Photoreversal kinetics of the I1 and I2 intermediates in the photocycle of photoactive yellow protein by double flash experiments with variable time delay

We investigated the kinetics of photoreversal from the I(1) and I(2) intermediates of photoactive yellow protein (PYP) by time-resolved optical absorption spectroscopy with double flash excitation. A first flash, at 430 nm, initiated the photocycle. After a variable time delay, the I(1) intermediate was photoreversed by a second flash, at 500 nm, or a mixture of I(2) and I(2)' intermediates was photoreversed by a second flash, at 355 nm. By varying the delay from 1 micros to 3 s, we were able to selectively excite the intermediates I(1), I(2), and I(2)'. The photoreversal kinetics of I(2) and I(2)' at 21 different delays and two wavelengths (340 and 450 nm) required two exponentials for a global fit with time constants of tau(1) = 57 +/- 5 micros and tau(2) = 380 +/- 40 micros (pH 6, 20 degrees C). These were assigned to photoreversal from sequential I(2) and I(2)' intermediates, respectively. The good agreement of the delay dependence of the two amplitudes, A(1) and A(2), with the time dependence of the I(2) and I(2)' populations provided strong evidence for the sequential model. The persistence of A(1) beyond delay times of 5 ms and its decay, together with A(2) around 500 ms, suggest moreover that I(2) and I(2)' are in thermal equilibrium. The wavelength dependence of the photoreversal kinetics was measured at 26 wavelengths from 510 to 330 nm at the two fixed delays of 1 and 10 ms. These data also required two exponentials for a global fit with tau(1) = 59 +/- 5 micros and tau(2) = 400 +/- 40 micros, in good agreement with the delay results. Photoreversal from I(2)' is slower than from I(2), since, in addition to chromophore protonation, the global conformational change has to be reversed. Our data thus provide a first estimate of about 59 micros for deprotonation and 400 micros for the structural change, which also occurs in the thermal decay of the signaling state but is obscured there since reisomerization is rate-limiting. The first step in photoreversal is rapid cis-trans isomerization of the chromophore, which we could not resolve, but which was detected by the instantaneous increase in absorbance between 330 and 380 nm. In agreement with this observation, the spectrum of the I(2)'(trans) intermediate, derived from the A(2) amplitude spectrum, has a much larger extinction coefficient than the spectrum of the I(2)'(cis) intermediate. With a first flash, at 430 nm, and a second flash, at 500 nm, we observed efficient photoreversal of the I(1) intermediate at a delay of 20 micros when most molecules in the cycle are in I(1). We conclude that each of the three intermediates studied can be reversed by a laser flash. Depending on the progression of the photocycle, reversal becomes slower with the time delay, thus mirroring the individual steps of the forward photocycle.     

Effect of modification of the length and flexibility of the acyl carrier protein-thioesterase interdomain linker on functionality of the animal fatty acid synthase

A natural linker of approximately 20 residues connects the acyl carrier protein with the carboxy-terminal thioesterase domain of the animal fatty acid synthase. This study examines the effects of changes in the length and amino acid composition of this linker on catalytic activity, product composition, and segmental motion of the thioesterase domain. Deletion of 10 residues, almost half of the interdomain linker, had no effect on either mobility of the thioesterase domain, estimated from fluorescence polarization of a pyrenebutyl methylphosphono moiety bound covalently to the active site serine residue, or functionality of the fatty acid synthase; further shortening of the linker limited mobility of the thioesterase domain and resulted in reduced fatty acid synthase activity and an increase in product chain length from 16 to 18 and 20 carbon atoms. Surprisingly, however, even when the entire linker region was deleted, the fatty acid synthase retained 28% activity. Lengthening of the linker, by insertion of an unusually long acyl carrier protein-thioesterase linker from a modular polyketide synthase, increased mobility of the thioesterase domain without having any significant effect on catalytic properties of the complex. Interdomain linkers could also be used to tether, to the acyl carrier protein domain of the fatty acid synthase, a thioesterase active toward shorter chain length acyl thioesters generating novel short-chain fatty acid synthases. These studies reveal that although truncation of the interdomain linker partially impacts the ability of the thioesterase domain to terminate growth of the acyl chain, the overall integrity of the fatty acid synthase is quite tolerant to moderate changes in linker length and flexibility. The retention of fatty acid synthesizing activity on deletion of the entire linker region implies that the inherent flexibility of the phosphopantetheine "swinging arm" also contributes significantly to the successful docking of the long-chain acyl moiety in the thioesterase active site.     

Phosphate solubilization and growth promotion by <Emphasis Type="Italic">Pseudomonas fragi</Emphasis> CS11RH1 (MTCC 8984), a psychrotolerant bacterium isolated from a high altitude Himalayan rhizosphere

Phosphate solubilization and growth promotion by Pseudomonas fragi CS11RH1 (MTCC 8984), a psychrotolerant bacterium isolated from a high altitude garlic rhizosphere from the Indian Himalayas, are reported here. The identity of the isolate was arrived on the basis of its biochemical features and sequencing of the 16S rRNA gene. The isolate grew and solubilized phosphate at temperatures ranging from 4 to 30°C. Besides solubilizing P it produced indole acetic acid (IAA) and hydrogen cyanide (HCN). Seed bacterization with the isolate significantly increased the percent germination, rate of germination, plant biomass and nutrient uptake of wheat seedlings. While Pseudomonas fragi is normally associated with the spoilage of dairy products stored at cold temperatures, this is an early report on the plant growth promoting ability of the bacterium.     

Effect of maternal micronutrients (folic acid, vitamin B12) and omega 3 fatty acids on liver fatty acid desaturases and transport proteins in Wistar rats

A disturbed fatty acid metabolism increases the risk of adult non-communicable diseases. This study examines the effect of maternal micronutrients on the fatty acid composition, desaturase activity, mRNA levels of fatty acid desaturases and transport proteins in the liver. Pregnant female rats were divided into 6 groups at 2 levels of folic acid both in the presence and absence of vitamin B(12). The vitamin B(12) deficient groups were supplemented with omega 3 fatty acid. An imbalance of maternal micronutrients reduces liver docosahexaenoic acid, increases Δ5 desaturase activity but decreases mRNA levels, decreases Δ6 desaturase activity but not mRNA levels as compared to control. mRNA level of Δ5 desaturase reverts back to the levels of the control group as a result of omega 3 fatty acid supplementation. Our data for the first time indicates that maternal micronutrients differentially alter the activity and expression of fatty acid desaturases in the liver.     

Determination of growth inhibitory action point of interferon gamma on WISH cells in cell cycle progression and the window of responsiveness of the cells to the interferon

We had earlier shown that human foetal epithelial cells (WISH), growth-inhibited by interferon gamma (IFNgamma), were reversibly detained at a point prior to DNA synthesis. In the present study, we determined the window of action of IFNgamma in the G1 phase duration and the exact point of detention of WISH cells in cell cycle progression with respect to the known points of detention by the inhibitors of DNA replication initiation (aphidicolin and carbonyl diphosphonate) and of activation of replication protein A (6-dimethylaminopurine), of which RPA activation being the earlier event compared to DNA replication initiation in cell cycle progression. WISH cells, which were released from IFNgamma-induced arrest, permeabilised and exposed independently to these inhibitors show that IFNgamma detains WISH cells prior to initiation of DNA synthesis. Further, exposure of IFNalpha-synchronized (at G0/G1) or mimosine-synchronized (at G1/S) WISH cells to IFNgamma, which was added at different time points post-release from the synchronizing agent, showed that the cells were promptly responsive to the growth inhibitory action of IFNgamma only during the first 11h in G1 phase. Taken together, these results suggest that IFNgamma inhibits growth of WISH cells by detaining them at a point prior to initiation of DNA synthesis and that the IFN acts within the first 11h in G1 phase of the cell cycle.     

Insertion of the human immunodeficiency virus CD4 receptor into the envelope of vesicular stomatitis virus particles.

Enveloped virus particles carrying the human immunodeficiency virus (HIV) CD4 receptor may potentially be employed in a targeted antiviral approach. The mechanisms for efficient insertion and the requirements for the functionality of foreign glycoproteins within viral envelopes, however, have not been elucidated. Conditions for efficient insertion of foreign glycoproteins into the vesicular stomatitis virus (VSV) envelope were first established by inserting the wild-type envelope glycoprotein (G) of VSV expressed by a vaccinia virus recombinant. To determine whether the transmembrane and cytoplasmic portions of the VSV G protein were required for insertion of the HIV receptor, a chimeric CD4/G glycoprotein gene was constructed and a vaccinia virus recombinant which expresses the fused CD4/G gene was isolated. The chimeric CD4/G protein was functional as shown in a syncytium-forming assay in HeLa cells as demonstrated by coexpression with a vaccinia virus recombinant expressing the HIV envelope protein. The CD4/G protein was efficiently inserted into the envelope of VSV, and the virus particles retained their infectivity even after specific immunoprecipitation experiments with monoclonal anti-CD4 antibodies. Expression of the normal CD4 protein also led to insertion of the receptor into the envelope of VSV particles. The efficiency of CD4 insertion was similar to that of CD4/G, with approximately 60 molecules of CD4/G or CD4 per virus particle compared with 1,200 molecules of VSV G protein. Considering that (i) the amount of VSV G protein in the cell extract was fivefold higher than for either CD4 or CD4/G and (ii) VSV G protein is inserted as a trimer (CD4 is a monomer), the insertion of VSV G protein was not significantly preferred over CD4 or CD4/G, if at all. We conclude that the efficiency of CD4 or CD4/G insertion appears dependent on the concentration of the glycoprotein rather than on specific selection of these glycoproteins during viral assembly.     

Siderophore Cross-Utilization Amongst Rhizospheric Bacteria and the Role of Their Differential Affinities for Fe3+ on Growth Stimulation Under Iron-Limited Conditions

The majority of bacteria isolated from rhizospheres of Arachis hypogea (Groundnut) and Vigna radiata (Mung bean) predominantly produced catechol-type siderophores except for a few fluorescent pseudomonads that produced hydroxamates in addition to catecholates. The rhizospheric isolates differed in their ability to cross-utilize siderophores produced by other rhizospheric isolates (heterologous); some were highly proficient at utilizing heterologous siderophores, while others were poor cross-utilizers. Isolate G9, which utilized hydroxamate as well as catecholate siderophores, was found to be an efficient siderophore cross-utilizer, while isolates G2 and G6 were poor-utilizers of catecholate and non-utilizers of hydroxamate siderophores. Growth stimulation of two isolates G9 and G6 was seen when grown in the presence of externally supplied heterologous siderophores, which they cross-utilized. The iron-regulated outer membrane protein (IROMP) profiles differed for the most cross-utilizer and the least cross-utilizer strains, but in both the cases no new outer membrane proteins (OMP) were induced in response to the exogenous siderophores supplied. The growth of the organisms in the presence of heterologous siderophores that they failed to cross-utilize led to growth inhibition in the case of isolate G9. This appears to be due to a lower affinity of the siderophore of G9 as compared to the exogenously supplied G6 siderophore. A simple method was devised to measure relative affinities of respective siderophores for iron based on CAS solution decolorization by the siderophore preparations. The effect on the growth of the differential affinities of the siderophores for iron and the interactions of the organisms through cross-utilization is also discussed.     

Differential recovery of membrane proteins after extraction by aqueous methanol and trifluoroethanol

Cell membrane proteome analysis is limited by inherent membrane hydrophobicity. Conventional membrane protein extraction techniques use detergents, chaotropes and organic acids that require sample clean-up or pH adjustment, and are associated with significant sample loss. We extracted membrane proteins from red blood cells (RBCs) using methanol (MeOH), trifluoroethanol (TFE) and urea, and identified membrane proteins using 2-D LC coupled with MALDI-TOF/TOF-MS. We show that organic solvents MeOH- and TFE-based methods have membrane protein analysis efficiencies comparable to urea, and are complementary for the recovery of both hydrophilic and hydrophobic peptides. The mean grand average of hydropathicity (GRAVY) value of identified peptides from the TFE-based method (-0.107) was significantly higher than that of the MeOH-based method (-0.465) (p<0.001). Sequential and adjunctive use of the organic solvents MeOH and TFE increases the number of proteins identified, and the confidence of their identification. We show that this strategy is effective for shotgun membrane proteome analysis.     

Interaction of benzanthrone with cytochrome P450: altered patterns of hepatic xenobiotic metabolism in rats

Benzanthrone, an anthraquinone dye intermediate, is commonly used for the synthesis of a number of polycyclic vat and disperse dyes. Our prior studies have shown that benzanthrone can be metabolized by rat hepatic microsomal cytochrome P450 (P450) (Biochem. Int., 18, 1989, 1237). In this study, the interaction of benzanthrone with rat hepatic microsomal P-450 and its effect on xenobiotic metabolism have been investigated. Parenteral administration of benzanthrone (40 mg/kg body weight) for 3, 7, or 21 days caused no change in the relative body weight or organ weight of rats. The levels of P450 were found to be reduced (33%-50%) in all the benzanthrone-exposed animals at all the time periods. In vitro addition of benzanthrone caused a spectral change with oxidized P450 and concentration-dependent reduction in the carbon monoxide spectrum of dithionite-reduced P450. The addition of benzanthrone to hepatic microsomes prepared from phenobarbital-treated rats resulted in spectral changes characterized by an absorbance maximum at 397 nm indicative of type I binding. In vitro addition of benzanthrone showed a concentration-dependent inhibition of hepatic aminopyrine N-demethylase (APD) and ethoxyresorufin-O-deethylase (ERD) activities with respective I50 values of 9.5 x 10(-4) and 8.0 x 10(-5) M. However, the inhibition of aryl hydrocarbon hydroxylase (AHH) even at the highest concentration of benzanthrone (10(-2) M), was of the order of only 29%. In vivo administration of benzanthrone also led to the inhibition of APD, AHH, and ERD activities at all treatment times although the magnitude of inhibition was of a lower order.(ABSTRACT TRUNCATED AT 250 WORDS)     

MutS recognition: Multiple mismatches and sequence context effects

Escherichia coli MutS is a versatile repair protein that specifically recognizes not only various types of mismatches but also single stranded loops of up to 4 nucleotides in length. Specific binding, followed by the next step of tracking the DNA helix that locates hemi-methylated sites, is regulated by the conformational state of the protein as a function of ATP binding/hydrolysis. Here, we study how various molecular determinants of a heteroduplex regulate mismatch recognition by MutS, the critical first step of mismatch repair. Using classical DNase I footprinting assays, we demonstrate that the hierarchy of MutS binding to various types of mismatches is identical whether the mismatches are present singly or in multiples. Moreover, this unique hierarchy is indifferent both to the differential level of DNA helical flexibility and to the unpaired status of the mismatched bases in a heteroduplex. Surprisingly, multiple mismatches exhibit reduced affinity of binding to MutS, compared to that of a similar single mismatch. Such a reduction in the affinity might be due to sequence context effects, which we established more directly by studying two identical single mismatches in an altered sequence background. A mismatch, upon simply being flipped at the same location, elicits changes in MutS specific contacts, thereby underscoring the importance of sequence context in modulating MutS binding to mismatches.