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Background: Hereditary palmoplantar keratodermas (PPK) represent a heterogeneous group of rare skin disorders with epidermal hyperkeratosis of the palms and soles, with occasional additional manifestations in other tissues. Mutations in at least 69 genes have been implicated in PPK, but further novel candidate genes and mutations are still to be found. Objectives: To identify mutations underlying PPK in a cohort of 64 patients. Methods: DNA of 48 patients was analysed on a custom-designed in-house panel for 35 PPK genes, and 16 patients were investigated by a diagnostic genetic laboratory either by whole-exome sequencing, gene panels or targeted single-gene sequencing. Results: Of the 64 PPK patients, 32 had diffuse (50%), 19 focal (30%) and 13 punctate (20%) PPK. None had striate PPK. Pathogenic mutations in altogether five genes were identified in 31 of 64 (48%) patients, the majority (22/31) with diffuse PPK. Of them, 11 had a mutation in AQP5, five in SERPINB7, four in KRT9 and two in SLURP1. AAGAB mutations were found in nine punctate PPK patients. New mutations were identified in KRT9 and AAGAB. No pathogenic mutations were detected in focal PPK. Variants of uncertain significance (VUS) in PPK-associated and other genes were observed in 21 patients that might explain their PPK. No suggestive pathogenic variants were found for 12 patients. Conclusions: Diffuse PPK was the most common (50%) and striate PPK was not observed. We identified pathogenic mutations in 48% of our PPK patients, mainly in five genes: AQP5, AAGAB, KRT9, SERPINB7 and SLURP1., QC 20220314

In this study the potential targeted use of zinc to inactivate proteinase inhibitors (PI) has been investigated as an alternative to the widely applied heat treatment used industrially for inactivation of PI. Zinc was utilized for the reduction of disulfide bonds leading to the structural changes in proteins, thus affecting the decreased affinity between PI and proteinases. The protein disulfide bond reduction mechanism was studied using a newly developed micellar electrokinetic capillary chromatography (MECC) with the glutathione redox reaction with dithiothreitol (DTT) as model system. This model proved efficient in monitoring the reduction of disulfide bonds in the Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). The use of zinc as a reductant resulted in a significant reduction of trypsin inhibitor activity (TIA) of 72% for KTI and 85% for BBI, highlighting zinc as a promising potential agent to reduce the activity of PI as an alternative to heat treatment.

Lipid-lowering therapies have been shown to improve cardiovascular outcome in a wide range of patients. The current guidelines recommend a graded approach to reduction in low-density lipoprotein cholesterol (LDL-C) proportional to the patient's risk, with the goal of achieving either a certain magnitude of reduction or a specific threshold of final LDL-C. Recent findings from a meta-analysis of numerous randomized trials suggest that more attention should be given to the baseline LDL-C of an individual patient. In this review, we discuss how the baseline LDL-C level may provide a means to better understand the results of recent cardiovascular outcome trials and the expected benefits of lipid-lowering therapies. The exact quantification of the clinical benefit associate with an intensified lipid-lowering therapy depends on the baseline LDL-C. Mortality is reduced in a log-linear fashion only when LDL-C > 100 mg/dL.

Serine proteinase inhibitors play important and diverse roles in biological processes such as coagulation, defense mechanisms, and immune responses. Here, we identified and characterized a Kunitz-type proteinase inhibitor, designated FcKuSPI, of the BPTI/Kunitz family of serine proteinase inhibitors from the hemocyte cDNA library of the shrimp Fenneropenaeus chinensis. The deduced amino acid sequence of FcKuSPI comprises 80 residues with a putative signal peptide of 15 amino acids. The predicted molecular weight of the mature peptide is 7.66 kDa and its predicted isoelectric point is 8.84. FcKuSPI includes a Kunitz domain containing six conserved cysteine residues that are predicted to form three disulfide bonds. FcKuSPI shares 44e53% homology with BPTI/Kunitz family members from other species. FcKuSPI mRNAwas expressed highly in the hemocytes and moderately in muscle in healthy shrimp. Recombinant FcKuSPI protein demonstrated anti-protease activity against trypsin and anticoagulant activity against citrated human plasma in a dose-dependent manner in in vitro assays.

Biljni inhibitori proteinaza aktivno učestvuju u odbrani biljaka od insekata štetočina inhibirajući insekatske digestivne proteinaze. In planta analiza ekspresije BvSTI gena koji kodira inhibitora serinskih proteinaza urađena je sa ciljem otkrivanja uloge ovog inhibitora u otpornosti biljaka šećerne repe na insekte, kao i radi utvrđivanja potencijala ovog gena kao pogodnog kandidat-gena koji bi se biotehnološkim metodama mogao uvesti u osetljive biljne genome, čime bi se povećala njihova otpornost prema insektima štetočinama. Ekspresija BvSTI gena praćena je kod tri genotipa šećerne repe koji se odlikuju umerenom otpornošću prema larvama štetočine korena Tetanops myopaeformis Roder, F1016, F1015 i UT-8, kao i kod jednog osetljivog genotipa, F1010. Kod svih otpornih genotipova mehaničko povređivanje indukovalo je ekspresiju ovog gena, a nivo transkripcije bio je povišen u poređenju sa nivoom kod osetljivog genotipa. Najintenzivniji odgovor na povređivanje zabeležen je kod otpornih genotipova F1016 i UT-8. U listovima osetljivog F1010, ali i trećeg otpornog genotipa F1015, registrovan je samo neznatni porast u intenzitetu BvSTI transkripcije, dok je u korenovima ova dva genotipa mehaničko povređivanje dovelo do blage početne supresije u aktivnosti BvSTI gena. Akumulacija BvSTI transkripata u listovima i korenovima otpornog F1016 i osetljivog F1010 kojima su se hranile larve Spodoptera frugiperda J.E. Smith pokazala je sličan obrazac ekspresije kod oba genotipa. U poređenju sa transkripcionim obrascima dobijenim nakon mehanilčkog povređivanja, ishrana insekata dovela je do znatno sporije indukcije i slabijeg intenziteta transkripcije. Analize na proteinskom nivou pokazale su da nakon povređivanja listova dolazi do akumulacije proteina veličine 30 kDa za koji su se vezala poliklonalna BvSTI specifična antitela kako kod otpornog F1016, tako i kod osetljivog F1010 genotipa. Aktivnost BvSTI inhibitora protiv tripsina pokazana je kod F1016 korenova i listova, kao i kod F101, Plant proteinase inhibitor genes are among the prime candidates suitable for insect resistance improvement in plants. Expression pattern of a sugar beet serine proteinase inhibitor gene, BvSTI, was characterized in response to mechanical and fall armyworm (Spodoptera frugiperda J.E. Smith) induced wounding. BvSTI expression was analyzed in three breeding lines moderately resistant to sugar beet root maggot (Tetanops myopaeformis Roder.) and in a susceptible line, F1010. Increased mechanical wounding induced levels of BvSTI expression were observed in all resistant lines as compared to F1010. The most intensive response to wounding was observed in resistant lines, F1016 and UT-8, with a maximum up to 4- and 2,5-fold increase of BvSTI transcript levels over non-wounded roots and leaves, respectively. In contrast, slight increase of BvSTI transcript levels in leaves and even an initial decrease in roots were observed in sensitive F1010, but also in the third resistant line, F1015. BvSTI transcript accumulation in F1016 and F1010 tissues wounded by FAW showed a similar gene expression pattern, but it was delayed and less intense than the response incited by abiotic wounding. On the protein level, BvSTI-specific polyclonal antibodies confirmed increased accumulation of the 30 kDa BvSTI protein in wounded leaves but not in roots of F1016 and F1010. Using trypsin inhibition assays, the activity of BvSTI was confirmed in F1016 roots and leaves and F1010 leaves. In F1010 roots BvSTI activity was completely lacking. To confirm the potential role of the BvSTI gene in defending mechanisms to insect pests in sugar beet the same analyzed germplasm were bioassayed for resistance to fall armyworm insects. Larvae fed sugar beet leaves from all three resistant germplasms (F1016, F1015 and UT-8) had significant reductions in larval weights as compared to larvae fed on sensitive F1010 leaves. The observed daily weight increase was also the highest in larvae from sensitive vs. resistant

Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly. A few conserved positions, e.g. six invariant cysteines were present in all domain sequences whereas the position of P1 amino acid, a determinant for substrate specificity, varied highly. A study with a single crayfish animal suggested that even at the individual level considerable sequence variability among hemocyte KPIs produced exist. Expression analysis of four crayfish KPI transcripts in hematopoietic tissue cells and different hemocyte types suggest that some of these KPIs are likely to be involved in hematopoiesis or hemocyte release as they were produced in particular hemocyte types or maturation stages only.

An exact algorithm for the reconstruction of the contact map (CM) of a protein structure from the sole knowledge of its principal eigenvector (PE) was presented. The algorithm yielded a unique contact map for all 221 globular proteins of PDBselect25 of length N ≤120. It was shown that the reconstructures map allow for the accurate reconstruction of the three-dimensional structure. The results showed that the reduced vectorial representation provided by the principal eigenvector of the contact map was equivalent to the protein structure itself.

Proteins of the serpin superfamily (similar to43 kDa) from mature cereal grains are in vitro suicide-substrate inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (<25 kDa), the biological functions of plant serpins are unknown. Expression studies of genes encoding members of three subfamilies of serpins (BSZx, BSZ4 and BSZ7) in developing grain and vegetative tissues of barley (Hordeum vulgare L.) showed that transcripts encoding BSZx, which inhibits distinct proteinases at overlapping reactive centres in vitro, were ubiquitous at low levels, but the protein could not be detected. EST analysis showed that expression of genes for serpins with BSZx-type reactive centres in vegetative tissues is widespread in the plant kingdom, suggesting a common regulatory function. For BSZ4 and BSZ7, expression at the protein level was highest in the maturing grain (greater than or equal to15 d post-anthesis), where these serpins were localized by immunomicroscopy to the central and peripheral starchy endosperm, subaleurone, and (at lower levels) to the aleurone. Serpins were also localized to the meristem and vascular tissues of roots, and to the phloem of coleoptiles and leaves. The identification of BSZ4 in vegetative tissues by western blotting was confirmed for the roots by purification and amino acid sequencing, and for the leaves by in vitro reactive-centre loop cleavage studies. Plant serpins are likely to use their irreversible inhibitory mechanism in the inhibition of exogenous proteinases capable of breaking down seed storage proteins, and in the defence of specific cell types in vegetative tissues.

A high incidence of alpha 1-antitrypsin (AAT) deficiency has been reported in patients with C-ANCA systemic vasculitis in association with antibodies against proteinase-3 (PR3). To clarify the role of AAT deficiency in the acute vasculitic process as well as in progression of the disease, we studied 84 patients with either C-ANCA or P-ANCA vasculitis with special reference to: (a) the AAT gene, (b) the phenotypic (Pi) variants and (c) the serum levels during both acute illness and remission. The PiZ gene was found in six patients (8% vs. 1.5% controls) irrespective of the type of autoantibodies (C-ANCA vs. P-ANCA). All PiZ patients displayed the ability to raise their AAT serum levels up to the normal range during acute illness. In contrast, 24 patients with the PiM phenotype presented low AAT serum levels during acute illness. In all these patients, the AAT levels returned to normal values during the remission. Low AAT levels were associated with low levels of C-reactive protein (PCR) (P < 0.001), with a less severe renal involvement or a minor risk of death, and, in one tested patient, with a novel point mutation (TCGA-->TCAA) at the enhancer-promoter region of the AAT gene. Low AAT serum levels did not correlate with either type/titre of autoantibody or distribution/severity of the vasculitis process. In the case-control study, high AAT levels emerged as a major determinant of progression towards end-stage renal failure [odds ratio 3 (95% CI 1.1-8.4)]. These results indicate: (a) a high incidence of the PiZ gene of AAT in systemic vasculitis irrespective of the type of autoantibodies; (b) a novel form of AAT deficiency associated with the normal PiM phenotype becoming manifest only during acute illness; (c) dysregulation of the acute-phase response affecting selectively AAT or both AAT and PCR; (d) correlation between low plasma levels of AAT and less severe renal involvement or risk of death