The optimization of PpSP15 purification from salivary glands in Iranian wild Phlebotomus papatasi (Diptera: Psychodidae).

BACKGROUND & OBJECTIVES: Sand fly saliva contains proteins that modulate the host immune system and it plays an important role in both blood feeding and the outcome of Leishmania infections. The profile of the salivary proteins was examined and analyzed from an endemic focus of zoonotic cutaneous leishmaniasis by wild P. papatasi to find local and suitable antigens as potential proteins for developing Leishmania vaccine alongside the development of a new extraction technique. METHODS: Specimens were caught from Bojnord, using funnel and CDC traps. Different methods of protein extraction were employed and a new technique was developed. The proteins were extracted from the salivary glands tissues with a lysis buffer. Purification was performed using RP-HPLC, with a linear gradient protocol from 0-60 % of acetonitrile. PpSP15 was characterized by SDS-PAGE. RESULTS: The concentration of extracted protein content was 0.5 and 0.03 µg/µl in chemical and physical methods, respectively. PpSP15 was isolated at a weight of 15kDa in 80-85 min of run time. SDS-PAGE was able to characterize PpSP15. The crude extract of the chemical method, revealed 15 separated bands, ranging from 11-100 KDa. Tajima D index was positive. INTERPRETATION & CONCLUSION: PpSP15 was characterized from Iranian specimens; it is a very highly hydrophobic protein of salivary glands among SP15- like proteins. The chemical method of extraction was found to be more effective than physical methods (P < 0.05). For developing a vaccine against leishmaniasis, depending on the location, choosing suitable proteins should be considered and an efficient extraction method should be used.

In vitro effect of some nucleoside reverse transcriptase inhibitors against HSV-1 replication.

OBJECTIVE: The current study was designed to investigate the effects of some nucleoside reverse transcriptase inhibitors (NRTIs) on HSV-1 infection. MATERIALS AND METHODS: Initially, the SwissTargetPrediction server was used to predict the interactions between HSV-1 thymidine kinase and acyclovir, stavudine, zidovudine, didanosine, and entecavir. The effect of each component on Vero cell viability was assessed by the MTT assay. After treatment, the cell supernatants were collected, and HSV-1 replication was analyzed by quantitative real-time PCR. RESULTS: The qPCR results revealed that viral titers were reduced 41, 40, 19, 44, and 31-fold in the presence of acyclovir, zidovudine, stavudine, didanosine, and entecavir, respectively. CONCLUSIONS: Our findings indicate that NRTIs significantly reduce HSV-1 replication in cell culture.

Effect of maternal morphine sulfate exposure on neuronal plasticity of dentate gyrus in Balb/c mice offspring.

This study carried out to evaluate the effects of maternal morphine exposure during gestational and lactation period on the neuronal cells of dentate gyrus in 18 and 32 days Balb/c mice offspring. In this experimental study 10 female mice were randomly allocated into cases and controls. In experimental group, animals were received morphine sulfate 10 mg/kg/body weight intraperitoneally during 7 days before mating, gestational period (GD0-21), 18 and 32 days after delivery. The control animals were received an equivalent volume normal saline. Cerebrum of six infant for each group were removed and stained with cresyl violet and monoclonal anti-neuronal nuclei (NeuN) antibody. Quantitative computer-assisted morphometric study was done on dentate gyrus of hippocampus. In the P18 mice, the numbers of granular cells in dentate gyrus medial blade and dentate gyrus lateral blade significantly reduced from 171.45 +/- 4.2 and 174.51 +/- 3.1 cells in control group to 153.32 +/- 2.8 and 151.23 +/- 3.2 cells in 10000 microm2 area of granular layer in treated group (p < 0.001). In P32 mice the numbers of granular cells in mb and lb of dentate gyrus significantly decreased from 155.31 +/- 4.1 and 153.77 +/- 3.4 in control group to 138.33 +/- 4.5 and 135.13 +/- 4.3 in treated group, respectively (p < 0.001). The granular layer thickness in mb and lb area of dentate gyrus significantly reduced in treated mice in compared to controls in P18 and P32 mice (p < 0.05). This study revealed that morphine administration before, during pregnancy and lactation period causes neuronal cells loss of dentate gyrus in 18 and 32 days old infant mice.

Gestational diabetes induced neuronal loss in CA1 and CA3 subfields of rat hippocampus in early postnatal life.

This study was conducted to determine the effect of gestational diabetes on the neuronal density of CA1 and CA3 subfields of the hippocampus in Wistar rat offspring. On day 1 of gestation, 10 dams were randomly allocated into two control and diabetic groups. Five animals in the diabetic group received 40 mg/kg/b.w. of streptozotocin (intraperitoneally) and the control animals were received normal saline. Six offspring of each of the gestational diabetics and controls were randomly selected in postnatal days 7 and 21. The infants were scarified and coronal sections were taken from the right dorsal hippocampus and stained with cresyl violet. The number of pyramidal cells per 10000 µm(2) area and the thickness of layers of hippocampus in CA1 and CA3 were evaluated. In postnatal day 7, the number of pyramidal neurons in CA1 significantly reduced from 118.82 ± 8.0 in the control group to 84.71 ± 3.3 neurons in gestational diabetic group, and in postnatal day 21 it significantly reduced from 112.71 ± 6.9 in the control group to 91.52 ± 8.5 in the gestational diabetic group. Also, the number of pyramidal cells of CA3 on postnatal day 7 significantly reduced from 90.33 ± 8.1 in the control group to 62.86 ± 7.2 in the gestational diabetic group, and in P21 the number of pyramidal cells significantly reduced from 78.33 ± 2.4 in the control group to 61.7 ± 9.5 cells in the diabetic group. In CA1 and CA3 the thickness of the pyramidal layer on postnatal days 7 and 21 non-significantly increased in gestational diabetics in comparison with the controls. This study showed that uncontrolled gestational diabetes reduces the pyramidal neurons of the hippocampus in rat offspring.

Vestibuloplasty: allograft versus mucosal graft.

The aim of the present study was to compare the application of alloderm and mucosal graft for vestibuloplasty. This randomized controlled trial with split mouth design was carried out on 20 edentulous patients. Patients underwent vestibuloplasty surgery with the Clark technique. Half of the prepared bed in each patient was covered with alloderm and the other half with mucosal graft. Vestibule depth (width of fixed tissue) and relapse in the two sides immediately after surgery, and 1, 3 and 6 months after surgery were measured and compared. Statistical analysis was carried out using the Kolmogorov-Smirnov, Student's paired t and Friedman tests. The width of the fixed tissue in the alloderm graft at 1, 3 and 6 month intervals was significantly lower than that in the autograft (P<0.05). The difference in relapse between the two grafts was not statistically significant at any time. The results of the study suggest that alloderm is as effective as mucosal grafts in vestibuloplasty.

Effect of Urtica dioica L. (Urticaceae) on testicular tissue in STZ-induced diabetic rats.

Urtica dioica L. (Stinging nettle) has already been known for a long time as a medicinal plant in the world. This histopathological and morphometrical study was conducted to determine the effects of the hydroalcoholic extract of Urtica dioica leaves on testis of streptozotocin-induced diabetic rats. Eighteen male Wistar rats were allocated to equally normal, diabetic and treatment groups. Hyperglycemia was induced by Streptozotocin (80 mg kg(-1)) in animals of diabetic and treatment groups. One week after STZ injection (80 mg kg(-1)), the rats of treatment group received the extract of U. dioica (100 mg/kg/day) IP for 28 days. After 5 weeks of study, all the rats were sacrificed and testes were removed and fixed in bouin and after tissue processing stained with H and E technique. Tubular cell disintegration, sertoli and spermatogonia cell vacuolization and decrease in sperm concentration in seminiferous tubules were seen in diabetic and treatment groups group in comparison with control. External Seminiferous Tubular Diameter (STD) and Seminiferous Epithelial Height (SEH) significantly reduced (p < 0.05) in the diabetic rats compared with controls and these parameters in the treatment group were similar to diabetics animals. This study showed that hydroalcoholic extract of Urtica dioica leaves, after induction of diabetes; has no treatment effect on seminiferous tubules alterations in streptozotocin-induced diabetic rats.

Cytotoxic activities of selected medicinal plants from Iran and phytochemical evaluation of the most potent extract.

Methanolic extract of 15 Iranian medicinal plants were prepared and tested for their cytotoxic activities against three cancer cell lines (MCF7, HepG2, WEHI164) and one normal cell line (MDBK). Some plants showed cytotoxic activities. The extract of Ferula szowitsiana root, which proved to be the most active, was chosen for further phytochemical studies. The major compounds of the most potent acetone extract were isolated. They were identified as chimgin and chimganin, two known monoterpenoids, by spectroscopic means. Their cytotoxic activity was evaluated in three cell lines. The results show that these compounds are responsible, at least in part, for the cytotoxic activity of this plant.

The granule cell density of the dentate gyrus following administration of Urtica dioica extract to young diabetic rats.

Urtica dioica L. Stinging nettle has long been known worldwide as a medicinal plant. To study the benefits of the nettle in diabetic encephalopathy, the granule cell density of the dentate gyrus of diabetic rats was studied following administration of Urtica dioica extract. A total of 24 male albino Wistar rats were allocated equally to normal, diabetic, preventive and treatment groups. Hyperglycaemia was induced by streptozotocin (80 mg/kg) in the animals of the diabetic and treatment groups. One week after injection of the streptozotocin the animals in the treatment group received a hydroalcoholic extract of Urtica dioica (100 mg/kg/day) for 4 weeks intraperitoneally. The rats of the preventive group received hydroalcoholic extract of U. dioica (100 mg/kg/day) IP for the first 5 days and an injection of streptozotocin (80 mg/kg) on the 6th day. After 5 weeks of study all the rats were sacrificed and coronal sections were taken from the dorsal hippocampal formation of the right cerebral hemispheres and stained with cresyl violet. The area densities of the granule cells were measured and compared in the four groups. The density was lower in the diabetic rats compared with the controls (p > 0.05). The preventive group showed lower cell density than the controls (p > 0.05). The densities in the treated rats were higher than in the diabetic rats (p > 0.05). Furthermore, the control and treated rats showed similar densities (p > 0.05). It seems that U. dioica extract can help compensate for granule cell loss in the diabetic rat dentate gyrus, which can ameliorate cognitive impairment in diabetes. However, preventive use of the extract showed no significant benefit.

Morphometric alterations of the rat spleen following formaldehyde exposure.

Formaldehyde is commonly used in the production of various industrial and medical products. At room temperature formaldehyde easily evaporates. Exposure to formaldehyde can be hazardous to human health. Studies show that the vapour can be the cause of clinical symptoms such as throat, eye, skin and nasal irritation. It can also decrease the production of IgM in the spleen cells. This study was designed to determine the morphometric changes to the spleen in rats when samples were exposed to formaldehyde for 18 weeks. A total of 28 albino Wistar rats aged 6-7 postnatal weeks were divided into the following three case groups according to their exposure to formaldehyde: E1 (2 h/day, 2 days/week), E2 (2 h/day, 4 days/week), E3 (4 h/day, 4 days/week) and one control group. When the exposure period had expired the animals were anaesthetised with chloroform. After cervical dislocation, the abdomen was dissected and spleen specimens were taken. These were sectioned and stained with the haematoxylin and eosin technique for morphometric study. Data was obtained from an Olympus light microscope and then analysed with SPSS (version 11.5) and one-way ANOVA test. The white pulp area and diameter and the marginal zone diameter were greater in group E3 than those in the other groups. The germinal centre area and diameter and the diameter of the periarterial lymphoid sheaths (PALS) were greater in group E2 than in other groups, although there was no significant difference between groups in the area of white pulp and the PALS diameter (p<0.05). This study showed that formaldehyde vapour can cause morphometric changes in the white pulp of the spleen in rats.

Formaldehyde exposure induces histopathological and morphometric changes in the rat testis.

Formaldehyde is a chemical which is traditionally used for fixing cadavers and routine histopathology techniques. It is vaporised during the dissection and practical study of a cadaver. Previous studies have shown that this vapour may cause clinical symptoms such as throat, eye, skin and nasal irritation. This study was designed to determine the histopathology and morphometrics of the rat testis when all the experimental animals were exposed to formaldehyde for 18 weeks. The study was performed in 2004 on 28 albino Wistar rats of 6-7 postnatal weeks. The rats were divided into three case groups (E1: 4 h/d, 4 d/w; E2: 2 h/d, 4 d/w; E3: 2 h/d, 2 d/w) and one control group. The testes specimens were sectioned at 5 microm and stained with the haematoxylin and eosin staining technique for histological and morphometrical studies. We found a severe decrease in germ cells associated with spermatogenesis arrest in the E1 group. A decrease in germ cells and a thickening of the basal membrane of the seminiferous tubules were seen in E2. Displacement of Sertoli and germinal cells were also found in the E3 group. The mean seminiferous tubular diameter and seminiferous epithelial height in the experimental groups were decreased in comparison with the control group and the differences were statistically significant (p < 0.05). The findings of this study revealed that chronic formaldehyde exposure can cause histopathological and morphometric changes to the seminiferous epithelium in rats and that these changes depend on the duration of the formaldehyde exposure.

Endoscopic ultrasound-guided fine needle aspiration of the pancreas: cytomorphological evaluation with emphasis on adequacy assessment, diagnostic criteria and contamination from the gastrointestinal tract.

OBJECTIVE: Endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) has been proved to be safe, efficient and reliable in the diagnosis of pancreatic lesions. This study evaluated specimen adequacy, diagnostic criteria of various pancreatic neoplasms and contamination from the gastrointestinal (GI) tract. METHODS: EUS-guided FNA of the pancreas and subsequent surgical resections performed at the University of California Irvine Medical Center during February 1996-October 2000 were retrospectively selected. Modified Papanicolaou staining method was used for immediate evaluation and cell block prepared. RESULTS: A total of 267 cases were available for review, including 147 (55.1%) positive/suspicious, 10 (3.7%) atypical, 96 (36.0%) negative and 14 (5.2%) unsatisfactory cases. Eighty-six (58.5%) positive/suspicious cases had histological confirmation and 12 (8.3%) had lymph node or distant metastases by cytology. Three atypical, two negative, and two unsatisfactory cases proved to have adenocarcinoma. Contamination from duodenum, stomach or pancreas was found in 77 positive/suspicious, three atypical and 90 negative cases. The sensitivity, specificity, diagnostic accuracy, positive and negative predictive values were 94.6%, 100%, 95.6%, 100%, 82% respectively. CONCLUSIONS: EUS FNA is efficient and accurate in the diagnosis of pancreatic neoplasms in adequate samples. Contamination from the GI tract should be well recognized to avoid misinterpretation.

Cytologic diagnosis of gastrointestinal stromal tumors of the stomach by endoscopic ultrasound-guided fine-needle aspiration biopsy: cytomorphologic and immunohistochemical study of 12 cases.

Gastrointestinal stromal tumor (GIST) is an uncommon tumor, which was usually diagnosed by endoscopic biopsy or surgical resection. This study evaluated the efficacy and accuracy of endoscopic ultrasound (EUS) -guided fine-needle aspiration (FNA) biopsy in the diagnosis of GIST and reported its cytomorphologic features. Twelve patients with gastric GIST were diagnosed through EUS-guided FNA. Immediate on-site evaluation and cytologic diagnoses were given in nine cases (75.0%) with an average of three passes. Cell blocks provided diagnostic material in three cases (25.0%). Spindle cells were present in the cytologic material in all cases. Two patients had subsequent surgical resections. Immunohistochemical (IHC) studies performed in cell blocks and two surgical specimens all supported the original diagnoses. In the two cases with surgical resections, IHC results in cell blocks were similar to that in the resected specimens. This study demonstrated that when combining smears and cell blocks, EUS-guided FNA is accurate and efficient in the diagnosis of GIST. IHC reactivity in cell blocks correlated with that of the main tumors.

Screening for medium chain acyl-CoA dehydrogenase deficiency using electrospray ionisation tandem mass spectrometry.

OBJECTIVE: To establish criteria for the diagnosis of medium chain acyl-CoA dehydrogenase (MCAD) deficiency in the UK population using a method in which carnitine species eluted from blood spots are butylated and analysed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). DESIGN: Four groups were studied: (1) 35 children, aged 4 days to 16.2 years, with proven MCAD deficiency (mostly homozygous for the A985G mutation, none receiving carnitine supplements); (2) 2168 control children; (3) 482 neonates; and (4) 15 MCAD heterozygotes. RESULTS: All patients with MCAD deficiency had an octanoylcarnitine concentration ([C8-Cn]) > 0.38 microM and no accumulation of carnitine species > C10 or < C6. Among the patients with MCAD deficiency, the [C8-Cn] was significantly lower in children > 10 weeks old and in children with carnitine depletion (free carnitine < 20 microM). Neonatal blood spots from patients with MCAD deficiency had a [C8-Cn] > 1.5 microM, whereas in heterozygotes and other normal neonates the [C8-Cn] was < 1.0 microM. In contrast, the blood spot [C8-Cn] in eight of 27 patients with MCAD deficiency > 10 weeks old fell within the same range as five of 15 MCAD heterozygotes (0.38-1.0 microM). However, the free carnitine concentrations were reduced (< 20 microM) in the patients with MCAD deficiency but normal in the heterozygotes. CONCLUSIONS: Criteria for the diagnosis of MCAD deficiency using ESI-MS/MS must take account of age and carnitine depletion. If screening is undertaken at 7-10 days, the number of false positive and negative results should be negligible. Because there have been no instances of death or neurological damage following diagnosis of MCAD deficiency in our patient group, a strong case can be made for neonatal screening for MCAD deficiency in the UK.

Characterization of a seventh family of immunoglobulin heavy chain VH gene segments in the channel catfish, Ictalurus punctatus.

A VH gene segment that could not be assigned to any of the six known VH gene families of the channel catfish was identified in a genomic clone containing VH gene segments. This gene segment (designated VH7.1) exhibited the structural features characteristic of vertebrate VH genes, specifically potential upstream regulatory sequences, a leader sequence split by an intron, a reading frame that could be readily divided into framework and complementarity determining regions, and a 3' recombination signal sequence. Two regions of nucleotide deletions coupled with degeneracy in the nonamer sequence indicate that this VH gene segment is a pseudogene. Genomic DNA restricted with different enzymes and hybridized under stringent conditions with probes derived from VH7.1 showed that 8-10 bands were present in Southern blots. Reverse transcriptase PCR approaches were used to determine if any of these related sequences were expressed. Sequence analysis of cloned PCR products indicates that different VH gene segments exhibiting > 80% similarity to germline VH7.1 are expressed. Multiple sequence alignments showed that the expressed cVH7a cDNA sequence shared less than 60% nucleotide similarity with representative cDNA sequences from the other known catfish VH gene families. These combined results thus fulfil the criteria for the definition of a new family of catfish VH gene segments. This newly defined, small VH family is designated VH7.