Valine-sensitive acetohydroxy acid synthases in Escherichia coli K-12: unique regulation modulated by multiple genetic sites.

Summary Spontaneous mutants (146) of Escherichia coli K-12 were selected that were resistant to inhibition of growth by 1.2 mM L-valine (Val^r). The Val^r isolates, containing acetohydroxy acid synthase resistant to feedback inhibition by L-valine (AHAS^r), were classed according to cotransduction of the mutation with leu. Several mutations resulting in an AHAS^r phenotype were found to be cotransducible with glyA. However, no mutations causing a Val^r phenotype were linked to ilv. AHAS activity was more closely examined in representatives of three classes of mutants with Val^r linked to leu, labeled ilv-660, ilv-661, and ilv-662. The ilvE503 allele in E. coli K-12, known to cause a two- to three-fold derepression of AHAS, was found to affect regulation of synthesis of both valine-sensitive AHAS (AHAS^s) and AHAS^r in the mutants containing ilv-660 and ilv-661, whereas it affected repression of AHAS^s, only, in the mutant containing ilv-662. Further, both AHAS^s and AHAS^r in the ilv-661 mutant were repressed by valine, whereas valine did not repress AHAS^r synthesis in the strain carrying ilv-660 and only partially repressed AHAS^r in the strain carrying ilv-662. Unexpectedly, AHAS^r synthesis in strains carrying ilv-660 or ilv-662 was repressible by leucine. The ilv-660 locus appears to be similar in position to ilvH and encodes a product that confers valine-sensitivity upon AHAS activity in the wild-type E. coli K-12. The ilv-660 and ilv-662 loci may normally encode products that influence both the feedback sensitivity of AHAS and control of AHAS biosynthesis.     

Purification and characterization of two isoenzymes of lipoxygenase from soybeans

A chromatographic procedure for the purification of two lipoxygenase isoenzymes (linoleate: O₂ oxidoreductase, EC 1.13.11.12.) from soybean is described. The procedure for the purification of isoenzyme L-1 includes optimalized extraction, ammonium sulfate fractionation, heat treatment and gradient elution from a CM-Sephadex C-50 column. The purification of L-2 includes ammonium sulfate fractionation, gelfiltration on Sephadex G-150 and gradient elution from a DEAE-cellulose column. Both isoenzymes L-1 and L-2 appear homogeneous after Disc-PAGE. The isoelectric points are 5.6 for L-1 and 5.8 for L-2. Molecular weights are estimated as 100,000 for L-1 as well as L-2 applying three different methods. Both isoenzymes contain 0.9 mol iron per mol protien. The estimated turn over numbers are 8,200 mol linoleate per mol enzyme and min for L-1 and 3,100 for L-2. Amino acid compositions determined after acid hydrolysis show marked differences between L-1 and L-2, particularly with respect to the amino acids Lys, Phe, Ser, Gly and Leu. L-1 posesses a total of 9 cysteine molecules, 6 of which are present as disulfide bonds. L-2 posesses a total of 8 cysteine molecules with only one disulfide bond.These results have been presented in part at the 13th ISF Congress in Marseille on 2nd September 1976     

Maximal activities of hexokinase, 6-phosphofructokinase,oxoglutarate dehydrogenase,and carnitine palmitoyltransferase in rat and avian muscles

The maximum activities of 6-phosphofructokinase and oxoglutarate dehydrogenase in muscle provide quantitative indices of the maximum capacities of anaerobic glycolysis and the Krebs cycle (i.e. the aerobic capacity) respectively. These activities were measured in red, white, and cardiac muscle of birds and the rat. The activities in the white pectoral muscle of the domestic fowl suggest that the Krebs cycle plus electron transfer could provide only about 1% of the rate of ATP production provided by anaerobic glycolysis whereas in pigeon pectoral muscle the predicted maximal rates from the two processes are similar. In contrast to domestic-fowl pectoral muscle, the white rat muscle, epitrochlearis, contains a significant activity of oxoglutarate dehydrogenase, which indicates that the Krebs cycle could provide about 12% of the maximum rate of ATP formation. This may be explained by a higher proportion of type-I and -IIA fibres in the rat muscle compared to the avian muscle. In the aerobic muscles of the rat the maximum activities of carnitine palmitoyl transferase indicate that fatty-acid oxidation could provide a high rate of ATP formation.     

Histological characterization of in vitro regenerated structures of Panax ginseng

Callus was induced from mature leaf lamina, petiole, stem, and main and branch root of Panax ginseng and maintained under different nutrient and light conditions. Heterogenous callus cultures differentiated organoids and/or embryoids. Embryoids that arose from callus cultures of leaf origin germinated into shoots. The occurrence of the early stages of different regenerated structures was proved on histological level.     

A new homeobox-leucine zipper gene from Arabidopsis thaliana

We have isolated a homeobox-containing gene from Arabidopsis thaliana using a degenerate oligonucleotide probe corresponding to the most conserved region of the homeodomain. This strategy has been used previously to isolate homeobox-containing genes from Caenorhabditis, and recently from A. thaliana. The Arabidopsis genes have an unusual structure in that they have a leucine zipper motif adjacent to the carboxy terminal region of the homeo domain, a feature not found in homeobox-containing genes isolated from animals. We report the isolation and primary structure of a new member of this Arabidopsis homeobox-leucine zipper gene family. This new member has the homeodomain and leucine-zipper motif similar to the two genes previously identified, but differs from these genes in the part corresponding to the carboxy terminus of the polypeptide, as well as in size and isoelectric point of the protein.     

Role of H-2 antigens in the host response to methylcholanthrene-induced tumors

H-2 loss variant sublines of a sarcoma (M-AS), induced by methylcholanthrene in an (A × A.SW)F₁ mouse, were used to study the role of the MHC products in the recognition of MC-TSTA. The two reciprocal variant sublines (M-A and M-S) were found to express the TSTA of the original tumor as shown by cross-reactions in graft rejection experiments performed in (A × A.SW)F₁ mice. In the A/Sn and A.SW mice the presence of the reciprocal parental H-2 antigens on the immunizing cells decreased the response against the tumor antigens. An admixture of lymphocytes derived from hyperimmune mice inhibited the outgrowth of the tumor cells. The growth inhibition was mediated by T cells and was H-2 restricted. Cells derived from hyperimmune syngeneic mice inhibited the outgrowth of the variant subline used for immunization but had no effect on the reciprocal variant subline.     

Activated T lymphocytes within human solid tumors

Summary The majority of lymphocytes separated from tumor cell suspensions were T cells. Conjugates of T lymphocytes and tumor cells were often seen. Variable numbers of T cells exhibited signs of activation such as the ability to form stable E rosettes and attachment to normal and malignant cells (a phenomenon designated natural attachment: NA). A proportion of T cells activated in vitro by allogeneic stimulation regularly exhibit these properties. The T cell-tumor conjugates in the suspensions may represent the NA phenomenon, but they could also be the product of T cells that adhere on the basis of specific recognition of cell surface antigens.Abbreviations BBS balanced salt solution - E rosettes rosettes formed with sheep erythrocytes - EA rosettes rosettes formed with ox erythrocytes coated with anti-ox IgG - FCS fetal calf serum - MLC mixed lymphocyte cultures - NA natural attachment - PBL peripheral blood lymphocytes - SRBC sheep erythrocytes - T lymphocytes thymusderived lymphocytes