Radiation causes increased production and decreased utilization of IL-2 in human mononuclear cells

The effects of radiation on the kinetics of Interleukin-2 (IL-2) production and utilization by mononuclear cells (MNCs) were studied. Mononuclear cells from normal, healthy individuals were subjected to various doses of radiation ranging from 0 to 2,000 rad and cultured in the presence of PHA. Supernatants from these cultures were harvested at various periods and their IL-2 contents determined by both the standard bioassay and ELISA. A radiation dose of 800 rad and higher had a marked effect on both IL-2 production and consumption. Although the supernatants from both the irradiated and non-irradiated MNCs contained maximal concentrations of IL-2 between 8 and 24 h of culture, the former had three times as much IL-2 as the latter. An increase in IL-2-mRNA levels was also noticed in irradiated, PHA-stimulated cells. Moreover, the supernatants from irradiated MNCs collected as late as 72 h after the initiation of culture contained more than 30% of the total IL-2 produced compared to less than 8% in supernatants from non-irradiated cells. Supernatants from non-irradiated cells incubated further with irradiated cells contained relatively higher quantities of IL-2 than those incubated continuously with non-irradiated cells. Supernatants from co-cultures of irradiated and non-irradiated MNCs contained less than expected amounts of IL-2 in two of the three subjects. Despite a difference in both the production and consumption of IL-2 between the irradiated and non-irradiated cells, there was no difference in their ability to generate IL-2 receptors. The results indicate that inactivation of radiosensitive suppressor T cells is associated with superinduction of IL-2 mRNA, increased production and decreased consumption of IL-2 by MNCs, thereby resulting in increased accumulation of IL-2.     

XRCC2 and XRCC3 Regulate the Balance between Short- and Long-Tract Gene Conversions between Sister Chromatids

Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DNA lesions associated with replication and is thought to be important for suppressing genomic instability. The mechanisms regulating the initiation and termination of SCR in mammalian cells are poorly understood. Previous work has implicated all the Rad51 paralogs in the initiation of gene conversion and the Rad51C/XRCC3 complex in its termination. Here, we show that hamster cells deficient in the Rad51 paralog XRCC2, a component of the Rad51B/Rad51C/Rad51D/XRCC2 complex, reveal a bias in favor of long-tract gene conversion (LTGC) during SCR. This defect is corrected by expression of wild-type XRCC2 and also by XRCC2 mutants defective in ATP binding and hydrolysis. In contrast, XRCC3-mediated homologous recombination and suppression of LTGC are dependent on ATP binding and hydrolysis. These results reveal an unexpectedly general role for Rad51 paralogs in the control of the termination of gene conversion between sister chromatids.DNA double-strand breaks (DSBs) are potentially dangerous lesions, since their misrepair may cause chromosomal translocations, gene amplifications, loss of heterozygosity (LOH), and other types of genomic instability characteristic of human cancers (7, 9, 21, 40, 76, 79). DSBs are repaired predominantly by nonhomologous end joining or homologous recombination (HR), two evolutionarily conserved DSB repair mechanisms (8, 12, 16, 33, 48, 60, 71). DSBs generated during the S or G₂ phase of the cell cycle may be repaired preferentially by HR, using the intact sister chromatid as a template for repair (12, 26, 29, 32, 71). Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DSBs, which has led to the proposal that SCR protects against genomic instability, cancer, and aging. Indeed, a number of human cancer predisposition genes are implicated in SCR control (10, 24, 45, 57, 75).HR entails an initial processing of the DSB to generate a free 3′ single-stranded DNA (ssDNA) overhang (25, 48, 56). This is coupled to the loading of Rad51, the eukaryotic homolog of Escherichia coli RecA, which polymerizes to form an ssDNA-Rad51 “presynaptic” nucleoprotein filament. Formation of the presynaptic filament is tightly regulated and requires the concerted action of a large number of gene products (55, 66, 68). Rad51-coated ssDNA engages in a homology search by invading homologous duplex DNA. If sufficient homology exists between the invading and invaded strands, a triple-stranded synapse (D-loop) forms, and the 3′ end of the invading (nascent) strand is extended, using the donor as a template for gene conversion. This recombination intermediate is thought to be channeled into one of the following two major subpathways: classical gap repair or synthesis-dependent strand annealing (SDSA) (48). Gap repair entails the formation of a double Holliday junction, which may resolve into either crossover or noncrossover products. Although this is a major pathway in meiotic recombination, crossing-over is highly suppressed in somatic eukaryotic cells (26, 44, 48). Indeed, the donor DNA molecule is seldom rearranged during somatic HR, suggesting that SDSA is the major pathway for the repair of somatic DSBs (26, 44, 49, 69). SDSA terminates when the nascent strand is displaced from the D-loop and pairs with the second end of the DSB to form a noncrossover product. The mechanisms underlying displacement of the nascent strand are not well understood. However, failure to displace the nascent strand might be expected to result in the production of longer gene conversion tracts during HR (36, 44, 48, 63).Gene conversion triggered in response to a Saccharomyces cerevisiae or mammalian chromosomal DSB generally results in the copying of a short (50- to 300-bp) stretch of information from the donor (short-tract gene conversion [STGC]) (14, 47, 48, 67, 69). A minority of gene conversions in mammalian cells entail more-extensive copying, generating gene conversion tracts that are up to several kilobases in length (long-tract gene conversion [LTGC]) (26, 44, 51, 54, 64). In yeast, very long gene conversions can result from break-induced replication (BIR), a highly processive form of gene conversion in which a bona fide replication fork is thought to be established at the recombination synapse (11, 36, 37, 39, 61, 63). In contrast, SDSA does not require lagging-strand polymerases and appears to be much less processive than a conventional replication fork (37, 42, 78). BIR in yeast has been proposed to play a role in LOH in aging yeast, telomere maintenance, and palindromic gene amplification (5, 41, 52). It is unclear to what extent a BIR-like mechanism operates in mammalian cells, although BIR has been invoked to explain telomere elongation in tumors lacking telomerase (13). It is currently unknown whether LTGC and STGC in somatic mammalian cells are products of mechanistically distinct pathways or whether they represent alternative outcomes of a common SDSA pathway.Vertebrate cells contain five Rad51 paralogs—polypeptides with limited sequence homology to Rad51—Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3 (74). The Rad51 paralogs form the following two major complexes: Rad51B/Rad51C/Rad51D/XRCC2 (BCDX2) and Rad51C/XRCC3 (CX3) (38, 73). Genetic deletion of any one of the rad51 paralogs in the mouse germ line produces early embryonic lethality, and mouse or chicken cells lacking any of the rad51 paralogs reveal hypersensitivity to DNA-damaging agents, reduced frequencies of HR and of sister chromatid exchanges, increased chromatid-type errors, and defective sister chromatid cohesion (18, 72, 73, 82). Collectively, these data implicate the Rad51 paralogs in SCR regulation. The purified Rad51B/Rad51C complex has been shown to assist Rad51-mediated strand exchange (62). XRCC3 null or Rad51C null hamster cells reveal a bias toward production of longer gene conversion tracts, suggesting a role for the CX3 complex in late stages of SDSA (6, 44). Rad51C copurifies with branch migration and Holliday junction resolution activities in mammalian cell extracts (35), and XRCC3, but not XRCC2, facilitates telomere shortening by reciprocal crossing-over in telomeric T loops (77). These data, taken together with the meiotic defects observed in Rad51C hypomorphic mice, suggest a specialized role for CX3, but not for BCDX2, in resolving Holliday junction structures (31, 58).To further address the roles of Rad51 paralogs in late stages of recombination, we have studied the balance between long-tract (>1-kb) and short-tract (<1-kb) SCR in XRCC2 mutant hamster cells. We found that DSB-induced gene conversion in both XRCC2 and XRCC3 mutant cells is biased in favor of LTGC. These defects were suppressed by expression of wild-type (wt) XRCC2 or XRCC3, respectively, although the dependence upon ATP binding and hydrolysis differed between the two Rad51 paralogs. These results indicate that Rad51 paralogs play a more general role in determining the balance between STGC and LTGC than was previously appreciated and suggest roles for both the BCDX2 and CX3 complexes in influencing the termination of gene conversion in mammals.     

The rate of oxygen utilization by cells

The discovery of oxygen is considered by some to be the most important scientific discovery of all time—from both physical-chemical/astrophysics and biology/evolution viewpoints. One of the major developments during evolution is the ability to capture dioxygen in the environment and deliver it to each cell in the multicellular, complex mammalian body—on demand, i.e., just in time. Humans use oxygen to extract approximately 2550 calories (10.4 MJ) from food to meet daily energy requirements. This combustion requires about 22 mol of dioxygen per day, or 2.5 × 10^− 4 mol s^− 1. This is an average rate of oxygen utilization of 2.5 × 10^− 18 mol cell^− 1 s^− 1, i.e., 2.5 amol cell^− 1 s^− 1. Cells have a wide range of oxygen utilization, depending on cell type, function, and biological status. Measured rates of oxygen utilization by mammalian cells in culture range from < 1 to > 350 amol cell^− 1 s^− 1. There is a loose positive linear correlation of the rate of oxygen consumption by mammalian cells in culture with cell volume and cell protein. The use of oxygen by cells and tissues is an essential aspect of the basic redox biology of cells and tissues. This type of quantitative information is fundamental to investigations in quantitative redox biology, especially redox systems biology.     

Psychometric Properties of the Problem Areas in Diabetes (PAID) Instrument in Singapore

Background

Emotional distress is an important dimension in diabetes, and several instruments have been developed to measure this aspect. The Problem Areas in Diabetes (PAID) scale is one such instrument which has demonstrated validity and reliability in Western populations, but its psychometric properties in Asian populations have not been examined.

Methods

This was a secondary analysis of data from patients with Type 2 diabetes mellitus recruited through convenience sampling from a diabetes specialist outpatient clinic in Singapore. The following psychometric properties were assessed: Construct validity through confirmatory factor analysis (CFA) and Rasch analysis, concurrent validity through correlation with related scales (Kessler Psychological Distress Scale, Diabetes Health Profile—psychological distress, Audit of Diabetes Dependent Quality of Life), reliability through assessment of internal consistency and floor and ceiling effects, and sensitivity by estimating effect sizes for known clinical and social functioning groups.

Results

203 patients with mean age of 45±12 years were analysed. None of the previously published model structures achieved a good fit on CFA. On Rasch analysis, four items showed poor fit and were removed. The abridged 16-item PAID mapped to a single latent trait, with a high degree of internal consistency (Cronbach ɑ 0.95), but significant floor effect (24.6% scoring at floor). Both 20-item and 16-item PAID scores were moderately correlated with scores of related scales, and sensitive to differences in clinical and social functioning groups, with large effect sizes for glycemic control and diabetes related complications, nephropathy and neuropathy.

Conclusion

The abridged 16-item PAID measures a single latent trait of emotional distress due to diabetes whereas the 20-item PAID appears to measures more than one latent trait. However, both the 16-item and 20-item PAID versions are valid, reliable and sensitive for use among Singaporean patients with diabetes.     

Metabolically engineered Escherichia coli as a tool for the production of bioenergy and biochemicals from glycerol

Currently, a variety of feedstock is utilized by metabolically engineered bacteria for the production of bioenergy and biochemicals. Recent studies have shown that glycerol can be used as an alternative feedstock for glucose, considering its higher availability, lower price, and high degree of reduction. Hence, this review focuses on recent developments in the bioconversion of glycerol to bioenergy (ethanol and hydrogen) and biochemicals (1,3-propanediol, 1,2-propanediol, 3-hydroxypropionic acid, succinic acid, lactic acid, polyhydroxyalkanoates and Lphenyl alanine) using metabolically engineered Escherichia coli.     

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events

Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked beta1-->3 to a beta-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.     

Construction of malate-sensing <Emphasis Type="Italic">Escherichia coli</Emphasis> by introduction of a novel chimeric two-component system

In an attempt to develop a high-throughput screening system for screening microorganisms which produce high amounts of malate, a MalKZ chimeric HK-based biosensor was constructed. Considering the sequence similarity among Escherichia coli (E. coli) MalK with Bacillus subtilis MalK and E. coli DcuS, the putative sensor domain of MalK was fused with the catalytic domain of EnvZ. The chimeric MalK/EnvZ TCS induced the ompC promoter through the cognate response regulator, OmpR, in response to extracellular malate. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as malate concentration increased. By using this strategy, various chimeric TCS-based bacteria biosensors can be constructed, which may be used for the development of biochemical-producing recombinant microorganisms.