Synthesis and properties of halogenated 7-deaza-2'-deoxyxanthosine and protected derivatives for oligonucleotide synthesis

The 7-bromo- (4a) and 7-iodo- (4b) derivatives of 7-deaza-2'-deoxyxanthosine (5) are prepared. Furthermore, the building blocks 6-8 of 7-deaza-2'-deoxyxanthosine (5) are synthesized and tested for their usage in oligonucleotide synthesis.     

Fast 3D motif search of EM density maps using a locally normalized cross-correlation function

Three-dimensional motif search is becoming increasingly important both in the search for molecular signatures within a tomographic reconstruction, at low resolution, and in the search for atomic structures within high-resolution cryo-EM maps of macromolecular complexes. The present work describes the implementation of a fast local correlation algorithm suitable for template matching in the SPIDER environment. Two examples are given, one in each of the areas of application: (i). within a 7.8A single-particle reconstruction of the Escherichia coli ribosome, four proteins and one RNA structure were located with high accuracy; (ii). within a cryo-tomogram of sarcoplasmic reticulum vesicles, ryanodine receptors were located in positions that agreed with expert knowledge.     

Monounsaturated PE does not phase-separate from the lipid raft molecules sphingomyelin and cholesterol: role for polyunsaturation?

We investigated interactions of the lipid raft molecules sphingomyelin (SM) and cholesterol (CHOL) in monolayers and bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylethanolamine (POPE) or 1-palmitoyl-2-docosahexaenoyl-sn-glycerophosphatidylethanolamine (PDPE) at 35 degrees C. Techniques employed were pressure-area (pi-A) isotherms generated from Langmuir-Blodgett films, solid-state (2)H and (31)P NMR spectroscopies, and differential scanning calorimetry (DSC). Condensation calculated from pi-A isotherms and reduction in the enthalpy of the gel-liquid-crystalline transition in DSC scans showed CHOL has a strong affinity for POPE, comparable to that observed between SM-CHOL. Order parameters derived from (2)H NMR spectra of the perdeuterated sn-1 chain of POPE-d(31) increased by >50% upon addition of equimolar CHOL to POPE-d(31)/SM (1:1 mol) bilayers. Close proximity of CHOL to POPE even in the presence of SM is indicated. Chemical shift anisotropy (Deltasigma(csa)) measured from (1)H-decoupled (31)P NMR spectra also implied intimate lipid mixing in POPE/SM/CHOL (1:1:1 mol). In contrast, pi-A isotherms and corroborating DSC studies of PDPE/SM (1:1 mol) indicate phase separation between SM and PDPE, which was maintained in the presence of CHOL. The cholesterol-associated increase in order of the perdeuterated sn-1 chain of PDPE determined by (2)H NMR was 2-fold less for PDPE-d(31)/SM/CHOL (1:1:1 mol) than POPE-d(31)/SM/CHOL (1:1:1 mol). Our findings support the notion that acyl chain dependent lateral phase separation occurs in the presence of a docosahexaenoic acid (DHA)-containing phospholipid (PDPE), but not an oleic acid-containing phospholipid (POPE). We propose that monounsaturated lipids do not promote formation of stable lipid rafts and that polyunsaturation may be important for raft stability.     

Short-term administration of conjugated linoleic acid reduces liver triglyceride concentration and phosphatidate phosphohydrolase activity in OLETF rats

The present study explored the short-term effects of dietary conjugated-linoleic acid (CLA) on liver lipid metabolism in starved/refed Otsuka Long Evans Tokushima Fatty (OLETF) rats. Male OLETF rats (12 weeks old) were starved for 24 hours, then refed for 48 hours with either a CLA diet [7.5% CLA and 7.5% Safflower oil (SAF)] or a SAF control diet (15% SAF). The results demonstrated a 30% reduction of hepatic triglyceride (TG) concentration in the CLA group when compared to the control group. Liver cholesterol concentration was also 26% lower in the CLA fed rats. The activity of mitochondrial carnitine palmitoyltransferase, the rate-limiting enzyme of fatty acid oxidation, was moderately elevated by 1.2-fold in the livers of the CLA group when compared to the control. In contrast, phosphatidate phosphohydrolase, the rate-limiting enzyme for TG synthesis, was found to be 20% lower in the livers of the CLA-fed rats. Therefore, dietary CLA evidently lowers liver lipid concentrations through a reduced TG synthesis and enhanced fatty acid oxidation in starved/refed OLETF rats.     

Molecular systematics and paleobiogeography of the South American sigmodontine rodents [published erratum appears in Mol Biol Evol 1998 Feb;15(2):224]

The murid rodent subfamily Sigmodontinae contains 79 genera which are distributed throughout the New World. The time of arrival of the first sigmodontines in South America and the estimated divergence time(s) of the different lineages of South American sigmodontines have been controversial due to the lack of a good fossil record and the immense number of extant species. The "early-arrival hypothesis" states that the sigmodontines must have arrived in South America no later than the early Miocene, at least 20 MYA, in order to account for their vast present-day diversity, whereas the "late-arrival hypothesis" includes the sigmodontines as part of the Plio-Pleistocene Great American Interchange, which occurred approximately 3.5 MYA. The phylogenetic relationships among 33 of these genera were reconstructed using mitochondrial DNA (mtDNA) sequence data from the ND3, ND4L, arginine tRNA, and ND4 genes, which we show to be evolving at the same rate. A molecular clock was calibrated for these genes using published fossil dates, and the genetic distances were estimated from the DNA sequences in this study. The molecular clock was used to estimate the dates of the South American sigmodontine origin and the main sigmodontine radiation in order to evaluate the "early-" and "late-arrival" scenarios. We estimate the time of the sigmodontine invasion of South America as between approximately 5 and 9 MYA, supporting neither of the scenarios but suggesting two possible models in which the invading lineage was either (1) ancestral to the oryzomyines, akodonts, and phyllotines or (2) ancestral to the akodonts and phyllotines and accompanied by the oryzomyines. The sigmodontine invasion of South America provides an example of the advantage afforded to a lineage by the fortuitous invasion of a previously unexploited habitat, in this case an entire continent.      

Chitinolytic enzymes: their contribution to basic and applied research

After cellulose, chitin is the second most abundant renewable resource available in nature. Marine invertebrates and fungal biomass are the two main sources of chitinous waste, which is commercially exploited. The enzymes involved in chitin degradation have been particularly well studied. Such enzymes have applications in ultrastructural studies, in the preparation of chitooligosaccharides which show anti-tumour activity, as biocontrol agents and in single-cell protein production. Here, the contribution chitin enzymology can make to basic and applied research is discussed.     

Prostaglandins E and F in uterine venous plasma in relation to peripheral plasma levels of progesterone and 20α-hydroxyprogesterone in the rat throughout pregnancy and parturition

Prostaglandins E and F in uterine venous plasma and progesterone (P) and 20α-hydroxyprogesterone (20α-OH-P) in peripheral plasma were measured by radioimmunoassays throughout pregnancy and parturition in the rat. E Prostaglandins are low (approx. 2 ng/ml) and maintain a more or less constant level throughout most of the pregnancy except just before parturition when they rise to 3.8 ng/ml on day 20. F Prostaglandin levels are always higher than E prostaglandins and show distinct peaks around day 5 (5 ng/ml), day 11 (7 ng/ml), and before parturition (8.4 ng/ml).Progesterone levels are higher than 20α-OH-P levels throughout most of the pregnancy (day 6–20); however, during early pregnancy (day 1–5) and before parturition more 20α-OH-P than P is present in peripheral blood.The possible role of uterine venous prostaglandin levels in altering the 20α-OH-P/P ratio during pregnancy and parturition is discussed.     

Cross-reactivity of metallothioneins from different origins with rabbit anti-rat hepatic metallothione in antibody.

Rat hepatic Cd-metallothionein was purified and isolated into its two components, metallothionein 1 and 2, by disc electrophoresis. Antibodies to metallothionein 2 were generated in rabbits. The antiserum reacted with the protein and formed a single precipitin band on a double diffusion plate. By ammonium sulfate precipitations, it was found that the antiserum cross-reacted with rat hepatic metallothionein 1. Cross-reactivity of the antiserum was also observed for components of rat renal Cd-metallothionein, rabbit hepatic Cd-metallothionein and human renal metallothionein.