Genetic diversity of four populations of Qualea grandiflora Mart. in fragments of the Brazilian Cerrado

We analyzed the genetic structure and diversity of Qualea grandiflora Mart., the most abundant woody species in the Brazilian Cerrado. Eight microsatellite loci were used to analyze samples from four populations subjected to different types of anthropic pressure, distributed throughout the state of São Paulo in the regions of Assis, Brotas, Itirapina and Pedregulho. Results indicated a mean number of 12 alleles per locus, but only six effective alleles. Alleles private to particular populations and rare alleles were also detected. An excess of homozygotes and moderate levels of inbreeding were observed. No clones were identified. All populations departed from Hardy–Weinberg equilibrium (p < 0.05). Spatial structure was observed in the distribution of specimens in distance classes ranging from 30 to 40 km and three genetic clusters were identified, with genotypes in the Pedregulho population differing from the others by up to 90 %. The influence of the Wahlund effect on the studied populations lies between 8.5 and 53.3 %. Estimates of effective population size were low (<10), and the minimum viable area for conservation in the short-, medium- and long-term was estimated to be between 4 and 184 ha. Gene flow was high enough to counter the effects of genetic drift. The genetic diversity and divergence between the studied populations indicated that the Pedregulho population should be considered an Evolutionary Significant Unit and a Management Unit.     

LC3B is indispensable for selective autophagy of p62 but not basal autophagy

Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs.     

Development of Multiplex PCR to Detect Five Pythium Species Related to Turfgrass Diseases

The objective of this study was to develop multiplex PCR detection method for five Pythium species associated with turfgrass diseases, Pythium aphanidermatum, Pythium arrhenomanes, Pythium graminicola, Pythium torulosum and Pythium vanterpoolii. Species‐specific primers and two common primers were designed based on the sequences of the internal transcribed spacer region of ribosomal DNA. Another primer set by which all organisms would be amplified in 18S rDNA was used as a positive control. When these total nine primers were applied to the multiplex PCR, all species were individually discriminated in the mixture of five species culture DNA. Furthermore, all five Pythium species were detected in naturally infected plants using the multiplex PCR.     

Polymerase chain reaction detection of Clostridium perfringens in feces from captive and wild chimpanzees, Pan troglodytes

Background For veterinary management of non‐human primates in captivity, and conservation of wild‐living primates, management of their health risks is necessary. Incidences of pathogenic bacteria in the fecal specimens are considered as one of the useful indicators for non‐invasive health monitoring. Methods We carried out the detection of Clostridium perfringens in feces from captive and wild chimpanzees by the rapid polymerase chain reaction method. Results The bacterium was detected in most fecal specimens (80%) in captive chimpanzees. Contrarily, the detection rate in the wild chimpanzees was low, with 23% (n = 12) of 53 fecal samples from the Bossou group, Guinea, and 1.2% (1/81) in the Mahale group, Tanzania. Conclusions These results show that the intestinal microflora differs between Pan populations under various living conditions, being influenced by their diet and environment.     

Anti-tumor and anti-invasive effects of diverse delta-alkyllactones: dependence on molecular side-chain length, action period and intracellular uptake

New delta-alkyllactones (DALs) with diverse side-chain lengths (184-254 Da), which are structurally different from the widespread, naturally occurring delta-lactones of higher molecular weight (348-439 Da), such as camptothecin and sultriecin, were chemically synthesized and analyzed for their carcinostatic activity. Of the DALs with 11, 12, 13, 14, or 16 carbon atoms, delta-hexadecalactone (DH16:0) was the most carcinostatic when administered to Ehrlich ascites tumor (EAT) cells at 37 degrees C for 20 h, and measured by the mitochondrial dehydrogenase-based WST-1 assay. Prolongation of the administration period to 72 h enhanced the carcinostatic activity more markedly for DH16:0 than for other DALs. The carcinostatic activity of DALs was unexpectedly augmented by increasing the number of carbon atoms, in contrast to the conventional view that carcinostatic activity is attenuated by the addition of carbon atoms to fatty acids. Intracellular accumulation of DH16:0, as analyzed by gas chromatography, was detected (1.5 Pg/cell), whereas other DALs studied were rarely found. The results indicate a close relationship between carcinostatic activity and intracellular accumulation. Invasion of human fibrosarcoma HT-1080 cells through the reconstituted basement membrane was inhibited by several DALs, even at doses as low as 5-10% of those necessary for carcinostatic activity, suggesting an invasive mechanism different from carcinostasis. The invasion-inhibitory activity was intensified by increasing the number of carbon atoms, in a manner similar to that for the carcinostatic activity. The lifespan of EAT-cell-transplanted mice was markedly prolonged with DH16:0, presumably due to excellent distribution throughout the body and tumor cells. Thus DH16:0 may be a potent anticancer agent, in term of its carcinostatic, anti-invasive, and lifespan-prolonging activities.     

Synthesis of hybrid molecules of caffeine and eudistomin D and its effects on adenosine receptors

Four hybrid molecules (1 and 12-14) of caffeine and eudistomin D, a beta-carboline alkaloid from a marine tunicate, were synthesized, and their affinity and selectivity for adenosine receptors A(1), A(2A), and A(3) were examined. It was found that all the compounds showed better potency as adenosine receptor ligands as compared with caffeine. Among them, a compound (13) possessing a nitrogen at the delta-position of the pyridine ring (delta-N type) showed the most potent affinity for adenosine receptor A(3) subtype, while N-methylation (14) of a pyrrole ring in 13 significantly lowered the potency as adenosine receptor ligands. Compounds (1 and 12) having a nitrogen at the beta-position of the pyridine ring (beta-N type) showed lower affinity than the corresponding delta-N type compounds (13 and 14), while compounds (10, 11, and 17) lacking a pyrrole ring between the pyridine and pyrimidine rings exhibited almost no affinity to the adenosine receptor subtypes examined.     

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

Lyconadins D and E, and complanadine E, new Lycopodium alkaloids from Lycopodium complanatum

Three new Lycopodium alkaloids, lyconadins D (1) and E (2), and complanadine E (3), were isolated from the club moss Lycopodium complanatum. Lyconadin D (1) was the first example of fastigiatine-type alkaloid isolated from Lycopodium complanatum. The structures and relative stereochemistry of 1-3 were elucidated on the basis of spectroscopic data. Complanadine E (3) enhanced mRNA expression for NGF.