NMR structure of stem-loop SL2 of the HIV-1 psi RNA packaging signal reveals a novel A-U-A base-triple platform

The genome of the human immunodeficiency virus type-1 (HIV-1) contains a stretch of approximately 120 nucleotides known as the psi-site that is essential for RNA packaging during virus assembly. These nucleotides have been proposed to form four stem-loops (SL1-SL4) that have both independent and overlapping functions. Stem-loop SL2 is important for efficient recognition and packaging of the full-length, unspliced viral genome, and also contains the major splice-donor site (SD) for mRNA splicing. We have determined the structure of the 19-residue SL2 oligoribonucleotide by heteronuclear NMR methods. The structure is generally consistent with the most recent of two earlier secondary structure predictions, with residues G1-G2-C3-G4 and C6-U7 forming standard Watson Crick base-pairs with self-complementary residues C16-G17-C18-C19 and A12-G13, respectively. However, residue A15, which is located near the center of the stem, does not form a predicted bulge, and residues A5 and U14 do not form an expected Watson-Crick base-pair. Instead, these residues form a novel A5-U14-A15 base-triple that appears to be stabilized by hydrogen bonds from A15-H61 and -H62 to A5-N1 and U14-O2, respectively; from A5-H61 to U14-O2, and from C16-H42 to U14-O2'. A kink in the backbone allows the aromatic rings of the sequential U14-A15 residues to be approximately co-planar, adopting a stable "platform motif" that is structurally similar to the A-A (adenosine) platforms observed in the P4-P6 ribozyme domain of the Tetrahymena group I intron. Platform motifs generally function in RNA by mediating long-range interactions, and it is therefore possible that the A-U-A base-triple platform mediates long-range interactions that either stabilize the psi-RNA or facilitate splicing and/or packaging. Residue G8 of the G8-G9-U10-G11 tetraloop is stacked above the U7-A12 base-pair, and the remaining tetraloop residues are disordered and available for potential interactions with either other RNA or protein components.     

Apparently unstable normal FMR1 alleles in nine developmentally delayed patients: implications for molecular diagnosis of the fragile X syndrome

The Fragile X syndrome is a common form of X-linked mental retardation, affecting approximately 1 in 4,000 males. Since the discovery of the FMR1 gene responsible for the syndrome, molecular, rather than cytogenetic, diagnosis of Fragile X syndrome has become the gold standard. Numerous molecular diagnostic centers worldwide use PCR and Southern blotting to characterize the size of the CGG repeats within the gene, expansion of which has been shown to be associated with the vast majority of cases of Fragile X syndrome. Instability of this repeat through successive generations has been demonstrated in many patients and has been associated with numerous factors, including repeat length and molecular structure of the repeat. Nine males with normal-size alleles that exhibit repeat length instability by the presence of a second normal length distinct band by repeated PCR analysis from peripheral lymphocytes are reported. Many hypotheses addressing the reason for this apparent instability were tested without elucidating the underlying molecular causes, including cytogenetic analysis, sequence analysis of the repeat locus, and analysis of flanking dinucleotide repeat loci. All patients exhibited a normal complement of sex chromosomes by cytogenetic and molecular analysis. These results from the widely used PCR analysis illustrate an interesting molecular phenomenon and raise many questions relating to the factors and mechanisms involved in trinucleotide instability as well as having implications for the diagnostic testing of the Fragile X syndrome.     

Production of a recombinant form of early pregnancy factor that can prolong allogeneic skin graft survival time in rats

Early pregnancy factor (EPF), an extracellular chaperonin 10 homologue, has immunosuppressive and growth factor properties. In order to carry out more extensive studies on the in vivo characteristics of EPF, a recombinant form of the molecule has been prepared. Recombinant human EPF (rEPF) was expressed in Escherichia coli using the plasmid pGEX-2T expression system. Potency of rEPF in vitro in the rosette inhibition test, the bioassay for EPF, was equivalent to that of native EPF (nEPF), purified from human platelets, and synthetic EPF (sEPF). However, the half-life of activity (50% decrease in the log value) in serum, following i.p. injection, was significantly decreased (3.2 h, compared with nEPF 6.2 days, sEPF 5.8 days). This was thought to be due to modification of the N-terminus of the recombinant molecule inhibiting binding to serum carrier proteins. Because EPF can modify Th1 responses, the ability of the recombinant molecule to suppress allogeneic graft rejection was investigated. Following skin grafts from Lewis rats to DA rats and vice versa, rEPF was delivered locally at the graft site and the effect on survival time of the allografts noted. Results demonstrated that rEPF treatment significantly prolonged skin graft survival time by as much as 55% in stringent models of transplantation across major histocompatibility barriers.     

The NMR structure of the nucleocapsid protein from the mouse mammary tumor virus reveals unusual folding of the C-terminal zinc knuckle

The nucleocapsid protein (NC) from the mouse mammary tumor virus (MMTV) has been overexpressed in Escherichia coli and purified to homogeneity for structural studies by nuclear magnetic resonance (NMR) spectroscopy. The protein contains two copies of a conserved zinc-coordinating "CCHC array" or "zinc knuckle" motif common to the nucleocapsid proteins of nearly all known retroviruses. The residues comprising and adjacent to the zinc knuckles were assigned by standard two-dimensional (1)H and three-dimensional (1)H-(15)N NMR methods; the rotational dynamic properties of the protein were determined from (15)N relaxation experiments, and distance restraints derived from the nuclear Overhauser effect (NOE) data were used to calculate the three-dimensional structure. The (1)H-(1)H NOE and (15)N relaxation data indicate that the two zinc knuckles do not interact with each other, but instead behave as independently folded domains connected by a flexible 13-residue linker segment. The proximal zinc knuckle folds in a manner that is essentially identical to that observed previously for the two zinc knuckles of the human immunodeficiency virus type 1 nucleocapsid protein and for the moloney murine leukemia virus nucleocapsid zinc knuckle domain. However, the distal zinc knuckle of MMTV NC exhibits a rare three-dimensional fold that includes an additional C-terminal beta-hairpin. A similar C-terminal reverse turn-like structure was observed recently in the distal zinc knuckle of the Mason-Pfizer monkey virus nucleocapsid protein [Gao, Y., et al. (1998) Protein Sci. 7, 2265-2280]. However, despite a high degree of sequence homology, the conformation and orientation of the beta-hairpin in MMTV NC is significantly different from that of the reverse turn in MPMV NC. The results support the conclusion that structural features of NC zinc knuckle domains can vary significantly among the different genera of retroviridae, and are discussed in terms of the recent and surprising discovery that MMTV NC can facilitate packaging of the HIV-1 genome in chimeric MMTV mutants.     

Low dynamic state of viral competition in a chronic avian hepadnavirus infection

The dynamic state of infection of 11 ducks with the duck hepatitis B virus was investigated. Chronic infections were established in newly hatched ducklings by inoculation with a mixture of wild-type virus and a mutant virus with a partial replication defect. As expected, the wild-type virus was rapidly enriched in the virus population during the spread of infection. Enrichment thereafter was correlated with normal growth of the liver, with the average mutant-to-wild-type ratio stabilizing for at least 2 months beyond the time at which the liver mass stabilized. Using experimentally determined growth rates for the mutant and wild-type viruses, we estimated that after the spread of infection, competition between the two virus strains was limited by the amount of replication required to infect new hepatocytes in the growing livers. The results suggest that, in a chronically infected liver, the selection of variants with a replication rate advantage is inefficient and that the emergence of such variants would depend on induced liver cell turnover, such as that occurring during chronic hepatitis.     

Microbial Characterization of Biological Filters Used for Drinking Water Treatment

The impact of preozonation and filter contact time (depth) on microbial communities was examined in drinking water biofilters treating Ohio River water which had undergone conventional treatment (coagulation, flocculation, sedimentation) or solutions of natural organic matter isolated from groundwater (both ozonated and nonozonated). With respect to filter depth, compared to filters treating nonozonated waters, preozonation of treated water led to greater differences in community phospholipid fatty acid (PLFA) profiles, utilization of sole carbon sources (Biolog), and arbitrarily primed PCR fingerprints. PLFA profiles indicated that there was a shift toward anaerobic bacteria in the communities found in the filter treating ozonated water compared to the communities found in the filter treating nonozonated settled water, which had a greater abundance of eukaryotic markers.     

The breeding productivity of dark-bellied brent geese and curlew sandpipers in relation to changes in the numbers of arctic foxes and lemmings on the Taimyr Peninsula, Siberia —

There were about three-year cycles in the populations of arctic foxes, and the breeding productivities of brent geese and curlew sandpipers on the Taimyr Peninsula, Russia, The populations of arctic foxes and lemmings changed in synchrony. The breeding productivities of the birds tended to be good when the arctic foxes were increasing in numbers and poor when the arctic foxes were decreasing. There was a negative relationship between arctic fox numbers (or occupied lairs) and the breeding productivity of brent geese in the following year. Although there was evidence of wide-spread synchrony In the lemming cycle across the Taimyr Peninsula, some localities showed differences, However, such sites would still have been influenced by the general pattern of fox abundance in the typical tundra zone of the Taimyr Peninsula, where most of the arctic foxes breed and from which extensive movements of foxes occur after a decline in lemming numbers. The results support a prey-switching hypothesis (also known as the alternative prey hypothesis) whereby arctic foxes, and other predators, feed largely on lemmings when these are abundant or increasing, but switch to birds when the lemming population is small or declining. The relationships between arctic foxes, lemmings and brent geese may be further influenced by snowny owls which create fox-exclusion zones around their nests, thus providing safe nesting areas for the geese.     

Effect of hypohydration on gastric emptying and intestinal absorption during exercise

Dehydration and hyperthermia may impair gastricemptying (GE) during exercise; the effect of these alterations onintestinal water flux (WF) is unknown. Thus the purpose of this studywas to determine the effect of hypohydration (~2.7% body weight) on GE and WF of a water placebo (WP) during cycling exercise (85 min, 65%maximal oxygen uptake) in a cool environment (22°C) and to alsocompare GE and WF of three carbohydrate-electrolyte solutions (CES)while the subjects were hypohydrated. GE and WF were determined simultaneously by a nasogastric tube placed in the gastric antrum andvia a multilumen tube that spanned the duodenum and the first 25 cm ofjejunum. Hypohydration was attained 12-16 h before experiments bylow-intensity exercise in a hot (45°C), humid (relative humidity 50%) environment. Seven healthy subjects (age 26.7 ± 1.7 yr,maximal oxygen uptake 55.9 ± 8.2 ml · kg¹ · min¹)ingested either WP or a 6% (330 mosmol), 8% (400 mosmol), or a 9%(590 mosmol) CES the morning following hypohydration. For comparison,subjects ingested WP after a euhydration protocol. Solutions (~2.0liters total) were ingested as a large bolus (4.6 ml/kg body wt) 5 minbefore exercise and as small serial feedings (2.3 ml/kg body wt) every10 min of exercise. Average GE rates were not different amongconditions (P > 0.05). Mean(±SE) values for WF were also similar(P > 0.05) for the euhydration (15.3 ± 1.7 ml · cm¹ · h¹)and hypohydration (18.3 ± 2.6 ml · cm¹ · h¹)experiments. During exercise after hypohydration, waterabsorption was greater (P < 0.05)with ingestion of WP (18.3 ± 2.6) and the 6% CES (16.5 ± 3.7),compared with the 8% CES (6.9 ± 1.5) and the 9% CES (1.8 ± 1.7). Mean values for final core temperature (38.6 ± 0.1°C),heart rate (152 ± 1 beats/min), and change in plasma volume(5.7 ± 0.7%) were similar among experimental trials. Weconclude that 1) hypohydration to~3% body weight does not impair GE or fluid absorption duringmoderate exercise when ingesting WP, and2) hyperosmolality (>400 mosmol)reduced WF in the proximal intestine.

     

Regulation of Insulin-Stimulated Glucose Transporter GLUT4 Translocation and Akt Kinase Activity by Ceramide

The sphingomyelin derivative ceramide is a signaling molecule implicated in numerous physiological events. Recently published reports indicate that ceramide levels are elevated in insulin-responsive tissues of diabetic animals and that agents which trigger ceramide production inhibit insulin signaling. In the present series of studies, the short-chain ceramide analog C₂-ceramide inhibited insulin-stimulated glucose transport by ~50% in 3T3-L1 adipocytes, with similar reductions in hormone-stimulated translocation of the insulin-responsive glucose transporter (GLUT4) and insulin-responsive aminopeptidase. C₂-ceramide also inhibited phosphorylation and activation of Akt, a molecule proposed to mediate multiple insulin-stimulated metabolic events. C₂-ceramide, at concentrations which antagonized activation of both glucose uptake and Akt, had no effect on the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) or the amounts of p85 protein and phosphatidylinositol kinase activity that immunoprecipitated with anti-IRS-1 or antiphosphotyrosine antibodies. Moreover, C₂-ceramide also inhibited stimulation of Akt by platelet-derived growth factor, an event that is IRS-1 independent. C₂-ceramide did not inhibit insulin-stimulated phosphorylation of mitogen-activated protein kinase or pp70 S6-kinase, and it actually stimulated phosphorylation of the latter in the absence of insulin. Various pharmacological agents, including the immunosuppressant rapamycin, the protein synthesis inhibitor cycloheximide, and several protein kinase C inhibitors, were without effect on ceramide’s inhibition of Akt. These studies demonstrate ceramide’s capacity to inhibit activation of Akt and imply that this is a mechanism of antagonism of insulin-dependent physiological events, such as the peripheral activation of glucose transport and the suppression of apoptosis.