Conformational properties of oxytocin in dimethyl sulfoxide solution: NMR and restrained molecular dynamics studies.

The conformation of oxytocin, the neurohypophyseal nonapeptide hormone, in solution in deuterated dimethyl sulfoxide has been determined by 1H-nmr. The structural determination is based on the experimental data set of nuclear Overhauser effect restraints. Obtained after the restrained molecular dynamics simulation on an initial structure of extended conformation, five resultant structures satisfy the experimental restraints well. These structures resemble that of the crystal structure of deamino-oxytocin, an analogue of oxytocin, in terms of a close correlation observed both at two beta-turn regions of the 20-membered tocin ring and at the tripeptide tail end. Based on this comparison and analysis of restrained molecular dynamics trajectories, we found that, although the turns are stabilized by the formation of hydrogen bonds, the oxytocin molecule possesses a slight twist in DMSO solution relative to the orientation of deamino-oxytocin in the crystalline state. Analyses of oxytocin conformation indicate that the tripeptide tail is more flexible than the tocin ring.     

Early stages of formation and dispersal of the temperate flora in the Boreal Region

Budantsev, L. Yu. (Komarov Botanical Institute, Prof. Popov Str. 2, 197376 St. Petersburg, Russia). Early stages of formation and dispersal of the temperate flora in the Boreal Region. Bot. Rev.58(1): 1–48, 1992.—The thesis of this review is that, as stated as early as 1908 by V. L. Komarov, the composition of a flora can be understood only as a process, or separate stage, in the context of migration in time and space of various floristic assemblages and their isolation, as induced by transformation of continental and ocean shapes, changes in climate, and the environment as a whole. Thus the formation of geofloras of the past was influenced by gradually changing environments that determined the spread, patterning, and spatial differentiation of floras and their evolution. Parallel to the more commonly-seen names of eras—Paleozoic, Mesozoic, and Cenozoic—we can speak of the Paleophytic, Mesophytic, and Cenophytic eras. Eras defined in these two ways (by faunistic or by floristic criteria) do not completely coincide. Generally, changes in the flora have, necessarily, preceded changes in the fauna. It is the Cenophytic with which this review is mostly concerned, the era of Angiosperm dominance. The movement of early subtropical and warm temperate floras in the Early Cenophytic, followed by temperate or even boreal floras, as the climate changes, is traced in detail. The regions discussed most fully are the Boreal-Atlantic and Boreal-Pacific, with emphasis on the Angaro-Beringian flora. The disappearance of archaic forms (e.g., cycadophytes) and the gradual predominance of angiosperms is documented. The movements of the floral assemblages in response to environmental changes are mapped and described. The early development and diversification of the boreal temperate flora is considered to have taken place mainly in Angaro-Beringia, associated with the invasive migration of tropical angiosperms from southeastern Asia.     

Methylation effects on the microdomain structures of phosphatidylethanolamine monolayers.

Increasing methylation of the headgroup in DPPE results in an increase of minimum area per molecule in highly compressed monolayers at the air-water interface. The shape of solid domains, as observed by epifluorescence microscopy, also exhibits marked changes upon increasing headgroup methylation. Branching domains are observed in DPPE and DP(Me)PE, whereas U-shaped or round domains are observed in DP(Me)2PE and DPPC under our experimental conditions. The domain shape is determined more by the headgroup methylatin than by the corresponding shift in critical temperatures, as shown by the study of PCs of different acyl chain moieties. In mixed lipid monolayers, PC (phosphatidylcholine) and PE (phosphatidylethanolamine) do not mix ideally, as indicated by the non-linear variation of the average area per molecule with composition, and by distinct domain shapes in LE/LC (liquid expanded/liquid condensed) coexisting phases representing PE-enriched or PC-enriched domains in those mixed monolayers.     

Initiation of replication in the Chinese hamster dihydrofolate reductase domain

Two-dimensional (2-D) gel analysis of replication intermediates in the Chinese hamster dihydrofolate reductase domain has suggested that nascent chains can initiate at any of a large number of sites scattered throughout a ～50 kb “initiation locus” (although the level of initiation detected at any given site within this region was relatively low). This result contrasts markedly with data from anin vitro strand switching assay suggesting that >80% of initiations occur within a single 500 bp fragment lying within the initiation locus. In an effort to reconcile these two disparate views of the initiation reaction, we have questioned the validity of our 2-D gel data in several ways. We show here that: 1) the number of replication bubbles detected in the DHFR locus in the early S period is markedly increased when the cells are released from a synchronizing agent that inhibits initiationper se, rather than from aphidicolin, which is a chain elongation inhibitor; 2) initiation in the DHFR domain occurs only during the first 90 min of the S period, as would be expected of an early-firing origin; 3) a pulse of³H-thymidine moves through the structures observed on 2-D gels with the kinetics expected ofbonafide replication intermediates; and 4) preparations of replication intermediates that are subsequently analyzed on 2-D gels appear, by electron microscopy, to represent the typical theta structures and single-forked molecules expected of bidirectional origins of replication; no unusual structures (e.g., microbubbles) were seen.     

Murine p53 inhibits the function but not the formation of SV40 T antigen hexamers and stimulates T antigen RNA helicase activity

We have characterized the effects of p53 on several biochemical activities of simian virus 40 (SV40) large tumor (T) antigen. While p53 induced a strong inhibition of the T antigen DNA helicase activity, surprisingly, its RNA helicase activity was stimulated. This supports the liklihood that the DNA and RNA helicase activities of T antigen reflect discrete functions. p53 did not significantly affect the ATP-dependent conversion of T antigen monomers to hexamers. However, the ability of these hexamers to assemble on a DNA fragment containing the viral origin was impaired by p53. Thus, these results suggest that p53 inhibits the function but not the formation of T antigen multimers. This conclusion was further supported by the observation that the addition of a purified p53:T antigen complex was as inhihitory as free p53 to the DNA helicase activity of free T antigen. Thus our data indicates that the targets of p53 inhibition are the functional units of T antigen, namely the hexamers.     

The 3′→5′ exonuclease associated with HeLa DNA polymerase epsilon

The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity.     

Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. Purification and stereochemical analysis of CDP-D-glucose oxidoreductase.

An NAD(+)-dependent CDP-D-glucose oxidoreductase which catalyzes the first step of the biosynthesis of CDP-ascarylose (CDP-3,6-dideoxy-L-arabino-hexose), converting CDP-D-glucose to CDP-4-keto-6-deoxy-D-glucose, was isolated from Yersinia pseudotuberculosis. A protocol consisting of DEAE-cellulose, Matrex Blue-A, hydroxylapatite, DEAE-Sephadex, Sephadex G-100, and NAD(+)-agarose column chromatography was used to purify this enzyme 6000-fold to homogeneity. This enzyme consists of two identical subunits, each with a molecular weight of 42,500. Using CDP-D-glucose as the substrate, the Km and Vmax of this catalysis were determined to be 222 microM and 8.3 mumols mg-1 min-1, respectively. Unlike most other oxidoreductases of its class which have a tightly bound NAD+, this highly purified CDP-D-glucose oxidoreductase showed an absolute requirement of NAD+ for its activity. Using chemically synthesized (6S)- and (6R)-CDP-D-[4-2H,6-3H]glucose as substrates, a stereochemical analysis showed this enzymatic reaction involves an intramolecular hydrogen migration from C-4 to C-6, and the displacement of C-6 hydroxyl group by the C-4 hydrogen occurs with inversion. Thus, despite the low cofactor affinity, this enzyme undergoes a mechanism consistent with that followed by other members of its type. Such a mechanistic and stereochemical convergency found for all sugar oxidoreductases so far characterized suggests the presence of a common progenitor of this class of enzyme.     

Molecular basis for the species selectivity of the neurokinin-1 receptor antagonists CP-96,345 and RP67580.

Two non-peptide substance P antagonists exhibit opposite rank orders of potency for the human and rat neurokinin-1 receptors. CP-96,345 shows selectivity for the human receptor, whereas RP67580 shows selectivity for the rat receptor. Amino acid sequence comparison of the two receptors reveals 22 divergent residues. To elucidate the molecular basis for the species selectivity of these antagonists, divergent residues in the human neurokinin-1 receptor were substituted by the rat homologs. Analysis of mutant receptors revealed that substitution of 2 residues (V116L and I290S) in the transmembrane domain of the human neurokinin-1 receptor is both necessary and sufficient to reproduce the antagonist binding affinities of the rat receptor. The nature of these substitutions and the magnitude of the changes in binding affinity suggest that residues 116 and 290 do not interact directly with the antagonist molecules. The present results support a model in which phylogenetically conserved residues interact directly with the antagonists, while phylogenetically divergent residues affect the local helical packing of the receptor. Such a change in local structure would lead to increased binding affinity for one class of antagonists and decreased affinity for another.     

The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions.

Accumulating evidence suggests that the matrix (MA) protein of retroviruses plays a key role in virus assembly by directing the intracellular transport and membrane association of the Gag polyprotein. In this report, we show that the MA protein of human immunodeficiency virus type 1 is also critical for the incorporation of viral Env proteins into mature virions. Several deletions introduced in the MA domain (p17) of human immunodeficiency virus type 1 Gag polyprotein did not greatly affect the synthesis and processing of the Gag polyprotein or the formation of virions. Analysis of the viral proteins revealed normal levels of Gag and Pol proteins in these mutant virions, but the Env proteins, gp120 and gp41, were hardly detectable in the mutant virions. Our data suggest that an interaction between the viral Env protein and the MA domain of the Gag polyprotein is required for the selective incorporation of Env proteins during virus assembly. Such an interaction appears to be very sensitive to conformational changes in the MA domain, as five small deletions in two separate regions of p17 equally inhibited viral Env protein incorporation. Mutant viruses were not infectious in T cells. When mutant and wild-type DNAs were cotransfected into T cells, the replication of wild-type virus was also hindered. These results suggest that the incorporation of viral Env protein is a critical step for replication of retroviruses and can be a target for the design of antiviral strategies.