Reproductive strategy of the European perch,Perca fluviatilis Linnaeus, 1758 (Osteichthyes: Percidae) in the Anzali wetland,southwest Caspian Sea

Temporal variation in reproductive traits of geographically distributed fish is supposed to take place in response to the spatial and environmental variations. With regard to the wide distribution of the European perch in the northern hemisphere, important reproductive traits such as the initiation and duration of the spawning activity are likely to vary in different latitudinal gradients. In this study, reproductive biology of the European perch, Perca fluviatilis, is described, based on 324 specimens caught in the Anzali wetland (southwest Caspian Sea) between June 2008 and May 2009. The gonadosomatic index, oocyte frequency distribution and histological examination suggested a long vitellogenic process (October to February) and a short spawning season (January and February). The size‐frequency distribution of the oocytes showed that this perch is a species with group‐synchronous ovarian development. Ovarian development occurred only in one clutch of oocytes (700–900 μm oocyte diameter) with no indication of maturation of any subsequent clutch in the spawning season. The average of (realized) fecundity (±SD) was estimated to be 16177 ± 5846 eggs in late vitellogenic stage, which was lower than the potential fecundity (17188 ± 6917 eggs). Histological examination of the gonads revealed the existence of atretic oocytes in early vitellogenic stages (October and November). This investigation highlights the temporal variation in the initiation and duration of the reproductive activity of the European perch in this region compared to other geographical regions. The results emphasize the necessity of specific temporal management in fishing of European perch based on spatial differences in reproductive biology.     

Dynamics of vitellogenin synthesis in juvenile giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin (Vg) mRNA expression and patterns of Vg and vitellin distribution in the hepatopancreas and ovary of juvenile Macrobrachium rosenbergii were examined using real-time RT-PCR and immunohistochemical methods. Eyestalk ablation was seen to induce rapid development of the gonads and Vg synthesis in females. In the female hepatopancreas, Vg mRNA expression was observed several days following ablation, after which levels increased gradually with increasing gonadosomatic index (GSI). Vitellin accumulation in the oocytes also increased with increasing Vg mRNA synthesis; expression was however negligible in the ovary. Hemolymph Vg levels in females ranged from 0.04 to 2.2 mg/ml. SDS PAGE/Western blotting analysis of hemolymph samples revealed that juvenile Vg was composed of 199 and 90 kDa subunits; the 102 kDa subunit present in adult female Vg (Okuno et al., 2002. J Exp Zool 292:417-429) could not be detected at any stage of vitellogenesis in juveniles. Vg was not detectable in non-ablated juveniles. The results of this study confirmed that the mode of involvement of eyestalk factors in regulating vitellogenesis is intrinsic to both juveniles and adults, and that a basic pattern of Vg synthesis and processing is conserved. However, the fact that juveniles are not able to produce the same Vg levels observed in adult females, and do not reach high GSI levels culminating in spawning suggests that other factors and physiological conditions specific to adult females are necessary to demonstrate full reproductive ability.     

Characterization and comparison of raft-like membranes isolated by two different methods from rat submandibular gland cells

Lipid rafts are defined as cholesterol and sphingolipid enriched domains in biological membranes. Their role in signalling and other cellular processes is widely accepted but the methodology used for their biochemical isolation and characterization remains controversial. Raft-like membranes from rat submandibular glands were isolated by two different protocols commonly described in the literature; one protocol was based on selective solubilization by Triton X-100 at low temperature and the other protocol consisted in extensive sonication. In both cases a low density vesicular fraction was obtained after ultracentrifugation in a sucrose density gradient. These fractions contained about 20% of total cholesterol but less than 8% of total proteins, and were more rigid than bulk membranes. Fatty acid analyses revealed a similar composition of raft-like membranes isolated by the two different methods, which was characterized by an enrichment in saturated fatty acids in detriment of polyunsaturated acids when compared with the whole cell membranes. Protein profile of detergent resistant membranes or raft-like membranes prepared by sonication was assessed by silver staining after SDS-PAGE and by MALDI-TOF. Both analyses provided evidence of a different protein composition of the Triton X-100 and sonication preparations. Immunoblot experiments revealed that raft-like membranes prepared by detergent extraction or sonication were free of Golgi apparatus or endoplasmic reticulum protein markers (β-COP and calnexin, respectively) and that they were not substantially contaminated by transferrin receptor (a non-raft protein). While caveolin-1 was highly enriched in raft-like membranes prepared by the two methods, the P2X₇ receptor was enriched in raft-like membrane fractions prepared by sonication, but almost undetectable in the detergent resistant membranes. It can be concluded that both methods can be used to obtain raft-like membranes, but that detergent may affect protein interactions responsible for their association with different membrane domains.     

Anti-apoptotic factor humanin is expressed in the testis and prevents cell-death in leydig cells during the first wave of spermatogenesis

Humanin (HN) is a 24 amino acids peptide with potent neuro-survival properties that protects against damage associated with Alzheimer's disease. In the present report, we have demonstrated by immunohistochemical analysis and Western blotting the pattern of expression of rat humanin (HNr) in the testis of 10- to 60-day-old rats. The Leydig cells of 10- and 40- day-old rats expressed this peptide at high levels; and in the testis of 60-day-old rats the expression of HNr expanded to include Leydig, endothelial, peritubular and germ cells. As monitored by Western blotting, HNr was released into the medium of cultures of Leydig cells isolated from 10-, 40-, and 60-days-old rats. HNr stimulated the incorporation of [(3)H]TdR into DNA of Leydig cells from 10-days-old rats, in a manner that indicated promotion of cell survival rather than an increase in the rate of cell multiplication. This peptide also enhanced steroidogenesis by cultured Leydig cells from 10- to 40-day-old rats both alone and synergistically with IGF-I. The expression of HNr in cultured Leydig cells increased in response to GH and IGF-I. In summary, we demonstrated here that HNr was expressed at all stages of maturation in the rat testis. This peptide promoted the survival of Leydig cells in culture and interacted with IGF-I to stimulate DNA synthesis and steroidogenesis. We propose that HNr is a novel testicular anti-apoptotic factor.     

Application of enriched <Superscript>137</Superscript>Ba tracer to mark juvenile Persian sturgeon (<Emphasis Type="Italic">Acipenser persicus</Emphasis>)

The present study evaluates the variations of ¹³⁷Ba abundance in pectoral fin spine of 1-month-old juvenile Persian sturgeon (Acipenser persicus) upon marking using the stable isotope approach. The marking of the fish was achieved by incorporation of ¹³⁷Ba²⁺ in the calcified lattice of the pectoral fin spine through substitution with structural Ca²⁺. This process was carried out by rearing juveniles in treatment tanks containing elevated concentrations of ¹³⁷Ba for 1, 3 and 5 days. The marked fish were then retained in natural abundance fresh and brackish waters, to evaluate the trend of exchange of ¹³⁷Ba from the fin spines. The abundance of ¹³⁷Ba in fin spines during marking and post-marking experiments were detected by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that a significant isotope mark can be obtained with no mortality and 100% marking rate on the first day of exposure to the isotope. The marked juveniles maintained their isotopic signature for at least 25 days. Statistical analysis of the obtained ¹³⁸Ba/¹³⁷Ba ratios demonstrated that the successful incorporation of ¹³⁷Ba²⁺ in pectoral fin spines provides an effective marking method for Persian sturgeon restocking programs.     

An ab initio molecular orbital study on the sequence-dependency of DNA conformation: an evaluation of intra- and inter-strand stacking interaction energy

The various nearest neighbor stacking interaction energies of stacked base pairs in the DNA double helix are calculated for both A- and B-type conformations using an ab initio molecular orbital method. It is demonstrated that the sequence-dependent conformational preference for A- or B-type results from the stacking interaction. In particular, the base sequence showing the highest preference for an A-type conformation is revealed as GC/GC, and the one with the next highest preference, AT/AT; for a B-type conformation, the respective sequences are CG/CG and CA/TG. The overall conformation of a DNA fragment is not determined by these particular sequences only but is influenced by all base pair steps. An intrinsically favorable conformation is predicted from the constituent stacking interaction.     

Development of a Solid Medium for Growth and Isolation of Axenic Microcystis Strains (Cyanobacteria)

Solid media on a base of B-12 or CB medium with agarose or agarose of low melting temperature were developed for the cultivation of Microcystis species. The media with 0.4% gel showed the highest number of CFU, and increasing the gel concentration resulted in a reduction of the number of CFU. There was no difference in the numbers of CFU between pour and spread plates made of the solid media. By using the solid media, 31 clones of Microcystis species were isolated from natural blooms in Lake Kasumigaura, and 5 axenic strains (1 of M. wesenbergii and 4 of M. aeruginosa) were established from the clones.     

Antioxidant and nitric oxide synthase activation properties of Auricularia auricula

In vitro evaluation of antioxidant activities of Auricularia auricula showed significant inhibition of lipid peroxidation, and potent hydroxyl radical scavenging activity when compared with standard drug catechin. IC5o value of crude, boiled and ethanolic extracts of A. auricula represented 403, 510, and 373 microg/ml respectively in case of hydroxyl radical scavenging activity and 310, 572 and 398 microg/ml respectively in case of lipid peroxidation. Furthermore, crude, boiled and ethanolic extracts also increase significantly nitric oxide production (664, 191 and 850 pmole/mg dry wt/hr respectively) over the control. The present results revealed that A. auricula had potential therapeutic use.     

Interleukin-17F affects cartilage matrix turnover by increasing the expression of collagenases and stromelysin-1 and by decreasing the expression of their inhibitors and extracellular matrix components in chondrocytes

Interleukin (IL)-17, a proinflammatory cytokine, is produced primarily by activated Th17 cells. IL-17 consists of six ligands that signal through five receptors (IL-17Rs); IL-17A and IL-17F share the highest homology in the family. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during cartilage remodeling whereas tissue inhibitor of metalloproteinases (TIMPs) inhibit the action of MMPs. In the present study, we examined the effect of IL-17F on the degradation and synthesis of the extracellular matrix in cartilage using human articular chondrocytes. We examined the effect of IL-17F on the expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and cyclooxygenases (COXs), as well as on prostaglandin E₂ (PGE₂) production. We also examined the indirect effect of PGE₂ on the above IL-17F-induced/reduced components using NS-398, a specific inhibitor of COX-2. Cells were cultured with or without IL-17F in the presence or absence of either an IL-17R antibody or NS-398 for up to 28 days. Expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and COXs at mRNA and protein levels was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. PGE₂ production was determined by ELISA. The expression of all types of IL-17Rs was detected in chondrocytes. However, IL-17RE expression was extremely low, compared with other IL-17Rs. The expression of MMP-1, MMP-3, MMP-13, and COX-2 as well as PGE₂ production were increased by addition of IL-17F, whereas the expression of IL-17RD, TIMP-2, TIMP-4, type II collagen, aggrecan, link protein, and COX-1 was decreased. The expression of IL-17RA, IL-17RB, IL-17RC, MMP-2, MMP-14, TIMP-1, and TIMP-3 was unaffected by addition of IL-17F. The IL-17R antibody blocked the stimulating/reducing effect of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, aggrecan, and link protein. NS-398 blocked the reducing effect of IL-17F on aggrecan expression, whereas it did not completely block the stimulating/reducing effects of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, and link protein. Our results suggest that IL-17F stimulates cartilage degradation by increasing the expression of collagenases (MMP-1 and -13) and stromelysin-1 (MMP-3) and by decreasing expression of their inhibitors (TIMP-2 and -4), type II collagen, aggrecan, and link protein in chondrocytes. Furthermore, our results suggest that the expression of aggrecan, link protein, and TIMP-4 decrease through the autocrine action of PGE₂ in chondrocytes.     

Passage-dependent changes in baboon endothelial cells--relevance to in vitro aging

In vitro cell culture system is a useful model for aging-related changes in a wide spectrum of biomedical research. In this study, we explored the passage and donor age-dependent changes in baboon macrovascular endothelial cells that are relevant to both in vitro cell culture aging models and experiments using cell culture techniques. We collected baboon femoral arterial samples from nine baboons ranging in age from 6 months to 30 years (equivalent to humans approximately 18 months to 90 years of age). We then cultured baboon femoral artery endothelial cells (BFAECs) in standard DMEM medium with 20% fetal calf serum with 1:3 split for subculture. Endothelial functions were documented by morphology, Dil-LDL uptake and expression of eNOS, MCP-1, vWF, VCAM-1, ICAM-1, and E-Selectin with or without cytokine stimulation. Most of the cells became nonmitotic after 30 population doublings, or 10 passages, when they became flattened, enlarged, and senescent. While it took approximately 3 days to reach confluence from three-dilution seeding at early passages (<6), confluence was not achieved even after 7 days of culture for cells after the 9th or 10th passage. There was a linear decline in eNOS expression with passage. However, this decline was significantly less in endothelial cells from a young baboon (6 months) than those from an old baboon (30 years). While basal expression of adhesion molecules was not changed with passaging, responses to cytokine stimulation appeared to be increased in later passaged cells. Our study has provided evidence for passage-related changes in key endothelial functions. The donor age-related differences in this in vitro aging process suggests that in vitro endothelial culture can serve as a biomarker for in vivo aging. Nonhuman primates can provide a model for investigating such aging-related biological characteristics.