A differential molecualr topography of the Pr and Pfr forms of native oat phytochrome as probed by fluoresence quenching

Tryptophan (Trp) surface topography of the red- and far-red-absorbing forms of phytochrome (Pr, Pfr) ofAvena sativa L. has been investigated by analyzing quenching of the two components of Trp fluorescence decay, in order to understand the differences in the two forms at the molecular level. Stern-Volmer kinetic analysis of the quenching data for two cationic surface quenchers, Cs⁺ and Tl⁺, showed strong quenching of the short component of the Pr fluorescence (Stern-Volmer constants,K _sv , 27.2 and 21.4 M⁻¹, respectively) relative to that of Pfr fluorescenceK _sv , 10.4 and 12.3 M⁻¹, respectively). The long component of the Trp fluorescence was quenched differentially by Cs⁺ and Tl⁺, withK _sv of 9.0 and 19.8 M⁻¹, respectively, for the Pr fluorescence andK _sv of 13.7 and 8.7 M⁻¹, respectively, for the Pfr fluorescence. The results indicate that the phytochrome Trp residues with short fluorescence lifetime are more accessible to the cationic surface quenchers than those with long fluorescence lifetime. The data, taken together with our earlier study (Singh et al. 1988, Biochim, Biophys. Acta936, 395–405), indicate that most, if not all the ten Trp residues of phytochrome, are fluorescent and exist in distinct groups differing in their topography and microenvironment, and the peptide segment containing Trp-774 and Trp-778 within the 55-kilodalton C-terminal domain of phytochrome also undergoes a subtle alteration in its surface topography during Pr→Pfr phototransformation. This paper is dedicated to Professor Hans Mohr in commemoration of his 60th birthday     

Linkage mapping of genes for resistance to leaf,stem and stripe rusts and ω-secalins on the short arm of rye chromosome 1R

Summary The genes controlling resistance to three wheat rusts, viz., leaf rust (Lr26), stem rust (Sr31) and stripe or yellow rust (Yr9), and -secalins (Sec1), located on the short arm of rye chromosome 1R, were mapped with respect to each other and the centromere. Analysis of 214 seeds (or families derived from them) from testcrosses between a 1BL.1RS/1R heterozygote and Chinese Spring ditelocentric 1BL showed no recombination between the genes for resistance to the three rusts, suggesting very tight linkage or perhaps a single complex locus conferring resistance to the three rusts. The rust resistance genes were located 5.4 ± 1.7 cM from the Sec1 locus, which in turn was located 26.1 ± 4.3 cM from the centromere; the gene order being centromere — Sec1 — Lr26/Sr31/Yr9 — telomere. In a second test-cross, using a different 1BL.1RS translocation which had only stem rust resistance (SrR), the above gene order was confirmed despite a very large proportion of aneuploids (45.8%) among the progeny. Furthermore, a map distance of 16.0 ± 4.8 cM was estimated for SrR and the telomeric heterochromatin (C-band) on 1RS. These results suggest that a very small segment of 1RS chromatin is required to maintain resistance to all three wheat rusts. It should be possible but difficult to separate the rust resistance genes from the secalin gene(s), which are thought to contribute to dough stickiness of wheat-rye translocation lines carrying 1RS.     

The meaning of interaction parameters in two-state protein complexes

The theory of interaction parameters has thus far been based on the free-energy relationships in the formation of ternary complexes formed between a pair of ligands and a protein molecule. The concept has been formulted in terms of a thermodynamic square comprised of the free protein, the two binary complexes, and the ternary complex. However, an increasing number of proteins have been found to exist as equilibrium mixtures of two macrostates. The equilibrium constants for such two-state transitions vary quite considerably between the various binary and ternary complexes of a given protein. We show here that the interpretations of interaction parameters in such two-state systems, requiring the use of a thermodynamic cube, are much more complex than those based on the classic thermodynamic square commonly employed. We demonstrate the use of enthalpies of interaction and heat capacities of interaction to analyze the source of observed free enerigies of interaction in such systems. Specifically, we find that measured negative interaction parameters may arise simply from the inability of a system to achieve all of the positive component effects anticipated by the conventional formulation.     

Studies on lactic dehydrogenase and sorbitol dehydrogenase release in relation to deep freezing of buffalo semen in certain extenders

Forty seminal ejaculates from five mature buffalo bulls (n=8 from each bull), exhibiting more than 70% initial sperm motility, were frozen in the following three extenders: egg-yolk sodium citrate glycerol (EYCG); tris-egg yolk glycerol (TYG); and citric acid whey glycerol (CAWG). The extenders were evaluated for the release of intracellular enzymes, lactic dehydrogenase (LDH) and sorbitol dehydrogenase (SODH) from the spermatozoa during freezing (in fresh semen, after dilution and after equilibration) and post freezing (24 h and 7 d after freezing. It was found that the release of LDH and SODH enzymes was significantly lower in TYG than in EYCG and CAWG extenders. The most critical stage, at which the enzyme release was maximal, was between equilibration and 24 h post freezing in all three extenders.     

Effect of uterine fluid on in vitro acrosome reaction of buffalo (Bubalus bubalis ) spermatozoa

The present study was undertaken to examine in vitro acrosome reaction in the uterine fluid of estrous buffalo. Successful acrosome reaction was achieved by incubating buffalo spermatozoa in 2% detoxified uterine fluid in Biggers Whitten Whittingham (BWW) medium, pH 7.4 at 37 degrees C and a sperm concentration of 36 x 10/ml. Neat uterine fluid has been found to be toxic to spermatozoa, hence we detoxified the uterine fluid at 56 degrees C for 30 min, which resulted in higher percentage of sperm motility, viability, and acrosome reaction. All the three stages of acrosome reaction i.e., acrosome swelling, vesiculation and shedding, were observed and they reached an apparent maximum at 4, 7 and 8 h of incubation, respectively. The significance of the findings in relation to the role of female reproductive tract in acrosome reaction is discussed.     

Ischemia-reperfusion injury: biochemical alterations in peroxisomes of rat kidney.

Exogenously supplied catalase, a peroxisomal enzyme, has been found to be of therapeutic value in ischemic injury. Therefore, we examined the effect of ischemic-reperfusion injury on the structure and function of kidney peroxisomes. Ischemic injury changed the density of peroxisomes from 1.21 g/cm3 (peak I) to a lighter density of 1.14 g/cm3 (peak II). The number of peroxisomes moving from the normal density population (peak I) to a lower density population (peak II) increased with an increase in ischemic injury. Latency experiments indicated both populations of peroxisomes to be of intact peroxisomes. Immunoblot analysis with antibodies against peroxisomal matrix and membrane proteins demonstrated that after 90 min of ischemia a significant number of matrix proteins were lost in the peak II population, suggesting that functions of these peroxisomes may be severally affected. Reperfusion following ischemic injury resulted in loss of peroxisomal matrix proteins in both peaks I and II, suggesting that peroxisomal functions may be drastically compromised. This change in peroxisomal functions is reflected by a significant decrease in peroxisomal catalase activity (35%) and beta-oxidation of lignoceric acid (43%) observed following 90 min of ischemia. The decrease in catalase activity was more pronounced in reperfused kidneys even after a shorter term of ischemic injury. Reperfusion restored the normal peroxisomal beta-oxidation in kidneys exposed up to 60 min of ischemia. However, 90 min of ischemia was irreversible as there was a further decrease in beta-oxidation upon reperfusion. The decrease in catalase activity during ischemia alone was due to the formation of an inactive complex, whereas during reperfusion, following 90 min of ischemia, inactivation and proteolysis or decreased synthesis of catalase contributed equally toward the injury. The observed changes in the structure and function of peroxisomes as a result of ischemic-reperfusion injury and the ubiquitous distribution of peroxisomes underlines the importance of this organelle in the pathophysiology of vascular injury in general.     

Purification, crystallization, and X-ray diffraction studies of lactotransferrin from buffalo colostrum.

Lactotransferrin is an iron-binding protein. It has been purified from buffalo colostrum. The purified lactotransferrin has been crystallized in 10% ethanol solution. The crystals are orthorhombic and the space group is P2(1)2(1)2(1) with unit cell dimensions a = 161.70 A, b = 155.75 A, c = 113.48 A. The asymmetric unit contains three molecules of the protein with a solvent content of about 59%. The crystals were stable in the X-ray beam and diffract beyond 3.5 A resolution. The native data have been collected and the structure determination is in progress.     

Functional properties of the anaerobic responsive element of the maize Adh1 gene

The functional properties of the anaerobic responsive element (ARE) of the maize Adh1 gene have been analysed using a transient expression assay in electroporated maize protoplasts. The ARE functions in both orientations although inversion of the ARE sequence relative to the TATA box element produces slightly weaker promoter activity under anaerobic conditions and elevated expression under aerobic conditions. Promoter activity under anaerobic conditions is proportional to the number of complete ARE sequences in the Adh1 promotor. The ARE contains two sub-regions and dimers of sub-region II are as efficient as the wild-type sequence in activating gene expression under anaerobic conditions. However, sub-region I dimers do not appear capable of inducing gene expression in response to anaerobic stress. We conclude that sub-region II is essential for anaerobic induction of gene expression. Reporter gene expression remains constant when the spacing between sub-regions of the ARE is increased up to at least 64 bp, but increased spacing of 136 bp or greater abolishes expression in both aerobic and anaerobic conditions, indicating that a close association of the two sub-regions is required both for anaerobic responsiveness and for maximal levels of aerobic gene expression. When the ARE is placed upstream of position –90 of the CaMV 35S promoter, the ARE produces a high level of expression in both aerobic and anaerobic conditions. The general enhancement of gene expression driven by the hybrid ARE/35S promoter in aerobic conditions requires an intact sub-region II motif since mutation or deletion of sub-region II from the hybrid promoter reduces the level of expression to that observed for the truncated 35S promoter alone. In addition, mutation of the sub-region I sequences in the ARE/35S hybrid promoter does not significantly reduce expression in aerobic conditions, relative to pARE/35S(-90), suggesting that sub-region I does not contribute to this general enhancer function.     

Uptake of volatilen-alkanes byPseudomonas PG-I

Using a bacterial speciesPseudomonas PG-1, evidence has been obtained which indicates that uptake ofn-pentane ton-octane by microbial cells takes place primarily from the gas phase either directly orvia the aqueous phase. Specific growth rate increased along with the increase in substrate concentration but above the alkane concentration of 0.3% by volume, specific growth rate decreased indicating substrate inhibition of growth. In the case of less volatile alkanes,n-nonane andn-decane, substrate transfer is predominantly through substrate solubilization system elaborated by the cells. EDTA, a strong inhibitor of hydrocarbon solubilization by the cells, inhibited growth on these two alkanes but had negligible effect on growth onn-pentane ton-octane.     

Fecundity,reproductive rate,longevity, and intrinsic rate of increase of an aphidiid parasitoidLysiphlebia mirzai

A life-table was constructed for a little known aphidiid waspLysiphlebia mirzai, a parasitoid of cereal aphid,Rhopalosiphum maidis. The female parasitoid survived 6.4 ± 1.17 (SD) days and oviposited intensively 4.0 ± 0.47 days. The total fecundity rate, R_t, was 169.2 ± 6.94 mummies/female and net reproductive rate, R_o, was 92.70 female offspring/female. The intrinsic total fecundity rate, r_t, and intrinsic rate of natural increase, r_m, the finite rate of total fecundity, λ^t, and finite rate of increase, λ^m, was 0.27048, 0.24155, 1.31059 and 1.27322 respectively. The mean generation time (18.75 days) and doubling time of the population (2.87 days) was slightly higher than other aphidiids studied so far. The proportion of female progenies decreased significantly on the successive oviposition days.