The effects of functionalization of carbon nanotubes on toxicological parameters in mice.

Carbon nanotubes (CNTs) have emerged as a new class of multifunctional nanoparticles in biomedicine, but their multiple in vivo effects remain unclear. Also, the impact of various functionalization types and duration of exposures are still unidentified. Herein, we report a complete toxicological study to evaluate the effects of single- and multiwalled carbon nanotubes (SWCNTs and MWCNTs) with either amine or carboxylic acid (COOH) surface functional groups. The results showed that significant oxidative stress and the subsequent cell apoptosis could be resulted in both acute and, mainly, in chronic intravenous administrations. Also, male reproductive parameters were altered during these exposures. The amino-functionalized CNTs had more toxic properties compared with the COOH functionalized group, and also, in some groups, the multiwalled nanotubes were more active in eliciting cytotoxicity than the single-walled nanotubes. Interestingly, the SWCNTs-COOH had the least alterations in most of the parameters. Evidently, it is concluded that the toxicity of CNTs in specific organs can be minimized through particular surface functionalizations.

Genetic screening in Iranian patients with retinoblastoma.

PurposeThe most common intraocular tumor in childhood, retinoblastoma, is largely associated with mutations in the RB1 gene. In the most comprehensive RB1 screening in Iran, we evaluated the RB1 mutations in 106 patients with retinoblastoma, including 73 bilateral (heritable) and 33 unilateral (sporadic) cases.Patients and methodsMutations were identified using amplification refractory mutation system (ARMS) PCR and direct sequencing of the 27 coding exons of RB1 and multiplex ligation-dependent probe amplification (MLPA).Results and ConclusionWe found 33 (31%) and 64 (60%) patients with sporadic unilateral and bilateral retinoblastoma, respectively as well as 9 (8.5%) cases with hereditary bilateral retinoblastoma. In total, we identified 52 causative RB1 mutations in 106 patients (global mutation rate of 49%). Of the 52 patients, 48 (92%) had sporadic and familial bilateral and 4 (8%) had sporadic unilateral RB. Therefore, the detection rate of RB1 mutations was 66% (48/73) and 12% (4/33) in bilateral and unilateral cases, respectively. Mutations were classified as nonsense in 31 (60%), missense in 1 (2%), large deletion in 11 (21%), small deletion in the 7 novel (15%) and splice site mutation in 2 (4%) patients with RB. Of 31 nonsense mutations, 23 (74%) occurred in the 11 Arginine codons of the RB1. Seven mutations (13%) were novel, and 45 (87%) had been previously reported. Thirty-three mutations were single-base substitutions leading to 31 nonsense amino acid changes and 2 splice site mutations in introns 12 and 16 of RB1. The altered 3D model structures of the RB1 novel mutant proteins are also predicted in this study.

Imerslund-Grasbeck syndrome in a 5-year-old Iranian boy.

Imerslund-Grasbeck syndrome (IGS) is a rare syndrome characterized by clinical symptoms and signs of Vitamin B12 deficiency and proteinuria. Our patient was a 5-year-old boy with pallor, lack of appetite, and low weight gain. Laboratory studies showed severe macrocytic anemia, normal reticulocyte count, negative direct coombs test, normal osmotic fragility, and autohemolysis test. He has had intermittent proteinuria since 3 years ago despite normal creatinine level and absence of hematuria or hypertension. Finally, based on low level of serum B12 vitamin and normal folate level accompanied by asymptomatic proteinuria, the diagnosis of IGS was made. Furthermore, his sister has had laboratory abnormalities without any symptoms. IGS responded to B12 replacement therapy dramatically but intermittent proteinuria persisted even after appropriate therapy.

Tsga10 expression correlates with sperm profiles in the adult formalin-exposed mice.

Testis-specific gene antigen10 (Tsga10), as a cytoskeletal protein in the sperm tail, impacts the sperm motility. This study investigates the correlation between sperm profile alterations and Tsga10 gene expression in adult mice exposed to formaldehyde (FA) and then treated with antioxidant effect of manganese (Mn2+ ). In this regard, we examined 35 NMRI adult male mice (6-8 weeks age) in 4 groups of control, sham, FA-exposed and FA+Mn2+ . The mice in FA+Mn2+ group were exposed to FA (10 mg kg-1 twice a day) for 2 weeks and treated with daily Mn2+ administration (5 mg kg-1 ) in the second week prior to sacrificing the mice for testis dissection. The right testis was dissected in each group and subjected to RNA extraction and cDNA syntheses for gene expression analysis by real-time PCR. The findings revealed that FA decreased sperm parameters and Tsga10 expression (52.6 ± 24.37%). However, the injected powerful manganese antioxidant improved sperm profile through overexpression of Tsga10 (121.6 ± 27.13%) under FA-induced stressful condition which proves the correlation between sperm profile and Tsga10 expression (P ≤ 0.05). This study also shows that Tsga10 expression protects sperm dysfunction in FA+Mn2+ group and resulting in better preservation of spermatozoa and improvement of male fertility.

In vitro toxicity assay of cisplatin on mouse acute lymphoblastic leukaemia and spermatogonial stem cells.

Testicular cancer is the most common cancer affecting men in reproductive age, and cisplatin is one of the major helpful chemotherapeutic agents for treatment of this cancer. In addition, exposure of testes cancer cells to cisplatin could potentially eliminate tumour cells from germ cells in patients. The aim of this study was to evaluate the effect of cisplatin on viability of mouse acute lymphoblastic leukaemia cell line (EL-4) and neonatal mouse spermatogonial cells in vitro. In this study, the isolated spermatogonial stem cells (SSC) and EL-4 were divided into six groups including control (received medium), sham (received DMSO in medium) and experimental groups which received different doses of cisplatin (0.5, 5, 10 and 15 µg ml(-1) ). Cells viability was evaluated with MTT assay. The identity of the cultured cells was confirmed by the expression of specific markers. Our finding showed that viability of both SSC and EL-4 cells was reduced with the dose of 15 µg/ml when compared to the control group (P ≤ 0.05). Also, the differences between the IC50 in doses 10 and 15 µg/ml at different time were significant (P ≤ 0.05). The number of TUNEL-positive cells was increased, and the BAX and caspase-3 expressions were upregulated in EL4 cells for group that received an effective dose of cisplatin). In conclusion, despite the dramatic effects of cisplatin on both cells, spermatogonial stem cells could form colony in culture.

Solid Pseudo-Papillary Tumor of the Pancreas in a 10 Year Old Girl.

Background Solid pseudo-papillary tumor of the pancreas (SPTP) is a rare disease with a low malignant potential. Though it shows low malignant potential 10% to 15% of the cases show aggressive behavior with metastatic involvement of the liver. The symptoms include abdominal discomfort and abdominal pain. It is very rare in early years of age. This is the case of a 10 year old girl with abdominal pain and her evaluation revealed solid pseudo papillary tumor of pancreas. In family history, her grandmother died because of pancreas cancer. The mass was excised and in her 6-month follow up she didn't have any problems. This case is presented to point out physicians that more attention to pseudo- papillary tumor can bring us significant improvement in the diagnosis of this pathology, though pseudo- papillary tumor is a rare pathologic condition in children.

Correlation between expression of CatSper family and sperm profiles in the adult mouse testis following Iranian Kerack abuse.

Illicit drug use can be an important cause of male infertility. The aim of this study was to investigate the effects of an Iranian illicit drug, Kerack, on sperm parameters, testicular structure and CatSper genes expression of mice. In this study, 25 male mice were divided into five groups consisting of control, sham and three experimental groups. All animal in experimental groups were addicted to Kerack for 7 days. These experimental groups include experimental I which was given Kerack at a dose of 5 mg/kg, experimental II, 35 mg/kg and experimental III, 70 mg/kg, intraperitoneally twice a day for a period of 35 days. Mice were then sacrificed and spermatozoas were removed from cauda epididymis and analyzed for count, motility, morphology (normal/abnormal) and viability. Right testes were removed, weighed and processed for light microscopic studies whereas left testes removed were subjected to total mRNA extraction for using in real-time PCR (RT-PCR). The results were analyzed by performing anova (Tukey's tests) and Pearson correlation coefficient. Sperm parameters and seminiferous epithelium thickness were decreased in experimental groups (dose-dependently) vs. sham and control groups (p < 0.05). RT-PCR results showed that CatSper 2, 3, 4 genes expressions were reduced with 35 and 70 mg/kg injected Kerack when compared with control testes (p ≤ 0.05). However, CatSper1 expression was only reduced with high dose injected Kerack (70 mg/kg) in comparison to control testes (p ≤ 0.05). This study shows the deleterious effects of Kerack used in Iran on testis structure and sperm parameters in general, and particularly sperm morphology in adult mouse. It could down-regulate the expression of CatSper genes, resulting in depression of sperm motility.

Antioxidant effect of manganese on the testis structure and sperm parameters of formalin-treated mice.

Manganese inhibits oxidative stress damage. The aim of this study was to investigate the protective role of manganese on testis structure and sperm parameters in adult mice exposed to formaldehyde (FA). Twenty adult male NMRI mice were selected and randomly divided into four groups: (i) control; (ii) sham; (iii) 'FA'-exposed group; and (iv) 'FA and manganese chloride'-exposed group. The FA-exposed groups received 10 mg kg(-1) FA daily for 14 days, and manganese chloride was just injected intraperitoneally 5 mg kg(-1) on 2nd weeks. Mice were sacrificed, and spermatozoa were collected from the cauda of the right epididymis and analysed for count, motility, morphology and viability. The other testicular tissues were weighed and prepared for histological examination upon removal. Seminiferous tubules, lumen diameters and epithelium thickness were also measured. The findings revealed that FA significantly reduced the testicular weight, sperm count, motility, viability and normal morphology compared with control group (P ≤ 0.05). In addition, seminiferous tubules atrophied and seminiferous epithelial cells disintegrated in the FA group in comparison with the control group (P ≤ 0.05). However, manganese improved the testicular structure and sperm parameters in FA-treated mice testes (P ≤ 0.05). According to the results, manganese may improve and protect mice epididymal sperm parameters and testis structure treated with FA respectively.

NF-kappaB-dependent gene expression of proinflammatory cytokines in T24 cells: possible role in interstitial cystitis.

Our previous report of predominant activation of nuclear transcription factor NF-kappaB in the bladder urothelium of interstitial cystitis (IC) patients suggests a potential role for this nuclear factor in the pathogenesis of the disease. Although NF-kappaB has been implicated in the pathogenesis of several inflammatory diseases, the downstream mechanism(s) by which it can mediate its effects are still fragmentary. In this study, we examined the role of this nuclear factor on the induction of proinflammatory cytokine gene expression in human bladder carcinoma T24 cells and further examined their corresponding protein levels in the urine of IC patients. T24 cells transduced with a dominant-negative super-repressor IkappaB mutant (pAxCAmIkappaB-M) or wild-type adenoviral vectors in the presence or absence of rhTNF-alpha. Transduction efficiency and ability of pAxCAmIkappaB-M to inhibit NF-kappaB activation were monitored by in situ reporter beta-galactosidase and gel mobility shift assays, respectively. Expression profile analysis of proinflammatory cytokines was measured in cells and urine of IC patients using RT-PCR and ELISA, respectively. The activation of NF-kappaB by rhTNF-alpha was associated with 27, eight, ten and sevenfold increases in the TNF-alpha, IL-1beta, IL-6 and IL-8 transcripts, respectively. In contrast, abrogation of the TNF-alpha-induced cytokine gene expression by an adenovirus super-repressor IkappaB mutant vector demonstrate that these effects were NF-kappaB-dependent. Interestingly, the NF-kappaB-induced expression of these transcripts correlates with increased protein levels of NF-kappaB-regulated proinflammatory factors in the urine of IC patients in comparison to controls. That these factors are capable of activating NF-kappaB in urothelial cells suggests a pivotal role for this nuclear transcription factor in the pathophysiology of the disease, possibly by inducing aberrant immune and inflammatory responses within the bladder of IC patients.

Evidence for a folded conformation of methionine- and leucine-enkephalin in a membrane environment.

Transfer of an aqueous-soluble peptide hormone or neurotransmitter such as [Met]- or [Leu]enkephalin (Tyr1-Gly2-Gly3-Phe4-Met5(Leu5)), to the lipid-rich environment of its membrane-embedded receptor protein may convert the peptide into a ("bioactive") conformation required for eliciting biological activity. We have examined by high-resolution nuclear magnetic resonance (NMR) spectroscopy the conformational parameters of free enkephalin in aqueous solution versus those of enkephalin bound to lysophosphatidylcholine micelles using two approaches: 1) exchange rates, line broadening, coupling constants, and chemical shift changes of enkephalin backbone peptide N-H protons were measured for free and membrane-bound peptide in H2O (360 MHz, pH 5.6, 20 degrees C). A selective upfield shift observed for the Met5(Leu5) N-H proton upon lipid binding was interpreted in terms of its incorporation into an intramolecular H-bond. 2) 13C chemical shift changes induced by the shift reagent praseodymium nitrate (Pr(NO3)3) were compared in the presence and absence of lipid micelles. Significant changes occurring in Gly2 carbon atoms in membrane-bound enkephalin suggested the relative proximity of this residue to the Pr3+ atom (bound to the Met5(Leu5) COOH-terminal carboxylate 4 residues away). These combined results, in conjunction with studies on the specific interactions of enkephalin substituents with the micelles (Deber, C. M., and Behnam, B. A., (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 61-65) suggest that enkephalin folds into an intramolecularly H-bonded beta-turn structure (with an H-bond between Gly2 C = O and Met5 NH) in the lipid environment. Such folding could facilitate the positioning of strategic residues in vivo as the hormone diffuses toward its receptor.

Role of membrane lipids in peptide hormone function: binding of enkephalins to micelles.

In the course of their biological function, peptide hormones must be transferred from an aqueous phase to the lipid-rich environment of their membrane-bound receptor proteins. We have investigated the possible influence of phospholipids in this process, using 360-MHz 1H and 90-MHz 13C NMR spectroscopy to examine the association of the opioid peptides [Met]- and [Leu]enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) with phospholipid micelles. Binding of peptides to lipid was monitored in NMR spectra by selective chemical shift movements (e.g., the Phe aromatic ring protons) and residue-specific line broadening (e.g., of Met/Leu carbonyl- and alpha-carbon resonances). Results established that the zwitterionic hormones associate hydrophobically both with a neutral lipid (lysophosphatidylcholine) and (also electrostatically) with a negative lipid (lysophosphatidylglycerol). An association constant of Ka = 3.7 X 10(1) M-1 was calculated for the hydrophobic binding of enkephalin to lysophosphatidylcholine. NMR data suggested that enkephalin binds to the lipid with Met/Leu, Phe, and likely Tyr side-chain substituents associated with nonpolar interior regions of the micelle, whereas the COOH-terminal carboxylate moiety of the peptide is located in the surface of the lipid particle. An "attraction-interaction" model is proposed for hormone-lipid association wherein negative lipids attract the hormone electrostatically, while site-specific hydrophobic contacts facilitate its entry, concentration, and orientation into the lipid phase.

Skin manifestations of mercury poisoning.

Eight patients with skin lesions attributed to methylmercury exposure are reported. The outbreak of poisoning occurred in Iraq as a result of the handling and ingestion of treated grain and home-made bread. The skin manifestations were rather rare and varied in severity and histological appearance.