Analysis ofras mutations in human melanocytic lesions: activation of theras gene seems to be associated with the nodular type of human malignant melanoma

We have analyzed the Ha-ras, Ki-ras and N-ras gene for point mutations at codons 12, 13 and 61 via restriction fragment length polymorphism/polymerase chain reaction analysis and subsequent direct sequencing in non-cultured fresh-frozen tissues of 16 superficial spreading melanomas (SSM), 13 nodular malignant melanomas (NMM), 2 lentigo malignant melanomas (LMM), 1 dysplastic nevus, 1 congenital nevus and 5 normal nevi from 38 patients. Mutations were found in 4 melanoma samples, all belonging to the nodular malignant type. Three of them were mutated in N-ras and one in the Ha-ras gene. Mutation in N-ras was also detected in the congenital nevus. All mutations were exclusively located at the first two base pairs of codon 61. No Ki-ras mutation was detected in any lesion. No mutation could be found in SSM and LMM in addition to dysplastic and normal nevi. The frequency ofras mutation in NMM was 31%, whereas in SSM it was 0%. Our study suggests (a) an association betweenras mutations (mainly N-ras) and the NMM as a subgroup of human melanoma; (b) that activation of Ki-ras is not involved in the pathogenesis of melanoma. The role of UV radiation in point mutations ofras genes in human melanoma is discussed. This investigation was supported by grant EV5V-CT92-0096     

Enhanced leukocyte binding by intestinal microvascular endothelial cells in inflammatory bowel disease

BACKGROUND & AIMS: Microvascular endothelial cells mediate leukocyte homing, angiogenesis, and inflammation and healing and show tissue- specific adhesion molecules and functions. The activation of human intestinal mucosal microvascular endothelial cells (HIMECs) was studied in vitro to uncover possible abnormalities associated with inflammatory bowel disease. METHODS: HIMECs were isolated from normal and inflammatory bowel disease mucosa and assessed for phenotypic and morphological features, proliferative response, leukocyte binding capacity, and adhesion molecule expression. RESULTS: Basal proliferation by HIMECs was less than that of human umbilical vein endothelial cells (HUVECs) but increased proportionally more in response to vascular endothelial growth factor. Proinflammatory stimuli induced an activated, spindle-shaped morphology in HIMEC monolayers. Compared with HUVECs, unstimulated HIMECs showed less adhesiveness for U937 and MOLT4 cells and neutrophils, but cytokines and lipopolysaccharide substantially increased the binding capacity of HIMECs. HIMECs derived from inflammatory bowel disease mucosa showed a markedly greater leukocyte-binding capacity than normal mucosal HIMECs. Patterns of intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and E-selectin messenger RNA expression were distinct in HIMECs, HUVECs, and mucosal mesenchymal cells. CONCLUSIONS: HIMECs represent differentiated endothelial cells with unique functional properties. Their dramatically enhanced capacity to bind leukocytes in inflammatory bowel disease suggests that HIMECs play an important role in initiating or maintaining inflammation. (Gastroenterology 1997 Jun;112(6):1895-907)     

Cystic fibrosis and the phenotypic expression of autosomal dominant polycystic kidney disease

Recent experiments in cultured cyst epithelial cells from kidneys of patients with autosomal dominant polycystic kidney disease (ADPKD) have shown that the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is present in the apical surface of these cells and mediates chloride (Cl-) and fluid secretion in vitro. To determine whether the presence of CF with the expression of mutated CFTR proteins modifies cyst formation in ADPKD, we studied a large family with both inherited diseases. ADPKD in this family is linked to PKD1. The family is composed of 26 members; 11 members with ADPKD, 4 members with CF, and 2 members with both diseases. Renal volumes measured by computerized tomography (CT), calculated creatinine clearances, and other clinical parameters in the family members with ADPKD and CF were compared with those in the family members with ADPKD alone, as well as to a large population of patients with ADPKD. The patients with CF and ADPKD, but not the CF heterozygote carriers with ADPKD, had less severe polycystic kidney and liver disease, as indicated by normal renal function; smaller renal volume, even when corrected for height and body surface area; and the absence of hypertension and liver cysts. These observations suggest that the coexistence of CF may reduce the severity of ADPKD.     

Mitotic disturbances and micronucleus induction in Syrian hamster embryo fibroblast cells caused by asbestos fibers.

Asbestos and other mineral fibers have long been known to induce lung cancer and mesothelioma. However, the primary mechanisms of fiber-induced carcinogenesis still remain unclear. We investigated the occurrence of mitotic disturbances induced by asbestos (amosite, crocidolite, chrysotile) in an in vitro approach using Syrian hamster embryo (SHE) fibroblast cells. The following endpoints were investigated: micronucleus formation as a result of mitotic disturbances and characterization of the induced micronucleus population by kinetochore staining and visualization of the spindle apparatus. Supravital UV-microscopy was used to analyze changes in interphase chromatin structure, impaired chromatid separation, and blocked cytokinesis. All three asbestos fiber types induced a high frequency of micronucleus formation in SHE cells (> 200/2000 cells) in a dose-dependent manner (0.1-5.0 micrograms/cm2), with a maximum between 48 hr and 66 hr exposure time. At higher concentrations (more than 5.0 micrograms/cm2) the micronucleus formation decreased again as a result of increased toxicity. Kinetochore staining of micronuclei revealed that 48 +/- 2% of asbestos-induced micronuclei reacted positively with CREST (antikinetochore) serum. Furthermore, spindle apparatus deformations occurred in cells with disturbed metaphases and anaphases, while the spindle fiber morphology appeared unchanged. Our results show that asbestos fibers may cause both loss and breakage of chromosomes in the absence of direct interaction with spindle fibers.     

Genotoxicity and cell transformation studies with sorbates in Syrian hamster embryo fibroblasts.

Sorbic acid, sodium sorbate and potassium sorbate were tested for their genotoxic potential in the Syrian hamster embryo (SHE) fibroblast micronucleus assay and the SHE cell transformation test in vitro. Sorbic acid and potassium sorbate showed no activity in either test system. When freshly prepared sodium sorbate solutions were used, no genotoxic or cell-transforming activity was detected. However, sodium sorbate as stored solution, which previously had been heated and sonicated to facilitate solubilization, yielded a positive response in both test systems. It is concluded that oxidation products of sodium sorbate that possess genotoxic and cell-transforming properties are formed under conditions of heating, sonication and storage.     

Risk factors for development of dehydration in children aged under five who have acute watery diarrhoea: a case-control study

Objective: To identify risk factors for development of dehydration in under five year olds with acute watery diarrhoea.Design: Hospital based unmatched case-control study.Setting: Diarrhoea Treatment Unit, Government Medical College Hospital, Nagpur, India.Participants: The study included 387 cases of diarrhoea having severe or moderate dehydration and 387 controls suffering from diarrhoea with mild or no dehydration.Risk factors: The study included infancy, female sex, religion, residing in urban slums or rural area, under nutrition, cessation of breast feeding during diarrhoeal episode, fluid intake decreased/stopped during diarrhoea, ORS not received, home available fluids (HAF) not received, both ORS and HAF not received, non-washing of hands by mother before preparation of food, after defaecation, after disposal of faeces, history of measles in the previous six months, frequency of stools >8/d, frequency of vomiting more than twice per day and temperature more than 99°F, as risk factors for development of dehydration.Statistical analysis: Univariate analysis included OR, 95% CI for OR and Chi-square test. Multivariate analysis was carried out by unconditional multiple logistic regression (MLR).Results: This study identified the significance of infancy, religion, severe undernutrition, non-washing of hands by mother before preparation of food, frequency of stool >8/d, frequency of vomiting >2/d, history of measles in previous six months, withdrawal of breast feeding during diarrhoea, withdrawal of fluids during diarrhoea and not giving ORS, HAF or both during diarrhoea, in the outcome of development of moderate or severe dehydration.Conclusions: Timely intervention in the preventable risk factors included in this study may prevent the development of moderate or severe dehydration in the children suffering form acute watery diarrhoea.