Methionine-oxidized horse heart cytochromec. III. Ascorbate reduction and the methionine-80-sulfur-iron linkage

The ascorbate reduction of the CT-cytochromes—two chemically generated forms of horse heart cytochrome c, F^III and F^II, with both methionines, 80 and 65, as methionine sulfoxides, no iron-sulfur linkage, and potentiometric and physiological oxidoreduction properties distinct from those of the native protein and one another (J. Pande et al., 1987)—has been investigated using a stopped-flow technique. The reaction was monitored at 550 nm, and studies were conducted in 10 mM phosphate +0.17 M NaCl buffer,pH 7.4. Both CT-cytochromes are reduced by triphasic profiles, a faster and an intermediate ascorbate-dependent reaction and a slow, ascorbate-independent process. Both CT-cytochromes contain three molecular forms in slow equilibrium, two reducing directly by reaction with ascorbate and a third through conversion to one of the reducible forms. Like the reaction of the native protein, the ascorbate dependence of both the rapid and the intermediate process is nonlinear, approaching saturation values at high concentrations. The ascorbate profiles of the pseudo-first-order reduction constants are typical of the model for the reduction reaction of the unmodified protein, binding followed by a first-order reduction reaction (Myer et al., 1980; Myer and Kumar, 1984), but with distinct kinetic parameters, the first-order reduction constants and the protein-ascorbate stability constants. It has been concluded that the functional-conformational differences between the two CT-cytochromes are not operational to any significant extent in the reduction reaction with ascorbate. The methionine-80-sulfur-iron linkage of the protein is not a crucial requirement for the ascorbate reduction of the protein. The mechanism of the reaction in the main is also insensitive to the replacement of Met-80-S from heme coordination and/or the associated conformational-oxidoreduction properties of the protein. Of the two aspects of the reaction, the efficiency of the electron-transfer reaction and the stability of the ascorbate dianion-protein complex, the former is dependent on the integrity of the structural-conformational state of the molecule.     

Generation of somatic variants of a B cell hybrid mediated by a non-cytolytic L3T4+ idiotype-specific T cell

We previously reported that idiotype (Id)-loss, stable somatic variants of a B cell hybrid, 2C3E1, are generated both in vitro and in vivo, after interaction of the Id-positive tumor cells with autologous Id-specific effector T cells. The present investigation was undertaken to elucidate further the nature and functional characteristics of the effector T cells. We report here that the idiotype-specific cells mediating the generation of Id- tumor variants are Thy1+ L3T4+ Lyt-2- cells, which respond to specific idiotypic stimulation by secreting IL-2 in vitro. No IL-2 is secreted in response to unrelated Ig or an Id/Ig-2C3E1 tumor variant. Furthermore, the Id-specific T cells exert strong suppressive effects on the expression of 2C3E1 Ig and the effects can be reversed by blocking the L3T4+ T cells with monoclonal anti-L3T4 antibody in vitro during the initial 3 days of co-culture. After 4 days, the T cell-mediated suppression of the 2C3E1-Id is irreversible. In addition to the in vitro studies we have determined that the administration of anti-L3T4 mAb to mice just before priming with idiotype-bearing tumor cells also abrogates the suppressive effects of the idiotype primed spleen cells on Ig expression of 2C3E1. To study the Id-specific effector T cells in more detail we have generated functional Id-specific L3T4+ T cell lines. These T cell lines have been shown to recapitulate the generation of Id- tumor variants that we observed with Id-primed spleen cells. It is concluded that L3T4+, Id-specific Ts cells are responsible for the generation of somatic variants of the B cell hybrid 2C3E1 and that the induction or selection of these variants progresses from a reversible phase to an irreversible phase.     

A model for cell type-specific differential gene expression during heterocyst development and the constitution of aerobic nitrogen fixation ability inAnabaena sp. strain PCC 7120

When deprived of combined nitrogen, aerobically-grown filaments ofAnabaena sp. strain PCC7120 differentiate specialized cells called the heterocysts. The differentiation process is an elaborate and well orchestrated programme involving sensing of environmental and developmental signals, commitment of cells to development, gene rearrangements, intricate DNA-protein interactions, and differential expression of several genes. It culminates in a physiological division of labour between heterocysts, which become the sole sites of aerobic nitrogen fixation, and vegetative cells, that provide photosynthate to the heterocysts in return for nitrogen supplies. We propose a model, to describe the chronology of the important events and to explain how cell type-specific differential gene expression is facilitated by DNA-protein interactions leading to the development of heterocysts and constitution of nitrogen-fixing apparatus inAnabaena.     

A 43 kDa DNA binding protein from the pea chloroplast interacts with and stimulates the cognate DNA polymerase.

A DNA binding protein with DNA polymerase 'accessory activity' has been identified and purified to apparent homogeneity from pea chloroplasts. This protein consists of a single subunit of 43 kDa and binds to DNA regardless of its base sequence and topology. It increases cognate DNA polymerase-primase activity in a dose dependent manner. Using solid phase protein-protein interaction trapping and co-immunoprecipitation techniques, the purified protein was found to associate with the chloroplast DNA polymerase. The chloroplast DNA polymerase also binds directly to the radioiodinated 43 kDa protein. The specific interaction between 43 kDa protein and chloroplast DNA polymerase results in the synthesis of longer DNA chains. The 43 kDa protein, present abundantly in the pea chloroplast, appears to increase processivity of the chloroplast DNA polymerase and may play an important role in the replication of pea chloroplast DNA.     

Approximate methods for estimating the pattern of nucleotide substitution and the variation of substitution rates among sites

We propose two approximate methods (one based on parsimony and one on pairwise sequence comparison) for estimating the pattern of nucleotide substitution and a parsimony-based method for estimating the gamma parameter for variable substitution rates among sites. The matrix of substitution rates that represents the substitution pattern can be recovered through its relationship with the observable matrix of site pattern frequences in pairwise sequence comparisons. In the parsimony approach, the ancestral sequences reconstructed by the parsimony algorithm were used, and the two sequences compared are those at the ends of a branch in the phylogenetic tree. The method for estimating the gamma parameter was based on a reinterpretation of the numbers of changes at sites inferred by parsimony. Three data sets were analyzed to examine the utility of the approximate methods compared with the more reliable likelihood methods. The new methods for estimating the substitution pattern were found to produce estimates quite similar to those obtained from the likelihood analyses. The new method for estimating the gamma parameter was effective in reducing the bias in conventional parsimony estimates, although it also overestimated the parameter. The approximate methods are computationally very fast and appear useful for analyzing large data sets, for which use of the likelihood method requires excessive computation.      

Refining the region of branchio-oto-renal syndrome and defining the flanking markers on chromosome 8q by genetic mapping.

Branchio-oto-renal syndrome (BOR) is an autosomal dominant disorder associated with external-, middle-, and inner-ear malformations, branchial cleft sinuses, cervical fistulas, mixed hearing loss, and renal anomalies. The gene for BOR was mapped to the long arm of chromosome 8q. Several polymorphic dinucleotide repeat markers were investigated for linkage in two large BOR families, and the region of localization was refined. Two-point linkage analysis yielded the maximum lod scores of 7.44 at theta = .03 and 6.71 at theta = .04, with markers D8S279 and D8S260, respectively. A multipoint analysis was carried out to position the BOR gene with a defined region using markers D8S165, D8S285, PENK, D8S166, D8S260, D8S279, D8S164, D8S286, D8S84, D8S275, D8S167, D8S273, and D8S271. Haplotype analysis of recombination events at these polymorphic loci was also performed in multigeneration BOR kindreds. The linkage analysis and analysis of recombination events identified markers that clearly flank the BOR locus. The order was determined to be D8S260-BOR-D8S279 at odds > 10(3):1 over the other possible orders. This flanking markers provide a resource for high-resolution mapping toward cloning and characterizing the BOR gene.     

Synthesis of intraspecific somatic hybrids of Solanum tuberosum: assessments of morphological, biochemical and nematode (Globodera pallida) resistance characteristics

In an attempt to produce novel agronomic traits, a number ofintraspecific somatic hybrid plants have been produced followingleaf mesophyll protoplast fusion between S. tuberosum dihaploidclones PDH 40 (possessing good tuber shape and yield) and PDH417 (possessing resistance to potato cyst nematode, G. pallida).PDH 417 protoplast-derived calli failed to regenerate plantsusing the described culture conditions preventing this parentaltype amongst the mass of regenerated fusion products. Initially,somatic hybrid plants were selected based on differential pigmentationin tuber sprouts and where possible on petal colour. Differentialmobility of patatin bands in electrophoresed tuber extractsfurther confinned hybridity. The intraspecific somatic hybridsalso showed different levels of resistance to G. pallida pathotypesPa2 and Pa3 in the somatic hybrid plants examined. Key words: Somatic hybridization, dihaploids, patatin, nematode resistance, Solanum tuberosum, potato     

Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.)

Efficient plant regeneration via somatic embryogenesis has been developed in chickpea cultivar C235. Leaf explants, on MS medium supplemented with 1.25 mg/l 2,4-D and 0.25 mg/l kinetin, yielded somatic embryos with high efficiency during dark incubation. MS medium supplemented with B₅ vitamins, 0.125 mg/l IBA and 2 mg/l BAP was found suitable for embryo maturation. The well formed embryos germinated into plantlets on basal B₅ medium supplemented with 0.25 mg/l BAP. Further development into healthy plantlets was obtained on basal B₅ medium. Hardened plantlets produced normal, fertile plants upon transfer to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-Benzyl-aminopurine - IAA IndoIe-3-acetic acid - IBA Indole-3-butyric acid - NAA 1-Naphthalene acetic acid - Kinetin 6-furfuryl aminopurine - Zeatin 6-(4-hydroxy-3-methylbut-2-enylamino)-purine     

Inhibition of G1 phase cyclin dependent kinases by transforming growth factor β1

Transforming growth factor β1 (TGFβ1) inhibits epithelial cell proliferation late in the G1 phase of the cell cycle. We examined the effect of TGFβ1 on known late G1 cell cycle regulators in an attempt to determine the molecular mechanism of growth inhibition by this physiological inhibitor. The results demonstrate the TGFβ1 inhibits the late G1 and S phase specific histone H1 kinase activity of p33^cdk2. This inhibitiion is not dur to TGFβ1's effect on p33^cdk2 synthesis, but rather due to its negative effect on the late G1 phosphorylation of p33^cdk2. It is also shown that TGFβ1 inhibits both late G1 cyclin A and cyclin E associated histon H1 kinase activities. The inhibitor has no effects on the synthesis of cyclin E but to inhibit the synthesis of cyclin A protein in a cell cycle dependent manner. If TGFβ1 is added to cells which have progressed futher than 8 hours into G1, then it is without inhibitory effect on cyclin A synthesis. These effect on TGFβ1 on late G1 cell cycle regulators correlate well with its inhibitory effects on cellular growth and suggest that these G1 cyclin dependent kinases might serve as targets for TGFβ1-mediated growth arrest.