A novel function for Sam68: enhancement of HIV-1 RNA 3' end processing

Both cis elements and host cell proteins can significantly affect HIV-1 RNA processing and viral gene expression. Previously, we determined that the exon splicing silencer (ESS3) within the terminal exon of HIV-1 not only reduces use of the adjacent 3' splice site but also prevents Rev-induced export of the unspliced viral RNA to the cytoplasm. In this report, we demonstrate that loss of unspliced viral RNA export is correlated with the inhibition of 3' end processing by the ESS3. Furthermore, we find that the host factor Sam68, a stimulator of HIV-1 protein expression, is able to reverse the block to viral RNA export mediated by the ESS3. The reversal is associated with a stimulation of 3' end processing of the unspliced viral RNA. Our findings identify a novel activity for the ESS3 and Sam68 in regulating HIV-1 RNA polyadenylation. Furthermore, the observations provide an explanation for how Sam68, an exclusively nuclear protein, modulates cytoplasmic utilization of the affected RNAs. Our finding that Sam68 is also able to enhance 3' end processing of a heterologous RNA raises the possibility that it may play a similar role in regulating host gene expression.     

Complementary expression patterns of retinoid acid-synthesizing and -metabolizing enzymes in pre-natal mouse inner ear structures

Retinoic acid (RA) plays a pivotal role in patterning and differentiation of the embryonic inner ear. Despite its documented effects during embryonic development, the cellular sites that synthesize or metabolize RA in the inner ear have yet to be determined. Here we describe the distribution of three synthesizing enzymes, retinaldehyde dehydrogenases 1, 2 and 3 (RALDH1, RALDH2 and RALDH3) and two catabolizing enzymes (CYP26A1 and CYP26B1) in the mouse inner ear at embryonic day 18.5 when active cell differentiation is underway. Two detection methods, radioactive and non-radioactive in situ hybridization, were employed to elucidate the tissue distribution and cellular localization of these enzymes, respectively. All of the five enzymes examined, with the exception of CYP26A1, were expressed in both vestibular and cochlear end organs. While expression of the three RALDHs was observed in various cell types, CYP26B1 expression was found only in supporting cells of the vestibular and cochlear end organs. In the cochlea, expression domains of RALDH1-3 and CYP26B1 were complementary to one another. These results reveal specific tissue- and cellular expression patterns of RA synthesizing and catabolizing enzymes in the pre-natal inner ear, and suggest that a precise control of RA concentrations in various cell types of the inner ear is achieved by the balance between RALDHs and CYP26B1 activities.     

Identification of the sea urchin 350-kDa sperm-binding protein as a new sialic acid-binding lectin that belongs to the heat shock protein 110 family: implication of its binding to gangliosides in sperm lipid rafts in fertilization

The 350-kDa sperm-binding protein (SBP), a species-specific sperm-binding protein, is localized in the vitelline layer of sea urchin eggs. In this study, we have shown for the first time that sperm gangliosides are ligands for the intact glycosylated SBP. Using recombinant fragments of the SBP, the N-terminal heat shock protein 110-like domain was shown to be responsible for the binding. The intact SBP could bind various gangliosides, and the binding was sialidase-sensitive and inhibited by sialyllactose, thus indicating that it is the sialic acid-binding protein. Calcium and magnesium ions were not required but they did enhance the binding activity of SBP. The observation that bacterially expressed recombinant SBP and the sialidase-treated intact glycosylated SBP lost divalent cation-dependent enhancement of binding activity suggests that the sialylated carbohydrate moieties of the SBP may be involved in this property. Furthermore, the SBP was shown to bind sperm lipid rafts, in which gangliosides are enriched, and this binding was lost upon sialidase treatment of the lipid rafts. Finally, liposomes containing the ganglioside specifically inhibited fertilization. Taken together, these results allow us to identify SBP as a member of a new class of sialic acid-binding lectin belonging to the Hsp110 family, and indicate that SBP may be involved in interaction of sperm with the vitelline layer of the egg.     

Treponemal phospholipids inhibit innate immune responses induced by pathogen-associated molecular patterns

Host innate immune responses to microbial components, known as pathogen-associated molecular patterns (PAMPs), are regulated and modified by cellular receptors and serum proteins, including Toll-like receptors (TLRs), CD14, and LPS-binding protein (LBP). We demonstrated that a treponemal membrane lipid inhibited PAMPs-induced immune responses. The chemical structure of the lipid was elucidated as a phosphatidylglycerol (PG) derivative, which is scarce in most mammalian tissues, but relatively abundant in treponemal membrane lipids. Natural and synthetic PG counterparts as well as related natural anionic phospholipids, phosphatidylinositol, phosphatidylserine, and cardiolipin, also demonstrated an inhibitory effect. Further, we noted that PG inhibited PAMPs-induced immune responses by blocking the binding of PAMPs with LBP and CD14. In addition, PG decreased proinflammatory cytokine production in serum of LPS-injected mice and depressed abscess formation in mice infected with treponemes. These results suggest that treponemal phospholipid interfere the function of LBP/CD14 and act as a modulator of innate immune responses.     

Preparation of recombinant rat eosinophil-associated ribonuclease-1 and -2 and analysis of their biological activities

Rat eosinophils contain eosinophil-associated ribonucleases (Ears) in their granules. Ears are thought to be synthesized as pre-forms and stored in the granules as mature forms. However, the N-terminal amino acid of mature Ear-1 and Ear-2 is still controversial. Therefore, we prepared two recombinant mature forms of Ear-1 and Ear-2 in which the N-terminal amino acids are Ser24 (S) [Ear-1 (S) and Ear-2 (S)] and Gln26 (Q) [Ear-1 (Q) and Ear-2 (Q)], and analyzed their biological activities by comparing them with those of pre-form Ear-1 and pre-form Ear-2. The four mature Ears showed RNase A activity as well as bovine pancreatic RNase A activity, but pre-Ear-1 and pre-Ear-2 showed no RNase A activity. Mature Ear-1 (Q) and mature Ear-2 (Q) showed more potent RNase A activity than mature Ear-1 (S) and mature Ear-2 (S), respectively. The RNase A activities of mature Ear-1 (Q) and mature Ear-2 (Q) were reduced by treatment at 96 degrees C for 20 min or with RNase inhibitor. The growth of Escherichia coli was inhibited by both pre-Ears and mature Ears in a concentration-dependent manner, and was almost completely suppressed at 1.0 microM. The bactericidal activities of mature Ear-1 (Q) and mature Ear-2 (Q) were not inhibited by RNase inhibitor, but was increased by treatment at 96 degrees C for 20 min.     

Chemical structure and immunobiological activity of lipid A from Prevotella intermedia ATCC 25611 lipopolysaccharide

The novel chemical structure and immunobiological activities of Prevotella intermedia ATCC 25611 lipid A were investigated. A lipopolysaccharide (LPS) preparation of P. intermedia was extracted using a phenol-chloroform-petroleum ether method, after which its purified lipid A was prepared by weak acid hydrolysis followed by chromatographic separations. The lipid A structure was determined by mass spectrometry and nuclear magnetic resonance to be a diglucosamine backbone with a phosphate at the 4-position of the non-reducing side sugar, as well as five fatty acids containing branched long chains. It was similar to that of Bacteroides fragilis and Porphyromonas gingivalis, except for the phosphorylation site. P. intermedia lipid A induced weaker cytokine production and NF-kappaB activation in murine cells via Toll-like receptor (TLR) 4 as compared to Escherichia coli synthetic lipid A (compound 506). Our results indicate that P. intermedia lipid A activates cells through a TLR4-dependent pathway similar to E. coli-type lipid A, even though these have structural differences.     

Telomerase inhibitors--oligonucleotide phosphoramidates as potential therapeutic agents

We have designed, synthesized, and evaluated using physical, chemical and biochemical assays various oligonucleotide N3'-->P5' phosphoramidates, as potential telomerase inhibitors. Among the prepared compounds were 2'-deoxy, 2'-hydroxy, 2'-methoxy, 2'-ribo-fluoro, and 2'-arabino-fluoro oligonucleotide phosphoramidates, as well as novel N3'-->P5' thio-phosphoramidates. The compounds demonstrated sequence specific and dose dependent activity with IC50 values in the sub-nM to pM concentration range.     

Structural and functional mutations of the perlecan gene cause Schwartz-Jampel syndrome, with myotonic myopathy and chondrodysplasia

Perlecan, a large heparan sulfate proteoglycan, is a component of the basement membrane and other extracellular matrices and has been implicated in multiple biological functions. Mutations in the perlecan gene (HSPG2) cause two classes of skeletal disorders: the relatively mild Schwartz-Jampel syndrome (SJS) and severe neonatal lethal dyssegmental dysplasia, Silverman-Handmaker type (DDSH). SJS is an autosomal recessive skeletal dysplasia characterized by varying degrees of myotonia and chondrodysplasia, and patients with SJS survive. The molecular mechanism underlying the chondrodystrophic myotonia phenotype of SJS is unknown. In the present report, we identify five different mutations that resulted in various forms of perlecan in three unrelated patients with SJS. Heterozygous mutations in two patients with SJS either produced truncated perlecan that lacked domain V or significantly reduced levels of wild-type perlecan. The third patient had a homozygous 7-kb deletion that resulted in reduced amounts of nearly full-length perlecan. Unlike DDSH, the SJS mutations result in different forms of perlecan in reduced levels that are secreted to the extracellular matrix and are likely partially functional. These findings suggest that perlecan has an important role in neuromuscular function and cartilage formation, and they define the molecular basis involved in the difference in the phenotypic severity between DDSH and SJS.     

Comparison of heat- and pressure-induced unfolding of ribonuclease a: the critical role of Phe46 which appears to belong to a new hydrophobic chain-folding initiation site

To clarify the structural role of Phe46 inside the hydrophobic core of bovine pancreatic ribonuclease A (RNase A), thermal and pressure unfolding of wild-type RNase A and three mutant forms (F46V, F46E, and F46K) were analyzed by fourth-derivative UV absorbance spectroscopy. All the mutants, as well as the wild type, exhibited a two-state transition during both thermal and pressure unfolding, and both T(m) and P(m) decreased markedly when Phe46 was replaced with valine, glutamic acid, or lysine. The strongest effect was on the F46K mutant and the weakest on F46V. Both unfolding processes produced identical blue shifts in the fourth-derivative spectra, indicating that the tyrosine residues are similarly exposed in the temperature- and pressure-induced unfolded states. A comparison of Gibbs free energies determined from the pressure and temperature unfoldings, however, gave DeltaG(p)/DeltaG(t) ratios (r) of 1.7 for the wild type and 0.92 +/- 0.03 for the mutants. Furthermore, the DeltaV value for each mutant was larger than that for the wild type. CD spectra and activity measurements showed no obvious major structural differences in the folded state, indicating that the structures of the Phe46 mutants and wild type differ in the unfolded state. We propose a model in which Phe46 stabilizes the hydrophobic core at the boundary between two structural domains. Mutation of Phe46 decreases protein stability by weakening the unfolding cooperativity between these domains. This essential function of Phe46 in RNase A stability indicates that it belongs to a chain-folding initiation site.     

Activation of microsomal epoxide hydrolase by interaction with cytochromes P450: kinetic analysis of the association and substrate-specific activation of epoxide hydrolase function

The kinetics of the association between cytochrome P450 (P450) and microsomal epoxide hydrolase (mEH) was studied by means of resonant mirror based on the principle of surface plasmon resonance. The dissociation equilibrium constants (K(D)) for the affinity of P450 enzymes for mEH were estimated by resonant mirror using an optical biosensor cell covalently bound to rat mEH. Comparable K(D) values were obtained for CYP1A1 and 2B1, and these were greater by one order of magnitude than that for the CYP2C11. To clarify the influences of P450 enzymes on the catalytic activity of mEH, the hydrolyzing activity for styrene oxide and benzo(a)pyrene-7,8-oxide [B(a)P-oxide] was analyzed in the presence or absence of P450s. Styrene oxide hydrolysis was activated by all P450s including the CYP1A, 2B, 2C, and 3A subfamilies. In agreement with the association affinity determined by resonant mirror, CYP2C11 tends to have enhanced activity for styrene oxide hydrolysis. On the other hand, B(a)P-oxide hydrolysis was enhanced by only CYP2C11 while CYP1A1 and CYP2B1 had no effect. These results suggest that (1) many P450 enzymes associate nonspecifically with mEH, (2) the CYP2C11 plays a greater role in the association/activation of mEH and (3) the P450-mediated activation of mEH depends upon the substrate of mEH.