Derivation and characterization of a cytocompatible scaffold from human testis.

STUDY QUESTION: Is it possible to derive a scaffold from human testis for the purpose of tissue engineering and regenerative medicine? SUMMARY ANSWER: We developed a method to produce a cytocompatible decellularized testicular matrix (DTM) while maintaining the native tissue-specific characteristics and components. WHAT IS KNOWN ALREADY: The potential benefits of tissue-specific scaffolds consisting of naturally-derived extracellular matrix (ECM) have been demonstrated using a wide variety of animal and human tissue sources. However, so far, testis scaffolds have never been considered for constructive remodelling purposes. STUDY DESIGN, SIZE, DURATION: Human cadaveric testicular tissue was exposed for 24 or 48 h to 1% Triton X-100 and/or 1% sodium dodecyl sulphate (SDS). Acellular samples were used for further scaffold characterization purposes. PARTICIPANTS/MATERIALS, SETTING, METHODS: The extent of decellularization was evaluated by histology. Confirmation of cell removal in DTM was done by a DNA quantification technique. Retention of testicular tissue-specific characteristics was evaluated by mass spectrometry, immunohistochemistry, Alcian blue staining and scanning electron microscopy. Soluble toxicity and testicular cell attachment was assessed to check the cytocompatibility of DTM scaffolds. MAIN RESULTS AND THE ROLE OF CHANCE: Histological analysis showed that DTM could be obtained by mechanical agitation in 1% SDS for 24 h. The resulting DTM was found to be clear of cells while retaining the typical three-dimensional structure and the major components of the native tissue scaffold, including collagen type I and IV, fibronectin, laminin and glycosaminoglycans. In addition, using proteomic analysis, we revealed numerous additional ECM proteins in DTM, indicating its complex nature. The mass spectrometry data were deposited to the ProteomeXchange with identifier PXD001524. Importantly, we demonstrated that DTM scaffolds are not cytotoxic, as evidenced by MTT assay not showing an aberrant fibroblast proliferation activity after indirect exposure, and support testicular cell attachment and infiltration. LIMITATIONS, REASONS FOR CAUTION: The functionality of human testicular cells in DTM needs to be investigated. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that the insights into the molecular composition of the testicular ECM provide new clues for the unravelling of its important yet poorly understood role in regulating testicular function, and DTM-based bioscaffolds are promising components for the development of human in vitro spermatogenesis as a treatment for various types of male fertility disorders.

C-peptide: a molecule balancing insulin states in secretion and diabetes-associated depository conditions.

Gradually, the C-peptide part of proinsulin has evolved from being viewed upon as a side product of insulin synthesis and secretion to being considered as a bioactive peptide with endocrine functions. Independent of these, its biophysical properties and peptide interactions point to still further roles of C-peptide, in particular regarding possible links to diabetes-related protein aggregations. Insulin, which can deposit at the injection sites in the treatment of diabetes, and islet amyloid polypeptide (IAPP), which can form amyloid fibrils in the islets of Langerhans in diabetes type 2, are kept nonaggregated by charge-based interactions with C-peptide at defined stoichiometries. It is possible that the conformational stabilization of insulin and IAPP by C-peptide may also counterbalance their aggregational tendencies at the high peptide concentrations in the pancreatic ß-cell secretory granules. The concentration imbalances of C-peptide, insulin, and IAPP from the hyperpeptidism early in T2DM patients and the insulin-only injections in T1DM patients may distort equilibria of these peptide interactions and promote protein aggregation. Additionally, the chaperone-like actions of C-peptide may increase bioavailability of insulin supplements given to T1DM patients and prevent the formation of insulin deposits. Similarly, peptide interactions may influence depository tendencies in additional peptide systems. In short, biophysical studies are relevant to establish all roles of peptide imbalances in T1DM and T2DM and associated depository diseases.

Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes.

Short-chain dehydrogenases/reductases (SDRs) constitute a large family of NAD(P)(H)-dependent oxidoreductases, sharing sequence motifs and displaying similar mechanisms. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, cofactor, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. Sequence identities are low, and the most conserved feature is an alpha/beta folding pattern with a central beta sheet flanked by 2 - 3 alpha-helices from each side, thus a classical Rossmannfold motif for nucleotide binding. The conservation of this element and an active site, often with an Asn-Ser-Tyr-Lys tetrad, provides a platform for enzymatic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases. The common mechanism is an underlying hydride and proton transfer involving the nicotinamide and typically an active site tyrosine residue, whereas substrate specificity is determined by a variable C-terminal segment. Relationships exist with bacterial haloalcohol dehalogenases, which lack cofactor binding but have the active site architecture, emphasizing the versatility of the basic fold in also generating hydride transfer-independent lyases. The conserved fold and nucleotide binding emphasize the role of SDRs as scaffolds for an NAD(P)(H) redox sensor system, of importance to control metabolic routes, transcription and signalling.

Medium- and short-chain dehydrogenase/reductase gene and protein families : the MDR superfamily.

The MDR superfamily with ~350-residue subunits contains the classical liver alcohol dehydrogenase (ADH), quinone reductase, leukotriene B4 dehydrogenase and many more forms. ADH is a dimeric zinc metalloprotein and occurs as five different classes in humans, resulting from gene duplications during vertebrate evolution, the first one traced to ~500 MYA (million years ago) from an ancestral formaldehyde dehydrogenase line. Like many duplications at that time, it correlates with enzymogenesis of new activities, contributing to conditions for emergence of vertebrate land life from osseous fish. The speed of changes correlates with function, as do differential evolutionary patterns in separate segments. Subsequent recognitions now define at least 40 human MDR members in the Uniprot database (corresponding to 25 genes when excluding close homologues), and in all species at least 10888 entries. Overall, variability is large, but like for many dehydrogenases, subdivided into constant and variable forms, corresponding to household and emerging enzyme activities, respectively. This review covers basic facts and describes eight large MDR families and nine smaller families. Combined, they have specific substrates in metabolic pathways, some with wide substrate specificity, and several with little known functions.

Zinc binding to peptide analogs of the structural zinc site in alcohol dehydrogenase: implications for an entatic state.

Zinc binding to the peptide replica and analogs to residues 93-115 of horse liver alcohol dehydrogenase (ADH) was examined by competition of the peptides and the chromophoric chelator 4-(2- pyridylazo)resorcinol for zinc and X-ray absorption fine structure analysis of the zinc ligands. In the enzyme, zinc is coordinated by four Cys residues. In the peptide replica, zinc is bound to three Cys and one His residue. A four-Cys zinc coordination is observed only when His is removed, leading to increased zinc stability. ADH crystal structures reveal that the epsilon-amino group of the conserved residue Lys323 is within H-bond distance of the backbone amide oxygens of residues 103, 105 and 108, likely stabilizing the zinc coordination in the enzyme. The peptide data thus indicate structural strain and increased energy in the zinc-binding site in the protein, characteristic of an entatic state, implying a functional nature for this zinc site.

Glomerulus proteome analysis with two-dimensional gel electrophoresis and mass spectrometry.

A proteome analysis of mammalian glomeruli was performed in mouse using two-dimensional-gel electrophoresis with separate Coomassie and silver staining and subsequent mass spectrometric identifications. Altogether, 414 protein spots were identified, revealing 232 different proteins representing a wide spectrum of activities, including enzymes (27%), cell-signalling proteins (22%), structural proteins (12%), protein folding and metabolism (13%), cell-growth-related proteins (6%), and replication, transcription and translation (4%). Only 53 of the proteins were detected in another proteome study, showing the value of analyses with different methodologies. However, 50 of the proteins were also identified in a proteome analysis of endothelial cells and 42 in one of glomerular mesangial cells, revealing distinct similarities between these tissues, but also unique differences. Finally, 80 of the proteins were not identified in a separate transcriptome analysis, while 10 of the present proteins were then indentifiable with genes implicated in glomerulus development and function, allowing direct correlation with expression data.

Differential protein expression in pancreatic islets after treatment with an imidazoline compound.

The effects of an imidazoline compound (BL11282) on protein expression in rat pancreatic islets were investigated with a proteomic approach. The compound increases insulin release selectively at high glucose concentrations and is therefore of interest in type 2 diabetes. Whole cell extracts from isolated drug-treated and native pancreatic rat islets were compared after separation by 2-D gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry; 15 proteins were selectively up-regulated and 7 selectively down-regulated in drug-treated islets. Of special interest among the differentially expressed proteins are those involved in protein folding (Hsp60, protein disulfide isomerase, calreticulin), Ca(2+) binding (calgizzarin, calcyclin and annexin I) and metabolism or signalling (pyruvate kinase, alpha enolase and protein kinase C inhibitor 1).

Cellular internalization of proinsulin C-peptide.

Proinsulin C-peptide is known to bind specifically to cell membranes and to exert intracellular effects, but whether it is internalized in target cells is unknown. In this study, using confocal microscopy and immunostained or rhodamine-labeled peptide, we show that C-peptide is internalized and localized to the cytosol of Swiss 3T3 and HEK-293 cells. In addition, transport into nuclei was found using the labeled peptide. The internalization was followed at 37 degrees C for up to 1 h, and was reduced at 4 degrees C and after preincubation with pertussis toxin. Hence, it is concluded to occur via an energy-dependent, pertussis toxin-sensitive mechanism and without detectable degradation within the experimental time course. Surface plasmon resonance measurements demonstrated binding of HEK-293 cell extract components to C-peptide, and subsequent elution of bound material revealed the components to be intracellular proteins. The identification of C-peptide cellular internalization, intracellular binding proteins, absence of rapid subsequent C-peptide degradation and apparent nuclear internalization support a maintained activity similar to that of an intracrine peptide hormone. Hence, the data suggest the possibility of one further C-peptide site of action.