Uncapping the N-terminus of a ubiquitous His-tag peptide enhances its Cu2+ binding affinity.

Metal complexes with an N-terminally free and N-terminally acetylated polyhistidine region of Echis ocellatus venom, with an interesting His-rich motif present in numerous metal binding proteins from all kingdoms of life (DHDHDHHHHHHPGSSV-NH2 and Ac-DHDHDHHHHHHPGSSV-NH2) show the role of the free amino group in the thermodynamic enhancement of Cu2+, Ni2+ and Zn2+ binding. In the studied sequences, Cu2+ can be coordinated by different sets of imidazole rings, and a 3-10 helix is detected in close proximity of Cu2+ binding sites. The complexes are more stable than those with a typical His6-tag, despite a similar copper(ii) coordination mode in both cases.

New composite materials prepared by calcium phosphate precipitation in chitosan/collagen/hyaluronic acid sponge cross-linked by EDC/NHS.

Nowadays, fabrication of composite materials based on biopolymers is a rising field due to potential for bone repair and tissue engineering application. Blending of different biopolymers and incorporation of inorganic particles in the blend can lead to new materials with improved physicochemical properties and biocompatibility. In this work 3D porous structures called scaffolds based on chitosan, collagen and hyaluronic acid were obtained through the lyophilization process. Scaffolds were cross-linked by EDC/NHS. Infrared spectra for the materials were made, the percentage of swelling, scaffolds porosity and density, mechanical parameters, thermal stability were studied. Moreover, the scaffolds were used as matrixes for the calcium phosphate in situ precipitation. SEM images were taken and EDX analysis was carried out for calcium and phosphorous content determination in the scaffold. In addition, the adhesion and proliferation of human osteosarcoma SaOS-2 cells was examined on obtained scaffolds. The results showed that the properties of 3D composites cross-linked by EDC/NHS were altered after the addition of 1, 2 and 5% hyaluronic acid. Mechanical parameters, thermal stability and porosity of scaffolds were improved. Moreover, calcium and phosphorous were found in each kind of scaffold. SEM images showed that the precipitation was homogeneously carried in the whole volume of samples. Attachment of SaOS-2 cells to all modified materials was better compared to unmodified control and proliferation of these cells was markedly increased on scaffolds with precipitated calcium phosphate. Obtained materials can provide the support useful in tissue engineering and regenerative medicine.

Northern pike (Esox lucius) collagen: Extraction, characterization and potential application.

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the scales of northern pike (Esox lucius) were extracted and characterized. It was the first time that this species was used as sources of collagen. FT-IR and amino acid analysis results revealed the presence of collagen. Glycine accounts for one-third of its amino acid residues and specific for collagen amino acid - hydroxyproline - is present in isolated protein. The content of imino acid: proline and hydroxyproline in ASC and PSC was similar (12.5% Pro and 6.5% Hyp). Both ASC and PSC were type I collagen. The denaturation temperature of ASC and PSC were 28.5 and 27°C, respectively. Thin collagen films were obtained by casting of collagen solution onto glass plates. The surface properties of ASC and PSC films were different - the surface of ASC collagen film was more polar and less rough than PSC and we can observe the formation of collagen fibrils after solvent evaporation. ASC films showed much higher tensile properties than PSC. The obtained results suggest that northern pike scales have potential as an alternative source of collagen for use in various fields.

Effects of different crosslinking methods on the properties of collagen-calcium phosphate composite materials.

The purpose of this study is the preparation and characterization of porous collagen/calcium phosphates (Col/CaP) composites. Collagen scaffolds with high porosity were prepared by freeze-drying technique. Col/CaP scaffold were created by new method--by deposition of calcium phosphate within collagen matrix in two steps using freeze-drying process before immersing samples in calcium solution. To find the optimal preparative method, we prepared diverse Col/CaP scaffolds using different collagen concentration and various crosslinking method: crosslinking with carbodiimide (EDC/NHS) and dehydrothermal treatment (DHT). This study explores the effect of the different crosslinking method on the properties of scaffolds, such as: microstructure (porosity and density), dissolution, water uptake, mechanical properties and collagenase degradation. The results obtained showed that crosslinking the scaffolds by either EDC/NHS or DHT have good mechanical and morphological properties compatible with their potential application in bone regeneration. The results demonstrated that properties of Col/CaP scaffolds changed significantly with different crosslinking method. However, while EDC/NHS increased the scaffolds' resistance to dissolution and degradation by collagenase, DHT decreased the swelling ratio and resistance to dissolution in PBS solution. Based on our study, 2% collagen concentration and EDC/NHS as crosslinking reagent are recommended to design the scaffold for use in bone engineering.

Functional divergence in gastrointestinal microbiota in physically-separated genetically identical mice.

Despite the fundamental contribution of the gut microbiota to host physiology, the extent of its variation in genetically-identical animals used in research is not known. We report significant divergence in both the composition and metabolism of gut microbiota in genetically-identical adult C57BL/6 mice housed in separate controlled units within a single commercial production facility. The reported divergence in gut microbiota has the potential to confound experimental studies using mammalian models.

Properties and modification of porous 3-D collagen/hydroxyapatite composites.

A freeze drying technique was used to form porous three-dimensional collagen matrixes modified by the addition of a variable amount of nano-hydroxyapatite. For chemical cross-linking EDC/NHS were used. Physical cross-linking was achieved by dehydrothermal treatment. Mechanical properties, morphology, dissolution, porosity, density, enzymatic degradation and swelling properties of materials have been studied after cross-linking. The density of scaffolds and its compressive modulus increased with an increasing amount of hydroxyapatite and collagen concentration in the composite scaffold, while the swelling ratio and porosity decreased. The studied scaffolds dissolved slowly in PBS solution. DHT cross-linked collagen matrices showed a much faster degradation rate after exposure to collagenase than the EDC cross-linked samples.

Photochemical behaviour of hydrolysed keratin.

An investigation into the influence of UV irradiation on keratin hydrolysates was carried out using UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR) and fluorescence spectroscopy. It was found that the absorption of keratin hydrolysates in solution increased during irradiation of the sample, most notably between 250-280 and 320-410 nm. The increase in absorbance in the region 320-410 was because of the new photoproducts formed during UV irradiation of keratin hydrolysates. The fluorescence of keratin hydrolysates was observed at 328 nm after excitation at 270 nm. UV irradiation caused fluorescence fading at 328 nm, and after 60 min of irradiation, a new broad weak band of fluorescence, attributable to new photoproducts, emerged in the UV wavelength region with emission maximum between 400 and 500 nm. FTIR spectroscopy results showed degradation of keratin under UV irradiation. A slight increase in oxidized sulphur species was also observed. The results obtained suggest that UV irradiation can be used as modifying agent for preparation of keratin hydrolysates for cosmetic applications.

Chemical and thermal cross-linking of collagen and elastin hydrolysates.

Chemical and thermal cross-linking of collagen soluble in acetic acid and elastin hydrolysates soluble in water have been studied. Solutions of collagen and elastin hydrolysates were treated using variable concentrations of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Moreover, diepoxypropylether (DEPE) has been used as cross-linking agent. Films made of collagen and elastin hydrolysates were also treated with temperature at 60°C and 100°C to get additional cross-links. The effect of cross-linking has been studied using FTIR spectroscopy, thermal analysis, AFM and SEM microscopy. Mechanical and surface properties of materials have been studied after cross-linking. It was found that thermal and mechanical properties of collagen and elastin materials have been altered after thermal treatment and after the reactions with EDC/NHS and/or DEPE. Surface properties of collagen materials after chemical cross-linking have been modified. Thermal and chemical cross-linking of collagen films lead to alteration of polarity of the surface.

[Biochemical markers of fibrosis in chronic hepatitis and liver cirrhosis of viral origin]. / Biochemiczne wykladniki wlóknienia w przewleklym zapaleniu watroby i w marskosci watroby o etiologii wirusowej.

OBJECTIVE: Usefulness of three selected extracellular matrix components as markers of hepatic fibrosis in patients with chronic viral hepatitis and post inflammatory liver cirrhosis. METHODS: 91 patients with chronic HBV or HCV infection were divided into four groups; chronic hepatitis--group O; cirrhosis classes A, B and C (Child-Pugh classification)--groups A, B and C, respectively. The serum levels of: N-terminal peptide of type III procollagen (PIIIP), laminin (LP1) and hyaluronic acid (HA) in these patients were measured using commercial radioimmunological and radiometric tests. RESULTS: The mean values of the investigated markers increased in patients with cirrhosis according to the stage of the disease (thus group A showed the least level while the highest was exhibited in group C). Between all the groups: O, A, B and C, HA was the marker showing the greatest difference. The only marker with a greater mean level in group O than group A, was PIIIP. Substantially higher levels of the investigated markers were found in the patients with HCV than in those with HBV, but only within group A. CONCLUSIONS: 1) The values of the studied markers increased in various degree with the cirrhosis progress. 2) LP1 and HA measurements have particular value in the clinical estimation of cirrhotic advancement. 3) HA measurement may be helpful in differentiating between cirrhosis and chronic hepatitis. 4) The course of hepatic fibrosis may be influenced by the etiological factor as the values of the studied markers are higher in patients with HCV than in patients with HBV, in the early stage of cirrhosis.

[Immunological disorders in patients with chronic viral hepatitis type C]. / Zaburzenia immunologiczne u chorych z przewleklym wirusowym zapaleniem watroby typu C.

UNLABELLED: Various immunological disorders have been observed frequently in patients with chronic viral hepatitis C. Hepatitis C virus has been implicated in the pathogenesis of: essential mixed cryoglobulinemia, cryoglobulinemic vasculitis glomerulonephritis, thrombocytopenia, porphyria cutanea tarda, autoimmune thyroiditis (among others). AIM OF THE STUDY: Was to determine the prevalence and clinical meaning of immunological disorders in HCV infected patients. METHODS: 93 HCV infected patients were studied with regard to the presence of cryoglobulins, autoantibodies, rheumatoid factor (RF) and circulating immunological complexes (CIC). RESULTS: 35 patients out of 93 (38%) had detectable cryoglobulins. Cryoglobulins in 90 cases were of type III and in 3--of type II. CIC /by immunoelectrophoresis of the PEG sediments/ were found in sera of 87 patients (93.5%). 38 persons (41%) had detectable rheumatoid factor. Antinuclear antibodies were found in sera of 15 patients (16%), anti-smooth muscles antibodies--in 21 persons (23%) and anti-LKM1 antibodies--in 5 (5%). Titers of autoantibodies were usually low. The most frequent clinical manifestations were arthralgia and skin changes. CONCLUSION: Immunological disorders /circulating immune complexes (93.5%), autoantibodies (60%), rheumatoid factor (41%), cryoglobulinemia type III (38%)/ are frequently found in HCV infected patients. Age, gender, histological score and clinical evidence of liver cirrhosis do not influence on rate of these abnormalities. Clinical manifestations of immunological disorders are usually mild.

[Effectiveness of antiviral treatment of patients with chronic hepatitis C (a Polish multicenter study)]. / Efektywnosc leczenia przeciwwirusowego przewleklego zapalenia watroby typu C (wieloosrodkowe badania pòlskie).

Interferon alpha (INF) is routine treatment in patients with chronic hepatitis C. Many controlled investigations were evaluated to establish the optimal schedule of treatment with sustained virological and biochemical response. Recently, multicentre meta-analyses suggest that combination therapy (INF + Ribavirin) was more effective than treatment with interferon alone. The aim of this study was to compare the efficacy of four schedules of antiviral treatment in 445 patients with chronic hepatitis C. Combination therapy (INF + Ribavirin) given for 6 mo. and monotherapy (INF) for 18 mo. were more effective than interferon alone given for 6 mo. Treatment with INF alone for 6 mo. was demonstrated to be insufficient.

The influence of GSTM1 and GSTT1 genotypes on the induction of sister chromatid exchanges and chromosome aberrations by 1,2:3,4-diepoxybutane.

Cytogenetic tests - chromosome aberrations (CA), sister chromatid exchanges (SCE) and micronuclei (MN) - are most often applied in biomonitoring of the genotoxicity of potentially carcinogenic chemicals in human cells. One of the extensively studied genotoxins is diepoxybutane (DEB) - reactive biometabolite of butadiene (BD). Several studies showed a high SCE induction in human lymphocytes exposed in vitro to various concentrations of DEB. DEB also proved to be a potent inducer of chromosome aberrations and micronuclei. A bimodal distribution of SCE frequency after in vitro DEB treatment was observed. The aim of the present study was to examine the ability of DEB to induce different individual cytogenetic response measured by SCE and CA frequency. The possible influence of genetic polymorphism has also been taken into account, by including donors representing positive or null GSTM1 and GSTT1 genotypes. Our study supported the earlier results showing that DEB is an effective inducer of SCEs and CAs, causing also the decrease in replication index (RI). DEB bioactivity measured by SCE induction - but not by CA test - was significantly higher in GSTT1 negative than in GSTT1 positive donors. GSTM1 polymorphism had no influence on these endpoints. The donors GSTT1-/GSTM1+ were shown to be slightly more sensitive to DEB than GSTT1-/GSTM1- individuals. There was also observed a unimodal distribution of DEB-induced SCEs and CAs in the group, despite the fact that the experiment was performed on the lymphocytes obtained from both GSTT1 positive and negative donors.

[The incidence of cavum septum pellucidum in preterm and term newborns]. / Wystepowanie jamy przegrody przezroczystej w osrodkowym ukladzie nerwowym u noworodków i wczesniaków.

We reviewed cranial sonographic studies done on 167 newborn (139 preterm and 28 term newborn infants) to determine the incidence of cavum septum pellucidum (CSP) and association with pregnancy, labor and newborn adaptation. A CSP was seen in 34.53% preterm (below 37 weeks of gestational age) and 14.29% term newborn. On basis of our results and included in literature we suggest, that this findings on ultrasound scan is not an unquestionably norm. Evolution of CSP in ultrasound examinations and neurodevelopment of children with CSP should be observed.

Electrical impedance tomography. Technical and experimental problems encountered in impedance spectroscopy in the alpha and beta dispersion regions.

The main experimental problems (temperature, electrode, polarisation, non-uniformity of electric field, non-homogeneity of biological material) of impedance measurements of biological tissue are discussed. Minimisation of the inherent sources of experimental error is proposed.

Electrical impedance tomography. Determination of impedance of tissue in the frequency range 10(-6)-20 MHz: preliminary results.

In order to characterise small samples of tissue by complex impedance measurements between 10(-6) and 20 MHz, an experimental set-up is proposed. This paper describes the methodology used and presents some preliminary results.

[Cytogenetic studies in patients with non-Hodgkin's lymphoma (nHL)]. / Badania cytogenetyczne u pacjentów z chloniakami nieziarniczymi (nHL).

Cytogenetic examinations were carried out in 24 untreated patients with non-Hodgkin, non-Burkitt lymphoma. 10-20 G-banded metaphases, obtained from short-term cultures of unstimulated lymph++ node, bone marrow and blood cells were analyzed in each case. In 18 patients only, the obtained metaphases were suitable for cytogenetic analysis. In 11 patients (group A) karyotype was normal or only single, +non-clonal aberrations were observed. In 7 patients (group B) clonal aberrations were found, among them, in 3-structural changes of chromosome 1, but with different breakpoints: 1p31, 1p31, 1p36. The group of patients with chromosomal aberrations showed statistically significantly shorter survival time than the group without aberrations (p = 0.04). In the former group more patients had low grade malignancy lymphoma. Our observations confirm those data from the literature which indicate that the presence of chromosomal aberrations is a factor of poor prognosis, independent of other clinical and histopathological prognostic factors in non-Hodgkin Lymphoma.

Prognostic significance of secondary cytogenetic changes and nonspecific cross-reacting antigen (NCA) in patients with Ph-positive chronic myeloid leukemia.

Cytogenetic analyses were carried out on peripheral blood and bone marrow cells of 31 chronic myeloid leukemia (CML) patients who presented with blastic, accelerated, or chronic phases. The percentage of cytoplasmic nonspecific cross-reacting antigen (cNCA, a marker of myelocytic differentiation)-containing cells was determined in the same blood or bone marrow samples. The patients were divided in two groups according to cytogenetic results: those with aberrations in addition to the Philadelphia chromosome (Ph1) and those with Ph1 only. Among the additional aberrations such changes, not typical of CML, were found: del(2)(p21), t(6;11)(q25;q23), and t(12;?)(p13;?). The survival time and the percentage of cNCA-positive cells of patients in blastic and accelerated phases were compared between the above-mentioned two groups of patients using the Student t test and the Kaplan-Meier estimator. The percentage of cNCA-positive cells was significantly lower and the survival time significantly shorter in the group of patients with additional aberrations. The probability of survival according to the Kaplan-Meier estimator was also lower for this group. These data suggest that the immunologically determined lower degree of maturity, that characterized cells bearing additional aberrations, coincides with and/or results in more rapid progression of the disease.