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The parathyroid hormone family of peptides: structure, tissue distribution, regulation, and potential functional roles in calcium and phosphate balance in fish.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two factors that share amino acid sequence homology and act via a common receptor. In tetrapods, PTH is the main endocrine factor acting in bone and kidney to regulate calcium and phosphate. PTHrP is an essential paracrine developmental factor present in many tissues and is involved in the regulation of ossification, mammary gland development, muscle relaxation, and other functions. Fish apparently lack an equivalent of the parathyroid gland and were long thought to be devoid of PTH. Only in recent years has the existence of PTH-like peptides and their receptors in fish been firmly established. Two forms of PTH, two of PTHrP, and a protein with intermediate characteristics designated PTH-L are encoded by separate genes in teleost fish. Three receptors encoded by separate genes in fish mediate PTH/PTHrP actions, whereas only two receptors have so far been found in terrestrial vertebrates. PTHrP has been more intensively studied than PTH, from lampreys to advanced teleosts. It is expressed in many tissues and is present in high concentration in fish blood. Administration of this peptide alters calcium metabolism and has marked effects on associated gene expression and enzyme activity in vivo and in vitro. This review provides a comprehensive overview of the physiological roles, distribution, and molecular relationships of the piscine PTH-like peptides.

Animals↗

Analysis of vaccinia virus temperature-sensitive I7L mutants reveals two potential functional domains.

As an approach to initiating a structure-function analysis of the vaccinia virus I7L core protein proteinase, a collection of conditional-lethal mutants in which the mutation had been mapped to the I7L locus were subjected to genomic sequencing and phenotypic analyses. Mutations in six vaccinia virus I7L temperature sensitive mutants fall into two groups: changes at three positions at the N-terminal end between amino acids 29 and 37 and two different substitutions at amino acid 344, near the catalytic domain. Regardless of the position of the mutation, mutants at the non-permissive temperature failed to cleave core protein precursors and had their development arrested prior to core condensation. Thus it appears that the two clusters of mutations may affect two different functional domains required for proteinase activity.

Amino Acid Sequence↗

Estrogen receptor-related receptor alpha impinges on the estrogen axis in bone: potential function in osteoporosis.

The orphan nuclear estrogen receptor-related receptor alpha (ERRalpha) is expressed by osteoblastic cells and plays a functional role in osteoprogenitor proliferation and differentiation. To dissect further the role of ERRalpha in bone, we investigated the effects of estrogen (E2) on ERRalpha both in vitro and in vivo. Chronic treatment of fetal rat calvaria cells with E2-stimulated bone nodule formation and up-regulated ERRalpha mRNA expression at early (10 h and d 8) but not later times in culture, suggesting a link between ERRalpha and E2 during osteoprogenitor proliferation. ERRalpha mRNA levels were significantly lower in ovariectomized adult rat bones vs. those of sham-operated rats early (1 d and 1 wk) post surgery, but levels returned to control levels thereafter. ERRalpha is also expressed in osteoclasts (tartrate-resistant acid phosphatase + multinucleated cells) in vivo and in vitro (RAW 264.7 cells) and ovariectomization lowered the OPG/receptor activator of nuclear factor kappaB ligand expression ratio. Down-regulation of ERRalpha expression via antisense treatment of rat calvaria cells not only inhibited osteogenesis but also increased adipocyte colony formation and changed the OPG/receptor activator of nuclear factor kappaB ligand ratio. These data suggest that ERRalpha is regulated by estrogen in bone in which it may play a functional role at several levels (osteoblasts, adipocytes, and osteoclasts) in E2 deficiency diseases such as osteoporosis.

Adipocytes↗

Expression and potential functions of G-protein alpha subunits in embryos of Xenopus laevis.

During early embryonic development, many inductive interactions between tissues depend on signal transduction processes. We began to test the possibility that G-proteins participate in the signal transduction pathways that mediate neural induction. The expression during Xenopus development of three G alpha subunits, G alpha 0, G alpha i-1 and G alpha s-1, was characterized. The three maternally expressed genes showed different expression patterns during early development. Whole-mount in situ hybridization revealed that all three genes were expressed almost exclusively in the gastrula ectoderm and predominantly in the neuroectoderm in the neurula embryo. In order to investigate the involvement of these proteins in neural induction, we overexpressed the G-protein alpha subunits by injecting the G alpha mRNAs into fertilized eggs. Overexpression of G alpha s-1 increased the ability of gastrula ectoderm to become induced to neural tissue approximately four-fold. Overexpression of G alpha 0 and G alpha i-1 had less pronounced effects on neural competence, and inhibition of the G alpha 0 and G alpha i-1 proteins by pertussis toxin did not change the neural competence of the exposed gastrula ectoderm. Overexpression of the G alpha 0 and G alpha i-1 genes did, however, inhibit the normal disappearance of the blastocoel during gastrulation, suggesting a role for these G-proteins in regulating this process. The data also suggest a specific role for the G alpha s subunit in mediating the initial phases of neural induction.

Animals↗

COX2 in a euryhaline teleost, Fundulus heteroclitus: primary sequence, distribution, localization, and potential function in gills during salinity acclimation.

In the kidneys of mammals, cyclooxygenase type 2 (COX2) is expressed in medullary interstitial cells, the macula densa and epithelial cells of the cortical thick ascending limb where it generates prostaglandins that regulate hormone secretion, inhibit ion transport, and support cell survival during salt loading and dehydration. In teleosts, the gills are in direct contact with an aquatic environment and are the dominant site of osmoregulation. During transfers between salinities, specialized cells in the gills (chloride cells) rapidly regulate NaCl secretion for systemic osmoregulation while they simultaneously are exposed to acute osmotic shock. This study was conducted to determine if COX2 is expressed in the gills, and if so, to evaluate its function in cellular and systemic osmoregulation. Degenerate primers, reverse transcription-PCR and rapid amplification of cDNA ends were used to deduce the complete cDNA sequence of a putative COX2 enzyme from the gills of the euryhaline killifish (Fundulus heteroclitus). The 2,738 base pair cDNA includes a coding region for a 610 amino acid protein that is over 70% identical to mammalian COX2. A purified antibody generated against a conserved region of mouse COX2 labeled chloride cells, suggesting that the enzyme may control NaCl secretion as an autocrine agent. Real-time PCR was then used to demonstrate that mRNA expression of the COX2 homologue was threefold greater in gills from chronic seawater killifish than in gills from chronic freshwater killifish. Expression of Na(+)/K(+)/2Cl(-) cotransporter and the cystic fibrosis transmembrane conductance regulator were also greater in seawater, suggesting that chronic COX2 expression in the gills is regulated in parallel to the key ion transporters that mediate NaCl secretion. Real-time PCR was also used to demonstrate that acute transfer from seawater to freshwater and from freshwater to seawater led to rapid, transient inductions of COX2 expression. Together with previous physiological evidence, the present molecular and immunological data suggest that constitutive branchial COX2 expression is enhanced in seawater, where prostaglandins can regulate NaCl secretion in chloride cells. Our data also suggest that branchial COX2 expression may play a role in cell survival during acute osmotic shock.

Acclimatization↗

The face that sank the Essex: potential function of the spermaceti organ in aggression.

'Forehead to forehead I meet thee, this third time, Moby Dick!' [Ahab (Melville, 1851)] Herman Melville's fictional portrayal of the sinking of the Pequod was inspired by instances in which large sperm whales sank whaling ships by ramming the ships with their heads. Observations of aggression in species of the four major clades of cetacean and the artiodactyl outgroup suggest that head-butting during male-male aggression is a basal behavior for cetaceans. We hypothesize that the ability of sperm whales to destroy stout wooden ships, 3-5 times their body mass, is a product of specialization for male-male aggression. Specifically, we suggest that the greatly enlarged and derived melon of sperm whales, the spermaceti organ, evolved as a battering ram to injure an opponent. To address this hypothesis, we examined the correlation between relative melon size and the level of sexual dimorphism in body size among cetaceans. We also modeled impacts between two equal-sized sperm whales to determine whether it is physically possible for the spermaceti organ to function as an effective battering ram. We found (i) that the evolution of relative melon size in cetaceans is positively correlated with the evolution of sexual dimorphism in body size and (ii) that the spermaceti organ of a charging sperm whale has enough momentum to seriously injure an opponent. These observations are consistent with the hypothesis that the spermaceti organ has evolved to be a weapon used in male-male aggression.

Aggression↗

Potential function for the ROS-generating activity of TRACP.

TRACP is an enzyme with unknown biological function. It is expressed primarily in bone-resorbing osteoclasts and activated macrophages. In addition to its phosphatase activity, TRACP is capable of generating reactive oxygen species (ROS). In resorbing osteoclasts, TRACP is localized in transcytotic vesicles transporting bone matrix degradation products from the resorption lacuna to a functional secretory domain in the basolateral membrane. ROS generated by TRACP are capable of destroying organic bone matrix components, suggesting that they may be targeted to further destroy initial matrix degradation products in the transcytotic vesicles. The transcytotic route of osteoclasts is analogous with the antigen presentation route of macrophages transporting endocytosed foreign material into cell surface for presentation to other cells of the immune system. Macrophages overexpressing TRACP have elevated levels of intracellular ROS. In alveolar macrophages, TRACP is colocalized with endocytosed Staphylococcus aureus, a pathogen whose clearance is reduced in TRACP-deficient mice, suggesting that ROS generated by TRACP may be targeted to destroy foreign material in the antigen presentation route of macrophages. These data suggest that the ROS generating activity of TRACP may have an important role both in bone resorption and in the immune defense system.

Acid Phosphatase↗

The expanded human kallikrein (KLK) gene family: genomic organisation, tissue-specific expression and potential functions.

The tissue kallikreins are serine proteases encoded by highly conserved multi-gene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13-26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5' or 3' untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25-44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.

Amino Acid Sequence↗

A biphasic pattern of 45Ca2+ uptake by mouse spermatozoa in vitro correlates with changing functional potential.

Mouse sperm capacitation in vitro, leading to hyperactivated motility, acrosomal exocytosis and rapid fertilization, takes approximately 120 min in a medium containing sufficient Ca2+. During that period, spermatozoa incubated in 45Ca2+ exhibited a biphasic pattern of Ca2+ uptake, with the first and lower peak occurring from 10 to 50 min and the second and higher peak from 60 to 90 min. When the exogenously supplied glucose was reduced from 5.56 mmol l-1 to 5.56 mumol l-1, the latter supporting capacitation but not fertilization, only the first peak of 45Ca2+ uptake was observed. Increasing the glucose to a millimolar concentration produced a second peak of uptake. We therefore propose that the first phase of 45Ca2+ uptake is associated with capacitation and the second phase with acrosomal exocytosis, which are both necessary prerequisites for fertilization. In micromolar glucose the rate of 45Ca2+ uptake during the first 30 min was 47% higher than in millimolar glucose, suggesting that the former conditions might promote a precocious rise in the intracellular Ca2+ concentration ([Ca2+]i) and hence accelerate capacitation. This hypothesis was confirmed by demonstrating both significantly accelerated transition from the uncapacitated F pattern of chlortetracycline (CTC) fluorescence to the capacitated B and AR patterns and significantly higher fertility in vitro in suspensions preincubated for 30 min in micromolar glucose, compared with those maintained continuously in millimolar glucose. These results suggest that an ATP-dependent mechanism, for example a Ca(2+)-ATPase, may be involved in maintaining a low [Ca2+]i. In micromolar glucose, available ATP would be limited and hence the ATPase activity would decline, allowing [Ca2+]i to rise.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗

Identification, cellular distribution and potential function of the metalloprotease-disintegrin MDC9 in the kidney.

The complex interactions of glomerular and tubular epithelial cells with the basal laminae play a critical role in renal function. Disruption of these interactions has been widely implicated in glomerular diseases and acute renal failure. MDC are a large family of membrane-bound proteins containing metalloprotease, disintegrin (integrin interaction sites), and cysteine-rich domains. Little information is available concerning the presence of MDC in the kidney or their role in renal pathophysiology. Using degenerate PCR primers for the conserved metalloprotease and disintegrin domains of this protein family, cDNA templates from tubules, whole glomeruli, and glomerular epithelial cells (GEC) yielded a single, 195-bp product, which on sequence analysis corresponded to a region in the disintegrin domain of MDC9. Northern analysis of poly(A)+ RNA from tubules, whole glomeruli, and GEC revealed a 3.9-kb transcript, identical to that of mouse MDC9. Using antibodies generated against a 21-amino acid peptide present in the metalloprotease domain of MDC9, Western analysis of concanavalin A-enriched glomerular microsomal extracts demonstrated both processed (76 kD) and unprocessed (116 kD) forms of MDC9, which upon reduction changed to the corresponding 84- and 124-kD forms. Histochemical studies revealed a basolateral localization of intrinsic MDC9 protein in renal cortical tubule cells and glomerular visceral epithelial cells, which colocalized with the beta1 integrin chain. Expression of green fluorescence protein MDC9 chimeric constructs in GEC or polarized Madin-Darby canine kidney epithelial cells revealed a similar punctate basolateral surface localization. Transient overexpression of the soluble disintegrin domain-green fluorescence protein chimera in GEC led to dramatic changes in cellular morphology with rounding and detachment from cell monolayers. These studies document the presence of MDC9 in renal epithelial cells and suggest an important role for MDC9 in renal epithelial cellular interactions with the basal lamina and adjoining cells.

ADAM Proteins↗

Sequence polymorphisms of potential functional relevance in the beta-globin gene locus.

The purpose of this review is to give a general up-date of the most recent developments concerning polymorphisms within the beta-globin gene cluster. The first polymorphisms to be identified were mostly restriction fragment length polymorphisms. They were important markers both for their use as diagnostic tools and for anthropological investigations. Although they have been associated with specific patterns of globin gene expression in the hemoglobinopathies, none have been demonstrated to have any intrinsic functional significance. More recently, additional single nucleotide variations and microsatellite-like polymorphic simple sequence repeats have been identified which are frequently located in trans-acting protein binding segments and hence might affect regulatory processes.

Anemia, Sickle Cell↗

Potential functions of plasma prorenin; regional activation and tissue extraction.

The fate of circulating inactive prorenin was examined in patients and volunteers. Prorenin was activated either by acid-dialysis with warming at pH 3.3 or with trypsin. The results were similar but omission of warming reduced the value by 13%. In 6 volunteers, 20 min forearm venous occlusion raised regional total (T) and inactive (I) plasma renin concentration (PRC) by 51% and 48% without change of active (A) renin. During intense forearm exercise the ratio APRC: IPRC did not change in muscle or skin venous blood. Body anaerobic exercise increased APRC 3.7-fold without change in IPRC. These procedures activate plasminogen but are without effect on prorenin. In 18 patient with stable angina, TPRC was lower in coronary sinus than arterial blood (p less than 0.001) but APRC was not affected. A-V differences were not detected across the leg. Prorenin is apparently stable in the circulation but extracted by the heart.

Adult↗

Genome-Wide Identification of the TIFY Family in Cannabis sativa L. and Its Potential Functional Analysis in Response to Alkaline Stress and in Cannabinoid Metabolism.

TIFY transcription factors play crucial regulatory roles in secondary metabolism and stress response. However, the expression patterns of the Cannabis sativa L. TIFY gene family under alkali stress, their involvement in cannabinoid metabolism, and their underlying genetic evolutionary mechanisms remain largely unexplored. In this study, we used bioinformatics approaches to conduct genome-wide identification and functional characterization of the C. sativa TIFY gene family. Fourteen TIFY genes were identified and mapped onto seven chromosomes. These genes were classified into four subfamilies: TIFY, JAZ, ZML, and PPD, with the JAZ subfamily further subdivided into five distinct branches. Collinearity analysis suggested that gene duplication events contributed to the expansion of the TIFY gene family in C. sativa. Weighted gene coexpression network analysis (WGCNA) revealed that CsJAZ2, CsJAZ3, and CsJAZ6 participated in the cannabinoid regulatory network. Cis-element analysis indicated that the promoter regions of TIFY genes were enriched in hormone- and stress-responsive elements. Furthermore, transcriptome and RT-qPCR analyses were conducted to examine gene expression patterns under alkaline stress (the RNA employed in RT-qPCR was extracted from the apical leaves of samples subjected to short-duration alkaline stress treatment). The results showed that CsJAZ5 and CsJAZ6 were downregulated, whereas CsPPD1, CsTIFY1, and CsZML1 were upregulated in response to alkali stress. In summary, CsJAZ5, CsPPD1, and CsTIFY1 may serve as candidate genes for the development of alkali-tolerant cultivars, while CsJAZ2 and CsJAZ3 may be valuable targets for enhancing cannabinoid production. This study provides important molecular insights and a theoretical basis for future research on the evolutionary dynamics and functional roles of TIFY transcription factors, particularly in stress adaptation and cannabinoid metabolism.

Cannabis↗

Sugar-nucleotide-binding and autoglycosylating polypeptide(s) from nasturtium fruit: biochemical capacities and potential functions.

Polypeptide assemblies cross-linked by S-S bonds (molecular mass>200 kDa) and single polypeptides folded with internal S-S cross-links (<41 kDa) have been detected by SDS/PAGE in particulate membranes and soluble extracts of developing cotyledons of nasturtium (Tropaeolum majus L.). When first prepared from fruit homogenates, these polypeptides were found to bind reversibly to UDP-Gal (labelled with [(14)C]Gal or [(3)H]uridine), and to co-precipitate specifically with added xyloglucan from solutions made with 67% ethanol. Initially, the bound UDP-[(14)C]Gal could be replaced (bumped) by adding excess UDP, or exchanged (chased) with UDP-Gal, -Glc, -Man or -Xyl. However, this capacity for turnover was lost during incubation in reaction media, or during SDS/PAGE under reducing conditions, even as the glycone moiety was conserved by autoglycosylation to form a stable 41 kDa polypeptide. Polyclonal antibodies raised to a similar product purified from Arabidopsis bound to all the labelled nasturtium polypeptides in immunoblotting tests. The antibodies also inhibited the binding of nasturtium polypeptides to UDP-Gal, the uptake of UDP-[(14)C]Gal into intact nasturtium membrane vesicles and the incorporation of [(14)C]Gal into nascent xyloglucan within these vesicles. This is the first direct evidence that these polypeptides facilitate the channelling of UDP-activated sugars from the cytoplasm through Golgi vesicle membranes to lumenal sites, where they can be used as substrates for glycosyltransferases to synthesize products such as xyloglucan.

Antibodies↗