In vitro BrdUrd incorporation of colorectal tumour tissue

Abstract. This study describes a novel in vitro method for the incorporation of the thymidine analogue, bromodeoxyuridine (BrdUrd), in fresh colorectal tumour tissue. Disaggregation by pronase, collagenase and DNAse resulted in high cell yields of viable single cell suspensions, representative of the original tumour, which could be infiltrated with BrdUrd. A modified ELISA identified optimal incubation times and BrdUrd concentrations. This technique has been used in preliminary studies to investigate two important areas intrinsic in the analysis of BrdUrd colorectal cell proliferation data: 1 to determine the effects of the individual constituents of the cell culture media, in particular glutamine, on BrdUrd incorporation in suspensions of colorectal cells and 2 to examine the denaturation step. This method will have wide applicability in investigations of cell proliferation status in both normal and diseased tissue.     

Distinction between carcinoma cells and mesothelial cells in serous effusions. Usefulness of immunohistochemistry

The usefulness of an immunoperoxidase battery to distinguish carcinomatous from benign effusions was examined. Cell block sections from 90 previously diagnosed effusions were stained with antibodies to Leu-M1, B72.3, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA) and vimentin. The 90 cases comprised 69 carcinomas (23 mammary, 16 ovarian, 10 pulmonary, 7 gastrointestinal [GI] and 13 others), 2 malignant mesotheliomas and 19 cases with reactive mesothelial cells only. EMA and vimentin were the most useful markers for distinguishing carcinoma cells from reactive mesothelial cells. EMA reacted with 86% of the carcinomas while vimentin reacted with 90% of the reactive cases. Leu-M1, B72.3 and CEA, although generally less sensitive than EMA, were also helpful in this regard. Additionally, the use of Leu-M1 and CEA together may help to distinguish pulmonary from GI carcinomas.     

Goniometric measurement of hip motion in cycling while standing

The purpose of this study was to develop an instrument for quantifying the motion of the hip relative to the bicycle while cycling in the standing position. Because of the need to measure hip motion on the road as well as in the laboratory, a goniometer which locates the hip using spherical coordinates was designed. The goniometer is presented first, followed by the development of the equations that enable the distance from the joint center to the pedal spindle to be determined. The orientation of this line segment is specified by calculating two angles referenced to the frame. Also outlined are the procedures used to both calibrate the goniometer and perform an accuracy check. The results of this check indicate that the attachment point of the goniometer to the rider can be located to within 2.5 mm of the true position. The goniometer was used to record the hip movement patterns of six subjects who cycled in the standing position on a treadmill. Representative results from one test subject who cycled at 6% grade and 25 km h-1 are presented. Results indicate that the bicycle is leaned from side to side with the frequency of leaning equal to the frequency of pedalling. Extreme lean angles are +/- 6 degrees. The distance from the hip to the pedal varies approximately sinusoidally with frequency equal to pedalling rate and amplitude somewhat less than crank arm length. The absolute elevation of the hip, however, exhibits two cycles for each crank cycle. Asymmetry in the plot of elevation over a single crank cycle indicates that the pelvis rocks from side to side and that the elevation of the pelvis midpoint changes. Extreme values of the pelvis rocking angle are +/- 12 degrees. Highest pelvis midpoint elevations, however, do not occur at the same crank angles as those angles at which the pelvis rocking is extreme. It appears that the vertical motion of the hips affects pedalling mechanics when cycling in the standing position.     

Age related toxic effects of endosulfan on certain enzymes of rat erythrocytes

The effect of oral administration of endosulfan (12.5 mg/kg body weight), daily for 4 days was investigated on erythrocytes of female rats of 4 different age groups i.e. 15, 30, 70 and 365 days old. Erythrocyte membrane Na+, K(+)-ATPase and Mg2(+)-ATPase activities were significantly inhibited in all the age groups of rats. However, percent inhibition was maximum in the youngest animals. A significant decrease in the activity of erythrocyte glutathione reductase was observed in 30 and 70 days old rats whereas a significant increase in the activity of glucose-6-phosphate dehydrogenase (G-6-PD) was observed in these groups. The increase in G-6-PD activity may be a physiological response to compensate for decrease in the reduced glutathione level which results from decrease in the activity of glutathione reductase.     

Peroxide Damage to the Eye Lens In Vitro Prevention by Pyruvate

The ability of pyruvate to protect the eye lens against physiological damage by hydrogen peroxide has been studied. The physiological damage was estimated in terms of a decrease in the ability of the lens to transport rubidium against an electrochemical gradient under organ culture conditions. Peroxide was either added directly to the culture medium or generated therein by incorporation of xanthine and xanthine oxidase. In both these cases, addition of pyruvate to the medium led to a greater accumulation of rubidium by the lens. The net accumulation of this cation in the presence of 1 to 5 mM pyruvate from the medium containing peroxide (0.2 to 0.45 mM) was very close to that observed in the absence of peroxide. The protective effect was thus substantial. The mechanism of the pyruvate effect has been discussed, and seems to be related to the scavenging of peroxide by pyruvate.     

Toxicity of the methyl isocyanate metabolite S-(N-methylcarbamoyl)GSH on mouse embryos in culture.

Methyl isocyanate, the chemical involved in the 1984 accident at Bhopal, India, forms a labile conjugate, S-(N-methylcarbamoyl)GSH (SMG), by way of a reversible reaction with GSH. We studied the toxicity of SMG on mouse embryos explanted on day 8 of gestation and cultured in rat serum for 42 hr. SMG caused concentration-dependent decreases in growth and development over the range 0.1-2 mM, without causing significant mortality. At a concentration of 2 mM, SMG completely arrested embryo development, but heartbeat was absent in only one of nine embryos at 42 hr. At a concentration of 0.25 mM, SMG reduced embryo size to 75% and protein content to 63% of the control; 18% of embryos failed to rotate. At this concentration (0.25 mM), which was selected for all other studies, spinal kinks and somite pair distortion in the region of the forelimb were evident in 38% of embryos; no other abnormalities were noted. DNA content of and thymidine incorporation by embryos and yolk sacs was reduced by SMG, although this was more pronounced in the yolk sac than in embryos. At subtoxic concentrations, the L-cysteine precursor (-)-2-oxo-4-thiazolidine-carboxylic acid did not, but GSH did, inhibit embryotoxicity of SMG. It is concluded that SMG exerts embryotoxic and dysmorphogenic effects and may contribute to systemic toxicity of methyl isocyanate.     

Measurement of Elastic Modulus of Collagen Type I Single Fiber

Collagen fibers are the main components of the extra cellular matrix and the primary contributors to the mechanical properties of tissues. Here we report a novel approach to measure the longitudinal component of the elastic moduli of biological fibers under conditions close to those found in vivo and apply it to type I collagen from rat tail tendon. This approach combines optical tweezers, atomic force microscopy, and exploits Euler-Bernoulli elasticity theory for data analysis. This approach also avoids drying for measurements or visualization, since samples are freshly extracted. Importantly, strains are kept below 0.5%, which appear consistent with the linear elastic regime. We find, surprisingly, that the longitudinal elastic modulus of type I collagen cannot be represented by a single quantity but rather is a distribution that is broader than the uncertainty of our experimental technique. The longitudinal component of the single-fiber elastic modulus is between 100 MPa and 360 MPa for samples extracted from different rats and/or different parts of a single tail. Variations are also observed in the fibril-bundle / fibril diameter with an average of 325±40 nm. Since bending forces depend on the diameter to the fourth power, this variation in diameter is important for estimating the range of elastic moduli. The remaining variations in the modulus may be due to differences in composition of the fibril-bundles, or the extent of the proteoglycans constituting fibril-bundles, or that some single fibrils may be of fibril-bundle size.     

Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC₅₀ values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H₂O₂) induced RBC haemolysis (IC₅₀ 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H₂O₂-induced damage; inhibited H₂O₂ induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations.