The leucine-rich repeat is a recently characterized structural motif used in molecular recognition processes as diverse as signal transduction, cell adhesion, cell development, DNA repair and RNA processing. We present here the crystal structure at 2.5 A resolution of the complex between ribonuclease A and ribonuclease inhibitor, a protein built entirely of leucine-rich repeats. The unusual non-globular structure of ribonuclease inhibitor, its solvent-exposed parallel beta-sheet and the conformational flexibility of the structure are used in the interaction; they appear to be the principal reasons for the effectiveness of leucine-rich repeats as protein-binding motifs. The structure can serve as a model for the interactions of other proteins containing leucine-rich repeats with their ligands.

A structural basis of the interactions between leucine-rich repeats and protein ligands.

Various tissues and cells in culture contain a specific inhibitor of DNase I (EC 3145) In this paper evidence is presented that this inhibitor is actin, one of the major structural proteins of muscle and nonmuscle cells (a) The inhibitor is a major cellular component constituting 5-10% of the soluble protein (b) It migrates with actin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, having a characteristic molecular weight of 42,000 (c) It has an amino-acid composition closely similar to that of actin (d) The peptide maps of the two proteins are nearly identical (e) Skeletal muscle actin inhibits the enzymatic activity of DNase I (f) DNase I-agarose affinity chromatography quantitatively retains purified skeletal muscle actin, and actin, specifically, from high-speed supernatants of whole cell extracts (g) An antibody to purified inhibitor protein from calf thymus, used in indirect immunofluorescence on cells grown in culture, stains a two-dimensional network of fibers similar to that seen with an actin-specific antibody The observation that actin can be isolated by DNase-agarose affinity chromatography provides a useful tool for the biochemical study of actin under different physiological conditions

https://www.pnas.org/content/pnas/71/12/4742.full.pdf

Actin Is the Naturally Occurring Inhibitor of Deoxyribonuclease I

Background: Gene silencing by abnormal methylation of promoter regions of regulatory genes is commonly associated with cancer. Silenced tumor suppressor genes are obvious targets for reactivation by methylation inhibitors such as 5-azacytidine (5-Aza-CR) and 5-aza-2-deoxycytidine (5Aza-CdR). However, both compounds are chemically unstable and toxic and neither can be given orally. We characterized a new demethylating agent, zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one], which is a chemically stable cytidine analog. Methods: We tested the ability of zebularine to reactivate a silenced Neurospora crassa gene using a hygromycin gene reactivation assay. We then analyzed the ability of zebularine to inhibit DNA methylation in C3H 10T1/2 Cl8 (10T1/2) mouse embryo cells as assayed by induction of a myogenic phenotype and in T24 human bladder carcinoma cells, using the methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay. We also evaluated the effects of zebularine (administered orally or intraperitoneally) on growth of EJ6 human bladder carcinoma cells grown in BALB/c nu/nu mice (five mice per group) and the in vivo reactivation of a methylated p16 gene in these cells. All statistical tests were two-sided. Results: In N. crassa, zebularine inhibited DNA methylation and reactivated a gene previously silenced by methylation. Zebularine induced the myogenic phenotype in 10T1/2 cells, which is a phenomenon unique to DNA methylation inhibitors. Zebularine reactivated a silenced p16 gene and demethylated its promoter region in T24 bladder carcinoma cells in vitro and in tumors grown in mice. Zebularine was only slightly cytotoxic to T24 cells in vitro (1 mM zebularine for 48 hours decreased plating efficiency by 17% [95% confidence interval (CI) = 12.8% to 21.2%]) and to tumor-bearing mice (average maximal weight change in mice treated with 1000 mg/kg zebularine = 11% [95% CI = 4% to 19%]). Compared with those in control mice, tumor volumes were statistically significantly reduced in mice treated with high-dose zebularine administered by intraperitoneal injection (P<.001) or by oral gavage (P<.001). Conclusions: Zebularine is a stable DNA demethylating agent and the first drug in its class able to reactivate an epigenetically silenced gene by oral administration. [J Natl Cancer Inst 2003;95:399–409]

Inhibition of DNA Methylation and Reactivation of Silenced Genes by Zebularine

In tissue engineering and regenerative medicine, a biomaterial provides mechanical support and biochemical signals to encourage cell attachment and modulate cell behaviour. Nature’s template for a biomaterial is the extracellular matrix (ECM). The ECM contains intrinsic biochemical and mechanical cues that regulate cell phenotype and function in development, in homeostasis and in response to injury. The use of ECM-based materials in biomedical research has advanced from coating cell culture plates with purified ECM components to the design of ECM-mimicking biomaterials and the engineering of decellularized tissues aimed at recapitulating the dynamics, composition and structure of the ECM. In this Review, we highlight important matrix properties and functions in the context of tissue engineering and regenerative medicine, consider techniques such as proteomics for the investigation of matrix structure and composition and discuss different engineering strategies for the design of matrix-mimicking biomaterials. Tissue, whole organ and cell culture decellularization approaches are examined for their potential to preserve the tissue-specific biochemical composition and ultrastructure of the ECM and for the development of biomaterials that promote the formation of functional tissues in clinical applications. Finally, we investigate challenges and opportunities of ECM biomaterials for the design of organotypic models to study disease progression, for the ex vivo creation of engineered tissue and for the clinical translation of functional tissue reconstruction strategies in vivo. The extracellular matrix is nature’s template for an ideal biomaterial to guide tissue homeostasis and repair. In this Review, matrix-mimicking biomaterials and decellularized matrices are discussed for their potential to reconstruct and repair tissues in vitro and in vivo.

Extracellular matrix-based materials for regenerative medicine

Acceleration of reactivation of reduced bovine pancreatic ribonuclease by a microsomal system from rat liver

Ribonuclease. V. Studies on the properties and distribution of ribonuclease inhibitor in the rat.

Ribonuclease. VII. Partial purification and characterization of a ribonuclease inhibitor in rat liver supernatant fraction.

The pharmacokinetics and metabolism of the anti-human T-lymphotrophic virus type III/lymphadenopathy-associated virus agent 2',3-dideoxycytidine have been examined in BDF1 mice and rhesus monkeys, with ancillary enzyme studies carried out on tissue derived from both the latter species and also from human subjects. For the pharmacokinetic studies, 2',3-dideoxycytidine and its catabolic product 2',3-dideoxyuridine have been separated and measured in plasma, urine, and cerebrospinal fluid by a reverse HPLC method. For metabolic studies, tritium-labeled drug (labeled in the 5- and 6-positions of the pyrimidine ring) has been employed, utilizing an ion exchange HPLC analytical method suitable for the separation of the parent nucleoside from its mono-, di-, and triphosphates in cell extracts and in tissue homogenates. The drug is rapidly cleared from plasma in a biphasic manner (terminal t 1/2 in BDF1 mice and rhesus monkeys of 67 min and 109 min, respectively) following an iv bolus dose of 325 mg/m2. This two-compartment open model is predictive of plasma concentrations during long term ip infusions in mice. Dideoxycytidine is predominantly excreted in the urine as unchanged parent compound, although a minor urinary metabolite (2,3-dideoxyuridine) is detected in the monkey but not in the mouse. Oral absorption of 2',3'-dideoxycytidine is rapid, with plasma levels approaching those seen after iv administration within 45 min in the mouse. Entry to the central nervous system is also rapid, but the cerebrospinal fluid to plasma AUC ratio after iv administration is only 0.026-0.040 in rhesus monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

The disposition and metabolism of 2',3'-dideoxycytidine, an in vitro inhibitor of human T-lymphotrophic virus type III infectivity, in mice and monkeys.

Purpose
The component separation technique for hernia repair results in significant wound morbidity due to the need for large undermining skin flaps. The endoscopic component separation technique allows for advancement of the abdominal wall while preserving the blood supply originating from the epigastric vessels. This study compares the outcomes following hernia repair utilizing these techniques.

The endoscopic component separation technique for hernia repair results in reduced morbidity compared to the open component separation technique.

Laparoscopic incisional–ventral hernia repair (LIVH) is used with increasing frequency for the morbidly obese and for complex and recurrent hernias. The experience of a single institution with this technique is reviewed and the findings and complications are presented. Data were collected retrospectively for a single surgeon’s series of patients undergoing LIVH at the institution described in this report. The review showed a complication rate of 15.2%, a recurrence rate of 2%, and a prosthetic infection rate of 2%. Patients with a body mass index greater than 30 cm/m2 accounted for 73% of the complications and made up 62.2% of the patients. The LIVH procedure may be safely performed with low complication and recurrence rates even for the obese, allowing ventral hernia repair to be performed safely with good results. The LIVH technique should be considered for the repair of all incisional and ventral hernias requiring repair with a mesh prosthesis.

Jay S. Roth

Papers

Ribonuclease. V. Studies on the properties and distribution of ribonuclease inhibitor in the rat.

Ribonuclease. VII. Partial purification and characterization of a ribonuclease inhibitor in rat liver supernatant fraction.

The disposition and metabolism of 2',3'-dideoxycytidine, an in vitro inhibitor of human T-lymphotrophic virus type III infectivity, in mice and monkeys.

The endoscopic component separation technique for hernia repair results in reduced morbidity compared to the open component separation technique.

Complications of laparoscopic incisional-ventral hernia repair: the experience of a single institution.