Broadly distributed chemical reactivity of natural antibodies expressed in coordination with specific antigen binding activity

Antibody (Ab) nucleophilic reactivity was studied using hapten and polypeptide antigens containing biotinylated phosphonate diester groups (covalently reactive antigen analogs, CRAs). Polyclonal IgG from healthy donors formed covalent adducts with a positively charged hapten CRA at levels superior to trypsin. Each of the 16 single chain Fv clones studied expressed a similar reactivity, indicating the V domain location of the nucleophiles and their broad distribution in diverse Abs. The formation of hapten CRA-Fv adducts was correlated with Fv proteolytic activity determined by cleavage of a model peptide substrate. Despite excellent nucleophilicity, proteolysis by IgG proceeded at lower rates than trypsin, suggesting that events occurring after nucleophilic attack on the substrate limit the rate of Ab proteolysis. The extracellular domain of the epidermal growth factor receptor with phosphonate diester groups at Lys side chains and a synthetic peptide corresponding to residues 421- 431 of human immunodeficiency virus glycoprotein (gp) 120 with the phosphonate diester at the C terminus formed covalent adducts with specific polyclonal and monoclonal Abs raised by immunization with epidermal growth factor receptor and synthetic gp120-(421- 436) devoid of phosphonate diester groups, respectively. Adduct formation was inhibited by extracellular domain of the epidermal growth factor receptor (exEGFB) and synthetic gp120-(421- 436) devoid of phosphonate groups, suggesting that the nucleophiles are located within the antigen binding sites. These results suggest the innate character of the Ab nucleophilic reactivity, its functional coordination with non-covalent adaptive binding interactions developing over the course of B cell maturation, and novel routes toward permanent inhibition of Abs.     

The GCN2 eIF2alpha kinase is required for adaptation to amino acid deprivation in mice

The GCN2 eIF2alpha kinase is essential for activation of the general amino acid control pathway in yeast when one or more amino acids become limiting for growth. GCN2's function in mammals is unknown, but must differ, since mammals, unlike yeast, can synthesize only half of the standard 20 amino acids. To investigate the function of mammalian GCN2, we have generated a Gcn2(-/-) knockout strain of mice. Gcn2(-/-) mice are viable, fertile, and exhibit no phenotypic abnormalities under standard growth conditions. However, prenatal and neonatal mortalities are significantly increased in Gcn2(-/-) mice whose mothers were reared on leucine-, tryptophan-, or glycine-deficient diets during gestation. Leucine deprivation produced the most pronounced effect, with a 63% reduction in the expected number of viable neonatal mice. Cultured embryonic stem cells derived from Gcn2(-/-) mice failed to show the normal induction of eIF2alpha phosphorylation in cells deprived of leucine. To assess the biochemical effects of the loss of GCN2 in the whole animal, liver perfusion experiments were conducted. Histidine limitation in the presence of histidinol induced a twofold increase in the phosphorylation of eIF2alpha and a concomitant reduction in eIF2B activity in perfused livers from wild-type mice, but no changes in livers from Gcn2(-/-) mice.     

The photoperiodic entrainment and induction of the circadian clock regulating seasonal responses in the migratory blackheaded bunting (Emberiza melanocephala)

The properties of the circadian photoperiodic oscillator have been investigated in detail only in the Japanese quail. While the study of the quail is clearly very important, one cannot simply assume that other species, especially passerines that seem to have a different circadian organization than quail, function the same way. The current set of experiments was conducted to understand the entrainment and photoinduction of the circadian photoperiodic oscillator in a passerine species, the blackheaded bunting (Emberiza melanocephala). The experimental paradigm used skeleton photoperiods with two light periods, the first called the “entraining light pulse” (E-pulse) and the second called the “inducing light pulse” (I-pulse). Three experiments were performed on photosensitive male birds (N=6-8/group). Experiment 1 investigated the effects of the temporal relationship between E- and I-pulses on photoperiodic induction. Buntings entrained to 8h:16h L:D for 4 wk were released into constant dim light (LL_dim, ∼1 lux). Beginning on subjective day 8, they received for 8 wk, E- and I-pulses only at alternate cycles. While I-pulse was 1 h and always began at zt 11.5, E-pulse varied in duration and timing (the 1h E-pulse beginning either at zt 0, zt 5, or zt 9, the 4h one beginning at zt 0 or zt 6, and the 10h one at zt 0; zeitgeber time 0=time of lights-on under 8h:16h L:D prior to release into LL_dim). A photoperiodic response was induced only when the E-pulse began at zt 0, and thus the beginning of E- and I-pulses were separated by 11.5 h. Experiment 2 determined whether the duration of the E-pulse influences the position of the photoinducible phase (φi) of the circadian photoperiodic oscillator. Birds were entrained to 1h:23h L:D or 10h:14h L:D for 2 wk, and then exposed to 1h I-pulse at zt 11.5, zt 15, or zt 18.5 for another 8 wk. Photoperiodic induction occurred at all 3 zts in birds entrained to 10 h but only at zt 11.5 in birds entrained to 1 h, which infers the circadian rhythm of photoinducibility (CRP) in buntings was re-entrained when I-pulse fell at zt 15 and after. The last experiment examined the possibility of the re-entrainment of the CRP to light pulses falling at zt 15 and after. Birds received 1h I-pulse for 8 wk at zt 15 following 2 wk of 2.5h:21.5h L:D or 3.5h:20.5h L:D, or at zt 21.5 or zt 22.5 following 2 wk of 10h:14h LD. Photoperiodic induction was consistent with the hypothesis of the re-entrainment of the CRP under these light-dark cycles. The I-pulse appeared to be interpreted as a “new dawn”, and so the photoperiodic induction was determined by the coincidence of φi with the E-pulse. These results suggest a phase-dependent action of light on the circadian oscillator regulating photoperiodic responses in the blackheaded bunting. This could be a useful strategy for a photoperiodic species to regulate its seasonal responses in nature.     

Murine coronavirus replication-induced p38 mitogen-activated protein kinase activation promotes interleukin-6 production and virus replication in cultured cells

Analyses of mitogen-activated protein kinases (MAPKs) in a mouse hepatitis virus (MHV)-infected macrophage-derived J774.1 cell line showed activation of two MAPKs, p38 MAPK and c-Jun N-terminal kinase (JNK), but not of extracellular signal-regulated kinase (ERK). Activation of MAPKs was evident by 6 h postinfection. However, UV-irradiated MHV failed to activate MAPKs, which demonstrated that MHV replication was necessary for their activation. Several other MHV-permissive cell lines also showed activation of both p38 MAPK and JNK, which indicated that the MHV-induced stress-kinase activation was not restricted to any particular cell type. The upstream kinase responsible for activating MHV-induced p38 MAPK was the MAPK kinase 3. Experiments with a specific inhibitor of p38 MAPK, SB 203580, demonstrated that MHV-induced p38 MAPK activation resulted in the accumulation of interleukin-6 (IL-6) mRNAs and an increase in the production of IL-6, regardless of MHV-induced general host protein synthesis inhibition. Furthermore, MHV production was suppressed in SB 203580-treated cells, demonstrating that activated p38 MAPK played a role in MHV replication. The reduced MHV production in SB 203580-treated cells was, at least in part, due to a decrease in virus-specific protein synthesis and virus-specific mRNA accumulation. Interestingly, there was a transient increase in the amount of phosphorylation of the translation initiation factor 4E (eIF4E) in infected cells, and this eIF4E phosphorylation was p38 MAPK dependent; it is known that phosphorylated eIF4E enhances translation rates of cap-containing mRNAs. Furthermore, the upstream kinase responsible for eIF4E phosphorylation, MAPK-interacting kinase 1, was also phosphorylated and activated in response to MHV infection. Our data suggested that host cells, in response to MHV replication, activated p38 MAPK, which subsequently phosphorylated eIF4E to efficiently translate certain host proteins, including IL-6, during virus-induced severe host protein synthesis inhibition. MHV utilized this p38 MAPK-dependent increase in eIF4E phosphorylation to promote virus-specific protein synthesis and subsequent progeny virus production. Enhancement of virus-specific protein synthesis through virus-induced eIF4E activation has not been reported in any other viruses.     

Bacterial superantigen exposure after resolution of influenza virus infection perturbs the virus-specific memory CD8(+)-T-cell repertoire

Heterologous viral infections have been shown to impact the preexisting memory CD8(+)-T-cell repertoire. Bacterial superantigens are products of common human pathogenic bacteria, including staphylococci and streptococci, that are potent T-cell-stimulatory molecules. In this report, we show that exposure to staphylococcal enterotoxin B, a bacterial superantigen, causes a selective functional deletion of cross-reactive influenza virus-specific CD8(+) memory T cells. This perturbation of the memory repertoire can have a significant impact on viral clearance after secondary challenge.     

Amino acid sequence of the 26 kDa subunit of legumin-type seed storage protein of common buckwheat (Fagopyrum esculentum Moench): molecular characterization and phylogenetic analysis

The paper describes the amino acid sequence of a 26 kDa basic subunit of 13S globulin of common buckwheat (Fagopyrum esculentum Moench). The protein has 93 and 75% sequence homology with 11S globulin of Coffea arabica and beta subunit of 11S globulin of Cucurbita pepo respectively. The subunit has the "globally conserved" N-terminal sequence consisting of Gly-Ile-Asp-Glu and the cysteine at P7' from the proteolytic processing site. A conserved 7 residue domain of Pro-His-Trp-Asn-Ile-Asn-Ala, characteristic of basic subunits of legumins from non-leguminous angiosperms, is also present in this protein. A distinguishing features of this subunit is the relatively high level of lysine and methionine.     

Distribution of Candida species in different clinical sources in Delhi, India, and proteinase and phospholipase activity of Candida albicans isolates

Eighty-five isolates of Candida recovered from three hundred and fifty diverse clinical sources, viz. respiratory tract (sputum, bronchial washing,bronchoalveolar lavage, tracheal aspirate), blood, urine, high vaginal swab, skin and plastic devices, were studied in detail for their morphological and biochemical characters. Seven species of Candida were identified, viz., C. albicans (45.8%), C. tropicalis (24.7%), C. parapsilosis (10.5%), C. krusei (7.0%), C. kefyr (7.0%), C. guilliermondii (3.5%), and C. glabrata (1.1%). C. albicans was the predominant species isolated from all clinical specimens, except blood from which C. krusei was most frequently (38.4%) recovered. Out of 39 isolates of C. albicans, 26 (66.6%) and 19 (48.7%) exhibited strong proteinase and phospholipase activity respectively. There was a higher prevalence of proteinase producing strains amongst the vaginal and skin isolates than that in urinary and respiratory isolates. Also a greater number of phospholipase producing strains was observed in the vaginal and urinary isolates than that in the respiratory and skin isolates.     

Biocontrol of collar rot disease of betelvine (Piper betle L.) caused by Sclerotium rolfsii by using rhizosphere-competent Pseudomonas fluorescens NBRI-N6 and P. fluorescens NBRI-N

Collar rot disease of betelvine (Piper betle L.) caused by Sclerotium rolfsii is difficult to control by conventional means by use of chemicals; therefore, use of biocontrol agents is desirable. In the present study, 186 bacterial strains of different morphological types were screened for their biocontrol activity against S. rolfsii under in vitro conditions. Two strains, Pseudomonas fluorescens NBRI-N6 and P. fluorescens NBRI-N, were selected for further studies because of their ability to inhibit the mycelial growth of the pathogen significantly. Spontaneous rifampicin-resistant (Rif) derivatives of P. fluorescens NBRI-N6 and P. fluorescens NBRI-N showing growth rate and membrane protein composition comparable to the wild type were selected to facilitate their monitoring in the rhizosphere. Field trials demonstrated that strain P. fluorescens NBRI-N6 was better than P. fluorescens NBRI-N in increasing the yield of betelvine significantly, whereas a consortium of the two strains controlled the disease more than either of the strains. The screening method should prove useful in identifying rhizosphere bacteria with the greatest potential for controlling diseases caused by phytopathogenic fungi.     

Aluminium-induced enhancement of ageing-related biochemical and electrophysiological parameters in rat brain regions

We investigated alterations in the ageing-related parameters: multiple-unit action potentials, Na(+), K(+)-ATPase activity, glutathione-s-transferase (GST) activity, glutathione peroxidase (GPx) activity, lipid peroxidation and lipofuscin contents in the brain regions cerebral cortex, striatum, hippocampus and thalamus, resulting from the chronic administration of aluminium chloride (AlCl3) in drinking water to rats of 6 and 12 months of age. Aluminium treatment significantly depressed Na(+), K(+)-ATPase, GST and GPx activities, elevated lipid peroxidation and lipofuscin contents, and produced intense epileptiform activity in the electroencephalograms of the studied brain regions together with a concomitant increase in the multiple-unit action potentials (MUA) indicating a vigorous neuronal epileptic hyperactivity. Taken together the aluminium-induced alterations in these parameters are indicative of an accelerated ageing process.