Fluoride-inhibited calcium ATPase of sarcoplasmic reticulum. Magnesium and fluoride stoichiometry.

The sarcoplasmic reticulum (SR) CaATPase is inactivated by fluoride in the presence of magnesium (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 5229-5235). The inactive complex is very stable and can be isolated free of other components by 48 h of dialysis at 4 degrees C (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 16990-16994). In this study, we used a fluoride-specific electrode to determine that the amount of tightly bound fluoride in the complex was 9.4 +/- 2 nmol mg-1 SR protein. The rate constant of inactivation was very similar to the rate constant of fluoride incorporation and varied directly as the square of the fluoride concentration. Luminal Ca2+ accelerated reactivation of the inhibited enzyme, and the rate constants of activity regain and fluoride release were very similar. Although required for inhibition, added magnesium did not accelerate reactivation. Analysis for magnesium using antipyrylazo III of the inhibited enzyme showed 4.1 +/- 0.4 nmol mg-1 SR protein. As there is much evidence in the literature supportive of an estimate of calcium pumps equal to approximately 4-5 nmol mg-1 SR protein, our results indicate that each inhibited enzyme contains two tightly bound fluorides and one tightly bound magnesium.     

How costly is clutch formation in the Audouin's Gull Larus audouinii?

During the Audouin's Gull's breeding season at the Ebro Delta in 1993, 24 fresh eggs from eight three-egg clutches (modal clutch-size) were collected at the peak of the laying period. Eggs were processed to obtain formalin-fixed yolks, which were halved and stained using the potassium dichromate method. Digitized images of the yolks were examined to assess the daily rates of yolk deposition. We used these data in combination with egg compositional analysis to build a model of energy demands during the formation of an average clutch in Audouin's Gull. To show how the different parameters of clutch formation affect the daily energy investment peak, we performed a simulation analysis in which the rapid yolk development (RYD) period, the follicle triggering interval (FTI), the laying interval (LI) and the albumen synthesis period (ASP) were allowed to vary simultaneously. In our sample, the mean RYD period was seven days with a range from six to eight days. There were no significant differences in yolk volume among eggs in a clutch, but albumen volume was significantly smaller in third eggs. According to our model the albumen synthesis of the a-egg coincides with the energy demand peak for clutch formation. This peak represents an increase by ca. 42% in female energy requirements. Values obtained from the simulation analysis showed that only the ASP of the a-egg and the RYD durations of the second and third follicles produced noticeable reductions in peak energy investment. We predict that in gulls, whose laying intervals seem to be kept constant, significant increases of the durations of the RYD periods of second and third eggs, or even significant reductions of yolk size of these eggs, may operate simultaneously to match the energy demands during clutch formation to the prevailing food conditions.     

Defensive strategies of soft corals (Coelenterata: Octocorallia) of the Great Barrier Reef

Summary The relationship between ichthyotoxicity and predation-related defensive functional morphology was examined in alcyonacean soft corals of the central and northern regions of the Great Barrier Reef (GBR), Australia. Approximately 170 specimens were assessed encompassing a number of genera within three families: 1) the Alcyoniidae (Lobophytum, Sarcophytum, Sinularia, Cladiella, Parerythropodium, and Alcyonium); 2) Neptheidae (Lemnalia, Paralemnalia, Capnella, Lithophyton, Nephthea, Dendronephthya, Scleronephthya, and Stereonephthya), and 3) Xeniidae (Anthelia, Efflatounaria, Cespitularia, Heteroxenia, and Xenia). Ichthyotoxicity data were derived from earlier studies which used Gambusia affinis Baird and Girard (Vertebrata, Pisces) as a test organism. These data were compared to morphological data collected from specimens in the field and laboratory. Three sets of statistical analyses were performed, each considering a progressively narrower group of taxa. The first included 68 specimens and considered 16 morphological characters in each, falling into the general categories of gross colony form, colony texture, presence of mucus, colony color, polyp retractility, and sclerite morphology and distribution. These were tested for independence against ichthyotoxicity data. The second set of analyses involved a more restricted morphological data set derived from 28 species of Sinularia in combination with 28 species within the Nephtheidae, comparing them to their respective toxicity ranks. The third analysis considered the previous two taxonomic groups separately in relation to their toxicity levels.The attempt to consider many morphological characters in a taxonomically diverse collection did not reveal any general association in the Alcyonacea between defensive morphology and toxicity, and those associations which did emerge were clearly erroneous. The second analysis, considering only Sinularia spp. and nephtheids, demonstrated a negative association between ichthyotoxicity and the morphological characters of a) polypary armament, b) microarmament of the individual polyp, and c) strong mineralization of the coenenchyme. The third analysis revealed that the negative association found between toxicity and the first two characters was derived entirely from the nephtheids while the association detected between toxicity and the third character was restricted to Sinularia. It is concluded that a relationship between toxicity and morphology can be demonstrated, but it is heavily dependent upon which specific morphological characters are being considered and at what level of taxonomic resolution the analysis is being performed. An approach utilizing many characters over many taxa is unlikely to yield significant, reliable, or meaningful results.Australian Institute of Marine Science Contribution Number 383     

Trivalent behavior during prophase I in male mice heterozygous for three Robertsonian translocations: an electron-microscopic study

A synaptonemal complex (SC) analysis was carried out in male mice heterozygous (CHT/+) for three Robertsonian translocations. All pachytene preparations studied showed the presence of three trivalents. At early pachytene, the nonhomologous centromeric regions of the acrocentric chromosomes were unpaired. Heterosynapsis subsequently took place with complete pairing of the trivalents. Association between one of the three trivalents and the sex vesicle was observed in 30.4% of the nuclei. Association between the unpaired regions of two trivalents was present in 14.4% of the cells, suggesting that the relationship between unpaired regions of structural rearrangements and the X-Y bivalent may simply reflect the tendency of unpaired regions to establish end-to-end associations or heterosynapses among them, which are usually resolved during the pachytene stage of prophase I. Since the sex bivalent always has unpaired regions, these associations often affect the sex chromosomes.     

Cytosolic free Ca2+ concentration and intracellular calcium distribution of Ca2+-tolerant isolated heart cells

The cytosolic free Ca2+ concentration of calcium-tolerant rat myocytes has been measured by the null point titration technique using arsenazo III as a Ca2+ indicator and digitonin to permeabilize the plasma membrane. The mean value obtained for 8 separate preparations was 270 +/- 35 nM. The distribution of releasable calcium between the mitochondrial and sarcoplasmic reticular compartments was measured by the successive additions of uncoupler and A23187 to cells pretreated with ruthenium red. The relative distribution of calcium in each pool was independent of the cell calcium content up to the maximum value of releasable calcium investigated (4.5 nmol/mg of cell dry weight) and was distributed in the approximate ratio of 2:1 in favor of the sarcoplasmic reticulum. The cells contained 1 nmol of calcium/mg of cell dry weight in a form nonreleasable by A23187, which was independent of the total cell calcium content as measured by atomic absorption spectroscopy. It is calculated that the calcium content of mitochondria in heart under physiological conditions is about 5 nmol/mg of mitochondrial protein. At this level, the mitochondria are likely to provide effective buffering of the cytosolic free Ca2+ concentration of quiescent heart cells. The corresponding intramitochondrial free Ca2+ is in a range above values needed to regulate the activity of Ca2+-dependent enzymes of the citric acid cycle in heart. The physiological calcium content of the sarcoplasmic reticulum in heart cells is estimated to be about 2.5 nmol/mg of cell dry weight, which is at least 5-fold greater than the amount of calcium release calculated to cause maximum tension development of cardiac muscle.     

Effects of branched chain alpha-ketoacids on the metabolism of isolated rat liver cells. I. Regulation of branched chain alpha-ketoacid metabolism.

alpha-Ketoisocaproate (ketoleucine) is shown to be metabolized to ketone bodies rapidly by isolated rat liver cells. Acetoacetate is the major end product and maximum rates were observed with 2 mM substrate. Studies with 2-tetradecylglycidic acid (an inhibitor of long chain fatty acid oxidation) showed that ketogenesis from alpha-ketoisocaproate and from endogenous fatty acids were additive. With alpha-ketoisocaproate present as soole substrate at 2 mM, leucine production was less than 10% of alpha-ketoisocaproate uptake and only 30% of the acetyl coenzyme A generated was oxidized in the citric acid cycle. Metabolism of alpha-ketoisocaproate was inhibited by fatty acids, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and pyruvate. Oxidation of acetyl-CoA generated from alpha-ketoisocaproate was suppressed by oleate and by pyruvate, but was enhanced by lactate. Metabolism between the different branched chain alpha-ketoacids was mutually competitive. When alpha-ketoisocaproate (2 mM) was added in the presence of high pyruvate concentrations (4.4 mM), flux through pyruvate dehydrogenase was decreased, and the proportion of total pyruvate dehydrogenase in the active form (PDHa) also fell. With lactate as substrate, PDHa was only 25% of total activity and was little affected by addition of alpha-ketoisocaproate. These data suggest that enhanced oxidation of acetyl-CoA from alpha-ketoisocaproate by lactate addition is caused by a low activity of pyruvate dehydrogenase combined with increased flux through the citric acid cycle in response to the energy requirements for gluconeogenesis. However, acetyl-CoA generation from pyruvate is apparently insufficiently inhibited by alpha-ketoisocaproate to cause a diversion of acetyl-CoA formed during alpha-ketoisocaproate metabolism from ketone body formation to oxidation in the citric acid cycle. Measurements of the cell contents of CoASH, acetyl-CoA, acid-soluble acyl-CoA, and acid-insoluble fatty acyl-CoA indicated that when the branched chain alpha-ketoacids were added as sole substrate, their oxidation was limited at a step distal to the branched chain alpha-ketoacid dehydrogenase. Acid-soluble acyl-CoA derivatives were depleted after oleate addition in the presence of alpha-ketoisocaproate, suggesting an inhibition of the branched chain alpha-ketoacid dehydrogenase by the elevation of the mitochondrial NADH/NAD+ ratio observed during fatty acid oxidation. This effect was not observed in the presence of oleate and 2-tetradecylglycidic acid.     

Cloning and nucleotide sequence of celA1, and endo-beta-1,4-glucanase-encoding gene from Streptomyces halstedii JM8.

The celA1 gene encoding an endo-beta-1,4-glucanase from a mesophilic actinomycete, strain JM8, identified as Streptomyces halstedii, was cloned and expressed in S. lividans JI66. From the nucleotide sequence of a 1.7-kb DNA fragment we identified an open reading frame of 963 nucleotides encoding a protein of 321 amino acids, starting at TTG (instead of ATG). The Cel1 mature enzyme is a protein of 294 amino acids (after signal peptide cleavage) and can be included in the beta-glycanase family B (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991). The Cel1 enzyme lacks a cellulose-binding domain as predicted by computer analysis of the sequence and confirmed by Avicel binding experiments. The promoter region of celA1 was identified by S1 mapping; the -35 region closely resembles those of housekeeping Streptomyces promoters. Three imperfectly repeated sequences of 15, 15, and 14 nucleotides were found upstream from celA1 [ATTGGGACCGCTTCC-(N85)-ATTGGGACCGCTTCC-(N2)-TGGGAGC GCTCCCA]; The 14-nucleotide sequence has a perfect palindrome identical to that found in several cellulase-encoding genes from Thermomonospora fusca, an alkalophilic Streptomyces strain, and Streptomyces lividans. This sequence has been implicated in the mechanism of induction exerted by cellobiose. Using an internal celA1 probe, we detected similar genes in several other Streptomyces species, most of them cellulase producers.     

Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis

RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells.     

The Crystal Structure and Small-Angle X-Ray Analysis of CsdL/TcdA Reveal a New tRNA Binding Motif in the MoeB/E1 Superfamily

Cyclic N⁶-threonylcarbamoyladenosine (‘cyclic t⁶A’, ct⁶A) is a non-thiolated hypermodification found in transfer RNAs (tRNAs) in bacteria, protists, fungi and plants. In bacteria and yeast cells ct⁶A has been shown to enhance translation fidelity and efficiency of ANN codons by improving the faithful discrimination of aminoacylated tRNAs by the ribosome. To further the understanding of ct⁶A biology we have determined the high-resolution crystal structures of CsdL/TcdA in complex with AMP and ATP, an E1-like activating enzyme from Escherichia coli, which catalyzes the ATP-dependent dehydration of t⁶A to form ct⁶A. CsdL/TcdA is a dimer whose structural integrity and dimer interface depend critically on strongly bound K⁺ and Na⁺ cations. By using biochemical assays and small-angle X-ray scattering we show that CsdL/TcdA can associate with tRNA with a 1:1 stoichiometry and with the proper position and orientation for the cyclization of t⁶A. Furthermore, we show by nuclear magnetic resonance that CsdL/TcdA engages in transient interactions with CsdA and CsdE, which, in the latter case, involve catalytically important residues. These short-lived interactions may underpin the precise channeling of sulfur atoms from cysteine to CsdL/TcdA as previously characterized. In summary, the combination of structural, biophysical and biochemical methods applied to CsdL/TcdA has afforded a more thorough understanding of how the structure of this E1-like enzyme has been fine tuned to accomplish ct⁶A synthesis on tRNAs while providing support for the notion that CsdA and CsdE are able to functionally interact with CsdL/TcdA.