Immortalization of human fibroblasts transformed by origin-defective simian virus 40.

Simian virus 40 (SV40)-mediated transformation of human diploid fibroblasts has provided an effective experimental system for studies of both "senescence" in cell culture and carcinogenesis. Previous interpretations may have been complicated, however, by the semipermissive virus-cell interaction. In earlier studies, we previously demonstrated that the human diploid fibroblast line HS74 can be efficiently transformed by DNA from replication-defective mutants of SV40 containing a deletion in the viral origin for DNA synthesis (SVori-). In the current study, we found that such SVori- transformants show a significantly increased life span in culture, as compared with either HS74 or an independent transformant containing an intact viral genome, but they nonetheless undergo senescence. We have clonally isolated six immortalized derivatives of one such transformant (SV/HF-5). Growth studies indicate that the immortalized cell lines do not invariably grow better than SV/HF-5 or HS74. Genetic studies involving karyotypic analysis and Southern analysis of integrated viral sequences demonstrated both random and nonrandom alterations. All immortalized derivatives conserved one of the two copies of SV40 sequences which expressed a truncated T antigen. These cloned SV40-transformed cell lines, pre- and postimmortalization, should be useful in defining molecular changes associated with immortalization.     

Ecological aspects of swidden cultivation among the Andoke and Witoto Indians of the Colombian Amazon

The investigation of crop and soil-crop conditions among Andoke and Witoto cultivators in southeast Colombia is used as a basis for assessing Geertz' (1963) model of swidden cultivation. In this respect, the extent to which maniocdominated swiddens in the study area simulate the structure and composition of the forest climax community is questioned. As Geertz (1963) indicates, an initial nutrient boost for crop cultivation results from the preliminary burning of forest debris, but weed competition, rather than progressive loss of soil fertility, is reported to be the primary cause of abandoning manioc cultivation after 2–3 years. While the Andoke and Witoto crop system remains adaptive at the individual field level, particularly in its constituent species, its fundamental adaptation is considered to be its integration into the broader field and fallow system that juxtaposes crop production with extended periods of forest regeneration.     

Localization of the receptor binding site of IFN-alpha 2b

The receptor binding site of IFN-alpha is not precisely known. To further characterize this site, mAb against IFN-alpha 2b were selected that block the binding of radiolabeled IFN-alpha 2b to its cell surface receptor. These antibodies also neutralized the anti-viral and anti-proliferative properties of IFN-alpha 2b. A subset of these antibodies (group 1) do not recognize IFN-alpha 2a, either in solid-phase immunoassays or functional assays, whereas a second subset (group 2), with no cross-reactivity with group 1, recognizes both IFN-alpha subtypes. Because IFN-alpha 2b and IFN-alpha 2a differ by only alpha Arg23-Lys23 substitution, group 1 antibodies must recognize an epitope within the receptor binding region of IFN-alpha 2b that includes Arg23. Group 2 antibodies recognize a separate and distinct epitope within the binding site that does not include Arg23.     

Electroporation and conjugal plasmid transfer to members of the genus Aquaspirillum

Electroporation methods and conjugal matings were used to transfer several plasmid vectors to Aquaspirillum dispar and Aquaspirillum itersonii. The incompatibility P class plasmid RP4 was conjugally transferred from Escherichia coli HB101 to these spirilla, and the transconjugants subsequently donated the molecule to plasmid-free E. coli and A. dispar strains via conjugal matings. High-voltage electrotransformation was used to transfer plasmids pUCD2, pSa151 and RP4 to A. dispar and A. itersonii, at efficiencies as high as 3×10⁴ transformants per μg plasmid DNA. RP4 DNA isolated from spirillum hosts, but not RP4 from E. coli cells was successfully transferred to A. dispar and A. itersonii by electrotransformation, suggesting that modification and/or restriction activity may be present in these Aquaspirillum species.     

EPR and EXAFS studies of glucose isomerase activation by divalent metals

The interactions of Mn²⁺ and Co²⁺ with glucose isomerases from three microbial sources have been studied using various direct physical methods. Co²⁺ was found to activate each enzyme, although the degree of activation varied significantly for enzymes from different organisms. EPR spectroscopy measurements revealed that dissimilarities in the coordination sphere of enzyme-bound Mn²⁺ accompanied the differences in enzyme activity. Variations in the EPR spectra of a nitroxide spin label coupled to two of the three isomerases, possibly near their active sites, were also observed. In no case was the EPR spectrum influenced by Co²⁺ addition, a result discordant with the hypothesis that Co²⁺ activates the enzyme by inducing a conformational change. The proximal biochemical environment of enzymebound Co²⁺ was also examined using EXAFS spectroscopy. This method showed that glucose causes notable changes in the ligand environment of the enzyme-bound metal, suggesting the formation of an enzyme-metal-substrate bridge complex. The significance of these results relative to possible reaction mechanisms is discussed.     

Transient electrical birefringence characterization of heavy meromyosin

Heavy meromyosin (HMM) and myosin subfragment 1 (S1) were prepared from myosin by using low concentrations of alpha-chymotrypsin. The light chain distribution in HMM was identical with that of myosin, within experimental error, when analyzed on 12% polyacrylamide gels after electrophoresis. Specific birefringences and birefringence decay times were measured by transient electrical birefringence in 5 mM KCl, 5 mM tris(hydroxymethyl)aminomethane (pH 7), and 1 mM MgCl2 at 4 degrees C under gentle conditions that reduced the CaATPase activity by less than 10%. For solutions of HMM, by use of electric field pulses shorter than 0.5 microseconds, the birefringence decay signal from the S1 portions of HMM could be resolved and the rotational motions of the S1 moieties observed directly. The rotation relaxation time, adjusted to 20 degrees C, was 0.34 microseconds; this is in quantitative agreement with previous hydrodynamic results obtained by using covalently attached probes. The assignment of the fast decay time obtained with HMM to the S1 portions was confirmed by birefringence decay measurements on free S1, for which the relaxation time was 0.13 microseconds, corrected to 20 degrees C. The specific birefringences for S1 and HMM, respectively, were 0.37 X 10(-6) and 12.8 X 10(-6) (cm/statvolt)2. Thus, for much longer electric field pulses, the signal from HMM is due almost entirely to its subfragment 2 (S2) portion, and its rotational dynamics can also be monitored directly by using electrical birefringence. The decay of the signal from the S2 portion could be adequately fit without evoking bending of the S2 portion of HMM other than at its junction with S1.     

DNA methylation is not increased in mouse-human somatic cell hybrids

The level of DNA methylation in three mouse-human cell lines that retained different human chromosomes and in the parental mouse and human lines has been determined by high-pressure liquid chromatography (HPLC). The level of methylation is similar in the hybrid and parental cells, indicating that interspecific somatic cell hybridization followed by preferential chromosome segregation can occur without an increase in overall DNA methylation.     

Isolation and identification of two isomeric trihydroxy octadecenoic acids with prostaglandin E-like activity from onion bulbs (Allium cepa)

Two fractions with prostaglandin E-like activity were isolated from onion (Allium cepa) by using XAD-2 adsorption, silicic acid column chromatography and thin layer chromatography. The fractions were analyzed by gas chromatography/mass spectrometry and were characterized as isomeric mixtures of 9,10,13-trihydroxy-11-octadecenoic and 9,12,13-trihydroxy-10-octadecenoic acid, which are lipoxygenase metabolites of linoleic acid. Bio-assay, for which cascade superfusion was used and the rabbit coeliac and mesenteric arteries and the rat fundus strip were employed as assay organs, was utilized to monitor the bio-active profile throughout the isolation procedures. The activity of 1 microgram of the pharmacologically active fractions T1 and T2 was found to be equivalent to that of respectively 1.33 and 0.63 ng of prostaglandin E2.     

Conserved sequences in both coding and 5'' flanking regions of mammalian opal suppressor tRNA genes.

The rabbit genome encodes an opal suppressor tRNA gene. The coding region is strictly conserved between the rabbit gene and the corresponding gene in the human genome. The rabbit opal suppressor gene contains the consensus sequence in the 3' internal control region but like the human and chicken genes, the rabbit 5' internal control region contains two additional nucleotides. The 5' flanking sequences of the rabbit and the human opal suppressor genes contain extensive regions of homology. A subset of these homologies is also present 5' to the chicken opal suppressor gene. Both the rabbit and the human genomes also encode a pseudogene. That of the rabbit lacks the 3' half of the coding region. Neither pseudogene has homologous regions to the 5' flanking regions of the genes. The presence of 5' homologies flanking only the transcribed genes and not the pseudogenes suggests that these regions may be regulatory control elements specifically involved in the expression of the eukaryotic opal suppressor gene. Moreover the strict conservation of coding sequences indicates functional importance for the opal suppressor tRNA genes.