Enzyme-induced posterior vitreous detachment in the rat produces increased lens nuclear pO2 levels.

It has been proposed that disruption of normal vitreous humor may permit O(2) to travel more easily from the retina to the center of the lens where it may cause nuclear cataract (Barbazetto, I.A., Liang, J., Chang, S., Zheng, L., Spector, A., Dillon, J.P., 2004. Oxygen tension in the rabbit lens and vitreous before and after vitrectomy. Exp. Eye Res. 78, 917-924; Harocopos, G.J., Shui, Y.B., McKinnon, M., Holekamp, N.M., Gordon, M.O., Beebe, D.C., 2004. Importance of vitreous liquefaction in age-related cataract. Invest. Ophthalmol. Vis. Sci. 45, 77-85). In the present study, we injected enzymes intravitreally into guinea pigs (which possess an avascular retina) and rats (which possess a vascular retina) to produce either vitreous humor liquefaction plus a posterior vitreous detachment (PVD) (with use of microplasmin) or vitreous humor liquefaction only (with use of hyaluronidase), and 1-2 weeks later measured lens nuclear pO(2) levels in vivo using a platinum-based fluorophore O(2) sensor (Oxford-Optronix, Ltd.). Experiments were also conducted in which the animals were allowed to breathe 100% O(2) following intravitreal injection with either microplasmin or hyaluronidase in order to investigate possible effects on O(2) exchange within the eye. Injection of guinea pigs with either of the two enzymes produced no significant differences in lens pO(2) levels 1-2 weeks later, compared to controls. However, for the rat, injection of microplasmin produced a 68% increase in O(2) level in the center of the lens, compared to the controls (5.6mm Hg increasing to 9.4mm Hg, p<0.05), with no corresponding effect observed following similar use of hyaluronidase. Treatment of guinea pigs with microplasmin dramatically accelerated movement of O(2) across the vitreal space when the animals were later allowed to breathe 100% O(2) (for example, O(2) traveled to a location directly behind the lens 5x faster than control; p<0.01); however, the effect following treatment with hyaluronidase was significantly less. When microplasmin-injected rats breathed 100% O(2), the time required for O(2) to reach the center of the lens was 3x faster than control (0.4 min compared to 1.4 min, p<0.01). The results have implication with regard to the occurrence of age-related PVD in the human, and a possible acceleration of maturity-onset nuclear cataract. In addition, enzymatic creation of a PVD to increase the rate of O(2) exchange within the vitreal space may have potential application for treatment of retinal ischemic disease.

Hydrofoils: optimum lift-off speed for sailboats.

For a hydrofoil sailboat there is a unique optimum lift-off speed. Before this speed is reached, if there are no parasitic vertical hydrofoil appendages, the submerged or partially submerged hydrofoils increase drag and degrade performance. As soon as this speed is reached and the hydrofoils are fully and promptly deployed, the performance of a hydrofoil-borne craft is significantly improved. At speeds exceeding optimum lift-off speed, partially submerged hydrofoils impair performance if there is no significant effect of loading on the hydrofoil lift-to-drag ratio.

Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.

Failure of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate to enhance sister chromatid exchange, mitotic segregation, or expression of mutations in Chinese hamster cells.

The potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was tested for its ability (a) to induce sister chromatid exchange, (b) to increase the rate of transition at the adenine phosphoribosyltransferase (apt) locus from the presumptive heterozygous state ((+/- to the homozygous state (-/ - or -), and (c) to enhance the frequency of mutations expressed after ultraviolet radiation mutagenesis. We have found no significant effect of TPA in any of these experiments. Sister chromatid exchange frequencies in both V79 and Chinese hamster ovary cells remained unchanged by TPA treatment under various conditions, a result inconsistent with the hypothesis that an important effect of TPA might be to increase the rate of chromosomal mitotic recombination (and hence segregation of recessive mutations) in a manner akin to increased chromatid recombination. We have also been unable to obtain evidence for mitotic recombination affecting the aprt locus in Chinese hamster ovary cells for which the rate of change to a high level of resistance to azaadenine was measured. The rate of 8.6 X 10(-7) mutation (and/or segregations) per cell generation assessed by fluctuation analysis was not increased by the continuous presence of TPA, 4 microgram/ml, in the medium. In the third set of experiments, mutant frequencies in Chinese hamster ovary cells after ultraviolet mutagenesis were measured for the markers ouabain resistance, thioguanine resistance, and azaadenine resistance, under conditions with and without pretreatment with TPA before mutant selection. No convincing enhancement in mutation expression was observed. In summary, these results argue that promotion by TPA does not proceed by a mechanism involving genetic recombination or the altered expression of newly mutated alleles.

Elevated pentose cycle and glucuronyltransferase in daunorubicin-resistant P388 cells.

Anthracycline resistance of P388 daunorubicin-resistant cells cannot be accounted for merely by differences in drug uptake and retention; protection against intracellular drug was also indicated. Cytotoxicity of daunorubicin may be partially due to the formation of free radicals and reactive oxygen species (hydrogen peroxide, hydroxyl radical, singlet oxygen, and superoxide anion radical). Protection against free radicals and peroxides is largely dependent upon the availability of reduced glutathione, which in turn requires NADPH for its continual regeneration. Pentose phosphate cycle (also called hexose monophosphate shunt) is known to provide NADPH for maintenance of glutathione. Activities of the two NADPH-producing dehydrogenases of the cycle, glucose-6-phosphate and 6-phosphogluconate dehydrogenase, were 40% higher (P less than 0.05) and activity of the cycle in intact cells was 2-fold higher in the resistant than the sensitive cells. The cycle was as active in these cells as it is known to be in macrophages, indicating a very effective protection against oxidative stress, free radicals, and alkylating electrophiles. Elevated activity of the pentose phosphate pathway in drug-resistant cells can represent a mechanism of resistance against multiple structurally unrelated drugs. Efflux of daunorubicin may be aided by further metabolism to glucuronides. Daunorubicinol, a known active metabolite of daunorubicin, can be metabolized to a glucuronide by the cells and eliminated into the surrounding medium. Glucuronidation of daunorubicinol was evidenced by (a) release of daunorubicinol following glucuronidase hydrolysis of media from cell incubations with 1.8 microM daunorubicin and (b) production of radioactive glucuronide when cell homogenates were incubated with UDP-[14C]glucuronic acid plus daunorubicinol. Glucuronyltransferase activity with a broad substrate specificity was found in the cells. Using model substrates, 1-naphthol and o-aminophenol, it was determined that glucuronyltransferase activity was 4 times higher in daunorubicin-resistant than -sensitive P388 cells. Elevated glucuronyltransferase could contribute to daunorubicin and multidrug resistance.

P-glycoprotein in human sarcoma: evidence for multidrug resistance.

Overexpression of an immunologically conserved, cell-surface glycoprotein (P-glycoprotein) is consistently associated with multidrug resistance in cell lines in vitro. A preliminary survey of specimens from 12 solid tumor types in our laboratories indicates significant overexpression of P-glycoprotein in some sarcomas. When tested by immunoblotting with monoclonal antibodies directed against P-glycoprotein; tumors from six of 25 sarcoma patients displayed elevated levels of P-glycoprotein. Three of the sarcoma patients exhibiting P-glycoprotein had not previously been exposed to chemotherapy, implying that overexpression of this marker and possible concomitant multidrug resistance may not depend only on selection during prior drug treatments. The P-glycoprotein overexpression in the sarcoma specimens is evidence for the presence of multidrug resistant cells in these tumors; thus, our data suggest that this mode of resistance may have clinical significance in sarcoma patients.

Hypoxia-induced sympathetic inhibition of the fetal plasma insulin response to hyperglycemia.

Large-for-delivery date babies, considered characteristic of diabetic pregnancy, are believed to result from fetal hyperinsulinemia. Paradoxically, infant birth weights tend to be low-for-delivery date in mothers with more severe diabetes. We tested the hypothesis that hypoxemia in such fetuses leads to sympathoadrenal stimulation and inhibition of insulin secretion; and, thus, produces a net reduction in the growth-promoting effects. Fetal sheep were prepared with chronic peripheral and adrenal cannulas. Fetal blood gases, lactate, norepinephrine, and epinephrine secretion rates; and plasma norepinephrine, glucose, and immunoreactive insulin concentrations were determined at 30-min intervals during a 2-h baseline period and a 4-h period of hyperglycemia divided into 2-h segments of hypoxemia (with and without alpha-blockade) and hyperoxia. Hypoxemia-hyperoxia sequences were varied randomly. Well-oxygenated fetuses responded to a threefold increase in glucose with a sixfold increase in plasma immunoreactive insulin. With hypoxemia, norepinephrine and epinephrine secretion were elevated and the insulin response was blocked. With hypoxemia and phentolamine blockade, the insulin response was enhanced with a 10-fold increase above baseline. In severe maternal diabetes with vascular disease or with poor control and very high glucose levels, the fetus is likely to be relatively hypoxemic. Our experiments suggest that in this situation, the fetal insulin response to hyperglycemia will be attenuated; this effect is mediated, at least partly, through sympathoadrenal stimulation.

Enhanced sensitivity to G418 of human KB cells adapted to certain media and sera.

Sensitivity to the neomycin derivative G418 was determined for a human cell line, KB 3-1, that had been adapted to six different combinations of media and sera. The results indicate that while the plating efficiency is similar for all conditions, the susceptibility to G418 can differ markedly depending on the particular combination of media and sera used. This suggests that in experiments using neomycin resistance as a selectable marker, conditions may be found where the amount of G418 required for selection and maintenance of transfected cell lines can be reduced, providing a significant savings.

Resistance of CHO cells expressing P-glycoprotein to cyclopropylpyrroloindole (CPI) alkylating agents.

Several new antitumor agents belonging to the class of minor groove binders that are able to form covalent bonds with DNA via a cyclopropylpyrroloindole (CPI) group are susceptible to a multidrug resistance (MDR) phenotype in Chinese hamster ovary (CHO) cells. The multidrug resistant CCHR-C5 cell line was 16-, 23- and 13-fold more resistant to the analogs U-73,975, U-77,779 and U-80,244, respectively, although its cytotoxic response to the parent compound CC-1065 was similar to the response of the drug-sensitive wild-type cells (AuxB1). For a sequence of MDR cell lines showing increasing expression of P-glycoprotein (Pgp) there were corresponding increments in the level of resistance to U-73,975, arguing that Pgp is the key determinant in resistance of the MDR cells to CPI agents. MDR cells treated with U-73,975 showed diminished generation of covalent adducts on DNA as well as increased resistance to cytotoxicity.

Variations in the response of sheep to experimental magnesium deficiency.

Hypomagnesaemia was induced in adult sheep by changing their diet from chaff given orally to a milk diet, low in Mg, infused via the abomasum. All sheep given the milk diet developed hypomagnesaemia and four out of 19 developed nervous convulsions similar to the natural disease grass tetany. Analysis of the sequential changes in plasma Ca, P and Mg revealed a striking positive correlation between Ca and Mg and hypocalcaemia thus appeared to be a general response to Mg deficiency. Many sheep appeared to adapt to the deficiency since after a period of about 11 days the decline in plasma Mg (and Ca) was either alleviated or reversed. However the four sheep which developed convulsions all showed a rapid and substantial decline in plasma Mg associated with a decline in plasma Ca.

Exploring the doctor-patient relationship: a sociocultural pilot study in a family practice residency.

Family physicians' growing attention to the nature of their patients' live should include the social and cultural factors that influence patient health and illness behavior. Patient visits to a family practice residency program were found to be influenced by the patients' beliefs about symptoms and the beliefs of their significant others, and symptom interference with valued activities. Data from physician-patient encounters suggest that physician attention to such sociocultural information as occupation and family structure may have positively influenced rapport. Results from this pilot study confirm the feasibility of observational research by physician-behavioral scientist teams in a primary care setting.

Classification and coding of psychosocial problems in family medicine.

Disease and problem classification systems for primary care have recognized that psychosocial problems are integrally related to more traditional medical problems which patients present to physicians. These classification systems remain inadequate for the description of primary care problems, especially as several disciplines interrelate in primary care, such as medicine, nursing, and social work. This paper presents a classification and coding system of psychosocial problems gleaned from a number of existing coding systems. The purpose of presenting it here is to contribute to a dialogue which will result in the establishment of a common psychosocial language for all health professionals. By so doing, progress in research, education, patient care, and administration in the psychosocial area will be facilitated.