Non‐spherical pearl layers in the Polynesian ‘black‐lipped’ Pinctada margaritifera: The non‐nacreous deposits compared to microstructure of the shell growing edge

Cultivated pearls frequently exhibit morphological irregularities making obvious that mineral deposition was irregularly distributed onto nucleus surface. Taking advantage of experimental cultivations with short durations (from 10 days to few months), these irregular deposits predating occurrence of the nacre were investigated in Polynesian pearls by biochemical characterizations and a series of physical methods. Diversity in the resulting data suggests that various in‐depth alterations of the biomineralization mechanism may have occurred during the grafting process, leading to diversity in the biochemical pathways to nacreous deposition. This allows a precise discussion of current views about pearl formation. The “reversed shell theory” is formally disproved through point to point comparison with development of the shell growing edge. Similarity of pearl formation with “regeneration” or “shell repair” is also discussed, emphasizing the differences between these concepts.     

Genetic diversity in seven populations of Nicaraguan teosinte (<Emphasis Type="Italic">Zea nicaraguensis</Emphasis> Iltis et Benz) as estimated by microsatellite variation

Teosintes are the closest relatives to modern maize, Zea mays L. ssp. mays. They are wild grasses with a native distribution area from Mexico to Nicaragua and represent an important genetic resource. However, the genetic diversity of Nicaraguan teosinte (Zea nicaraguensis Iltis et Benz) has not yet been determined. This teosinte species has decreased in the last 25 years and now must be regarded as an endangered species. An analysis of the genetic diversity of Zea nicaraguensis was carried out in a total of 240 individuals from seven populations. Eleven Simple Sequences Repeat (SSR) primer pairs were used. A total of 42 alleles were found, the range of alleles per locus was 2–5 (mean 3.8) and the numbers of genotypes varied between primers. The primer Bnlg 1538 showed the highest value, with 45 genotypes through all populations. The genetic diversity observed (Ho) between all populations varied from 0.51 to 0.63, with an average of 0.563. One of the populations had as many as 40 alleles. Analysis of molecular variance (AMOVA) revealed that most of the variation was within population, at a significantly high level (P < 0.001). Rare alleles were detected in all populations, but unique alleles were only found in four populations. These results are highly relevant when developing conservation strategies and show that preserving populations in their natural habits is highly important.     

Heparanase and Syndecan-4 Are Involved in Low Molecular Weight Fucoidan-Induced Angiogenesis

Induction of angiogenesis is a potential treatment for chronic ischemia. Low molecular weight fucoidan (LMWF), the sulfated polysaccharide from brown seaweeds, has been shown to promote revascularization in a rat limb ischemia, increasing angiogenesis in vivo. We investigated the potential role of two heparan sulfate (HS) metabolism enzymes, exostosin-2 (EXT2) and heparanase (HPSE), and of two HS-membrane proteoglycans, syndecan-1 and -4 (SDC-1 and SDC-4), in LMWF induced angiogenesis. Our results showed that LMWF increases human vascular endothelial cell (HUVEC) migration and angiogenesis in vitro. We report that the expression and activity of the HS-degrading HPSE was increased after LMWF treatment. The phenotypic tests of LMWF-treated and EXT2- or HPSE-siRNA-transfected cells indicated that EXT2 or HPSE expression significantly affect the proangiogenic potential of LMWF. In addition, LMWF increased SDC-1, but decreased SDC-4 expressions. The effect of LMWF depends on SDC-4 expression. Silencing EXT2 or HPSE leads to an increased expression of SDC-4, providing the evidence that EXT2 and HPSE regulate the SDC-4 expression. Altogether, these data indicate that EXT2, HPSE, and SDC-4 are involved in the proangiogenic effects of LMWF, suggesting that the HS metabolism changes linked to LMWF-induced angiogenesis offer the opportunity for new therapeutic strategies of ischemic diseases.     

Chitinolytic Bacteria-Assisted Conversion of Squid Pen and Its Effect on Dyes and Pigments Adsorption

The aim of this work was to produce chitosanase by fermenting from squid pen, and recover the fermented squid pen for dye removal by adsorption. One chitosanase induced from squid pen powder (SPP)-containing medium by Bacillus cereus TKU034 was purified in high purification fold (441) and high yield of activity recovery (51%) by ammonium sulfate precipitation and combined column chromatography. The SDS-PAGE results showed its molecular mass to be around 43 kDa. The TKU034 chitosanase used for the chitooligomers preparation was studied. The enzyme products revealed that the chitosanase could degrade chitosan with various degrees of polymerization, ranging from 3 to 9, as well as the chitosanase in an endolytic manner. Besides, the fermented SPP was recovered and displayed a better adsorption rate (up to 99.5%) for the disperse dyes (red, yellow, blue, and black) than the water-soluble food colorants, Allura Red AC (R40) and Tartrazine (Y4). The adsorbed R40 on the unfermented SPP and the fermented SPP was eluted by distilled water and 1 M NaOH to confirm the dye adsorption mechanism. The fermented SPP had a slightly higher adsorption capacity than the unfermented, and elution of the dye from the fermented SPP was easier than from the unfermented. The main dye adsorption mechanism of fermented SPP was physical adsorption, while the adsorption mechanism of unfermented SPP was chemical adsorption.     

Reproducible infection model for Clostridium perfringens in broiler chickens

Experiments were carried out to establish an infection and disease model for Clostridium perfringens in broiler chickens. Previous experiments had failed to induce disease and only a transient colonization with challenge strains had been obtained. In the present study, two series of experiments were conducted, each involving four groups of chickens with each group kept in separate isolators. A coccidial vaccine given at 10 times the prescribed dosage was used to promote the development of necrotic enteritis. In the first experiment, cultures of C. perfringens were mixed with the feed at day 9, 10, 11, and 12, and the coccidial vaccine was given at day 10, whereas in the second experiment, C. perfringens cultures were mixed with the feed at day 17, 18, 19, and 20, and the coccidial vaccine was given at day 18. Chickens were examined at day 9, 11, 12, and 15 (Experiment 1), and at day 17, 18, 20, and 24 (Experiment 2). There was no mortality in any of the groups; however, chickens in the groups receiving both coccidial vaccine and C. peifringens developed the subclinical form of necrotic enteritis, demonstrated by focal necroses in the small intestine, whereas chickens in control groups or groups receiving only coccidial vaccine or only C. perfringens cultures developed no necroses. The results underline the importance of predisposing factors in the development of necrotic enteritis.     

Localization of Insulin‐Like Growth Factor‐I (IGF‐I) and IGF‐I Receptor (IGF‐IR) in Equine Testes

The insulin‐like growth factor‐I (IGF‐I) is a key regulator of reproductive functions. IGF‐I actions are primarily mediated by IGF‐IR. The main objective of this research was to evaluate the presence of IGF‐I and IGF‐I Receptor (IGF‐IR) in stallion testicular tissue. The hypotheses of this study were (i) IGF‐I and IGF‐IR are present in stallion testicular cells including Leydig, Sertoli, and developing germ cells, and (ii) the immunolabelling of IGF‐I and IGF‐IR varies with age. Testicular tissues from groups of 4 stallions in different developmental ages were used. Rabbit anti‐human polyclonal antibodies against IGF‐I and IGF‐IR were used as primary antibodies for immunohistochemistry and Western blot. At the pre‐pubertal and pubertal stages, IGF‐I immunolabelling was present in spermatogonia and Leydig cells. At post‐pubertal, adult and aged stages, immunolabelling of IGF‐I was observed in spermatogenic cells (spermatogonia, spermatocyte, spermatid, and spermatozoa) and Leydig cells. Immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at the pre‐pubertal stage. The immunolabelling becomes stronger as the age of animals advance through the post‐pubertal stage. Strong immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at post‐puberty, adult and aged stallions; and faint labelling was seen in spermatocytes at these ages. Immunolabelling of IGF‐I and IGF‐IR was not observed in Sertoli cells. In conclusion, IGF‐I is localized in equine spermatogenic and Leydig cells, and IGF‐IR is present in spermatogonia, spermatocytes and Leydig cells, suggesting that the IGF‐I may be involved in equine spermatogenesis and Leydig cell function as a paracrine/autocrine factor.     

Quantitative requirement of Penaeus kerathurus for a natural unprocessed diet

Juvenile Penaeus kerathurus (average weight = 0.64 g) were fed three different daily amounts (40%, 60% and 100% of biomass) of an unprocessed and unsupplemented diet composed of 43% anchovy, 33% shrimp head and 24% squid, whose suitability for P. kerathurus had been previously assessed. The experiment was performed with an animal density of 30/m² at 23° C for a period of 180 days. From the results obtained, it is concluded that, of the three feeding rates employed, 100% of biomass was suitable for animals weighing 0.64–1.8 g. For animals 1.8–4.3 g a feeding rate of 60% was appropriate, while a reduction to 40% seemed satisfactory for animals over 4.3 g in this experiment.     

Antioxidant properties and phytochemical characteristics of extracts from Lactuca indica

Lactuca indica (Compositae) is an edible wild vegetable, used as a folk medicine in anti-inflammatory, antibacterial, and other medications in Asia. This is the first scientific evaluation of the chemopreventive therapeutic properties of L. indica using five antioxidation assay systems. An extract from L. indica was found to possess significant free radical scavenging activity, effectively protecting phix174 supercoiled DNA against strand cleavage and reducing oxidative stress in human promyelocytic leukemia HL-60 cells. Moreover, extracts of L. indica almost totally inhibited nitric oxide production and the mRNA expression of inducible nitric oxide synthase, at a dosage of 100 microg/mL, in LPS-stimulated macrophage RAW264.7 cells. Bioactivity-guided chromatographic fractionation and metabolite profiling coupled with spectroscopic analyses revealed that the six phenolic compounds, that is, protocatechulic acid (1), methyl p-hydroxybenzoate (2), caffeic acid (3), 3,5-dicaffeoylquinic acid (4), luteolin 7-O-beta-glucopyranoside (5), and quercetin 3-O-beta-glucopyranoside (6), are the major antioxidative constituents in the L. indica extract.