Generation of twitch tension in frog atrial fibers by Na/Ca exchange

Electrical and mechanical responses of frog atrial trabeculae were studied simultaneously using the double-sucrose gap method. Action potentials and twitch tension could be successively generated in fibers in which the slow inward calcium channel current was not observed. As a rule, this could be obtained in the course of a long experiment (3 to 4 hours). Peak tension was shown to increase monotonically with membrane potential in these preparations. In preparations with the slow inward current the total peak tension could be separated into two components. The first component (tonic) monotonically increased with the membrane potential and was probably related to Na/Ca exchange (Horackova 1984). The potential dependency of the second (phasic) component correlated with that of the slow inward calcium current. Only the tonic but not the phasic component could be observed in preparations without the presence of the slow inward calcium current. The tonic component prevailed when both the slow inward current and phasic tension were greatly reduced by nifedipine. Long experiments, long depolarizing clamp pulses, a metabolic inhibitor 2,4-dinitrophenol, inhibitors of Na/K pump ouabain and AR-L57, toxins promoting intracellular sodium accumulation (aconitine, scorpion toxin) were all shown to increase the tonic tension, but not the slow inward current; they induced a transition from biphasic tension-voltage curve into a monotonically increasing one. We concluded that these procedures and agents greatly stimulate Ca influx via Na/Ca exchange. These results show that Na/Ca exchange can function as a reserve system of Ca2+ used for contraction, thus supporting the heart function, especially under unfavourable metabolic conditions.     

Comparison of different bioreactor systems for the production of high titer retroviral vectors

Improved, human-based packaging cell lines allow the production of high-titer, RCR-free retroviral vectors. The utility of these cell lines for the production of clinical grade vectors critically depends on the definition of optimal conditions for scaled-up cultures. In this work, a clone derived from the TE Fly GALV packaging cell (Duisit et al. Hum. Gene Ther. 1999, 10, 189) that produces high titers of a lacZ containing retroviral vector with a Gibbon Ape Leukemia Virus envelope glycoprotein was used. This clone can produce (2-5) x 10(6) PFU cm(-3) in small scale cultures and has been evaluated for growth and vector production in different reactor systems. The performances of fixed bed reactors [CellCube (Costar) and Celligen (New Brunswick)] and stirred tank reactors [microcarriers and clump cultures] were compared. The cells showed a higher apparent growth rate in the fixed bed reactor systems than in the suspension systems, probably as a result of the fact that aggregation and/or formation of clumps led to a reduced viability and reduced growth of cells in the interior of the clumps. As a consequence, the final cell density and number were in average 3- to 7-fold higher in the fixed bed systems in comparison to the suspension culture systems. The average titers obtained ranged from 0.5 to 2.1 x 10(7) PFU cm(-3) for the fixed bed and microcarrier systems, while the clump cultures produced only (2-5) x 10(5) PFU cm(-3). The differences in titers reflect cell densities as well as specific viral vector production rates, with the immobilization and microcarrier systems exhibiting an at least 10-fold higher production rate in comparison to the clump cultures. A partial optimization of the culture conditions in the Celligen fixed bed reactor, consisting of a 9-fold reduction of the seeding cell density, led to a 5-fold increased vector production rate accompanied by an average titer of 3 x 10(7) PFU cm(-3) (maximum titer (4-5) x 10(7) PFU cm(-3)) in the fixed bed reactor. The performance evaluation results using mathematical models indicated that the fixed bed bioreactor has a higher potential for retroviral vector production because of both the higher reactor productivity and the lower sensitivity of productivity in relation to the changes in final retrovirus titer in the range of 3 x 10(6) to 15 x 10(6) PFU cm(-3).     

Comparative organization of follicle, accessory cells and spawning anlagen in dynamic semelparous clutch manipulators, the urochordate Oikopleuridae

BACKGROUND INFORMATION: The urochordate appendicularians play a key trophic role in marine ecosystems and are the second largest component of zooplankton after copepods. Part of their success is due to their ability to undergo rapid population blooms in response to changes in primary productivity. Nonetheless, the reproductive biology of this important group remains poorly understood. RESULTS: In the present study, we investigated the organization of male and female germ and accessory somatic cells in the Oikopleuridae. We found that the structure of the ovary had been previously misconstrued as consisting of germ and accessory 'cells' interspersed together, whereas, in fact, the germline exists as a giant transparent syncytium. Somatic follicle cells, integral to regulation of the temporal progression of gametogenesis, could be classified into three types in females and two in males, and we characterized functional gap junctions between follicle cells and the germline syncytium in both sexes. The number of follicle cells per oocyte produced was much reduced in comparison with many commonly studied model organisms. We further identified a novel anlagen that permits spawning of the animal via rupture of the gonad wall, which is obligatory for the release of oocytes, but optional for the release of sperm that usually occurs via the spermiduct. CONCLUSIONS: The organization of the female germline in the Oikopleuridae shares some features of meroistic oogenesis with the arthropod Drosophila, but the process of synchronous oogenesis in these semelparous organisms remains quite distinctive with respect to that previously characterized in the animal kingdom and certainly within the chordate phylum.     

Equine lymphocyte antigens in four major Belgian horse populations. Contribution to serology and antigen distribution

158 Belgian Saddlebreds, 130 Belgian Trotters, 108 Belgian Draft horses and 92 Shetland ponies have been typed for serologically defined antigens at the ELA and ELY systems. Gene frequencies were estimated in each breed for the internationally established ELA, ELY-1 and ELY-2 alleles as well as for locally assigned additional ELA markers and for subtypes of ELA-W3, W9 and W11. The distribution of ELA alleles was in agreement with the expected Hardy-Weinberg equilibrium for the 4 horse breeds described here. Differences in gene frequencies between these main Belgian horse populations were observed.     

Alcaloïdes des ecorces de racines de Crioceras dipladeniiflorus

Eight alkaloids have been isolated from the root bark of Crioceras dipladeniiflorus. Five of them are new: Δ¹⁴-vincamine (I) epi-16 Δ¹⁴-vincamine (II) 12-methoxy-δ ¹⁴-vincamine (V) apo Δ¹⁴- vincamine (VI), and ditabersonine (VII), a dimeric alkaloid; the three others are known, voaphylline (III), tabersonine, (IV), and vobtusine (VIII).     

The grapevine fleshless berry mutation. A unique genotype to investigate differences between fleshy and nonfleshy fruit

In flowering plants, fruit morphogenesis is a distinct process following fertilization resulting in the formation of a specialized organ associated with seeds. Despite large variations in types and shapes among species, fleshy fruits share common characteristics to promote seed dispersal by animals such as organ growth and metabolite accumulation to attract animal feeding. The molecular biology of fruit ripening has received considerable attention, but little is known about the determinism of early fruit morphogenesis and why some fruits are fleshy while others lack flesh. We have identified in grapevine (Vitis vinifera) a mutation we have named fleshless berry (flb) that reduces by 20 times the weight of the pericarp at ripening without any effect on fertility or seed size and number. The flb mutation strongly impaired division and differentiation of the most vacuolated cells in the inner mesocarp. The timing of ripening was not altered by the mutation although the accumulation of malic acid in the green stage was noticeably reduced while sucrose content (instead of hexoses) increased during ripening. The mutation segregates as a single dominant locus. These results indicate that the Flb- mutant is suitable material to advance our understanding of the genetic and developmental processes involved in the differentiation of an ovary into a fruit.     

An Arginine-Rich Motif in the ORF2 capsid protein regulates the hepatitis E virus lifecycle and interactions with the host cell

Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation likely to promote regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. It serves as maturation site of glycosylated ORF2 by furin, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes.     

Alcaloides des feuilles du Crioceras dipladeniiflorus

Three alkaloids have been isolated from leaves of Crioceras dipladeniiflorus. Two of them, 12-methoxy-voaphylline and criophylline are new and their structures have been determined. The third has been identified as andrangine an alkaloid isolated recently from another Apocynaceae Craspidospermum verticillatum.     

Genetic and physical mapping of the grapevine powdery mildew resistance gene, Run1, using a bacterial artificial chromosome library

Resistance to grapevine powdery mildew is controlled by Run1, a single dominant gene present in the wild grapevine species, Muscadinia rotundifolia, but absent from the cultivated species, Vitis vinifera. Run1 has been introgressed into V. vinifera using a pseudo-backcross strategy, and genetic markers have previously been identified that are linked to the resistance locus. Here we describe the construction of comprehensive genetic and physical maps spanning the resistance locus that will enable future positional cloning of the resistance gene. Physical mapping was performed using a bacterial artificial chromosome (BAC) library constructed using genomic DNA extracted from a resistant V. vinifera individual carrying Run1 within an introgression. BAC contig assembly has enabled 20 new genetic markers to be identified that are closely linked to Run1, and the position of the resistance locus has been refined, locating the gene between the simple sequence repeat (SSR) marker, VMC4f3.1, and the BAC end sequence-derived marker, CB292.294. This region contains two multigene families of resistance gene analogues (RGA). A comparison of physical and genetic mapping data indicates that recombination is severely repressed in the vicinity of Run1, possibly due to divergent sequence contained within the introgressed fragment from M. rotundifolia that carries the Run1 gene.     

A detailed physical map of the porcine major histocompatibility complex (MHC) class III region: Comparison with human and mouse MHC class III regions

A detailed physical map of the porcine MHC class III region on Chr 7 was constructed with a panel of probes in a series of hybridizations on genomic pulsed field gel electrophoresis (PFGE) Southern blots. A precise organization of the 700-kb segment of DNA between G18 and BAT1 can now be proposed, with more than 30 genes mapped to it. Comparison of this region with homologous regions in human and mouse showed only minor differences. The biggest difference was observed in the CYP21/C4 locus with only one CYP21 gene and one C4 gene found, whereas in human and mouse these genes are duplicated. These results show the class III region is very well conserved between pig, human, and mouse, in contrast with the class I and class II regions, which seem more prone to rearrangements. Received: 13 October 1995 / Accepted: 19 January 1996