Site-specific conjugation of fibroblast growth factor 2 (FGF2) based on incorporation of alkyne-reactive unnatural amino acid.

Recent advances in site-specific protein modification include the increasingly popular incorporation of unnatural amino acid(s) using amber codon, a method developed by Schultz and coworkers. In this study, we employ this technique to introduce propargyllysine (PrK) in human fibroblast growth factor 2 (FGF2). Owing to an alkyne moiety in its side chain, PrK is compatible with Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC). We successfully tested CuAAC-mediated conjugation of FGF2 with two compounds - a fluorophore carboxyrhodamine 110 or a cytotoxic drug monomethyl auristatin E (MMAE). In the case of the MMAE conjugate we improved the initial poor conjugation yield to achieve nearly 100% efficiency after extensive optimization. The detergent-based optimization approach may help overcome problems with the CuAAC reaction yield for protein modification with hydrophobic compounds, such as MMAE.

RSK2 regulates endocytosis of FGF receptor 1 by phosphorylation on serine 789.

FGFR1 (fibroblast growth factor receptor 1) regulates many key cellular responses including proliferation, migration and differentiation through activation of signaling pathways. Irregularities in FGFR1 signaling have been implicated in several pathological conditions, including human cancer. In order to discover novel regulators of FGFR1 signaling, we performed yeast two-hybrid screens and identified RSK2 (p90 ribosomal S6 kinase 2) as a potential FGFR1 interaction partner. RSK2 belongs to the family of serine/threonine kinases that are activated through the Ras-MAPK signal transduction pathway. Both in vitro and in vivo experiments confirmed the interaction and we show that phosphorylated RSK2 binds to and phosphorylates serine 789 in the C-terminal tail of FGFR1. Inhibition of RSK2 activity led to prolonged tyrosine transphosphorylation of FGFR1. Furthermore, prevention of FGFR1 phosphorylation by inhibition of RSK2 activity or mutation of serine 789 to alanine reduced FGFR1 endocytosis and ubiquitination explaining mechanistically the prolonged signaling activity. We propose a novel regulatory mechanism whereby activated RSK2 directly interacts with and phosphorylates FGFR1, thereby modulating receptor signaling through regulation of endocytosis.

BCR expression is decreased in meningiomas showing loss of heterozygosity of 22q within a new minimal deletion region.

Neurofibromin 2 (NF2), located on chromosome arm 22q, has been established as a tumor suppressor gene involved in meningioma pathogenesis. In our study, we investigated 149 meningiomas to determine whether there are additional tumor suppressor genes localized on chromosome 22q, apart from NF2, that might be involved in meningioma pathogenesis. The LOH analysis on chromosome 22q identified two regions of deletion: the first one, which is limited to the NF2 gene locus, and the second one, which is outside this location. The new minimal deletion region (MDR) included the following genes: BCR (breakpoint cluster region), RAB36 (a member of RAS oncogene family), GNAZ [guanine nucleotide binding protein (G protein), alpha-z polypeptide], and RTDR1 (rhabdoid tumor deletion region gene 1). The expression levels of all these genes, including NF2, were subsequently analyzed by quantitative real-time polymerase chain reaction. We observed a significantly lowered expression level of NF2 in meningiomas with 22q loss of heterozygosity (LOH) within NF2 region compared to the one in meningiomas with 22q retention of heterozygosity (ROH, P<0.05). Similarly, BCR showed a significantly lowered expression in meningiomas with 22q LOH within the new MDR compared to cases with 22q ROH (P<0.05). Our data, together with the already published information considering BCR function suggest that BCR can be considered as a candidate tumor suppressor gene localized on chromosome 22q which may be involved in meningioma pathogenesis.

Elimination of wild-type P53 mRNA in glioblastomas showing heterozygous mutations of P53.

We screened 50 glioblastomas for P53 mutations. Five glioblastomas showed heterozygous mutations, while three were putatively heterozygous. Six of these eight glioblastomas showed elimination of wild-type P53 mRNA. These results strongly suggest that some sort of mechanism(s) favouring mutated over wild-type P53 mRNA exists in glioblastoma cells with heterozygous mutations of this gene.

[The risk of inhalable wood dust: assessment of workers exposure wood working factories]. / Il rischio da polveri di legno inalabili: valutazione dell'esposizione professionale nelle falegnamerie.

The International Agency for Research on Cancer RC) has classified wood dust as carcinogenic to humans based on demiological and experimental evidence. Exposure to wood dust may use respiratory and dermal symptoms and diseases. The aim of this work was to estimate occupational exposure to inhalable wood dust adopting the formal procedure described by UNI EN 689/97. The exposure of 23 workers in three different working day was measured. In total, 69 personal air samplings were carried out at five wood working factories. Inhalable fraction of airborne dust was collected on 5 microm pore size, 25 mm diameter PVC filters utilizing the IOM samplers. The quantity of the wood dust was determined with gravimetric method. The results show that about 13% of the exposure values exceed the limit of 5 mg/m3 specified by the Italian Law Decree 66/2000 and about 48% of personal exposures are lower then the limit value. Prevention measures, technological solutions and personal protection equipment should be adopted in order to reduce worker's exposure.

Structure-function relationships in serine protease-bovine pancreatic trypsin inhibitor interaction.

We report our progress in understanding the structure-function relationships for the interaction between BPTI and serine proteases. We focused on extensive mutagenesis of four crucial positions from the protease binding loop of BPTI. Selected variants were characterized by determination of association constants, stability parameters and structures of protease-inhibitor complexes.

Molecular abnormalities in pediatric embryonal brain tumors--analysis of loss of heterozygosity on chromosomes 1, 5, 9, 10, 11, 16, 17 and 22.

Embryonal tumors, the most common group of malignant brain tumors in childhood, are heterogeneous and have been associated with a large number of genetic abnormalities. The aim of this study was to comprehensively analyze loss of heterozygosity (LOH) on regions harboring suppressor genes (PTCH2, PTCH1, APC, PTEN, DMBT1, SUFU, AXIN1, hSNF5/INI1) and to study chromosomal regions in which deletions have been described most frequently (1p, 1q, 11p, 16p, 17p). Twenty-nine children (17 male and 12 female), aged from 1 year 13 years were included in this study. There were 24 medulloblastomas (MB) and 5 supratentorial primitive neuroectodermal tumors (sPNET). Tissue samples from 29 primary and 11 recurrent tumors were analyzed according to the LOH standard procedures, which were extended to include fluorescence in situ hybridization for detection of isochromosome 17q (i(17q)) and direct sequencing ofTP53 exon 4. LOH on 17p was found in 15 out of 29 tumors. FISH analysis identified the presence of i(17q) in 16 tumors. Comparison of LOH analysis and the FISH data indicated that alterations of 17p were related to be the introduction of an i(17q) formation. LOH on 10q and 9q was observed in 4 and 2 cases, respectively, and was associated with alterations of chromosome 17. These results indicated a connection between alterations of PTCH/SHH genes and abnormalities of chromosome 17. A deleted region on 22q, covering the hSNF5/INI1 locus, was observed in 3 tumors. Progression of the molecular changes occurred in 1 case of recurrent medulloblastoma. LOH on 10q and 17p was found in both primary and recurrent tumor, while losses on 11p, 16p, and 16q occurred only in the recurrent tumor. No evidence of alteration in TP53 exon 4 was identified.

Molecular heterogeneity of meningioma with INI1 mutation.

BACKGROUND: INI1 (hSNF5) mutations are linked to rhabdoid tumours, but mutations in meningiomas with hot spot mutations in position 377 have also been reported. AIMS: To analyse the INI1 gene in meningioma. METHODS: Exons 1, 4, 5, and 9 of the INI1 gene were analysed by the polymerase chain reaction and direct sequencing in 80 meningiomas. For all cases, western blotting of the INI1 protein was performed. RESULTS: Only one of the 80 samples showed a cytosine insertion in codon 376. This mutation changed the open reading frame in almost the whole exon 9 and resulted in a longer hSNF5 protein. Complex analysis of the above described tumour sample by western blotting, DNA sequencing, and loss of heterozygosity (LOH) analysis showed that this particular meningioma consisted of heterogeneic cellular components. One of these components had a mutated INI1 gene, whereas in the other component INI1 was intact. CONCLUSIONS: INI1 mutation is a rare event in the molecular pathology of meningiomas. It is possible for the INI1 gene to be mutated in only a proportion of meningioma cells.

[Polish version of Temperament and Character Inventory (TCI): the analysis of reliability]. / Polska wersja Inwentarza Temperamentu i Charakteru (TCI): analiza rzetelnosci.

The reliability of every main scale of the Polish version of Temperament and Character Inventory (TCI) was proven on the group of 144 students.

Polymorphisms in the dopamine, serotonin, and norepinephrine transporter genes and their relationship to temperamental dimensions measured by the Temperament and Character Inventory in healthy volunteers.

There is evidence for an association between polymorphisms of monoamine transporter genes and temperamental personality traits. Recent findings have shown that interaction of allelic variants of the different genes may contribute to the personality factors. We studied the association between temperamental personality dimensions measured with the Temperament and Character Inventory (TCI) and polymorphisms of the dopamine (DAT), norepinephrine (NET) and serotonin (5-HTT) transporter genes in 127 healthy Polish volunteers. There were no significant differences between means of TCI temperamental dimensions (novelty seeking, reward dependence, persistence and harm avoidance) and the transporter genes compared by ANOVA. There were some significant associations between genotypes and TCI subdimensions. Individuals carrying the A9/A9 DAT genotype have lower RD4 scores (dependence vs. independence) than A10/A10 individuals (3.0 +/- 1.4 vs. 3.5 +/- 1.3); p = 0.01. Examining 5-HTT gene promoter polymorphism, heterozygous individuals (l/s) and individuals with 44-bp deletion (s/s) scored significantly lower in the HA1 subdimension (anticipatory worry and pessimism vs. uninhibited optimism; 4.3 +/- 2.3 vs. 5.5 +/- 2.6) in comparison with individuals without deletion (l/l); p = 0.021. The NET transporter gene polymorphism showed no significant association with any of the temperamental TCI subdimensions.

Accumulation of cadmium in and its effect on bank vole tissues after chronic exposure.

Cadmium is one of many metals that are not physiologically or biochemically essential to organisms. This element is extremely dangerous as it is easily absorbed and remains in tissues for a long time. Long exposure to high doses of cadmium may cause biochemical and functional changes in some critical organs. In this study, wheat grains contaminated with cadmium chloride were used to test the influence of cadmium on male bank voles (Clethrionomys glareolus). Doses used in the experiment were environmentally realistic: 0.25 microg g-1 (control), 15 microg g-1, and 40 microg g-1 cadmium (dry weight). The animals were given cadmium-contaminated food and clean water ad libitum for 3 and 6 months. After these exposures, the animals were killed and the kidneys, liver, and testes from each vole were collected for analyses. The concentrations of Cd, Cu, Zn, and Fe in the tissues were determined with an atomic absorption spectrophotometer. The formalin-fixed testes, kidneys, and part of the liver were embedded in paraffin and then stained with hematoxylin and eosin. Cadmium accumulation in the tissues was directly proportional to dose. The highest cadmium concentrations were found in the kidneys of animals fed the highest dose of cadmium. Histological examination of the tissues revealed some pathological changes in the structure of kidneys, liver, and testes.

[The study of the relationship between cholesterol and lipid concentration and suicidal behavior in patients with schizophrenia affective illness]. / Badania zaleznosci miedzy stezeniem cholesterolu i lipidów a zachowaniami samobójczymi u chorych na schizofrenie i choroby afektywne.

In 143 patients (63 male, 80 female), admitted to the Department of Adult Psychiatry, University of Medical Sciences in Poznan, throughout the period from 1 October 95-31 March 96, with the diagnosis of schizophrenia (46 patients), endogenous depression (79 patients) or mania (18 patients), serum concentration of total cholesterol, LDL and HDL cholesterol, triglycerides and total lipids was estimated during the first days of admission. The occurrence of suicidal behaviors (thoughts, tendencies or acts) in these patients during the period of 3 months preceding the admission was also determined. The occurrence of suicidal behavior was found in 74 patients (30 male, 44 female). The persons revealing suicidal behaviors had significantly lower concentrations of total cholesterol, LDL cholesterol, triglycerides and total lipids compared with patients without such behaviors. This relationship was observed in all diagnostic groups as well as in both younger (below 32 years) and older (over 40 years) groups of patients. The results obtained confirm previous reports on the association between low cholesterol concentration and an increased risk of suicidal behavior in patients with psychiatric disorders. Authors discuss possible mechanisms and also clinical implications of this finding.

[The typological analysis of males with alcohol dependence disorder]. / Analiza typologiczna mezczyzn z zespolem uzaleznienia od alkoholu.

The aim of our study was the typological analysis of male alcoholics that was conducted on the basis of the K-means cluster analysis. 296 hospitalized men were examined after a duration of drinking. The results of the statistical analysis let us isolate two and then three types of alcoholics. When dividing the examined into two clusters it appeared that those who belonged to the 1st type had shorter history of addiction, became dependent later and in their case coexistence of psychiatric disorders, somatic diseases and the genetic loading for alcoholism were found less frequently than those from the 2nd type. Thus, early age of dependence onset was connected with the family alcoholism and worse clinical course-i.e.-co-occurrence of psychiatric disorders and somatic diseases. When dividing the patients into three clusters the following variables that differentiate the examined from three types of alcoholics were found: (a)-1st type: late age of dependence onset, rare load of family alcoholism, psychiatric disorders, somatic diseases; (b)-2nd and 3rd type: early age of dependence onset, the load of family-alcoholism, co-occurrence of psychiatric disorders and somatic diseases; (c)-3rd type: patients from the 3rd type were dependent longer than those from the 2nd type. Longer duration of the disease was connected with more frequent occurrence of psychiatric disorders and somatic diseases.

Population genetic data determined for five different single locus minisatellite probes.

We report on the population genetic data (frequencies of restriction fragments, heterozygosity rates, and mutation rates) obtained by analysis of approximately 1100 Hinfl-digested DNAs from West Germans. Probe G3 detects a common 1.7 kb DNA fragment showing a population frequency of about 13%. All the other fragments detected with probes MS1, MS31, MS43, G3 and YNH24 show frequencies of less than 8%. These data suggest that single locus DNA probes can provide valuable information for parentage evaluation and individualization.

BamHI polymorphism of locus D2S44 in a West German population as revealed by VNTR probe YNH24.

BamHI polymorphism at the VNTR locus D2S44 was investigated, concentrating on band frequencies, mutation rate and confirmation of Mendelian inheritance. In this series 39 restriction fragments showing frequencies less than 10% could clearly be distinguished. No mutations could be observed and the Mendelian character of inheritance is beyond reasonable doubt.

Bank voles as monitors of environmental contamination by heavy metals. A remote wilderness area in Poland imperiled.

Concentrations of heavy metals (Cd, Pb, Cu, Zn, Fe) were determined in the tissues of bank voles collected in polluted forest sites in southern Poland and in Bialowieza National Park. The highest cadmium concentrations were found in livers (0.9-12.8 microg g(-1)) and kidneys (3.2-29.6 microg g(-1)), whereas the lowest were in bones (0.2-0.9 microg g(-1)). The lead concentrations found in kidneys and bones ranged from 9.5 to 40.0 microg g(-1). The ranges of zinc, iron and copper concentrations in the tissues of animals were: 137-343 microg g(-1), 206-1017 microg(-1) and 12.0-73.8 microg(-1), respectively. Some of the differences between the levels found in various sites were statistically significant.