Non-enzymatic glycation of antithrombin III in vitro

Non-enzymatic glycation of antithrombin III (AT-III) has been proposed as a significant contributor to the increased incidence of thrombo-occlusive events in diabetics. AT-III, isolated from normal human plasma by means of heparin affinity and ion-exchange chromatography, was incubated with 0-0.5 M glucose in neutral phosphate buffer at 37 degrees C. The extent of non-enzymatic glycation could be monitored by uptake of radioactivity as well as by binding to a phenylboronate affinity resin, which effectively retards AT-III containing ketoamine-linked glucose. Non-enzymatically glycated AT-III (approx. 1 mol glucose/mol protein) bound heparin nearly as efficiently as non-glycated AT-III. The two AT-III preparations were equally active in inhibiting thrombin cleavage of chromogenic substrate. Following incubation with [14C]glucose, structural analyses of cyanogen-bromide-cleaved peptides of enzymatically glycated AT-III showed that the [14C]glucose adducts were distributed over many sites on the molecule. This lack of specificity contrasts with the restricted sites of modification on hemoglobin, albumin and ribonuclease A, and explains why non-enzymatic glycation of AT-III has little if any effect on its function.     

Clonal variations in keratin : Intermediate filament expression by human somatic cell hybrids

The intermediate filament composition of differentiated vertebrate cells provides a stable phenotype which appears to be specifically regulated in each cell type. In order to analyse the regulation of intermediate filament expression we have constructed human somatic cell hybrids from the fusion of the HeLa-derived cell line HEB7A and a normal human diploid fibroblast, GM2291. These parental cells differ with respect to the presence or absence of keratin intermediate filaments. Isolation of independently arising clones produced two classes of hybrids. One class expresses keratin in a stable manner and the other class lacks keratin altogether. Indirect immunofluorescence of hybrid cells using antikeratin antiserum demonstrates that there are variations in the intensity and organization of cytoskeletal keratin staining. SDS-PAGE comparisons of cell extracts from these hybrids indicates that there are quantitative differences in the relative amounts of individual keratin polypeptides as well. These clonal variations have allowed us to begin assessing the consequences of genetic interactions between cell types that are normally capable of closely regulating different subsets of intermediate filament genes.     

Segregation of mitochondrial DNA in human somatic cell hybrids

Summary The maintenance of mtDNA has been examined in human intraspecific hybrid cells constructed from the fusion of HEB7A, a HeLa tumor cell line carrying the mitochondrially coded chloramphenical (CAP) resistance mutation, and GM 2291, a limited lifespan human diploid fibroblast which is CAP sensitive. These two cells can be distinguished by a polymorphism in a site for the restriction endonuclease, HaeIII. Independently isolated clones of hybrid cells were characterized for their growth properties (either normal limited lifespan or transformed and immortal). Whole cell DNA preparations were made from each hybrid, digested with HaeIII, and the resultant fragments were detected by hybridization to ³²P labelled mouse mtDNA as probe. Experiments with mixtures of HEB7A and GM2291 DNA reveal that HEB7A mtDNA can be detected when it constitutes as little as 5% of the total cell mtDNA.The results indicate that the HEB7A mtDNA is lost from most hybrids, and when it does persist it is usually a minor component of total mtDNA. The addition of CAP at the time of fusion slightly increases the quantity of HEB7A mtDNA, but not enough to confer CAP resistance. Furthermore, five limited lifespan hybrids contained no detectable HEB7A mtDNA, while three transformed hybrids contained varying quantities of HEB7A mtDNA, suggesting that retention of this tumor form of mtDNA is associated with tumor growth behavior. These results suggest that cytoplasmic genetic incompatibility occurs in intraspecific hybrids.     

Using patient and general practice characteristics to explain variations in cervical smear uptake rates.

OBJECTIVES--To produce practice and patient variables for general practices from census and family health services authority data, and to determine the importance of these variables in explaining variation in cervical smear uptake rates between practices. DESIGN--Population based study examining variations in cervical smear uptake rates among 126 general practices using routine data. SETTING--Merton, Sutton, and Wandsworth Family Health Services Authority, which covers parts of inner and outer London. MAIN OUTCOME MEASURE--Percentage of women aged 25-64 years registered with a general practitioner who had undergone a cervical smear test during the five and a half years preceding 31 March 1992. RESULTS--Cervical smear uptake rates varied from 16.5% to 94.1%. The estimated percentage of practice population from ethnic minority groups correlated negatively with uptake rates (r = -0.42), as did variables associated with social deprivation such as overcrowding (r = -0.42), not owning a car (r = -0.41), and unemployment (r = -0.40). Percentage of practice population under 5 years of age correlated positively with uptake rate (r = 0.42). Rates were higher in practices with a female partner than in those without (66.6% v 49.1%; difference 17.5% (95% confidence interval 10.5% to 24.5%)), and in computerised than in non-computerised practices (64.5% v 50.5%; 14.0% (6.4% to 21.6%)). Rates were higher in larger practices. In a stepwise multiple regression model that explained 52% of variation, five factors were significant predictors of uptake rates: presence of a female partner; children under 5; overcrowding; number of women aged 35-44 as percentage of all women aged 25-64; change of address in past year. CONCLUSIONS--Over half of variation in cervical smear uptake rates can be explained by patient and practice variables derived from census and family health services authority data; these variables may have a role in explaining variations in performance of general practices and in producing adjusted measures of practice performance. Practices with a female partner had substantially higher uptake rates.     

The sigma compounds 1,3-di-o-tolylguanidine and N-allylnormetazocine inhibit agonist-stimulated inositol phospholipid metabolism in bovine adrenal medullary cells

Muscarine stimulated a concentration-dependent accumulation of [³H]inositol phosphates in bovine adrenal medullary cells preloaded with [³H]inositol. This muscarinic activation of inositol phospholipid metabolism was fully inhibited by the -ligand 1,3-di-o-tolylguanidine (DTG) with an IC₅₀ of approximately 45 M. Higher concentrations (100 M) of (+) N-allylnormetazocine (SKF-10047) also partially inhibited this response. A concentration of DTG sufficient to fully inhibit the muscarinic response also produced a significant partial inhibition of [³H]inositol phosphate accumulation in response to histamine but not to angiotensin II. These data demonstrate that -compounds inhibit agonist-stimulated inositol phospholipid metabolism in bovine adrenal medullary cells, with a degree of selectivity towards the muscarinic response.     

Single-dose murine monoclonal antibody ricin A chain immunotoxin in the treatment of metastatic melanoma: a phase I trial.

To determine the maximally tolerated dose of a ricin A chain-conjugated antimelanoma antibody (XomaZyme-Mel), 20 patients with metastatic melanoma were treated with escalating doses of the murine immunotoxin given as single intravenous infusion over 30 minutes. The starting dose was 0.6 mg/kg and was escalated in five groups to a maximum of 1.6 mg/kg. The maximally tolerated dose was 1.25 mg/kg as three of six patients treated at 1.6 mg/kg developed unacceptable toxicity. The dose-limiting toxicity consisted of profound fatigue, myalgias, and arthralgias. These occurred within 4 days and resolved in 7 to 10 days. Other non-dose-limiting toxicities encountered consisted of hypoalbuminemia, weight gain, peripheral edema, mild hypotension, and flu-like syndrome; the severity of these was also dose related. In addition, two allergic reactions occurred, one severe. There was one durable complete response of 12+ months' duration and one brief mixed response lasting 3 months. We conclude that the maximum tolerated single dose of XomaZyme-Mel is 1.25 mg/kg. Phase I studies evaluating 1.25 mg/kg given in multiple doses at 2- to 4-week intervals and phase II studies to determine the response rate of a single 1.25 mg/kg dose are warranted.     

Aeration: A simple method to control vitrification and improve in vitro culture of rare australian plants

Summary Aeration of tissue cultured rare Australian plantsConostylis wonganensis S.D. Hopper (Haemodoraceae);Diplolaena andrewsii Ostenf.;Drummondita ericoides Harvey (Rutaceae);Eremophila resinosa F. Muell. (Myoporaceae);Eucalyptus ‘graniticola’ (Myrtaceae);Lechenaultia pulvinaris C. Gardner (goodeniaceae); andSowerbaea multicaulis E. Pritzel (Liliaceae) has been found to reduce vitrification in sensitive species as well as significantly improving shoot quality and transfer to soil in most study species. A simple 7-mm hole with a double-layer insert of filter paper in the polypropylene screw lids of the culture vessel decreased shoot vitrification over a 4-wk culture period. The method has implications for facilitating the tissue culture of other rare Australian plants and reducing the occurrence of this developmental abnormality.     

Cytoplasmic inheritance in mammalian tissue culture cells.

A series of intraspecific, interspecific and interorder somatic cell cybrids and hybrids have been prepared by fusions in which one of the parents contained the cytoplasmically inherited marker for chloramphenicol (CAP) resistance. A clear relationship has been established between the expression of the CAP-resistant (CAP-R) determinants in the fusion products and the genetic homology of the parents. With increased genetic divergence, the acceptability of the CAP-R mitochondria decreased. Intraspecific cybrids and hybrids of the same strain were stable for the CAP-R marker, while those between strains were stable only in CAP. Intergeneric mouse-hamster cybrids occurred at a high frequency but were unstable in CAP, while CAP suppressed hybrid formation 100-fold. Interorder cybrids (CAP-R human X CAP-S mouse) occurred either at a moderate frequency and were stable at a low frequency and were unstable in CAP. Interorder hybrids could only be formed by challenging HAT-selected hybrids with CAP or by direct selection in ouabain and CAP. Reciprocal interorder crosses between CAP-R mouse and CAP-S human cells were unsuccessful. Interspecific cybrids contain only the chromosomes of the CAP-S parent. Interspecific hybrids selected directly in CAP segregated the chromosomes of the CAP-S parent, while hybrids selected in HAT and then CAP segregated those of the CAP-R parent. The mitochondrial DNA(mtDNA) of all mouse-human cybrids and most HAT and then CAP-selected hybrids contain only the mtDNA of the CAP-S mouse parent. However, preliminary evidence suggests that one of these hybrids contains both mouse and human mtDNA sequences.