Analysis of candidate antagonists of IAP-mediated caspase inhibition using yeast reconstituted with the mammalian Apaf-1-activated apoptosis mechanism

We have reconstituted the Apaf-1-activated apoptosis mechanism in Sacchromyces cerevisiae such that the presence of a constitutively active form of Apaf-1 together with both Caspase-9 and Caspase-3 results in yeast death. This system is a good model of the Apaf-1-activated pathway in mammalian cells: MIHA (XIAP/hILP), and to a lesser degree MIHB (c-IAP1/HIAP2) and MIHC (c-IAP-2/HIAP1) can inhibit caspases in this system, and protection by IAPs (inhibitor of apoptosis) can be abrogated by coexpression of the Drosophila pro-apoptotic proteins HID and GRIM or the mammalian protein DIABLO/Smac. Using this system we demonstrate that unlike DIABLO/Smac, other proteins which interact with mammalian IAPs (TAB-1, Zap-1, Traf-1 and Traf-2) do not act to antagonise IAP- mediated caspase inhibition.     

Discovering lactic acid bacteria by genomics

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, nvironmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram–positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.     

Activation of phosphoinositide 3-kinase gamma by Ras

BACKGROUND: Type I phosphoinositide 3-kinases are responsible for the hormone-sensitive synthesis of the lipid messenger phosphatidylinositol(3,4,5)-trisphosphate. Type IA and IB subfamily members contain a Ras binding domain and are stimulated by activated Ras proteins both in vivo and in vitro. The mechanism of Ras activation of type I PI3Ks is unknown, in part because no robust in vitro assay of this event has been established and characterized. Other Ras effectors, such as Raf and phosphoinositide-phospholipase Cepsilon, have been shown to be translocated into the plasma membrane, leading to their activation.RESULTS: We show that posttranslationally lipid-modified, activated N-, H-, K-, and R-Ras proteins can potently and substantially activate PI3Kgamma when using a stripped neutrophil membrane fraction as a source of phospholipid substrate. We have found GTPgammaS-loaded Ras can significantly (6- to 8-fold) activate PI3Kgamma when using artificial phospholipid vesicles containing their substrate, and this effect is a result of both a decrease in apparent Km for phosphatidylinositol(4,5)-bisphosphate and an increase in the apparent Vmax. However, neither in vivo nor in the two in vitro assays of Ras activation of PI3Kgamma could we detect any evidence of a Ras-dependent translocation of PI3Kgamma to its source of phospholipid substrate.CONCLUSIONS: Our data suggest that Ras activate PI3Kgamma at the level of the membrane, by allosteric modulation and/or reorientation of the PI3Kgamma, implying that Ras can activate PI3Kgamma without its membrane translocation. This view is supported by structural work that has suggested binding of Ras to PI3Kgamma results in a change in the structure of the catalytic pocket.     

Founder mutation R245H of Sanfilippo syndrome type A in the Cayman Islands

Sanfilippo A syndrome is an autosomal recessive lysosomal storage disease. This disease was reported in the Cayman Islands population with carrier frequency of 1/7 to 1/10 in the West Bay district of Grand Cayman. The carrier testing of Sanfilippo A disease for families at risk was carried out using the thermal characteristics of sulfamidase activity. In the present study, a search for mutations in the sulfamidase gene in an index family was performed. In addition, 77 individuals, relatives of children with Sanfilippo A syndrome, were also studied by single-strand conformation polymorphism (SSCP), restriction fragment-length polymorphism (RFLP) analyses, and sequencing. A single mutation, G746A (R245H), was found in the family, with the patient being homozygous and both parents and 1 of the 3 siblings being carriers. Among the 77 family members of the patient with Sanfilippo syndrome, the same mutation was found among carriers of the disease. The finding of a single mutation supports the idea of a founder effect, which facilitates accurate carrier identification of Sanfilippo A syndrome in the population of Cayman Islands.     

A variance-stabilizing transformation for gene-expression microarray data

MOTIVATION: Standard statistical techniques often assume that data are normally distributed, with constant variance not depending on the mean of the data. Data that violate these assumptions can often be brought in line with the assumptions by application of a transformation. Gene-expression microarray data have a complicated error structure, with a variance that changes with the mean in a non-linear fashion. Log transformations, which are often applied to microarray data, can inflate the variance of observations near background. RESULTS: We introduce a transformation that stabilizes the variance of microarray data across the full range of expression. Simulation studies also suggest that this transformation approximately symmetrizes microarray data.     

Nucleotide variation,haplotype structure,and association with end-stage renal disease of the human interleukin-1 gene cluster

A dense gene-based SNP map was constructed across a 360-kb region containing the interleukin-1 gene cluster (IL1A, IL1B, and IL1RN), focusing on IL1RN. In total, 95 polymorphisms were confirmed or identified primarily by direct sequencing. Polymorphisms were precisely mapped to completed BAC and genomic sequences spanning this region. The polymorphisms were typed in 443 case-control subjects from Caucasian and African American groups. Consecutive pair-wise marker linkage disequilibrium was not strictly correlated with distance and ranged from D'=0.0079 to 1.000 and D'=0.0521 to 1.0000 in Caucasians and African Americans, respectively. Single markers and haplotypes in IL1 cluster genes were evaluated for association with end-stage renal disease (ESRD). Eleven SNPs show some evidence of association with ESRD, with the strongest associations in two IL1A variants, one SNP, rs1516792-3, in intron 5 (p=0.0015) and a 4-bp insertion/deletion within the 3'UTR, rs16347-2 (p=0.0024), among African Americans with non-T2DM-associated ESRD.     

Some possible codes for encrypting data in DNA

Three codes are reported for storing written information in DNA. We refer to these codes as the Huffman code, the comma code and the alternating code. The Huffman code was devised using Huffman's algorithm for constructing economical codes. The comma code uses a single base to punctuate the message, creating an automatic reading frame and DNA which is obviously artificial. The alternating code comprises an alternating sequence of purines and pyrimidines, again creating DNA that is clearly artificial. The Huffman code would be useful for routine, short-term storage purposes, supposing – not unrealistically – that very fast methods for assembling and sequencing large pieces of DNA can be developed. The other two codes would be better suited to archiving data over long periods of time (hundreds to thousands of years).     

Finite element thermal analysis of bone cement for joint replacements

A finite element technique was developed to investigate the thermal behavior of bone cement in joint replacement procedures. Thermal tests were designed and performed to provide the parameters in a kinetic model of bone cement exothermic polymerization. The kinetic model was then coupled with an energy balance equation using a finite element formulation to predict the temperature history and polymerization development in the bone-cement-prosthesis system. Based on the temperature history, the possibility of the thermal bone necrosis was then evaluated. As a demonstration, the effect of cement mantle thickness on the thermal behavior of the system was investigated. The temperature profiles in the bone-cement-prosthesis system have shown that the thicker the cement, the higher the peak temperature in the bone. In the 7 mm thick cement case, a peak temperature of over 55 degrees C was predicted. These high temperatures occurred in a small region near the bone/cement interface. No damage was predicted in the 3 mm and 5 mm cement mantle thickness cases. Although thermal damage was predicted in the bone for the 7 mm mantle thickness case, the amount of thermal necrosis predicted was minimal. If more cement is used in the surgical procedure, more heat will be generated and the potential for thermal bone damage may rise. The systems should be carefully selected to reduce thermal tissue damage when more cement is used. The methodology developed in this paper provides a numerical tool for the quantitative simulation of the thermal behavior of bone-cement-prosthesis designs.