Role of ATP and enzyme-bound nascent peptides in the control of elongation for mycobacillin synthesis.

The presence of a Zn2+-dependent acid p-nitrophenyl phosphatase (EC 3.1.3.2) in bovine liver was described. The enzyme was purified to apparent homogeneity and migrates as a single band during electrophoresis on polyacrylamide gel. The enzyme requires Zn2+ ions for catalytic activity, other bivalent cations have little or no effect. The enzyme, of Mr 118,000, optimum pH 6-6.2 and pI 7.4-7.5, was inhibited by EDTA, tartrate, adenine and ATP, but not by fluoride. The common phosphate esters are poor substrates for the enzyme, which hydrolyses preferentially p-nitrophenyl phosphate and o-carboxyphenyl phosphate. The Zn2+-dependent acid p-nitrophenyl phosphatase of bovine liver was different from the high-Mr acid phosphatases previously detected in mammalian tissues.     

Changes in polyamines and related enzymes with loss of viability in rice seeds

Putrescine, spermidine and spermine of high vigour, low vigour and non-viable (classes 1, 2 and 3 respectively) seeds of Oryza sativa increased with loss of viability. The largest concentration of spermine was found in non-viable embryos. Spermine was absent in the husks of all the three categories of seeds. Arginine decarboxylase was greatest in high vigoured seeds and its activity gradually declined with loss of viability. However, diamine oxidase and polyamine oxidase activities gradually increased with the loss of viability of the seeds while DNA, RNA and protein contents decreased. The total content of polyamines increased on kinetin treatment but declined on ABA treatment. DNA, RNA and protein followed the same trend as polyamines. The polyamine contents increased by ca 3- and 4-fold, respectively, in high vigoured and low vigoured seeds on 10^?4 M kinetin treatment. The activity of ADC followed the same change as that of the polyamines in both cases, but the reverse was observed for the activities of diamine and polyamine oxidases.     

Regulation of fibrinogen assembly. Transfection of Hep G2 cells with B beta cDNA specifically enhances synthesis of the three component chains of fibrinogen

Previous studies indicated that synthesis of B beta chain may be a rate-limiting factor in the production of human fibrinogen since Hep G2 cells contain surplus pools of A alpha and gamma but not of B beta chains, and fibrinogen assembly commences by the addition of preformed A alpha and gamma chains to nascent B beta chains attached to polysomes. To test whether B beta chain synthesis is rate limiting Hep G2 cells were transfected with B beta cDNA, and its effect on fibrinogen synthesis and secretion was measured. Two sets of stable B beta cDNA-transfected Hep G2 cells were prepared, and both cell lines synthesized 3-fold more B beta chains than control cells. The B beta-transfected cells also synthesized and secreted increased amounts of fibrinogen. Transfection with B beta cDNA not only increased the synthesis of B beta chain but also increased the rate of synthesis of the other two component chains of fibrinogen and maintained surplus intracellular pools of A alpha and gamma chains. Transfection with B beta cDNA did not affect the synthesis of albumin, transferrin, or anti-chymotrypsin and had a small inhibitory effect on the synthesis of C-reactive protein. Taken together these studies demonstrate that increased B beta chain synthesis specifically causes increased production of the other two component chains of fibrinogen and that unequal and surplus amounts of A alpha and gamma chains are maintained intracellularly.     

Effect of temperature on the spectral properties of coenzyme F420 and related compounds.

The uv-visible spectra of 7,8-didemethyl-8-hydroxy-5-deazaflavin-5'-phosphoryllactyl glutamate (coenzyme F420), a naturally occurring 5-deazaflavin derivative, in three different buffers changed with a rise in temperature; the effect on the extinction coefficient at 420 nm (epsilon 420) was as follows: In phosphate-buffered solutions at pH less than 7.5, the epsilon 420 increased (at pH 5.0 for a temperature shift from 15 to 60 degrees C, delta epsilon 420 was +87%), but between pH 7.5 and 8, epsilon 420 changed very little. At pH greater than 8.0 in phosphate- or borate-buffered solutions, epsilon 420 decreased slightly. In morpholineethanesulfonic acid (Mes)-buffered F420 solutions at pH 5 and 5.5, epsilon 420 changed very little, whereas at pH 6-8, the epsilon 420 decreased. Absorbance of F420 at 401 nm in phosphate buffer at pH 5 to 9 was not significantly affected by temperature. Changes in epsilon 420 due to temperature change corresponded to changes in the pKa of 8-OH of the deazaflavin molecule; studies with adenylated F420 showed that the 8-OH of F420 was responsible for these changes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transfer of Brassica tournefartii (TT) genes to allotetraploid oilseed Brassica species (B. juncea AABB, B. napus AACC, B. carinata BBCC): homoeologous pairing is more pronounced in the three-genome hybrids (TACC, TBAA, TCAA, TCBB) as compared to allodiploids (TA, TB, TC)

For the transfer of genes from B. tournefortii (TT) to the allotetraploid oilseed brassicas, B. juncea AABB, B. carinata BBCC and B. napus AACC, B. tournefortii was first crossed with the three basic diploid species, B. campestris (AA), B. nigra (BE) and B. oleracea (CC), to produce the allodiploids TA, TB and TC. These were tetraploidized by colchicine treatment to produce the allotetraploids TTAA, TTBB and TTCC, which were further crossed with B. juncea and B. napus to produce three-genome hybrids with substitution-type genomic configurations: TACC, TBAA and TCAA. These hybrids along with another hybrid TCBB produced earlier, the three allodiploids, their allotetraploids and the four diploid parent species were studied for their male meiotic behaviour. The diploid parent and the allotetraploids (TTAA, TTBB and TTCC) showed regular meiosis although the pollen viability was generally low in the allotetraploids. In the allodiploids (TA, TB and TC) only some end-to-end associations were observed without any clearly discernible chiasmata or exchange points. Chromosomes involved in end-to-end associations were randomly distributed at the metaphase/anaphase-I stages. In contrast, the three-genome hybrids (TACC, TBAA, TCAA and TCBB) showed normal bivalents whose number exceeded the expected bivalent values. Bivalents arising out of homoeologous pairing were indistinguishable from normal pairs by their disjunction pattern but could be distinguished on the basis of the heteromorphy of the homoeologous chromosomes. The three-genome hybrids could be backcrossed to allotetraploid oilseed brassicas as they had some fertility. In contrast, the allodiploids could neither be selfed nor back-crossed. On the basis of their meiotic stability, in terms of more pronounced homoeologous pairing and fertility for backcrossing, the three-genome configurations provide the best possible situation for the introgression of alien genes from the secondary gene pool to the allotetraploid oilseed crops B. juncea, B. napus and B. carinata.     

Synthesis of hexaploid (AABBCC) somatic hybrids: a bridging material for transfer of ‘tour’ cytoplasmic male sterility to different Brassica species

Most of the alloplasmic cytoplasmic male sterility (CMS) systems are known to be associated with a number of floral abnormalities that result from nuclear-cytoplasmic incompatibilities. One such system, tour, which is derived from Brassica tournefortii, induces additional floral abnormalities and causes chlorosis in Brassica spp. While the restorer for this CMS has been reported to be present in B. napus, in B. juncea, where the abnormalities are more pronounced, no restorer has yet been identified. Rectification of these floral abnormalities through mitochondrial recombinations and chloroplast replacement might result in the improvement of this CMS system. As organelle recombinations can possibly be achieved only by somatic cell hybridization, fusion experiments were carried out between hygromycin-resistant B. juncea AABB carrying tour cytoplasm and phosphinotricin-resistant, normal B. oleracea CC to generate AABBCC hexaploid somatic hybrids. The presence of selectable marker genes facilitated the selection of hybrids in large numbers. The resulting hybrids showed wide variation in floral morphology and organelle composition. Regenerants with normal, male-sterile flowers having recombinant tour-or oleracea-type mitochondria and oleracea-type chloroplasts were obtained. Hybrids with male-fertile flowers were also obtained that had recombined tour mitochondria. The AABBCC hexaploid hybrids synthesized in the present study were successfully utilized as a bridging material for transferring variability in the organelle genome simultaneously to all the digenomic Brassica species, and all of these hybrids are now being stabilized through repeated backcrosses to the allopolyploid crop brassicas.     

Identification and characterization of bacterial endophytes of rice

We isolated seven different bacteria from rice seedlings grown from surface sterilized seeds. Three were associated with the rice seed husk and the other four were growing endophytically within the seed. Microscopic studies revealed that the endophytes were concentrated in the root stele region. Some of the bacteria exhibited strong anti-fungal activity against Rhizoctonia solani, Pythium myriotylum, Guamannomyces graminis and Heterobasidium annosum.     

Molecular dynamics simulation of oligosaccharides containing N-acetyl neuraminic acid

αD -N-acetyl neuraminic acid (Neu5Ac, sialic acid) is a commonly occurring carbohydrate residue in various cell surface glycolipids and glycoproteins. This residue is linked terminally or internally to Gal residues via an α(2 → 3) or α(2 → 6) linkage. In the cell surface receptor, sialyl-Lewis^X, a terminal α(2 → 3) linkage is present. Previous studies from our laboratory have shown that in solution Lewis^X adopts a relatively rigid structure. In order to model the Neu5Ac residue, vacuum molecular dynamics of this monosaccharide were compared with simulations that explicitly include solvent water. The dynamical average of the monosaccharide conformation obtained from the two simulations was similar. Vacuum calculations for the disaccharide Neu5Ac α(2 → 3) Gal β-O-methyl show that a number of low energy minima are accessible to this disaccharide. Molecular dynamics simulations starting from the low energy minima show conformational transitions with a time scale of 10–50 ps among several of the minima while large barriers between other minima prevent transitions on the time scale studied. Simulations of this disaccharide in the presence of solvent show fewer conformational transitions, illustrating a dampening effect of the solvent that has been observed in some other studies. Our results are most consistent with an equilibrium among multiple conformations for the Neu5Ac α(2 → 3) Gal β linkage. © 1994 John Wiley & Sons, Inc.     

Somatic hybrids with substitution type genomic configuration TCBB for the transfer of nuclear and organelle genes from Brassica tournefortii TT to allotetraploid oilseed crop B. carinata BBCC

Oilseed crop Brassica carinata BBCC is a natural allotetraploid of diploid species B. nigra BB and B. oleracea CC. To transfer the nuclear and organelle genes in a concerted manner from an alien species, B. tournefortii TT, to B. carinata, we produced somatic hybrids with genomic configuration TCBB using B. nigra and B. oleracea stocks that carried selectable marker genes. B. tournefortii TT was sexually crossed with hygromycin-resistant B. oleracea CC. Protoplasts isolated from shoot cultures of hygromycin-resistant F₁ hybrids of B. tournefortiixB. oleracea TC were fused with protoplasts of kanamycin-resistant B. nigra BB. In two different fusion experiments 80 colonies were obtained through selection on media containing both hygromycin and kanamycin. Of these, 39 colonies regenerated into plants. Analysis of 15 regenerants by random amplified polymorphic DNA (RAPD) markers showed the presence of all three genomes, thereby confirming these to be true hybrids. Restriction fragment length polymorphism (RFLP) analysis of organelle genomes with heterologous chloroplast (cp)and mitochondrial (mt) DNA probes showed that the chloroplast genome was inherited from either of the two parents while mitochondrial genomes predominantly showed novel configurations due to either rearrangements or intergenomic recombinations. We anticipate that the TCBB genomic configuration will provide a more conducive situation for recombination between the T and C genomes during meiosis than the TTCCBB or TCCBB type configurations that are usually produced for alien gene transfer. The agronomic aim of producing TCBB hybrids is to transfer mitochondrial genes conferring cytoplasmic male sterility and nuclear genes for fertility restoration from B. tournefortii to B. carinata.     

Cellular affinity is the only deciding factor for microcarrier selection for the cultivation of anchorage dependent cells

Summary Vero and BHK-21 cells are not attached and spreaded equally on the same microcarrier. BHK-21 cells require a microenvironment similar to the mammalian tissue for attachment and spreading. Cellular affinity, which control adhesion to the surface, varied with the growth phase, membrane integrity and the type of microcarrier.