Congenital afibrinogenemia: intracellular retention of fibrinogen due to a novel W437G mutation in the fibrinogen Bbeta-chain gene

Congenital afibrinogenemia is a rare autosomal recessive coagulation disorder characterised by hemorrhagic manifestations of variable entity and by severe plasma fibrinogen deficiency. Among the 31 afibrinogenemia-causing mutations so far reported, only 2 are missense mutations and both are located in the fibrinogen Bbeta-chain gene. Direct sequencing of the fibrinogen gene cluster in two afibrinogenemic Iranian siblings revealed a novel homozygous T>G transversion in exon 8 (nucleotide position 8025) of the fibrinogen Bbeta-chain gene. The resulting W437G missense mutation involves a highly conserved amino acid residue, located in the C-terminal globular D domain. The role of the W437G amino acid substitution on fibrinogen synthesis, folding, and secretion was assessed by in vitro expression experiments in COS-1 cells, followed by qualitative and quantitative analyses of intracellular and secreted mutant fibrinogen. Results of both pulse-chase experiments and enzyme-linked immunosorbent assays demonstrated intracellular retention of the mutant W437G fibrinogen and marked reduction of its secretion. These data, besides elucidating the pathogenetic role of the W437G mutation in afibrinogenemia, underline the importance of the Bbeta-chain D domain in fibrinogen folding and secretion.     

New insight into the molecular mechanisms of two-partner secretion

Two-partner secretion (TPS) systems, which export large proteins to the surface and/or extracellular milieu of Gram-negative bacteria, are members of a large superfamily of protein translocation systems that are widely distributed in animals, plants and fungi, in addition to nearly all groups of Gram-negative bacteria. Recent intense research on TPS systems has provided new insight into the structure and topology of the outer membrane translocator proteins and the large exoproteins that they secrete, the interactions between them, and mechanisms for retention of some of the secreted proteins on the bacterial surface. Evidence for secretion-dependent folding of mature exoproteins has also been obtained. Together, these findings provide a deeper understanding of the molecular mechanisms underlying these simple but elegant secretion systems.     

Growth hormone secretion: molecular and cellular mechanisms and in vivo approaches

Growth hormone (GH) release is under the direct control of hypothalamic releasing hormones, some being also produced peripherally. The role of these hypothalamic factors has been understood by in vitro studies together with such in vivo approaches as stalk sectioning. Secretion of GH is stimulated by GH-releasing hormone (GHRH) and ghrelin (acting via the GH secretagogue [GHS] receptor [GHSR]), and inhibited by somatostatin (SRIF). Other peptides/proteins influence GH secretion, at least in some species. The cellular mechanism by which the releasing hormones affect GH secretion from the somatotrope requires specific signal transduction systems (cAMP and/or calcium influx and/or mobilization of intracellular calcium) and/ or tyrosine kinase(s) and/or nitric oxide (NO)/cGMP. At the subcellular level, GH release (at least in response to GHS) is accomplished by the following. The GH-containing secretory granules are moved close to the cell surface. There is then transient fusion of the secretory granules with the fusion pores in the multiple secretory pits in the somatotrope cell surface.     

Cellular and molecular mechanisms controlling melatonin release by mammalian pineal glands

1. The pineal gland is regulated primarily by photoperiodic information attaining the organ through a multisynaptic pathway initiated in the retina and the retinohypothalamic tract. 2. Norepinephrine (NE) released from superior cervical ganglion (SCG) neurons that provide sympathetic innervation to the pineal acts through alpha1- and beta 1- adrenoceptors to stimulate melatonin synthesis and release. 3. The increase in cyclic AMP mediated by beta 1-adrenergic activation is potentiated by the increase in Ca2+ flux, inositol phospholipid turnover, and prostaglandin and leukotriene synthesis produced by alpha 1-adrenergic activation. 4. Central pinealopetal connections may also participate in pineal control mechanisms; transmitters and modulators in these pathways include several neuropeptides, amino acids such as gamma-aminobutyric acid (GABA) and glutamate, and biogenic amines such as serotonin, acetylcholine, and dopamine. 5. Secondary regulatory signals for pineal secretory activity are several hormones that act on receptors sites on pineal cells or at any stage of the neuronal pinealopetal pathway.     

Different effects of aging on the opioid mechanisms controlling gonadotropin and cortisol secretion in man

The present study was undertaken in order to assess the influence of aging on the endogenous opioid control of gonadotropin and adrenocorticotropin/cortisol secretion in man. For this purpose, the capability of the opioid antagonist naloxone to increase circulating levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and cortisol was tested in male subjects of different ages. Thirty normal men were randomly chosen and divided into 3 groups by age: group I = 22-40 years (n = 10); group II = 41-59 years (n = 10); group III = 62-80 years (n = 10). Since the men of group III showed higher basal serum gonadotropin concentrations than the subjects of group I and group II, we selected from a large population a fourth group of elderly men with normal basal LH and FSH levels: group IV = 61-82 years (n = 7). All subjects were tested for 120 min during the intravenous administration of naloxone (4 mg given in an intravenous bolus at time 0, plus 10 mg infused for 2 h). Control tests with normal saline instead of naloxone were performed in all groups. All subjects had similar blood testosterone and cortisol levels, whereas LH and FSH concentrations were significantly higher in group III than in groups I, II and IV. Naloxone increased plasma cortisol concentrations by 50% in all groups. The cortisol secretory response followed a similar pattern regardless of age.(ABSTRACT TRUNCATED AT 250 WORDS)     

The molecular mechanisms of quantum mediator secretion in the synapse

The modern condition of knowledge about the molecular mechanisms underlying the quantal transmitter release in the central and the peripheric synapses is analysed. The data about the synaptic vesicles types, their forming, transporting to the sites of release at the nerve endings, exo- and endocytosis processes are presented. Ultrastructural and molecular organization of active zone of nerve ending and transmitter release morphofunctional unit--secretosome, which includes synaptic vesicle, exocytosis protein complex and calcium channels, are described. The basic proteins involved in the exo- and endocytosis and their interactions during transmitter release are examined. The role of the intracellular buffer systems, calcium micro- and macrodomains in the quantal transmitter secretion are considered. The reasons of the active zones functional non-uniformity and plasticity and factors reduced transmitter release in the active zone to the single quantum are analysed.     

Platelet fibrinogen: subcellular localization by means of immunofluorescent studies in normals and in congenital afibrinogenemia.

We have studied the site of fibrinogen localization in normal platelets and in the platelets of a patient with congenital afibrinogenemia (CA). The methods employed were: direct immunofluorescence technique (DIT) and indirect immunofluorescence technique (IIT). By means of the DIT normal platelets were shown to have a clear peripheral staining. Such staining disappeared after treatment with proteolytic enzymes and after specific blocking experiments. Such peripheral staining of platelets was absent in congenital afibrinogenemia even after fibrinogen infusion. By means of the IIT platelets were shown to have a considerable amount of fibrinogen. Such protein was demonstrated to represent an important part of platelet surface, since intact platelets were able to absorb completely a specific antifibrinogen antiserum.     

Apoptosis: molecular mechanisms

Apoptosis is a genetically programmed cell death that is required for morphogenesis during embryogenic development and for tissue homeostasis in adult organisms. In most cases, apoptosis involves cytochrome c release from mitochondria. In the cytosol, cytochrome c combines with APAF-1 in the presence of ATP to activate caspase-9 that, in turn, activates effectors caspases such as caspase-3. Bcl-2 and related proteins control cytochrome c release from the mitochondria whereas IAP (for Inhibitor of APoptosis) molecules modulate the activity of caspases. Plasma membrane receptors such as Fas (CD95, APO-1), characterized by a so-called "death domain" in their cytoplasmic domain, can activate the caspase cascade through adaptator molecules such as FADD (Fas-Associated protein with a Death Domain). Dysregulation of the apoptotic machinery plays a role in the pathogenesis of various diseases and molecules involved in cell death pathways are potential therapeutic targets in immunologic, neurologic, cancer, infectious and inflammatory diseases.     

Studies on the assembly and secretion of fibrinogen.

cDNAs of fibrinogen A alpha and gamma chains were individually subcloned into a eukaryotic expression vector by using the polymerase chain reaction. Triple cotransfection into COS cells of the two plasmids together with a B beta chain expression plasmid, constructed as described previously (Danishefsky, K.J., Hartwig, R., Banerjee, D., and Redman, C. (1990) Biochim. Biophys. Acta 1048, 202-208), resulted in the secretion of complete fibrinogen into the media and the formation of four additional intracellular complexes which we also showed to be present in the hepatocyte cell line Hep 3B. The complexes, which have Mr = 232, 150, 135, and 128 (x 10(-3) conform with the Mr expected for A alpha B beta gamma 2, B beta gamma 2 and gamma 3, respectively. A A mechanism of assembly is proposed based on the assumption that all these complexes are precursors of complete fibrinogen. Each of the expressed fibrinogen chains in transfected COS cells interacts noncovalently with binding protein (BiP, GRP 78), but not to the same extent; gamma chain binds less BiP than the A alpha and B beta chains. Assembly of fibrinogen is not absolutely required for its secretion. In addition to complete fibrinogen, the conditioned media of hepatocytes and of transfected COS cells contained free A alpha, free gamma, and two of the above-mentioned complexes, A alpha gamma 2 and A alpha B beta gamma 2.     

Central peptidergic mechanisms controlling reproductive hormone secretion: novel methodology reveals a role for the natriuretic peptides.

A variety of neural factors can influence reproductive hormone secretion by neuromodulatory actions within the hypothalamus or neuroendocrine actions within the anterior pituitary gland. Passive immunoneutralization and antagonist administration protocols have suggested physiological roles for a number of these factors; however, both experimental approaches have severe technical limitations. We have developed novel methodology utilizing cytotoxin cell targeting with neuropeptides linked to the toxic A chain of the plant cytotoxin ricin. With this methodology we can target and destroy in vivo or in vitro cells bearing receptors for that peptide. Ricin A chain conjugated to atrial natriuretic peptide (ANP), a neuropeptide known to pharmacologically inhibit luteinizing hormone-releasing hormone (LHRH) release, was injected into the cerebroventricular system of intact, cycling rats and ovariectomized rats. Cytotoxin conjugate treatment significantly lengthened the estrous cycle. In ovariectomized rats the luteinizing hormone surge induced by steroid priming was completely inhibited. LHRH content of the median eminences of these rats was not significantly altered. These data suggest that ANP binding to clearance receptors in the hypothalamus displaces the C-type natriuretic peptide (CNP) from the shared clearance receptor, making more CNP available to inhibit LHRH release. In the absence of cells bearing the clearance receptor all available CNP binds to the ANPR-B receptor and exerts its effect via an inhibitory interneuron, since LHRH fibers are spared by this treatment.     

Beckwith-Wiedemann syndrome: multiple molecular mechanisms

Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth condition with an increased risk of developing embryonic tumours, such as Wilms' tumour. The cardinal features are abdominal wall defects, macroglossia and gigantism. BWS is generally sporadic; only 10-15% of cases are familial. A variety of molecular aberrations have been associated with BWS. The only mutations within a gene are loss-of-function mutations in the CDKN1C gene, which codes for an imprinted cell-cycle regulator. CDKN1C mutations appear to be particularly associated with umbilical abnormalities, but not with increased predisposition to Wilms' tumour. In the remaining BWS subgroups, a disturbance of the tight epigenetic regulation of gene expression (patUPD 11p, microdeletions or epimutations) seems to be the cause of the syndrome. Here we describe the clinical presentation of BWS and its dissociation from phenotypically overlapping overgrowth syndromes. We then review the current concepts of causative molecular genetic and epigenetic mechanisms, and discuss future directions of research.     

Organogenesis: molecular mechanisms of tubulogenesis

As organisms have evolved in size and complexity, tubular systems have developed to enable the efficient transport of substances into and out of tissues. These tubular systems are generated using strategies that are based on common elements of cell behaviour, including cell polarization, tube migration to target sites, cell-fate diversification and localization of specialized cells to different regions of the tube system. Using examples from both invertebrate and vertebrate systems, this review highlights progress in understanding these basic principles and briefly discusses the possible evolution of strategies to regulate the morphogenesis of tubular systems.     

Cancer cachexia: the molecular mechanisms

Cancer cachexia is a syndrome characterised by a marked weight loss, anorexia, asthenia and anaemia. In fact, many patients who die with advanced cancer suffer from cancer cachexia. The cachectic state is invariably associated with the presence and growth of the tumour and leads to a malnutrition status due to the induction of anorexia or decreased food intake. In addition, the competition for nutrients between the tumour and the host leads to an accelerated starvation state which promotes severe metabolic disturbances in the host, including hypermetabolism which leads to an increased energetic inefficiency. Although, the search for the cachectic factor(s) started a long time ago, and although many scientific and economic efforts have been devoted to its discovery, we are still a long way from knowing the whole truth. The main aim of the present review is to summarise and evaluate the different catabolic mediators (both humoural and tumoural) involved in cancer cachexia since they may represent targets for future promising clinical investigations.