The effect of calcium oxalate crystallization kinetics on the kinetics of calcium uptake and calcium ATPase activity of sarcoplasmic reticulum vesicles

The ionophore A23187 is a potent inhibitor of oxalate supported calcium uptake if added before uptake is initiated by ATP and is a much weaker inhibitor of uptake once uptake has been initiated. This observation is shown to be due to a failure of oxalate to capture the transported calcium at the beginning of uptake because the rate of calcium oxalate crystallization is initially slow, thereby allowing the ionophore to release the accumulated calcium. This hypothesis is supported by the observation that calcium oxalate crystallization shows a lag phase which is absent when calcium oxalate seeds are in the reaction system. Once calcium uptake has progressed, calcium oxalate seeds are present in the sarcoplasmic reticulum and calcium oxalate crystallization proceeds sufficiently rapidly that the ionophore cannot compete successfully for calcium. That A23187 and oxalate compete for intravesicular ionic calcium is shown by the stimulation which each produces in ATPase activity and by the dependence of ionophore activity on oxalate concentration.The failure of calcium oxalate crystallization to reach equilibrium during the early phase of calcium uptake caused us to examine whether at any time during calcium uptake, crystallization reaches equilibrium. Skeletal sarcoplasmic reticulum accumulated calcium at such a high rate that oxalate, in concentrations up to 20mM, was unable to clamp intravesicular calcium at equilibrium values. The lower rate of calcium accumulation by cardiac sarcoplasmic reticulum and/or perhaps its greater permeability to oxalate apparently allows intravesicular calcium to be clamped by oxalate.     

Substrate and metal ion binding to carbamate kinase: NMR and EPR studies

Metal ion and substrate binding to carbamate kinase from Streptococcus faecalis was studied by nuclear magnetic resonance (NMR) and electron paramagnetic resonance using Mn²⁺ as the paramagnetic probe. The enzyme binds Mn²⁺ weakly (K_D = 0.45 ± 0.05 mm) with a stoichiometry of one per two subunits. However, in the presence of nucleotides, tighter binding of Mn²⁺ was observed with K_D = 44 ± 4 μm in the presence of ADP and K_D = 23 ± 4 μm with ATP present. Proton relaxation rate enhancement studies were conducted on water molecules interacting with ternary enzyme-Mn²⁺-nucleotide and binary enzyme-Mn²⁺ complexes. Mn²⁺ bound to carbamate kinase enhances the proton relaxation rate of water giving a binary enhancement value of ?_b = 9.3 ± 0.4. When enzyme-Mn²⁺ was titrated with ADP or ATP, a bell-shaped titration curve was obtained typical of many other enzyme-Mn²⁺-nucleotide ternary complexes. Computer fits to the titration data gave ternary enhancement values of ?_t^ADP = 14 ± 1 and ?_t^ATP = 19 ± 1. The dissociation constants for Mn-ADP and Mn-ATP binding to carbamate kinase were also obtained from these data analyses and are K₁ = 2.5 ± 0.5 μm and K₁ = 50 ± 8 μm, respectively. Therefore, these data demonstrate the formation of a ternary enzyme-metal-nucleotide bridge complex at the nucleotide substrate site of carbamate kinase. Distance measurements were conducted by NMR techniques with ¹³C-enriched carbamate and demonstrate that carbamate is 4–8 Å from enzyme-bound Mn²⁺. Thus carbamate binds near the metal-nucleotide substrate site of carbamate kinase.     

Modification of an essential arginine of carbamate kinase

Carbamate kinase from Streptococcus faecalis is inactivated by butanedione in borate buffer, which implies the presence of an essential arginine at the active site of the enzyme. The inactivation reaction is first order in [butanedione] and a replot of the inactivation rate data infers that one arginine is modified. The enzyme is protected against inactivation by ADP, ATP, the metal-nucleotides and carbamyl phosphate but not by carbamate. Amino acid analyses reveal that one of three arginines is modified by butanedione in the absence of protecting agents, and the binding of ADP to the enzyme prevents modification. Thus, analysis of the data suggest that (i) substrate binding to arginine and (ii) protein conformational changes at the active site are responsible for protection of an essential arginine against modification by butanedione.     

Seasonal and annual variability in the phytoplankton community of the Raunefjord,west coast of Norway from 2001–2006

Changes in phytoplankton community composition potentially affect the entire marine food web. Because of seasonal cycles and inter-annual variations in species composition, long-term monitoring, covering many sequential years, is required to establish a baseline study and to reveal long-term trends. The current study describes the phytoplankton biomass variations and species composition in relation to hydrographic and meteorological conditions in the Raunefjord, western Norway, over a 6-year period from 2001 to 2006. The extent of inflow or upwelling in the fjord varied from year to year and resulted in pronounced differences in water column stability. The annual phytoplankton community succession showed some repeated seasonal patterns, but also high variability between years. Two to four diatom blooms were observed per year, and the spring blooms occurring before water column stratification in March were dominated by Skeletonema marinoi and Chaetoceros socialis, and other Chaetoceros and Thalassiosira spp. Blooms of the haptophytes Phaeocystis pouchetii and Emiliania huxleyi were irregular and in some years totally absent. Although E. huxleyi was present all year round it appeared in bloom concentrations only in 2003, when the summer was warm and the water column characterized by high surface temperatures and pronounced stratification. The annual average abundance of both diatoms and flagellates increased during the six years. Despite the high variation from year to year, our investigation provides valuable knowledge about annual phytoplankton community patterns in the region, and can be used as a reference to detect possible future changes.     

Hypo-osmotic shock and the role of freshwater input on the post-bloom recovery of Cyanothece sp. in Lake St Lucia,South Africa

Modern anthropogenic modifications to aquatic environments, specifically hydrodynamic alterations, play a major role in cyanobacterial bloom potential. In shallow-water ecosystems, salinity is a driver of microbial communities and increases in salinity may facilitate the bloom potential of competitive cyanobacteria. This study investigated the osmotic response and mortality rate of a persistent bloom-forming (18 months) cyanobacterium, Cyanothece sp., isolated from Lake St Lucia (South Africa), after sudden hypo-osmotic shock. Laboratory experiments were performed with Cyanothece sp. cells to observe osmotic responses and to determine mortalities during salinity reductions. In general, Cyanothece sp. cells conformed to the external medium at all salinity levels used in the treatments, reducing their intracellular osmolality in response to salinity downshifts. There were limits to this, however, as successful downshift was not observed during the freshwater exposure treatment. Highest mortality rates occurred in cultures that were grown at high salinities (i.e. 180, 240 and 300 units), as well as in treatments with large salinity downshifts (e.g. 120 salinity unit reduction and direct freshwater exposure). Cyanothece sp. has been shown to sustain bloom status for long periods. Its ability to tolerate high salinities and respond to sudden salinity downshifts is an important factor in its bloom success. However, it is still susceptible to reductions in salinity that may occur due to heavy rainfall and floods, which highlights the importance that freshwater plays in the bloom ecology of a persistent bloom-forming halotolerant cyanobacterium, as freshwater inputs markedly influence the physiology of the cells and their survival ability.     

Population structure,relative growth and morphological sexual maturity of the snapping shrimp Alpheus brasileiro Anker, 2012 (Caridea: Alpheidae) from the south-eastern coast of Brazil

The present study investigated the population structure of the snapping shrimp Alpheus brasileiro. We tested the hypotheses that the sex ratio of the population differs from the expected 1:1 and that the growth pattern differs between adults and juveniles and between sexes. Reproductive period, juvenile recruitment and morphological sexual maturity were estimated. Samples were collected in two periods. The first sampling occurred bimonthly from March 2013 to January 2014. The second sampling occurred monthly from April 2015 to March 2016. The population structure was determined based on the size-frequency distribution of different demographic categories. To analyse the relative growth, we measured the second pleuron length, major cheliped propodus length, major cheliped propodus width, major cheliped propodus height, appendix interna length, appendix masculina length. The results showed that the sex ratio did not differ from 1:1 (male:female). Reproduction and juvenile recruitment were continuous throughout the study period. The analysis of relative growth confirmed the hypothesis that males and females, and juveniles and adults, have different growth patterns, indicating distinct strategies of energy allocation. The estimated carapace length at the onset of morphological sexual maturity was 4.9?mm in males and 4.7?mm in females. The results obtained in this study are probably related to the monogamous social behaviour of A. brasileiro. Monogamy can influence demographic parameters such as sex ratio and the size at which males and females reach morphological sexual maturity.     

Comparative study on the surface ultrastructures of fertilized eggs of three Sebastes species

Fertilized eggs of Sebastes schlegelii, Sebastes pachycephalus and Sebastes hubbsi are morphologically similar under stereomicroscope. However, under the scanning electron microscope, significant differences in the ultrastructures of the egg surface among the three species were observed, and herein, a taxonomic key was proposed for future applications. A clustering analysis based on the ultrastructures of the egg surface and the diameter of the oil globule suggested that among the three species, S. hubbsi and S. pachycephalus were more genetically similar, while S. schlegelii had diverged earlier. The results agree with the conclusions drawn from morphological and molecular analyses on adult samples of the same species.     

Age and growth differences in two populations of the edible marine gastropod Buccinanops globulosus

Buccinanops globulosus is an edible marine gastropod that is being captured by artisanal fishermen without management regulations. As basic knowledge on population features is required in case a sustained commercial exploitation of this species is established, we estimated and compared the age and growth of B. globulosus in two populations separated by 16?km, inhabiting similar physical environments but different anthropic influence. Our results, based on stable oxygen isotope analysis and best fitted models by likelihood ratio tests, detected differences in age and growth between both samplings. Maximum shell marks suggest one year difference between populations. Richards was chosen as the best fitting model for both sampling sites and significant differences were observed between them. Human activities could be causing the age reduction among other irreversible effects on the population under anthropic influence. This should be considered when developing sustainable management regulations for this fishery resource, especially those based on shell/age size.     

Seasonal and geographic variations of the sterols from the starfish asterias vulgaris (verrill)

The levels of the major sterols of the starfish $\text{Asterias vulgaris}$ collected at one location in Nova Scotia varied considerably from month to month. After spawning in June the levels of the sterols in the starfish were very low, but a rapid assimilation of dietary sterols allowed the total sterol level to increase approximately two-hundred-fold to the annual maximum in July. The levels of a few minor sterols were unaffected by the spawning process, and during this period they emerged as the major components of the sterol mixture. The sterol mixtures from samples collected at different locations were compared.     

Reproductive biology and space–time modelling of spawning for sailfish Istiophorus platypterus in the western Atlantic Ocean

This study presents data and estimates reproductive parameters of the sailfish Istiophorus platypterus (Shaw, 1792) in the western Atlantic, useful for future stock assessments of the species in the Atlantic Ocean. During the period 2006–2011, a total of 1132 fish were sampled by scientific observers on board Venezuelan and Brazilian longline vessels and from artisanal fisheries based out of Cumaná, Puerto La Cruz and Playa Verde, Venezuela and Cabo Frio, Rio de Janeiro, Brazil. The ovaries of 543 females were classified into five ovarian development phases based on microscopic analysis of the most advanced group of oocytes. Generalized additive models were applied to the gonadal index and sex ratio data in order to assess spatio-temporal trends of the reproductive activity and identify the spawning areas. Space–time effect was a significant factor contributing to changes in sex ratio and reproductive activity. Spawning activity was observed in two distinct areas. On the south-east Brazilian coast spawning peaked during December–February, while in the south-eastern Caribbean Sea spawning occurred between March and September, with its peak in March and April. Using a Bayesian approach, size at 50% maturity was estimated to be 146.12?cm (credibility interval of 95%, 138.45–152.09?cm). Batch fecundity ranged from 436,800 to 2,492,500 hydrated oocytes per female. This study confirms multiple aggregation spawning for this species in the western Atlantic Ocean.     

XX/XY sex chromosome system and chromosome markers in the snake eel Ophisurus serpens (Anguilliformes: Ophichtidae)

We report evidence of an XX/XY sex chromosome system in the snake eel Ophisurus serpens (Anguilliformes: Ophichthidae). We characterized the male and female karyotypes by C-, replication- and HaeIII-bandings. The 45S and 5S ribosomal gene families were located using dual fluorescence in situ hybridization, which showed that the 5S rDNA sites were present on the X chromosome, beside an autosome pair. FISH with a telomeric peptide nucleic acid probe enabled recognition of Interstitial Telomeric Sequences (ITSs), likely remnants of chromosomal rearrangements, in five chromosome pairs, including the rDNA-bearing ones. Possible mechanisms of the origin of sex chromosomes in this species are discussed, considering the presence of a sex-linked marker and ITSs.     

Alien barnacle Amphibalanus amphitrite epizoic on two native oyster species in the southern Gulf of Mexico: spatio-temporal variability and current status of its epibiosis

The study examines the current invasive status of the barnacle Amphibalanus amphitrite on natural populations of two native oyster species (the eastern oyster Crassostrea virginica and the mangrove oyster C. rhizophorae) in a estuary of the southern Gulf of Mexico. The spatio-temporal variability of the frequency of occurrence, density and coverage of A. amphitrite on shells of each oyster species was examined and compared with those of other native congeners (A. eburneus, A. venustus and A. improvisus) with which it coexists. These three variables were significantly higher in C. rhizophorae than in C. virginica and were within the range recorded for its congeners. The density of this invasive barnacle varied significantly at spatial and temporal scales. Its highest overall mean density occurred in February (at temperatures 26–27°C and salinities 29–32). Multivariate analyses showed a separation of Amphibalanus assemblages into three main groups according to variations in their composition and abundance among sampling sites and dates. It was also determined that salinity and sedimentation rate were the variables that best explain the distribution of the Amphibalanus species within the estuary. The limited presence of A. amphitrite on C. virginica reefs is likely due to (amongst other factors) the relatively higher sedimentation loads (370–5709?g?m^?2?d^?1) and lower salinities (9–23) recorded in these sites. This study can act as a reference point for the subsequent monitoring of this invasive species, since possible increases in its population density and competitive ability could threaten the integrity of oyster populations and other organisms in the southern Gulf of Mexico.     

Time of the preovulatory LH surge in the gilt and sow relative to the onset of behavioral estrus

Two experiments were conducted to study the time of occurrence of the preovulatory LH surge in pigs. Sampling every ten minutes in six cycling gilts before and after onset of standing estrus revealed the preovulatory surge began from 8 hr before to 12 hr after the lordosis reflex was elicited. Three of six gilts initiated the preovulatory LH release coincident with the onset of estrus. Data from 28 postpartum sows, with samples drawn every six hours commencing with the onset of estrus, indicated maximum LH levels were present at the first observance of estrus. Six of the 28 sows had an LH peak 18-24 hr after the onset of estrus.     

Experimental allergic encephalomyelitis and cellular immunity in the Lewis rat

The encephalitogenic difference between purified guinea pig and bovine myelin proteins in the Lewis rat is reflected by the two molecules' lack of crossreactivity in the migration inhibition test. Peritoneal exudate cells from rats injected with guinea pig or bovine derived myelin basic protein in Freund's complete adjuvant demonstrate substantial migration inhibition to the sensitizing antigen but little inhibition when cultured in the presence of the other basic protein. The cellular reactivity to guinea pig basic protein is present throughout the induction phase of Experimental Allergic Encephalomyelitis and persists after the recovery of the rats from the paralytic state. Substantial cellular reactivity is also demonstrated to bovine basic protein even though this molecule shows minimal encephalitogenic activity in the Lewis rat. Minimal lymphocyte transformation could be demonstrated to either of the basic proteins, although the immune cells react strongly to the plant mitogen phytohemagglutinin and to a Mycobacterium tuberculosis antigen.     

Dopamine and norepinephrine in the alimentary tract changes after chemical sympathectomy and surgical vagotomy

The aim of this study was to examine the distribution of dopamine and norepinephrine in the proximal alimentary tract of the rat and to assess the contributions of sympathetic and vagal fibers to the tissue concentrations of both catecholamines. Tissues were extracted in perchloric acid and the catecholamines were separated by high pressure liquid chromatography and detected electrochemically. In untreated rats (controls) both catecholamines were concentrated in the gastric muscle but norepinephrine levels were 6-8 times higher (corpus, dopamine 35 +/- 7 ng . g-1, norepinephrine 265 +/- 50 ng . g-1, mean +/- SE, n = 6). In the mucosa norepinephrine concentrations were 10-12 times higher (corpus, dopamine 12 +/- 3 ng . g-1, norepinephrine 140 +/- 26 ng . g-1). Chemical sympathectomy (6 hydroxydopamine, 100 mg . kg-1 ip 3 days) significantly reduced dopamine concentrations in muscle and norepinephrine in muscle, mucosa, pylorus and duodenum. In all tissues the effects on norepinephrine were greater. Surgical vagotomy significantly reduced dopamine concentrations in the gastric muscle, but not the mucosa. Norepinephrine concentrations in the stomach of vagotomized rats were significantly reduced only in the pylorus. Differences in the relative concentrations of dopamine and norepinephrine in gastric tissues of the normal rat and differences in the effects of sympathectomy and vagotomy suggest that dopamine and norepinephrine exist, to an extent, in separate populations of cells and that dopamine is not merely a precursor of norepinephrine. Gastric mucosal dopamine, which was mainly unaffected by either treatment, may exist in APUD cells.     

Reconstitution of the purified calmodulin-dependent (Ca2+ + Mg2+)-ATPase from smooth muscle

The purified calmodulin dependent (Ca2+ + Mg2+)-ATPase (CaMg ATPase) from porcine antral smooth muscle transports Ca2+ after reconstitution in lipid vesicles indicating that this enzyme is indeed a Ca2+-transport ATPase. For CaMg ATPase reconstituted in asolectin vesicles a good correlation was found between the time course of Ca2+ accumulation and the corresponding changes in CaMg ATPase activity. The ATPase activity was stimulated 8-fold by A23187, which further indicates a tight coupling between ATP hydrolysis and Ca2+ transport. Asolectin vesicles with incorporated enzyme accumulated Ca2+ with a ratio approaching one Ca2+ ion transported for each ATP hydrolyzed. For CaMg ATPase reconstituted in phosphatidylcholine vesicles on the other hand, Ca2+ transport and CaMg ATPase were poorly coupled as is shown by the approximately 3.5 fold stimulation by A23187. The activity of the CaMg ATPase when reconstituted in asolectin vesicles was stimulated 1.25 fold by calmodulin while in phosphatidylcholine a value of 4.25 was obtained. The CaMg ATPase activity of the enzyme reconstituted either in asolectin or phosphatidylcholine was, after its stimulation by A23187, still further stimulated by detergent by a factor of 5.