A small set of cellular genes, termed proto-oncogenes, appear to be frequent targets for genetic alterations leading to cancer. One family of such oncogenes, the ras gene family, has been directly implicated in the malignant process in both animals and humans. The normal ras gene product, a 21, 000-molecular-weight protein, exhibits GTP and GDP binding as well as an associated GTPase activity 14, 30, 34 ; . Mutations that commonly activate the transforming properties of ras genes involve point mutations around codon 12 or 61 4, 13, ; . It has been shown that microinjection of activated ras p21 into quiescent fibroblasts results in transient morphological transformation and cell proliferation 6, 11, 33 ; . Microinjection of the unaltered proto-oncogene p21 failed to produce a response. Thus, microinjection of activated p21 into quiescent cells provides an excellent system for examining the rapid biological effects of this transforming protein on normal cells 1 ; . One of the earliest effects of a variety of growth factors on quiescent cells is a rapid influx of Na + into the cell. Stimulation of Na + influx results from activation of a membranebound Na + H antiporter and leads to cytoplasmic alkalinization 16, 20, 25, ; . There is recent evidence that increased intracellular pH pHj ; may be an important determinant for the initiation of DNA synthesis 19 ; . In this study we have developed a novel system for monitoring pHi in single cells following the microinjection of ras p21 to assess the role of pHi changes in mediating the mitogenic effect of our single-cell assay system, we examined the recovery of pH, in NIH 3T3 cells following an acid load induced by transient treatment of cells with NH4Cl 3, 23 ; . Exposure of cells to NH4Cl results in a rapid intracellular alkalinization because of NH4 entry. When the external NH4 is removed, the NH4 accumulated within the cell is forced to leave the cell as NH3, loading the cells with an excess of protons. Following an NH4-induced acid load, pH, recovery was rapid and complete in different cells in 3 to min -70% of cells responded, n 50 ; Fig. 1B ; . Since phorbol esters have been shown to induce a rapid elevation of pH, in fibroblasts 5 ; and lymphocytes 24 ; , we next tested the response of pHi in single cells to treatment with TPA ; . An increase in pH1 0.2 to 0.3 pH units ; was noticed within 90 s in more than 90% of cells treated with TPA Fig. 1C ; . The magnitude and kinetics of pH, changes observed in single cells in the above experiments agree with the results of previous reports which measured the sum of pH-dependent fluorescence in larger numbers of cells 5, 16, 20, ; . We next measured fluorescence in single cells prior to and following microinjection of ras p21. The p21 species used for microinjection in these experiments were purified from Escherichia coli with expression vectors containing the PL promoter of phage lambda. The H-ras proto-oncogene as well as the transforming v-H-ras oncogene containing a glycine-to-arginine substitution at amino acid position 12 and an alanine-to-threonine substitution at position 59 were expressed and purified to apparent homogeneity as described before 10 ; . The activated p21 used in these studies induced both morphological transformation and DNA synthesis in 60% of quiescent mouse NIH 3T3 cells after microinjection, as assayed by 3H-thymidine uptake and autoradiography. A similar concentration of the unaltered proto-oncogene p21 elicited no response under the same conditions Table 1 ; . Microinjection of -106 molecules of oncogenic ras p21 per cell produced a characteristic response in NIH 3T3 cells and rat-1 cells loaded with BCECF. Fluorescence intensity increased 1 to 2 min following injection and reached a plateau at -10 min; this level was sustained over the 30- to 60-min observation period Fig. 2A ; . In -5% of responsive cells a more rapid response, which reached a maximum in 2 to min, was noted Fig. 2C ; . The resting pH of NIH 3T3 cells was found to be 7.01 0.12, and we calculated the mean p21-induced increase in pH, in responsive cells to be -0.17 pH units. Approximately 60%o of injected cells showed the pH alteration, a response rate and ketorolac.
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OBJECTIVE -- To evaluate the clinical effects of a comprehensive prepackaged meal plan, incorporating the overall dietary guidelines of the American Diabetes Association and other national health organizations, relative to those of a self-selected diet based on exchange lists in free-living individuals with type 2 diabetes. RESEARCH DESIGN AND METHODS -- A total of 202 women and men BMI 42 kg m2 ; whose diabetes was treated with diet alone or an oral hypoglycemic agent were enrolled at 10 medical centers. After a 4-week baseline period, participants were randomized to a nutrient-fortified prepared meal plan or a self-selected exchange-list diet for 10 weeks. On a caloric basis, both interventions were designed to provide 5560% carbohydrate, 2030% fat, and 1520% protein. At intervals, 3-day food records were completed, and body weight, glycemic control, plasma lipids, and blood pressure were assessed. RESULTS -- Food records showed that multiple nutritional improvements were achieved with both diet plans. There were significant overall reductions in body weight and BMI, fasting plasma glucose and serum insulin, fructosamine, HbA1c, total and LDL cholesterol, and blood pressure P 0.001 or better for all ; . In general, differences in major end points between the diet plans were not statistically significant. CONCLUSIONS -- Glycemic control and cardiovascular risk factors improve in individuals with type 2 diabetes who consume diets in accordance with the American Diabetes Association guidelines. The prepared meal program was as clinically effective as the exchange-list and indapamide. TAG NUMBER Q 14 3. REGULATION The ASC must establish a program for identifying and preventing infections, maintaining a sanitary environment, and reporting the results to appropriate authorities. GUIDANCE TO SURVEYORS Interpretive Guidelines: 416.44 a ; 3 ; Since there is a risk of nosocomial infection there must be an active surveillance program of specific measures for prevention, early detection, control, education, and investigation of infectious and communicable diseases in ASCs. There must be a mechanism to evaluate the program s ; and take corrective action. The ASC should institute the most current recommendations of The Centers for Disease Control CDC ; and Prevention relative to the specific infection s ; and communicable disease s ; . Survey Procedures and Probes: 416.44 a ; 3 ; Annotate on the survey report form if the written policies and procedures do not contain, at a minimum: o Methods to minimize sources and transmission of infection, including adequate surveillance techniques such as; o o o o Assessing the risk for infections and communicable diseases; Identifying patients at risk for infections and communicable diseases; Educating health care workers about infectious and communicable diseases; Screening health care workers; Providing a safe environment consistent with the most current CDC recommendations; Providing treatment measures consistent with the most current CDC recommendation for the identified infection and or communicable disease; and Providing for program evaluation and revision of program, when indicated.

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