Generation of the O630 photointermediate of bacteriorhodopsin is controlled by the state of protonation of several protein residues

The last stages of the photocycle of the photosynthetic pigment all-trans bacteriorhodopsin (bR570), as well as its proton pump mechanism, are markedly pH dependent. We have measured the relative amount of the accumulated O630 intermediate (Phir), as well as its rise and decay rate constants (kr and kd, respectively), over a wide pH range. The experiments were carried out in deionized membrane suspensions to which varying concentrations of metal cations and of large organic cations were added. The observed pH dependencies, s-shaped curves in the case of Phir and bell-shaped curves for kr and kd, are interpreted in terms of the titration of three protein residues denoted as R1, R2, and R3. The R1 titration is responsible for the increase in Phir, kr, and kd upon lowering the pH from pH approximately 9.5 to 7. At low pH Phir exhibits a secondary rise which is attributed to the titration of a low pKa group, R2. After reaching a maximum at pH approximately 7, kr and kd undergo a decrease upon decreasing the pH, which is attributed to the titration of R3. All three titrations exhibit pKa values which decrease upon increasing the salt concentration. As in the case of the Purple (bR570) if Blue (bR605) equilibrium, divalent cations are substantially more effective than monovalent cations in shifting the pKa values. Moreover, bulky organic cations are as effective as small metal cations. It is concluded that analogously to the Purple if Blue equilibrium, the salt binding sites which control the pKa values of R1, R2, and R3 are located on, or close to, the membrane surface. Possible identifications of the three protein residues are considered. Experiments with the E204Q mutant show that the mutation has markedly affected the R2 (Phir) titration, suggesting that R2 should be identified with Glu-204 or with a group whose pKa is affected by Glu-204. The relation between the R1, R2 and R3 titrations and the proton pump mechanism is discussed. It is evident that the pH dependence of Phir is unrelated to the measured pKa of the group (XH) which releases the proton to the extracellular medium during the photocycle. However, since the same residue may exhibit different pKa values at different stages of the photocycle, it cannot be excluded that R2 or R3 may be identified with XH.     

Predatory efficiency of the water bug Sphaerodema annulatum on mosquito larvae (Culex quinquefasciatus) and its effect on the adult emergence

The daily number of IV instar larva of Culex quinquefasciatus killed, rate of pupation and adult emergence was noted in presence of the predatory water bug Sphaerodema annulatum for a period of seven consecutive days, experimentally, in the laboratory. The rate of IV instar larva killed by the water bugs on an average was 65.17 per day. The rate of pupation ranged between 7.6 and 48 in control while in presence of water bugs it ranged between 6 and 35. The rate of adult emergence in control experiments varied between 1.4 and 4.8 per day, which was reduced to only 0.4-28.8 per day in case of the water bugs. The results clearly indicate that the water bugs on its way of predation reduces the rate of pupation and adult emergence of Cx. quinquefasciatus significantly which calls for an extensive field trials.     

Cold-shock induced high-yield protein production in Escherichia coli

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.     

AP endonuclease-independent DNA base excision repair in human cells

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.     

Reciprocal CD40 signals through p38MAPK and ERK-1/2 induce counteracting immune responses

Macrophages play host to Leishmania major, a parasite that causes leishmaniasis in 500,000 people annually. Macrophage-expressed CD40, a costimulatory molecule, induces interleukin-12 (IL-12)-dependent and interferon-gamma (IFN-gamma)-dependent host-protective immune responses to Leishmania and other intracellular pathogens. Paradoxically, IL-10, another CD40-induced cytokine in macrophages, promotes Leishmania infection. How CD40 signaling regulates the secretion of these two counteractive cytokines remains unknown. Here we show that weak CD40 signals induce extracellular stress-related kinase-1/2 (ERK-1/2)-dependent IL-10 expression, whereas stronger signals induce p38 mitogen-activated protein kinase (p38MAPK)-dependent IL-12 production. p38MAPK and ERK-1/2 therefore have counter-regulatory actions. Leishmania skews CD40 signaling toward ERK-1/2, inducing IL-10, which inhibits activation of CD40-induced p38MAPK and expression of inducible nitric oxide synthase-2 (iNOS-2) and IL-12. ERK-1/2 inhibition or IL-10 neutralization restores CD40-induced p38MAPK activation and parasite killing in macrophages and the BALB/c mouse, a susceptible host. These data uncover a new immune evasion strategy, whereby Leishmania differentially modulates CD40-engaged, reciprocally functioning signaling modules, and provide a new conceptual framework for immune homeostasis.     

The fragile X syndrome repeats form RNA hairpins that do not activate the interferon-inducible protein kinase, PKR, but are cut by Dicer

We show here that under physiologically reasonable conditions, CGG repeats in RNA readily form hairpins. In contrast to its DNA counterpart that forms a complex mixture of hairpins and tetraplexes, r(CGG)₂₂ forms a single stable hairpin with no evidence for any other folded structure even at low pH. RNA with the sequence (CGG)₉AGG (CGG)₁₂AGG(CGG)₉₇, found in a fragile X syndrome pre-mutation allele, forms a number of different hairpins. The most prominent hairpin forms in the 3′ part of the repeat and involves the 97 uninterrupted CGG repeats. In contrast to the CUG-RNA hairpins formed by myotonic dystrophy type 1 repeats, we found no evidence that CGG-RNA hairpins activate PKR, the interferon-inducible protein kinase that is activated by a wide range of double-stranded RNAs. However, we do show that the CGG-RNA is digested, albeit inefficiently, by the human Dicer enzyme, a step central to the RNA interference effect on gene expression. These data provide clues to the basis of the toxic effect of CGG-RNA that is thought to occur in fragile X pre-mutation carriers. In addition, RNA hairpins may also account for the stalling of the 40S ribosomal subunit that is thought to contribute to the translation deficit in fragile X pre-mutation and full mutation alleles.     

Sandwich microgravimetric immunoassay: sensitive and specific detection of low molecular weight analytes using piezoelectric quartz crystal

A multi-channel sandwich microgravimetric immunoassay (sMIA), using the quartz crystal microbalance (QCM) principle, has been developed to quantify low molecular weight substances in standard solutions. An antigen is sandwiched between two antigen-specific antibodies: the first antibody is coated on the quartz crystal surface and the second antibody is used for the detection of analyte. The concentration of low molecular weight antigen (insulin was used in this study, M _r6000 Da) was correlated with the shift of resonant frequency of QCM system before and after second antibody binding to insulin. The developed assay is highly specific showing low cross-reactivity, and is sensitive to approx. 1 ng insulin ml^–1 with a linear response for insulin from 10 g ml^–1 to 10 ng ml^–1 in standard solutions. The technique may also be applied for the detection of other small biomolecules.     

Simple sequence repeat (SSR) markers linked to the Ligon lintless (Li(1)) mutant in cotton

Ligon lintless (Li(1)) is a monogenic, dominant mutant in cotton, whose expression results in extreme reductions in fiber length on mature seed. The objectives of this research were to compare fiber initiation between the Li(1) mutant and TM-1 to reveal the fiber initiation differences between normal and mutant phenotypes, to develop a linkage map of simple sequence repeat (SSR) markers with the Li(1) locus, and to identify the chromosomal location of the Li(1) locus. Comparative scanning electron microscopy studies of fiber development in a normal TM-1 genotype and the near-isogenic Li(1) mutant at 1 and 3 days postanthesis revealed little differences between the two during early stages of development, suggesting that Li(1) gene expression occurs later, probably during the elongation phase. Thirty-eight SSR loci were found to be polymorphic between TM-1 and Li(1) and were used for mapping in an F(2) population. Twenty-two SSR loci, along with Li(1), were located on eight linkage groups, covering a total genetic distance of 218.3 cM. Analysis of individual monosomic and monotelodisomic plants indicated that two SSR loci (MP4030 and MP673) from the Li(1) linkage group were located on chromosome 22.