Conformationally Restricted Nucleocyclitols: a Study into their Conformational Preferences and Supramolecular Architecture in the Solid State

With the intent of probing the feasibility of employing annulation as a tactic to engender axial rich conformations in nucleoside analogues, two adenine-derived, ``conformationally restricted'' nucleocylitols, 9 and 10, have been conceptualized as representatives of a hitherto unexplored class of nucleic acid base-cyclitol hybrids. A general synthetic strategy, with an inherent scope for diversification, allowed rapid functionalization of indane and tetralin to furnish 9 and 10 respectively in fair yield. Single-crystal X-ray diffraction analysis revealed that the two nucleocyclitols under study, though homologous, present completely dissimilar modes of molecular packing, marked, in particular, by the nature of involvement of the adenynyl NH2 group in the supramolecular assembly. In addition, the crystal structures of 9 and 10 also exhibit two different conformations of the functionalized cyclohexane ring. Thus, while the six-membered carbocycle in cyclopenta-annulated 9 exists in the expected chair (C) conformation that in cyclohexaannulated 10, which crystallizes as a dihydrate, shows an unusual twist-boat (TB) conformation. From a close analysis of the (HNMR)-H-1 spectroscopic data recorded for 9 and 10 in CD3OD, it was possible to put forth a putative explanation for the uncanny conformational preferences of crystalline 9 and 10.

Positional Preferences of Polypurine/Polypyrimidine Tracts in Saccharomyces cerevisiae Genome: Implications for cis Regulation of Gene Expression

The complete genome of the baker's yeast S. cerevisiae was analyzed for the presence of polypurine/polypyrimidine (poly[pu/py]) repeats and their occurrences were classified on the basis of their location within and outside open reading frames (ORFs). The analysis reveals that such sequence motifs are present abundantly both in coding as well as noncoding regions. Clear positional preferences are seen when these tracts occur in noncoding regions. These motifs appear to occur predominantly at a unit nucleosomal length both upstream and downstream of ORFs. Moreover, there is a biased distribution of polypurines in the coding strands when these motifs occur within open reading frames. The significance of the biased distribution is discussed with reference to the occurrence of these motifs in other known mRNA sequences and expressed sequence tags. A model for cis regulation of gene expression is proposed based on the ability of these motifs to form an intermolecular triple helix structure when present within the coding region and/or to modulate nucleosome positioning via enhanced histone affinity when present outside coding regions.

Amino acid interaction preferences in proteins

Understanding the key factors that influence the interaction preferences of amino acids in the folding of proteins have remained a challenge. Here we present a knowledge-based approach for determining the effective interactions between amino acids based on amino acid type, their secondary structure, and the contact based environment that they find themselves in the native state structure as measured by their number of neighbors. We find that the optimal information is approximately encoded in a 60 x 60 matrix describing the 20 types of amino acids in three distinct secondary structures (helix, beta strand, and loop). We carry out a clustering scheme to understand the similarity between these interactions and to elucidate a nonredundant set. We demonstrate that the inferred energy parameters can be used for assessing the fit of a given sequence into a putative native state structure.

Discovering user preferences by using time entries in click-through data to improve search engine results

The search engine log files have been used to gather direct user feedback on the relevancy of the documents presented in the results page. Typically the relative position of the clicks gathered from the log files is used a proxy for the direct user feedback. In this paper we identify reasons for the incompleteness of the relative position of clicks for deciphering the user preferences. Hence, we propose the use of time spent by the user in reading through the document as indicative of user preference for a document with respect to a query. Also, we identify the issues involved in using the time measure and propose means to address them.

Expression of GC-C, a receptor-guanylate cyclase, and its endogenous ligands uroguanylin and guanylin along the rostrocaudal axis of the intestine

Members of the receptor-guanylate cyclase (rGC) family possess an intracellular catalytic domain that is regulated by an extracellular receptor domain. GC-C, an intestinally expressed rGC, was initially cloned by homology as an orphan receptor. The search for its Ligands has yielded three candidates: STa (a bacterial toxin that causes traveler's diarrhea) and the endogenous peptides uroguanylin and guanylin. Here, by performing Northern and Western blots, and by measuring [I-125]STa binding and STa-dependent elevation of cGMP levels, we investigate whether the distribution of GC-C matches that of its endogenous ligands in the rat intestine. We establish that 1) uroguanylin is essentially restricted to small bowel; 2) guanylin is very low in proximal small bowel, increasing to prominent levels in distal small bowel and throughout colon; 3) GC-C messenger RNA and STa-binding sites are uniformly expressed throughout the intestine; and 4) GC-C-mediated cGMP synthesis peaks at the proximal and distal extremes of the intestine (duodenum and colon), but is nearly absent in the middle (ileum). These observations suggest that GC-C's activity may be posttranslationally regulated, demonstrate that the distribution of GC-C is appropriate to mediate the actions of both uroguanylin and guanylin, and help to refine current hypotheses about the physiological role(s) of these peptides.

A Ca2+-sensitive cyclic-3',5'-nucleotide phosphodiesterase from sheep lung modulated by a tightly bound endogenous calmodulin.

Highly purified sheep lung cyclic-3',5'-nucleotide phosphodiesterase was sensitive to Ca2+/EGTA but insensitive to exogenous calmodulin. The Ca2+-sensitivity was inhibited by trifluoperazine. Heat-treated enzyme could activate a calmodulin-deficient phosphodiesterase, suggesting the presence of endogenous calmodulin in sheep lung cyclic-3',5'-nucleotide phosphodiesterase, possibly associated with the enzyme in a Ca2+-independent manner.

Effect of Neutralization of Endogenous Follicle Stimulating Hormone (FSH) or Luteinizing Hormone (LH) on Ovarian Lipids in the Hamster: A Histochemical and Biochemical Evaluation

The effect of neutralizing FSH or LH on ovarian lipids in the cycling hamster was studied. In the normal cycling hamster on the day of proestrus, histochemical examination revealed the presence of sudanophilic lipids in the granulosa cells of the follicles and in the interstitium. A clear reduction in the intensity of lipid staining was observed on proestrus in the ovary of hamsters treated with FSH antiserum on the previous proestrus. Similar treatment with antiserum to LH, on the other hand, caused an accumulation of lipids in these structures. Estimation of the free and esterified fractions of cholesterol and triglycerides in the nonluteal tissue of the ovary of hamsters on proestrus following treatment with FSH antiserum on the previous proestrus revealed a significant reduction in all 3 lipid components. Even a short term deprivation of FSH caused a similar reduction in these lipids in the ovary. In contrast, treatment with LH antiserum either on the previous proestrus or on the previous day (diestrus-2) resulted in an enhancement in esterified cholesterol and triglycerides, while it caused a reduction in the free cholesterol fraction of the ovary on proestrus.It is suggested that though treatment with antisera to either FSH or LH causes a disruption in follicular maturation, their effect on lipid metabolism is different. A positive role for FSH and LH in maintaining normal sterol and triglyceride levels in the nonluteal ovarian tissue of cycling hamster is indicated.

Effect of altering endogenous gonadotrophin concentrations on the kinetics of testicular germ cell turnover in the bonnet monkey (Macaca radiata)

The role of FSH and diurnal testosterone rhythms in specific germ cell transformations during spermatogenesis were investigated using DNA flow cytometry and morphometry of the seminiferous epithelium of the adult male bonnet monkey (Macaca radiata), the endogenous hormone levels of which were altered by two different protocols. (1) Active immunization of five monkeys for 290 days using ovine FSH adsorbed on Alhydrogel resulted in the neutralization of endogenous FSH, leaving the LH and diurnal testosterone rhythms normal. (2) Desensitization of the pituitary gonadotrophs of ten monkeys by chronically infusing gonadotrophin-releasing hormone analogue, buserelin (50 micrograms/day release rate), via an Alzet pump implant (s.c.) led to a 60-80% reduction in LH and FSH as well as total abolition of testosterone rhythms. The basal testosterone level (3.3 +/- 2.0 micrograms/l), however, was maintained in this group by way of an s.c. testosterone silicone elastomer implant. Both of the treatments caused significant (P < 0.01) nearly identical reduction in testicular biopsy scores, mitotic indices and daily sperm production rates compared with respective controls. The germ cell DNA flow cytometric profiles of the two treatment groups, however, were fundamentally different from each other. The pituitary-desensitized group exhibited a significant (P < 0.001) increase in 2C (spermatogonial) and decrease in 1C (round spermatid) populations while S-phase (preleptotene spermatocytes) and 4C (primary spermatocytes) populations were normal, indicating an arrest in meiosis caused presumably by the lack of increment in nocturnal serum testosterone. In contrast, in the FSH-immunized group, at day 80 when the FSH deprivation was total, the primary block appeared to be at the conversion of spermatogonia (2C) to cells in S-phase and primary spermatocytes (4C reduced by > 90%). In addition, at this time, although the round spermatid (1C) population was reduced by 65% (P < 0.01) the elongate spermatid (HC) population showed an increase of 52% (P < 0.05). This, taken together with the fact that sperm output in the ejaculate is reduced by 80%, suggests a blockade in spermiogenesis and spermiation. Administration of booster injections of oFSH at time-points at which the antibody titre was markedly low (at days 84 and 180) resulted in a transient resurgence in spermatogenesis (at day 180 and 228), and this again was blocked by day 290 when the FSH antibody titre increased.

Comparative studies on the effects of specific immunoneutralization of endogenous FSH or LH on testicular germ cell transformations in the adult bonnet monkey (Macaca radiata)

PROBLEM: It is yet to be determined clearly whether the two hormones FSH and T act synergistically in the same cell type-the Sertoli cells-to control overall spermatogenesis or influence independently the transformation of specific germ cell types during spermatogenesis in the adult mammal. METHOD: Adult male bonnet monkeys specifically deprived of either FSH or LH using immunoneutralization techniques were monitored for changes in testicular germ cell transformation by DNA flow cytometry. RESULTS: FSH deprivation caused a significant reduction (>40%; P < 0.05) in [H-3] thymidine incorporation into DNA of proliferating 2C (spermatogonial) cells, a marked inhibition (>50%) in the transformation of 2C to primary spermatocytes (4C) and a concomitant, belated reduction (50%) in the formation of round spermatids (1C). In contrast, specific LH/T deprivation led to an immediate arrest in the meiotic transformation of 4C to 1C/HC leading to an effective and significant block (<90%; P < 0.01) in sperm production. CONCLUSION: Thus, LH rather than FSH deprivation has a more pronounced and immediate effect as the former primarily blocks meiosis (4C --> 1C/HC) which controls production of spermatids. These data provide evidence for LH/T and FSH regulating spermatogenic process in the adult primate by primarily acting at specific germ cell transformation steps.

Conformational preferences of α-functionalised keten-S,N-acetals: Potential role of SO and SS interactions in solution

PMR spectra of carbonyl compounds 2a-k reveal significant variations in the population of E and Z isomers on changing the solvent from CDCl3 to DMSO-d6. In non-polar media, the intramolecular N-H…. O hydrogen bonded form is exclusively observed. In DMSO-d6, the alternative Z form is also populated. A similar conformational switch is also noted in the corresponding thiones. Different interpretations are critically analysed. The most consistent explanation is suggested to involvean interplay of N-H….X hydrogen bonding and S…X attractive interaction (X = O,S) in these systems. Ab initio calculations support this interpretation.

Susceptibility of sperm chromatin to acid denaturation in situ: A study in endogenous FSH-deprived adult male bonnet monkeys (Macaca radiata)

Acid denaturation of calf thymus DNA in vitro followed by acridine orange (AO) binding induced a 112% increase in the emission of red, a 58% decrease in green, and a consequential decrease in the ratio of green:red fluorescences from 1.7 to 0.9. This metachromatic property of AO on binding to DNA following acid denaturation was utilized to study the susceptibility of normal and ovine follicle-stimulating hormone (oFSH) actively immunized bonnet monkey spermatozoa voided throughout the year. For analyses, the scattergram generated by the emission of red and green fluorescences by 10,000 AO-bound sperm from each semen sample was divided into 4 quadrant zones representing percentage cells fluorescing high green-low red (Q1), high green-high red (Q2), low green-low red (Q3) and low green-high red. (Q4). Normal monkey sperm obtained during the months of July-December exhibited 76, 13, and 11% cells in Q2, Q3, and Q4 quadrants, respectively. However, during January-June, when the females of the species are markedly subfertile, noncycling, and amenorrhoeic, the spermatozoa ejaculated by the male monkeys exhibited 38, 39, and 23% sperm in Q2, Q3, and Q4, respectively, the differences being highly significant (p < .01-.001). FSH deprivation induced significant shifts in fluorescence emissions, from respective controls, with 39, 33, and 28% cells in Q2, Q3, and Q4, respectively, during July-December, and 15, 48, and 37% sperm in Q2, Q3, and Q4 quadrants, respectively, during January-June. It is postulated that the altered kinetics of germ cell transformations and the deficient spermiogenesis observed earlier following FSH deprivation in these monkeys may have induced the enhanced susceptibility to acid denaturation in sperm.

Amino acid interaction preferences in helical membrane proteins

Membrane proteins are involved in a number of important biological functions. Yet, they are poorly understood from the structure and folding point of view. The external environment being drastically different from that of globular proteins, the intra-protein interactions in membrane proteins are also expected to be different. Hence, statistical potentials representing the features of inter-residue interactions based exclusively on the structures of membrane proteins are much needed. Currently, a reasonable number of structures are available, making it possible to undertake such an analysis on membrane proteins. In this study we have examined the inter-residue interaction propensities of amino acids in the membrane spanning regions of the alpha-helical membrane (HM) proteins. Recently we have shown that valuable information can be obtained on globular proteins by the evaluation of the pair-wise interactions of amino acids by classifying them into different structural environments, based on factors such as the secondary structure or the number of contacts that a residue can make. Here we have explored the possible ways of classifying the intra-protein environment of HM proteins and have developed scoring functions based on different classification schemes. On evaluation of different schemes, we find that the scheme which classifies amino acids to different intra-contact environment is the most promising one. Based on this classification scheme, we also redefine the hydrophobicity scale of amino acids in HM proteins.