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CgPG21 is involved in the degradation of the cell wall during the secretory cavity formation in Citrus grandis 'Tomentosa' fruits.

CgPG21 is mainly located in the cell wall, participates in the intercellular layer degradation of the cell wall during the formation of secretory cavity in the intercellular space-forming and lumen-expanding stages. The secretory cavity is a common structure in Citrus plants and is the main site for synthesis and accumulation of medicinal ingredients. The secretory cavity is formed in lysogenesis, when epithelial cells enter a process of programmed cell death. Pectinases are known to be involved in degradation of the cell wall during the cytolysis of secretory cavity cells, but the changes in cell structure, the dynamic characteristics of cell wall polysaccharides and the related genes regulating cell wall degradation are unclear. In this study, electron microscopy and cell wall polysaccharide-labeling techniques were used to study the main characteristics of cell wall degradation of the secreting cavity of Citrus grandis 'Tomentosa' fruits. At the same time, the full CDS length of the pectinase gene CgPG21 was cloned, encoding a protein composed of 480 amino acids. CgPG21 is mainly located in the cell wall, participates in the degradation of the intercellular layer of the cell wall during the development of the secretory cavity, and plays an important role in the formation of the secretory cavity in the intercellular space-forming and lumen-expanding stages. With the development of secretory cavity, the cell wall polysaccharides of epithelial cells gradually degrade. CgPG21 is mainly involved in the intercellular layer degradation.

Vitellogenin of the solitary bees Centris tarsata and Centris analis (Hymenoptera: Apidae): cDNA structural analysis and gene expression

Vitellogenin (Vg) plays a vital role in the reproduction of oviparous species, as the precursor of the major egg storage protein. In bees, most of the studies of this protein rely on honey bees (Apis mellifera), and very little is known in solitary species. The solitary bees Centris tarsata and C. analis were chosen to characterize vg cDNA and better understand its expression. The Vg-deduced amino acid sequences from these bees have typical Vg features found in other hymenopterans. Besides its main site of synthesis, the fat body, transcripts of vg were also observed in ovaries. Males also showed expression of vg, although in lower levels compared with females. During development, vg expression was observed in late larvae, pupae, and female adults. The highest amounts of vg transcripts were observed in egg-laying females. Expression of vg increased rapidly in a few hours after female adult emergence, which indicates that its gene expression does not depend on mating to be triggered.

Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands.

The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands.

The Fibrinolytic Status in Liver Diseases.

The liver is the main site of synthesis and/or clearance of proteins involved in fibrinolysis. Therefore, chronic liver disease, including cirrhosis, leads to altered plasma levels of fibrinolytic proteins. Historical studies using in vitro clot lysis assays suggested that patients with chronic liver disease had accelerated fibrinolysis. Subsequent studies measured levels of individual pro- and antifibrinolytic proteins and showed that levels of tissue-type plasminogen activator are elevated. Plasma levels of plasminogen activator inhibitor-1 may also be altered, which leads to a shift in balance in the fibrinolytic system. Despite the fact that a more recent study using a plasma clot lysis assay challenged the existence of hyperfibrinolysis, other recent studies detected hyperfibrinolysis in a considerable number of patients with cirrhosis. Therefore, it is now recognized that hyperfibrinolysis may occur in 30 to 50% of patients with end-stage liver disease. A causal role of hyperfibrinolysis in bleeding is difficult to establish because also other concomitant changes in hemostasis occur. Treatment of hyperfibrinolysis consists of the use of fibrinolysis inhibitors, such as tranexamic acid. In this review we summarize current insights of the role of the liver in fibrinolysis, changes in fibrinolytic proteins, the potential clinical implications, and management of hyperfibrinolysis in liver disease.

CarbonQuest: Unfolding the Map of Seed Metabolism

In view of our rapid growing demands on vegetable oil, enhancing fatty acid synthesis in maize (Zea mays) represents tremendous nutritional and economic benefits. In corn kernels, the endosperm and the embryo are the main site for synthesis and accumulation of starch and oil, respectively. So far, breeding efforts to achieve elevated oil content in maize resulted in smaller endosperm and therefore lower yield. We hypothesize that the key to increasing oil content in maize kernels without affecting the yield lies on directly changing their carbon metabolism. To test our hypothesis, we compared the intracellular metabolite levels in maize embryos from two different maize lines, Alex synthetic (Alex) and LH59, which accumulate 48 and 34% of oil, respectively. The comparative metabolomics highlighted the metabolites and pathways that were active in maize embryos and important for oil production. The contribution of each pathway to fatty acid synthesis in terms of carbon, reductant and energy provision was assessed by measuring the carbon flow through the metabolic network (13C‐Metabolic Flux Analysis) in developing Alex and LH59 embryos to build maps of carbon flow through central metabolism. Our approach combined mathematical modeling with biochemical quantification to identify metabolic bottlenecks in fatty acid synthesis in maize embryos. Transgenic maize plants that we generated to overexpress a lead gene increased embryo fatty acid content by 35% without affecting the size of the kernel. This study hence describes the combination of innovative tools that will pave the way for controlling seed composition in important food crops.

Histological features, starch accumulation and sprouting in the early root development of Jacaranda ulei (Bignoniaceae)

The plant species occurring in the savanna region of the Cerrado biome in Brazil present typical morphological and physiological adaptations to a dry climate with seasonal occurrence of wildfires. In this study, the histological features of the root system, the main sites of synthesis and storage of starch and the initial phases of the bud development were characterized in Jacaranda ulei. The anatomical features observed in the root system of J. ulei are related to the needs of the species to survive in the Cerrado. The histochemical analyses demonstrated high synthesis of glucose and glycoprotein after the third day of in vitro culture, in the proximal cells of the cortical parenchyma of the exoderm. Meristematic primordia were observed in the ninth day and the beginning of the meristem formation was observed after 21 days of in vitro culture. Jacaranda ulei displays morphological, anatomical and storage features typical from resprouter species. However, it may be vulnerable to unsustainable exploitation. Considering the importance of this species for local people, more studies regarding its therapeutic properties should be performed, including the planning of appropriate programs for the species management and the production of selected clones through in vitro micropropagation.

An intimate liaison: spatial organization of the endoplasmic reticulum–mitochondria relationship

Organelle localization is often crucial to properly modulate cellular functions and signalling cascades. For example, the distribution of organelles in axons is crucial for their function and is dysregulated in several diseases. Similarly, relative positioning of two or more organelles is also important to perform certain specialized processes. Perhaps, the best-known form of interorganellar organization is that between endoplasmic reticulum (ER) and mitochondria. Close communication between these two compartments has been observed for a long time. Recent evidence suggests that this is the basis for a bidirectional communication regulating a number of physiological processes ranging from mitochondrial energy and lipid metabolism to Ca(2+) signalling and cell death. The recent discovery of some of the molecular mediators of the tethering already allowed to extend the function of this paradigmatic spatial organization to previously unexpected functions, and will foster future research to explore it in cellular signalling cascades as well as in disease.

Molecular characterization of the alpha subunit of complement component C8 (GcC8α) in the nurse shark ( Ginglymostoma cirratum)

Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of α, β, and γ subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8α and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8γ-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4% identity with human and Xenopus C8α respectively. Southern blot analysis showed GcC8α exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8α orthologs and as a sister taxa to the Xenopus.

Overexpression of HFE in HepG2 cells reveals differences in intracellular distribution and co-localization of wt- and mutated forms

Liver is the primary target organ of Hereditary Hemochromatosis Type I, with the HFE mutations C282Y and H63D recognized as markers of this iron-overload disease. Hepatocytes are also the main site of synthesis of HFE. However, most early studies of overexpression of HFE were done in non-hepatic, non-HFE-expressing, cell lines. Here we report the setting up of a stable transfection model of wt- and mutant-HFE (H63D and C282Y) proteins in a hepatic cell line (HepG2), the analysis of its intracellular distribution and the effect of diferric transferrin on HFE localization. The C282Y mutant is retained in the ER, whereas HFE-wt and H63D co-localize with TfR1 exclusively in early recycling endosomes. Holotransferrin induces a re-localization of wt- and H63D-HFE, from early recycling endosomes to the cytoplasmic membrane. In conclusion our results establish the HepG2 cell line as a valuable model for the study of HFE.

Electron Microscopy Analysis of the Nucleolus ofTrypanosoma cruzi

The nucleolus is the main site for synthesis and processing of ribosomal RNA in eukaryotes. In mammals, plants, and yeast the nucleolus has been extensively characterized by electron microscopy, but in the majority of the unicellular eukaryotes no such studies have been performed. Here we used ultrastructural cytochemical and immunocytochemical techniques as well as three-dimensional reconstruction to analyze the nucleolus of Trypanosoma cruzi, which is an early divergent eukaryote of medical importance. In T. cruzi epimastigotes the nucleolus is a spherical intranuclear ribonucleoprotein organelle localized in a relatively central position within the nucleus. Dense fibrillar and granular components but not fibrillar centers were observed. In addition, nuclear bodies resembling Cajal bodies were observed associated to the nucleolus in the surrounding nucleoplasm. Our results provide additional morphological data to better understand the synthesis and processing of the ribosomal RNA in kinetoplastids.

Differential Gonadotropin-Releasing Hormone (GnRH) and GnRH Receptor Messenger Ribonucleic Acid Expression Patterns in Different Tissues of the Female Rat across the Estrous Cycle

GnRH, the main regulator of reproduction, is produced in a variety of tissues outside of the hypothalamus, its main site of synthesis and release. We aimed to determine whether GnRH produced in the female rat pituitary and ovaries is involved in the processes leading to ovulation. We studied the expression patterns of GnRH and GnRH receptor (GnRH-R) in the same animals throughout the estrous cycle using real-time PCR. Hypothalamic levels of GnRH mRNA were highest at 1700 h on proestrus, preceding the preovulatory LH surge. No significant changes in the level of hypothalamic GnRH-R mRNA were detected, although fluctuations during the day of proestrus are evident. High pituitary GnRH mRNA was detected during the day of estrus, in the morning of diestrus 1, and at noon on proestrus. Pituitary GnRH-R displayed a similar pattern of expression, except on estrus, when its mRNA levels declined. Ovarian GnRH mRNA levels increased in the morning of diestrus 1 and early afternoon of proestrus. Here, too, GnRH-R displayed a somewhat similar pattern of expression to that of its ligand. To the best of our knowledge, this is the first demonstration of a GnRH expression pattern in the pituitary and ovary of any species. The different timings of the GnRH peaks in the three tissues imply differential tissue-specific regulation. We believe that the GnRH produced in the anterior pituitary and ovary could play a physiological role in the preparation of these organs for the midcycle gonadotropin surge and ovulation, respectively, possibly via local GnRH-gonadotropin axes.

Expression and localization of lung surfactant protein A in human tissues.

Lung surfactant protein A (SP-A) is a collectin produced by alveolar type II cells and Clara cells. It binds to carbohydrate structures on microorganisms, initiating effector mechanisms of innate immunity and modulating the inflammatory response in the lung. Reverse transcriptase-polymerase chain reaction was performed on a panel of RNAs from human tissues for SP-A mRNA expression. The lung was the main site of synthesis, but transcripts were readily amplified from the trachea, prostate, pancreas, and thymus. Weak expression was observed in the colon and salivary gland. SP-A sequences derived from lung and thymus mRNA revealed the presence of both SP-A1 and SP-A2, whereas only SP-A2 expression was found in the trachea and prostate. Monoclonal antibodies were raised against SP-A and characterized. One of these (HYB 238-4) reacted in Western blotting with both reduced and unreduced SP-A, with N-deglycosylated and collagenase-treated SP-A, and with both recombinant SP-A1 and SP-A2. This antibody was used to demonstrate SP-A in immunohistochemistry of human tissues. Strong SP-A immunoreactivity was seen in alveolar type-II cells, Clara cells, and on and within alveolar macrophages, but no extrapulmonary SP-A immunoreactivity was observed. In contrast to lung surfactant protein D (SP-D), which is generally expressed on mucosal surfaces, SP-A seems to be restricted to the respiratory system.

Localization of lung surfactant protein D on mucosal surfaces in human tissues.

Lung surfactant protein-D (SP-D), a collectin mainly produced by alveolar type II cells, initiates the effector mechanisms of innate immunity on binding to microbial carbohydrates. A panel of mRNAs from human tissues was screened for SP-D mRNA by RT-PCR. The lung was the main site of synthesis, but transcripts were readily amplified from trachea, brain, testis, salivary gland, heart, prostate gland, kidney, and pancreas. Minor sites of synthesis were uterus, small intestine, placenta, mammary gland, and stomach. The sequence of SP-D derived from parotid gland mRNA was identical with that of pulmonary SP-D. mAbs were raised against SP-D, and one was used to locate SP-D in cells and tissues by immunohistochemistry. SP-D immunoreactivity was found in alveolar type II cells, Clara cells, on and within alveolar macrophages, in epithelial cells of large and small ducts of the parotid gland, sweat glands, and lachrymal glands, in epithelial cells of the gall bladder and intrahepatic bile ducts, and in exocrine pancreatic ducts. SP-D was also present in epithelial cells of the skin, esophagus, small intestine, and urinary tract, as well as in the collecting ducts of the kidney. SP-D is generally present on mucosal surfaces and not restricted to a subset of cells in the lung. The localization and functions of SP-D indicate that this collectin is the counterpart in the innate immune system of IgA in the adaptive immune system.

Serum α-Fetoprotein in Newborns

α-Fetoprotein (AFP) is a single chain α-globulin with a molecular weight of ∼70 000. AFP is produced in the developing fetus by both the yolk sac and the fetal liver. At 12 weeks after conception the yolk sac degenerates and the fetal liver becomes the main site of synthesis. After 14 weeks of gestation (peak value of AFP), AFP synthesis decreases until parturition (1). The observation that AFP synthesis does not entirely cease at birth may be explained on the basis of transient production sustained by the presence of fetal hepatocytes (1). Very few, contradictory, and incomplete data regarding the determination of reference values for AFP in healthy newborns, correlated to birth weight (BW) and/or gestational age (GA), are available (1)(2)(3)(4)(5)(6)(7). Yet the usefulness of serum AFP as a diagnostic marker for the detection and/or differentiation of a great number of infantile diseases is well established, especially for some forms of malignant tumors and liver disorders (8)(9). Apparently healthy newborns (n = 150; 71 males and 79 females) admitted to our Neonatal Intensive Care Unit (Servizio di Patologia Neonatale, Universita di Genova, Italia) were included in the study. Single capillary blood specimens were collected with parental consent within 24 h of birth, when blood collection for other clinical testing was carried out. All subjects were delivered naturally. Subjects who were delivered by cesarean section were excluded from the …

Gastrin and Cancer

Publisher Summary This chapter describes the biology of the gastrin system and the involvement of gastrin in the development of various forms of cancer. The chapter presents new analytical methods for the measurement of gastrin in clinical oncology. Gastrin is a well-studied hormone that is discovered as a gastric acid-stimulating factor in extracts of antral mucosa. The elaborate biosynthetic pathway of gastrin in its main site of synthesis—the antral G cells—is now well described (B12, H5, 52, SIO). The pathway explains the molecular heterogeneity of gastrin in tissue and in circulation (52, R11, Y1). The active site homology of gastrin with cholecystokinin (CCK) poses large analytical problems because the active site is strongly immunogenic and both CCK and gastrin receptors may bind peptides having common sequences. Consequently, evaluation of antibody and receptor binding of gastrin and studies of the carcinogenic effects of gastrin require due consideration of possible interactions and cross-reactivity with CCK peptides. In normal mammalian organisms, gastrin is generally produced by the antral G cells (L1 and R2). The chapter concludes with a discussion of the expression and synthesis of antral and extra-antral gastrin.

Postnatal expression of opioid genes in rat brain

Developmentally regulated expression of opioid genes in rat brain was studied by Northern blot hybridization, using cloned rat genes as probes. Total RNA was isolated from the rat medulla oblongata, hypothalamus and striatum at 11 stages of development from birth to postnatal day 28. The steady-state levels of opioid precursor mRNAs in the above tissues were determined by hybridization to 32P-labeled opioid DNA probes and densitometric scanning of the appropriate bands. Analysis of the relative steady-state concentrations of opioid mRNAs has shown that the tissue distribution and size of these mRNAs during postnatal development of the CNS are similar to the pattern described in the adult. However, up to 50-fold changes in the relative mRNA concentrations were observed. These changes are tissue-specific, characterized by a peak within the postnatal stage of development, and are followed by a build-up of mRNA levels characteristic of the adult. Moreover, comparison of the changes in mRNA levels of proenkephalin to those of prodynorphin at their main site of synthesis in the brain, namely the striatum, revealed striking similarities in pattern. The association of this phenomenon with a possible role of opioid genes in development is suggested.

Segmental synthesis and actions of prostaglandins along the nephron.

The sites of synthesis and action of prostaglandins (PGs) along the renal tubule are examined. We focused our attention on experiments performed on well-defined nephron segments, using direct quantitative measurements of prostaglandin synthesis by radio- or enzyme-immunoassay. On the other hand, we selected, among the described effects of PGs, those obtained on precisely defined tubular segments. Among PGs, PGE2 synthesis is largely predominant all along the tubule. Its main sites of synthesis are the medullary collecting tubule and, to a lesser extent, the cortical collecting tubule and the thin limb of Henle's loop. Synthesis of PGE2 is amplified approximately tenfold in the presence of an excess exogenous substrate, arachidonic acid, compared with values measured without addition of substrate. Other eicosanoids have roughly the same distribution along the tubule as PGE2. Their rate of synthesis is, however, much less than that of PGE2, approximately 20-fold lower for PGF2 alpha and 6-keto-PGF1 alpha, and 100-fold lower for thromboxane B2 (TxB2). This contrasts with glomerular PG synthesis, where the difference between the production of PGE2 and other eicosanoids is much less marked. Most studies agree that antidiuretic hormone (ADH) and kinins augment PGE2 synthesis, whereas corticosteroids decrease it, at least in the collecting tubule. Direct effects of PGE2 have been described mainly in the medullary thick ascending limb and collecting tubule. They generally consist of a decrease in transepithelial potential difference and reabsorptive rates of water and solutes, in particular sodium and chloride. However, whatever the solute or tubular segment concerned, some studies failed to find such effects. The bulk of evidence suggests that ADH and PGs interact in kidney tubular cells. It is generally accepted that PGs antagonize the hydrosmotic effects of ADH in the collecting tubule. The mechanisms underlying these complex interactions are still under discussion: they probably involve several types of receptors and pathways for ADH action, which intervene in the modulation of both PG synthesis and cyclic nucleotides, and several types of PG receptors, either stimulatory or inhibitory to adenylate cyclase.

Immunohistochemical location of HPL, SP1 and β-HCG in normal placentas of varying gestational age

Sixty-four placentas at various gestational ages were examined by immunohistochemical stains for HPL, SP1 and beta-HCG according to a modified PAP method (Sternberger 1970). Syncytiotrophoblast cell layer was identified as the main site of synthesis. Extravillous immunohistochemical reactions for HPL and SP1 (but not for beta-HCG) were found in X-cells of the basal plate and in the intervillous trophoblast islands. These cell types would thus seem to be derived from trophoblast. Hofbauer-cells of villous connective tissue stained specifically for beta-HCG apparently because of HCG phagocytosis. The intensity of staining for HPL, SP1 and beta-HCG was evaluated semiquantitatively in the syncytiotrophoblast cell layer at various gestational weeks. The maximum of staining for beta-HCG was found in the uniform syncytiotrophoblast layer of immature intermediate villi in early pregnancy (7-14 weeks), HPL and SP1 had their peak of staining reaction at 32-36 weeks of gestation. Increasing maturation results in a subspecialisation of the villous surface: epithelial plates (allowing feto maternal exchange) and nucleated portions of syncytiotrophoblast (which are the main site of hormone production and endocrine activity).

Cell-free synthesis of ornithine aminotransferase of rat liver.

Mitochondrial ornithine aminotransferase was synthesized in a rabbit reticulocyte cell-free system using polysomal mRNA from rat liver. Upon analyzing the primary translation product by electrophoresis and fluorography, we found that ornithine aminotransferase is synthesized not as a large precursor but as one with its authentic size. Free ribosomes were found to be the main site of synthesis of ornithine aminotransferase.