Missense mutations in the beta-myosin heavy chain (beta-MHC) gene cause hypertrophic cardiomyopathy (HCM). As normal and mutant beta-MHCs are expressed in slow-twitch skeletal muscle of HCM patients, we compared the contractile properties of single slow-twitch muscle fibers from patients with three distinct beta-MHC gene mutations and normal controls. Fibers with the 741Gly->>Arg mutation (near the binding site of essential light chain) demonstrated decreased maximum velocity of shortening (39% of normal) and decreased isometric force generation (42% of normal). Fibers with the 403Arg->>Gln mutation (at the actin interface of myosin) showed lowered force/stiffness ratio (56% of normal) and depressed velocity of shortening (50% of normal). Both the 741Gly->>Arg and 403Arg->>Gln mutation-containing fibers displayed abnormal force-velocity relationships and reduced power output. Fibers with the 256Gly->>Glu mutation (end of ATP-binding pocket) had contractile properties that were indistinguishable from normal. Thus there is variability in the nature and extent of functional impairments in skeletal fibers containing different beta-MHC gene mutations, which may correlate with the severity and penetrance of the disease that results from each mutation. These functional alterations likely constitute the primary stimulus for the cardiac hypertrophy that is characteristic of this disease.

Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm⁵U₃₄), 5-methoxycarbonylmethyluridine (mcm⁵U₃₄), and 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U₃₄) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNA(Lys)(s²UUU), tRNA(Gln)(s²UUG), and tRNA(Glu)(s²UUC), which in a wild-type background contain the mcm⁵s²U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U₃₄. Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm⁵s²U nucleoside in tRNA(Lys)(mcm⁵s²UUU), tRNA(Gln)(mcm⁵s²UUG), and tRNA(Glu)(mcm⁵s²UUC). These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U₃₄ are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants.

Using the techniques of gap misrepair mutagenesis and site-directed mutagenesis, we have generated two novel quinolone resistance mutations of the Escherichia coli DNA gyrase A protein. DNA sequencing showed these mutations to be Ser-83----Ala and Gln-106----Arg. The mutant proteins were overproduced and purified, and their enzymatic properties were analyzed and compared with those of the wild-type enzyme. With ciprofloxacin and other quinolones, the inhibition of DNA supercoiling, relaxation, and decatenation and the induction of DNA cleavage were investigated for both wild-type and mutant enzymes. In each assay, the mutant enzymes were found to require approximately 10 times more drug to inhibit the reaction or induce cleavage than was the wild-type enzyme. However, the Ca2(+)-directed DNA cleavage reaction was indistinguishable for wild-type and mutant gyrases. We discuss models for the gyrase-mediated bactericidal effects of quinolone drugs.

We have isolated a gene that can encode yeast tRNA(CAGGln). When present on a multicopy plasmid, this gene suppresses the phenotype of a number of amber mutants, but has no effect on the ocher mutants tested. We therefore conclude that the anticodon CUG in tRNA(CAGGln) can decode the amber codon UAG by G-U mispairing, possibly by wobble base-pairing in the first codon position. This represents the second example we have observed in this laboratory of nonsense suppression in yeast by natural tRNA(Gln), involving G-U mispairing in the first codon position. Replacing the genomic copy of the cloned gene with a disrupted tRNA gene results in recessive lethality in heterozygous diploids and is lethal to haploid cells. This lethality can be rescued by transformation of cells with a single copy plasmid containing the tRNA(CAGGln) gene. Thus, the gene encoding tRNA(CAGGln) is apparently essential for viability in yeast, suggesting that it is normally present as a single copy gene.

The lung cancer mortality rate in Xuan Wei County is among the highest in China and has been attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NER) plays a key role in reversing DNA damage from exposure to environmental carcinogens, such as PAHs, that form bulky DNA adducts. We studied single nucleotide polymorphisms (SNPs) and their corresponding haplotypes in 6 genes (ERCC1, ERCC2/XPD, ERCC4/XPF, ERCC5/XPG, RAD23B and XPC) involved in NER in a population-based case-control study of lung cancer in Xuan Wei. A total of 122 incident primary lung cancer cases and 122 individually matched controls were enrolled. Three linked SNPs in ERCC2 were associated with lung cancer with similar ORs; e.g., persons with the Gln allele at codon 751 had a 60% reduction of lung cancer (OR = 0.40, 95% CI 0.18-0.89). Moreover, one haplotype in ERCC2 was associated with a decreased risk of lung cancer (OR = 0.40, 95% CI 0.19-0.85) compared to the most common haplotype. In addition, subjects with one or 2 copies of the Val allele at codon 249 of RAD23B had a 2-fold increased risk of lung cancer (OR = 1.91, 95% CI 1.12-3.24). In summary, our results suggest that genetic variants in genes involved in the NER pathway may play a role in lung cancer susceptibility in Xuan Wei. However, due to the small sample size, additional studies are needed to evaluate these associations within Xuan Wei and in other populations with substantial environmental exposure to PAHs.

A murine model of genital infection with a thymidine kinase-deficient (tk-) strain of herpes simplex virus type 2 (HSV-2) was utilized to examine the development of the local T cell response in the genital mucosa and draining genital lymph nodes (gLN). HSV-specific cytokine-secreting T cells were detected in the gLN 4 days postintravaginal inoculation but not in the urogenital tract or spleen until 5 days postinoculation, suggesting the cellular immune response originates in the gLN. More CD4+ than CD8+ gLN T cells were detected by flow cytometric analysis following primary vaginal inoculation and the majority of HSV-specific gLN T cells detected by ELISPOT were CD4+ and Th1-like based on secretion of IFN gamma and not IL-4 or IL-5. A similar population of HSV-specific memory T cells persisted in the genital tract 2 months following HSV-2 tk- genital inoculation. These data suggest that the urogenital cellular immune response elicited in mice following genital inoculation with HSV-2 tk- is predominantly CD4+ and Th1-like, resembling that observed in humans. The results of this study are important for the rational design of vaccines capable of inducing protective immunity in the genital tract.

To determine the effects of different amino acids on the structure and stability of an alpha-helix in the context of a globular protein, all 19 naturally-occurring amino acids were substituted for Ser44 in phage T4 lysozyme. A more restricted set of nine replacements was also made for Val131. Ser44 and Val131 are two of a very limited number of possible sites in T4 lysozyme that are well within alpha-helices, are solvent-exposed and relatively free of interactions with neighboring residues, and are not involved in crystal contacts. High resolution structures for the majority of the mutants, some of which crystallized non-isomorphously with wild-type, were determined. With the exception of proline, the amino acid substitutions caused little if any perturbation of the alpha-helix backbone. Also the beta-branched residues Thr, Val and Ile show no indication of either side-chain or backbone distortion. Therefore, other than proline, there is no evidence that differences in helix propensities are associated with different amounts of strain introduced into the helix. For reference, and also to allow estimates of side-chain entropy, a survey was made of side-chain conformations in 100 well-refined protein structures. As noted previously all side-chains within alpha-helices strongly avoid the g- conformation (chi 1 approximately 60 degrees). This restricts the beta-branched residues Thr, Val and Ile to a single conformer (g+, chi 1 approximately -60 degrees). Asp, Asn, Met and Ser within helices also overwhelmingly prefer the g+ conformation. For Arg, Cys, Gln, Glu, Leu and Lys the t (chi 1 approximately 180 degrees) and g+ conformers are populated roughly equally. Only the aromatic residues, His, Tyr, Trp and Phe prefer the t conformation. These preferences are the same whether the side-chain is buried or solvent-exposed. In general, the side-chain conformations adopted by the residues substituted at positions 44 and 131 correspond to the most commonly observed conformation for the same amino acid in helices in known protein structures. The changes in protein stability for the replacements at site 131 in general agree well with those at site 44 (correlation r = 0.97), suggesting that these may be representative of substitutions at fully solvent-exposed sites in the middle of alpha-helices. The free energy values also agree quite well with those observed for equivalent replacements in a number of soluble alpha-helical model peptides and with data from "host-guest" studies and statistical surveys (r = 0.69 to 0.93).(ABSTRACT TRUNCATED AT 400 WORDS)

In response to epidermal injury, Parasilurus asotus, a catfish, secreted a strong antimicrobial peptide into the epithelial mucosal layer. The molecular mass of the antimicrobial peptide, named parasin I, was 2000.4 Da, as determined by matrix-associated laser desorption ionization mass spectrometry. The complete amino acid sequence of parasin I, which was determined by automated Edman degradation, was Lys-Gly-Arg-Gly-Lys-Gln-Gly-Gly-Lys-Val-Arg-Ala-Lys-Ala-Lys-Thr-Arg-Ser- Ser. Eighteen of the 19 residues in parasin I were identical to the N-terminal of buforin I, a 39-residue antimicrobial peptide derived from the N-terminal of toad histone H2A [Kim et al. (1996) Biochem. Biophys. Res. Commun. 229, 381-387], which implies that parasin I was cleaved off from the N-terminal of catfish histone H2A. Parasin I showed strong antimicrobial activity, about 12-100 times more potent than magainin 2, against a wide spectrum of microorganisms, without any hemolytic activity. Circular dichroism spectra of parasin I indicated a structural content of 11% alpha-helix, 33% beta-sheet, and 56% random coils. The beta-sheet axial projection diagram of parasin I showed an amphipathic structure. Our results indicate that the catfish may produce parasin I from its histone H2A by a specific protease upon injury to protect against invasion by microorganisms.

The crystal structure of the glutamine-binding protein (<em>Gln</em>BP) complexed with its ligand (<em>Gln</em>) was determined and refined to 1.94 A resolution. This ellipsoidal protein has two globular domains and is approximately 52 Ax40 Ax35 A in size. The glutamine ligand is located in the cleft between the two domains and stablized by hydrogen bondings and ionic interactions with Asp10, Gly68, Thr70, Ala67, Asp157, Arg75, Lys115, Gly119 and His156. The aliphatic portion of the glutamine ligand is sandwiched in a hydrophobic pocket formed between Phe13 and Phe50 and has 21 van der Waals contacts with <em>Gln</em>BP. Lys115 and His156, that are unique to <em>Gln</em>BP among amino acid binding proteins, apparently contribute to the ligand binding specificity of <em>Gln</em>BP. Asp10 is within 3 A of Lys115. These two residues are over 10 A apart in the ligand-free form of the <em>Gln</em>BP. In addition, <em>Gln</em>BP-<em>Gln</em> exhibits a large-scale movement of the two hinges connecting the two globular domains upon ligand binding. The most significant changes are 41.1 degrees in the phi angle of Gly89 and 34.3 degrees in the psi angle of Glu181 from the first and the second hinge of the protein, respectively. Besides the original six hydrogen bonds, three extra hydrogen bonds can be observed between the two hinge strands upon ligand binding. A hydrogen bond network connects the large domain to the second hinge and a second hydrogen bond network coalesces the small domain to the same strand, both via interaction with the glutamine ligand. Although the two strands of the hinge connecting the domains do not directly participate in the ligand binding, <em>Gln</em>183 and Tyr185 from the second hinge may be involved in the cascade of the conformational change that is induced by ligand binding.

The substrate recognition determinants of Ca2+-calmodulin-dependent protein kinase (CaMK) IV and CaMKIIalpha were investigated using peptide substrates modeled on the amino acid sequence encompassing Ser-9 of synapsin I. For both kinases, hydrophobic residues (Leu or Phe) at the -5 position, are well tolerated, whereas non-hydrophobic residues (Arg, Ala, or Asp) decrease Vmax/Km by 55- to >4000-fold. At the -3 position, substitution of Ala for Arg leads to decreases of 99- and 343- fold in Vmax/Km for CaMKIV and CaMKIIalpha, respectively. For both kinases, the nature of the residues occupying the -4, -1, and + 4 positions exerts relatively little influence on phosphorylation kinetics. CaMKIV and CaMKIIalpha respond differently to substitutions at the -2 and +1 positions. Substitution of Arg at the -2 position with non-basic residues (Gln or Ala) leads to 6-fold decreases in Vmax/Km for CaMKIV, but 17-28-fold increases for CaMKIIalpha. Additionally, peptides containing Leu, Asp, or Ala at the +1 position are phosphorylated with similar efficiencies by CaMKIV, whereas the Leu-substituted peptide is preferred by CaMKIIalpha (by a factor of 5.8-9.7-fold). Thus, CaMKIV and CaMKIIalpha preferentially phosphorylate substrates with the motifs: Hyd-X-Arg-X-X-Ser*/Thr*, and Hyd-X-Arg-NB-X-Ser*/Thr*-Hyd, respectively, where Hyd represents a hydrophobic, X any, and NB a non-basic amino acid residue. The different specificities of the two kinases may contribute to their targeting to distinct physiological substrates during Ca2+-dependent cellular events.

Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.

The importance of amino acid side-chains in helix stability has been investigated by making a series of mutations at the N-caps, C-caps and internal positions of the solvent-exposed faces of the two alpha-helices of barnase. There is a strong positional and context dependence of the effect of a particular amino acid on stability. Correlations have been found that provide insight into the physical basis of helix stabilization. The relative effects of Ala and Gly (or Ser) may be rationalized on the basis of solvent-accessible surface areas: burial of hydrophobic surface stabilizes the protein as does exposure to solvent of unpaired hydrogen bond donors or acceptors in the protein. There is a good correlation between the relative stabilizing effects of Ala and Gly at internal positions with the total change in solvent-accessible hydrophobic surface area of the folded protein on mutation of Ala----Gly. The relationship may be extended to the N and C-caps by including an extra term in hydrophilic surface area for the solvent exposure of the non-intramolecularly hydrogen-bonded main-chain CO, NH or protein side-chain hydrogen bonding groups. The requirement for solvent exposure of the C-cap main-chain CO groups may account for the strong preference for residues having positive phi and psi angles at this position, since this alpha L-conformation results in the largest solvent exposure of the C-terminal CO groups. Glycine in an alpha L-conformation results in the greatest exposure of these CO groups. Further, the side-chains of His, Asn, Arg and Lys may, with positive phi and psi-angles, form a hydrogen bond with the backbone CO of residue in position C -3 (residues are numbered relative to the C-cap). The preferences at the C-cap are Gly much greater than His greater than Asn greater than Arg greater than Lys greater than Ala approximately Ser approximately greater than Asp. The preferences at the N-cap are determined by hydrogen bonding of side-chains or solvent to the exposed backbone NH groups and are: Thr approximately Asp approximately Ser greater than Gly approximately Asn greater than Gln approximately Glu approximately His greater than Ala greater than Val much greater than Pro. These general trends may be obscured when mutation allows another side-chain to become a surrogate cap.(ABSTRACT TRUNCATED AT 400 WORDS)

Many tumor cells are fueled by altered metabolism and increased glutamine (Gln) dependence. We identify regulation of the L-glutamine carrier proteins SLC1A5 and SLC38A2 (SLC1A5/38A2) by the ubiquitin ligase RNF5. Paclitaxel-induced ER stress to breast cancer (BCa) cells promotes RNF5 association, ubiquitination, and degradation of SLC1A5/38A2. This decreases Gln uptake, levels of TCA cycle components, mTOR signaling, and proliferation while increasing autophagy and cell death. Rnf5-deficient MMTV-PyMT mammary tumors were less differentiated and showed elevated SLC1A5 expression. Whereas RNF5 depletion in MDA-MB-231 cells promoted tumorigenesis and abolished paclitaxel responsiveness, SLC1A5/38A2 knockdown elicited opposing effects. Inverse RNF5(hi)/SLC1A5/38A2(lo) expression was associated with positive prognosis in BCa. Thus, RNF5 control of Gln uptake underlies BCa response to chemotherapies.

Compositional analysis of streptococcal C5a peptidase (SCPA) cleavage products from a synthetic peptide corresponding to the 20 C-terminal residues of C5a demonstrated that the target cleavage site is His-Lys rather than Lys-Asp, as previously suggested. A C5a peptide analog with Lys replaced by Gln was also subject to cleavage by SCPA. This confirmed that His-Lys rather than Lys-Asp is the scissile bond. Cleavage at histidine is unusual but is the same as that suggested for a peptidase produced by group B streptococci. Native C5 protein was also resistant to SCPA, suggesting that the His-Lys bond is inaccessible prior to proteolytic cleavage by C5 convertase. These experiments showed that the streptococcal C5a peptidase is highly specific for C5a and suggest that its function is not merely to process protein for metabolic consumption but to act primarily to eliminate this chemotactic signal from inflammatory foci.

The acquisition of nutrients requires tight regulation to ensure optimal supply while preventing accumulation to toxic levels. Ammonium transporter/methylamine permease/rhesus (AMT/Mep/Rh) transporters are responsible for ammonium acquisition in bacteria, fungi, and plants. The ammonium transporter AMT1;1 from Arabidopsis thaliana uses a novel regulatory mechanism requiring the productive interaction between a trimer of subunits for function. Allosteric regulation is mediated by a cytosolic C-terminal trans-activation domain, which carries a conserved Thr (T460) in a critical position in the hinge region of the C terminus. When expressed in yeast, mutation of T460 leads to inactivation of the trimeric complex. This study shows that phosphorylation of T460 is triggered by ammonium in a time- and concentration-dependent manner. Neither Gln nor l-methionine sulfoximine-induced ammonium accumulation were effective in inducing phosphorylation, suggesting that roots use either the ammonium transporter itself or another extracellular sensor to measure ammonium concentrations in the rhizosphere. Phosphorylation of T460 in response to an increase in external ammonium correlates with inhibition of ammonium uptake into Arabidopsis roots. Thus, phosphorylation appears to function in a feedback loop restricting ammonium uptake. This novel autoregulatory mechanism is capable of tuning uptake capacity over a wide range of supply levels using an extracellular sensory system, potentially mediated by a transceptor (i.e., transporter and receptor).

Excessive free radical formation has been implicated as a causative factor in neurotoxic damage associated with exposures to a variety of metals, including manganese (Mn). It is well established that Mn accumulates in astrocytes, affecting their ability to indirectly induce and/or exacerbate neuronal dysfunction. The present study examined the effects of Mn treatment on the following endpoints in primary astrocyte cultures: (1) oxidative injury, (2) alterations in high-energy phosphate (adenosine 5'-triphosphate, ATP) levels, (3) mitochondrial inner membrane potential, and (4) glutamine uptake and the expression of glutamine transporters. We quantified astrocyte cerebral oxidative damage by measuring F(2)-isoprostanes (F(2)-IsoPs) using stable isotope dilution methods followed by gas chromatography-mass spectrometry with selective ion monitoring. Our data showed a significant (p < 0.01) elevation in F(2)-IsoPs levels at 2 h following exposure to Mn (100 microM, 500 microM, or 1 mM). Consistent with this observation, Mn induced a concentration-dependent reduction in ATP and the inner mitochondrial membrane potential (DeltaPsi(m)), measured by the high pressure liquid chromatography method and the potentiometric dye, tetramethyl rhodamine ethyl ester, respectively. Moreover, 30 min of pretreatment with Mn (100 microM, 500 microM, or 1 mM) inhibited the net uptake of glutamine (GLN) ((3)H-glutamine) measured at 1 and 5 min. Expression of the messenger RNA coding the GLN transporters, SNAT3/SN1 and SNAT1, was inhibited after 100 and 500 microM Mn treatment for 24 h. Our results demonstrate that induction of oxidative stress, associated mitochondrial dysfunction, and alterations in GLN/glutamate cycling in astrocytes represent key mechanisms by which Mn exerts its neurotoxicity.

An immune selection procedure was employed in order to isolate p53 binding sites from mouse genomic DNA. Two DNA clones capable of tight specific interaction with wild type p53 were subjected to further characterization. In both cases, the p53 binding regions displayed a high degree of sequence homology with the consensus binding site defined for human genomic DNA. One of the clones was found to be derived from the LTR of a retrovirus-like element (a member of the GLN family). The region encompassing the GLN LTR p53 binding site could confer p53 responsiveness upon a heterologous promoter. Furthermore, the expression of the endogenous, chromosomally integrated GLN elements was significantly induced upon activation of wild type p53 in cells harboring a temperature sensitive p53 mutant. Finally, it was demonstrated that p53 - MDM2 complexes fail to bind tightly to such a p53 binding site. This may contribute to the inhibition by MDM2 of p53-mediated transcriptional activation.

Corazonin, a new cardioaccelerating peptide, has been isolated from the corpora cardiaca of the American cockroach, Periplaneta americana, and its structure determined to be Glp-Thr-Phe-Gln-Tyr-Ser-Arg-Gly-Trp-Thr-Asn-amide. The peptide stimulated heart beat at concentrations as low as 0.2 nM, which makes it the most potent insect cardioactive neuropeptide.

Susceptibility to autoimmune hepatitis in white patients is associated with the human leukocyte antigen class II antigens DR3 and DR4. To analyze the molecular basis of these associations, we used oligonucleotide probes to determine the DRB, DQA and DQB hypervariable nucleotide sequences in 119 patients with autoimmune hepatitis and 177 matched controls. DRB3*0101, which encodes DR52a, predisposed patients most strongly to the disease. It was present in 58% of patients and 25% of controls (corrected P < 0.000005), whereas DQA1*0101 and 0102 conferred protection in males only. The DR4 subtype, DRB1*0401, was raised in the DRB3*0101-negative patients; 81% possessed either DRB3*0101 or DRB1*0401, compared with 42% of controls (corrected P < 0.0000001). These alleles encode the amino acid sequence Leu-Leu-Glu-Gln-Lys-Arg at positions 67 to 72 of the DR beta polypeptide, which was present in 94% of patients and 64% of controls (corrected P < 0.000001) and in all patients who tested positive for autoantibodies to the hepatic asialoglycoprotein receptor. The patients with DRB1*0401 had less severe disease, relapsed less frequently and were first seen significantly later in life than those patients with DRB3*0101; and whereas a single copy of DRB1*0401 predisposed to autoimmune hepatitis, DRB3*0101-associated susceptibility had a dose-related effect. These data provide evidence that specific residues in the DR beta polypeptides predispose to autoimmune hepatitis in white patients and genes linked to DRB3*0101 and DRB1*0401 may determine two clinically distinct disease patterns.

Toll-like receptor 3 (TLR3) and cytosolic RIG-I-like helicases (RIG-I and MDA5) sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN) through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF), and mitochondrial antiviral signaling protein (MAVS), respectively. Previously, we demonstrated that hepatitis A virus (HAV), a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3C(pro), that is derived by auto-processing of the P3 (3ABCD) segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C)-stimulated dimerization of IFN regulatory factor 3 (IRF-3), IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3C(pro) protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3C(pro) and downstream 3D(pol) sequence, but not 3D(pol) polymerase activity. Cleavage occurs at two non-canonical 3C(pro) recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3D(pol) sequence modulates the substrate specificity of the upstream 3C(pro) protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3C(pro). HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.

OBJECTIVE

To determine whether premature infants who have necrotizing enterocolitis (NEC) have deficiencies in glutamine (GLN) and arginine (ARG), which are essential to intestinal integrity.

METHODS

A 4-month prospective cohort study of serum amino acid and urea levels in premature infants was done. Serum amino acid and urea levels were measured by high-pressure liquid chromatography and enzymatic methods, respectively, on samples obtained on days of life 3, 7, 14, and 21.

RESULTS

Infants in the control (n = 32) and NEC groups (n = 13) were comparable for birth weight, gestational age, and Apgar scores. NEC began on mean day of life 14.5 (95% CI, day of life 11 to 18). Median values of GLN were 37% to 57% lower in the NEC group on days 7, 14, and 21 compared with those in the control group (P <.05). On days 7 and 14, median values of ARG, GLN, alanine, lysine, ornithine, and threonine were decreased 36% to 67% (P <.05) in the NEC group. Total nonessential amino and total essential amino acids were 35% to 50% lower in the NEC group on days 7 and 14 (P <.05). Infants in the NEC group had significant reductions in GLN and ARG 7 days before the onset of NEC.

CONCLUSIONS

Infants who have NEC have selective amino acid deficiencies including reduced levels of GLN and ARG that may predispose to the illness.

BACKGROUND

The disease gene for hypertrophic cardiomyopathy (HCM) has been identified as the beta-myosin heavy chain (beta-MHC) gene in some HCM families. We describe extensive clinical evaluations in two kindreds with two distinct point mutations in the beta-MHC gene.

RESULTS

We used single-strand confirmation polymorphism (SSCP) gel analysis of polymerase chain reaction-amplified products capturing each of the 40 beta-MHC gene exons to identify distinct missense mutations in two HCM kindreds. Clinical, ECG, and echocardiographic studies were performed in the two kindreds: kindred 2755 with amino acid 908Leu----Val mutation and kindred 2002 with amino acid 403Arg----Gln mutation. The morphological appearances of HCM were similar in these two kindreds. However, the two kindreds differed with respect to disease penetrance, age of onset of disease, and incidence of premature sudden death. Twelve of 31 adults (greater than or equal to 17 years) with the disease gene in kindred 2755 did not have left ventricular hypertrophy (LVH), and only five of these had ECG abnormalities. Thus, the disease penetrance in adults with this mutation was only 61%. None of 11 children aged less than 16 years had LVH. The 908 mutation was associated with a low incidence of cardiac events: Only two sudden deaths and one syncope occurred in 46 individuals with the mutant allele. In contrast, LVH was present in all 11 adults in kindred 2002 with the 403 mutation (100% disease penetrance). In addition, three of four affected children were symptomatic and had clinical evidence of HCM. The disease in this kindred was severe and resulted in six premature sudden deaths. Seven additional patients had syncope or presyncope.

CONCLUSIONS

In some kindreds, the HCM disease gene is more prevalent than indicated by echocardiography and ECG. Some point mutations may be associated with a more malignant prognosis. Preclinical identification of children with mutations associated with a high incidence of sudden death and syncope provides the opportunity to evaluate efficacy of early therapeutic interventions.

X-ray repair cross-complementing group 1 (XRCC1) and xeroderma pigmentosum group D (XPD) are mainly involved in base excision repair (BER) and nucleotide excision repair (NER) of DNA repair pathways, respectively. Polymorphisms of DNA repair gene XRCC1 and XPD has recently been identified, and there is a growing body of evidence that these polymorphisms may have some phenotypic significance. To investigate the role of XRCC1 polymorphisms (codon 194 and codon 399) and XPD polymorphism (codon 751) in lung cancer, a population-based case-control study of 109 lung cancer patients and 109 healthy control subjects (individually matched on age and gender) in a Chinese population was conducted. XRCC1 and XPD genotypes were identified using PCR-restriction fragments length polymorphism technique. Conditional logistic regression analysis revealed that XRCC1 codon 194Trp/Trp genotype was associated with a borderline increased risk of lung cancer [adjusted odd ratio (OR) = 3.06; confidence interval (CI) 0.94-9.92]. The XPD 751 Lys allele (combined Lys/Lys and Lys/Gln genotypes) was associated with a significantly increased risk of lung cancer (OR = 3.19; CI 1.01-10.07). The risk of lung cancer increased more than additive interaction (adjusted OR = 8.77; CI 1.47-52.31) for the individuals with both putative high-risk genotypes of XRCC1 194 Trp/Trp and XPD 751 Lys allele. Our results suggested that the genotypes of XRCC1 194Trp/Trp and XPD 751 Lys allele might be the risk genotypes for lung cancer in Chinese population.

Cells expressing the Glu 27 beta 2-adrenoceptor polymorphism show attenuated down-regulation of the receptor after long-term exposure to agonist. We studied beta 2-adrenoceptor genotype for the Gln/Glu 27 polymorphism and airway reactivity in 65 patients with mild to moderate asthma. Glu 27 homozygotes had a four-fold higher geometric mean methacholine PD20 (provocative dose) than individuals who were homozygous for the wild-type (Gln 27) form of the receptor; heterozygotes had an intermediate value (3.23, 0.86, 1.96 mumol, respectively). These data suggest that beta 2-adrenoceptor genotype is important in the establishment of the asthmatic phenotype.