Measurement by HPLC of Desferrioxamine-Available Iron in Rabbit Kidneys to Assess the Effect of Ischaemia on the Distribution of Iron Within the Total Pool

A method for the determination of desferrioxamine-available iron in tissue fractions is described which involves incubation with desferrioxamine, extraction of desferrioxamine and its iron-bound form, ferrioxamine, and quantitation of these two forms of the drug by reversed-phase hplc analysis. Chelatable iron levels in the 1-10µMolar region could be accurately and reproducibly measured using this technique.

The desferrioxamine-available iron levels in both the cortex and medulla of rabbit kidneys were significantly elevated (up to 2-fold) after the organs had been subjected to 2 hours warm ischaemia or 24 hours cold storage at 0°C In hypertonic citrate solution. There was no change in the total iron content of the tissues under these circumstances and thus a redistribution of intracellular iron to more available pools had presumably taken place as a result of ischaemia. This redistribution of iron may be an important factor in the initiation of peroxidative damage to cell membranes upon reperfusion of the organ with oxygen.     

Guanylyl cyclase is a heat-stable enterotoxin receptor.

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.     

A molecular basis for familial hypertrophic cardiomyopathy: an alpha/beta cardiac myosin heavy chain hybrid gene

An alpha/beta cardiac myosin heavy chain (MHC) hybrid gene is coinherited with familial hypertrophic cardiomyopathy (FHC) in one kindred. FHC is a disease of the heart muscle characterized by a thickening of the left ventricular wall with myocyte and myofibrillar disarray that is inherited as an autosomal dominant trait. We demonstrate here and in the accompanying article that the cardiac MHC genes, which encode integral myofibrillar components, are mutated in all affected individuals from two unrelated families with FHC. In one kindred, an unequal crossover event during meiosis may have produced the alpha/beta cardiac MHC hybrid gene that is present in affected individuals. We conclude that mutations in the cardiac MHC genes can cause FHC.     

Desensitization of the Neurokinin 1 Receptor Is Mediated by the Receptor Carboxy-Terminal Region, but Is Not Caused by Receptor Internalization

Abstract: The carboxy-terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy-terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5-trisphosphate response) when preexposed for 1 min to 1 µ M neurokinin, indicating a role for the carboxy-terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 n M ) indicated that ∼75–80% of the receptors were internalized in each cell line after 10 min at 37°C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 µ M substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31-8220, staurosporine, and Zn²⁺ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate-limiting in neurokinin 1 receptor desensitization in this system.     

The molecular analysis ofbrown eye color mutations isolated from geographically discrete populations ofDrosophila melanogaster

A large proportion of spontaneous mutations inDrosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessivebrown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caughtDrosophila melanogaster females,bw mutations were isolated from seven separate geographic sites distributed among Japan, California, Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in thebw gene. The remainder are inseparable from wild-typebw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneousbw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.     

Effects of substrate limitation on product distribution and H2/CO2 ratio inKlebsiella pneumoniae during anaerobic fermentation of glycerol

Product formation during anaerobic degradation of glycerol byKlebsiella pneumoniae DSM 2026, under glycerol limitation and glycerol excess in continugius cultures, has been investigated. Major and minor products and by-products as well as gaseous products were measured. The results indicated a positive correlation between specific glycerol uptake and most product formation rates under glycerol limitation. The production of 1,3-propanediol, lactate, formate, acetate, succinate and the by-products of anaerobic glycerol degradation byK. pneumoniae, acetoin and 2,3-butanediol, was favoured by glycerol excess, while hydrogen generation and ethanol formation were best under glycerol limitation. It was also found that under glycerol limitation the rate of hydrogen evolution was generally higher than the CO₂ production rate while under excess glycerol the reverse was true. Hence, on the basis of the ratio of the specific rates of evolution of H₂ and CO₂ (q _{H 2}/q _{CO 2}), it is possible to infer the existence of glycerol limitation. On the basis of the carbon and available electron balances, which are independent of metabolic pathways, the data are consistent. The NADH₂ balance, which took into consideration the pathways of product formation, was also tested to check the validity of the assumed pathways and to check critically the consistency of the data. Good balances were also obtained.[     

Cell-substrate adhesion and metastatic potential of cultured mesothelioma cells induced by asbestos

Cell-substrate adhesion was quantified for two cultured mesothelioma cell lines (epitheliomatus and sarcomatous) on glass, fibronectin and laminin substrates. Interference reflection microscopy (IRM) was used to image the adhesion patterns of cells and a grey level analysis was employed to quantify adhesion. Sarcomatous cells demonstrated marked adhesion to glass and fibronectin-coated substrates but not to laminin-coated substrate, with the greatest adhesion occurring on the fibronectin-coated surface. This adhesion was accompanied by cytoplasmic spreading. By contrast, epitheliomatous cells showed little tendency to adhere to any of the substrates and only showed significant spreading when in contact with the laminin substrate (P < 0.01). A bioassay was used to determine the metastatic potential of each of the cell lines. Via the intravenous route, the sarcomatous cells killed the host rats in 24.7 ± 1.5 (S.D.) days compared to 27.3 ± 0.9 (S.D.) days for the epitheliomatous cells (P < 0.01). After subcutaneous inoculation of tumour cells, the sarcomatous cells killed the host rats in 54.7 ± 0.7 (S.D.) days compared to 48.5 ± 0.5 (S.D.) days for the epitheliomatous cells (P < 0.01). We conclude that the results of the metastasis bioassays were consistent with the predicted behavior of these cell lines based on their ability to adhere to substrates in the in vitro adhesion assays.     

A robust fed-batch feeding strategy for optimal parameter estimation for baker's yeast production

Parameter identification of structured models is often a problem in biotechnology, because the poor data situation and the number of unknown parameters only allow for inaccurate estimates. But often only a subset of all kinetic parameters of the model are of interest for production purposes, e.g. for fed-batch cultivation. These parameters should be estimated with a given accuracy. In addition, the experiments for information acquisition with respect to these parameters should be as simple as possible and should consider some practical restrictions. In this contribution a fed-batch feeding strategy is proposed to allow for an accurate estimation of yield and of critical growth rate of baker's yeast. The feeding also allows for economic and stereotyped use of staff and equipment and is therefore suitable for routine use in screening of strains and media. The overall pattern is similar to that one, usually used in production scale to minimize errors by limited model validity. After an initial phase for achieving a reproducible state three different growth rates are adjusted to cover the range of possible critical growth rates. From biomass and ethanol measurements yield and critical growth rate can be estimated with an accuracy of about 2.1%. The fermentation pattern ends up with a constant feeding rate to simulate a limited oxygen transfer rate and to allow for an uptake of residual sugar and ethanol before a dough test can be carried out. Beside experimental results simulations and sensitivity analyses are shown.List of Symbols P ethanol concentration - S substrate concentration - S _f substrate concentration in feed - T fermentation time - V fermenter volume - X biomass concentration - C measurement error covariance matrix - F Fisher information matrix - X state variables - Y output variables - X _p state sensitivity functions with respect to parameters - Y _p output sensitivity functions - e eigenvectors - k vector of limitation and inhibition parameters - n number of observations - q _in feeding stream - q _b stream for samples and ammonia feed - r vector of specific turnover rates - y vector of yields - specific weight - eigenvalues - specific growth rate - _set exponent in exponential feeding - standard deviation Dedicated to the 65th birthday of Professor Fritz Wagner.A. O. Ejiofor and B. O. Solomon are grateful to the Alexander von Humboldt Stiftung for granting them fellowships and to GBF for providing all the materials necessary for their successful research stay in Germany.