Multiligand specificity of pathogen-associated molecular pattern-binding site in peptidoglycan recognition protein

The peptidoglycan recognition protein PGRP-S is an innate immunity molecule that specifically interacts with microbial peptidoglycans and other pathogen-associated molecular patterns. We report here two structures of the unique tetrameric camel PGRP-S (CPGRP-S) complexed with (i) muramyl dipeptide (MDP) at 2.5 Å resolution and (ii) GlcNAc and β-maltose at 1.7Å resolution. The binding studies carried out using surface plasmon resonance indicated that CPGRP-S binds to MDP with a dissociation constant of 10⁻⁷ m, whereas the binding affinities for GlcNAc and β-maltose separately are in the range of 10⁻⁴ m to 10⁻⁵ m, whereas the dissociation constant for the mixture of GlcNAc and maltose was estimated to be 10⁻⁶ m. The data from bacterial suspension culture experiments showed a significant inhibition of the growth of Staphylococcus aureus cells when CPGRP-S was added to culture medium. The ELISA experiment showed that the amount of MDP-induced production of TNF-α and IL-6 decreased considerably after the introduction of CPGRP-S. The crystal structure determinations of (i) a binary complex with MDP and (ii) a ternary complex with GlcNAc and β-maltose revealed that MDP, GlcNAc, and β-maltose bound to CPGRP-S in the ligand binding cleft, which is situated at the interface of molecules C and D of the homotetramer formed by four protein molecules A, B, C, and D. In the binary complex, the muramyl moiety of MDP is observed at the C-D interface, whereas the peptide chain protrudes into the center of tetramer. In the ternary complex, GlcNAc and β-maltose occupy distinct non-overlapping positions belonging to different subsites.     

Paternal reciprocal translocation t(11;16)(p13;q24.3) in a Silver-Russel syndrome patient

We describe a 7-month-old male child with Silver-Russel syndrome (SRS) phenotype, presented with two major clinical features: low birth weight, short stature, and minor features, such as macrocephaly, clinodactyly, essential for the diagnosis of SRS. Routine cytogenetic studies with GTG-banding showed 46,XY,t(11;16)(p13;q24.3). Fluorescence in situ hybridisation (FISH) with single copy probes BAC (11p13) and PAC (16q24.3), showed a reciprocal translocation. Chromosomal analysis of the mother was normal and the phenotypically normal father had apparently identical translocation t(11;16)(p13;q24.3). The disruption of growth factor genes at 11p and 16q breakpoint regions due to reciprocal translocation in the father might have caused SRS phenotype in the child.     

Characterisation of senescence-induced changes in light harvesting complex II and photosystem I complex of thylakoids of Cucumis sativus cotyledons: age induced association of LHCII with photosystem I

Structure and function of chloroplasts are known to after during senescence. The senescence-induced specific changes in light harvesting antenna of photosystem II (PSII) and photosystem I (PSI) were investigated in Cucumis cotyledons. Purified light harvesting complex II (LHCII) and photosystem I complex were isolated from 6-day non-senescing and 27-day senescing Cucumis cotyledons. The chlorophyll a/b ratio of LHCII obtained from 6-day-old control cotyledons and their absorption, chlorophyll a fluorescence emission and the circular dichroism (CD) spectral properties were comparable to the LHCII preparations from other plants such as pea and spinach. The purified LHCII obtained from 27-day senescing cotyledons had a Chl a/b ratio of 1.25 instead of 1.2 as with 6-day LHCII and also exhibited significant changes in the visible CD spectrum compared to that of 6-day LHCII, indicating some specific alterations in the organisation of chlorophylls of LHCII. The light harvesting antenna of photosystems are likely to be altered due to aging. The room temperature absorption spectrum of LHCII obtained from 27-day senescing cotyledons showed changes in the peak positions. Similarly, comparison of 77K chlorophyll a fluorescence emission characteristics of LHCII preparation from senescing cotyledons with that of control showed a small shift in the peak position and the alteration in the emission profile, which is suggestive of possible changes in energy transfer within LHCII chlorophylls. Further, the salt induced aggregation of LHCII samples was lower, resulting in lower yields of LHCII from 27-day cotyledons than from normal cotyledons. Moreover, the PSI preparations of 6-day cotyledons showed Chl a/b ratios of 5 to 5.5, where as the PSI sample of 27-day cotyledons had a Chl a/b ratio of 2.9 suggesting LHCII association with PSI. The absorption, fluorescence emission and visible CD spectral measurements as well as the polypeptide profiles of 27-day cotyledon-PSI complexes indicated age-induced association of LHCII of PSII with PSI obtained from 27-day cotyledons. We modified our isolation protocols by increasing the duration of detergent Triton X-100 treatment for preparing the PSI and LHCII complexes from 27-day cotyledons. However, the PSI complexes isolated from senescing samples invariably proved to have significantly low Chl a/b ratio suggesting an age induced lateral movement and possible association of LHCII with PSI complexes. The analyses of polypeptide compositions of LHCII and PSI holocomplexes isolated from 6-day control and 27-day senescing cotyledons showed distinctive differences in their profiles. The presence of 26-28 kDa polypeptide in PSI complexes from 27-day cotyledons, but not in 6-day control PSI complexes is in agreement with the notion that senescence induced migration of LHCII to stroma lamellae and its possible association with PSI. We suggest that the migration of LHCII to the stroma lamellae region and its possible association with PSI might cause the destacking and flattening of grana structure during senescence of the chloroplasts. Such structural changes in light harvesting antenna are likely to alter energy transfer between two photosystems. The nature of aging induced migration and association of LHCII with PSI and its existence in other senescing systems need to be estimated in the future.     

Aging induced changes in photosynthetic electron transport of detached barley leaves

Primary leaf segments of 11-day-old seedlings of barley (Hordtumvulgare L. cv IB 65) were floated on distilled water in darknessat 25°C to induce senescence. This stress induced agingbrings significant loss in the total content of pigments, proteinsand nucleic acids (DNA, RNA) of the leaves and of chloroplastsisolated from the senescing leaves. Of the three macromolecularcomponents, RNA content of theisolated chloroplasts was foundmost susceptible to stress-induced aging. Loss of DCPIP Hill activity of the isolated chloroplasts couldbe correlated, in a general way, with the loss of pigments,proteins and nucleic acids of the leaves and chloroplasts isolatedfrom them. However, during the stress period, the ability ofdifferent exogenous electron donors like MnCl₂ and diphenylcarbazide(DPC) to feed electrons to Photo System II (PS II) was foundto be different. MnCl₂ supported photoreduction of DCPIP onlyup to the fourth day, whereas DPC sustained its ability to donateelectrons up to the seventh day of incubation of the leavesin darkness. These results suggest a sequential alteration ofthe sites in the electron-transport chain between H₂O and PSII reaction centers of chloroplasts during dark-induced senescence.Kinetin not only prevented the loss of pigments and proteinsduring senescence, but also preserved the integrity of the electron-transportchain. (Received November 15, 1975; )     

High-throughput fluorescent tagging of full-length Arabidopsis gene products in planta

We developed a high-throughput methodology, termed fluorescent tagging of full-length proteins (FTFLP), to analyze expression patterns and subcellular localization of Arabidopsis gene products in planta. Determination of these parameters is a logical first step in functional characterization of the approximately one-third of all known Arabidopsis genes that encode novel proteins of unknown function. Our FTFLP-based approach offers two significant advantages: first, it produces internally-tagged full-length proteins that are likely to exhibit native intracellular localization, and second, it yields information about the tissue specificity of gene expression by the use of native promoters. To demonstrate how FTFLP may be used for characterization of the Arabidopsis proteome, we tagged a series of known proteins with diverse subcellular targeting patterns as well as several proteins with unknown function and unassigned subcellular localization.     

GnRH-II analogs for selective activation and inhibition of non-mammalian and type-II mammalian GnRH receptors

Recently, we identified three types of non-mammalian gonadotropin-releasing hormone receptors (GnRHR) in the bullfrog (designated bfGnRHR-1-3), and a mammalian type-II GnRHR in green monkey cell lines (denoted gmGnRHR-2). All these receptors responded better to GnRH-II than GnRH-I, while mammalian type-I GnRHR showed greater sensitivity to GnRH-I than GnRH-II. In the present study, we designed new GnRH-II analogs and examined whether they activated or inhibited non-mammalian and mammalian type-II GnRHRs. [D-Ala6]GnRH-II, with D-Ala substituted for Gly6 in GnRH-II, increased inositol phosphate (IP) production in cells stably expressing non-mammalian GnRHRs more effectively than native GnRH-II. However, it exhibited lower activity for mammalian type-I GnRHR than GnRH-I itself. Trptorelix-1, a GnRH-II antagonist, inhibited GnRH-induced IP production in cells expressing non-mammalian GnRHRs more effectively than Cetrorelix, a GnRH-I antagonist. Trptorelix-1, however, had lower potency for mammalian type-I GnRHR than Cetrorelix. Ligand-receptor binding assays revealed that [D-Ala6]GnRH-II and Trptorelix-1 have higher affinities for non-mammalian GnRHRs but lower affinities for mammalian type-I GnRHR than GnRH-II and Cetrorelix, respectively. Moreover, [D-Ala6]GnRH-II and Trptorelix-1 had a higher affinity for gmGnRHR-2 than GnRH-II and Cetrorelix, respectively. These results indicate that [D-Ala6]GnRH-II and Trptorelix-1 are highly effective agonist and antagonist, respectively, for non-mammalian and type-II mammalian GnRHRs.     

Compact and ordered collapse of randomly generated RNA sequences

As the raw material for evolution, arbitrary RNA sequences represent the baseline for RNA structure formation and a standard to which evolved structures can be compared. Here, we set out to probe, using physical and chemical methods, the structural properties of RNAs having randomly generated oligonucleotide sequences that were of sufficient length and information content to encode complex, functional folds, yet were unbiased by either genealogical or functional constraints. Typically, these unevolved, nonfunctional RNAs had sequence-specific secondary structure configurations and compact magnesium-dependent conformational states comparable to those of evolved RNA isolates. But unlike evolved sequences, arbitrary sequences were prone to having multiple competing conformations. Thus, for RNAs the size of small ribozymes, natural selection seems necessary to achieve uniquely folding sequences, but not to account for the well-ordered secondary structures and overall compactness observed in nature.     

I n Vivo Evaluation of 5-ASA Colon-Specific Tablets Using Experimental-Induced Colitis Rat Animal Model

Colonic drug delivery is intended not only for local treatment in inflammatory bowel disease (IBD) but also for systemic delivery of therapeutics. Intestinal myeloperoxidase (MPO) determination could be used to estimate the average level of inflammation in colon as well as to determine the efficacy of drugs to be used in the treatment of inflammatory bowel diseases or study the specificity of dosage forms to be used for colonic targeting of anti-inflammatory drugs. Colonic prodrug sulfasalazine (SASP) gets metabolized to give 5-aminosalicylic acid (5-ASA), which is the active portion of SASP. However, when given orally, 5-ASA is absorbed in upper part of gastrointestinal tract (GIT) and not made available in colon. In the present study, colon-targeted delivery of 5-ASA was achieved by formulating tablets with two natural polymers namely guar gum and pectin using compression coating method. Colonic specificity of 5-ASA tablets (prepared using guar gum and pectin as polymers) was evaluated in vitro using simulated fluids mimicking in vivo environment as well as in vivo method using chemically (2,4,6-trinitrobenzenesulfonic acid and acetic acid)-induced colitis rat model. Both colon-specific formulations of 5-ASA (guar gum and pectin) were observed to be more effective in reducing inflammation in chemically induced colitis rat models when compared to colon-specific prodrug sulfasalazine as well as conventional 5-ASA administered orally.KEY WORDS: colitis, colon-specific drug delivery, myeloperoxidase     

Soil microorganisms regulate extracellular enzyme production to maximize their growth rate

Biogeochemistry - Soil carbon cycling and ecosystem functioning can strongly depend on how microbial communities regulate their metabolism and adapt to changing environmental conditions to improve...