Construction and characterization of two infectious molecular clones of encephalomyocarditis virus.

We constructed and characterized two infectious molecular clones of encephalomyocarditis (EMC) virus. Both constructs, pDL and pDA, were assembled from five overlapping cDNA clones derived from the diabetogenic variant of EMC virus (EMC-D) and from two synthetic oligonucleotide cartridges. pDA contained a single point mutation at position 1720 within the "puff" region of capsid protein 1AB that was derived from the nondiabetogenic variant of EMC virus (EMC-B). This point mutation resulted in an amino acid substitution of arginine (EMC-B) for lysine (EMC-D). Our construction illustrates two novel findings: (i) that the problem of stably cloning long poly(C) tracts of EMC virus can be circumvented by the use of a shortened, synthetic, poly(dC-dG) oligonucleotide cartridge, and (ii) that a single point mutation in the puff region of the capsid protein 1AB leads to change in its electrophoretic mobility and to a change in the plaque size of recombinant virus.     

Isolation and partial characterization of an unusual human immunodeficiency retrovirus from two persons of west-central African origin.

An unusual human retrovirus was isolated from two patients with persistent generalized lymphadenopathy who originate from West-Central Africa and are currently residing in Belgium. Although the virus shared a number of the same biological and morphological properties as human immunodeficiency retrovirus type 1 (HIV-1) and HIV-2, significant antigenic differences could be demonstrated. Several of the viral proteins also differed in molecular weight from the corresponding HIV-1 and HIV-2 proteins. Partial chemical cleavage of the most highly conserved viral proteins resulted in patterns which differed from those of HIV-1 and HIV-2. Furthermore, nucleic acid hybridization experiments were capable of discriminating between the virus types. Sequence analysis of the viral U3 region revealed a unique enhancer organization not found in other immunodeficiency viruses. The data indicated that the new isolate is more closely related to HIV-1 than to HIV-2 but clearly differs in a number of important respects.     

Two better cell lines for making hybridomas expressing specific T cell receptors

Two variants of the AKR thymoma BW5147 have been isolated which can no longer express functional TCR alpha- and beta-chains. By generating hybridomas with these variant fusion lines, TCR of any normal T lymphocyte, including TCR-gamma/delta, can be studied at a clonal level, without interference of the BW5147-derived receptor chains. In this study one of the variants has been useful in identifying the reactivity to allogeneic MHC Ag of BW5147 itself.     

Herbicide Chlorsulfuron Decreases Assimilate Transport Out of Treated Leaves of Field Pennycress (Thlaspi arvense L.) Seedlings

Treatment of field pennycress (Thlaspi arvense L.) leaves with the herbicide chlorsulfuron resulted in a decrease in the export of assimilate. Twelve hours after a spot application of 1 microgram, assimilate translocation was 70% of that in control leaves. In excised leaves treated with chlorsulfuron the total amounts of sugars and free amino acids were 150 and 170%, respectively, of the amounts in control leaves, 30 hours after herbicide treatment. The amount of sucrose was 247% of that in control leaves. The increase in the concentration of sucrose in the chlorsulfuron-treated leaves, combined with the absence of an effect of chlorsulfuron on carbon dioxide fixation, suggests that the decrease in assimilate transport is not due to an effect on the synthesis of assimilates, but rather to an effect on their movement out of the leaves. Supplying branched-chain amino acids to the field pennycress seedlings prior to the application of chlorsulfuron prevented the occurrence of the effects described.     

Body growth in Atlantic walruses (Odobenus rosmarus rosmarus) from Greenland

Morphometric data were collected from 105 Atlantic walruses ( Odobenus rosmarus rosmarus ) in northwestern Greenland in the periods 1977–78 and 1989–91. Of these 21 walruses were subjected to a detailed study on body composition.
The asymptotic maximum standard body length of Atlantic walruses in NW Greenland was 269 cm for females and 314 cm for males. This is similar to Pacific walruses, but significantly longer than Atlantic walruses from Hudson Bay. Despite this, walruses from NW Greenland apparently do not attain the same total body mass as Pacific walruses ( O.r.divergens ).
The asymptotic maximum body weights for walruses in NW Greenland were estimated to be 720 kg for females and 1114 kg for males.
Total body surface area was proportional to the two-thirds power of total body weight.
The percentage proportion of blubber and viscera were both negatively correlated to body mass, while skin and muscles constituted a nearly fixed proportion. On average, blubber constituted 19% of total body mass of adult females, 15% of adult males and 24% of subadults of both genders. The average walrus consisted of 18% blubber, 12% skin, 12% viscera and 58% blood, muscle and skeleton. Muscles were estimated to constitute 44% of total body weight.
Allometric functions for weight of internal organs relative to body mass are presented.     

REPRODUCTION IN MALE ATLANTIC WALRUSES (ODOBENUS ROSMARUS ROSMARUS) FROM THE NORTH WATER (N BAFFIN BAY)

Age at sexual maturity and timing of the mating season were determined in male Atlantic walruses ( Odobenus rosmarus rosmarus , L.) from the "North Water" subpopulation in northern Baffin Bay. Testes and epididymides of 174 male walruses (between 0 and 30 yr old) from NW Greenland (1987–1990) were studied macroscopically and a subset of 57 specimens was analyzed microscopically. In physically mature bulls ( i.e. , ≥12 yr old), sperm or apparently ripe spermatids were found between 9 November and 12 July. In younger walruses these signs of fertility were found in a few specimens (7–11 yr old) collected between 9 January and 28 May. The mating season seems to peak in January—April. The youngest sexually mature individual was 7 yr old and the oldest apparently immature individual was 13 yr old. Average age of sexual maturity was 10.9 yr (95% C.I.: 9–6–12.2 yr) and all were sexually mature by the time they were 14 yr old. The non-spermiogenetic testes and epididymides showed accelerated growth between about the 5–6th and about the 12–15th year of life, indicating that sexual maturation occurs during these years. The length of the baculum increased gradually until about 12–15 yr of age, when physical maturity was reached.     

An approach to integrated antibody production: Coupling of fluidized bed cultivation and fluidized bed adsorption

Continuous culture may be an efficient way of producing proteins which are susceptible to secondary processing in the course of a fermentation process. Short residence times in these systems support the production of correctly assembled proteins by avoiding substrate limitations and product inhibitions and also minimize the contact of sensitive bioproducts with degrading enzymes. Thus products of increased stability and integrity are obtained from continuous processes. The downstream process following continuous culture has to be adapted to the specific conditions of continuous fermentations, e.g. large liquid volumes and diluted process solutions. In this paper an approach is shown how a fluidized bed adsorption as first recovery operation may be coupled directly to a continuous production. Immobilized hybridoma cells are cultivated in porous glass microcarriers in a continuous fluidized bed process, the cell containing harvest is purified by fluidized bed adsorption using an agarose based cation exchange matrix. By this coupled mode of operation the large biomass containing harvest volume resulting from the continuous cultivation may be applied directly to a fluidized chromatographic matrix without prior clarification, leading to a particle free and initially purified product solution of reduced volume. In an experimental setup a bench-scale fluidized bed bioreactor of 25 ml carrier volume was coupled to a fluidized bed adsorption column operated with 300 ml of adsorbent. This configuration yielded up to 20 mg of monoclonal antibody per day in a cell free solution at fourfold concentration and fivefold purification. The process was run for more than three weeks with consistent product output.The help of H. Schmitz, A. Bader, J. Gätgens and M. Halfar during the experiments is gratefully acknowledged. This work was partially funded by the ministry of science and research of the Federal Republic of Germany within the project Stoffumwandlung mit Biokatalysatoren.