an 8.5-MHz linear array to detect moderate or severe intestinal ischemia in a porcine model. This study compares the ability of the 8.5-MHz linear array with a prototype miniature 20-MHz ultrasound (US) imaging probe in detecting small bowel ischemia. Five piglets were studied in which vascular clamps were applied to isolated jejunal pedicles, then released sequentially at hourly intervals to induce ischemia from 0 to 6 hours. After 24 hours of reperfusion, the tissue was removed and examined with both the 8.5-MHz linear array and the 20-MHz probe. A histologic examination also was done. The acoustical criteria used for interpretation were presence or absence of folds, number of echo layers, relative thickness of layers and homogeneity and continuity of layers. The 8.5-MHz system predicted the duration of ischemia with a kappa value of 0.66 +/- 0.03, whereas the 20-MHz system had a kappa value of 0.49 +/- 0.03. Both systems were able to distinguish normal or mild ischemia from moderate or severe ischemia with sensitivity and specificity rates of at least 94%. Both 8.5- and 20-MHz US systems detected intestinal ischemia in vitro. Further studies are indicated to determine the ideal frequency and design for a US system that can be used clinically.

Surgical infections in patients with chronic renal failure.

Patients with chronic renal failure and uremia have impaired host defenses and wound healing that can lead to an increased risk of infection in addition to a frequent need for surgical procedures with synthetic grafts and catheters. Antibiotic therapy plus timely surgical intervention in removal of infected grafts and catheters is crucial for infection control and patient survival. Other surgical infections, such as wound problems, intraabdominal infections, fungal infections, diabetic foot ulcers, and necrotizing soft tissue infections must be attended to promptly.

Diastolic blood pressure is related to urinary sodium excretion in hypertensive Chinese patients.

We studied 70 Hong Kong Chinese patients with untreated hypertension and 47 normotensive controls. Blood pressure measurements and 24-h urine collection were performed for each patient, and were repeated 12 weeks later in 14 hypertensive patients who remained untreated. Twenty-two hypertensive patients underwent ambulatory blood pressure monitoring. The primary hypothesis tested was a correlation between diastolic blood pressure and 24-h urinary sodium excretion. In the hypertensive patients, diastolic blood pressure correlated with 24-h urinary sodium excretion (r=0.41, p<0.001), even after adjustment for age, gender, body mass index, ethanol intake and season (r=0.34, p=0.02). In normotensive controls, diastolic blood pressure did not correlate with sodium excretion (r=0.21, p=0.16). A correlation between diastolic blood pressure and sodium excretion was also observed in the patients who underwent ambulatory blood pressure monitoring (r=0.47, p=0.026), and in repeat measurements in untreated patients (r=0.60, p=0.02). Systolic blood pressure did not correlate with sodium excretion, although it increased with patient age (0.6+/-0.1 mmHg/year, p<0.001). In a multiple regression analysis with diastolic blood pressure as the dependent variable, the regression coefficient was 0.06+/-0.02 mmHg/mmol Na. The regression coefficients for ambulatory diastolic blood pressure and diastolic pressure repeated at 12 weeks were 0.07+/-0.03 and 0. 09+/-0.04 mmHg/mmol Na, respectively. Urinary sodium excretion was related to diastolic blood pressure in our hypertensive patients, accounting for 17% of the variance of diastolic blood pressure.

A salvage technique for continuous ambulatory peritoneal dialysis catheters with exit-site infections.

Catheter infection is a major complication of continuous ambulatory peritoneal dialysis (CAPD) therapy for end-stage renal disease. Catheter exist-site infections were treated with a new surgical technique consisting of dissection and removal of the existing catheter in the subcutaneous layer, insertion of a catheter connector and new catheter piece, and creation of a new subcutaneous tunnel. The new surgery can be performed on an outpatient basis and allows for the continuation of CAPD, thereby avoiding the cost associated with inpatient admission and interim hemodialysis.

Use of ultrasound to detect intestinal wall ischemia in piglets.

To evaluate the use of ultrasound (US) to detect intestinal wall ischemia, we isolated segments of jejunum on single vascular pedicles in five piglets. We sequentially clamped these segments in intervals of 0 to 6 h, reperfused them for 24 h, and then examined them in vitro histologically and with an 8.5 MHz US scan. All segments were grossly viable except those with 6 h of ischemia. Histologically, mild submucosal edema developed after 1 to 2 h of ischemia; after 3 to 4 h, mucosal necrosis, loss of folds, worsening submucosal edema, and prominent neutrophilic infiltration occurred; after 5 to 6 h, severe mucosal necrosis with hemorrhage and submucosal edema was present. Ultrasonically, we saw five wall layers in the control group corresponding to mucosa, submucosa, muscularis propria, and subserosal fat. After mild (1 to 2 h) ischemia, all layers were present except for a discontinuity in layer 3. After moderate (3 to 4 h) ischemia, the five layers persisted but with a markedly thickened submucosal layer, reduced echogenicity, and flattened mucosal folds. With severe (5 to 6 h) ischemia, we observed a loss of all normal layers with no discernible architecture. Using these US criteria, blinded observers were able to differentiate normal/mild from moderate/severe ischemia with a sensitivity and specificity of 100%. These data suggest that US can differentiate, in vitro, normal from moderate and severe degrees of intestinal wall ischemia that correlates well with the histological appearance. Endoscopic US or surgically implantable US probes can potentially help diagnose clinical intestinal wall ischemia.

Experimental use of high-frequency ultrasound to image bowel wall after porcine intestinal transplantation.

A means to monitor intestinal allografts will be crucial for the future success of small bowel transplantation. We have previously demonstrated the ability of high-frequency ultrasound (US) to diagnose porcine intestinal ischemia in vitro. The aim of this study was to compare the histologic appearance of normal porcine small bowel versus bowel undergoing acute rejection, using 8.5-MHz US images. We allowed porcine heterotopic small bowel allograft transplants to reject and then removed, at scheduled intervals from postoperative day 0 to 12, specimens of both the transplanted bowel and the native bowel. We examined the tissues in vitro with an 8.5-MHz linear array US system then studied them histologically. Histologically, the earliest changes of rejection occurred at days 4 to 5, with mild submucosal edema, endotheliitis, and vasculitis affecting the small vessels; the mucosa remained normal. By days 7 to 8, the submucosal endotheliitis became more prominent, with focal thrombosed small vessels; the mucosa now appeared abnormal with flattened villi, erosions, and necrosis. By days 10 to 12, marked submucosal edema, vasculitis, endotheliitis, and necrotic mucosa were present. Ultrasonically, normal intestinal wall has five wall layers, corresponding to mucosa, submucosa, muscularis propria, and subserosal fat. The US criteria for abnormality were loss of folds, decreased numbers of echo layers, discontinuity of layers, and a homogeneous appearance. Using these US criteria, blinded observers differentiated normal from abnormal bowel wall after transplantation with a sensitivity of 84% and a specificity of 81%. Most of the errors occurred with the day 4 and 5 specimens, which appeared nearly normal on US.(ABSTRACT TRUNCATED AT 250 WORDS)

Organ donation and culture: a comparison of Asian American and European American beliefs, attitudes, and behaviors.

The well-known gap between organ-donor supply and demand in the United States is particularly acute for Asian Americans. Lower participation in organ donation programs by Asian Americans has been hypothesized as one explanation for this observation. This study finds that, relative to European Americans, Asian Americans hold more negative attitudes toward and participate less frequently in a large, urban organ-donor program. The study also hypothesizes and test possible reasons for subcultural differences in attitudes toward donation. Two cultural belief constructs hypothesized to more strongly predict Asian American attitudes and behaviors appear to impact both groups equally. Reasons for these results along with public policy implications and future research directions are discussed.

Pancreas transplantation for diabetic patients in Hawaii.

Diabetes mellitus is a common disease affecting a large population in Hawaii. Over the past 20 years, pancreas transplantation has evolved into a viable therapeutic option for selected patients with diabetes mellitus. This report describes the first combined pancreas-kidney transplant performed in Hawaii on June 28, 1993 on a patient with juvenile-onset diabetes mellitus and diabetic nephropathy. The patient has remained off insulin and off dialysis since the transplant. The history, indications, techniques, and potential complications related to this procedure are discussed.

The first non-heart-beating organ donor in Hawaii--medical and ethical considerations.

The shortage of organ donors remains a major obstacle in transplantation in Hawaii. Some patients die while waiting for a life-saving organ. Across the nation, "marginal" donors, including non-heart-beating donors are used. The authors describe the first successful non-heart-beating organ donor transplant in Hawaii, and include medical and ethical considerations.