Demonstration of pH control in a commercial immobilized glucose isomerase

The synthesis of a variety of important biochemicals involves multistep enzyme-catalyzed reactions. In many cases, the optimal operating pH is much different for the individual enzymatic steps of such synthesis reactions. Yet, it may be beneficial if such reaction steps are combined or paired, allowing them to occur simultaneously, in proximity to one another, and at their respective optimal pH. This can be achieved by separating the micro-environments of the two steps of a reaction pathway using a thin urease layer that catalyzes an ammonia-forming reaction. In this article, the pH control system in a commercial immobilized glucose (xylose) isomerase pellet, which has an optimal pH of 7.5, is demonstrated. This system allows the glucose isomerase to have near its optimal pH activity when immersed in a bulk solution of pH 4.6. A theoretical analysis is also given for the effective fraction of the immobilized glucose isomerase, which remains active when the bulk pH is at 4.6 in the presence of 20 mM urea versus when the bulk pH is at its optimal pH of 7.5. Both theoretical and experimental results show that this pH control system works well in this case. (c) 1996 John Wiley & Sons, Inc.     

Advantages of the scanning ion microscopy for mapping halogen corticoids in normal and transformed cells in culture

Summary— The intra-cellular distribution of eight halogen glucocorticoids was investigated by ion microscopy in two cellular varieties of cultured non-cancer cells (fibroblast 3T3) and cancer cells (human breast tumor cells MCF-7). Two types of ion microscopy helped to determine this distribution, a direct imaging ion microscope (SMI 300) with low spatial resolution, and a scanning ion microscope (IMS4F), featuring high resolution, serving to obtain maps representing the intra-cellular distribution of the fluorine elements and drugs present in these monolayer cultured cells. The fluorine images representative of the drugs containing fluorine showed that these drugs are essentially concentrated in the cell nuclei. In these nuclei, the distribution of these drugs is different from that of heterochromatin and of the nucleolus.     

Familial Site-specific Ovarian Cancer Is Linked to BRCA1 on 17q12-21

In a study of nine families with “site-specific” ovarian cancer (criterion: three or more cases of epithelial ovarian cancer and no cases of breast cancer diagnosed at age <50 years) we have obtained evidence of linkage to the breast-ovarian cancer susceptibility gene, BRCA1 on 17q12-21. If the risk of cancer in these families is assumed to be restricted to the ovary, the best estimate of the proportion of families linked to BRCA1 is .78 (95% confidence interval .32–1.0). If predisposition to both breast and ovarian cancer is assumed, the proportion linked is 1.0 (95% confidence interval .46–1.0). The linkage of familial site-specific ovarian cancer to BRCA1 indicates the possibility of predictive testing in such families; however, this is only appropriate in families where the evidence for linkage to BRCA1 is conclusive.     

Involvement of an N-Acetylglucosaminidase in Autolysis of Propionibacterium freudenreichii CNRZ 725

Propionibacterium freudenreichii plays an important role in Swiss cheese ripening (it produces propionic acid, acetic acid, and CO₂). Moreover, autolysis of this organism certainly contributes to proteolysis and lipolysis of the curd because intracellular enzymes are released. By varying external factors, we determined the following conditions which promoted autolysis of both whole cells and isolated cell walls of P. freudenreichii CNRZ 725: (i) 0.1 M potassium phosphate buffer (pH 5.8) at 40°C and (ii) 0.05 to 0.1 M KCl at 40°C. We found that early-exponential-phase cells possessed the highest autolytic activity. It should be emphasized that the pH of Swiss cheese curd (pH 5.5 to 5.7) is near the optimal pH which we determined. Ultrastructural observations by electron microscopy revealed a 16-nm-thick homogeneous cell wall, as well as degradation of the cell wall that occurred concomitantly with cell autolysis. In the presence of 0.05 M potassium chloride, there was a great deal of isolated cell wall autolysis (the optical density at 650 nm decreased 77.5% ± 7.3% in 3 h), and one-half of the peptidoglycan material was released. Finally, the main autolytic activity was due to an N-acetylglucosaminidase activity.     

Effect of ischemia/reperfusion sequence on cytosolic iron status and its release in the coronary effluent in isolated rat hearts

The hypothesis that oxygen-derived free radicals play an important role in myocardial ischemic and reperfusion injury has received a lot of support. In the presence of catalytic amounts of transition metals such as iron, superoxide anions, and hydrogen peroxide can be transformed into a highly reactive hydroxyl radical °OH (Haber-Weiss reaction). In view of this, we have undertaken this study to investigate whether iron is involved in the reperfusion syndrome and therefore could aggravate free radicals injury. Coronary effluent iron concentrations and cardiac cytosolic iron levels were evaluated in rat hearts subjected to an ischemia/reperfusion sequences. In the case of total ischemia, iron concentration in coronary effluents peaked immediately in the first sample collected upon reperfusion. However, in the case of partial ischemia, iron concentration in coronary effluents peaked rather exclusively during ischemia period. Cardiac cytosolic iron level augmented significantly after 30 min of total ischemia and non significantly in the other ischemia protocols compared to perfused control hearts. It also appears that the iron released is not protein-bound, and could therefore have a marked catalytic activity. The results of the present study suggest that in the oxygen paradox, iron plays an important role in inducing alterations during reoxygenation.     

A protein involved in co-ordinated regulation of inorganic carbon and glucose metabolism in the facultative photoautotrophic cyanobacteriumSynechocystis PCC6803

The involvement of a gene ofSynechocystis PCC6803,icfG, in the co-ordinated regulation of inorganic carbon and glucose metabolism, was established. TheicfG gene codes for a 72 kDa protein, which shows no homology with those registered in data libraries. Expression oficfG required glucose, the actual inducer probably being glucose-6-phosphate, and was independent of light and of the external inorganic carbon concentration. Mutants carrying an inactivated copy oficfG were constructed. Their growth characteristics were identical to those of the wild type under all regimes except in limiting inorganic carbon with glucose being present either before or after the transfer to the limiting conditions. These conditions completely prevented growth, both in the light and in the dark. The inhibition could be relieved by several intermediates of the tricarboxylic acid cycle. Assays of various enzymic activities related to inorganic carbon uptake and to its assimilationvia either the Calvin cycle or phosphoenolpyruvate carboxylase did not reveal the level of action of IcfG. Possible models include a blockage of the assimilation of both carbon sources in the absence of IcfG, or the inhibition of Ci incorporation route(s) essential under limiting inorganic carbon conditions, even when glucose is present, and even in the dark.     

Drosophila melanogaster acetylcholinesterase: Identification and expression of two mutations responsible for cold- and heat-sensitive phenotypes

Ace ^IJ29 and Ac ^IJ40 are cold- and heat-sensitive variants of the gene coding for acetylcholinesterase in Drosophila melanogaster. In the homozygous condition, these mutations are lethal when animals are raised at restrictive temperatures, i.e., below 23° C for Ace ^IJ29 or above 25° C for Ace ^IJ40. The coding regions of the gene in these mutants were sequenced and mutations changing Ser³⁷⁴ to Phe in Ace ^IJ29 and Pro⁷⁵ to Leu in Ace ^IJ40 were found. Acetylcholinesterases bearing these mutations were expressed in Xenopus oocytes and we found that these mutations decrease the secretion rate of the protein most probably by affecting its folding. This phenomenon is exacerbated at restrictive temperatures decreasing the amount of secreted acetylcholinesterase below the lethality threshold. In parallel, the substitution of the conserved Asp²⁴⁸ by an Asn residue completely inhibits the activity of the enzyme and its secretion, preventing the correct folding of the protein in a non-conditional manner.     

Modulation of drug-induced cytotoxicity by a bispecific monoclonal antibody that recognizes the epidermal growth factor receptor and doxorubicin

A hybrid hybridoma secreting a bispecific hybrid mAb (bsmAb), which recognizes both the epidermal growth factor receptor (EGF-R) and the drug doxorubicin, was produced by somatic hybridization of two hybridomas. The bsmAb obtained was able to retarget doxorubicin cytotoxicity in vitro specifically on EGF-R-positive cells exerting at the same time an antidotal effect on cells that did not overexpress the EGF-R. Distribution studies in mice indicate that the bsmAb selectively delivers the drug to tumour cells and modifies doxorubicin biodistribution with a statistically significant decrease of drug concentration in the intestine, which is the main target of early anthracycline toxicity. In keeping with this finding is the remarkable antidotal activity exerted by bsmAb in mice treated with doxorubiein, which is proved by retardation in loss of body weight and mortality. The effectiveness on tumour growth of the mAb followed by the administration of doxorubicin appears to be equal to that of the drug alone; however, the bsmAb exerts a remarkable antidotal activity.