The effect of high temperature and high atmospheric CO2 on carbohydrate changes in bell pepper (Capsicum annuum) pollen in relation to its germination

Pollen viability and germination are known to be sensitive to high temperature (HT). However, the mode by which high temperature impairs pollen functioning is not yet clear. In the present study, we investigated the effect of high temperature on changes occurring in carbohydrate of bell pepper (Capsicum annuum L. cv. Mazurka) pollen in order to find possible relations between these changes and pollen germination under heat stress. When pepper plants were maintained under a moderate HT regime (32/26 degrees C, day/night) for 8 days before flowers have reached anthesis, pollen count at anthesis was similar to that found in plants grown under normal temperatures (NT 28/22 degrees C). However, the in vitro germination, carried out at 25 degrees C, of pollen from HT plants was greatly reduced. This effect matched the marked reduction in the number of seeds per fruit in the HT plants. Maintaining the plants at high air CO2 concentration (800 &mgr;mol mol-1 air) in both temperature treatments did not affect the in vitro germination of pollen from NT plants, but restored germination to near the normal level in pollen from HT plants. Under NT conditions, starch, which was negligible in pollen at meiosis (8 days before anthesis, A-8) started to accumulate at A-4 and continued to accumulate until A-2. From that stage until anthesis, starch was rapidly degraded. On the other hand, sucrose concentration rose from stage A-4 until anthesis. Acid invertase (EC 3.2.1.26) activity rose parallel with the increase of sucrose. In pollen from HT plants, sucrose and starch concentrations were significantly higher at A-1 pollen than in that of NT plants. Under high CO2 conditions, the sucrose concentration in the pollen of HT plants was reduced to levels similar to those in NT pollen. In accordance with the higher sucrose concentration in HT pollen, the acid invertase activity in these pollen grains was lower than in NT pollen. The results suggest that the higher concentrations of sucrose and starch in the pollen grains of HT plants may result from reduction in their metabolism under heat stress. Elevated CO2 concentration, presumably by increasing assimilate availability to the pollen grain, may alleviate the inhibition of sucrose and starch metabolism, thereby increasing their utilization for pollen germination under the HT stress. Acid invertase may have a regulatory role in this system.     

How prevalent is functional alternative splicing in the human genome?

Comparative analyses of ESTs and cDNAs with genomic DNA predict a high frequency of alternative splicing in human genes. However, there is an ongoing debate as to how many of these predicted splice variants are functional and how many are the result of aberrant splicing (or 'noise'). To address this question, we compared alternatively spliced cassette exons that are conserved between human and mouse with EST-predicted cassette exons that are not conserved in the mouse genome. Presumably, conserved exon-skipping events represent functional alternative splicing. We show that conserved (functional) cassette exons possess unique characteristics in size, repeat content and in their influence on the protein. By contrast, most non-conserved cassette exons do not share these characteristics. We conclude that a significant portion of cassette exons evident in EST databases is not functional, and might result from aberrant rather than regulated splicing.     

The pp60c-Src inhibitor PP1 is non-competitive against ATP

The C7-cyclitol 2-epi-5-epi-valiolone is the first precursor of the cyclitol moiety of the -glucosidase inhibitor acarbose in Actinoplanes sp. SE50. The 2-epi-5-epi-valiolone becomes phosphorylated at C7 by the ATP dependent kinase AcbM prior to the next modifications. Preliminary data gave evidences that the AcbO protein could catalyse the first modification step of 2-epi-5-epi-valiolone-7-phosphate. Therefore, the AcbO protein, the encoding gene of which is also part of the acbKMLNOC operon, was overproduced and purified. Indeed the purified protein catalysed the 2-epimerisation of 2-epi-5-epi-valiolone-7-phosphate. The chemical structure of the purified reaction product was proven by nuclear magnetic resonance spectroscopy to be 5-epi-valiolone-7-phosphate.     

Cystathionine- -synthase gene transfer and 3-deazaadenosine ameliorate inflammatory response in endothelial cells

Although elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with increased inflammation and vascular remodeling, the mechanism of Hcy-mediated inflammation and vascular remodeling is unclear. The matrix metalloproteinases (MMPs) and adhesion molecules play an important role in vascular remodeling. We hypothesized that HHcy induces inflammation by increasing adhesion molecules and matrix protein expression. Endothelial cells were supplemented with high methionine, and Hcy accumulation was measured by HPLC. Nitric oxide (NO) bioavailability was detected by a NO probe. The protein expression was measured by Western blot analysis. MMP-9 activity was detected by gelatin-gel zymography. We demonstrated that methionine supplement promoted upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) through increased Hcy accumulation. In addition, increased synthesis of collagen type-1 was also observed. MMP-9 gene expression and protein activity were increased in methionine supplement groups. 3-Deazaadenosine (DZA), an adenosine analogue, prevented high methionine-induced ICAM-1 and VCAM-1 expression and collagen type-1 synthesis. Transfection of endothelial cells with cystathionine-β-synthase (CBS) gene construct, which converts Hcy to cystathionine, reduced Hcy accumulation in high methionine-fed cells. CBS gene transfection reduced the inflammatory response, as evident by attenuated ICAM-1 and VCAM-1 expression. Furthermore, collagen type-1 expression and MMP-9 activity were dramatically attenuated with CBS gene transfection. These results suggested that methionine supplement increased Hcy accumulation, which was associated with inflammatory response and matrix remodeling such as collagen type-1 synthesis and MMP-9 activity. However, in vitro DZA and CBS gene therapy successfully treated the HHcy-induced inflammatory reaction in the methionine metabolism pathway. extracellular matrix; matrix metalloproteinase-9; intercellular cell adhesion molecule-1; vascular cell adhesion molecule-1; collagen type-1; hyperhomocysteinemia     

The birth of new exons: mechanisms and evolutionary consequences

A significant amount of literature was dedicated to hypotheses concerning the origin of ancient introns and exons, but accumulating evidence indicates that new exons are also constantly being added to evolving genomes. Several mechanisms contribute to the creation of novel exons in metazoan genomes, including whole gene and single exon duplications, but perhaps the most intriguing are events of exonization, where intronic sequences become exons de novo. Exonizations of intronic sequences, particularly those originating from repetitive elements, are now widely documented in many genomes including human, mouse, dog, and fish. Such de novo appearance of exons is very frequently associated with alternative splicing, with the new exon-containing variant typically being the rare one. This allows the new variant to be evolutionarily tested without compromising the original one, and provides an evolutionary strategy for generation of novel functions with minimum damage to the existing functional repertoire. This review discusses the molecular mechanisms leading to exonization, its extent in vertebrate genomes, and its evolutionary implications.