Various Tissues Of Vertebrates Research Articles

Identification and structure-based engineering of a dipeptidase CpPepD from Clostridium perfringens for the synthesis of l-carnosine

l-Carnosine (l-Car), an endogenous dipeptide presents in muscle and brain tissues of various vertebrates, has a wide range of application values. The enzymatic preparation of l-Car is a promising synthetic method because it avoids the protection and deprotection steps. In the present study, a dipeptidase gene (CpPepD) from Clostridium perfringens with high l-Car synthetic activity was cloned and characterized. In an effort to improve the performance of this enzyme, we carried out site saturation mutagenesis using CpPepD as the template. By the o-phthalaldehyde (OPA)-derived high throughput screening method, mutant A171S was obtained with 2.2-fold enhanced synthetic activity. The enzymatic properties of CpPepD and mutant A171S were investigated. Under the optimized conditions, 63.94 mM (14.46 g L−1) or 67.02 mM (15.16 g L−1) l-Car was produced at the substrate concentrations of 6 M β-Ala and 0.2 M l-His using wild-type or mutant A171S enzyme, respectively. Although the mutation enhanced the enzyme activity, the reaction equilibrium was barely affected.

The first report of prostacyclin and its physiological roles in insects

Prostaglandins (PGs) mediate physiological processes of insects as well as mammals. Prostaglandin I2 (PGI2) is a relatively well-known eicosanoid with potent hormone-like actions on various tissues of vertebrates, however, its presence and biosynthetic pathway have not been described in insects. This study demonstrated that fat bodies of the lepidopteran species, Spodoptera exigua, contained ~ 3.6 pg/g PGI2. To identify its biosynthetic pathway, a PGI2 synthase gene of S. exigua (Se-PGIS) was predicted from a transcriptome of S. exigua; 25.6% homology with human PGIS was demonstrated. Furthermore, a predicted three-dimensional structure of Se-PGIS was demonstrated to be 38.3% similar to the human PGIS ortholog, including catalytic residues. Se-PGIS was expressed in all developmental stages of S. exigua and most abundant larval and adult stages; immune challenging of larvae significantly up-regulated these expression levels. The inducible expression of Se-PGIS expression was followed by a greater than four-fold increase in the concentration of PGI2 in fat bodies 10 h after immune challenge. RNA interference (RNAi) against Se-PGIS was performed by injecting double-stranded RNA (dsRNA). Under these RNAi conditions, cellular immune responses (e.g., hemocyte-spreading behavior, nodulation, phenoloxidase activity) were not affected by bacterial challenge. The addition of PGI2 to larvae treated with an eicosanoid biosynthesis inhibitor did not rescue the immunosuppression. Interestingly, PGI2 injection significantly suppressed nodule formation in response to bacterial challenge. In addition to the negative effect of PGI2 against immunity, the Se-PGIS-RNAi treatment significantly interfered with immature development and severely impaired oocyte development in female adults; the addition of PGI2 to RNAi-treated females significantly recovered oocyte development. Se-PGIS RNAi treatment also impaired male fertility by reducing fecundity after mating with untreated females. These results suggest that PGI2 acts as a negative regulator of immune responses initiated by other factors and mediates S. exigua development and reproduction.

Kinetic study of hepatic β-glucuronidase in the Indian major carp, Labeo rohita (Hamilton)

Liver is the metabolic factory and contains several valuable enzymes that catalyze biochemical reactions. β-glucuronidase is one of the well-known lysozymes that participates in the carbohydrate metabolism in the tissues of various vertebrates. In the present study, an attempt was made to study the kinetic properties of hepatic β-glucuronidase of the Indian major carp (IMC), Labeo rohita. It was observed that the enzyme activity increased largely at pH 5 (0.1M acetate buffer) when exposed to 38°C. However, the maximum activity was noticed at 52°C and later it started declined up to 70°C. It was also observed that with time the enzyme activity increased until substrate was completely used up. It has been concluded that it is a heat stable enzyme and cannot be destroyed at room temperature. Enzyme activity was observed to increase in response to increase in enzyme and substrate concentrations. The reaction velocity maxima (Vmax) and Michaelis constant (Km) were recorded using Lineweaver-Burk plot that was 18.182μg/h and 2.907mM, respectively.

Secretoneurin stimulates goldfish pituitary luteinizing hormone production

Secretoneurin (SN), a 33-34 amino acid neuropeptide is derived from endoproteolysis of secretogranin II (SgII), a member protein of the chromogranin family. SN is widely distributed in various tissues of vertebrates especially in pituitary and hypothalamus, and is a potential new hormone. In vivo, i.p. injection of SN increased luteinizing hormone (LH) release in goldfish pretreated with the dopamine antagonist domperidone. In 6-h static incubation of goldfish pituitary fragments, 10 and 100 nM but not 1 nM concentrations of goldfish SN had a direct stimulatory effect to increase LH release by 2.3- and 1.5-fold (p<0.05), respectively. In addition, 500 nM SN induced a 2.6-fold increase in LHbeta subunit messenger RNA (mRNA) levels in pituitary fragments, regardless of whether LHbeta mRNA levels were expressed relative to 18S ribosomal RNA or beta-actin mRNA. We suggest that the stimulatory actions of SN on LH release may be a part of a paracrine or autocrine feedback loop in the pituitary.

The lipocalin α 1-microglobulin binds heme in different species

The lipocalin α 1-microglobulin (α 1m), found in plasma and tissues of various vertebrates, is brown, forms complexes with other proteins and has immunomodulatory effects in vitro, but the physiological function is not yet established. Human α 1m was recently shown to bind heme and, after cleavage of a C-terminal tetrapeptide, initiate heme degradation, thus suggesting a heme-scavenger function. In this work the heme-binding of α 1m was characterized using heme immobilized on agarose beads, spectrophotometry, and electrophoresis. α 1m, both in plasma and in purified form, displayed a concentration-dependent binding to heme–agarose. The apparent dissociation-constant was estimated to be around 2 × 10 −6 M for both free α 1m and the IgA–α 1m complex. Incubation with free heme resulted in two forms of α 1m with different electrophoretic mobility. α 1m, identified on Western blotting, was found in eluates from heme–agarose after incubation with human biological fluids as well as sera from non-human species, indicating evolutionary conservation of the heme-binding property. Heme-binding could be instrumental for isolating new α 1m-homologues.

Expression of platelet-derived growth factor in the developing cochlea of rats

ObjectivePlatelet-derived growth factor (PDGF) is involved in the control of cell proliferation, differentiation and survival in various tissues of vertebrates. However, little is known about the expression of PDGF in the developing cochlea of rodents.Material and MethodsWe examined the expression of PDGF family genes in the developing cochlear tissue of rats using microarrays and tested their role in the proliferation of progenitor hair cells using cellular and molecular biology techniques.ResultsIt was found that the genes for PDGF-A, PDGF receptor (R)-α and PDGFR-β were highly expressed in the rapidly growing otocyst on embryonic Days 12–14 and weakly expressed thereafter. Reverse transcriptase polymerase chain reaction demonstrated the expression of PDGF-C and confirmed the expression of PDGF-A and PDGFR-α and -β in the developing cochlear tissue of rats and the cultured progenitor hair cells. Inhibition of the expression of PDGFs in the cultured progenitor hair cells with antisense oligonucleotides reduced the DNA synthesis.ConclusionPDGFs and their receptors may play a role in the proliferation of developing cochlear hair cells.

Nuclear estrogen binding sites in the liver of the newt Pleurodeles waltl

Previous investigations of the liver estrogen-specific binding in the newt, Pleurodeles waltl, have identified in the cytosol fraction, only from normal males, a new middle-affinity estrogen-binding component (MEBC) displaying the properties of type II sites reported in various tissues of vertebrates. The present work demonstrates that MEBC sites are not unique to the male but are also present in the nuclei of female animals. However, comparative study between males and females of liver nuclear sites under various extraction conditions shows sex-linked differences in the subnuclear localization. The relationships between the association states of MEBC in the nuclear compartment according to sex and their presence or absence in the cytosol fraction are discussed.

Insulin-like growth factors I and II are present in the skeletal tissues of ten vertebrates

Previous studies have shown that insulin-like growth factor I and insulin-like growth factor II (IGF I and IGF II) constitute an important family of skeletal growth-regulating peptides. We undertook this study to determine if, as in the case of sera in various animals, IGF I and IGF II are conserved in the skeletal tissues of various vertebrates. Skeletal tissues of ten animals representing five of the six vertebrate classes were studied: monkey, dog, sheep, adult mice, neonatal mice, chicken, lizard, frog, trout, and shark. The skeletal tissues were pulverized and demineralized with 10% ethylenediaminetetraacetic acid (pH 7.0) to extract the soluble matrix proteins. The tissue extracts were concentrated, dialized, and subjected to specific human IGF assays. IGF I and IGF II were measured with a radioimmunoassay and a radioreceptor assay, respectively. We found that 1) there are detectable human IGF I- and II-like substances in all extracts, 2) IGF values obtained in the skeletal extracts were not caused by binding protein artifacts, 3) in general there is more IGF II than IGF 1, and 4) the skeletal tissue levels of the IGFs are comparable with their respective serum levels. We conclude that, in the skeletal tissues of vertebrates, the IGFs are conserved and may be important regulators of osteogenesis.

ウシカエル骨格筋のリボヌクレアーゼ

It has been known that several RNases occur in various tissues of vertebrates, although the role in the RNA metabolism of individual RNase is obscure. To study a possible mechanism involved in the regulation of RNase activity in the muscle during bullfrog metamorphosis, it was attempted to characterize a RNase from bullfrog muscle showing its activity in neutral region.The neutral RNase in the cytosol fraction from bullfrog muscle occurs in two forms, one is a free form and the other a complex (latent form) of the RNase and its inhibitor. As compared with those of bullfrog liver, it was found that in the muscle cytosol fraction the greater part of the RNase is present as the complex and an excess amount of the RNase inhibitor as a free form. The partially purified RNase from the muscle cytosol fraction resembled to that of the liver in enzymatic properties.

Concentration of uridine diphosphate sugars in various tissues of vertebrates

1. 1. UDP-glucose, UDP-N-acetylglucosamine and UDP-glucuronic acid concentrations in various tissues of vertebrates were measured; changes in UDP-sugar quantity during the prenatal and postnatal development of pigs were also studied. 2. 2. Compared to UDP-glucose and UDP-N-acetylglucosamine a larger amount of UDP-glucuronic acid in guinea-pig and pig liver as well as in guinea-pig small intestine was established. No UDP-glucuronic acid was found in the spleen, pancreas, pituitary gland, testis and oviduct of the hen. 3. 3. In rat small intestine, rat and guinea-pig pancreas, anterior lobe of the pituitary gland of the calf, isthmus and magnum of the oviduct of the hen the quantity of UDP-N-acetylglucosamine is considerably higher than that of UDP-glucose. In all other cases the UDP-glucose concentration predominated over that of the other two UDP-sugars. 4. 4. In the early stages of pig embryogenesis a large liver UDP-glucose and UDP-N-acetylglucosamine concentration was found, which decreased five to seven times several days before birth.

Fine structure of the resorptive cells in the teeth of frogs

AbstractTeeth undergoing resorption were dissected from the maxillae of frogs and prepared for electron microscopy using standard methods and methods designed to minimize demineralization. The resorptive cells overlapped one another, forming palisades. A central ruffled border was surrounded by a relatively structureless cytoplasmic “seal.” Crystals of mineral were present in the folds of the ruffled borders, and demineralized collagen fibrils were seen in the adjacent matrix in sections treated to prevent demineralization. The surfaces of the cells opposite the ruffled borders displayed numerous small villi.The cytoplasm of the resorptive cells contained a variety of lysosomes in various stages of development, mitochondria, scattered vesicles of endoplasmic reticulum and free ribosomes. Well developed Golgi zones were frequently seen, and occasional sections passed through centrioles. Two centrioles were commonly seen in such sections, but never more. Occasional patches of cytoplasm contained mitochondria and rough‐surfaced endoplasmic reticulum, resembling “blast” cell cytoplasm.We concluded that the resorptive cells of several mineralized tissues of various vertebrates are morphologically, indistinguishable.

The Occurrence of Angiostrongylus cantonensis in Frogs of New Caledonia with Observations on Paratenic Hosts of Metastrongyles

Twenty-three of 43 frogs (Hyla aurea) from market gardens in Noumea, New Caledonia were found to harbor infective-stage larvae of Angiostrongylus cantonenisis in their tissues. The larvae were encapsulated in the wall of the alimentary tract, the mesentery, liver, and somatic musculature; they were infective for rats. Sea snakes (Laticauda colubrina) were experimentally infected with third-stage A. cantonensis larvae and in these hosts the larvae survived and remained infective for rats for at least 1 month. The use of paratenic hosts by A. cantonensis and other metastrongyles is discussed. Very little is known as to the extent and significance of the role played by paratenic hosts in the transmission of metastrongylid infections in nature. The Hobmaiers (1935) were the first to note the use of such hosts among the metastrongyles when they demonstrated that amphibians, reptiles, birds, and mammals could serve as experimental hosts for larvae of the cat lungworm, Aelurostrongylus abstrusus. Subsequently, it has been shown that other metastrongyle larvae will enter the tissues of various vertebrates that possibly may serve as paratenic hosts in nature (Gerichter, 1949; Seneviratna, 1959; Anderson, 1962). The discovery that Angiostrongylus cantonensis, a parasite of rats, may infect man and cause eosinophilic meningitis has served to focus attention upon the biology of this nematode in particular, and upon metastrongyles in general. Thus far, infectivestage A. cantonensis larvae have been reported in nature from such paratenic hosts as planarians (Alicata, 1962), shrimp (Alicata and Brown, 1962; Rosen et al., 1967) and land crabs (Alicata, 1964b). In addition to these natural infections there have been successful experimental infections of shrimp (Wallace and Rosen, 1966), fish (Wallace and Rosen, 1967) and pigs and calves (Alicata, 1964a). Observations reported in this paper further extend the paratenic host range for A. cantonensis to amphibians and reptiles. Received for publication 16 February 1968. * Present address: Division of Infectious and Tropical Diseases, School of Public Health, UCLA, Los Angeles, California 90024. RESULTS 1. Natural infection in frogs In the course of surveying market gardens in Noumea, New Caledonia for mollusks and other invertebrates harboring nematode larvae frogs infected with A. cantonensis were found on several occasions. Hyla aurea (Less.) was the frog collected. This species was originally introduced from Australia and is the only known species of frog to occur in New Caledonia. It is relatively small, 5 to 7 cm long, and may weigh from 5 to 27 g. Examination of the stomach contents of these amphibians revealed that while insects probably comprise the bulk of their diet the remnants of mollusk shells as well as the bodies of smaller frogs were not uncommon findings. From mid-1966 to April 1967 a total of 43 frogs (29 females, 14 males) were examined for infection with A. cantonensis. In each instance, except as subsequently noted, the entire frog was examined for the presence of larvae by digestion in 0.5% pepsin in 0.7% HC1 and by pressing the tissues between thick glass slides. The frog was killed, skinned and the mesentery was examined for the presence of encapsulated larvae. Selected organs and tissues were then removed and divided into four groups for digestion: (1) alimentary tract and mesentery, (2) liver, (3) rear legs, and (4) rest of the carcass. Following digestion for 2 to 3 hr the digests were allowed to sediment in a modified Baermann funnel for an additional 2 to 3 hr prior to examination for larvae. Tissues that were examined for larvae

Metastatic Calcification Induced by Hytakerol in Chickens Infected With Plasmodium gallinaceum

INTRODUCTIONMETASTATIC calcification can be produced experimentally in the tissues of various vertebrates by the administration of high doses of such compounds as parathormone and irradiated ergosterol derivatives. A modification of this response by a malarial infection was first described by von Brand and Holtz (1933) in a study with canaries infected with Plasmodium paercox that were treated with an irradiated ergosterol derivative. The infected specimens were more susceptible to calcification than the uninfected ones. This finding prompted Mercado and von Brand (1962) to test the modifying action of a mammalian species of malaria, P. berghei, on the calcification produced by another ergosterol derivative. Their results were quite different from those obtained by von Brand and Holtz, infected treated rats being markedly less susceptible to calcification than the uninfected treated ones. These contradictory results led to the present investigation in which the calcifying effect of the dihydrotachysterol concentrate used in…