Paternal cyclophosphamide treatment causes postimplantation loss via inner cell mass-specific cell death.

Treatment of the father with the anticancer alkylating agent cyclophosphamide has negative effects on embryonic development in the rat. Four-week treatment of male rats with a low dose of cyclophosphamide causes a dramatic, dose-dependent increase in postimplantation death of the progeny. Several recent studies have indicated that the paternal genome is required for the development of the extraembryonic tissues. Thus, the purpose of this study was to determine which tissues of the implanting embryo were affected by paternal exposure to cyclophosphamide. Male Sprague-Dawley rats were given cyclophosphamide (6 mg/kg/day) or saline by gavage and bred to untreated female rats after 4 weeks of treatment. Pregnant female rats were killed on day 7 of gestation, and implantation sites were dissected from the uterus, fixed, embedded in Epon for semithin serial sectioning, and stained for subsequent light microscopy. Strikingly, many of the implantation sites of affected embryos sired by treated males displayed an apparently normal trophectoderm enclosing a region of dying cells, containing dark-stained pyknotic nuclei. Very few or no inner cell mass-derived embryonic cells were present in these implantation sites. Therefore, there is a selective death of inner cell mass-derived cells in day 7 implantation sites obtained from the progeny of cyclophosphamide-treated males. The results of this study suggest that treatment of the male with cyclophosphamide can affect paternal genes specifically required for development of the inner cell mass cells of the embryo, without an apparent effect on those genes required for normal trophectoderm.     

Mammalian carbamyl phosphate synthetase (CPS). DNA sequence and evolution of the CPS domain of the Syrian hamster multifunctional protein CAD

Glutamine-dependent carbamoyl-phosphate synthetase (EC 6.3.5.5) catalyzes the first step in de novo pyrimidine biosynthesis. The mammalian enzyme is part of a 240-kDa multifunctional protein which also has the second (aspartate carbamoyltransferase, EC 2.1.3.2), and third (dihydroorotase, EC 3.5.2.3) activities of the pathway. Shigesada et al. (Shigesada, K., Stark, G.R., Maley, J.A., and Davidson, J.N. (1985) Mol. Cell Biol. 175, 1-7) produced a truncated cDNA clone from a Syrian hamster cell line that contained most of the coding region for this protein. We have completed sequencing this clone, known as pCAD142. The cDNA insert contained all of the coding region for the glutaminase (GLN) and carbamyl phosphate synthetase (CPS) domains but lacked a short amino-terminal segment. By comparing the primary structure of the mammalian chimera to monofunctional proteins we have identified the borders of the functional domains. The GLN domain is 21 kDa, close to the size of the functionally similar polypeptide products of the Escherichia coli pabA and hisH genes. The domain has the three regions of homology common to trpG-type glutamine amidotransferases, as well as a fourth region specific to the carbamyl phosphate synthetases. The CPSase domain is similar to other reported CPSases in size (120 kDa), primary structure (37-67% amino acid identity), and homology between its amino and carboxyl halves. Analysis of the nucleotide and amino acid sequence identities among the various carbamyl phosphate synthetases suggests that the gene fusion which joined the GLN and CPS domains was an early event in the evolution of eukaryotic organisms and that the Saccharomyces cerevisiae enzyme consisting of separate subunits arose by defusion from an ancestral multifunctional protein.     

Operculum closing as a defence against predatory leeches in four British freshwater prosobranch snails

The defence reaction of operculum closing in response to the presence of the molluscivorous leech Glossiphonia complanata (L.) and the non-molluscivorous Erpobdella octoculata (L.) was studied in four species of freshwater prosobranch gastropod. Bithynia tentaculata (L.) and Valvata piscinalis (Müller) can distinguish between the leeches, reacting only to G. complanata. V. piscinalis is capable of a greater degree of distance chemoreception of the leech ‘scent’. Valvata cristata Müller and Potamopyrgus jenkinsi (Smith) did not react to either leech. V. cristata may not be a potential prey item for G. complanata, while P. jenkinsi is fed on by the leech, but is a relative newcomer to the freshwater fauna. Animal Ecology Research Group, Department of Zoology, University of Oxford Commonwealth Forestry Institute, Department of Plant Sciences, University of Oxford     

Calcium and proton activities in rat cardiac mitochondria. Effect of matrix environment on behaviour of fluorescent probes.

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.     

The receptors for prolactin and growth hormone are localized in the same region of human chromosome 5

Prolactin receptor (PRLR) and growth hormone receptor (GHR) are encoded by members of a gene family containing regions of identical sequences. To determine their chromosomal locations, cDNA probes for these genes were used. Analysis of hybridization to several somatic cell hybrids, together with hybridization in situ to metaphase chromosomes, resulted in the assignment of the loci for both receptors to human chromosome 5 in the region p13----p14. Thus, these proteins may be encoded by a cluster of related genes that evolved from a common ancestral gene, in a manner consonant with that of their ligands.     

A novel extracellular carbohydrase produced by Lipomyces tetrasporus

Summary The ascosporogenous yeast Lipomyces tetrasporus produced an unusual extracellular carbohydrase. It was purified to homogeneity using ammonium sulphate precipitation and DEAE Bio-gel A ion-exchange chromatography. While retaining highest activity on low-molecular-weight saccharides such as maltose and nigerose, it displays considerable activity towards polymeric substrates including soluble starch. It is particularly unusual in that it also hydrolyses dextran and has a very high affinity for this substrate. The enzyme has an exo-lytic mode of action with the only hydrolysis product, glucose, being released in the -anomeric form. Optimum activity occurs at pH 4.5 and at 50°C. It is a glycoprotein, and has an M _r value of 150 000 (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) — 183 000 (fast protein liquid chromatography) and a pI of 6.0. Offprint requests to: C. T. Kelly