Recombinant hirudin: kinetic mechanism for the inhibition of human thrombin

Recombinant hirudin variant-2(Lys47 ), was found to be a competitive inhibitor of human α-thrombin with respect to peptidyl p-Miitroanilide substrates. These results contrast with those of Degryse and coworkers that suggest that recombinant hirudin variant-2(Lys47) inhibited thrombin by a non-competitive mechanism [Degryse et al. (1989) Protein Engng, 2, 459-465], γ-Thrombin, which can arise from α-thrombin by autolysis, was shown to have an affinity for recombinant hirudin variant-2(Lys47) that was four orders of magnitude lower than that of α-thrombin. It was demonstrated that the apparent noncompetitive mechanism observed previously was probably caused by a contamination of the thrombin preparation by γ-thrombin. Comparison of the inhibition of α-thrombin by recombinant hirudins variant-2(Lys47) and variant-1, which differ from one another in eight out of 65 amino acids, indicated that the two variants have essentially the same kinetic parameter

Recombinant human interleukin-12 is the second example of a C-mannosylated protein

The β-chain of human interleukin 12 (IL-12) contains at position 319-322, the sequence Trp-x-x-Trp. In human RNase 2 this is the recognition motif for a new, recently discovered posttranslational modification, i.e., the C-glycosidic attachment of a mannosyl residue to the side chain of tryptophan. Analysis of C-terminal peptides of recombinant IL-12 (rHuIL-12) by mass spectrometry and NMR spectroscopy revealed that Trp-319β is (partially) C-mannosylated. This finding was extended by in vitro mannosylation experiments, using a synthetic peptide derived from the same region of the protein as an acceptor. Furthermore, human B-lymphoblastoid cells, which secrete IL-12, were found to contain an enzyme that carries out the C-mannosylation reaction. This shows that nonrecombinant IL-12 is potentially C-mannosylated as well. This is only the second report on a C-mannosylated protein. However, the occurrence of the C-mannosyltransferase activity in a variety of cells and tissues, and the presence of the recognition motif in many proteins indicate that more C-mannosylated proteins may be foun

Caveolin-1 interacts with the chaperone complex TCP-1 and modulates its protein folding activity

Abstract.: We report that caveolin-1, one of the major structural protein of caveolae, interacts with TCP-1, a hetero-oligomeric chaperone complex present in all eukaryotic cells that contributes mainly to the folding of actin and tubulin. The caveolin-TCP-1 interaction entails the first 32 amino acids of the N-terminal segment of caveolin. Our data show that caveolin-1 expression is needed for the induction of TCP-1 actin folding function in response to insulin stimulation. Caveolin-1 phosphorylation at tyrosine residue 14 induces the dissociation of caveolin-1 from TCP-1 and activates actin folding. We show that the mechanism by which caveolin-1 modulates TCP-1 activity is indirect and involves the cytoskeleton linker filamin. Filamin is known to bind caveolin-1 and to function as a negative regulator of insulin-mediated signaling. Our data support the notion that the caveolin-filamin interaction contributes to restore insulin-mediated phosphorylation of caveolin, thus allowing the release of active TCP-

Considerations for long-term clinical performance of partial restorations:exploring the pathway to success

Restorative dentistry has evolved significantly, emphasizing the importance of partial restorations in preserving natural tooth structure. Advances in adhesive systems play a pivotal role in extending their longevity and promoting minimally invasive dentistry. The second chapter of the thesis, a systematic review, revealed that intracoronal gold restorations outperform indirect resin composites, while lithium disilicate shows promise as a dental material. Laboratory studies on direct composite and lithium disilicate partial restorations in chapters 3, 4, and 5 show good results, which argue for minimal invasive preparation. The use of immediate dentin sealing improves the fracture strength of the restorations. The clinical studies included in this thesis report long-term follow-up. A retrospective clinical study in chapter 6, spanning 15 years, highlights the remarkable longevity of extensive direct resin composite restorations after replacing amalgams. A long-term randomized clinical trial in chapter 7 showcases the suitability of both direct and indirect resin composites for restoring maxillary premolars with cusp-replacing restorations. Chapter 8 focuses on long-term partial indirect lithium disilicate restorations, exhibiting excellent clinical survival over 7.5 years, with specific patient factors impacting their longevity. Chapter 9 highlights the medium-term clinical excellence of indirect lithium disilicate restorations, where no influence of preparation characteristics on the survival and success of the restorations was seen. Chapter 10 discusses the individual chapters, the connections, and correlations between the studies, as well as the limitations in the aforementioned chapters. Additionally, recommendations for future research were made, and some considerations for the daily application of partial restorations were formulated

The Influence of Dentin Wall Thickness and Adhesive Surface in Post and Core Crown and Endocrown Restorations on Central and Lateral Incisors

Purpose: The main purpose of this study was to determine the influence of dentin wall thickness (DWT) and adhesive surface on the fracture strength and failure mode in maxillary incisors restored with post and core crowns or endocrowns. Methods and Materials: Forty-eight sound maxillary incisors were selected and randomly divided into four groups (n=12): lateral incisor endocrown, lateral incisor post and core, central incisor endocrown, and central incisor post and core. All specimens obtained an endodontic treatment and were decoronated (2 mm ferrule remained). Chamfer outlines ended at the cementoenamel junction (outline in dentin). Dentin wall thickness (mm) was measured on 12 points per sample using a modified digital calliper. Fiber posts and cores were placed in two groups, and an immediate dentin sealing was applied on exposed dentin in all groups before taking digital impressions. Digital impressions were analyzed and the adhesive surface (mm(2)) was measured. Indirect restorations were made of lithium disilicate (IPS e.max, computer-aided design/computer-aided manufacturing). The restorations were luted after surface conditioning the crowns and teeth. Thermocyclic aging was performed (10,000 times in baths of 5 degrees C and 55 degrees C) and the specimens were loaded until fracture. Fractures were specified on failure mode and repairability, and they were analyzed with one-way ANOVA, chi(2)-test, and linear regression analysis in SPSS (alpha=0.05). Results: There was no significant difference in fracture strength and failure mode between all groups. Endocrown restorations on central incisors had significantly more repairable fractures than the post and core crowns. Regression analyses showed a statistically significant positive correlation between DWT/adhesive surface and fracture strength in the post and core groups. Conclusions: Both endocrowns and post and core crowns on the central and lateral incisors obtained clinically applicable fracture strengths. In the central incisor groups, the endocrown restorations had significantly more repairable failures. When the walls were thicker, or when the adhesive surface was larger, higher fracture strengths were obtained in the post and core groups

Accuracy of SenseWear Pro2 armband to predict resting energy expenditure in childhood obesity.

OBJECTIVE: We evaluate the accuracy of the SenseWear Pro2 Armband (SWA) in estimating resting energy expenditure (REE) in children and adolescents with obesity, using indirect calorimetry (IC) as a reference. DESIGN AND METHODS: REE was assessed using both the SWA and IC in 40 obese subjects (26 M/14 F, age 11.5±2.57 years, z-score BMI 3.14±0.53). The agreement between methods was assessed by the Bland-Altman procedure. The relationship between REE assessments and patients' characteristics was also analyzed. RESULTS: SWA- and IC-derived estimates of REE showed a significant correlation (r=0.614; P<0.001), but the SWA overestimated mean REE by 13% (P<0.001). Age and kg of fat-free mass (kgFFM) were significantly correlated with both REE estimation by SWA (r=0.434 and r=0.564; respectively) and IC (r=0.401 and r=0.518; respectively). Only kgFFM was demonstrated to be the main predictor factor of REE variability (r2 79% SWA; 75% IC). CONCLUSIONS: The SWA overestimated mean REE in childhood obesity, suggesting that the SWA and IC are not yet interchangeable methods. This would require improving the SWA by developing better algorithms for predicting REE and, probably, bias in each individual REE could be reduced by an adjustment for subjects' kgFFM

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling, in inflammation and in vascular biology. The human family includes 19 members that can be sub-grouped on the basis of their known substrates, namely the aggrecanases or proteoglycanases (ADAMTS1, 4, 5, 8, 9, 15 and 20), the procollagen N-propeptidases (ADAMTS2, 3 and 14), the cartilage oligomeric matrix protein-cleaving enzymes (ADAMTS7 and 12), the von-Willebrand Factor proteinase (ADAMTS13) and a group of orphan enzymes (ADAMTS6, 10, 16, 17, 18 and 19). Control of the structure and function of the extracellular matrix (ECM) is a central theme of the biology of the ADAMTS, as exemplified by the actions of the procollagen-N-propeptidases in collagen fibril assembly and of the aggrecanases in the cleavage or modification of ECM proteoglycans. Defects in certain family members give rise to inherited genetic disorders, while the aberrant expression or function of others is associated with arthritis, cancer and cardiovascular disease. In particular, ADAMTS4 and 5 have emerged as therapeutic targets in arthritis. Multiple ADAMTSs from different sub-groupings exert either positive or negative effects on tumorigenesis and metastasis, with both metalloproteinase-dependent and -independent actions known to occur. The basic ADAMTS structure comprises a metalloproteinase catalytic domain and a carboxy-terminal ancillary domain, the latter determining substrate specificity and the localization of the protease and its interaction partners; ancillary domains probably also have independent biological functions. Focusing primarily on the aggrecanases and proteoglycanases, this review provides a perspective on the evolution of the ADAMTS family, their links with developmental and disease mechanisms, and key questions for the future