Expression Patterns and Potential Biological Roles of Dip2a

Disconnected (disco)-interacting protein 2 homolog A is a member of the DIP2 protein family encoded by Dip2a gene. Dip2a expression pattern has never been systematically studied. Functions of Dip2a in embryonic development and adult are not known. To investigate Dip2a gene expression and function in embryo and adult, a Dip2a-LacZ mouse model was generated by insertion of β-Gal cDNA after Dip2a promoter using CRISPR/Cas9 technology. Dip2a-LacZ mouse was designed to be a lacZ reporter mouse as well as a Dip2a knockout mouse. Heterozygous mice were used to study endogenous Dip2a expression and homozygotes to study DIP2A-associated structure and function. LacZ staining indicated that Dip2a is broadly expressed in neuronal, reproductive and vascular tissues, as well as in heart, kidney, liver and lung. Results demonstrate that Dip2a is expressed in ectoderm-derived tissues in developing embryos. Adult tissues showed rich staining in neurons, mesenchymal, endothelial, smooth muscle cells and cardiomyocytes by cell types. The expression pattern highly overlaps with FSTL1 and supports previous report that DIP2A to be potential receptor of FSTL1 and its protective roles of cardiomyocytes. Broad and intense embryonic and adult expression of Dip2a has implied their multiple structural and physiological roles.     

Lésions des organes génitaux externes par arme a feu: a propos de six observations

Patients and Methods

This is a retrospective study of patients admitted with gunshot injuries to the external genital organs to the urology departments of Aristide Le Dantec Hospital, Grand Yoff General Hospital and Dakar Principal Hospital, between January 1998 and January 2003. The following parameters were studied: patients’ age and gender, interval between accident and admission, clinical features, complementary investigations and treatment, and early and late outcomes.

Results

Only males were observed and the mean age of these patients was 37.7 years (range: 22–52 years). Lesions were accidental in five cases. Two cases of testicular wounds, two cases of corpus cavernosum injuries, two cases of urethral injury, and one case of perineal wound were observed. After excision of the lesions, complementary procedures consisted of: orchidectomy in two cases, corpus cavernosum — albuginea suture in two cases and end-to-end urethral suture in one case. Perineoscrotal wounds were sutured three days after debridement. All patients received antibiotics and antitetanus serum. No death was observed during the immediate follow-up. No case of urethral stricture or erectile dysfunction was observed with a follow-up of 18 months. Semen analysis was normal in the two cases of orchidectomy.     

Subtelomeric proteins negatively regulate telomere elongation in budding yeast

The Tbf1 and Reb1 proteins are present in yeast subtelomeric regions. We establish in this work that they inhibit telomerase-dependent lengthening of telomere. For example, tethering the N-terminal domain of Tbf1 and Reb1 in a subtelomeric region shortens that telomere proportionally to the number of domains bound. We further identified a 90 amino-acid long sequence within the N-terminal domain of Tbf1 that is necessary but not sufficient for its length regulation properties. The role of the subtelomeric factors in telomere length regulation is antagonized by TEL1 and does not correlate with a global telomere derepression. We show that the absence of TEL1 induces an alteration in the structure of telomeric chromatin, as defined biochemically by an increased susceptibility to nucleases and a greater heterogeneity of products. We propose that the absence of TEL1 modifies the organization of the telomeres, which allows Tbf1 and Reb1 to cis-inhibit telomerase. The involvement of subtelomeric factors in telomere length regulation provides a possible mechanism for the chromosome-specific length setting observed at yeast and human telomeres.     

Stabilization of the E* Form Turns Thrombin into an Anticoagulant

Previous studies have shown that deletion of nine residues in the autolysis loop of thrombin produces a mutant with an anticoagulant propensity of potential clinical relevance, but the molecular origin of the effect has remained unresolved. The x-ray crystal structure of this mutant solved in the free form at 1.55 Å resolution reveals an inactive conformation that is practically identical (root mean square deviation of 0.154 Å) to the recently identified E* form. The side chain of Trp²¹⁵ collapses into the active site by shifting >10 Å from its position in the active E form, and the oxyanion hole is disrupted by a flip of the Glu¹⁹²–Gly¹⁹³ peptide bond. This finding confirms the existence of the inactive form E* in essentially the same incarnation as first identified in the structure of the thrombin mutant D102N. In addition, it demonstrates that the anticoagulant profile often caused by a mutation of the thrombin scaffold finds its likely molecular origin in the stabilization of the inactive E* form that is selectively shifted to the active E form upon thrombomodulin and protein C binding.Serine proteases of the trypsin family are responsible for digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis, and immunity (1). Activation of the protease requires the transition from a zymogen form (2) and formation of an ion pair between the newly formed amino terminus of the catalytic chain and the side chain of the highly conserved residue Asp¹⁹⁴ (chymotrypsinogen numbering) next to the catalytic Ser¹⁹⁵. This ensures substrate access to the active site and proper formation of the oxyanion hole contributed by the backbone N atoms of Ser¹⁹⁵ and Gly¹⁹³ (3). The zymogen → protease conversion is classically associated with the onset of catalytic activity (3, 4) and provides a useful paradigm for understanding key features of protease function and regulation.Recent kinetic (5) and structural (6, 7) studies of thrombin, the key protease in the blood coagulation cascade (8), have drawn attention to a significant plasticity of the trypsin fold that impacts the function of the enzyme in a decisive manner. The active form of the protease, E, coexists with an inactive form, E*, that is distinct from the zymogen conformation (9). The E* form features a collapse of the 215–217 β-strand into the active site and a flip of the peptide bond between residues Glu¹⁹² and Gly¹⁹³ that disrupts the oxyanion hole. Importantly, the ion pair between Ile¹⁶ and Asp¹⁹⁴ remains intact, suggesting that E* is not equivalent to the zymogen form of the protease and that the E*-E equilibrium is established after the conversion from the zymogen form has taken place. Indeed, existing structures of the zymogen forms of trypsin (10), chymotrypsin (11), and chymase (12) feature a broken Ile¹⁶–Asp¹⁹⁴ ion pair but no collapse of the 215–217 β-strand. Stopped-flow experiments show that the E*-E conversion takes place on a time scale of <10 ms (5), as opposed to the much longer (100–1000 ms) time scale required for the zymogen-protease conversion (13, 14).The E* form is not a peculiarity of thrombin. The collapse of the 215–217 β-strand into the active site is observed in the inactive form of αI-tryptase (15), the high temperature requirement-like protease (16), complement factor D (17), granzyme K (18), hepatocyte growth factor activator (19), prostate kallikrein (20), and prostasin (21). A disrupted oxyanion hole is observed in complement factor B (22) and the arterivirus protease Nsp4 (23). The most likely explanation for the widespread occurrence of inactive conformations of trypsin-like proteases is that the E*-E equilibrium is a basic property of the trypsin fold that fine tunes activity and specificity once the zymogen → protease conversion has taken place (9).The new paradigm established by the E*-E equilibrium has obvious physiological relevance. In the case of complement factors, kallikreins, tryptase, and some coagulation factors must be kept to a minimum until binding of a trigger factor ensues. Stabilization of E* may afford a resting state of the protease waiting for action, as seen for other systems (24–28). For example, factor B is mostly inactive until binding of complement factor C3 unleashes catalytic activity at the site where amplification of C3 activation is most needed prior to formation of the membrane attack complex (29). Indeed, the crystal structure of factor B reveals a conformation with the oxyanion hole disrupted by a flip of the 192–193 peptide bond (22), as observed in the E* form of thrombin (6, 7).The allosteric equilibrium as shown in Scheme 1, involves the rates for the E* → E transition, k₁, and backward, k₋₁, that define the equilibrium constant r = k₋₁/k₁ = [E*]/[E] (5). The value of k_cat/K_m for an enzyme undergoing the E*-E equilibrium is as shown in Equation 1 (30), where s_E is the value of s for the E form, and obviously s_E* = 0. Likewise, the binding of an inhibitor to the enzyme undergoing the E*-E equilibrium is shown in Equation 2, where K_E is the value of the equilibrium association constant K for the E form, and K_E* = 0. As the value of r increases upon stabilization of E*, the values of s and K in Equations 1 and 2 decrease without limits. Hence, stabilization of E* has the potential to completely abrogate substrate hydrolysis (s → 0) or inhibitor binding (K → 0). However, binding of a suitable cofactor could restore activity by triggering the E* → E transition. This suggests a simple explanation for the anticoagulant profile observed in a number of thrombin mutants that have poor activity toward all physiological substrates but retain activity toward the anticoagulant protein C in the presence of the cofactor thrombomodulin (31–34). Here we report evidence that stabilization of E* provides a molecular mechanism to turn thrombin into an anticoagulant.     

Secretin, glucagon, gastric inhibitory polypeptide, parathyroid hormone, and related peptides in the regulation of the hypothalamus- pituitary-adrenal axis

Secretin, glucagon, gastric inhibitory polypeptide (GIP), and parathyroid hormone (PTH) belong, together with vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase (AC)-activating polypeptide, to a family of peptides (the VIP-secretin-glucagon family), which also includes growth hormone-releasing hormone and exendins. All the members of this peptide family possess a remarkable amino-acid sequence homology, and bind to G-protein-coupled receptors, whose signaling mechanism primarily involves AC/protein kinase A and phospholipase C/protein kinase C cascades. VIP and pituitary AC-activating polypeptide play a role in the regulation of the hypothalamus-pituitary-adrenal (HPA) axis, and in this review we survey findings that also other members of the VIP-secretin-glucagon family may have the same function. Secretin and secretin receptors are expressed in the hypothalamus and pituitary gland, and secretin inhibits adrenocorticotropic hormone (ACTH) release. No evidence is available for the presence of secretin receptors in adrenal glands, but secretin selectively depresses the glucocorticoid response to ACTH of dispersed zona fasciculata-reticularis (ZF/R) cells. Glucagon and glucagon-like peptide-1 are contained in the hypothalamus, and all the components of the HPA axis are provided with glucagon and glucagons-like-1 receptors. These peptides exert a short-term inhibitory effect on stress-induced pituitary ACTH release and depress the ZF/R cell response to ACTH by inhibiting the AC/protein kinase A cascade; they also stimulate hypothalamic arginine-vasopressin release. GIP receptors are present in the ZF/R of the normal adrenals, and are particularly abundant in some types of adrenocortical adenomas and hyperplasias. GIP, through the activation of the AC/protein kinase A cascade, evokes a sizeable glucocorticoid secretagogue effect, leading to the identification of a food/GIP-dependent Cushing's syndrome. PTH and PTH-related protein are expressed in the hypothalamus and pituitary gland, and PTH and PTH-related protein receptors in all the components of the HPA axis. Both peptides enhance ACTH and arginine-vasopressin release, as well as stimulate aldosterone and glucocorticoid secretion of dispersed zona glomerulosa and ZF/R cells, respectively. The involvement of growth hormone-releasing hormone and exendins in the functional regulation of the HPA axis has not yet been extensively investigated.