BCN Checkpoint: same-day confirmation of reactive HIV rapid test with Point Of Care HIV-RNA accelerates linkage to care and reduces anxiety.

BACKGROUND: The introduction in 2006 of the rapid HIV test by BCN Checkpoint in a non-clinical setting has been a successful step forwards in the uptake of testing. Nevertheless, HIV serostatus should be reported as HIV positive only when a reactive result has been tested again using a different assay (WHO guidelines 2015). The standard confirmation test has been the Western Blot (WB) test. However confirmation results take around 7 days to come back. AIMS: This study explores the possibility of Point of Care PCR testing for a same-day confirmation. MATERIALS AND METHODS: Between March 2015 and September 2016 a POC PCR test (Xpert® HIV-1 Qual) was performed in parallel to the Western Blot test after a reactive HIV rapid test (Alere Determine™ HIV-1/2 Ag/Ab Combo and Alere™ HIV Combo). HIV confirmed positive cases received emotional support by peers, were informed and prepared for treatment initiation and rapidly linked to HIV clinic. RESULTS: During the study period 11 455 tests were performed to 7163 clients. A total of 249 reactive rapid HIV tests were found. For analysis a total of 33 cases were excluded due to the lack of PCR and/or WB test. Results of comparison of the 216 cases showed 194 concordant positive confirmations and 14 concordant negative results. In three cases PCR was positive and WB negative. In five cases PCR was negative and WB positive. CONCLUSION: The POC PCR assay is easy to use and feasible in a community-based center. Reducing time for confirmation to 90 min has been possible in 91.2% (197/216) of cases with positive PCR result. In cases of a negative PCR result an additional test (WB, Elisa or PCR quantitative) was needed to distinguish false positive results (6.5%) from viral load results below level of detection (2.3%). Clients expressed satisfaction with same-day confirmation and less anxiety.

The shell effect on the room temperature photoluminescence from ZnO/MgO core/shell nanowires: exciton-phonon coupling and strain.

The room temperature photoluminescence from ZnO/MgO core/shell nanowires (NWs) grown by a simple two-step vapor transport method was studied for various MgO shell widths (w). Two distinct effects induced by the MgO shell were clearly identified. The first one, related to the ZnO/MgO interface formation, is evidenced by strong enhancements of the zero-phonon and first phonon replica of the excitonic emission, which are accompanied by a total suppression of its second phonon replica. This effect can be explained by the reduction of the band bending within the ZnO NW core that follows the removal of atmospheric adsorbates and associated surface traps during the MgO growth process on one hand, and a reduced exciton-phonon coupling as a result of the mechanical stabilization of the outermost ZnO NW monolayers by the MgO shell on the other hand. The second effect is the gradual increase of the excitonic emission and decrease in the defect related emission by up to two and one orders of magnitude, respectively, when w is increased in the ∼3-17 nm range. Uniaxial strain build-up within the ZnO NW core with increasing w, as detected by x-ray diffraction measurements, and photocarrier tunneling escape from the ZnO core through the MgO shell enabled by defect-states are proposed as possible mechanisms involved in this effect. These findings are expected to be of key significance for the efficient design and fabrication of ZnO/MgO NW heterostructures and devices.

A "SYDE" effect of hierarchical phosphorylation: possible relevance to the cystic fibrosis basic defect.

The motif "SYDE", incorporating the protein kinase CK2 consensus sequence (S-x-x-E) has been found to be phosphorylated at both its serine and tyrosine residues in several proteins. Of special interest is the case of cystic fibrosis Transmembrane-conductance Regulator (CFTR), where this motif is close to the residue (F508), whose deletion is the by far commonest cause of cystic fibrosis. Intriguingly, however, CFTR S511 cannot be phosphorylated by CK2 to any appreciable extent. Using a number of peptide substrates encompassing the CFTR "SYDE" site we have recently shown that: (1) failure of CK2 to phosphorylate the S(511)YDE motif is due to the presence of Y512; (2) CK2 readily phosphorylates S511 if Y512 is replaced by a phospho-tyrosine; (3) the Src family protein tyrosine kinase Lyn phosphorylates Y512 in a manner that is enhanced by the deletion of F508. These data, in conjunction with the recent observation that by inhibiting CK2 the degradation of F508delCFTR is reduced, lead us to hypothesize that the hierarchical phosphorylation of the motif SYDE by the concerted action of protein tyrosine kinases and CK2 is one of the mechanisms that cooperate to the premature degradation of F508delCFTR.

Encapsulated engineered myoblasts can cure Hurler syndrome: preclinical experiments in the mouse model.

Mucopolysaccharidosis type I (MPSI) is an autosomic recessive, lysosomal storage disorder due to the deficit of the enzyme α-L-iduronidase (IDUA). The disease accounts for a general impairment of tissue and organ functions, mainly including heart disease, corneal clouding, organomegaly, skeletal malformations and joint stiffness. Neurological deterioration affects the severe forms. Both haemopoietic stem cell transplantation and enzyme replacement therapy can be applied to the treatment of the disorder; however, they both present several limitations. Thus, the search for alternative strategies to complement the present procedures is highly desirable. A murine myoblast cell line engineered to overexpress IDUA was generated and enclosed in alginate microcapsules, which were intra-peritoneally implanted in the MPSI mouse model. Plasma and tissue enzyme activity induced by the treatment and urinary and tissue glycosaminoglycan content were monitored in the animals, progressively sacrificed up to 4 months after implantation. Significant induction of enzyme activity and reduction of glycosaminoglycan accumulation were detected in the implanted animals, complete normalization of deposits was achieved in two animals. Intra-peritoneal implantation of alginate microcapsule confirms to be a valid approach as an endogenous enzyme replacement procedure.

Portable single-lead electrocardiogram device is accurate for QTc evaluation in hospitalized patients.

BACKGROUND: Many commonly used drugs can prolong the QTc interval (QTc), which can lead to potentially life-threatening arrhythmias. In the current era of the COVID-19 pandemic, it is worth mentioning that the disease itself and several drugs used for its treatment have been associated with QTc prolongation. OBJECTIVE: To evaluate the agreement and clinical precision of a portable single-lead electrocardiogram (ECG) device to measure the QTc interval compared to the standard 12-lead ECG. METHODS: In sequential tests, QTc of ECG recordings obtained with the KardiaMobile (KM-1L) device (AliveCor, San Francisco, CA) were compared to QTc obtained with conventional 12-lead ECG. Agreement was evaluated using Bland-Altman plots and Lin's concordance coefficient. Consistency between the 2 devices in determining QTc prolongation (QTc ≥470 ms in males or ≥480 ms in females) was evaluated with kappa statistics. RESULTS: A total of 128 patients with a presumed or confirmed diagnosis of COVID-19 admitted to a university hospital were included. QTc intervals measured with KM-1L were similar to QTc measured with conventional ECG (442.45 ± 40.5 vs 441.65 ± 40.3 ms, P = .15). Bland-Altman analysis showed no significant difference in QTc values (average difference of -0.797, 95% limits of agreement:-13.179; 11.585). Lin's concordance coefficient showed an excellent agreement (0.988, P < .001). Concordance between the 2 devices for determining QTc prolongation was excellent (kappa >0.90). CONCLUSION: ECG recordings obtained with KM-1L allow an accurate QTc interval assessment. Considering its simplicity of use, this approach has advantages over conventional ECG and can provide an alternative for the evaluation of QTc in hospitalized patients, during the current time of the COVID-19 pandemic and beyond.

A white paper on a neurodevelopmental framework for drug discovery in autism and other neurodevelopmental disorders.

In the last decade there has been a revolution in terms of genetic findings in neurodevelopmental disorders (NDDs), with many discoveries critical for understanding their aetiology and pathophysiology. Clinical trials in single-gene disorders such as fragile X syndrome highlight the challenges of investigating new drug targets in NDDs. Incorporating a developmental perspective into the process of drug development for NDDs could help to overcome some of the current difficulties in identifying and testing new treatments. This paper provides a summary of the proceedings of the 'New Frontiers Meeting' on neurodevelopmental disorders organised by the European College of Neuropsychopharmacology in conjunction with the Innovative Medicines Initiative-sponsored AIMS-2-TRIALS consortium. It brought together experts in developmental genetics, autism, NDDs, and clinical trials from academia and industry, regulators, patient and family associations, and other stakeholders. The meeting sought to provide a platform for focused communication on scientific insights, challenges, and methodologies that might be applicable to the development of CNS treatments from a neurodevelopmental perspective. Multidisciplinary translational consortia to develop basic and clinical research in parallel could be pivotal to advance knowledge in the field. Although implementation of clinical trials for NDDs in paediatric populations is widely acknowledged as essential, safety concerns should guide each aspect of their design. Industry and academia should join forces to improve knowledge of the biology of brain development, identify the optimal timing of interventions, and translate these findings into new drugs, allowing for the needs of users and families, with support from regulatory agencies.

Gene therapy of Hunter syndrome: evaluation of the efficiency of muscle electro gene transfer for the production and release of recombinant iduronate-2-sulfatase (IDS).

Mucopolysaccharidosis type II (MPSII) is an inherited disorder due to a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The disease is characterized by a considerable deposition of heparan- and dermatan-sulfate, causing a general impairment of physiological functions. Most of the therapeutic protocols proposed so far are mainly based upon enzyme replacement therapy which is very expensive. There is a requirement for an alternative approach, and to this aim, we evaluated the feasibility of muscle electro gene transfer (EGT) performed in the IDS-knockout (IDS-ko) mouse model. EGT is a highly efficient method of delivering exogenous molecules into different tissues. More recently, pre-treatment with bovine hyaluronidase has shown to further improve transfection efficiency of muscle EGT. We here show that, by applying such procedure, IDS was very efficiently produced inside the muscle. However, no induced IDS activity was measured in the IDS-ko mice plasma, in contrast to matched healthy controls. In the same samples, an anticipated and rapidly increasing immune response against the recombinant protein was observed in the IDS-ko vs control mice, although reaching the same levels at 5 weeks post-injection. Additional experiments performed on healthy mice showed a significant contribution of hyaluronidase pre-treatment in increasing the immune response.

Sorting of striatal and cortical interneurons regulated by semaphorin-neuropilin interactions.

Most striatal and cortical interneurons arise from the basal telencephalon, later segregating to their respective targets. Here, we show that migrating cortical interneurons avoid entering the striatum because of a chemorepulsive signal composed at least in part of semaphorin 3A and semaphorin 3F. Migrating interneurons expressing neuropilins, receptors for semaphorins, are directed to the cortex; those lacking them go to the striatum. Loss of neuropilin function increases the number of interneurons that migrate into the striatum. These observations reveal a mechanism by which neuropilins mediate sorting of distinct neuronal populations into different brain structures, and provide evidence that, in addition to guiding axons, these receptors also control neuronal migration in the central nervous system.

Cell migration from the ganglionic eminences is required for the development of hippocampal GABAergic interneurons.

GABAergic interneurons have major roles in hippocampal function and dysfunction. Here we provide evidence that, in mice, virtually all of these cells originate from progenitors in the basal telencephalon. Immature interneurons tangentially migrate from the basal telencephalon through the neocortex to take up their final positions in the hippocampus. Disrupting differentiation in the embryonic basal telencephalon (lateral and medial ganglionic eminences) through loss of Dlx1/2 homeobox function blocks the migration of virtually all GABAergic interneurons to the hippocampus. On the other hand, disrupting specification of the medial ganglionic eminence through loss of Nkx2.1 homeobox function depletes the hippocampus of a distinct subset of hippocampal interneurons. Loss of hippocampal interneurons does not appear to have major effects on the early development of hippocampal projection neurons nor on the pathfinding of afferrent tracts.

Milk-derived bioactive peptides protect against oxidative stress in a Caco-2 cell model.

Milk and milk-derived products are a relevant source of bioactive peptides, which are also potential components of functional foods. Bioactive peptides exert multiple actions including an antioxidant role. In the present paper, four synthetic peptides (NPYVPR, AVPYPQR, KVLPVPEK, and ARHPHPHLSFM), corresponding to milk-derived peptides were studied. Although with different features, as revealed by RP-HPLC chromatography and MS analysis, the obtained peptides were shown to be taken up by Caco-2 cells arranged in an epithelial monolayer formation. The four peptides were all able to preserve cell viability against induced oxidative stress indicating that they might have a role in the control of oxidative stress. Therefore, an estimation of total thiols and glutathione content was performed after cell treatment with oxidants like hydrogen peroxide (H2O2) or tert-butyl hydroperoxide (TbOOH). The peptides were able to prevent the decrease of both total thiols and glutathione induced by H2O2 or TbOOH, and, in addition, they showed a protective effect on the thiol-related antioxidant enzymes thioredoxin reductase and glutathione reductase. Finally, they caused a decrease of ROS production induced by TbOOH in Caco-2 cells. The reported results highlight the relevant antioxidant role played by bioactive peptides in cells, which adds to other previously known properties.

Protein kinase CK2 phosphorylates and upregulates Akt/PKB.

Treatment of Jurkat cells with specific inhibitors of protein kinase CK2 induces apoptosis. Here we provide evidence that the anti-apoptotic effect of CK2 can be at least partially mediated by upregulation of the Akt/PKB pathway. Such a conclusion is based on the following observations: (1) inhibition of CK2 by cell treatment with two structurally unrelated CK2 inhibitors induces downregulation of Akt/PKB, as judged from decreased phosphorylation of its physiological targets, and immunoprecipitate kinase assay; (2) similar results are observed upon reduction of CK2 catalytic subunit by the RNA-interference technique; (3) Akt/PKB Ser129 is phosphorylated by CK2 in vitro and in vivo; (4) such a phosphorylation of activated Akt/PKB correlates with a further increase in catalytic activity. These data disclose an unanticipated mechanism by which constitutive phosphorylation by CK2 may be required for maximal activation of Akt/PKB.

Evolution of the basal ganglia in tetrapods: a new perspective based on recent studies in amphibians.

It has been postulated frequently that the fundamental organization of the basal ganglia (BG) in vertebrates arose with the appearance of amniotes during evolution. An alternative hypothesis, however, is that such a condition was already present in early anamniotic tetrapods and, therefore, characterizes the acquisition of the tetrapod phenotype rather than the anamniotic-amniotic transition. Re-examination of the BG organization in tetrapods in the light of recent findings in amphibians strongly supports the notion that elementary BG structures were present in the brain of ancestral tetrapods and that they were organized according to a general plan shared today by all extant tetrapods.

Basal ganglia organization in amphibians: evidence for a common pattern in tetrapods.

The results of recent studies investigating the connections and chemoarchitecture of the basal forebrain of amphibians provide strong evidence that tetrapod vertebrates share a common pattern of basal ganglia organization. This pattern includes the existence of dorsal and ventral striatopallidal systems, reciprocal connections between the striatopallidal complex and the diencephalic and mesencephalic basal plate (striato-nigral and nigro-striatal projections), and descending pathways from the striatopallidal system to the midbrain tectum and reticular formation. The connectional similarities are parallelled by similarities in the distribution of chemical markers of striatal and pallidal structures such as dopamine, substance P and enkephalin. Moreover, studies of development and expression of homeobox genes have given further support to the notion that both amniotic and anamniotic tetrapods have a common pattern of basal ganglia organization. A new nomenclature of basal forebrain structures in amphibians is proposed which reflects our current understanding of basal ganglia organization in this class of vertebrates.

An analysis of the substrate specificity of insulin-stimulated protein kinase-1, a mammalian homologue of S6 kinase-II.

The specificity determinants for insulin-stimulated protein kinase-I (ISPK-1) have been investigated with synthetic peptides based on naturally-occurring protein phosphoacceptor sequences. Peptides (Arg-Arg-Xaa-Ser-Xaa) that fulfill the consensus sequence for cyclic-AMP-dependent protein kinase (PK-A) are also phosphorylated readily by ISPK-1. The phosphorylation efficiency is improved by increasing the number of N-terminal arginine residues and by moving the arginyl cluster one residue further away from the serine, the nonapeptide (Arg)4-Ala-Ala-Ser-Val-Ala being the best substrate among all the short peptides tested (Km = 15 microM). Conversely, the substitution of either Thr for Ser or Lys for Arg is detrimental. Likewise, two flanking Pro residues and an Arg immediately N-terminal to the Ser act as negative specificity determinants. While the specificity of ISPK-1 shows several similarities to that of PK-A, including an absolute requirement for basic residues on the N-terminal side of the target Ser, it differs in several other respects including (1), the detrimental effect of a Lys for Arg substitution which is still compatible with some phosphorylation by ISPK-1, but not PK-A; (2), the presence of C-terminal acidic residues which are tolerated very well by ISPK-1, but are detrimental to PK-A; (3), the effect of substituting Phe for Val in the peptide Arg-Arg-Ala-Ser-Val-Ala, which improves the efficiency of phosphorylation by PK-A (lowering the Km 4-fold), but has no effect on phosphorylation by ISPK-1. These differences in peptide substrate specificity may account in part for the different rates of phosphorylation of physiological substrates for ISPK-1 and PK-A, such as the G subunit of protein phosphatase-1.

Synthetic peptide substrates for casein kinase 2. Assessment of minimum structural requirements for phosphorylation.

Unlike the peptides SAEAAA and SEEAAA which are not substrates for casein kinase 2 (CK-2) their analogs SAAEAE and SAAEAA are still significantly phosphorylated. Their Km values, however, (13.3 and 18.9 mM, respectively) are almost two orders of magnitude higher than that of SEEEEE and their Vmax values are 3- and 14-fold lower than that of SAAEEE. The peptide ESEEEEE, but not ASEEEEE, is a slightly better substrate than SEEEEE, while both RSEEEEE and SEEEKE are very poor substrates compared to ASEEEEE and SEEEAE, respectively. SAAEAE is much more responsive to polylysine stimulation and polyphosphate inhibition than is SEEEEE. Taken together these data show that a single acidic residue at the third position from the C-terminal side of the phosphorylatable amino acid represents not only a necessary, but also a sufficient condition for site recognition by CK-2. Optimal phosphorylation efficiency, however, requires an extended C-terminal cluster of several acidic residues, and can be compromised by the presence of only a basic residue either inside the acidic cluster or adjacent to the N-terminal side of the phosphoacceptor amino acid. The structure of the phosphoacceptor site can greatly influence the efficacy of substrate-directed effectors of CK-2.

Resistance of initiation factor 2 (eIF-2alpha) kinases to staurosporine: an approach for assaying the kinases in crude extracts.

We studied the effect of staurosporine on two well characterised mammalian eIF-2alpha kinases, the heme-regulated translational inhibitor (HRI), and interferon-inducible double-stranded RNA-activated protein kinase (PKR). Both pure eIF-2 and a synthetic peptide used to measure the activity of purified or immunoprecipitated enzymes (sequence ILLSELSRRRIRAI) were phosphorylated with purified enzymes and crude preparations of tissues or cells in the presence of the inhibitor. In the presence of 0.25 microM staurosporine (a concentration which completely inhibits a wide range of Ser/Thr protein kinases), the phosphorylation of eIF-2alpha by HRI and PKR was not inhibited. The lack of response of eIF-2alpha kinases to staurosporine allowed us to measure PKR activity in salt washed postmicrosomal supernatants without previous purification of the enzyme. In the presence of poly(I):poly(C), the PKR activator, we detected both an increased phosphorylation of eIF-2alpha and an increment in the autophosphorylation of PKR. We also confirmed an induction of PKR in cultured neuronal cells after treatment with interferon. The results obtained following phosphorylation of the synthetic peptide with crude extracts are less conclusive. Although its phosphorylation is specific because it displaces eIF-2 phosphorylation, and the presence of staurosporine prevents its phosphorylation by other serine/threonine kinases, it is a rather poor substrate for PKR.

Serum G-CSF levels in patients undergoing G-CSF/chemotherapy mobilized peripheral stem cell harvest and predictors of neutrophil and platelet recovery.

Granulocyte colony-stimulating factor (G-CSF)/Chemotherapy mobilized peripheral blood progenitors are an effective source of stem cells which affords rapid and complete hematopoietic engraftment after myeloablative chemotherapy regimens. The dose of G-CSF most commonly used for mobilization is 5 micrograms/kg. We measured G-CSF levels in patients with chemosensitive malignancies undergoing peripheral stem cell harvest to determine whether there was a relationship between serum G-CSF levels and the yield of CD34+ hematopoietic progenitors. Peripheral blood stem cells (PBSCs) were mobilized by chemotherapy followed by G-CSF (5 micrograms/kg) started 24 hours after completion of chemotherapy. PBSCs were collected by daily leukapheresis during G-CSF stimulation once the WBC had recovered to 1.0 x 10(9)/L, with 10 liters of blood processed using a Fenwall CS 3000. G-CSF levels were monitored daily before and after leukapheresis. CD34+ cells from daily leukapheresis collections were determined in 11 patients. Immunophenotyping analyses of CD34+ and non-CD34+ cells for surface antigens CD38+, HLA-DR, CD71+, CD61+ and CD42a+ were performed on these daily leukapheresis. The mean (SD) number of days to neutrophil recovery (NR: > or = 0.5 x 10(9)/L) after stem cell reinfusion was 9.2 (1.92). The corresponding values for platelet recovery (PR: > or = 20 x 10(9) L) were 8.1 (2.39) days. Using multiple regression analyses, the best predictors for NR were: last G-CSF (R2 = 0.21); last G-CSF and CD34+ (R2 = 0.67); last G-CSF, CD34+ and number of chemotherapy cycles (R2 = 0.72).(ABSTRACT TRUNCATED AT 250 WORDS)

Exploring the role of MKK7 in excitotoxicity and cerebral ischemia: a novel pharmacological strategy against brain injury.

Excitotoxicity following cerebral ischemia elicits a molecular cascade, which leads to neuronal death. c-Jun-N-terminal kinase (JNK) has a key role in excitotoxic cell death. We have previously shown that JNK inhibition by a specific cell-permeable peptide significantly reduces infarct size and neuronal death in an in vivo model of cerebral ischemia. However, systemic inhibition of JNK may have detrimental side effects, owing to blockade of its physiological function. Here we designed a new inhibitor peptide (growth arrest and DNA damage-inducible 45ß (GADD45ß-I)) targeting mitogen-activated protein kinase kinase 7 (MKK7), an upstream activator of JNK, which exclusively mediates JNK's pathological activation. GADD45ß-I was engineered by optimizing the domain of the GADD45ß, able to bind to MKK7, and by linking it to the TAT peptide sequence, to allow penetration of biological membranes. Our data clearly indicate that GADD45ß-I significantly reduces neuronal death in excitotoxicity induced by either N-methyl-D-aspartate exposure or by oxygen-glucose deprivation in vitro. Moreover, GADD45ß-I exerted neuroprotection in vivo in two models of ischemia, obtained by electrocoagulation and by thromboembolic occlusion of the middle cerebral artery (MCAo). Indeed, GADD45ß-I reduced the infarct size when injected 30 min before the lesion in both models. The peptide was also effective when administrated 6 h after lesion, as demonstrated in the electrocoagulation model. The neuroprotective effect of GADD45ß-I is long lasting; in fact, 1 week after MCAo the infarct volume was still reduced by 49%. Targeting MKK7 could represent a new therapeutic strategy for the treatment of ischemia and other pathologies involving MKK7/JNK activation. Moreover, this new inhibitor can be useful to further dissect the physiological and pathological role of the JNK pathway in the brain.

Chemical synthesis and structural characterization of the RGD-protein decorsin: a potent inhibitor of platelet aggregation.

Decorsin is a 39-residue RGD-protein crosslinked by three disulfide bridges isolated from the leech Macrobdella decora belonging to the family of GPIIb-IIIa antagonists and acting as a potent inhibitor of platelet aggregation. Here we report the solid-phase synthesis of decorsin using the Fmoc strategy. The crude polypeptide was purified by reverse-phase HPLC in its reduced form and allowed to refold in the presence of glutathione. The homogeneity of the synthetic oxidized decorsin was established by reverse-phase HPLC and capillary zone electrophoresis. The results of amino acid analysis after acid hydrolysis of the synthetic protein, NH2-terminal sequencing and mass determination (4,377 Da) by electrospray mass spectrometry were in full agreement with this theory. The correct pairing of the three disulfide bridges in synthetic decorsin was determined by a combined approach of both peptide mapping using proteolytic enzymes and analysis of the disulfide chirality by CD spectroscopy in the near-UV region. Synthetic decorsin inhibited human platelet aggregation with an IC50 of approximately 0.1 microM, a figure quite similar to that determined utilizing decorsin from natural source. In particular, the synthetic protein was 2,000-fold more potent than a model RGD-peptide (e.g., Arg-Gly-Asp-Ser) in inhibiting platelet aggregation. Thermal denaturation experiments of synthetic decorsin, monitored by CD spectroscopy, revealed its high thermal stability (Tm approximately 74 degrees C). The features of the oxidative refolding process of reduced decorsin, as well as the thermal stability of the oxidized species, were compared with those previously determined for the NH2-terminal core domain fragment 1-41 or 1-43 from hirudin. This fragment shows similarity in size, pairing of the three disulfides and three-dimensional structure with those of decorsin, even if very low sequence similarity. It is suggested that the less efficient oxidative folding and the enhanced thermal stability of decorsin in respect to those of hirudin core domain likely can be ascribed to the presence of the six Pro residues in the decorsin chain, whereas none is present in the hirudin domain. The results of this study indicate that decorsin can be obtained by solid-phase methodology in purity and quantities suitable for structural and functional studies and thus open the way to prepare by chemical methods novel decorsin derivatives containing unusual amino acids or even non-peptidic moieties.

Activation of protein phosphatase-1 isoforms and glycogen synthase kinase-3 beta in muscle from mdx mice.

Three Protein Phosphatase-1 (PP1) isoforms (PP1 alpha, PP1 gamma-1 and PP1 delta) are found in skeletal muscle. These are bound to regulatory subunits, such as inhibitor 2 (I2) in the cytosol and G in the glycogen and microsomal fractions. In vitro, the PP1-12 complex is activated by Glycogen Synthase Kinase-3 (GSK-3 or FA). We investigated the activities and protein levels of the three PP1 isoforms and of GSK-3 in muscle of mdx dystrophic mice. PP1 was assayed as phosphorylase phosphatase, in the presence of 5 nM okadaic acid (which inhibits PP2A). Peptide antibodies were produced and used to investigate PP1 alpha, PP1 gamma-1 and PP1 delta. GSK-3 was assayed using a previously described peptide. This was synthesized in a pre-phosphorylated from, which avoids the additional use of Casein Kinase II. Higher PP1 activity was assayed in the cytosol from mdx rather than from control muscles. Immunoprecipitation indicated that only PP1 alpha and PP1 gamma-1 were more active. This was most likely due to enzyme activation, since the immunodetected proteins were unchanged. On the other hand, the immunodetected PP1 delta was lower in the glycogen and microsomal fractions from mdx muscle. GSK-3 was more active in the mdx extract Selective immunoprecipitation of GSK-3 alpha and GSK-3 beta indicated that both isoforms were activated. In the case of GSK-3 beta, the immunodetected protein was also increased. The changes described herein may be related to the pathological events occurring in the mdx muscle. These include increased protein degradation and turnover, and fibre regeneration. In fact, the decreased PP1 delta may be due to protein degradation and the increased GSK-3 may be the consequence of increased protein turnover or regeneration. The apparent correlation between the increased PP1 alpha and PP1 gamma-1 activities and the increased GSK-3 may agree with the hypothesis that GSK-3 activates the newly synthesized PP1.