Ursodeoxycholic and chenodeoxycholic bile acids attenuate systemic and liver inflammation induced by lipopolysaccharide in rats.

Bacterial lipopolysaccharide (LPS) induces general inflammation, by activating pathways involving cytokine production, blood coagulation, complement system activation, and acute phase protein release. The key cellular players are leukocytes and endothelial cells, that lead to tissue injury and organ failure. The aim of this study was to explore the anti-inflammatory, antioxidant, and cytoprotective properties of two bile acids, ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) in LPS-induced endotoxemia in rats. The experiment involved six distinct groups of Wistar rats, each subjected to different pretreatment conditions: control and LPS groups were pretreated with propylene glycol, as a bile acid solvent, while the other groups were pretreated with UDCA or CDCA for 10 days followed by an LPS injection on day 10. The results showed that both UDCA and CDCA reduced the production of pro-inflammatory cytokines: TNF-α, GM-CSF, IL-2, IFNγ, IL-6, and IL-1ß and expression of nuclear factor-κB (NF-κB) induced by LPS. In addition, pretreatment with these bile acids showed a positive impact on lipid profiles, a decrease in ICAM levels, an increase in antioxidant activity (SOD, |CAT, GSH), and a decrease in prooxidant markers (H2O2 and O2-). Furthermore, both bile acids alleviated LPS-induced liver injury. While UDCA and CDCA pretreatment attenuated homocysteine levels in LPS-treated rats, only UDCA pretreatment showed reductions in other serum biochemical markers, including creatine kinase, lactate dehydrogenase, and high-sensitivity troponin I. It can be concluded that both, UDCA and CDCA, although exerted slightly different effects, can prevent the inflammatory responses induced by LPS, improve oxidative stress status, and attenuate LPS-induced liver injury.

Untreated PKU patients without intellectual disability: SHANK gene family as a candidate modifier.

Phenylketonuria (PKU) is an inborn error of metabolism caused by variants in the phenylalanine hydroxylase (PAH) gene and it is characterized by excessively high levels of phenylalanine in body fluids. PKU is a paradigm for a genetic disease that can be treated and majority of developed countries have a population-based newborn screening. Thus, the combination of early diagnosis and immediate initiation of treatment has resulted in normal intelligence for treated PKU patients. Although PKU is a monogenic disease, decades of research and clinical practice have shown that the correlation between the genotype and corresponding phenotype is not simple at all. Attempts have been made to discover modifier genes for PKU cognitive phenotype but without any success so far. We conducted whole genome sequencing of 4 subjects from unrelated non-consanguineous families who presented with pathogenic mutations in the PAH gene, high blood phenylalanine concentrations and near-normal cognitive development despite no treatment. We used cross sample analysis to select genes common for more than one patient. Thus, the SHANK gene family emerged as the only relevant gene family with variants detected in 3 of 4 analyzed patients. We detected two novel variants, p.Pro1591Ala in SHANK1 and p.Asp18Asn in SHANK2, as well as SHANK2:p.Gly46Ser, SHANK2:p.Pro1388_Phe1389insLeuPro and SHANK3:p.Pro1716Thr variants that were previously described. Computational analysis indicated that the identified variants do not abolish the function of SHANK proteins. However, changes in posttranslational modifications of SHANK proteins could influence functioning of the glutamatergic synapses, cytoskeleton regulation and contribute to maintaining optimal synaptic density and number of dendritic spines. Our findings are linking SHANK gene family and brain plasticity in PKU for the first time. We hypothesize that variant SHANK proteins maintain optimal synaptic density and number of dendritic spines under high concentrations of phenylalanine and could have protective modifying effect on cognitive development of PKU patients.

UGT1A1 (TA)n Promoter Genotype: Diagnostic and Population Pharmacogenetic Marker in Serbia.

The UGT1A1 enzyme is involved in the metabolism of bilirubin and numerous medications. Unconjugated hyperbilirubinemia, commonly presented as Gilbert syndrome (GS), is a result of decreased activity of the UGT1A1 enzyme, variable number of TA repeats in the promoter of the UGT1A1 gene affects enzyme activity. Seven and eight TA repeats cause a decrease of UGT1A1 activity and risk GS alleles, while six TA repeats contribute to normal UGT1A1 activity and non-risk GS allele. Also, the UGT1A1 (TA)n promoter genotype is recognized as a clinically relevant pharmacogenetic marker. The aim of this study was to assess diagnostic value of UGT1A1 (TA)n promoter genotyping in pediatric GS patients. Correlation of the UGT1A1 (TA)n genotypes and level of unconjugated bilirubin at diagnosis and after hypocaloric and phenobarbitone tests in these patients was analyzed. Another aim of the study was to assess pharmacogenetic potential of UGT1A1 (TA)n variants in Serbia. Fifty-one pediatric GS patients and 100 healthy individuals were genotyped using different methodologies, polymerase chain reaction (PCR) followed by acrylamide electrophoresis, fragment length analysis and/or DNA sequencing. Concordance of the UGT1A1 (TA)n promoter risk GS genotypes with GS was found in 80.0% of patients. Therefore, UGT1A1 (TA)n promoter genotyping is not a reliable genetic test for GS, but it is useful for differential diagnosis of diseases associated with hyperbilirubinemia. Level of bilirubin in pediatric GS patients at diagnosis was UGT1A1 (TA)n promoter genotype-dependent. We found that the frequency of pharmacogenetic relevant UGT1A1 (TA)n promoter genotypes was 63.0%, pointing out that UGT1A1 (TA)n promoter genotyping could be recommended for preemptive pharmacogenetic testing in Serbia.

Genetic characterization of GSD I in Serbian population revealed unexpectedly high incidence of GSD Ib and 3 novel SLC37A4 variants.

Glycogen storage disease (GSD) type I is inborn metabolic disease characterized by accumulation of glycogen in multiple organs. We analyzed 38 patients with clinical suspicion of GSD I using Sanger and next-generation sequencing (NGS). We identified 28 GSD Ib and 5 GSD Ia patients. In 5 patients, GSD III, VI, IX, cholesteryl-ester storage disease and Shwachman-Diamond syndrome diagnoses were set using NGS. Incidences for GSD Ia and GSD Ib were estimated at 1:172 746 and 1:60 461 live-births, respectively. Two variants were identified in G6PC gene: c.247C>T (p.Arg83Cys) and c.518T>C (p.Leu173Pro). In SLC37A4 gene, 6 variants were detected. Three previously reported variants c.81T>A (p.Asn27Lys), c.162C>A (p.Ser54Arg) and c.1042_1043delCT (p.Leu348Valfs*53) accounted for 87% of all analyzed alleles. Computational, transcription studies and/or clinical presentation in patients confirmed pathogenic effect of 3 novel variants: c.248G>A (p.Gly83Glu), c.404G>A (p.Gly135Asp) and c.785G>A (p.Ser263Glyfs*33 or p.Gly262Asp). In the cohort, hepatomegaly, hypoglycemia and failure to thrive were the most frequent presenting signs of GSD Ia, while hepatomegaly and recurrent bacterial infections were clinical hallmarks of GSD Ib. All GSD Ib patients developed neutropenia while 20.6% developed inflammatory bowel disease. Our study revealed the highest worldwide incidence of GSD Ib. Furthermore, description of 3 novel variants will facilitate medical genetic practice.

Prospective study on diagnostic and prognostic significance of postoperative FDG PET/CT in recurrent colorectal carcinoma patients: comparison with MRI and tumor markers.

Current guidelines for follow-up after resection of colorectal cancer (CRC) recommend regular measurements of carcinoembryogenic antigen (CEA) and imaging tests. Multidetector computed tomography (MDCT) and magnetic resonance imaging (MRI) are currently primary imaging modalities, while the role of fluorine-18-fluoro-deoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT), which is recommended in patients with negative MDCT and increased CEA, is still uncertain. Our aim was to compare diagnostic performance and prognostic significance of 18F-FDG PET/CT with MRI and tumor markers CEA and carbohydrate antigen 19-9 (CA 19-9) in detection of recurrent CRC. This prospective study included 35 patients with resected CRC, referred to 18F-FDG PET/CT examination for suspected recurrence. During median follow-up of 24.4±1.5 months 18F-FDG PET/CT and MRI results and tumor marker levels were compared with findings of histopathological examination or with results of clinical and imaging follow-up. Management plan before the 18F-FDG PET/CT scan was considered and compared to the final treatment decision. The sensitivity, specificity, positive and negative predictive value and accuracy of 18F-FDG PET/CT and MRI in detection of recurrent colorectal cancer in patient-based analysis were 92.6%, 75%, 92.6%, 75% and 88.6%, and 65.4%, 66.7%, 85%, 40% and 65.7%, respectively. In lesion-based analysis the sensitivity of 18F-FDG PET/CT and MRI was 83.1% and 68.2%, respectively. The overall accuracy of CEA and CA 19-9 in recurrence detection was 48.6% and 54.3%, respectively. PET/CT induced therapy alterations in 13/35 (37.1%) patients. Progression was observed in 16/35 patients during follow-up, with significantly lower risk of progression in patients with treatment changes based on PET findings (Multivariate Cox regression; p=0.017). In addition, elevated CA 19-9 levels in time of PET scan and male gender carried significantly higher risk of progression (p=0.007 and p=0.016, respectively). Kaplan-Meier Log rank test showed significantly longer progression-free survival time in patients who had treatment plan changed based on PET/CT (p=0.046). We can conclude that 18F-FDG PET/CT showed better sensitivity and accuracy compared to MRI in detection of recurrent colorectal cancer, with much better sensitivity compared to CEA and CA 19-9. Patients with treatment changes based on 18F-FDG PET/CT had significantly better prognosis and longer progression-free survival, while elevated values of CA 19-9 and male gender were associated with worse prognosis.

Molecular and phenotypic characteristics of seven novel mutations causing branched-chain organic acidurias.

Specific mitochondrial enzymatic deficiencies in the catabolism of branched-chain amino acids cause methylmalonic aciduria (MMA), propionic acidemia (PA) and maple syrup urine disease (MSUD). Disease-causing mutations were identified in nine unrelated branched-chain organic acidurias (BCOA) patients. We detected eight previously described mutations: p.Asn219Tyr, p.Arg369His p.Val553Glyfs*17 in MUT, p.Thr198Serfs*6 in MMAA, p.Ile144_Leu181del in PCCB, p.Gly288Valfs*11, p.Tyr438Asn in BCKDHA and p.Ala137Val in BCKDHB gene. Interestingly, we identified seven novel genetic variants: p.Leu549Pro, p.Glu564*, p.Leu641Pro in MUT, p.Tyr206Cys in PCCB, p.His194Arg, p.Val298Met in BCKDHA and p.Glu286_Met290del in BCKDHB gene. In silico and/or eukaryotic expression studies confirmed pathogenic effect of all novel genetic variants. Aberrant enzymes p.Leu549Pro MUT, p.Leu641Pro MUT and p.Tyr206Cys PCCB did not show residual activity in activity assays. In addition, activity of MUT enzymes was not rescued in the presence of vitamin B12 precursor in vitro which was in accordance with non-responsiveness or partial responsiveness of patients to vitamin B12 therapy. Our study brings the first molecular genetic data and detailed phenotypic characteristics for MMA, PA and MSUD patients for Serbia and the whole South-Eastern European region. Therefore, our study contributes to the better understanding of molecular landscape of BCOA in Europe and to general knowledge on genotype-phenotype correlation for these rare diseases.

Metabolic activity of breast cancer metastatic lesions on positron emission tomography/computed tomography: comparison with histological and biological characteristics of primary tumor.

Higher intensity of FDG uptake on PET/CT in primary tumor is seen in patients with IDC compared to ILC, also in high grade tumours, tumours with negative ER and higher Ki67 values, while data are inconsistent in case of relation between primary tumor's PgR and HER2 expression with its metabolic activity levels. On account of the lack of studies that include research of breast cancer metastatic lesion metabolism level and its relation to tumor histology and biology, our goal was to investigate the association of metastatic lesions' glucose metabolism level on PET/CT with different histological and biological characteristics of primary tumor. In a total number of N=100 patients, highest SUVmax values for each patient were used in testing difference between metastatic metabolic activity in patients with different tumor histology, grade, ER, PgR and HER2 status, subtype, as well in testing relation of Ki67 index to metastasis' metabolism level. In testing difference between histological types of breast cancer, SUVmax values were also compared separately for each specific anatomical site (regional and distant lymph nodes, bones and liver). No difference was found regarding metastatic SUVmax values in patients with primary IDC (n=55, median SUVmax 9.70) and ILC (n=34, median SUVmax 7.20) independently of anatomic site, and for each of analysed sites separately. No difference was found as well between SUVmax detected in metastasis in patients with different grade (grade II: n=58, median SUVmax 7.70; grade III: n=12, median SUVmax 10.20), ER (59 positive, median SUVmax 8.50; 22 negative, median SUVmax 8.05), PgR (55 positive, median SUVmax 8.50; 23 negative, median SUVmax 7.80), and HER2 (14 positive, median SUVmax 6.84; 51 negative, median SUVmax 8.63) expression in primary tumor, and between patients with different tumor subtype. Ki67 was also not associated with tumor metastatic SUVmax values (n=11, rs = -0.21, p=0.53). We conclude that there is no association of primary breast cancer histological type, grade, ER, PgR, HER2 and Ki67 expression with metabolic activity in metastasis detected on PET/CT.

Molecular genetic study of congenital adrenal hyperplasia in Serbia: novel p.Leu129Pro and p.Ser165Pro CYP21A2 gene mutations.

PURPOSE: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease characterized by impaired adrenal steroidogenesis and most often caused by CYP21A2 gene mutations. For the first time, we reported complete spectrum and frequency of CYP21A2 gene mutations in 61 unrelated patients with classical and non-classical CAH from Serbia. METHODS: Direct DNA sequencing of whole CYP21A2 gene and polymerase chain reaction with sequence-specific primers for detection of CYP21A1P/CYP21A2 chimeras were combined. RESULTS: We identified 18 different pathogenic alleles-two of them novel. Mutation detection rate was highest in patients with salt-wasting form of CAH (94.7%). The most prevalent mutation was intron 2 splice site mutation, c.290-13A/C>G (18.5%). Other mutation frequencies were: CYP21A1P/CYP21A2 chimeras (13%), p.P30L (13%), p.R356W (11.1%), p.G110fs (7.4%), p.Q318X (4.6%), p.V281L (4.6%), p.I172N (2.8%), p.L307fs (2.8%), p.P453S (1.9%), etc. Mainly, frequencies were similar to those in Slavic populations and bordering countries. However, we found 6.5% of alleles with multiple mutations, frequently including p.P453S. Effects of novel mutations, c.386T>C (p.Leu129Pro) and c.493T>C (p.Ser165Pro), were characterized in silico as deleterious. The effect of well-known mutations on Serbian patients' phenotype was as expected. CONCLUSIONS: The first comprehensive molecular genetic study of Serbian CAH patients revealed two novel CYP21A2 mutations. This study will enable genetic counseling in our population and contribute to better understanding of molecular landscape of CAH in Europe.

Oestradiol Treatment Counteracts the Effect of Fructose-Rich Diet on Matrix Metalloproteinase 9 Expression and NFκB Activation.

Fructose-rich diet induces metabolic changes similar to those observed in metabolic syndrome. Among other matrix metalloproteinases, MMP-9 has an important role in adverse cardiac remodelling and might have a role in the development of cardiovascular disorders associated with metabolic syndrome. The changes of MMP-9 expression could be mediated via the NFκB pathway. In this study we investigated the effect of fructose-rich diet on MMP-9 expression in the heart of male and female rats, along with the effect of fructose-rich diet and oestradiol on MMP-9 expression in ovariectomized females. We further assessed the effect of fructose-rich diet and oestradiol on NFκB activation, measured as the level of p65 phosphorylation at Ser 276. The results showed that the diet regime did not affect the heart mass. Higher MMP-9 gene expression was found in cardiac tissue of male rats fed the fructose-rich diet than in females on the same diet regime. In ovariectomized females, fructose-rich diet upregulated MMP-9 protein and mRNA expression in the heart, as well as phosphorylation of the p65 subunit of NFκB at Ser 276. Oestradiol replacement therapy reverted these changes in the heart of ovariectomized females. This study has shown that oestradiol could revert the early molecular changes in MMP-9 expression induced by fructose-rich diet that occurred before cardiac hypertrophy development by decreasing phosphorylation of the NFκB p65 subunit at Ser 276.

Expression and cellular distribution of glucose transporters and alpha subunits of Na+/K+-ATPase in the heart of fructose-fed female rats: the role of estradiol.

Remarkable parallels are observed between glucose transporters (GLUT) and subunits of Na+/K+-ATPase, which deal with insulin regulation, tissue specificity, intracellular distribution and function of these proteins. To test our hypothesis that similarities also exist in alteration of cardiac GLUTs and alpha subunit isoforms of the pump in insulin resistance, animal model of fructose rich diet was exploited. The role of estradiol in regulation of GLUTs and Na+/K+-ATPase in insulin resistance context was studied as well. Cardiac protein expression, as well as insulin-regulated cellular localization of GLUT4, GLUT1, and α1 and α2 subunits of the pump were analyzed by Western blot. Fructose rich diet increased plasma insulin level and HOMA index, while estradiol treatment reversed both parameters to the control level. We did not observe obvious similarities in the pattern of alterations of GLUT1/α1 subunit of the pump, as well as GLUT4/α2 subunit, related to diet or hormone treatment. Considering alterations in expression and cellular localization of GLUTs and the pump subunits, fructose rich diet jeopardized cardiac glucose transport in some extent, but in contrast, stimulated Na+/K+-ATPase function. Estradiol treatment opposed the fructose diet biochemical action and the effect on cardiac GLUTs, but was inefficient concerning the changes of cardiac Na+/K+-ATPase subunits. Changes of the cardiac molecules can be mediated by alterations in the level of insulin and nonesterified fatty acids, induced by the diet and hormone treatment.

Interaction between insulin and estradiol in regulation of cardiac glucose and free fatty acid transporters.

The estrogen binding to specific extranuclear receptors (ER) activates several intracellular pathways that are activated by insulin as well. Moreover, insulin and estradiol (E2) influence cardiac energy substrates, blood glucose and free fatty acids (FFAs), and both hormones exert cardio-beneficial effects. In view of these facts, we suggest that cross-talk between their signaling pathways might have an important role in regulation of cardiac energy substrate transport. Ovariectomized rats were treated with insulin, estradiol (E2), or their combination 20, 30, or 40 min before analysis of blood glucose and FFA level, as well as cardiac plasma membranes (PM) and low density microsomes (LDM) content of glucose (GLUT4 and GLUT1) and FFA (CD36) transporters. Insulin, given alone, or in combination with E2, decreased plasma glucose level at all time points, but did not influence FFA level, while E2 treatment itself did not change glucose and FFA concentration. Insulin increased PM GLUT4 and GLUT1 content 30 and 40 min after treatment and the increases were partially accompanied by decrease in transporter LDM content. E2 increased PM content and decreased LDM content only of GLUT4 at 30 min. Insulin generally, and E2 at 20 min increased CD36 content in PM fraction. Both hormones decreased CD36 LDM content 20 min after administration. Effect of combined treatment mostly did not differ from single hormone treatment, but occasionally, particularly in distribution of GLUT4, combined treatment emphasized single hormone effect, suggesting that insulin and E2 act synergistically in regulation of energy substrate transporters in cardiac tissue.

Chronic isolation stress predisposes the frontal cortex but not the hippocampus to the potentially detrimental release of cytochrome c from mitochondria and the activation of caspase-3.

Mitochondria are central integrators and transducers of proapoptotic signals for neuronal apoptosis. The tumor suppressor protein p53 can trigger apoptosis independently of its transcriptional activity, through subcellular translocation of cytochrome c and caspase activation. To define better the proapoptotic role of p53 under various stress conditions, we investigated the protein levels of p53 and cytochrome c in mitochondrial and cytosolic fractions, as well as caspase-3 activation and apoptosis, in the prefrontal cortex and hippocampus of male Wistar rats subjected to acute, chronic, or combined stressors. Mitochondrial p53 can suppress the antioxidant enzyme MnSOD, so its activity was also determined. In the prefrontal cortex, but not in hippocampus, increased protein levels of p53 were found in mitochondria, leading to cytochrome c release into cytoplasm, activation of caspase-3, and apoptotic cell death following combined stressors. Decreased mitochondrial MnSOD activity following combined stressors in both brain structures indicated a state of oxidative stress. This suggests that chronic isolation stress compromises mitochondrial MnSOD activity in both the prefrontal cortex and the hippocampus but likely results in mitochondrial-triggered proapoptotic signaling mediated by a transcription-independent p53 mechanism only in the prefrontal cortex. Thus, our data demonstrate a tissue-specific (prefrontal cortex vs. hippocampus) response to applied stressors.

The cortical stab injury induces beading of fibers expressing ecto-nucleoside triphosphate diphosphohydrolase 3.

The ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), an enzyme involved in degradation of extracellular adenosine triphosphate (ATP), is expressed on nerve fibers in different brain regions, including cortex. Here we studied the expression and role of this enzyme after unilateral cortical stab injury in rats. In cortical sections of control rats, NTPDase3 immunoreactivity was associated with two types of fibers: thin processes, occasionally with small mushroom-like protrusions and slightly thicker fibers with more pronounced and more frequent varicosities, whereas immunopositive neuronal perycaria were never observed. Although NTPDase3-positive thin processes and thicker fibers, by general appearance, size and shape, could be dendrites and axons, respectively, they were never immunopositive for microtubule associated protein-2 or neurofilament H subunit. Cortical stab injury induced rapid (within 4 hours) focal varicose swelling that evolved over time to prominent beading of NTPDase3-positive fibers. The NTPDase3-positive fibers in all experimental groups also abundantly express NTPDase1, ecto-5'-nucleotidase and P2X2 receptor channels. Because the brain injury causes a massive ATP release, it is reasonable to conclude that purinoreceptors and ectonucleotidases play an important role in the process of neuritic beading.

Modern treatment of invasive carcinoma of the uterine cervix.

Treatment of invasive cervical carcinoma is determined by the clinical disease stage. Microinvasive carcinoma of the uterine cervix, due to its limited metastatic potential, is usually curable with non-radical treatment. There are no standard approaches to the treatment of stage Ib-IIa carcinoma of the uterine cervix. Both radical surgery and radical radiotherapy are utilized with similar efficacy but with different associated morbidity and complications. Stage Ib1 was commonly treated with radical hysterectomy plus pelvic lymphadenectomy. Laparoscopically-assisted radical vaginal hysterectomy demonstrated similar efficacy and recurrence rates for this disease stage. In cases where fertility is to be preserved, radical vaginal trachelectomy is a valid option for small cervical cancers. Among the papers dealing with bulky cervical disease (stages Ib-IIa) a great deal of disagreement is evident. Some oncologic centres prefer primary surgery with postoperative radiotherapy, with or without chemotherapy, while others prefer primary chemoradiotherapy. Moreover, as a possible alternative, neoadjuvant chemotherapy followed by radical surgery is recommended for stage Ib2 disease. Simultaneous chemoradiation is being introduced as a new standard for advanced cancer, since it has been clearly demonstrated that it can prolong disease-free and overall survival. The treatment of recurrent carcinoma depends on the type of previous treatment, site and extent of recurrent disease, and on the disease-free period and general health of the patient. In conclusion, the decision on the treatment approach for invasive carcinoma of the uterine cervix should be individualized, based on numerous factors, such as disease stage, general health of the patient, cancer-related factors, in order to choose the best approach with minimal complications.

Insulin signaling in the liver and uterus of ovariectomized rats treated with estradiol.

We used rat hepatic and uterine tissues to examine the impact of estradiol (E2) on insulin (INS) signaling. Ovariectomized (OVX) female Wistar rats were treated with E2 (20 microg/kg b.wt., i.p.) and used for the experiment 6h after E2 administration. To highlight E2 effects on tyrosine phosphorylation of INS receptor (IR) and INS receptor substrates (IRSs) and IRSs association with p85 subunit of phosphatidylinositol 3-kinase (PI3-K) in the context of INS signaling, E2-treated OVX rats were also injected with INS (20 IU, i.p.), 30 min before the experiment. Treatment with E2 did not change the levels of plasma INS and glucose (Glu). However, it significantly decreased the free fatty acid (FFA) level and increased uterine weight. Furthermore, the results show that E2 had no effect on the content of hepatic IR protein, but significantly increased IR protein content in the uterus and decreased IR tyrosine phosphorylation in both the liver and uterus. Compared to the control, hepatic IRS-1 and IRS-2 were significantly decreased and increased, respectively, after E2 treatment. Protein content of both molecules, IRS-1 and IRS-2, was increased in uterine tissue after E2 administration. Protein content of the p85 subunit of PI3-K and that of protein kinase B (Akt) were increased in the uterus, with no changes in the liver. The results suggest that E2 treatment induces tissue-specific changes in INS signaling. The consequences of E2 treatment on INS signaling molecules are more apparent in the uterus, but their physiological relevance for INS action is probably greater in the liver.

Molecular and phenotypic characteristics of patients with phenylketonuria in Serbia and Montenegro.

Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism in Caucasians. PKU is caused by mutations in the gene encoding phenylalanine hydroxylase (PAH) enzyme. Here, we report the spectrum and the frequency of mutations in the PAH gene and discuss genotype-phenotype correlation in 34 unrelated patients with PKU from Serbia and Montenegro. Using both polymerase chain reaction-restriction fragment length polymorphism and 'broad-range' denaturing-gradient gel electrophoresis/DNA sequencing analysis, 19 disease-causing mutations were identified, corresponding to mutation detection rate of 97%. The most frequent ones were L48S (21%), R408W (18%), P281L (9%), E390G (7%) and R261Q (6%), accounting for 60% of all mutant alleles. The genotype-phenotype correlation was studied in homozygous and functionally hemizygous patients. We found that the most frequent mutation, L48S, was exclusively associated with the classical (severe) PKU phenotype. The mutation E390G gave rise to mild PKU. For the mutation R261Q, patients had been recorded in two phenotype categories. Considering allele frequencies, PKU in Serbia and Montenegro is heterogeneous, reflecting numerous migrations over the Balkan Peninsula.

Pattern of glial fibrillary acidic protein expression following kainate-induced cerebellar lesion in rats.

In the present study glial fibrillary acidic protein (GFAP) expression was assessed following intravermian injection of kainic acid (KA) or physiological saline to adult rat cerebellum. After 2- to 30-day recovery period, free-floating sections cut with a microtome were obtained and were proccessed for immunocytochemistry against GFAP. Injection of both kainate and physiological saline elicited significant astrogliotic reaction, i.e. in the area around the lesion thick GFAP-positive Bergmann fibers with typical orientation appeared in the molecular and hypertrophied astrocytes abundantly appeared in the granular layer. However, following kainate intoxication lesion was not surrounded by typical demarcation glial scar during 30-day recovery period in contrast to the appearance of usual glial scar in the group injected with physiological saline, as early as 7-day postlesion. Preserved spatial organization of Bergmann fibers and the absence of typical demarcating glial scar after kainate-induced cerebellar lesion suggest distinct pattern of astrogliosis that presents an interesting model system to study the importance of glial scar in the recovery after ischemic brain insults.

Developmental profile of NTPDase activity in synaptic plasma membranes isolated from rat cerebral cortex.

In the present study the developmental profile of ATP-hydrolyzing activity promoted by NTPDase 1, its kinetic properties and the enzyme protein abundance associated with synaptic plasma membrane from rat cerebral cortex were characterized. NTPDase 1 activity increased from birth to day 30; afterwards it decreased and remained unchanged from adulthood (90 days) to senescence (365 days). Kinetic analysis revealed that enzyme exhibited the highest specific activity at day 30 and highest apparent affinity for ATP at day 365; however, V(max)/K(m) values remained unchanged for each age studied. Immunoblot analysis demonstrated that relative abundance of NTPDase 1 is highest at day 15 during ontogeny. The discrepancy between maximum enzyme activity and maximum enzyme protein abundance indicates that NTPDase 1 may have an additional role during development.

Spatio-temporal changes in neurofilament proteins immunoreactivity following kainate-induced cerebellar lesion in rats.

1. Spatio-temporal changes in phosphorylated (pNFP) and nonphosphorylated (npNFP) neurofilament proteins were assessed immunocytochemicaly in adult rat cerebellum, 2-30 days following unilateral injection of kainic acid (KA) or physiological saline (s.c.). 2. Analysis of the staining intensity and pattern demonstrated that injection of both KA and physiological saline elicited significant and long-lasting increase of pNFP and npNFP immunoreactivity, at the ipsilateral, and to lesser extent at the contralateral side of lesion. 3. Kainate intoxication induced abundant expression of pNFP and npNFP in cerebellar white matter, as well as in all layers of perilesioned cortex. Higher pNFP expression was evidenced in the Purkinje cell layer, particularly at cell bodies, initial segments, and proximal dendrites, which normally do not contain pNFP. In addition, synaptophysin immunocytochemistry was used as a marker of synaptogenesis and plasticity. 4. Spatio-temporal pattern of NFP and synaptophysin expression suggests that perilesioned cortex undergoes dynamic changes following brain demage and possess a reparative capacity to abridge the consequences of brain trauma.

Ecto-ATPase and ecto-ATP-diphosphohydrolase are co-localized in rat hippocampal and caudate nucleus synaptic plasma membranes.

Enzymes that hydrolyze extracellular ATP, i.e. ecto-ATPase and ecto-ATP diphosphohydrolase (ATPDase), can be differentiated by ability of the latter to hydrolyze ADP and by slightly different kinetic properties of the two enzymes. Synaptic plasma membrane fractions isolated from rat hippocampus and caudate nucleus exhibit ADP-hydrolyzing activity, as revealed by the enzyme assay, and the presence of ecto-ATPase protein, as revealed by immunological identification on Western blot. These findings indicate that both enzymes are co-expressed in the synaptic membrane compartment of hippocampal and caudate nucleus neurons. Kinetic analysis was performed to determine the relative contribution of each enzyme to the total ATP-hydrolyzing activity, while an inhibition study was carried out in order to exclude the interference of other nonspecific ATPase and phosphatase activities. Based on the kinetic properties, sensitivity to inhibitors and V(ATP)/V(ADP) ratio of about 2, we concluded that a substantial portion of ATP-hydrolyzing activity in both synaptic membrane preparations can be ascribed to the catalytic action of ATPDase. On the other hand, the highest catalytic efficacy when ATP is the substrate and the greater abundance of ecto-ATPase protein in caudate nucleus preparation suggest that the relative contribution of ecto-ATPase to the total ATP-hydrolyzing activity in the caudate nucleus is higher than in the hippocampus.