Beta-thalassemia mutations in Indonesia and their linkage to beta haplotypes.

A total of 72 chromosomes from 36 Indonesian patients, 23 with beta-thalassemia major and 13 with Hb E-beta-thalassemia, were analyzed by specific oligonucleotide hybridization after DNA amplification. Thirteen had the beta E mutation (codon 26 GAG----AAG). Of the 59-beta-thalassemic chromosomes, 32 were of the variant IVS-1 nt5 (G----C). Seven had the mutation IVS-2 nt654 (C----T), one had the mutation codon 41/42 (deletion CTTT), and one had the mutation codon 17 (AAG----TAG). Another six with the mutation IVS-1 nt1 (G----T), one with the mutation IVS-1 nt1 (G----A), four with the mutation codon 15 (TGG----TAG), one with a mutation codon 30 (AGG----ACG), and one with a mutation codon 35 (deletion C) were first identified by direct sequencing of a patient's genomic DNA followed by further hybridizing other patients' DNA with the appropriate oligonucleotide probes. Five did not carry the common mutations previously described in Asian populations. The four most prevalent mutations encountered made up 83% of the total number of beta-thalassemic chromosomes studied. The most common mutation, IVS-1 nt5 (G----C), was mostly associated with two different haplotypes.     

Ribosomal Protein Genes RPS10 and RPS26 Are Commonly Mutated in Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA), an inherited bone marrow failure syndrome characterized by anemia that usually presents before the first birthday or in early childhood, is associated with birth defects and an increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart, and urinary system defects that are present in ∼30%–50% of patients. DBA has been associated with mutations in seven ribosomal protein (RP) genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, and RPS7, in about 43% of patients. To continue our large-scale screen of RP genes in a DBA population, we sequenced 35 ribosomal protein genes, RPL15, RPL24, RPL29, RPL32, RPL34, RPL9, RPL37, RPS14, RPS23, RPL10A, RPS10, RPS12, RPS18, RPL30, RPS20, RPL12, RPL7A, RPS6, RPL27A, RPLP2, RPS25, RPS3, RPL41, RPL6, RPLP0, RPS26, RPL21, RPL36AL, RPS29, RPL4, RPLP1, RPL13, RPS15A, RPS2, and RPL38, in our DBA patient cohort of 117 probands. We identified three distinct mutations of RPS10 in five probands and nine distinct mutations of RPS26 in 12 probands. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knockdown of RPS10 or RPS26 expression with siRNAs, which indicates that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins in rRNA processing.     

Gout. Epidemiology of gout

Gout is the most prevalent form of inflammatory arthropathy. Several studies suggest that its prevalence and incidence have risen in recent decades. Numerous risk factors for the development of gout have been established, including hyperuricaemia, genetic factors, dietary factors, alcohol consumption, metabolic syndrome, hypertension, obesity, diuretic use and chronic renal disease. Osteoarthritis predisposes to local crystal deposition. Gout appears to be an independent risk factor for all-cause mortality and cardiovascular mortality and morbidity, additional to the risk conferred by its association with traditional cardiovascular risk factors.     

Enhanced circulating half-life and antitumor activity of a site-specific pegylated interferon-alpha protein therapeutic

Recombinant interferon alpha-2 (IFN-alpha2) has proven useful for treating a variety of human cancers and viral diseases. IFN-alpha2 has a short circulating half-life in vivo, which necessitates daily or thrice weekly administration to patients. It is possible to extend the circulating half-life of IFN-alpha2 by random modification of lysine residues in the protein with polyethylene glycol (PEG); however, such preparations have heterogeneous structures and low specific activities, and may not provide optimal therapeutic benefits to patients. A long-acting, site-specific, monoPEGylated IFN-alpha2 protein has now been created by targeted attachment of a 20 kDa or a 40 kDa maleimide-PEG to a cysteine analogue of IFN-alpha2, M111C. In vitro bioactivities of the purified 20 kDa and 40 kDa PEG-M111C proteins were within 2- to 3-fold of those of wild type IFN-alpha2 and 7- to 10-fold better than that of a 40 kDa PEG IFN-alpha2 protein created using nontargeted, amine-PEGylation methodology. The 20 kDa and 40 kDa PEG-M111C proteins demonstrated 26- to 38-fold longer half-lives, respectively, than IFN-alpha2 following subcutaneous administration to rats. The 20 kDa PEG M111C protein inhibited growth of human NIH:OVCAR-3 cells transplanted into nude mice by >90%, as measured by tumor size, tumor weight, and number of animals with detectable tumors at necropsy, and was significantly more effective than a comparable dose of IFN-alpha2. These data extend our previous findings that bioactivity of IFN-alpha2 can be largely preserved by targeted attachment of PEG moieties to nonessential sites in the protein and provide evidence that site-specific PEGylated IFN-alpha2 proteins possess enhanced tumoricidal properties in vivo.     

Random amplified polymorphic DNA markers reveal genetic variation in the symbiotic fungus of leaf-cutting ants

RAPD markers were used to examine the degree of genetic variation within the putatively asexual basidiomycete fungus (Lepiotaceae: provisionally named Leucoagaricus gongylophorus) associated with the leaf-cutting ant species Atta cephalotes. We analyzed fungal isolates from ant nests in two geographically distant sites, two isolates from Panama and five isolates from Trinidad. Ten decamer primers were used to amplify total DNA from these seven fungal isolates, and RAPD banding patterns were compared. Genetic similarity among isolates was determined by pair-wise comparisons of the shared number of DNA bands on an agarose gel. There was considerable genetic variation among isolates of the symbiotic fungus even within sites. Pairs of fungal isolates from the two different sites shared an average of only 36% of the bands in their RAPD profiles, while pairs from the within sites shared an average of 72% of the bands. RAPD markers may be useful for further investigation of the genetic structure of the fungal symbiont within species of leaf-cutting ants.     

Has gene duplication impacted the evolution of Eutherian longevity?

One of the greatest unresolved questions in aging biology is determining the genetic basis of interspecies longevity variation. Gene duplication is often the key to understanding the origin and evolution of important Eutherian phenotypes. We systematically identified longevity‐associated genes in model organisms that duplicated throughout Eutherian evolution. Longevity‐associated gene families have a marginally significantly higher rate of duplication compared to non‐longevity‐associated gene families. Anti‐longevity‐associated gene families have significantly increased rate of duplication compared to pro‐longevity gene families and are enriched in neurodegenerative disease categories. Conversely, duplicated pro‐longevity‐associated gene families are enriched in cell cycle genes. There is a cluster of longevity‐associated gene families that expanded solely in long‐lived species that is significantly enriched in pathways relating to 3‐UTR‐mediated translational regulation, metabolism of proteins and gene expression, pathways that have the potential to affect longevity. The identification of a gene cluster that duplicated solely in long‐lived species involved in such fundamental processes provides a promising avenue for further exploration of Eutherian longevity evolution.     

Cardiac effects of endothelin-1 (ET-1) and related C terminal peptide fragment: increased inotropy or contribution to heart failure?

The contrasting pattern of cardiac inotropy induced by human peptide endothelin-1 (ET-1) has not been satisfactorily explained. It is not clear whether ET-1 is primarily responsible for increased myocardial ET-1 expression and release with resultant inotropic effects, or for the induction of myocardial hypertrophy and heart failure. There are at least two subtypes of endothelin receptors (ET(A) and ET(B)) and the inotropic effects of ET-1 differ depending on the receptor involved. Along with some other groups, we reported significant subtype-ET(B) endothelin receptor down-regulation in human cardiac cells preincubated with endothelin agonists (Drímal et al. 1999, 2000). The present study was therefore designed to clarify the subtype-selective mechanisms underlying the inotropic response to ET-1 and to its ET(B)-selective fragment (8-21)ET-1 in the isolated rat heart. The hearts were subjected to (1-21)ET-1 and to (8-21)ET-1, or to 30 min of stop-flow ischemia followed by 40 min of reperfusion, both before and after selective blockade of endothelin receptors.The present study revealed that both peptides, ET-1 and its (8-21)ET-1 fragment, significantly reduced coronary blood flow in nmolar and higher concentrations. The concomitant negative inotropy and chronotropy were marked after ET-1, while the infusion of the ET-1(8-21) fragment produced a slight but significant positive inotropic effect. Among the four endothelin antagonists tested in continuous infusion only the non-selective PD145065 and ET(B1/B2) selective BQ788 (in molar concentrations) slightly reduced the early contractile dysfunction of the heart induced by ischemia, whereas ET(A)-selective PD155080 partially protected the rat heart on reperfusion.     

Side reactions in the synthesis of phosphotyrosine-containing peptides

Summary A series of phosphopeptides Tyr(PO₃H₂)-Val-Pro-Xxx-Leu (Xxx=Met, Met(O), Nle, Dab or Cys), derived from the native platelet-derived growth factor- receptor (PDGF-) sequence, has been prepared to study their interaction with the src-homology 2 (SH2) domains of the p85 subunit of PI3 kinase. The phosphopeptides were synthesized using Fmoc methodology incorporating N-Boc dibenzyl-protected phosphotyrosine (Boc-Tyr[PO₃(Bzl)₂]) as the N-terminal amino acid, since the benzyl groups can be removed during resin cleavage with TFA. Only peptides containing methionine were found to exist partially as S-benzyl sulfonium salts after TFA cleavage from the resin. The desired peptide could be obtained from the S-benzyl sulfonium salt by hydrogenolysis.     

Addition of a prominent epitope affects influenza A virus-specific CD8+ T cell immunodominance hierarchies when antigen is limiting

A reverse genetics strategy was used to insert the OVA peptide (amino acid sequence SIINFEKL; OVA(257-264)) into the neuraminidase stalk of both the A/PR8 (H1N1) and A/HKx31 (H3N2) influenza A viruses. Initial characterization determined that K(b)OVA257 is presented on targets infected with PR8-OVA and HK-OVA without significantly altering D(b) nucleoprotein (NP)366 presentation. There were similar levels of K(b)OVA257- and D(b)NP366-specific CTL expansion following both primary and secondary intranasal challenge. Interestingly, while variable, the presence of the immunodominant K(b)OVA257-specific response resulted in diminished D(b) acidic polymerase224- and K(b) basic polymerase subunit 1(703)-, but not D(b)NP366-specific responses and didn't alter endogenous influenza A virus-specific immunodominance hierarchies. However, challenging PR8-OVA-primed mice with HK-OVA via the i.p. route, and thereby limiting Ag dose, led to a reduction in the magnitude of all the influenza A virus-specific responses measured. A similar reduction in CTL response to native epitopes was also seen following primary respiratory HK-OVA infection of mice that received substantial numbers of K(b)OVA257-specific TCR transgenic T cells. Thus, during the course of infection, the generation of individual virus-specific CTL responses is independently regulated. However, in cases in which Ag is limiting, or high precursor frequency, the presence of immunodominant CTL responses can impact on the magnitude of other specific populations. Therefore, depending on both the size of the T cell precursor pool and the mode of Ag presentation, the addition of a major epitope can diminish the size of endogenous, influenza-specific CD8+ T cell responses, although never to the point that these are totally compromised.