Carbon fractions in the rhizosphere of pea inoculated with 2,4 diacetylphloroglucinol producing and non-producing Pseudomonas fluorescens F113

The aim of this work was to determine the effect of wild type and functionally modified Pseudomonas fluorescens strains on C fractions in the rhizosphere of pea. The lac ZY marked F113 strain produces the antibiotic 2,4 diacetylphloroglucinol (DAPG) useful in plant disease control. The modified strain of F113 was represented in production of DAPG, creating the DAPG negative strain F113 G22. The F113 treatment resulted in a significantly lower shoot/root ratio. The F113 G22 treatment had a significantly greater indigenous and total fluorescent Pseudomonas population than the control and F113 (DAPG₊) treatment. Both strains significantly increased the water soluble carbohydrates and the total water soluble carbon in the pea rhizosphere soil. Strain F113 significantly increased the soil protein content relative to the control, but not in relation to the F113 G22 treatment. The F113 treatment had a significantly greater organic acid content than the control and F113 G22 treatments, whilst the F113 G22 treatment was also significantly greater than the control. Both inocula resulted in significantly lower phosphate contents than the control. The F113 inocula significantly increased alkaline phosphatase, sulphatase and urease activities, and reduced β glucosidase activities indicating increased carbon availability. Both inocula increased C availability ; however, antibiotic production by strain F113 reduced the utilisation of organic acids released from the plant resulting in differing effects of the two strains on nutrient availability, plant growth, soil enzyme activities and Pseudomonas populations.     

Phaseolus (Fabaceae) in Archaeology: AMS

Beans of several species were domesticated in tropical America thousands of years ago, to be combined with maize and other crops in highly successful New World agricultural systems. Radiocarbon dates on charcoal associated with Phaseolus in archaeological sites, in Mexico and Peru indicated the presence of domesticated beans as early as 10 000 years ago. However, direct dates on the beans and pods themselves by accelerator mass spectrometry (AMS) do not provide evidence for the cultivation in Mexico of common beans, P. vulgaris, and teparies, P. acutifolius, before about 2500 B.P. in the Tehuacán Valley, and of common beans about 1300 years ago in Tamaulipas and 2100 years ago in the Valley of Oaxaca. AMS dates support the presence in the Peruvian Andes of domesticated common beans by about 4400 B.P. and lima beans by about 3500 B. P. and lima beans by about 5600 B.P. in the coastal valleys of Peru. The late appearance of common and lima beans in the Central Highlands of Mesoamerica supports the importance of missing evidence that may be obtained from prehistoric agricultural sites in western Mexico and in Central America which are located within the range of the wild populations of these species. Additionally, biochemical studies of subsamples of the dated specimens should be carried out in order to extend the molecular evidence for the independent domestication of North and South American common beans.     

Preservation of duplicate genes by complementary, degenerative mutations

The origin of organismal complexity is generally thought to be tightly coupled to the evolution of new gene functions arising subsequent to gene duplication. Under the classical model for the evolution of duplicate genes, one member of the duplicated pair usually degenerates within a few million years by accumulating deleterious mutations, while the other duplicate retains the original function. This model further predicts that on rare occasions, one duplicate may acquire a new adaptive function, resulting in the preservation of both members of the pair, one with the new function and the other retaining the old. However, empirical data suggest that a much greater proportion of gene duplicates is preserved than predicted by the classical model. Here we present a new conceptual framework for understanding the evolution of duplicate genes that may help explain this conundrum. Focusing on the regulatory complexity of eukaryotic genes, we show how complementary degenerative mutations in different regulatory elements of duplicated genes can facilitate the preservation of both duplicates, thereby increasing long-term opportunities for the evolution of new gene functions. The duplication-degeneration-complementation (DDC) model predicts that (1) degenerative mutations in regulatory elements can increase rather than reduce the probability of duplicate gene preservation and (2) the usual mechanism of duplicate gene preservation is the partitioning of ancestral functions rather than the evolution of new functions. We present several examples (including analysis of a new engrailed gene in zebrafish) that appear to be consistent with the DDC model, and we suggest several analytical and experimental approaches for determining whether the complementary loss of gene subfunctions or the acquisition of novel functions are likely to be the primary mechanisms for the preservation of gene duplicates. For a newly duplicated paralog, survival depends on the outcome of the race between entropic decay and chance acquisition of an advantageous regulatory mutation.Sidow 1996(p. 717) On one hand, it may fix an advantageous allele giving it a slightly different, and selectable, function from its original copy. This initial fixation provides substantial protection against future fixation of null mutations, allowing additional mutations to accumulate that refine functional differentiation. Alternatively, a duplicate locus can instead first fix a null allele, becoming a pseudogene.Walsh 1995 (p. 426) Duplicated genes persist only if mutations create new and essential protein functions, an event that is predicted to occur rarely.Nadeau and Sankoff 1997 (p. 1259) Thus overall, with complex metazoans, the major mechanism for retention of ancient gene duplicates would appear to have been the acquisition of novel expression sites for developmental genes, with its accompanying opportunity for new gene roles underlying the progressive extension of development itself.Cooke et al. 1997 (p. 362)     

A disease-associated polymorphism alters splicing of the human CD45 phosphatase gene by disrupting combinatorial repression by heterogeneous nuclear ribonucleoproteins (hnRNPs)

Alternative splicing is typically controlled by complexes of regulatory proteins that bind to sequences within or flanking variable exons. The identification of regulatory sequence motifs and the characterization of sequence motifs bound by splicing regulatory proteins have been essential to predicting splicing regulation. The activation-responsive sequence (ARS) motif has previously been identified in several exons that undergo changes in splicing upon T cell activation. hnRNP L binds to this ARS motif and regulates ARS-containing exons; however, hnRNP L does not function alone. Interestingly, the proteins that bind together with hnRNP L differ for different exons that contain the ARS core motif. Here we undertake a systematic mutational analysis of the best characterized context of the ARS motif, namely the ESS1 sequence from CD45 exon 4, to understand the determinants of binding specificity among the components of the ESS1 regulatory complex and the relationship between protein binding and function. We demonstrate that different mutations within the ARS motif affect specific aspects of regulatory function and disrupt the binding of distinct proteins. Most notably, we demonstrate that the C77G polymorphism, which correlates with autoimmune disease susceptibility in humans, disrupts exon silencing by preventing the redundant activity of hnRNPs K and E2 to compensate for the weakened function of hnRNP L. Therefore, these studies provide an important example of the functional relevance of combinatorial function in splicing regulation and suggest that additional polymorphisms may similarly disrupt function of the ESS1 silencer.     

Combinatorial control of signal-induced exon repression by hnRNP L and PSF

Cells can regulate their protein repertoire in response to extracellular stimuli via alternative splicing; however, the mechanisms controlling this process are poorly understood. The CD45 gene undergoes alternative splicing in response to T-cell activation to regulate T-cell function. The ESS1 splicing silencer in CD45 exon 4 confers basal exon skipping in resting T cells through the activity of hnRNP L and confers activation-induced exon skipping in T cells via previously unknown mechanisms. Here we have developed an in vitro splicing assay that recapitulates the signal-induced alternative splicing of CD45 and demonstrate that cellular stimulation leads to two changes to the ESS1-bound splicing regulatory complex. Activation-induced posttranslational modification of hnRNP L correlates with a modest increase in the protein's repressive activity. More importantly, the splicing factor PSF is recruited to the ESS1 complex in an activation-dependent manner and accounts for the majority of the signal-regulated ESS1 activity. The associations of hnRNP L and PSF with the ESS1 complex are largely independent of each other, but together these proteins account for the total signal-regulated change in CD45 splicing observed in vitro and in vivo. Such a combinatorial effect on splicing allows for precise regulation of signal-induced alternative splicing.     

Epitope analysis of GAD65 binding in both cord blood and at the time of clinical diagnosis of childhood type 1 diabetes.

The GAD65 epitope immunoglobulin binding pattern in cord blood of children (n=37), who later developed type 1 diabetes at 3.2-14.9 years of age, was analyzed. First, the binding at diagnosis was compared with that in the cord blood serum. The next comparison was between the cord blood serum and the mothers' serum taken at delivery. Basal GAD65 binding levels were determined in Protein A Sepharose-based radiobinding assays with (35)S-labeled human and rat GAD65, rat GAD67 and GAD65/67 fusion proteins representing N-terminal (N), middle (M) and C-terminal (C) epitopes. In the first comparison, 28/37 children had GAD65 binding above 2.44 relative units (RU) (upper three quartiles), representing a marked increase from birth in the binding to human GAD65 (p<0.0001), rat GAD65 (p<0.0001), N- (p=0.04), M- (p<0.0001), C- (p=0.001), and M + C-epitopes (p<0.0001), but not to rat GAD67. At birth, 9/37 had GAD65 binding above 1.56 RU (upper quartile) demonstrating that their binding of human (35)S-GAD65 was higher in cord blood than in the mother (p=0.008). Higher cord blood binding was also observed for the N- (p=0.02) terminal epitope but not for rat GAD65, rat GAD67, and the remaining epitopes. These data suggest that differences in the epitope GAD65 binding between mother and child at birth are limited. In contrast, the epitope pattern at diagnosis differed from that at birth, supporting the view that disease-associated epitopes develop between birth and diagnosis.     

The origin of subfunctions and modular gene regulation

Evolutionary explanations for the origin of modularity in genetic and developmental pathways generally assume that modularity confers a selective advantage. However, our results suggest that even in the absence of any direct selective advantage, genotypic modularity may increase through the formation of new subfunctions under near-neutral processes. Two subfunctions may be formed from a single ancestral subfunction by the process of fission. Subfunction fission occurs when multiple functions under unified genetic control become subdivided into more restricted functions under independent genetic control. Provided that population size is sufficiently small, random genetic drift and mutation can conspire to produce changes in the number of subfunctions in the genome of a species without necessarily altering the phenotype. Extensive genotypic modularity may then accrue in a near-neutral fashion in permissive population-genetic environments, potentially opening novel pathways to morphological evolution. Many aspects of gene complexity in multicellular eukaryotes may have arisen passively as population size reductions accompanied increases in organism size, with the adaptive exploitation of such complexity occurring secondarily.     

Determining Glomerular Filtration Rate in Homozygous Sickle Cell Disease: Utility of Serum Creatinine Based Estimating Equations

Background

Various estimating equations have been developed to estimate glomerular filtration rate (GFR) for use in clinical practice. However, the unique renal physiological and pathological processes that occur in sickle cell disease (SCD) may invalidate these estimates in this patient population. This study aims to compare GFR estimated using common existing GFR predictive equations to actual measured GFR in persons with homozygous SCD. If the existing equations perform poorly, we propose to develop a new estimating equation for use in persons with SCD.

Methods

98 patients with the homozygous SS disease (55 females: 43 males; mean age 34±2.3 years) had serum measurements of creatinine, as well as had GFR measured using ^99mTc-DTPA nuclear renal scan. GFR was estimated using the Modification of Diet in Renal Disease (MDRD), Cockcroft-Gault (CG), and the serum creatinine based CKD-EPI equations. The Bland-Altman limit of agreement method was used to determine agreement between measured and estimated GFR values. A SCD-specific estimating equation for GFR (JSCCS-GFR equation) was generated by means of multiple regression via backward elimination.

Results

The mean measured GFR±SD was 94.9±27.4 mls/min/1.73 m² BSA, with a range of 6.4–159.0 mls/min/1.73 m². The MDRD and CG equations both overestimated GFR, with the agreement worsening with higher GFR values. The serum creatinine based CKD-EPI equation performed relatively well, but with a systematic bias of about 45 mls/min. The new equation developed resulted in a better fit to our sickle cell disease data than the MDRD equation.

Conclusion

Current estimating equations, other than the CKD-EPI equation, do not perform very accurately in persons with homozygous SS disease. A fairly accurate estimating equation, suitable for persons with GFR >60 mls/min/1.73 m² has been developed from our dataset and validated within a simulated dataset.     

Craniometric variation in the Eurasian badger, Meles meles

A multivariate examination of cranial variation within and between European populations of Meles meles (L.) revealed that populations from Ireland, Great Britain, Norway, and the Slovak Republic could be differentiated both by cranial form and by the degree of sexual dimorphism exhibited. Irish material was characterized by low sexual dimorphism, particularly when compared to Slovak specimens. Badgers from the British Isles had larger skulls than other samples and were more similar to each other than they were to badgers from mainland Europe. Size played a greater role in differentiating samples of female badgers than it did in males. Significant variation occurred within the British Isles, with individual samples being highly differentiable. There was, however, little relationship between morphological similarity and geographic proximity. We contend that macrogeographical (between-country) variation in the species is primarily determined by historical factors and adaptation to current conditions, while microgeographic (within-country) variation is a result of selectively neutral processes.