Site-directed mutagenesis of the arginine-glycine-aspartic acid sequence in osteopontin destroys cell adhesion and migration functions

Osteopontin (OPN) is a secreted calcium-binding phosphoprotein produced in a variety of normal and pathological contexts, including tissue mineralization and cancer. OPN contains a conserved RGD (arg-gly-asp) amino acid sequence that has been implicated in binding of OPN to cell surface integrins. To determine whether the RGD sequence in OPN is required for adhesive and chemotactic functions, we have introduced two site-directed mutations in the RGD site of the mouse OPN cDNA, in which the RGD sequence was either deleted or mutated to RGE (arg-gly-glu). In order to test the effect of these mutations on OPN function, we expressed control and mutated mouse OPN in E. coli as recombinant glutathione-S-transferase (GST)-OPN fusion proteins. Control mouse GST-OPN was functional in cell adhesion assays, supporting attachment and spreading of mouse (malignant PAP2 ras-transformed NIH 3T3, and, to a lesser extent, normal NIH 3T3 fibroblasts) and human (MDA-MB-435 breast cancer, and normal gingival fibroblast) cells. In contrast, neither of the RGD-mutated OPN proteins ("delRGD" or "RGE") supported adhesion of any of the cell lines, even when used at high concentrations or for long assay times. GRGDS (gly-arg-gly-asp-ser) peptides inhibited cell adhesion to intact GST-OPN, as well as to fibronectin and vitronectin. In chemotaxis assays, GST-OPN promoted directed cell migration of both malignant (PAP2, MDA-MB-435) and normal (gingival fibroblast, and NIH 3T3) cells, while RGD-mutated OPN proteins did not. Together these results suggest that the conserved RGD sequence in OPN is required for the majority of the protein's cell attachment and migration-stimulating functions.     

Rhinotillexomania: psychiatric disorder or habit?

BACKGROUND: Conditions once considered bad habits are now recognized as psychiatric disorders (trichotillomania, onychopagia). We hypothesized that nose picking is another such "habit," a common benign practice in most adults but a time-consuming, socially compromising, or physically harmful condition (rhinotillexomania) in some. METHODS: We developed the Rhinotillexomania Questionnaire, mailed it to 1000 randomly selected adult residents of Dane County, Wisconsin, and requested anonymous responses. The returned questionnaires were analyzed according to age, sex, marital status, living arrangement, and educational level. Nose picking was characterized according to time involved, level of distress, location, attitudes toward self and others regarding the practice, technique, methods of disposal, reasons, complications, and associated habits and psychiatric disorders. RESULTS: Two hundred fifty-four subjects responded. Ninety-one percent were current nose pickers although only 75% felt "almost everyone does it"; 1.2% picked at least every hour. For 2 subjects (0.8%), nose picking caused moderate to marked interferences with daily functioning. Two subjects spent between 15 and 30 minutes and 1 over 2 hours a day picking their nose. For 2 others, perforation of the nasal septum was a complication. Associated "habits" included picking cuticles (25%), picking at skin (20%), biting fingernails (18%), and pulling out hair (6%). CONCLUSION: This first population survey of nose picking suggests that it is an almost universal practice in adults but one that should not be considered pathologic for most. For some, however, the condition may meet criteria for a disorder-rhinotillexomania.     

Pixel driven implementation of filtered backprojection for reconstruction of fan beam SPECT data using a position dependent effective projection bin length

Filtered backprojection is commonly implemented as a pixel driven algorithm in which the density is reconstructed on an array of grid points that are usually associated with the centres of square image pixels. In fan beam geometry, this conventional pixel driven approach using two-bin linear interpolation leads to an inefficient use of the projection data due to magnification at the projection line. For typical count limited SPECT data, this results in increased reconstructed image noise. We propose an alternative type of pixel driven algorithm that makes efficient use of projection data by averaging over all bins within a position dependent "effective' projection bin interval. The effective bin interval is equivalent to the image pixel length magnified on the projection line. This heuristic method leads to more complete use of the projection data and results in reconstructed images with superior noise properties compared with the conventional method while presenting similar spatial resolution characteristics.     

Strength of silicone breast implants

Rupture and leakage are recognized problems associated with silicone breast implants. Data are scarce about the durability of the silicone shell, and the life span of this device is unknown. The purpose of this study was to investigate the strength of silicone breast implants. Thirty implant shells were subjected to mechanical testing. Twenty-nine of the shells were tested after explanation, and one unused implant served as a control to validate the testing method. Implantation time varied from 4 months to 20 years, and all shells were tested, regardless of condition. Fourteen implant shells were intact, eight were leaking, and seven were ruptured. All ruptured implants had been in place for 10 years or longer. The breaking force of all excised shell specimens ranged from 2.6 to 22.4 N (0.6 to 5.0 lb.). Specimens from the control "high performance" shell required 15.5 to 25.6 N (3.5 to 5.8 lb) of force to fail. The weakest group was from thin-shelled implants between 10 and 16 years of age. More than half these specimens failed with less than 1 lb of force. The average breaking force of ruptured shell material was less than that of intact shells. A comparison of strength data in this study with manufacturers' data suggests that breaking force is dependent on implant type, shell thickness, and implantation time.     

Betacyanins from plants and cell cultures of Phytolacca americana

Betacyanins from cell cultures of Phytolacca americana were characterized and compared with those of the stems and ripening fruits of the plant. Whereas in fruits prebetanin (betanin 6'-O-sulphate) and its isoform predominate, in the stem and cell cultures feruloylated derivatives occur as the major components. These were rigorously identified by various spectroscopic techniques (DAD-HPLC, NMR, LC-MS and electrospray MS-MS) and carbohydrate analyses as betanidin 5-O-[(5"-O-E-feruloyl)-2'-O-beta-D-apiofuranosyl] -beta-D-glucopyranoside, a new betacyanin of higher plants, and betanidin 5-O-(6'-O-E-feruloyl)-beta-D-glucopyranoside (lampranthin II), together with their isoforms.     

Propofol blocks voltage-gated potassium channels in human T lymphocytes

The effect of propofol (PR) on voltage-gated potassium channels (KV) in human T lymphocytes (TL) was studied using the patch-clamp technique in the whole-cell configuration. PR was found to reversibly block the KV channels in a dose-dependent manner with a half-blocking concentration of approximately 40 microM. The decrease in the peak current caused by PR was voltage-independent. The activation time constant of the whole-cell potassium currents remained unaffected upon PR treatment, whereas both the rate and extent of the inactivation process were increased, indicating the "open channel block" mechanism. The PR half-blocking concentration was of the same order of magnitude as PR blood concentrations employed in anesthesia. Taking into account the extensive use of PR and the important role of KV channels in human TL, these results suggest a need for investigations into the effect of PR on TL cell-function regulation.     

Adhesions: pathogenesis and prevention-panel discussion and summary

This article summarizes the discussions of the faculty and chairpersons on four major topics on postsurgical adhesions examined at the symposium, "Adhesions: Pathogenesis and Prevention". These topics are: 1) clinical significance; 2) pathogenesis; 3) research status and directions; and 4) recommendations for reduction or prevention. Abdominal postsurgical adhesions develop following trauma to the mesothelium, which is damaged often by surgical handling and instrument contact, foreign materials such as sutures and glove dusting powder, desiccation, and overheating. Postoperative adhesions occur after most surgical procedures and can result in serious complications, including intestinal obstruction, infertility, and pain. A long-term and unpredictable problem, postoperative adhesions impact the surgical workload and hospital resources, resulting in considerable health care expenditures. Although understanding of the pathogenesis of adhesions has improved recently, the molecular mechanisms involved continue to be delineated. Adhesions result from the normal peritoneal wound healing response and develop in the first five to seven days after injury. Adhesion formation and adhesion-free re-epithelialization are alternative pathways, both of which begin with coagulation which initiates a cascade of events resulting in the buildup of fibrin gel matrix. If not removed, the fibrin gel matrix serves as the progenitor to adhesions by forming a band or bridge when two peritoneal surfaces coated with it are apposed. The band or bridge becomes the basis for the organization of an adhesion. Protective fibrinolytic enzyme systems of the peritoneum, such as the plasmin system, can remove the fibrin gel matrix. However, surgery dramatically diminishes fibrinolytic activity. The pivotal events determining whether the pathway taken is adhesion formation or re-epithelialization are therefore the apposition of two damaged surfaces and the extent of fibrinolysis. Research in postsurgical adhesion formation and prevention abounds in a variety of avenues of investigation, including: 1) identification on a molecular level of the components involved in adhesiogenesis and their interactions; 2) clarification of the role of fibrin and fibrinolysis in adhesion formation; 3) standardization of design in preclinical and clinical studies of adhesion formation and prevention; 4) delineation of the relationship between adhesion formation and adhesive complications; and 5) elucidation of efficient, site-specific methods of prophylactic drug delivery. Currently, it seems logical to focus preventive research on development of barriers, fibrinolytic drugs, and selected agents such as phospholipids. The major strategies for adhesion prevention or reduction are adjusting surgical practice and applying adjuvants. Surgeons should adjust their major practices by: 1) becoming aware of the potential adhesive complications of a procedure; 2) minimizing the invasiveness of surgery; and 3) minimizing surgical trauma, ischemia, exposure to intestinal contents, introduction of foreign material into the body, and the use of talc- or starch-containing gloves. Available adjuvants include a newly developed by hyaluronic acid-phosphate-buffered saline solution applied intraoperatively to protect peritoneal surfaces from indirect surgical trauma and three mechanical barriers. One of these, a bioresorbable membrane consisting of hyaluronic acid and carboxymethylcellulose, has demonstrated efficacy and safety in both general and gynecological surgery. The other two barriers, one made of expanded polytetrafluoroethylene and one developed from oxidized regenerated cellulose, are indicated only for use in gynecological surgery.     

Reliability of using random urine samples for "spot" determination of fractional excretion of electrolytes in cats

OBJECTIVE: To determine whether the "spot" method of determining fractional excretion (FE) of electrolytes in cats is accurate. ANIMALS: 5 clinically normal young adult female cats. PROCEDURE: Cats were acclimated to metabolism cages, and 2 consecutive 72-hour collections of urine were made to determine FE of total calcium, potassium, total magnesium, sodium, and phosphorus by conventional methods, using endogenous creatinine clearance as an estimate of glomerular filtration rate. During collections, small samples of urine were obtained by cystocentesis at 8 AM, 3 PM, and 9 PM for determination of FE of the electrolytes by use of the "spot" method. RESULTS: Values from "spot" determinations were highly variable, compared with 72-hour values, with a high percentage falling outside the range of mean +/- 2 SD for 72-hour FE values. CONCLUSIONS AND CLINICAL RELEVANCE: The "spot" method for determining FE is not precise, and if used, caution and judgement should be exercised in interpretation of the results.     

Development and validation of a geriatrics knowledge test for primary care residents

This study reports the development and preliminary validation of an instrument to measure geriatrics knowledge of primary care residents. A 23-item test was developed using questions selected from the American Geriatrics Society's Geriatrics Review Syllabus. Ninety-six internal medicine and family practice residents, 14 geriatrics fellows, and 11 geriatrics faculty members participated in the study. Findings support the reliability (Cronbach's alpha = 0.66) and validity (content and "known groups") of this short test. Predictive validity and sensitivity of the test to changes in knowledge will have to be further explored as residents progress through their training.     

Neurotic disturbances, depression and anxiety disorders in the population living in the vicinity of overhead high-voltage transmission line 400 kV. Epidemiological pilot study

Numerous reports suggest a relationship between the increased incidence of depressive and neurotic symptoms in humans and the exposure to extremely low frequency electromagnetic field (EMF) at the place of residence. Our study was conducted in one of the Cracow suburbs with low socio-economic status, adjacent to an easement containing two 400 kV high-voltage transmission lines (distance up to 50 m from the line). The population investigated comprised 70 persons (35 males and 35 females). This group was exposed to EMF from 1986. A control group (n = 37), non-exposed to EMF, was also tested. The EMF intensity was measured by special devices. The following assessments instruments were used: Anamnestic Data Questionnaire, Neurotic Symptoms Questionnaire "O" Spielberger Self-Rating Questionnaire, and Beck-Self-Rating Scale. The measurements of the electric field levels taken at the front walls of investigated buildings averaged much higher than normal values of safety exposure. Our investigation showed the increased psychopathological values in all clinical tests. The difference between the group exposed to EMF and the control population was statistically significant. The results of our study did not support a possible cause-and-effect relationship between EMF and psychopathology observed. Some other factors (noise) can be responsible for the data obtained. The hypothesis that EMF produced by external electric power lines may enhance the occurrence of neurotic symptoms and depression, requires further clinical and experimental investigations.