Molecular Biological Properties Research Articles

Structure, diversity and expression of the TCRδ chains in the Mexican axolotl

Mammals and birds have two major populations of T cells, based on the molecular composition and biological properties of their antigen receptors (TCR). alpha beta T cells recognize antigenic peptides linked to major histocompatibility complex (MHC) molecules, and gamma delta T cells recognize native peptide or non-peptide antigens independently of MHC. Very little is known about gamma delta T cells in ectothermic vertebrates. We have cloned and characterized the TCRdelta chains of an urodele amphibian, the Mexican axolotl (Ambystoma mexicanum). The Cdelta domain is structurally similar to its mammalian homologues and the transmembrane domain is very well conserved. Four of the six Valpha regions that can associate with Calpha (Valpha2, Valpha3, Valpha5 and Valpha6) can also associate with Cdelta, but no specific Vdelta regions were found. This suggests that the axolotl TRD locus is nested within the TRA locus, as in mammals, and that this organization has been present in all tetrapod vertebrates and in the common ancestor of Lissamphibians and mammals, for over 400 million years. Two Jdelta regions were identified, but no Ddelta segments were clearly recognized at the Vdelta-Jdelta junctions. This results in shorter and less variable CDR3 loops than in other vertebrates and the size range of the Vdelta-Jdelta junctions is similar to that of mammalian immunoglobulin light chains. Equivalent quantities of TRD mRNA were found in the lymphoid organs, and in the skin and the intestines of normal and thymectomized axolotls. The analysis of several Valpha/delta6-Cdelta and Vbeta7-Cbeta junctions showed that both the TCRdelta and the TCRbeta chains were limited in diversity in thymectomized axolotls.

Biochemistry, Cellular and Molecular Biology, and Physiological Roles of the Iodothyronine Selenodeiodinases

The goal of this review is to place the exciting advances that have occurred in our understanding of the molecular biology of the types 1, 2, and 3 (D1, D2, and D3, respectively) iodothyronine deiodinases into a biochemical and physiological context. We review new data regarding the mechanism of selenoprotein synthesis, the molecular and cellular biological properties of the individual deiodinases, including gene structure, mRNA and protein characteristics, tissue distribution, subcellular localization and topology, enzymatic properties, structure-activity relationships, and regulation of synthesis, inactivation, and degradation. These provide the background for a discussion of their role in thyroid physiology in humans and other vertebrates, including evidence that D2 plays a significant role in human plasma T3 production. We discuss the pathological role of D3 overexpression causing “consumptive hypothyroidism” as well as our current understanding of the pathophysiology of iodothyronine deiodination during illness and amiodarone therapy. Finally, we review the new insights from analysis of mice with targeted disruption of the Dio2 gene and overexpression of D2 in the myocardium.

Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases.

The goal of this review is to place the exciting advances that have occurred in our understanding of the molecular biology of the types 1, 2, and 3 (D1, D2, and D3, respectively) iodothyronine deiodinases into a biochemical and physiological context. We review new data regarding the mechanism of selenoprotein synthesis, the molecular and cellular biological properties of the individual deiodinases, including gene structure, mRNA and protein characteristics, tissue distribution, subcellular localization and topology, enzymatic properties, structure-activity relationships, and regulation of synthesis, inactivation, and degradation. These provide the background for a discussion of their role in thyroid physiology in humans and other vertebrates, including evidence that D2 plays a significant role in human plasma T(3) production. We discuss the pathological role of D3 overexpression causing "consumptive hypothyroidism" as well as our current understanding of the pathophysiology of iodothyronine deiodination during illness and amiodarone therapy. Finally, we review the new insights from analysis of mice with targeted disruption of the Dio2 gene and overexpression of D2 in the myocardium.

Genomic organization of the dysferlin gene and novel mutations in Miyoshi myopathy.

Mutations in the skeletal muscle gene dysferlin cause two autosomal recessive forms of muscular dystrophy: Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B). The purpose of this study was to define the genomic organization of the dysferlin gene and conduct mutational screening and a survey of clinical features in 21 patients with defined molecular defects in the dysferlin gene. Genomic organization of the gene was determined by comparing the dysferlin cDNA and genomic sequence in P1-derived artificial chromosomes (PACs) containing the gene. Mutational screening entailed conformational analysis and sequencing of genomic DNA and cDNA. Clinical records of patients with defined dysferlin gene defects were reviewed retrospectively. The dysferlin gene encompasses 55 exons spanning over 150 kb of genomic DNA. Mutational screening revealed nine novel mutations associated with MM. The range of onset in this patient group was narrow with a mean of 19.0 +/- 3.9 years. This study confirms that the dysferlin gene is mutated in MM and LGMD2B and extends understanding of the timing of onset of the disease. Knowledge of the genomic organization of the gene will facilitate mutation detection and investigations of the molecular biologic properties of the dysferlin gene.

Pemphigoid gestationis: A review

Pemphigoid gestationis is a rare autoimmune vesiculobullous skin disease closely related to the pemphigoid group of blistering disorders. It is unique in that it is most frequently associated with pregnancy. Diagnosis is made on the basis of the presence of a subepidermal vesicle on routine histologic examination and of linear deposition of the third component of complement along the basement membrane zone of perilesional skin. Abnormal expression of major histocompatibility complex class II molecules in the placenta may trigger the initiating immunologic event in this organ, which is followed by cross-reactivity with an antigen present in the skin and the characteristic cutaneous signs of the disease. The immunogenetics of this disorder are interesting. A role for the involvement of major histocompatibility complex class II antigens seems likely because of the association with human leukocyte antigens DR3 and DR4. Major histocompatibility complex class III associations have also been observed, as has a high frequency of anti–human leukocyte antigen antibodies. We review the current understanding of the molecular biologic and immunogenetic properties of this disease and discuss treatment and potential impact on the fetus. (Am J Obstet Gynecol 2000;183:483-91.)

Biochemical and Molecular Biological Properties and Human Defect of TDP (thiamin diphosphate) -dependent 2-Oxo Acid Dephdrogenases

Biochemical and Molecular Biological Properties and Human Defect of TDP (thiamin diphosphate) -dependent 2-Oxo Acid Dephdrogenases

Targeted Gene Delivery: A Two-Pronged Approach

The success of gene therapy relies on the ability of gene delivery systems to selectively deliver therapeutic genes to a sufficient number of target cells yielding expression levels that impact the diseased state. The gene delivery systems can be divided into two classes: viral and nonviral (or plasmid DNA-based). The present gene delivery technology being used in clinics today can be considered first generation, in that they possess the ability to transfect or infect target cells through their inherent chemical, biochemical, and molecular biological properties. Relying on these sole properties, however, limits therapeutic applications. Expansion of therapeutic applications or increased effectiveness of current therapies can be achieved by increasing the number of cells and cell types susceptible to gene transfer. This can be achieved through physical targeting and molecular biological targeting. Physical targeting relies on the attachment to the delivery vehicle of ligands that bind to cell surface receptors unique to the target cells. Molecular biological targeting refers to selective expression of the therapeutic gene by the target cell through the use of selective promoters. Selective expression can be further achieved by the use of expression systems controlled by extrinsic induction molecules. This review will describe in detail the advances that have been made in each of these areas of gene targeting.

Biologic Activities of the Contact Factors In Vivo

Three plasma proteins, namely, Factor XII (Hageman factor), prekallikrein (Fletcher factor), and high molecular weight kininogen (Williams-Fitzgerald-Flaujeac factor), have been shown to be required for the activation of the surface-mediated pathway, or “contact system.” In vitro, such as in the activated partial thromboplastin test or on exposure to artificial surfaces, factor XII (FXII) is autoactivated to factor XIIa, which cleaves plasma prekallikrein (PK) by limited proteolysis into the active enzyme, plasma kallikrein (Fig. 1). In contrast, in vivo, high molecular weight kininogen (HK) binds to the endothelial cell surface associated with PK in a noncovalent complex. PK is activated by an endothelial cell cysteine protease to kallikrein which cleaves prourokinase to urokinase and factor XII to factor XIIa (Fig. 2). Thus, on cells, PK activation initiates the contact system, while on artificial surfaces, FXII is the initiating molecule. HK is also the substrate of plasma kallikrein, which cleaves to yield kinin-free HK (HKa) and bradykinin (Fig. 1). Although the genetically determined deficiencies of FXII, PK, and HK display abnormal in vitro plasma coagulation, none of these deficiencies is associated with a hemorrhagic diathesis. However, Factor XII, PK and HK display a wide variety of important interactions with other plasma proteins. In vitro, activated factor XII activates factor XI, factor VII and the C1 component of the complement system. Plasma kallikrein activates prourokinase (Fig. 1), prorenin, and factor XII, and cleaves HK, releasing bradykinin. Each contact protein interacts with blood and vascular cells. Factor XII downregulates a monocyte Fc receptor, releases IL-1 and IL-6 from monocytes and macrophages, and stimulates neutrophils, Plasma kallikrein activates neutrophils and potentiates fibrinolysis on endothelial cells. HK inhibits adhesion of neutrophils to artificial surfaces, enhances cellular fibrinolysis, and inhibits thrombin-induced platelet activation. Knowledge of these biochemical and cellular reactions has led to an understanding of the role of contact proteins in inflammation, fibrinolysis, cell adhesion, platelet activation and angiogenesis, and an appreciation of their participation in various pathophysiologic conditions. The molecular biology, structure and biochemical properties of FXII, PK and HK have recently been comprehensively reviewed. This review will focus on their participation in vivo in pathophysiologic processes including hypotension, inflammation, fibrinolysis, cell adhesion, angiogenesis and thrombosis.

Low-molecular-weight heparins: pharmacologic profile and product differentiation

The interchangeability of low-molecular-weight heparins (LMWHs) has been the subject of discussion since these products were first introduced for the prophylaxis of deep vein thrombosis. Experimental evidence now exists to show that LMWHs differ from each other in a number of characteristics. Products have been differentiated on the basis of molecular weight and biologic properties, but only limited information derived from the clinical setting is available. Potency has been described on the basis of anti-Factor Xa activity, but at equivalent anti-Xa activities, the anti-Factor IIa activity of different products shows marked variations. At the relatively small doses used for the management of postsurgical deep vein thrombosis, the effect of these interproduct differences may be relatively minor, but as LMWHs are developed for therapeutic use at much higher doses, such differences may become clinically important. Variations in safety and efficacy reported in clinical trials of LMWHs may reflect the known differences in their molecular composition and pharmacologic properties.

Male breast cancer: a review of the literature.

Although breast cancer is uncommon in men, it can cause significant morbidity and mortality. The current review was undertaken to determine whether strategies applied for the evaluation and treatment of breast cancer in females are appropriate in male breast cancer. Male breast cancer has biological differences compared with female breast cancer, including a high prevalence in certain parts of Africa, a higher incidence of oestrogen receptor positivity and more aggressive clinical behaviour. It responds to hormonal manipulation and chemotherapy, but optimal treatment regimens in males are unknown. Male breast cancer remains an uncommon disease. Most of our current knowledge regarding its biology, natural history and treatment strategies has been extrapolated from its female counterpart. Much research is needed to further characterise the molecular biological properties of male breast tumours and their prognostic significance, and to devise treatment strategies, including optimal chemotherapy regimens.

Molecular and pharmacological analysis of cyclic nucleotide-gated channel function in the central nervous system

Most functional studies of cyclic nucleotide-gated (CNG) channels have been confined to photoreceptors and olfactory epithelium, in which CNG channels are abundant and easy to study. The widespread distribution of CNG channels in tissues throughout the body has only recently been recognized and the functions of this channel family in many of these tissues remain largely unknown. The molecular biological and pharmacological properties of the CNG channel family are summarized in order to put in context studies aimed at probing CNG channel functions in these tissues using pharmacological and genetic methods. Compounds have now been identified that are useful in distinguishing CNG channel activated pathways from cAMP/cGMP dependent-protein kinases or other pathways. The ways in which these interact with CNG channels are understood and this knowledge is leading to the identification of more potent and more specific CNG channel subtype-specific agonists or antagonists. Recent molecular and genetic analyses have identified novel roles of CNG channels in neuronal development and plasticity in both invertebrates and vertebrates. Targeting CNG channels via specific drugs and genetic manipulation (such as knockout mice) will permit better understanding of the role of CNG channels in both basic and higher orders of brain function.

Human alpha-endosulfine, a possible regulator of sulfonylurea-sensitive KATP channel: molecular cloning, expression and biological properties.

Sulfonylureas are a class of drugs commonly used in the management of non-insulin-dependent diabetes mellitus. Their therapeutic action results primarily from their ability to inhibit ATP-sensitive potassium (KATP) channels in the plasma membrane of pancreatic beta cells and thereby stimulate insulin release. A key question is whether an endogenous ligand for the KATP channel exists that is able to mimic the inhibitory effects of sulfonylureas. We describe here the cloning of the cDNA encoding human alpha-endosulfine, a 13-kDa peptide that is a putative candidate for such a role. alpha-Endosulfine is expressed in a wide range of tissues including muscle, brain, and endocrine tissues. The recombinant protein displaces binding of the sulfonylurea [3H]glibenclamide to beta cell membranes, inhibits cloned KATP channel currents, and stimulates insulin secretion. We propose that endosulfine is an endogenous regulator of the KATP channel, which has a key role in the control of insulin release and, more generally, couples cell metabolism to electrical activity.

The structure of the cofactor-binding fragment of the LysR family member, CysB: a familiar fold with a surprising subunit arrangement

CysB is a tetrameric protein of identical subunits (M(r) = 36,000) which controls the expression of genes associated with the biosynthesis of cysteine in bacteria. CysB is both an activator and a repressor of transcription whose activity is responsive to the inducer N-acetylserine; thiosulphate and sulphide act as anti-inducers. CysB is a member of the LysR family of prokaryotic transcriptional regulatory proteins which share sequence similarities over approximately 280 residues including a putative helix-turn-helix DNA-binding motif at their N terminus. The aims of the present study were to explore further the complex molecular biology and curious ligand binding properties of CysB and to provide structural insights into the LysR family of proteins. The crystal structure of a dimeric chymotryptic fragment of Klebsiella aerogenes CysB comprising residues 88-324, has been solved by multiple isomorphous replacement and multi-crystal averaging and refined against data extending to 1.8 A resolution. The protein comprises two alpha/beta domains (I and II) connected by two short segments of polypeptide. The two domains enclose a cavity lined by polar sidechains, including those of two residues whose mutation is associated with constitutive expression of the cysteine regulon. A sulphate anion and a number of well ordered water molecules have been modelled into discrete electron-density peaks within this cavity. In the dimer, strands beta B from domain I and strands beta G from domain II come together so that a pair of antiparallel symmetry-related 11-stranded twisted beta-pleated sheets is formed. The overall structure of CysB(88-324) is strikingly similar to those of the periplasmic substrate-binding proteins. A similar fold has also been observed in the cofactor-binding domain of Lac repressor, implying a structural relationship between the Lac repressor and LysR families of proteins. In contrast to Lac repressor, in CysB the twofold axis of symmetry that relates the monomers in the dimer is perpendicular rather than parallel to the long axis of the cofactor-binding domain. This seems likely to place the DNA-binding domains at opposite extremes of the molecule possibly accounting for CysB's extended DNA footprints.

Recent advances in antiviral research : identification of inhibitors of the herpesvirus proteases

Major advances have been reported in the last two years regarding the molecular biology and structural properties of the herpesvirus proteases. X-ray diffraction studies have enabled several groups to solve the structure of the human cytomegalovirus protease. Fluorescence-based substrate assays have also been recently reported. These substrates exhibit sufficient kinetic and sensitivity properties to enable high-throughput screening efforts dedicated toward the discovery of protease inhibitors. Three classes of inhibitors have been reported recently: nonpeptidic aryl trifluoromethylketones; alternate substrate inhibitors (benzoxazinones/azalactones); and thiol-modifying inhibitors. The thiol-modifying class offers a unique opportunity to discover inhibitors specific to the human cytomegalovirus protease, as this protease requires reduced cysteine residues for its enzymatic activity.

Relationships between cytoskeletal proteins and the toxic effects of drugs.

Many cell functions and aspects of cell configuration are linked with changes in cytoskeletal proteins, by intracellular signal transduction. Histochemical and pathophysiological changes caused by toxins or drugs affect on dynamic aspects of cytoskeletal proteins. Here we review that biochemical and molecular biological properties of the microtubules and microfilaments and the relationships between these cytoskeletal proteins and the toxic effects of drugs and toxins. The intracellular signaling pathway makes use of cytoskeletal proteins.

Anterior cruciate ligament reconstruction with the patellar tendon augmented by absorbable artificial materials. A histological and molecular biological study in rabbits

An experimental study was performed to investigate the histological and molecular biological properties of reconstructed anterior cruciate ligaments (ACL) using the patellar tendon augmented by absorbable artificial material in rabbits. Thirty rabbits underwent ACL reconstruction with the patellar tendon (non-augmented group), and 30 with the patellar tendon augmented by a polyglactin 910 mesh (augmented group). They were respectively sacrificed at at 1, 2, 3, 4, 6, 8 and 12 weeks after surgery for histological and molecular biological examination. Histologically, the fibrocytes were disappeared in deep portion of transplanted graft at 2 weeks after surgery in both groups. The graft was replaced with granulated tissues at 6 weeks in the augmented group, but it took 8 weeks in the non-augmented group. At 12 weeks, the graft showed hypocellularity with regularly oriented fibers in the augmented group, while it still showed hypercellularity with disoriented fibers in the non-augmented group. Molecular biologically, after the reconstruction at one week, the relative radioactivity in the graft was 1.05 in the non-augmented group, and 2.10 in the augmented group. Additionally the level of the alpha 2 (I) collagen mRNA was also significantly higher in the augmented group than that in the non-augmented group. These results suggested that a polyglactin 910 mesh had an effect on the process of reorganization and remodeling in the transplanted patellar tendon during the post-operative period in rabbit.

The mammalian myosin heavy chain gene family.

Myosin is a highly conserved, ubiquitous protein found in all eukaryotic cells, where it provides the motor function for diverse movements such as cytokinesis, phagocytosis, and muscle contraction. All myosins contain an amino-terminal motor/head domain and a carboxy-terminal tail domain. Due to the extensive number of different molecules identified to date, myosins have been divided into seven distinct classes based on the properties of the head domain. One such class, class II myosins, consists of the conventional two-headed myosins that form filaments and are composed of two myosin heavy chain (MYH) subunits and four myosin light chain subunits. The MYH subunit contains the ATPase activity providing energy that is the driving force for contractile processes mentioned above, and numerous MYH isoforms exist in vertebrates to carry out this function. The MYHs involved in striated muscle contraction in mammals are the focus of the current review. The genetics, molecular biology, and biochemical properties of mammalian MYHs are discussed below. MYH gene expression patterns in developing and adult striated muscles are described in detail, as are studies of regulation of MYH genes in the heart. The discovery that mutant MYH isoforms have a causal role in the human disease familial hypertrophic cardiomyopathy (FHC) has implemented structure/function investigations of MYHs. The regulation of MYH genes expressed in skeletal muscle and the potential functional implications that distinct MYH isoforms may have on muscle physiology are addressed.

Mechanisms of Venous Leakage: A Prospective Clinicopathological Correlation of Corporeal Function and Structure

Purpose We investigated the pathophysiology of structurally based corporeal veno-occlusive dysfunction. Materials and Methods We prospectively evaluated 24 impotent patients (mean age plus or minus standard error 46 plus/minus 3 years) who had exposure to vascular risk factors and/or disorders inducing diffuse trabecular structure alterations and who underwent penile prosthesis insertion. Preoperative indexes of veno-occlusive function (flow to maintain, venous outflow resistance and pressure decay measurements using repeat dosing pharmacocavernosometry) were correlated with postoperative erectile tissue computer assisted color histomorphometry (percent trabecular smooth muscle to total erectile tissue area). To develop further study findings and correlate histomorphometric findings with molecular biological properties molecular biological studies (ribonuclease protection analysis, reverse transcription-polymerase chain reaction assay for expression of transforming growth factor-beta 1 messenger [m] ribonucleic acid [RNA] and protein affinity labeling techniques for specific transforming growth factor-beta receptors) were performed in representative patients with high (39 to 43 percent), intermediate (30 to 37 percent) and low (13 to 29 percent) trabecular smooth muscle content (normal 42 to 50 percent). Results Flow to maintain, venous outflow resistance and pressure decay values significantly correlated with trabecular smooth muscle cell content (r = -0.89, 0.82 and -0.85, respectively). In the high, intermediate and low smooth muscle content subgroups flow to maintain, venous outflow resistance and pressure decay values were 1 to 5, 9 to 30 and 50 to 120 ml. per minute, 17 to 84, 3 to 9 and 1 to 2 mm. Hg/ml. per minute, and 40 to 60, 48 to 80 and 110 to 120 mm. Hg decrease in 30 seconds from 150 mm. Hg, respectively. There were no significant differences in patient age or prevalence of risk factors among the 3 subgroups. Patients representative of all 3 subgroups had transforming growth factor-beta 1 mRNA, auto-induction of transforming growth factor-beta 1 mRNA and induction and/or increased availability of all 3 types of transforming growth factor-beta receptors. Conclusions The pathophysiology of structurally based corporeal veno-occlusive dysfunction is related to elevated corporeal connective tissue content. Based on our data and those in the literature corporeal fibrosis is hypothesized to develop secondary to abnormalities in the regulation of normal collagen synthesis and degradation, most likely associated with adverse influences of chronic ischemia.

Fragile X syndrome. I. An overview on its genetic mechanism

A large body of literature has accumulated within the last decade concerning the fragile X syndrome, the most common cause of X-linked mental retardation. The first article of this review summarizes the peculiar genetic mechanisms and molecular biology properties (eg, unstable DNA triplet repeats), which have been characterized since the detection of the FMR-1 gene in 1991. However, the most important question concerning the function of the FMR-1 gene is still an unresolved issue and is in need of future research. The second article of this review addresses the clinical picture, neuropsychological functioning and psychopathological characteristics of pre- and full mutation carriers.

The role of lectins in plant defence

Recent progress in the search for the physiological role of plant lectins supports the idea that some of these proteins are involved in the defence mechanisms of the plant. To place the evidence in favour of such a defensive role in a broad perspective, a short overview is given of the most important plant pathogens and predators. In addition, the solutions that plants have developed to resist the continuous threat of a hostile environment are briefly discussed in relation to the protective role of proteins in general. The presumed involvement of plant lectins in defence mechanisms is first inferred from an analysis of the biochemical, physiological, cellular biological and molecular biological properties of plant lectins. Subsequently, the available experimental evidence for the involvement of lectins in the plant's defence against viruses, bacteria, fungi and herbivorous invertebrates and vertebrates is discussed in some detail. Since the defensive role of plant lectins is determined largely by their ability to recognize and bind foreign glycans, a brief discussion is given of how the basically protective properties of these proteins can be exploited for histochemical applications in biological and biomedical research.