Micro-electrode array recordings reveal reductions in both excitation and inhibition in cultured cortical neuron networks lacking Shank3.

Numerous risk genes have recently been implicated in susceptibility to autism and schizophrenia. Translating such genetic findings into disease-relevant neurobiological mechanisms is challenging due to the lack of throughput assays that can be used to assess their functions on an appropriate scale. To address this issue, we explored the feasibility of using a micro-electrode array (MEA) as a potentially scalable assay to identify the electrical network phenotypes associated with risk genes. We first characterized local and global network firing in cortical neurons with MEAs, and then developed methods to analyze the alternation between the network active period (NAP) and the network inactive period (NIP), each of which lasts tens of seconds. We then evaluated the electric phenotypes of neurons derived from Shank3 knockout (KO) mice. Cortical neurons cultured on MEAs displayed a rich repertoire of spontaneous firing, and Shank3 deletion led to reduced firing activity. Enhancing excitation with CX546 rescued the deficit in the spike rate in the Shank3 KO network. In addition, the Shank3 KO network produced a shorter NIP, and this altered network firing pattern was normalized by clonazepam, a positive modulator of the GABAA receptor. MEA recordings revealed electric phenotypes that displayed altered excitation and inhibition in the network lacking Shank3. Thus, our study highlights MEAs as an experimental framework for measuring multiple robust neurobiological end points in dynamic networks and as an assay system that could be used to identify electric phenotypes in cultured neuronal networks and to analyze additional risk genes identified in psychiatric genetics.

Adsorption of heavy metal ions by sawdust of deciduous trees.

The adsorption of heavy metal ions from synthetic solutions was performed using sawdust of beech, linden and poplar trees. The adsorption depends on the process time, pH of the solution, type of ions, initial concentration of metals and the sawdust concentration in suspension. The kinetics of adsorption was relatively fast, reaching equilibrium for less than 20 min. The adsorption equilibrium follows Langmuir adsorption model. The ion exchange mechanism was confirmed assuming that the alkali-earth metals from the adsorbent are substituted by heavy metal ions and protons. On lowering the initial pH, the adsorption capacity decreased, achieving a zero value at a pH close to unity. The maximum adsorption capacity (7-8 mg g(-1) of sawdust) was achieved at a pH between 3.5 and 5 for all the studied kinds of sawdust. The initial concentration of the adsorbate and the concentration of sawdust strongly affect the process. No influence of particles size was evidenced. A degree of adsorption higher than 80% can be achieved for Cu(2+) ions but it is very low for Fe(2+) ions, not exceeding 10%.

Copper electrowinning from acid mine drainage: a case study from the closed mine "Cerovo".

Copper removal from acid mine drainage originating from closed copper mine "Cerovo" RTB Bor, Serbia and containing approximately 1.3 g dm(-3) of copper and a very small amount of Fe2+/Fe3+ ions, has been successfully performed by the direct electrowinning method using either a porous copper sheet or carbon felt as the cathode. A cell with a fluidised bed of inert turbulent promoters, also used in this study, may be considered as unacceptable for the purpose view, having a cell voltage between 12 and 14 V. The cells used in the electrowinning experiments were compared in terms of cell voltage, pH and copper concentration. The results showed that it is possible to remove copper successfully from the mine waters with a high degree of electrowinning--higher than 92% and with a satisfactorily average current efficiency (>60%). Depending on the process time and the applied current, a final copper concentration less than 0.1 g dm(-3) was achieved. The specific energy consumption was approximately 7 kWh kg(-1) of deposited copper. A dense copper deposit was obtained when a three-dimensional electrode was used.

Moisture and gamma-ray irradiation effects on the mechanical properties of carbon fibre-reinforced plastics.

The effects of gamma-irradiation and moisture absorption on the mechanical properties of carbon fibres-epoxy resin composites were studied. The properties dominated by the matrix and fibre-matrix interface (interlaminar and in-plane shear strength) were measured at room temperature using standard tests. These tests were carried out before and after exposures to gamma irradiation and before and after immersion in water at 80 degrees C during 21 days. The dosage of gamma irradiation was up to 11.7 MGy. The micrographs of surfaces fractured in performed tests were observed on a scanning electron microscope. They were analyzed with consulting the stated effects on mechanical properties and the measured values of the glass transition temperature of tested coupons before and after irradiation and immersion in water. The obtained results show that moisture and irradiation, if they act one after the other, have a significant influence on the degradation of matrix-dominated mechanical properties of the tested carbon-epoxy composite.

Periodontal conditions and oral hygiene in rural population of post-war Vukovar region, Croatia in correlation to stress.

OBJECTIVE: The greatest burden of oral diseases is on the socially marginalized population, such as those living in small villages and war areas. The aim of this study was to assess the periodontal conditions of people in post-war area villages in relation to oral hygiene, habits and war stress. METHODS: The study was conducted on a sample of 282 subjects (mean age 41.5 +/- 17.8) in seven Vukovar villages using the questionnaire and clinical periodontal examination. RESULTS: Five years after the war in Croatia the population was still very poor, low educated, and had a low level of periodontal health and oral hygiene habits. The level of periodontal disease and attachment loss tended to increase with age and physical activity and decrease with education level, higher frequency of tooth brushing and toothbrush replacement, dental visits and utilisation of auxiliary devices. Subjects exposed to any kind of war stress had a significantly worse periodontal status and more excluded sextants than those who were not exposed to such stress experience (p<0.05). Better periodontal conditions were found in individuals that were refugees, than those who were in war, wounded or lost a dear person (p<0.05). Individuals who were in war rarely brushed their teeth, visited dentist and changed toothbrushes, but more frequently drank alcohol and smoked, in comparison to other groups, especially those who have not been exposed to war stress. CONCLUSIONS: Specific socio-economic and psychological conditions in post-war areas could be significant risk factors for poor periodontal conditions.

Prevalence of gingivitis in 6- to 11-year-old Croatian children.

OBJECTIVE: The purpose of this study was to assess the prevalence of gingivitis in 6- to 11-year-old children in two urban settlements of Zagreb, Croatia. PARTICIPANTS: Total number of 1025 children was included in the study. The Gingival Bleeding Index was used to determine the condition of the gingiva on mesiobuccal and buccal surfaces of six selected teeth, totaling to 12 surfaces. RESULTS: Forty-three percent of the examined children had healthy gingival tissue, 12% showed bleeding from one surface, 6% from two, 5% from three, 17% from four, and 17% from five to eight surfaces. CONCLUSIONS: Gingivitis was prevalent, but slight to moderate. It is important to instruct and control schoolchildren in excellent oral hygiene in order to prevent gingivitis.

Screening of fixed prosthodontic dentures after five years of use in relation to material and construction.

The aim of this study was to screen patients with fixed prosthodontic appliances that were in oral cavity for a period of 5 years or more and to assess clinically and radiologically root caries, gingival recession, pocket formation, alveolar ridge resorption, as well as gingival (GI) and plaque index (PI). The aim also was to find out the differences between materials and constructions, between abutment and non-abutment teeth, and to find out the need for replacement. A total of 260 patients and their orthopantomograms were examined, with a total of 2,265 teeth, 610 being bridge abutments and 246 being crowns. The most frequent were metal+ acrylic veneer crowns or bridges. Root caries was found under the abutments in 10-20%; however abutments with ceramic crowns had the lowest percentage of caries (p < 0.01). Alveolar ridge resorption, pocket formation deeper than 3 mm and gingival recession of various degree was found in 50% of the cases, again with the lowest percentage of ceramic-fused-to-metal appliances (p < 0.01). Pocket depth was registered in significantly higher percentage in metal-acrylic veneer appliances compared to natural teeth (p < 0.01), while there was no significant difference between metal-ceramic appliances and natural teeth (p > 0.05). Although the worst findings were recorded for metal- + acrylic veneer crowns for PI, no significant difference existed between crowns of different material or non-abutment teeth (p > 0.05). There was statistically significant difference between abutments with metal + acrylic veneer crowns, full metal crowns, metal ceramic crowns and non-abutments for GI scores. Higher percentage of scores 0 and 1 was recorded for metal ceramic crowns and non-abutments and significantly higher percentage of scores 2 and 3 was recorded for metal + acrylic veneer crowns and full metallic crowns. Almost 50% of metal-ceramic abutments had no pathologic findings. Almost 30% of the patients needed replacement, or even some abutments to be extracted and therefore a new prosthodontic appliance.

Model of interstitial pressure as a result of cyclical changes in the capillary wall fluid transport.

Reported interstitial pressures range from -8 to +6 mm Hg in different tissues and from <-20 mm Hg in burned tissue or more than +30 mm Hg in tumors. We have tried to link interstitial pressure to the here proposed cyclical changes in the fluid transport across the capillary wall. In the presented model interstitial pressure is considered as an average of pressures in numerous pericapillary spaces. A single pericapillary pressure is a dynamic difference between the net outward (hydraulic pressure+interstitial colloid osmotic pressure) and inward (plasma colloid oncotic pressure) forces. Hence, dominating net outward forces would result in a positive pericapillary interstitial pressure, while stronger inward forces would produce negative pressures in the pericapillary space. All interruptions of blood flow leave some blood in capillaries with a normal oncotic pressure and no hydrostatic pressure that might act as a strong absorber of interstitial fluid until the blood flow is reestablished. Model assumptions for the systemic circulation capillaries include (a) precapillary sphincters can almost entirely stop the capillary flow, (b) only a minority of sphincters are normally open in the tissue, and (c) hydrostatic pressures in unperfused capillaries are similar to the pressures at their venous ends. The key proposal is that capillaries with closed precapillary sphincters along their entire length have low hydrostatic pressure of 10 to 15 mm Hg. This pressure cannot force filtration, so these capillaries reabsorb interstitial fluid from the pericapillary space along their entire length. In the open capillaries, hydrostatic pressure filtrates fluid to the pericapillary space along most of their length. Fluid enters, moves some 20 or 30 micrometers away and back to be reabsorbed at the same point. Closed periods are periods of intense fluid reabsorption, while the short open periods refill the space with fresh fluid. It can be calculated that subcutaneous tissue interstitial pressure values might develop if the closed periods are 1.14 to 2.66 times longer than the open periods. Positive interstitial pressures observed in some organs might develop if open periods are longer than the closed periods. High interstitial colloid pressure in lungs makes both perfused and unperfused capillaries absorptive, resulting in more negative values of lung interstitial pressure. The same model is used to explain interstitial pressure values in tumors, burned tissue and intestinal villi.

Gastropathy and diarrhea in diabetic patients: the presence of helicobacteriosis and PAS-positive vascular deposits in gastric and duodenal mucosa.

AIM: To determine the presence of vascular periodic acid-Schiff's reagent (PAS) positive deposits in the gastric and duodenal mucosa of diabetic patients and controls. METHODS: Forty-six poorly controlled diabetic patients with digestive symptoms, aged 23 to 63 years (32 type I patients on insulin therapy with a mean diabetes duration of 14.2 +/- 3.1 years (mean +/- SE) and 13 type II patients with a mean diabetes duration of 7.2 +/- 2.3 years) were included. Of these, 17 had mainly gastropathic symptoms while 13 had diarrhea, and the remaining 16 patients had nonspecific symptoms. Forty control individuals of similar age and gender were included. Biopsy specimens were taken from areas of grossly normal gastric and duodenal mucosa. RESULTS: Gastric mucosa samples were pathological in 38 of 46 diabetic patients (18 cases of chronic active H. pylori antral gastritis, 13 cases of chronic active H. pylori pangastritis and 7 cases of nonspecific chronic gastritis). Duodenal mucosa samples were pathological in 32/46 diabetic patients. In the control group, 21 of 40 gastric samples (5 cases of chronic active H. pylori antral gastritis, 3 cases of chronic active H. pylori pangastritis and 13 cases of H. pylori-negative chronic gastritis) and 12/40 duodenal samples were pathological. Both helicobacteriosis and gastric and duodenal mucosa pathologies were significantly (p < 0.01) more common in diabetic patients than in controls. No significant associations were found between histological findings of gastric mucosa and of duodenal mucosa in diabetics and the control group. PAS-positive material in the vascular wall of gastric (16/46 vs. 2/40 in controls) and duodenal mucosa specimens (25/46 vs. 5/40 in controls) was significantly more common among diabetics (p = 0.001 for gastric and p < 0.001 for duodenal mucosa). No significant association was found between the presence of gastropathy or diarrhea compared to the presence of neuropathy, retinopathy, nephropathy or the type of diabetes. CONCLUSION: Endoscopic specimens from the gastroduodenum of diabetic patients revealed a large quantity of PAS positive vascular deposits, probably reflecting the condition of the mucosal vessels in our patients.

Trimerization of cell adhesion molecule L1 mimics clustered L1 expression on the cell surface: influence on L1-ligand interactions and on promotion of neurite outgrowth.

Several studies indicate that cell adhesion molecules have to be clustered on the cell surface to engage in adhesive functions. We investigated adhesive functions of clustered versus monomeric L1 extracellular parts in vitro to distinguish how clustering affects ligand binding and promotion of neurite outgrowth. Trimeric L1 was recombinantly expressed and covalently assembled by the cartilage matrix protein's coiled-coil domain. Trimeric L1 has an apparent molecular mass of approximately 380 kDa in the nonreduced form and approximately 130 kDa in the reduced form. Rotary shadowing electron micrographs of trimeric L1 revealed a rod-like shape terminating in three globular domains. Monomeric L1 assumes a horseshoe shape of domains Ig I-IV followed by a rod-like structure consisting of Ig V and VI and fibronectin type III 1-5. Circular dichroism measurements showed that the secondary structure consists of beta-sheets. Trimeric L1 binds to itself, to monomeric L1, to laminin-1, and to alpha5beta1 integrin in a concentration-dependent manner. In contrast, binding of monomeric L1 could only be saturated with itself but not with laminin-1 and with alpha5beta1 integrin. Promotion of neurite outgrowth from PC12 cells cultured on adsorbed trimeric L1 was increased by 100%, whereas on monomeric L1 the increase was only 50% over the control value. Promotion of neurite outgrowth from PC12 cells was specifically inhibited in a concentration-dependent manner by a polyclonal antibody against L1. These findings show that clustering of only three extracellular domains increases considerably L1's binding affinity to different ligands and enhances neurite outgrowth, suggesting that adhesive functions of L1 on the cell surface depend on cluster formation.

Homophilic adhesion by cadherins.

Cadherins mediate cell-cell adhesion through homophilic interactions. High-resolution structures have greatly enhanced our understanding of this phenomenon over the past few years. Nonetheless, some of the original concepts about cadherin interactions need revision, with the new structural and additional mutagenesis data currently available. Furthermore, in vivo studies on cadherins have provided supplementary information.

A new crystal structure, Ca2+ dependence and mutational analysis reveal molecular details of E-cadherin homoassociation.

Electron microscopy of ECADCOMP, a recombinant E-cadherin ectodomain pentamerized by the assembly domain of cartilage oligomeric matrix protein, has been used to analyze the role of cis-dimerization and trans-interaction in the homophilic association of this cell adhesion molecule. The Ca2+ dependency of both interactions was investigated. Low Ca2+ concentrations (50 microM) stabilized the rod-like structure of E-cadherin. At medium Ca2+ concentration (500 microM), two adjacent ectodomains in a pentamer formed cis-dimers. At high Ca2+ concentration (>1 mM), two cis-dimers from different pentamers formed a trans-interaction. The X-ray structure of an N-terminal domain pair of E-cadherin revealed two molecules per asymmetric unit in an intertwisted X-shaped arrangement with closest contacts in the Ca2+-binding region between domains 1 and 2. Contrary to previous data, Trp2 was docked in the hydrophobic cavity of its own molecule, and was therefore not involved in cis-dimerization of two molecules. This was supported further by W2A and A80I (a residue involved in the hydrophobic cavity surrounding Trp2) mutations in ECADCOMP which both led to abrogation of the trans- but not the cis-interaction. Structural and biochemical data suggest a link between Ca2+ binding in the millimolar range and Trp2 docking, both events being essential for the trans-association.

All-trans retinol, vitamin D and other hydrophobic compounds bind in the axial pore of the five-stranded coiled-coil domain of cartilage oligomeric matrix protein.

The potential storage and delivery function of cartilage oligomeric matrix protein (COMP) for cell signaling molecules was explored by binding hydrophobic compounds to the recombinant five-stranded coiled-coil domain of COMP. Complex formation with benzene, cyclohexane, vitamin D3 and elaidic acid was demonstrated through increases in denaturation temperatures of 2-10 degreesC. For all-trans retinol and all-trans retinoic acid, an equilibrium dissociation constant KD = 0.6 microM was evaluated by fluorescence titration. Binding of benzene and all-trans retinol into the hydrophobic axial pore of the COMP coiled-coil domain was proven by the X-ray crystal structures of the corresponding complexes at 0.25 and 0.27 nm resolution, respectively. Benzene binds with its plane perpendicular to the pore axis. The binding site is between the two internal rings formed by Leu37 and Thr40 pointing into the pore of the COMP coiled-coil domain. The retinol beta-ionone ring is positioned in a hydrophobic environment near Thr40, and the 1.1 nm long isoprene tail follows a completely hydrophobic region of the pore. Its terminal hydroxyl group complexes with a ring of the five side chains of Gln54. A mutant in which Gln54 is replaced by Ile binds all-trans retinol with affinity similar to the wild-type, demonstrating that hydrophobic interactions are predominant.

Structure-based redesign of the catalytic/metal binding site of Cfr10I restriction endonuclease reveals importance of spatial rather than sequence conservation of active centre residues.

According to the crystal structure of Cfr10I restriction endonuclease the acidic residues D134, E71 and E204 are clustered together and presumably chelate metal ion(s) at the active site. Indeed, investigation of the DNA cleavage properties of substitutional mutants of Cfr10I D134A, E71Q, E71A and E204Q reveals that D134, E71 and E204 residues are essential for cleavage activity, supporting their active site function. Structural comparison indicates that the D134 residue of Cfr10I spatially overlaps with aspartate residues D91 and D74, from the invariant active site motifs 90PDX19EAK and 73PDX15DIK of EcoRI and EcoRV, respectively. However, structural studies in conjunction with mutational analyses suggest that the sequence motif 133PDX55KX13E corresponds to the active site of Cfr10I, but differs from canonical active site motifs of EcoRI and EcoRV. According to the crystal structure of Cfr10I the serine S188 residue from the 188SVK sequence motif is a spatial equivalent of the acidic residue from the (E/D)XK-part of the active site motif, which is conserved between EcoRI and EcoRV. Site-directed mutagenesis experiments of Cfr10I, however, revealed that the S188 was not so important for catalysis while the E204 residue located 2.8 A away indeed was essential for cleavage, suggesting that the glutamate E204 rather than the S188 residue contributes to the metal binding site in Cfr10I. In addition, model-building studies suggest that mutual interchange of the E204 and S188 residues should lead only to minor positional differences of the carboxylate residues of glutamate side-chains. The double mutant S188E/E204S was therefore prepared by site-directed mutagenesis where the active site motif 133PDX55KX13E of Cfr10I was changed to a canonical motif 133PDX53EVK, which is similar to that of EcoRI and EcoRV. Interestingly, the double mutant S188E/E204S of Cfr10I with redesigned active site structure, exhibited 10% of Wt cleavage activity in a gamma DNA cleavage assay. Thus, structure guided redesign of the catalytic/metal binding site of Cfr10I, provides novel experimental evidence to suggest that spatial rather than sequence conservation plays the dominant role in the formation of restriction enzyme active sites.

Sequence analysis suggests the presence of an IG-like domain in the N-terminal region of alpha-dystroglycan which was crystallized after mutation of a protease susceptible site (Arg168-->His).

Recently, we demonstrated that the N-terminal region of mouse alpha-dystroglycan represents an autonomously folding globular domain, organized into at least two subdomains (Brancaccio et al., Eur. J. Biochem. 246, 166-172, 1997). We have now found a similarity between a part of the alpha-dystroglycan N-terminal sequence (approximately from position 80 to 180) and several protein sequences belonging to the immunoglobulin kappa family. Moreover, we have recombinantly expressed and purified a 31 kDa protein fragment which matches the entire alpha-dystroglycan N-terminal globular domain. To prevent the action of bacterial endogenous proteases and/or thrombin, which cleaves the protein into two fragments at an Arg-Ala trypsin-sensitive site in positions 168-169, we have introduced a single mutation (Arg168-->His), thus making the whole domain more stable and suitable for crystallization. Crystals of this mutant protein were obtained by vapor diffusion using the hanging drop technique, and they diffract to 0.28 nm Bragg spacing.

Structure of dihydrodipicolinate synthase of Nicotiana sylvestris reveals novel quaternary structure.

DHDPS is the first enzyme unique to the lysine biosynthetic pathway in plants and bacteria and catalyses the formation of (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. It is feedback-regulated in plants by L-lysine. The crystal structure of Nicotiana sylvestris DHDPS with and without inhibitory lysine bound to the enzyme has been solved to a resolution of 2.8 A. The molecule is a homotetramer composed of a dimer of dimers. Comparison with the structure of Escherichia coli DHDPS showed a novel quaternary structure by a profound rearrangement of the dimers forming the tetramer. The crystal structure of the enzyme in the presence of L-lysine revealed substantial changes. These changes together with the novel quaternary structure provide a structural basis for the strong inhibition of plant DHDPS enzymes by L-lysine.

[Results of a multi-year study of persons with transplanted kidneys]. / Rezultati visegodisnjeg ispitivanja osoba s transplantisanim bubregom.

The authors review their results of five year follow up of renal transplantation. They summarised the results of treatment with three immunosuppressive regimens. There is an analysis of rejection episodes, recurrent glomerulonephritis, infections, surgical complications, a median graft as well as patient survival and other patterns of complications of renal transplantation.

[Urinary infections in patients with transplanted kidneys]. / Urinarne infekcije kod bolesnika s transplantisanim bubregom.

The aim of this study was to evaluate urinary infection frequency in kidney transplanted patients. Infection frequency was assessed in 116 patients (81 males and 35 females; mean age 36.2 years) after renal transplantation. During four year follow-up 41% patients suffered one or more episodes of infection. Bacterial infection caused by Escherichia coli were most frequent. The control group consisted of healthy medical staff and the investigations showed significantly over frequency of infection.

[Clinical and morphologic features in patients with acute nephritis syndrome]. / Klinicke i morfoloske odlike bolesnika s akutnim nefritisnim sindromom.

Acute glomerulonephritis is a distinct clinical entity, more frequently found in younger age. We report 69 patients with AcGN (25 female and 44 male) mean age 26 years (range 15-58). The disease is clinically characterized with hypertension (57%), edema (59%) and oliguria (35%). Urine analysis showed microhaematuria/proteinuria (36%) and micro/macrohaematuria alone in 89%, while azothaemia was observed in 16% pts, and decreased serum complement levels in one third of patients, more often decrease of C3 (33%) than C4 (15%). Initial infection of the upper respiratory tract was seen in 65%, pneumonia in 8%. In 25% of pts. there were no data of previous infection. Cultures of pharyngeal smear revealed. Streptococcus only in 2 pts. Elevated AST titer was found in 32% pts. Eleven kidney biopsies were made, and histological examination showed 2 normal findings, 6 mesangioproliferative GH, 2 endocapillary GN and 1 membranoproliferative GN. Follow ups have showed urinary abnormalities in 25% of pts., without developing renal failure.

[Methylprednisolone pulse therapy in the early phases of lupus nephritis]. / Dejstvo pulsne terapije metilprednozolonom kod ranih faza lupusnog nefritisa.

Renal involvement i.e. lupus nephritis (LN) in systemic lupus erythematosus (SLE) mainly determines course and outcome of the disease. Recognition of early manifestations of LN makes adequate therapy possible, with very good therapeutic results. We report 7 patients from a group of 150 SLE patients under our permanent control, 4 female and 3 male, mean age 21 years. All of them had signs of LN: proteinuria 7/7 haematuria 4/7 without azotaemia. Renal biopsy was performed in 6 pts, and histological finding was: class II 1 pt, class IV 3 pts and class V in 2 pts. In 4 pts tubulointerstitial changes were noted, while all showed immunofluorescent deposits of immunoglobulins and complement. Methylprednisolone "pulse" therapy (1000 mg, i.v., 3 days) followed by tapering of the steroid dose was given. Reduction of proteinuria and disappearance of haematuria were observed in all patients. During follow up, kidney function remained normal.