AFLP markers as a tool to reconstruct complex relationships: A case study in Rosa (Rosaceae)

The genus Rosa has a complex evolutionary history caused by several factors, often in conjunction: extensive hybridization, recent radiation, incomplete lineage sorting, and multiple events of polyploidy. We examined the applicability of AFLP markers for reconstructing (species) relationships in Rosa, using UPGMA clustering, Wagner parsimony, and Bayesian inference. All trees were well resolved, but many of the deeper branches were weakly supported. The cluster analysis showed that the rose cultivars can be separated into a European and an Oriental cluster, each being related to different wild species. The phylogenetic analyses showed that (1) two of the four subgenera (Hulthemia and Platyrhodon) do not deserve subgeneric status; (2) section Carolinae should be merged with sect. Cinnamomeae; (3) subsection Rubigineae is a monophyletic group within sect. Caninae, making sect. Caninae paraphyletic; and (4) there is little support for the distinction of the five other subsections within sect. Caninae. Comparison of the trees with morphological classifications and with previous molecular studies showed that all methods yielded reliable trees. Bayesian inference proved to be a useful alternative to parsimony analysis of AFLP data. Because of their genome-wide sampling, AFLPs are the markers of choice to reconstruct (species) relationships in evolutionary complex groups.     

A novel terrestrial fish habitat inside emergent logs

Reports of new habitats for a major group of organisms are rare. Fishes display diverse adaptations for temporary (amphibious) existence on land, but to our knowledge, none have ever been reported regularly living inside emergent logs. Here, we show that the mangrove killifish, Kryptolebias marmoratus, a species previously known to emerse (leave the water) regularly, is now known to emerse and aggregate in large numbers inside decaying mangrove logs that have been "galleried" by terrestrial insects. This behavior has now been documented in both Belize, Central America, and Florida, U.S.A., populations and represents the first known case of fishes entering terrestrial woody material. The dense packing of fish in the narrow log galleries may imply a novel social context in which intraspecific aggressive behaviors are reduced, possibly mediated by the physiological limitations imposed within this restrictive habitat.     

Virulence and antibiotic susceptibility of Aeromonas spp. isolated from drinking water

Aeromonas isolates from tap water, mineral water, and artesian well water were investigated for their ability to produce different potential virulence factors or markers such as hemolysins, cytotoxins, phospholipase, DNase, hydrophobicity and their ability to adhere to epithelial cells and to abiotic surfaces. The susceptibility to antibiotics of Aeromonas isolates was also examined. Majority of the isolates displayed hemolytic activity against sheep erythrocytes, while only 7 of the 23 Aeromonas strains displayed DNase activity and 4 of the 23 Aeromonas strains tested were regarded as positive for phospholipase production. Most of the isolates showed cytotoxic activities in culture filtrate dilutions at titer of 1/8 or lower. No general relation between the strain isolated and the ability to interact with epithelial cells could be established. Using the bacterial adherence to hydrocarbons method, most of the strains were classified as highly hydrophilic. All five Aeromonas jandaei strains isolates, 9 of the 12 Aeromonas sp strains and four of the five Aeromonas hydrophila were multidrug resistant. The most active antimicrobial was ciprofloxacin (susceptible in 100% of the isolates), and the least active antibiotic was ampicillin (resistance in 92% of the isolates). The majority of the isolates tested were not killed by chlorine at 1.2 mg/l. Whether the high tolerance to chlorine of Aeromonas isolates can be linked to greater virulence is not know.     

Comparative study of catalase-peroxidases (KatGs)

Catalase-peroxidases or KatGs from seven different organisms, including Archaeoglobus fulgidus,Bacillus stearothermophilus, Burkholderia pseudomallei, Escherichia coli, Mycobacterium tuberculosis, Rhodobacter capsulatus and Synechocystis PCC 6803, have been characterized to provide a comparative picture of their respective properties. Collectively, the enzymes exhibit similar turnover rates with the catalase and peroxidase reactions varying between 4900 and 15,900 s⁻¹ and 8-25 s⁻¹, respectively. The seven enzymes also exhibited similar pH optima for the peroxidase (4.25-5.0) and catalase reactions (5.75), and high sensitivity to azide and cyanide with IC₅₀ values of 0.2-20 μM and 50-170 μM, respectively. The K_Ms of the enzymes for H₂O₂ in the catalase reaction were relatively invariant between 3 and 5 mM at pH 7.0, but increased to values ranging from 20 to 225 mM at pH 5, consistent with protonation of the distal histidine (pKa approximately 6.2) interfering with H₂O₂ binding to Cpd I. The catalatic k_cat was 2- to 3-fold higher at pH 5 compared to pH 7, consistent with the uptake of a proton being involved in the reduction of Cpd I. The turnover rates for the INH lyase and isonicotinoyl-NAD synthase reactions, responsible for the activation of isoniazid as an anti-tubercular drug, were also similar across the seven enzymes, but considerably slower, at 0.5 and 0.002 s⁻¹, respectively. Only the NADH oxidase reaction varied more widely between 10⁻⁴ and 10⁻² s⁻¹ with the fastest rate being exhibited by the enzyme from B. pseudomallei.     

IL-4/Stat6 activities correlate with apoptosis and metastasis in colon cancer cells

IL-4-induced Stat6 signaling is active in a variety of cell types and plays a role in cell proliferation/growth and resistance to apoptosis. Using EMSA, we identified differential IL-4/Stat6 activities in colorectal cancer cell lines, HT-29 being active Stat6^high phenotype and Caco-2 being defective Stat6^null phenotype, respectively. Active Stat6^high HT-29 cells exhibited resistance to apoptosis by flowcytometry and aggressive metastasis by Transwell assay compared with defective Stat6^null Caco-2 cells. Comparing one another using RT-PCR, Stat6^high HT-29 cells expressed more mRNA of anti-apoptotic and pro-metastatic genes Survivin, MDM2, and TMPRSS4, while Stat6^null Caco-2 cells expressed more mRNA of pro-apoptotic and anti-metastatic genes BAX, CAV1, and P53, respectively. This is the first study describing correlations of IL-4/Stat6 activities with apoptosis and metastasis in colon cancer. These findings, together with the observation of constitutive Stat6 activation in many human malignancies, suggest that Stat6 activities could be a biomarker for cancer cell’s invasive/metastatic capability.     

Identification of candidate regulators of multipotency in human skeletal progenitor cells

Stem cell differentiation is controlled intrinsically by dynamic networks of interacting lineage-specifying and multipotency genes. However, the relationship between internal genetic dynamics and extrinsic regulation of internal dynamics is complex and, in the case of skeletal progenitor cell differentiation, incompletely understood. In this study we elucidate a set of candidate markers of multipotency in human skeletal progenitor cells by systematic study of the relationships between gene expression and environmental stimulus. We used full genome cDNA microarrays to explore gene expression profiles in skeletal progenitor enriched populations derived from adult human bone marrow, minimally cultured in basal, osteogenic, chondrogenic, and adipogenic lineage-specifying culture conditions. We then used a variety of statistical clustering procedures to identify a small subset of genes which are related to these stromal lineages but are specific to none. For a selection of 11 key genes, conclusions of the microarray study were confirmed using quantitative real-time PCR.     

The contribution of naturally occurring polymorphisms in altering the biochemical and structural characteristics of HIV-1 subtype C protease

Fourteen subtype B and C protease variants have been engineered in an effort to study whether the preexistent baseline polymorphisms, by themselves or in combination with drug resistance mutations, differentially alter the biochemical and structural features of the subtype C protease when compared with those of subtype B protease. The kinetic studies performed in this work showed that the preexistent polymorphisms in subtype C protease, by themselves, do not provide for a greater level of resistance. Inhibition analysis with eight clinically used protease inhibitors revealed that the natural polymorphisms found in subtype C protease, in combination with drug resistance mutations, can influence enzymatic catalytic efficiency and inhibitor resistance. Structural analyses of the subtype C protease bound to nelfinavir and indinavir showed that these inhibitors form similar interactions with the residues in the active site of subtype B and C proteases. It also revealed that the naturally occurring polymorphisms could alter the position of the outer loops of the subtype C protease, especially the 60's loop.     

Novel phospholipolytic activities associated with electronegative low-density lipoprotein are involved in increased self-aggregation

Electronegative low-density lipoprotein (LDL(-)) is a minor LDL subfraction present in plasma with increased platelet-activating factor acetylhydrolase (PAF-AH) activity. This activity could be involved in the proinflammatory effects of LDL(-). Our aim was to study the presence of additional phospholipolytic activities in LDL(-). Total LDL was fractionated into electropositive (LDL(+)) and LDL(-) by anion-exchange chromatography, and phospholipolytic activities were measured by fluorometric methods. Phospholipolytic activity was absent in LDL(+) whereas LDL(-) presented activity against lysophosphatidylcholine (LPC, 82.4 +/- 34.9 milliunits/mg of apoB), sphingomyelin (SM, 53.3 +/- 22.5 milliunits/mg of apoB), and phosphatidylcholine (PC, 25.7 +/- 4.3 milliunits/mg of apoB). LDL(-), but not LDL(+), presented spontaneous self-aggregation at 37 degrees C in parallel to phospholipid degradation. This was observed in the absence of lipid peroxidation and suggests the involvement of phospholipolytic activity in self-aggregation of LDL(-). Phospholipolytic activity was not due to PAF-AH, apoE, or apoC-III and was not increased in LDL(+) modified by Cu (2+) oxidation, acetylation, or secretory phospholipase A 2 (PLA 2). However, LDL(-) efficiently degraded phospholipids of lipoproteins enriched in LPC, such as oxidized LDL or PLA 2-LDL, but not native or acetylated LDL. This finding supports that LPC is the best substrate for LDL(-)-associated phospholipolytic activity. These results reveal novel properties of LDL(-) that could play a significant role in its atherogenic properties.     

Neck Circumference as a Simple Screening Measure for Identifying Overweight and Obese Patients

Objective: There are numerous methods of assessing overweight and obesity. We undertook an observational study to test a method of identifying overweight or obese patients solely by measuring the circumference of the neck. Research Methods and Procedures: A test sample and a second validation sample included 979 subjects (460 men and 519 women), who visited a family medicine clinic in a southern Israeli urban district for any reason between the randomly chosen months of January and September 1998. Main outcome included neck, waist, and hip circumferences; body mass index (BMI); and waist:hip ratio measures. Results: Pearson's correlation coefficients indicated a significant association between neck circumference (NC) and: BMI (men, r = 0.83; women, r = 0.71; each, p < 0.0001), age (men, r = 0.33; women, r = 0.36; each, p < 0.0001), weight (men, r = 0.7; women, r = 0.81; each, p < 0.0001), waist circumference (men, r = 0.86; women, r = 0.85; each, p < 0.0001), hip circumference (men, r = 0.62; women, r = 0.56; each, p < 0.0001), and waist:hip ratio (men, r = 0.66; women, r = 0.87; each, p < 0.0001). NC ≥37 cm for men and ≥34 cm for women were the best cutoff levels for determining the subjects with BMI ≥25.0 kg/m² using the receiver output curve analysis. In the validation unrelated group, the test characteristics were excellent with 98% sensitivity, 89% specificity, and 94% accuracy for men, and 100% sensitivity, 98% specificity, and 99% accuracy for women. NC ≥39.5 cm for men and ≥36.5 cm for women were the best cutoff levels for determining the subjects with BMI ≥30 kg/m² using the receiver output curve analysis. In the validation unrelated group, the test characteristics were excellent with 93% sensitivity, 90% specificity, and 91% accuracy for men, and 93% sensitivity, 98% specificity, and 97% accuracy for women. Discussion: NC measurement is a simple and time‐saving screening measure that can be used to identify overweight and obese patients. Men with NC <37 cm and women with NC <34 cm are not to be considered overweight. Patients with NC ≥37 cm for men and ≥34 cm for women require additional evaluation of overweight or obesity status.