Artemin is an RNA-binding protein with high thermal stability and potential RNA chaperone activity.

Encysted embryos of the crustacean, Artemia franciscana, are among the most stress-resistant of all multicellular eukaryotes, due in part to massive amounts of p26, a small heat shock protein, that acts as a molecular chaperone. These embryos contain equally large amounts of another protein called artemin, of previously unknown function, that we report on here. Its thermal stability allows large-scale purification in about a day, using ammonium sulfate fractionation and incubation at 70 degrees C for 7 min, followed by gel filtration. The latter yields an artemin-RNA complex from which the pure protein, apo-artemin, was obtained by anion-exchange chromatography. We evaluated the possibility that artemin acts as a molecular chaperone for proteins, but obtained no evidence for that in vitro. The association of RNA with apo-artemin occurs at high temperatures and, although it is not yet clear whether artemin has a specific role as an RNA chaperone, it does bind non-polyadenylated RNAs which are then translated in vitro. Artemin-RNA is thermostable, some molecules resisting destruction after 30 min at 90 degrees C. The first order rate constant for denaturation and aggregation of artemin-RNA at 85 degrees C is 8.5 x 10(-3)min(-1), which compares well with other thermostable proteins of similar size ( approximately 500 kDa) such as the ferritins with which artemin has amino acid sequence similarity. The amount of artemin extracted from embryos that had been stored dry, under laboratory conditions, since 1951 is comparable to the amount in contemporary embryos, indicating its stability in situ, and supporting the in vitro heating studies.

Yolk platelets in artemia embryos: are they really storage sites of immature mitochondria?

We have used semi-quantitative polymerase chain reaction (PCR) technology to determine the mitochondrial DNA (mtDNA) content of yolk platelets isolated from embryos of the brine shrimp, Artemia franciscana, and ultrastructural analysis of yolk platelet formation to determine whether these organelles contain mitochondria as reported previously. Using six different isolation and purification protocols, we found one yolk platelet preparation to be devoid of mtDNA, while four yolk platelet preparations contained mtDNA ranging from 16.4 to 85 pg/10(6) yolk platelets. One preparation contained 600 pg mtDNA per 10(6) yolk platelets. Based on our PCR analyses, the mtDNA component of Artemia yolk platelets represented 0.16-4.5% of the total DNA isolated from the platelets. We calculated that Artemia yolk platelets contain, on average, approximately 1.78 molecules of mtDNA/platelet. Direct analysis of mtDNA in "free" mitochondria isolated from yolk platelet-free preparations of Artemia embryos and newly hatched larvae yielded 0.76-0.80 ng/animal. Based on these values, the mtDNA content of yolk platelets was approximately 0.2% of total mtDNA in Artemia embryos. Microscopic analysis of yolk platelet formation during oogenesis in Artemia failed to show the inclusion of mitochondria during the assemblage of yolk platelets. The "mitochondria-like" structures that appear in yolk platelets during their utilization lack the well defined inner and outer membranes characteristic of mitochondria making it unlikely that the yolk platelet inclusions are mitochondria. Our results from PCR technology and ultrastructure analysis demonstrate that mtDNA in yolk platelets of Artemia franciscana embryos is a minor component of the total mtDNA in the embryo, and they fail to support the notion that yolk platelets in Artemia are a major source of immature mitochondria for development.

Characterization of a novel heterodimeric cathepsin L-like protease and cDNA encoding the catalytic subunit of the protease in embryos of Artemia franciscana.

Embryos and larvae of the brine shrimp, Artemia franciscana, contain a novel cathepsin L-like cysteine protease (ACP) composed of 28.5- and 31.5-kDa subunits. Both subunits of the ACP are glycosylated, and seven isoforms of the protease were identified by isoelectric focusing with pI values ranging from 4.6 to 6.2. Several clones containing sequences coding for the 28.5-kDa subunit of the ACP were isolated from an Artemia embryo cDNA library in lambda ZAP II. One clone of 1229 bp, with an open reading frame of 1014 bp, was sequenced and found to contain 50-65% amino acid sequence identity with several members of the cathepsin L subfamily of cysteine proteases. The mature protein predicted from this sequence consisted of 217 amino acids with a mass of 23.5 kDa prior to post-translational modifications. The mature protein showed 68.6% amino acid sequence identity with human cathepsin L and 73.9% identity with cathepsin L-like proteases from Sarcophaga. peregrina and Drosophila melanogaster. The full-length cDNA clone analyzed in this study (pCP-3b) was renamed AFCATL1 (A. franciscana Cathepsin L1) and the sequence has been deposited in the Genbank database, accession number AF147207. Northern blot analyses identified a single transcript of about 1.4 kb in both embryos and young larvae of Artemia. Southern blot analyses of Artemia genomic DNA treated with various restriction endonucleases indicated a single gene for the ACP. The catalytic subunit of the ACP was tightly associated with a 31.5-kDa protein, which may localize the protease to nonlysosomal sites in embryos and larvae.

Diguanosine nucleotide metabolism and the survival of artemia embryos during years of continuous anoxia.

Encysted embryos of the primitive crustacean, Artemia franciscana, are remarkably resistant to a variety of harsh environmental conditions, including continuous anoxia for periods of years at physiological temperatures and water contents. Previous study produced no evidence of an ongoing anoxic metabolism, suggesting that these embryos remained viable in spite of the lack of detectable free energy flow and biosynthesis. That seeming violation of a major axiom of cell biology and biochemistry prompted us to re-examine the nucleotide pool of encysted embryos during prolonged anoxia. We found that the nucleotide Gp(4)G, present initially in very large amounts, decreased slowly as anoxia continued over the 5.6-year period examined. Studies on other nucleotides and associated enzymes, including results from previous papers, provide a plausible metabolic pathway leading to the provision of ATP and GTP to meet the needs of endergonic processes in anoxic embryos. Exactly what those processes are is not obvious. One possibility involves the extensive anoxia-induced nuclear translocation of the stress protein, molecular chaperone p26, whose large molecular mass (approximately 500 kDa) most likely requires a supply of free energy to cross the nuclear envelope. Support for this possibility comes from our finding here that p26 is also a GTPase.

Potential role in development of the major cysteine protease in larvae of the brine shrimp Artemia franciscana.

Encysted embryos and larvae of the brine shrimp Artemia franciscana contain a cysteine protease which represents over 90% of the protease activity in these organisms. We have used immunocytochemical methods to determine the localization and potential role of the cysteine protease in development of young larvae. In prenauplius larvae, there is intense staining for the protease on the basal side of the epidermal layer in the posterior region and diffuse staining for the protease throughout the embryo. In first instar larvae, cysteine-protease staining becomes intense in the midgut-forming area where a reticulum-like pattern emerges in cells with an abundance of yolk platelets. Cysteine-protease staining in second instar larvae becomes intense in the apical side of epidermal cells and in the basal and apical zones of midgut cells. Subcellular localization of the protease in the epidermis and midgut of young larvae using immunogold electron microscopy suggests that most is located in the cytosol and extracellular matrix adjacent to these cells. Addition of cysteine-protease inhibitors to the growth medium, especially the fluoromethyl ketone Z-Phe-Ala-CH2F, inhibits growth and segmentation of the thorax. Collectively, these observations suggest that the major cysteine protease in embryos and larvae functions in yolk utilization, as a hatching enzyme, in apolysis during the molt cycle, and as a digestive enzyme when the swimming larvae begin to feed.

Extensive intracellular translocations of a major protein accompany anoxia in embryos of Artemia franciscana.

Cells of encysted gastrula embryos of the crustacean Artemia franciscana exhibit extraordinary stability during prolonged anoxia. We find that they contain an abundant protein (referred to as "26-kDa protein") that undergoes translocation to the nucleus during anoxia. The reverse translocation rapidly occurs when anoxic embryos are returned to aerobic conditions. A similar translocation appears to take place in embryos exposed to 42 degrees C aerobic heat shock and prolonged exposure to low temperature (0-2 degrees C), and in diapause embryos. Gel filtration and Western immunoblotting indicate that the 26-kDa protein is translocated to other cellular compartments and may also be associated with a wide variety of "soluble" proteins during anoxia. This protein makes up roughly 15% of the total nonyolk embryo protein and is, by far, most abundant in the encysted embryo stage of the life cycle. The hypothesis is advanced that the 26-kDa protein may play the role of a metabolic regulator and/or a protective molecular chaperone during prolonged anoxia and other forms of stress.

Characterization of parvalbumin cDNA clones and gene expression in normal and dystrophic mice of strain 129 ReJ.

Parvalbumin is a calcium-binding protein found in fast-twitch skeletal muscles and selected cells in the brain. In several dystrophic mutants in the mouse, the parvalbumin content of skeletal muscles and brain is reduced and this deficiency appears to correlate with the inability of these mice to handle enhanced calcium uptake associated with the dystrophic process. In this study, two overlapping cDNA clones of 392 and 1268 base pairs were isolated from a mouse cDNA library in lambda gt11, characterized, and used as probes to study the involvement of the parvalbumin gene and its expression in various tissues of dystrophic mice of strain 129 ReJ. Southern blot analyses of restriction fragments of genomic DNA from normal and dystrophic mice indicate the same number and size of parvalbumin-specific gene fragments observed by other researchers, suggesting that the size of the Pva gene is the same in both normal and dystrophic mice of strain 129 ReJ. Northern blot analyses of total RNA from hind-limb muscles using cloned parvalbumin cDNA as probes revealed an abundant 800-nucleotide mRNA with lesser amounts of a 1000-nucleotide mRNA transcript in both normal and dystrophic mice of strain 129 ReJ. The amount of these mRNAs was reduced by 65-77% in dystrophic muscles preparations and was similar to the levels of beta-actin mRNA in these animals. These results suggest that parvalbumin gene expression is not down regulated in dystrophic mice of strain 129 ReJ.

Purification and partial characterization of thiol protease inhibitors from embryos of the brine shrimp Artemia.

Thiol protease inhibitor (TPI) proteins in embryos of the brine shrimp Artemia were purified to apparent homogeneity and several of their properties were studied. Four protein fractions containing thiol protease inhibitor activity were obtained by high performance liquid chromatography of Artemia embryo proteins on a C-18 reverse-phase column and these were designated as TPI-1a, -1b, -2, and -3. Acrylamide gel electrophoresis showed that TPI-1a and TPI-1b each consisted of two bands of 11.8 and 13.6 kilodaltons (kDa), while TPI-2 and TPI-3 consisted of only one band of 12.5 kDa. Isoelectric focusing experiments demonstrated that TPI-3 contained one band at pH 5.3, while both TPI-1b and TPI-2 yielded bands at pH 5.2 and 5.3. TPI-1a did not yield any major bands. Amino acid composition analyses of the Artemia TPI proteins showed them to be remarkably similar to one another. All were rich in valine and aspartic and glutamic acids, and devoid of cysteine. Partial trypsin digestion of TPI-1b, TPI-2, and TPI-3 yielded several peptides with identical mobilities on a reverse-phase column and several other peptides with different mobilities, suggesting that the multiple forms of Artemia TPIs may have originated from the same parental protein. N-terminal amino acid sequence analyses of TPI-3 suggest that Artemia TPI proteins are members of the type I cystatin family of protease inhibitors.

Age-related changes and tissue distribution of parvalbumin in normal and dystrophic mice of strain 129 ReJ.

In murine muscular dystrophy, hindlimb muscle contains a functionally defective thiol protease inhibitor (TPI) which has been implicated in the onset and progression of the disease in mice. More recently, this protease inhibitor has been identified as parvalbumin, a calcium binding protein. In this study, a polyclonal antibody against mouse muscle parvalbumin was used to study the concentration and distribution of this protein in normal and dystrophic male mice at various ages. Immunodetection assays were used to screen extracts of hindlimb, forelimb, brain, heart, lung, liver, and kidney in 60-day-old normal and dystrophic male mice for parvalbumin content. Parvalbumin was detected in relatively high amounts in both hindlimb and forelimb muscle extracts, while much lower concentrations were detected in brains of normal and dystrophic animals. No parvalbumin was detected in the lung, liver, heart, or kidney extracts using the immunoassay. With aging, the parvalbumin concentration in hindlimb muscle of normal mice remained fairly constant for 90 days, whereupon the level increased at 120 days. In contrast, the parvalbumin concentration in hindlimb muscle of dystrophic mice decreased steadily with age to about 22%% of normal animals at 120 days. The parvalbumin content was also reduced in dystrophic brain.

Immunodetection of thiol proteinase levels in various populations of Artemia cysts and during development.

An immunodetection assay on Western blots has been used to determine the thiol proteinase content and composition in cysts from 12 populations of the brine shrimp Artemia. Our results showed no differences in the subunit composition of the thiol proteinase among cysts from eight bisexual strains and four parthenogenic strains, and confirmed an earlier finding that the proteinase is composed of two subunits of 25.9 and 31.5 kilodaltons. In contrast, we found that Artemia cysts from parthenogenic strains contain 17.1 ng/cyst of the thiol proteinase, while cysts from bisexual strains contain 8.2 ng/cyst of the thiol proteinase. Also, there was a good linear correlation (r = 0.863; p less than 0.001) between the thiol proteinase content and cyst mass. Embryo fractionation experiments showed that 82% of the thiol proteinase was in the cytosol, while 14 and 4%, respectively, were in the nuclei/yolk platelets and mitochondria/lysosome fractions. Measurements of the thiol proteinase content of developing Artemia embryos showed that the proteinase content was relatively constant during early development, suggesting that the activity of the thiol proteinase gene(s) may be constitutive and not developmentally regulated in Artemia embryos.

Thiol protease-thiol protease inhibitor imbalance in cardiac tissue of ageing cardiomyopathic hamsters.

In cardiomyopathic hamsters myofibrillar lesions and loss of muscle specific proteins occur in the heart early in life, and it has been suggested that intracellular proteases are involved in the process of muscle necrosis. In the age-dependent study reported here, we observed that cardiac tissue of dystrophic hamsters contains 30-100% more thiol protease activity than normal hamsters, with the greatest differences found in hearts of older animals. As well, the ratio of thiol protease activity in dystrophic hearts compared to normal hearts increases by 1.5-fold and 2.7-fold, respectively in the lysosomal and non-lysosomal fractions between 1.4 and 14 months of age. Cathepsin B accounted for over 90% of the thiol protease activity in the lysosomal fraction of both normal and dystrophic hamster hearts. In contrast, cardiac tissue of 3 to 14 month old dystrophic hamsters contains 20-45% less thiol protease inhibitor activity than hearts from age-matched normal hamsters. These results demonstrate that at an early age (1.4 to 3 months) an imbalance occurs between the thiol protease activity and thiol protease inhibitor content of cardiac tissue of dystrophic hamsters, which becomes more severe in older animals and associated with progressive myofibrillar lesions and tissue necrosis.

Suppression by cathepsin L inhibitors of the invasion of amnion membranes by murine cancer cells.

Cysteine proteinases, particularly cathepsins B and L, have been strongly implicated in fostering metastasis in mice. In this work four different inhibitors of cysteine proteinases have been shown to inhibit the invasion of the human amnion by murine melanoma and mammary carcinoma cells in vitro. Two of the inhibitors are synthetic peptides [ZPhePheCHN2 (benzyloxycarbonyl-L-phenylalanyl-L-phenylalanyldiazomethane) and ZPheAlaCH2F [3-(N-benzyloxycarbonylphenylalanylamido)-DL-1-fluoro-2-butanone]] and two are thiol protease inhibitors (TPIn, TPId) isolated from the skeletal muscle of the hind limbs of normal and dystrophic mice, respectively. The inhibitors (ZPhePheCHN2, TPId), with apparent selectivity for cathepsin L, blocked invasion as effectively as inhibitors (ZPheAlaCH2F, TPIn) effective on both cathepsins. The data reveal that in these cell lines the cysteine proteinases contribute significantly to the invasive capacity of the cells, but to a lesser extent than do the metalloproteinases. We suggest that the cysteine proteinases facilitate the action of metalloproteinases (collagenase, gelatinase, and stromelysin), possibly by activating them, by inactivating the tissue inhibitor of metalloproteinases, and/or by making basement membrane matrix more accessible.

High molecular weight kininogen, the extracellular inhibitor of thiol proteases, is deficient in hamsters with muscular dystrophy.

High molecular weight kininogen has been shown to be the principal plasma inhibitor of cellular thiol proteases including cathepsins B, H and L and calpains 1 and 2. Since these same enzymes have been reported to be elevated in animals with muscular dystrophy, we studied plasmas from hamsters with muscular dystrophy and compared these to normal hamster plasma. The ability of plasma to inhibit purified platelet calpain was assayed and found to be 62% of normal. Since low molecular weight kininogen can also inhibit calpain, the coagulant activity of kininogen, an activity unique for high molecular weight kininogen, was determined in dystrophic hamster plasma and found to be 69% of normal in close agreement with the calpain inhibitory activity. The contribution of the other plasma calpain inhibitor alpha 2-macroglobulin appeared small since inactivation with methylamine did not alter the ability to inhibit calpain in either normal or dystrophic plasma. We conclude that there is a selective deficiency of plasma high molecular weight kininogen in dystrophic hamsters, an abnormality which could play a role in the pathogenesis of this disorder.

Thiol protease and cathepsin D activities in selected tissues and cultured cells from normal and dystrophic mice.

Thiol protease and cathepsin D activities were studied in extracts from hindlimb muscle of 60-day-old normal and dystrophic mice, strain 129 ReJ, and from cultured normal and dystrophic cells. Total thiol protease activity in dystrophic muscle extracts was 3.5 times higher than in normal muscle extracts, while cathepsin D, activity was 2.2 times greater in dystrophic muscle compared with normal muscle. Activation (pH 4.5, 30 degrees C) of latent thiol protease activity in extracts of muscle occurred concomitant with the inactivation or dissociation of endogenous protease inhibitors. Thiol protease assays revealed a higher ratio of active to inactive protease activity in extracts from dystrophic muscle than from normal muscle. Cultured myoblasts (L69/1) were found to contain 30-fold more thiol protease(s) and 6-fold more cathepsin D activity than whole muscle. Cells established from dystrophic muscle and grown in culture for periods up to 6 months were more responsive to thiol protease activation conditions than similar cultures derived from normal muscle. From data on the rate and extent of thiol protease activation in extracts from dystrophic cells and hindlimb muscle compared with normal tissue, it appears that cells and tissues from dystrophic mice contain a lower level of protease inhibitors than cells and tissues from normal mice.

Evidence for a defective thiol protease inhibitor in skeletal muscle of mice with hereditary muscular dystrophy.

The thiol protease inhibitor (TPI-d) from hind-limb skeletal muscle of dystrophic 60-day-old male mice (strain 129/ReJ/dy) has been purified to apparent homogeneity and compared with the thiol protease inhibitor (TPI-n) from hind-limb skeletal muscle of normal 60-day-old male littermates. While both TPI-d and TPI-n displayed identical properties on sodium dodecyl sulfate-polyacrylamide gels (14,800 relative mass), analytical isoelectric focusing gels (pI 4.5), and high performance liquid chromatography columns, TPI-d was unable to inhibit papain and cathepsin B after purification by isoelectric focusing. However, a component in the purified TPI-d preparation with an isoelectric point of 4.9 initially masked the functional state of TPI-d, using papain when assayed with the test proteases papain and cathepsins H and L. This inhibitory component was absent from TPI-n preparations. Pure TPI-d was also unable to inhibit in vitro myosin hydrolysis by cathepsin B, whereas TPI-n completely blocked cathepsin B catalyzed myosin hydrolysis. Given the central role of the thiol proteases, especially cathepsin B, in intracellular protein metabolism and the possibility that uncontrolled thiol protease activity in muscle leads to muscle protein breakdown and dystrophy, our data suggest that a modified (defective) thiol protease inhibitor (TPI-d) may be (one of) the end product(s) of the dystrophy gene in mice with the hereditary form of the disease.

Purification and characterization of a cytosol protease from dormant cysts of the brine shrimp Artemia.

A thiol protease has been isolated and purified from the postribosomal fraction of encysted embryos of the brine shrimp Artemia using a six-step procedure. The purified enzyme has a molecular weight of 55,000 +/- 4,200 and is composed of subunits of Mr 31,500 +/- 559 and 25,867 +/- 1,087. Isoelectric focusing revealed two discrete bands, one at pH 4.6 and the other at pH 5.1. The protease appears to be a member of the thiol group of proteases based on its inhibition by leupeptin, antipain, chymostatin, Ep-475, and several other thiol protease inhibitors. The enzyme was stimulated by heavy metal chelators and thiol reagents. At pH 3.5-4.0 the thiol protease hydrolyzed a wide range of proteins including bovine serum albumin, hemoglobin, Artemia embryo soluble proteins, Artemia lipovitelline, and protamine, whereas at pH 6.0-6.5 the enzyme showed a high degree of specificity for Artemia elongation factor 2 and lipovitelline alpha 1. The total amount of protease activity in crude homogenates of Artemia embryos decreased by about 50% during the first 24 h of development, while the amount of free, active enzyme decreased proportionally for 9 h of development then remained constant during the next 26-27 h of development. These changes in protease activity appear to reflect changing levels of an endogenous protease inhibitor during development.

Absence of detectable 5-methylcytosine in DNA of embryos of the brine shrimp, Artemia.

DNA from three developmental stages of the brine shrimp, Artemia was found to be undermethylated compared to DNA from calf thymus and salmon sperm. Using high-performance liquid chromatography we found that DNA hydrolysates from both dormant cysts and prefeeding nauplii contain less than 1 residue of 5-methylcytosine per 59 kilobases, placing Artemia DNA in the "insect type" category. The absence of detectable 5-methylcytosine in DNA of developing Artemia supports the view that methylation status alone cannot account for regulation of transcription in protostomes, and that DNA methylation may be more common among deuterostomes.

Distribution of elongation factor 2 between particulate and soluble fractions of the brine shrimp Artemia during early development.

The ADP-ribosylation of elongation factor 2 (EF-2) in vitro was used to quantitate EF-2 and to determine its subcellular distribution in extracts of Artemia embryos at different stages of development. In extracts from dormant cysts of Artemia 40-45% of EF-2 is complexed to macromolecules smaller than ribosomes, whereas the remainder is soluble or free in the cytosol. During early development the amount of "complexed" EF-2 decreases markedly concomitant with an increase in the pool of soluble EF-2. Complexed EF-2 was found to be associated with macromolecules which sediment at 16S-20S and 40S-50S and not with monoribosomes or polyribosomes as reported for mammalian systems. The data show that the decrease in complexed EF-2 is associated with the resumption of development in Artemia.

Recovery of quenched radioactivity from thin-layer chromatographic plates. An improved assay for cGMP phosphodiesterase in Myxococcus xanthus.

Ammonium bicarbonate was found useful in extracting a variety of radiolabeled compounds from thin-layer chromatographic plates. The technique significantly increased the sensitivity of the assay for cyclic nucleotide phosphodiesterases. This method was used to show unequivocally, the presence of cGMP phosphodiesterase in vegetative cells of Myxococcus xanthus.