The fermented non-digestible fraction of common bean (Phaseolus vulgaris L.) triggers cell cycle arrest and apoptosis in human colon adenocarcinoma cells.

Cancer is a leading cause of death worldwide with colorectal cancer (CRC) ranking as the third contributing to overall cancer mortality. Non-digestible compounds such as dietary fiber have been inversely associated with CRC in epidemiological in vivo and in vitro studies. In order to investigate the effect of fermentation products from a whole non-digestible fraction of common bean versus the short-chain fatty acid (SCFAs) on colon cancer cells, we evaluated the human gut microbiota fermented non-digestible fraction (hgm-FNDF) of cooked common bean (Phaseolus vulgaris L.) cultivar Negro 8025 and a synthetic mixture SCFAs, mimicking their concentration in the lethal concentration 50 (SCFA-LC50) of FNDF (hgm-FNDF-LC50), on the molecular changes in human colon adenocarcinoma cells (HT-29). Total mRNA from hgm-FNDF-LC50 and SCFA-LC50 treated HT-29 cells were used to perform qPCR arrays to determine the effect of the treatments on the transcriptional expression of 84 genes related to the p53-pathway. This study showed that both treatments inhibited cell proliferation in accordance with modulating RB1, CDC2, CDC25A, NFKB and E2F genes. Furthermore, we found an association between the induction of apoptosis and the modulation of APAF1, BID, CASP9, FASLG, TNFR10B and BCL2A genes. The results suggest a mechanism of action by which the fermentation of non-digestible compounds of common bean exert a beneficial effect better than the SCFA mixture by modulating the expression of antiproliferative and pro-apoptotic genes in HT-29 cells to a greater extent, supporting previous results on cell behavior, probably due to the participation of other compounds, such as phenolic fatty acids derivatives and biopetides.

Surveillance for severe community-associated methicillin-resistant Staphylococcus aureus infection.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has rapidly emerged in the USA as a cause of severe infections in previously healthy persons without traditional risk factors. We describe the epidemiology of severe CA-MRSA disease in the state of Georgia, USA and analyse the risk of death associated with three different clinical syndromes of CA-MRSA disease - pneumonia, invasive disease, and skin and soft-tissue infections (SSTIs). A total of 1670 cases of severe CA-MRSA disease were reported during 2005-2007. The case-fatality rate was 3.4%; sex and race of fatal and non-fatal cases did not differ significantly. While CA-MRSA pneumonia and invasive disease were less common than SSTIs, they were about 15 times more likely to result in death [risk ratio 16.69, 95% confidence interval (CI) 10.28-27.07 and 13.98, 95% CI 7.74-25.27, respectively]. When controlling for age and the presence of other clinical syndromes the odds of death in patients manifesting specific severe CA-MRSA syndromes was highest in those with pneumonia (odds ratio 11.34). Possible risk factors for severe CA-MRSA SSTI and pneumonia included the draining of lesions without medical assistance and an antecedent influenza-like illness.

Comparison of the cost of short flights in a nectarivorous and a non-nectarivorous bird.

Although most birds are accustomed to making short flights, particularly during foraging, the flight patterns during these short periods of activity differ between species. Nectarivorous birds, in particular, often spend time hovering, while non-nectarivorous birds do not. The cost of short flights is likely therefore to differ between nectarivorous and non-nectarivorous birds because of the different energetic contributions of different flight types to the behaviour. The 13C-labelled bicarbonate technique was used to measure the energy cost of short flights in the nectarivorous Palestine sunbird Nectarinia osea (mean mass 6.17+/-0.16 g, N=8) and the non-nectarivorous starling Sturnus vulgaris (mean mass 70.11+/-1.11 g, N=9). The technique was initially calibrated in five individuals for each species at temperatures ranging from 1 to 35 degrees C, by comparing the isotope elimination rate to the metabolic rate measured simultaneously by indirect calorimetry. The cost for short intermittent flight was then measured by encouraging birds to fly between two perches at either end of a narrow corridor (perch distance for sunbirds, 6 m; for starlings, 5 m), and measuring the amount of isotope eliminated during the flight. The isotope elimination rate was interpolated onto the calibration equation to predict flight cost, as a direct calibration could not be performed during flight. Mean energy expenditure during flight was 1.64+/-0.32 W in sunbirds, while in starlings the flight costs averaged 20.6+/-0.78 W. Energy cost of flight relative to basal metabolic rate was substantially greater in the starling than the sunbird. Phylogenetic analysis of different modes of flight in these and additional species suggests that differences in flight behaviour may cause these elevated costs in slow flying non-nectarivores such as starlings, compared to birds that are more prone to short intermittent flights like the sunbirds.

Effect of freezing on the activity of catalase in apple flesh tissue.

Catalase (CAT, EC 1.11.1.6) activity was measured in flesh tissue of six apple cultivars (Malus domestica Borkh. cvs. Braeburn, Gala, Jonagold, McIntosh, Red Delicious, and Spartan). Activity of CAT was determined for fresh and frozen tissue of the same fruit. Freezing resulted in reductions of 50 to 90% in CAT activity compared with the activity measured in crude extracts from fresh tissues. The rate of freezing had an impact on the level of reduction of CAT activity, with slower freezing procedures leading to greater losses in activity. Six additives to the extraction buffer were tested to evaluate their potential to reduce the inactivation of CAT from frozen tissue, but only EDTA and Tween 20 showed any benefit. However, EDTA and Tween 20 provided only partial recovery in CAT activity. In contrast, crude extracts prepared from fresh tissue showed no appreciable loss in CAT activity after frozen storage for two weeks at -80 degrees C. Gel electrophoresis and immunological analysis indicated that the loss in CAT activity from tissue freezing could be attributed to loss of both the tetrameric CAT enzyme structure and total CAT protein. The implications of using freezing to preserve apple tissue samples prior to catalase activity analysis is discussed.

Fast apple (Malus x domestica) and tobacco (Nicotiana tobacum) leaf polyphenol oxidase activity assay for screening transgenic plants.

A spectrophotometric assay method for the analysis of polyphenol oxidase (PPO), in apple and tobacco leaves, has been optimized to increase efficiency in the screening of large numbers of transgenic plants. Crude protein extracts from leaf punches were prepared in a FastPrep homogenizer. The addition of Triton X-100 during extraction resulted in 44 and 74% increases in the PPO activity recovered, from apple and tobacco, respectively. The enzyme kinetics differed markedly between apple and tobacco. Apple leaf PPO was isolated in a latent state and was activated by the addition of SDS. In contrast, tobacco PPO activity was inhibited by SDS, particularly at acidic pH. Apple PPO showed a pronounced pH optimum around pH 6, whereas the pH profile for tobacco PPO was much flatter, with a broad optimum around pH 4. The calculated Km' value for apple PPO, using 4-methylcatechol as substrate, was 8.1, and for tobacco the Km was 4.3. The PPO reaction was strongly inhibited by tropolone, a Cu competitor, and restored by the addition of Cu2+. Several factors affecting variability in leaf PPO activity levels in plants are discussed.

Optimal foraging and beyond: how starlings cope with changes in food availability.

Foraging adaptations include behavioral and physiological responses, but most optimal foraging models deal exclusively with behavioral decision variables, taking other dimensions as constraints. To overcome this limitation, we measured behavioral and physiological responses of European starlings Sturnus vulgaris to changes in food availability in a laboratory environment. The birds lived in a closed economy with a choice of two foraging modes (flying and walking) and were observed under two treatments (hard and easy) that differed in the work required to obtain food. Comparing the hard with the easy treatment, we found the following differences. In the hard treatment, daily amount of work was higher, but daily intake was lower. Even though work was greater, total daily expenditure was smaller, partly because overnight metabolism was lower. Body mass was lower, but daily oscillation in body mass did not differ. Feces' caloric density was lower, indicating greater food utilization. Energy expenditure rate expressed as multiples of basal metabolic rate (BMR) increased during the working period from 3.5 x BMR (easy) to 5.2 x BMR (hard), but over the 24-h period, it was close to 2.4 x BMR in both treatments. We also found that rate of expenditure during flight was very high in both treatments (52.3 W in easy and 45.5 W in hard), as expected for short (as opposed to cruising) flights. The relative preferences between walking and flying were incompatible with maximizing the ratio of energy gains per unit of expenditure (efficiency) but compatible with maximizing net gain per unit of time during the foraging cycle (net rate). Neither currency explained the results when nonforaging time was included. Time was not a direct constraint: the birds rested more than 90% of the time in both treatments. Understanding this complex picture requires reasoning with ecological, physiological, and cognitive arguments. We defend the role of optimality as an appropriate tool to guide this integrative perspective.

A cherry protein and its gene, abundantly expressed in ripening fruit, have been identified as thaumatin-like.

A 29-kD polypeptide is the most abundant soluble protein in ripe cherry fruit (Prunus avium L); accumulation begins at the onset of ripening as the fruit turns from yellow to red. This protein was extracted from ripe cherries and purified by size-exclusion and ion-exchange chromatography. Antibodies to the purified protein were used to screen a cDNA library from ripe cherries. Numerous recombinant plaques reacted positively with the antibodies; the DNA sequence of representative clones encoded a polypeptide of 245 amino acid residues. A signal peptide was indicated, and the predicted mature protein corresponded to the purified protein in size (23.3 kD, by mass spectrometry) and isoelectric point (4.2). A search of known protein sequences revealed a strong similarity between this polypeptide and the thaumatin family of pathogenesis-related proteins. The cherry thaumatin-like protein does not have a sweet taste, and no antifungal activity was seen in preliminary assays. Expression of the protein appears to be regulated at the gene level, with mRNA levels at their highest in the ripe fruit.

Meckel's diverticulum in Amsterdam: experience in 136 patients.

The object of this study was to establish the relation of symptomatic diverticula to the age and gender of the patients and to the presence of ectopic tissue. A total of 136 patients with surgically treated diverticula were collected from the medical charts of five Amsterdam hospitals; 51 had undergone resection because of diverticulum-related symptoms and 85 during laparotomy for other causes. Obstruction was the predominant symptom (39%) in the 51 symptomatic patients. Hemorrhage, perforation, diverticulitis, and intussusception were the other symptoms (12-14% each). Obstruction occurred mainly in patients under age 10 years and perforation in patients 10 to 30 years old. All symptoms, hemorrhage excepted, occurred two to four times more in men. Hemorrhage and perforation were associated with the presence of ectopic gastric tissue. We concluded that symptoms caused by Meckel's diverticula are mainly due to the presence of bands or ectopic gastric tissue. The symptoms manifest at an early age (77% in those under age 30) and predominantly in males. Diverticula found incidentally in patients younger than 30 years should be resected. In the older patients, resection is indicated if ectopic gastric tissue is suspected. Diverticular bands can simply be cut.

Role of Helicobacter pylori in the pathogenesis of complications of Meckel's diverticula.

OBJECTIVE: To assess the possible role of colonisation of ectopic gastric mucosa in Meckel's diverticula by Helicobacter pylori in causing inflammation, ulceration, perforation and bleeding. DESIGN: Retrospective study. SETTING: Three hospitals in Amsterdam, The Netherlands. MATERIAL: Specimens of 65 diverticula, 49 of which had been resected incidentally, and 16 of which had been thought to be the presenting feature. MAIN OUTCOME MEASURE: The presence of H. pylori in gastric mucosa. RESULTS: 19 Diverticula contained ectopic tissue, 18 gastric and one pancreatic tissue. Gastric tissue was found in 10 of the diverticula removed incidentally, and 8 of those that were thought to be symptomatic. In 5 of the 8 there were signs of complications that might have been related directly to the presence of gastric tissue (perforation--n = 3; bleeding--n = 1; and peptic stenosis--n = 1), and none contained H. pylori. H. pylori was found in only one of the 18 diverticula, in which there were also signs of gastritis. CONCLUSION: H. pylori has no role in the pathogenesis of the complications of Meckel's diverticula.

Psoriatic alopecia: acute and chronic hair loss in 47 patients with scalp psoriasis.

Symptomatic hair loss and alopecia were seen in psoriatic lesions of the scalp in 47 patients. Remarkably, in 66% of the cases it was an inaugural manifestation, and in 36% the scalp was exclusively involved. Therefore 34% of the patients presented with a primary manifestation of isolated scalp psoriasis. Hair loss varied in intensity from protracted to moderate and massive (36% in tufts). It presented as acute (51%), chronic (36%) or chronic recurrent (13%). Thirteen patients (28%) became aware of the hair loss with the beginning of therapy. The alopecia was found to be circumscribed in 75% of the cases and diffuse in 25%. In 2 cases psoriatic alopecia also manifested itself at sites other than the scalp. The telogen count was found to be increased up to 25-86% in the florid stage. Examinations under the light microscope showed a patchy perifollicular lymphohistiocytic infiltrate in the upper and middle dermis with adnexotropia in several cases. This infiltrate can alter the follicle epithelium and may lead to a granulomatous foreign-body reaction with destruction of the hair follicle. After topical antipsoriatic treatment, most of the reexamined patients showed complete hair regrowth, while 5 developed a residual scarring. Therefore, in the patient with circumscribed or diffuse symptomatic alopecia, with or without scarring, psoriatic alopecia should be considered.

Specific truncations of an acetolactate synthase gene from Brassica napus efficiently complement ilvB/ilvG mutants of Salmonella typhimurium.

The expression of an acetolactate synthase (ALS) gene isolated from the cruciferous plant Brassica napus was investigated in Salmonella typhimurium. Using an expression plasmid containing the highly active trc (trp-lac) promoter, several plant ALS constructs were made containing successive in-frame truncations from the 5' end of the coding region. Functional complementation by these plant ALS constructs of a S. typhimurium mutant devoid of ALS enzymic activity was assayed on minimal medium. Truncations which eliminated a large portion of the transit peptide coding sequence proved to act as efficient ALS genes in the bacterial host. Truncations close to the putative processing site of the plant protein were inactive in the complementation test. A full length copy of the gene, including the entire transit peptide coding region, was also inactive. The efficiency of the complementation, estimated by comparison to the growth rate of wild-type S. typhimurium, was found to correlate with levels of ALS activity in the transformed bacteria. Specific mutations, known to produce herbicide resistance in plants, were introduced into the truncated ALS coding sequence by site-directed mutagenesis. When expressed in bacteria these constructs conferred a herbicide resistance phenotype on the host. The potential of this system for mutagenesis and enzymological studies of plant proteins is discussed.

Isolation, expression and phylogenetic inheritance of an acetolactate synthase gene from Brassica napus.

An acetolactate synthase gene was isolated and characterized from Brassica napus. This B. napus acetolactate synthase gene encodes a deduced polypeptide sequence of 637 amino acids which is 85% homologous to the corresponding proposed gene product from Arabidopsis thaliana. Peptide domains recently associated with herbicide resistance/sensitivity are conserved between the two sequences. From Southern analysis we conclude that the gene isolated is one member of a multigene acetolactate synthase gene family comprising four or five members. A probe spanning the presumptive transit peptide sequence of this gene was shown by Southern analysis to hybridize to a unique sequence in the B. napus genome. This unique probe was used to analyse DNA from B. campestris and B. oleracea, the presumed progenitors of B. napus. On the basis of restriction fragment length polymorphism, we conclude that the B. napus gene isolated here originated in B. campestris. Total acetolactate synthase-homologous transcripts were analysed in a variety of B. napus tissues, and showed preferential accumulation in rapidly growing material. The genomic clone was mutated in vitro at codon 173 to replace a proline residue with serine. This was re-introduced into plants, using Agrobacterium vectors, producing a herbicide-resistant phenotype which is characteristic of the predicted gene product.

Use of protein blotting to study the DNA-binding properties of histone H1 and H1 variants.

Sub-types of histone H1 have been observed in a variety of tissues from several organisms. One of the best characterized H1 variants is H5 from avian erythrocytes. Several lines of evidence suggest that H5 has a greater affinity for DNA than H1 and is thus thought to account, in part, for the highly condensed and transcriptionally repressed state of avian erythrocyte chromatin. In trout there is an analogous erythrocyte-specific H1 variant, previously termed 'H5' [B.L.A. Miki and J.M. Neelin (1975) Can. J. Biochem. 53, 1158-1169). Using a sensitive and rapid protein-blotting procedure which is specific amongst the histones for histone H1 and its variants, we compared DNA-binding properties of the trout erythrocyte histone 'H5' and chicken H5. By increasing the NaCl concentration of the binding buffer, a gradual decrease in the amount of DNA that bound to chicken H1, trout H1 and trout erythrocyte 'H5' variant was observed, such that at concentrations above 0.37 M, negligible amounts of DNA were bound. By contrast, chicken H5 bound a significantly greater amount of DNA even at a concentration of 0.4 M NaCl. Based on the DNA-binding, properties, we conclude that the trout erythrocyte variant 'H5' is more closely related to H1 than to H5. By assaying the DNA-binding affinity of calf thymus H1 DNA-binding affinity of calf thymus H1 peptide fragments, generated by protease and chemical cleavage, and the sperm-specific H1 variants of the annelid, Platynereis dumerilii, which possess greatly shortened C-terminal tails, we conclude that a domain that includes a very small portion of the C-terminal tail and part of the globular domain is sufficient for the binding of H1 to DNA.

Synthesis of messenger-like RNA in avian erythrocyte nuclei.

Cell ghosts have been prepared from mature chicken erythrocytes using 0.05% saponin. Such preparations are capable of incorporating label from [3H]UTP and provide a system, where the nucleus is permeable to nucleotides and macromolecules, for studying the low-level RNA synthesis characteristic of these cells. RNase A (50 micrograms/ml) eliminated all radioactivity binding to DE-81 filters, indicating that the product was RNA; and DNase (10 micrograms/ml) and actinomycin D (10 micrograms/ml) each inhibited UMP incorporation by 70%, suggesting that the synthesis was DNA-dependent. Polymerization was inhibited 90% by 0.1 microgram/ml alpha-amanitin, and maximum synthesis occurred in the presence of high salt (0.175 M KCl) and Mn2+ (0.5 mM). Polyacrylamide gel electrophoresis indicated that the newly synthesized RNA was heterogeneous in size, having a distribution from 5 to 60 S with a significant fraction migrating as 8-12 S. Approximately 15% of the total RNA was bound by an oligo(dT)-cellulose column, suggesting that some RNA processing was occurring, although attempts to detect the incorporation of label from [alpha-32P]GTP into a 5'-cap structure were unsuccessful. In comparison to RNA synthesis in reticulocyte nuclei, both the rate and extent of transcription in erythrocyte nuclei were much reduced. Moreover, about 25-30% of the reticulocyte nascent RNA was released from the nuclei during a 60-min incubation, while no release was observed for the erythrocyte nuclei. Hybridization of radiolabeled RNA to excess chicken DNA indicated that the majority (80%) of the in vitro transcripts were complementary to unique sequence DNA (C0t1/2 = 4.5 X 10(3)). When RNA synthesized by either erythrocyte or reticulocyte nuclei was hybridized to cDNA complementary to reticulocyte polysomal mRNA, about 8% of the reticulocyte nuclear RNA but less than 1% of the erythrocyte nuclear RNA were resistant to RNase A digestion. Taken together, these data suggest that nuclei prepared by saponin lysis of chicken erythrocytes synthesize messenger-like RNA via endogenous polymerase II activity. A fraction of this RNA is polyadenylated but contains few, if any, globin sequences or other transcripts found on reticulocyte polysomes.