Cell volume increase and extracellular Ca2+ are needed for hyposmotically induced prolactin release in tilapia

In thetilapia (Oreochromis mossambicus), as in many euryhalineteleost fish, prolactin (PRL) plays a central role in freshwater adaptation, acting on osmoregulatory surfaces to reduce ion and waterpermeability and increase solute retention. Consistent with theseactions, PRL release is stimulated as extracellular osmolality isreduced both in vivo and in vitro. In the current experiments, aperfusion system utilizing dispersed PRL cells was developed forpermitting the simultaneous measurement of cell volume and PRL release.Intracellular Ca²⁺ was monitored using fura 2-loaded cellsunder the same conditions. When PRL cells were exposed to hyposmoticmedium, an increase in PRL cell volume preceded the increase in PRLrelease. Cell volume increased in proportion to decreases of 15 and30% in osmolality. However, regulatory volume decrease was clearlyseen only after a 30% reduction. The hyposmotically induced PRLrelease was sharply reduced in Ca²⁺-deleted hyposmoticmedium, although cell volume changes were identical to those observedin normal hyposmotic medium. In most cells, a rise in intracellularCa²⁺ concentration ([Ca²⁺]_i)during hyposmotic stimulation was dependent on the availability ofextracellular Ca²⁺, although small transient increases in[Ca²⁺]_i were sometimes observed uponintroduction of Ca²⁺-deleted media of the same or reducedosmolality. These results indicate that an increase in cell size is acritical step in the transduction of an osmotic signal into PRL releaseand that the hyposmotically induced increase in PRL release is greatlydependent on extracellular Ca²⁺.

     

Rat ghrelin stimulates growth hormone and prolactin release in the tilapia,Oreochromis mossambicus

Recently, ghrelin (Ghr), a new peptide which specifically stimulates growth hormone (GH) release from the pituitary, was identified in the rat and human stomach. Ghrelin has been shown to stimulate GH release by acting through a growth hormone secretagogue (GHS) receptor in the rat. The present study describes the in vitro effect of rat Ghr on the release of GH and two forms of prolactin (PRL(177) and PRL(188)) in the tilapia, Oreochromis mossambicus. Rat Ghr stimulated the release of GH in a dose-related manner after 8 and 24 hr of incubation. Rat Ghr also significantly stimulated the release of PRL(177) and PRL(188) in a dose-related manner after 24 hr. Rat Ghr had no effect on the pituitary content of GH or PRL(188), but significantly increased PRL(177) content. These results show for the first time that rat Ghr significantly stimulates GH and PRL release in teleosts, and suggest that Ghr and a GHS receptor are present in fish.     

Effects of environmental salinity and 17alpha-methyltestosterone on growth and oxygen consumption in the tilapia, Oreochromis mossambicus

Effects of environmental salinity and 17α-methyltestosterone (MT) on growth and oxygen consumption were examined in the tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to one of four groups: FW, MT treatment in FW, seawater (SW) and MT treatment in SW. All treatment groups were fed to satiation three times daily. The fish reared in SW (both control and MT-treated groups) grew significantly larger than either group in FW from day 43 throughout the experiment (195 days). The fish fed with MT added to their feed grew significantly larger than their respective controls from day 85 in FW and in SW until the end of the experiment. The routine metabolic rate (RMR) was determined monthly from month 2 (day 62) to month 5 (day 155). A significant negative correlation was seen between RMR and body mass in all treatment groups. Among fish of the same age, the SW-reared tilapia had significantly lower RMRs than the FW-reared fish. The MT-treated fish in SW showed significantly lower RMRs than the SW control group at months 3–5, whereas MT treatment in FW significantly increased the RMR at month 3. Comparison of regression lines between RMR and body mass indicates that MT treatment in FW caused a significant increase in oxygen consumption at a given mass of the fish, whereas MT treatment was without effect on RMR in SW-reared fish. These results clearly indicate that SW-rearing and MT treatment accelerate growth of tilapia, and that RMR decreases as fish size increased. It is also likely that the increased RMR and growth in MT-treated tilapia in FW may be due to the metabolic actions of MT, although the reason for the absence of MT treatment in SW is unclear.     

A biophysical study of the interaction of the lipopeptide antibiotic iturin A with aqueous phospholipid bilayers

Iturin A is a lipopeptide extracted from the culture media of Bacillus subtilis which shows a strong antifungal action. The interaction of iturin A with multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) induced structures which did not sediment during centrifugation. Electron microscopy after negative staining showed that, at 30 mol%, iturin A/DMPC vesicles were visible but smaller than those formed by pure DMPC. Thermograms of DMPC/iturinA obtained after differential scanning calorimetry, at low concentrations of iturin A, were interpreted as indicating the presence of two laterally separated phases, one formed by pure phospholipid and the other by lipopeptide-phospholipid complexes, these two separated phases being already detected even at low concentrations such as 2 mol%. Fluorescence quenching experiments showed that the D-Tyr residue of the lipopeptide was fully accessible to the aqueous medium, indicating that the polar part of iturin A is located outside of the membrane hydrophobic palisade. It was concluded that the membrane barrier properties are likely to be damaged in the area where the lipid complexes are accumulated, due to structural fluctuations, and this may be one of the bases of its biological activity. Iturin-A was also able to greatly destabilize dielaidoylphosphatidylethanolamine (DEPE) membranes in the fluid form, producing a new structure which had a poor correlation in X-ray diffraction, and in 31P NMR spectroscopy gave rise to a spectrum containing a double isotropic signal. Iturin A was shown to induce DEPE to adopt phases other than H(II) inverted hexagonal, underlining that this lipopeptide is capable of modifying the curvature of the membrane, which may also be important in explaining the tendency of iturin A to create small vesicles and which may be another of the bases of its biological activity.     

Rapid Rather than Gradual Weight Reduction Impairs Hemorheological Parameters of Taekwondo Athletes through Reduction in RBC-NOS Activation

Purpose

Rapid weight reduction is part of the pre-competition routine and has been shown to negatively affect psychological and physiological performance of Taekwondo (TKD) athletes. This is caused by a reduction of the body water and an electrolyte imbalance. So far, it is unknown whether weight reduction also affects hemorheological properties and hemorheology-influencing nitric oxide (NO) signaling, important for oxygen supply to the muscles and organs.

Methods

For this purpose, ten male TKD athletes reduced their body weight by 5% within four days (rapid weight reduction, RWR). After a recovery phase, athletes reduced body weight by 5% within four weeks (gradual weight reduction, GWR). Each intervention was preceded by two baseline measurements and followed by a simulated competition. Basal blood parameters (red blood cell (RBC) count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean cellular hemoglobin and mean cellular hemoglobin concentration), RBC-NO synthase activation, RBC nitrite as marker for NO synthesis, RBC deformability and aggregation parameters were determined on a total of eight investigation days.

Results

Basal blood parameters were not affected by the two interventions. In contrast to GWR, RWR decreased activation of RBC-NO synthase, RBC nitrite, respective NO concentration and RBC deformability. Additionally, RWR increased RBC aggregation and disaggregation threshold.

Conclusion

The results point out that a rapid weight reduction negatively affects hemorheological parameters and NO signaling in RBC which might limit performance capacity. Thus, GWR should be preferred to achieve the desired weight prior to a competition to avoid these negative effects.     

The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles

Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles.Bacterial lipoproteins (Lpps)¹ are a subset of membrane proteins that are covalently modified with a lipidic moiety at their N-terminal cysteine residue. It is commonly reported that Lpps of Gram-positive bacteria are processed by two key enzymes; the prolipoprotein diacylglyceryl transferase (Lgt) and the lipoprotein signal peptidase (Lsp). The Lgt enzyme recognizes a so-called lipobox motif in the C-terminal region of the signal peptide of a premature lipoprotein and transfers a diacylglyceryl moiety to the cysteine residue of the lipobox (1), (2). Subsequently, the Lsp enzyme cleaves the signal peptide resulting in a mature Lpp (3), (4). Nevertheless, recent reports have suggested that N-acylation occurs in bacteria that lack the Gram-negative homologous apolipoprotein N-acyltransferase (Lnt) gene responsible for this modification (5, 6), and that Lpp N-terminal could also be modified with an acetyl group in some Gram-positive (7).Lpps have been described as virulence factors because they play critical roles in membrane stabilization, nutrient uptake, antibiotic resistance, bacterial adhesion to host cells, protein maturation and secretion and many of them still have unknown function (8). Several studies have suggested that bacterial Lpps are pathogen-associated molecular patterns (PAMPs) sensed by the mammalian host through Toll-like receptor 2 (TLR2) heterodimerized with TLR1 or TLR6 to induce innate immunity activation and to control adaptive immunity (9–12). TLR2 plays a critical role in the host response to the Gram-positive bacteria Staphylococcus aureus (13) and Streptococcus agalactiae (14). Although TLR2 has been considered a receptor for various structurally unrelated PAMPs, recent studies have suggested that, via their lipid moiety, bacterial Lpps function as the major, if not the sole, ligand molecules responsible for TLR2 activation (15). Although Gram-negative Lpps have been widely studied, little information is available for Gram-positive Lpps (16) and the ways they are released into the bacterial extracellular compartment and reach the host immune system remain unclear.We focused our attention on Lpps release by Streptococcus pyogenes. This Gram-positive bacterium is an important human pathogen that causes a wide range of diseases from superficial and self-limiting infection, e.g. pharyngitis and impetigo, to more systemic or invasive diseases like necrotizing fasciitis and septicemia (17). Understanding the role of bacterial Lpps in mediating innate and acquired immunity can be instrumental for the therapy and prophylaxis of human S. pyogenes infections. In this study, we showed that in S. pyogenes Lpps are released into the growth medium within vesicle-like structures in minute amounts. Conditions weakening the bacterial cell wall, such as the addition of sublethal concentrations of penicillin to the bacterial growth medium enhanced this phenomenon and allowed the recovery of sufficient material to enable an in-depth characterization. Proteomic analysis of the vesicles revealed that they were almost exclusively constituted of Lpps. A total of 28 Lpps were identified, representing more than 72% of the Lpps predicted from the genome of the strain under investigation. In addition, multiple transmembrane domain proteins were not found in abundance associated to the vesicles, indicating that vesicles were not representative of the bacterial membrane. We defined these vesicles as Lipoprotein-rich Membrane Vesicles (LMVs).Common characteristics are shared between the LMVs and the ExPortal described for the first time by Rosch and Caparon (18). This asymmetric and distinct membrane microdomain has been reported to be enriched in anionic phospholipids and acts in promoting the biogenesis of secreted proteins by coordinating interactions between nascent unfolded secretory proteins and the accessory factors required for their maturation (19–21). An association between ExPortal and peptidoglycan synthesis has also been reported (22). Similarly, LMVs are enriched in anionic phosphatidylglycerol, enzymes involved in protein maturation/secretion and cell wall biogenesis, suggesting that LMVs might derive from the ExPortal. Finally, we showed that LMVs do not induce TLR2 activation, indicating that the Lpps did not act as PAMPs when integrated into the LMVs.     

Relationship between Urinary Level of Phytate and Valvular Calcification in an Elderly Population: A Cross-Sectional Study

Pathological calcification generally consists of the formation of solid deposits of hydroxyapatite (calcium phosphate) in soft tissues. Supersaturation is the thermodynamic driving force for crystallization, so it is believed that higher blood levels of calcium and phosphate increase the risk of cardiovascular calcification. However several factors can promote or inhibit the natural process of pathological calcification. This cross-sectional study evaluated the relationship between physiological levels of urinary phytate and heart valve calcification in a population of elderly out subjects. A population of 188 elderly subjects (mean age: 68 years) was studied. Valve calcification was measured by echocardiography. Phytate determination was performed from a urine sample and data on blood chemistry, end-systolic volume, concomitant diseases, cardiovascular risk factors, medication usage and food were obtained. The study population was classified in three tertiles according to level of urinary phytate: low (<0.610 μM), intermediate (0.61–1.21 μM), and high (>1.21 μM). Subjects with higher levels of urinary phytate had less mitral annulus calcification and were less likely to have diabetes and hypercholesterolemia. In the multivariate analysis, age, serum phosphorous, leukocytes total count and urinary phytate excretion appeared as independent factors predictive of presence of mitral annulus calcification. There was an inverse correlation between urinary phytate content and mitral annulus calcification in our population of elderly out subjects. These results suggest that consumption of phytate-rich foods may help to prevent cardiovascular calcification evolution.     

Large-Scale Patterns of Turnover and Basal Area Change in Andean Forests

General patterns of forest dynamics and productivity in the Andes Mountains are poorly characterized. Here we present the first large-scale study of Andean forest dynamics using a set of 63 permanent forest plots assembled over the past two decades. In the North-Central Andes tree turnover (mortality and recruitment) and tree growth declined with increasing elevation and decreasing temperature. In addition, basal area increased in Lower Montane Moist Forests but did not change in Higher Montane Humid Forests. However, at higher elevations the lack of net basal area change and excess of mortality over recruitment suggests negative environmental impacts. In North-Western Argentina, forest dynamics appear to be influenced by land use history in addition to environmental variation. Taken together, our results indicate that combinations of abiotic and biotic factors that vary across elevation gradients are important determinants of tree turnover and productivity in the Andes. More extensive and longer-term monitoring and analyses of forest dynamics in permanent plots will be necessary to understand how demographic processes and woody biomass are responding to changing environmental conditions along elevation gradients through this century.     

Increase in Red Blood Cell-Nitric Oxide Synthase Dependent Nitric Oxide Production during Red Blood Cell Aging in Health and Disease: A Study on Age Dependent Changes of Rheologic and Enzymatic Properties in Red Blood Cells

Aim

To investigate RBC-NOS dependent NO signaling during in vivo RBC aging in health and disease.

Method

RBC from fifteen healthy volunteers (HC) and four patients with type 2 diabetes mellitus (DM) were separated in seven subpopulations by Percoll density gradient centrifugation.

Results

The proportion of old RBC was significantly higher in DM compared to HC. In both groups, in vivo aging was marked by changes in RBC shape and decreased cell volume. RBC nitrite, as marker for NO, was higher in DM and increased in both HC and DM during aging. RBC deformability was lower in DM and significantly decreased in old compared to young RBC in both HC and DM. RBC-NOS Serine¹¹⁷⁷ phosphorylation, indicating enzyme activation, increased during aging in both HC and DM. Arginase I activity remained unchanged during aging in HC. In DM, arginase I activity was significantly higher in young RBC compared to HC but decreased during aging. In HC, concentration of L-arginine, the substrate of RBC-NOS and arginase I, significantly dropped from young to old RBC. In DM, L-arginine concentration was significantly higher in young RBC compared to HC and significantly decreased during aging. In blood from healthy subjects, RBC-NOS activation was additionally inhibited by N⁵-(1-iminoethyl)-L-Ornithine dihydrochloride which decreased RBC nitrite, and impaired RBC deformability of all but the oldest RBC subpopulation.

Conclusion

This study first-time showed highest RBC-NOS activation and NO production in old RBC, possibly to counteract the negative impact of cell shrinkage on RBC deformability. This was even more pronounced in DM. It is further suggested that highly produced NO only insufficiently affects cell function of old RBC maybe because of isolated RBC-NOS in old RBC thus decreasing NO bioavailability. Thus, increasing NO availability may improve RBC function and may extend cell life span in old RBC.