Isolation of copper thionein from rat liver

The inducible Cu-binding protein from adult rat liver previously referred to as Cu-chelatin has been purified and shown to be Cu-thionein. The Cu-protein was purified to homogeneity by gel filtration and thiopropyl-Sepharose chromatography. The Cu-thionein exhibited an amino acid composition similar but not identical to that of the two forms of rat liver Cd,Zn-thionein. The polypeptide-chain molecular weight of Cu-thionein was indistinguishable from that of Cd,Zn-thionein. The identification of the Cu-protein as metallothionein was substantiated by the complete immunological cross-reactivity with antisera prepared against purified rat liver Cd,Zn-thionein. Purified Cu-thionein bound 9–11 g atoms of Cu per mole of protein in an electron paramagnetic resonance nondetectable form. The $\text{Cu}\text{Zn}$ ratio of the protein is about 100. Ion-exchange chromatography resolved the Cu-protein into three polymorphic forms which differed from the polymorphism of Cd,Zn-thionein.     

Developmental ability of bovine embryos after nuclear transfer based on the nuclear source: in vivo versus in vitro

Bovine nuclear transfer embryos reconsitituted from in vitro-matured recipient oocyte cytoplasm and different sources of donor nuclei (in vivo, in vitro-produced or frozen-thawed) were evaluated for their ability to develop in vitro. Their cleavage rate and blastocyst formation are compared with those of control IVF embryos derived from the same batches of in vitro-matured oocytes that were used for nuclear transfer and were co-cultured under the same conditions on bovine oviducal epithelial cell monolayers for 7 d. Using fresh donor morulae as the source of nuclei resulted in 30.2% blastocyst formation (150 497 ), which was similar to that of control IVM-IVF embryos (33.8% blastocysts, 222 657 ). When IVF embryos were used as the source of nuclei for cloning, a slightly lower blastocyst formation rate (22.6%, 41 181 ) was obtained but not significantly different from that using fresh donor morulae. Nuclear transfer embryos derived from vitrified donor embryos showed poor development in vitro (7.1%, 11 154 ). No difference in morphology or cell number was observed after 7 d of co-culture between blastocysts derived from nuclear transfer or control IVF embryos. The viability of 34 in vitro-developed nuclear transfer blastocysts was tested in vivo and resulted in the birth of 11 live calves (32.3%).     

Analysis of integrated ground squirrel hepatitis virus and flanking host DNA in two hepatocellular carcinomas.

We cloned the integrated ground squirrel hepatitis B virus (GSHV) sequences from two hepatomas showing a single viral insertion. The GSHV inserts shared structural features with integrated DNAs of other hepadnaviruses. Insertional activation of a cellular gene appears unlikely: the integrated GSHV sequences lacked the known viral enhancers and were not expressed in the tumors, and we found no evidence for the presence of a gene at the integration site. Our results, together with those earlier studies, suggest that GSHV does not behave as an extensive insertional mutagen, in sharp contrast with the closely related woodchuck hepatitis virus. GSHV may thus cause carcinogenesis by more indirect mechanisms, as does the human hepatitis B virus.     

Carbon Source Requirements for Exopolysaccharide Production by Lactobacillus casei CG11 and Partial Structure Analysis of the Polymer

Exopolysaccharide production by Lactobacillus casei CG11 was studied in basal minimum medium containing various carbon sources (galactose, glucose, lactose, sucrose, maltose, melibiose) at concentrations of 2, 5, 10, and 20 g/liter. L. casei CG11 produced exopolysaccharides in basal minimum medium containing each of the sugars tested; lactose and galactose were the poorest carbon sources, and glucose was by far the most efficient carbon source. Sugar concentrations had a marked effect on polymer yield. Plasmid-cured Muc^- derivatives grew better in the presence of glucose and attained slightly higher populations than the wild-type strain. The values obtained with lactose were considerably lower for both growth and exopolysaccharide yield. The level of specific polymer production per cell obtained with glucose was distinctively lower for Muc^- derivatives than for the Muc⁺ strain. The polymer produced by L. casei CG11 in the presence of glucose was different from that formed in the presence of lactose. The polysaccharide produced by L. casei CG11 in basal minimum medium containing 20 g of glucose per liter had an intrinsic viscosity of 1.13 dl/g. It was rich in glucose (76%), which was present mostly as 2- or 3-linked residues along with some 2,3 doubly substituted glucose units, and in rhamnose (21%), which was present as 2-linked or terminal rhamnose; traces of mannose and galactose were also present.     

Characterisation of the extractable pectins and hemicelluloses of the cell wall of glasswort, Salicornia ramosissima

Cell walls of glasswort (Salicornia ramosissima Woods), a halophytic Chenopodiaceae, prepared as alcohol-insoluble solids, were found to be rich in arabinose, galacturonic acid, glucose and proteins, and contained 0·7% ferulic acid and 3·8% acetic acid. Pectic and hemicellulosic polysaccharides were extracted by cyclohexanediaminotetraacetic acid, hot dilute acid, cold dilute alkali and concentrated alkali (twice), with yields of 2·9, 19·1, 4·7, 7·4 and 1·9% of the alcohol-insoluble solids, respectively. Protein-rich material precipitated upon dialysis. The dialysed fractions were fractionated by ion-exchange chromatography, and the main fractions were analysed by gel-filtration and glycosyl linkage analysis. The hot acid extract contained 46·2% arabinose and 28·9% galacturonic acid, with high degrees of methylation and acetylation (65 and 45, respectively). It could be fractionated into a low-molecular-weight arabinan rich in ferulic acid, and a pectic fraction still relatively rich in neutral sugars. The concentrated alkali extracts were rich in xylose (33·4 and 23·6%, respectively). They were separated by ion-exchange chromatography into a fucogalactoxyloglucan and a glucuronoarabinoxylan.     

The development and evolution of actin-containing organelles during spermiogenesis of a primitive nematode

Summry— Spermatogenesis in the primitive marine nematode Sphaerolaimus hirsutus (Chromadoria, Sphaerolaimidae) was investigated by examining the ultrastructure and cytochemistry. Spermatozoa are lenticular cells of about 15 μm in diameter and are devoid of flagellum and acrosome as in other nematodes. In spermatocytes, dictyosomes produced transient structures, the fibrous body-membranous organelle complexes (FB-MO). In spermatids, the FBs were arranged as cartwheel spokes, with the FBs in the centre and the MOs at the periphery. The FBs were first made up of parallel fibres and surrounded by a membrane, then, in a later stage, showed a dense central structure with a surrounding vermiculate region and were devoid of membrane. The FBs contain actin as shown by immunofluorescence using a monoclonal anti-actin antibody and by affinity cytochemistry using fluorescent phalloidin. MOs contained mainly F-actin as shown by their labelling by phalloidin. In spermatozoa, the MOs were no longer peripheral but arranged on a ring in the central region of the cell and the FBs disappeared to form the cytoskeleton of the cell outer region. It was assumed, by analogy with the ultrastructure of other nematodes, that this cytoskeleton was made up of major-sperm-protein (MSP). Labelling of spermatids of Caenorhabditis elegans also revealed the presence of actin, but cells and actin spots were very can be distinguished. In the few species in which it has been studied (C elegans and Ascaris suum), MSP is thought to constitute in spermatozoa a motile cytoskeleton excluding the presence of actin. However, the present study of Sphaerolaimus shows that the actin cytoskeleton is present during nematode spermiogenesis.     

Proline metabolism and NAD kinase activity in soybean calli during short- and long-term exposures to light and NaCl

Calli of soybean (Glycine max Merr.) cv. Maple Arrow grew better and accumulated more proline when cultured for 5 d on 70 mM NaCl under darkness than at light. This rapid proline accumulation in salinized soybean calli appeared to play a protective role rather than to be a cause of growth failure. Throughout a 28 d-culture cycle (in control and NaCl-treated calli exposed to light or darkness), we followed the possible relationships between the proline contents and the activities of enzymes of proline biosynthesis [ornithine transaminase; NAD(P)H-pyrroline-5-carboxylate reductase], of proline catabolism [NAD(P) proline dehydrogenase], and of NAD kinase responsible of variations in NADP(H) contents. Enzyme activities of proline metabolism and NAD kinase were clearly light- and NaCl-regulated; nevertheless, relationships between enzyme activities and proline content existed only in calli grown for short-term under darkness and in presence of NaCl. The ornithine transaminase route, which was particularly enhanced in these calli during the first days of salt application, seemed to be involved in the initial proline accumulation in soybean. This revised version was published online in July 2006 with corrections to the Cover Date.     

A new hepadnavirus endemic in arctic ground squirrels in Alaska.

We present evidence for a novel member of the hepadnavirus family that is endemic in wild arctic ground squirrels (Spermophylus parryi kennicotti) in Alaska. This virus, designated arctic squirrel hepatitis virus (ASHV), was initially detected in the livers of animals bearing large hepatic nodules by nucleic acid hybridization with hepadnavirus probes and in plasma by cross-reactivity with antibodies to hepadnavirus surface and core antigens. The complete nucleotide sequence of the 3,302-bp-long ASHV genome was determined and compared with those of ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus (WHV); all sequences were organized into four open reading frames, designated pre-C/C, pre-S/S, pol, and X. Despite roughly equivalent variability among the three rodent hepadnaviruses (around 16% base and 19% amino acid exchanges), ASHV appeared to be more closely related to GSHV than to WHV in phylogenetic analysis. Accordingly, preliminary studies of the pathology of ASHV infection suggested that ASHV may be a less efficient oncogenic agent than WHV. About one-third of aged animals maintained in captivity, including virus-infected as well as uninfected squirrels, developed large liver nodules, consisting of hepatocellular adenomas or carcinomas or nonmalignant lesions characterized by drastic microvesicular steatosis. ASHV-infected arctic ground squirrels may serve as a new model with which to analyze the contribution of hepadnavirus- and host-specific determinants to liver pathology and tumorigenesis.     

In vitro androgenesis and segregation distortion inBrassica napus L.: spontaneous versus colchicine-doubled lines

A total of 750 plantlets were regenerated from 1,400 embryos produced through microspore cultures from one F₁ plant of the cross Darmor-bzh x Yudal. Fifty-three percent of the regenerants were evaluated by flow cytometric analysis, which revealed that 31% were spontaneous diploid (SD), 63% were still haploid and the remaining 6% plants had other ploidy levels. Available segregation data (266 markers) produced on this androgenic progeny were used to study the interference between segregation distortion and the mode of chromosome doubling of androgenc lines. On the basis of the present results it is not possible to conclude that distortions are peculiar to one type of regenerated plant; only a difference in the intensity of the bias might be assumed.