Insoluble wild-type and protease-resistant mutant prion protein in brains of patients with inherited prion disease.

We studied prion proteins (PrP) in skin and brains of Libyan Jews carrying the E200K mutation who died of familial Creutzfeldt-Jakob disease (CJD). Unexpectedly, studies with brain showed that PrP molecules encoded both by the wild-type (wt) and mutant alleles exhibit altered properties characteristic of the prion protein associated with prion diseases (PrPSc). Using monospecific antisera, we found that wtPrP was insoluble in the brains of three patients who were heterozygous for the E200K mutation, whereas mutant PrP was both insoluble and protease-resistant. Our results argue that both wild-type and mutant PrP undergo conformational changes and are particularly intriguing, because the normal isoform PrPc is soluble in nondenaturing detergents and is readily digested by proteases, whereas PrPSc is insoluble and resistant to proteolytic digestion. Our findings indicate that insoluble wtPrP represents a conformational intermediate, the first to be identified, within a pathway in which PrPc is converted to PrPSc.

Effect of ghrelin on protein kinase C-ε and protein kinase C-δ gene expression in the pulmonary arterial smooth muscles of chronic hypoxic rats.

INTRODUCTION: Protein kinase C (PKC), can be activated in pulmonary arterial smooth muscle cells during hypoxia, leading to hypoxic pulmonary vasoconstriction (HPV). Studies are going on to detect the strict PKC isoform involved in the phenomenon. It has been shown that ghrelin, a 28-amino-acid peptide, may protect lungs from HPV side effects, to some extent. The aim of study was to evaluate the effect of exogenous ghrelin on PKC-ε and PKC-δ gene expression during chronic hypoxia. MATERIAL AND METHODS: Twenty-four adult male Wistar rats were divided randomly in 3 groups. Hypoxic rats with saline or ghrelin treatment were placed in a normobaric hypoxic chamber for 2 weeks. Controls remained in room air. PKC-ε and PKC-δ gene expression was measured by real-time RT-PCR. RESULTS: Morphometric analysis showed that ghrelin reversed the hypoxia induced pulmonary artery wall thickness. In hypoxic animals, there was a 2- and 4-fold increment in PKC-ε and PKC- δ gene expression, respectively. Ghrelin treatment reduced the overexpression of PKC-ε and PKC-δ to control animals' value. CONCLUSION: Ghrelin by decreasing the expression of PKC-ε and PKC-δ in hypoxic animals reduces the HPV. Although more studies are needed, it could be an honest deduction that ghrelin affects HPV in a multifunctional manner and might be used as a therapeutic agent in the future.

Gill lesions and mortality in common carp (Cyprinus carpio) with a dense bloom of Heterosigma-like algae in Khuzestan province.

BACKGROUND: Widespread common carp (Cyprinus carpio) mortalities have been recorded in Khuzestan province fish farms in recent years. In summer of 2017 two cases of harmful algal bloom were encountered that led to massive mortality in common carp in Khuzestan, Iran. AIMS: The aim of this study was to identify the possible etiologic agent of two mortalities with characteristic symptoms of gill lesions due to harmful algae. METHODS: Water samples were collected and 5 moribund fishes were examined by histophatologic, scanning electron microscopic and PCR examination. RESULTS: In wet smear preparations, a lot of algal cells and fragments, and sloughed, necrotic epithelial cells were observed between the lamellae. In histopathologic examination of gills, hyperplasia, necrosis and algal cells surrounded by hyperplastic cells were seen in tissue sections. No inflammatory cell aggregation was noticed. In scanning electron microscopic examination the algae was found attached to the gill surface (cell diameter: 8.5 ± 4.2 µm) with 2 equal flagella. CONCLUSION: Phytoplankton analysis using direct microscopy and electron microscopy, morphologically resembling Heterosigma was identified, however, in PCR tests, Heterosigma analysis showed negative results, therefore the causative agent was called "Heterosigma-like" algae.

Marker analysis of cloned populations of human monocytes.

The presence of myelomonocytic progenitor cells in human peripheral blood was used for the analysis of cloned populations of human monocytes. Colonies of granulocytes and macrophages were obtained by plating peripheral blood mononuclear cells (PBM) in methylcellulose containing medium in the presence of medium conditioned by nonstimulated PBM (CM). Following 20-25 days of incubation, most colonies were found to consist of cells with monocyte-macrophage morphology. Cloned populations of monocytes were tested for several monocyte membrane markers and compared to noncloned adherent monocytes. HLA-DR, 63D3, LeuM2 antigens and Fc receptors were expressed on cells from individual colonies in similar proportions to their expression on noncloned monocytes. Some colonies were uniform in their negative expression of the 63D3 antigen, as were the noncloned monocytes. Although the clonality of cells tested was not directly proven, these results indicated that at least for some monocyte markers, heterogeneous expression was obtained in monoclonal populations of monocytes. It is possible, however, that testing of additional markers and functions may reveal homogeneous clones of monocytes and suggest the existence of stable subsets.

A new myelomonoblastic cell line (M20): analysis of properties, differentiation, and comparison with other established lines of similar origin.

A new myelomonoblastic cell line (M20) was established from the peripheral blood of a ten-year-old child with acute myeloblastic leukemia, using an improved method for supporting the initial stages of cell proliferation. The addition of irradiated macrophage monolayers to the proliferating cells appeared to overcome the deterioration of the primary cultures and enable them to continue proliferating until they became independent of this environment. The cell line that developed consisted of myeloblasts and promyelocytes characterized by light and scanning electron microscopy, cytochemistry, and enzymatic activities. The cells expressed Fc receptors and WT1 antigens but did not exhibit HLA-DR, HMA1, Epstein-Barr virus nuclear antigen, and surface Ig. The M20 cells produced colonies when cultured in semisolid medium and secreted lysozyme, prostaglandin E2, and interleukin 1. An attempt was also made to analyse the position of the M20 cells in the scheme of differentiation of the myelomonocytic lineage using different approaches. Treatment of the cells with 12-O-tetradecanoyl phorbol 13-acetate induced their adherence to plastic surfaces and partial maturation to macrophages as judged by morphological criteria, cytochemistry, and enzyme activities. However, comparison of the M20 cells to other well-established myelomonoblastic cell lines did not reveal any pattern suggesting a possible relationship between surface markers, cell function, and differentiation pathway of the various cell lines tested. Establishment of additional cell lines and identification of new markers may assist in defining the mechanisms involved in normal differentiation and malignant transformation of this cell lineage. In addition, such cell lines may also provide a tool for the quantitative recovery of a variety of monokines.

Differential allelic expression of PrP mRNA in carriers of the E200K mutation.

Creutzfeldt-Jakob disease (CJD) linked to the E200K mutation of the prion protein (PrP) gene presents with a wide range of age at disease onset. Since most patients are heterozygous for the mutation, we tested whether differential expression of mutant versus wild-type (wt) PrP may affect the age at disease onset in carriers of the mutation. We measured wt and mutant PrP protein and mRNA in Epstein-Barr virus (EBV)-transformed B cells of either E200K CJD patients or healthy E200K carriers. Our results suggests that while in most healthy carriers the expression of wt PrP was higher than that of E200K PrP, most of the E200K CJD patients express equal levels of both PrP proteins. Similar results were obtained for either PrP protein or PrP mRNA. These results suggest that preferential expression of PrP from the wt allele may modulate the outbreak of the disease in carriers of prion mutations. This notion is consistent with the results obtained in transgenic mice carrying a human PrP gene, which suggest that endogenous PrP protects mice from contracting scrapie after inoculation with human CJD brain. Similar mechanisms may prevail in other inherited diseases with variable phenotypes.

Detection of 14-3-3 protein in the CSF of genetic Creutzfeldt-Jakob disease.

The 14-3-3 protein, a protein involved in signal transduction, is present in the CSF of patients with Creutzfeldt-Jakob disease (CJD) and not in patients with other dementing diseases. We show here that this is also true for patients with E200K CJD, but not for healthy carriers of the mutation.

Presence of prion protein in peripheral tissues of Libyan Jews with Creutzfeldt-Jakob disease.

The prion protein (PrP) gene on chromosome 20 encodes a protein designated PrPC. An abnormal, protease-resistant isoform of PrPC, denoted PrPCJD or PrPSc, is present in the brains of patients with Creutzfeldt-Jakob disease (CJD). In Libyan Jews, CJD segregates with a point mutation at codon 200 of the PrP gene, resulting in the substitution of lysine for glutamate. In the present study, we examined the presence of PrP in fibroblasts and leukocytes derived from eight CJD patients with the codon 200 mutation. In cultured fibroblasts as well as in leukocytes, there was a significant increase in PrP as judged by immunocytochemistry in addition to immunoblotting. Most of the PrP in fibroblasts and leukocytes could be released from the external surface by phosphatidylinositol-specific phospholipase C, a property characteristic of PrPC. In leukocytes only, part of the protein was protease resistant, resembling PrPCJD. The concentration of PrP mRNA was similar in fibroblast lines derived from controls and CJD patients. These results suggest that in CJD patients carrying a mutation at codon 200 of the PrP gene, the metabolism of PrP, rather than PrP synthesis, is abnormal.

The initial slope of human tumor cell survival curves: its modification by the oxic cell sensitizer beta-arabinofuranosyladenine.

The initial slope of the survival curve, which is a characteristic of each tumor cell line, varies with the histological group of the tumor. It is one of the factors on which clinical radioresponsiveness depends. The DNA dependant DNA polymerase inhibitor beta-ara A acts as an oxic cell sensitizer. This study was carried out on human tumor cell lines to look for a correlation between the degree of radiosensitization induced by beta-ara A and the radiosensitivity of a given cell line. Six human tumor cell lines with different radiosensitivities were used (the survival rate at 2 Gy and D ranged from 20 to 73% and from 1.2 to 3.2 Gy, respectively). beta-ara A had a major toxic effect on all cell lines but this varied greatly from one cell line to another and was concentration dependant; this toxic effect was taken into account when calculating the surviving fractions. For all cell lines, beta-ara A acted as an oxic radiosensitizer and the radiosensitization was concentration dependant. Analysis of the survival curves of the 6 cell lines using the linear quadratic model showed that concentrations of beta-ara A between 200 and 1000 microM induced an increase in the linear component while the quadratic component underwent no systematic change. The sensitizing enhancement ratio (SER) measured from the Ds ratios, varied greatly from one line to another. For example, at a concentration of 500 microM, the extreme values of Ds ratios were 1.5 and 2.6. The radiosensitization is greater, the higher the radiosensitivity of the cell line studied during exponential growth. The results do not favor the use of beta-ara A in the treatment of intrinsically radioresistant human tumors.

Development of macrophage and granulocyte colonies from human peripheral blood.

The presence of macrophage and granulocyte progenitor cells in the human peripheral blood enabled the establishment of colonies from this accessible tissue and obviated the need for bone marrow to achieve this task. We have developed a method of obtaining reproducible growth of macrophage/granulocyte colonies from human peripheral blood. Colonies of macrophages and granulocytes were obtained by plating peripheral blood mononuclear cells (PBM) in methylcellulose containing medium in the presence of medium conditioned by nonstimulated PBM (CM). At early stages of colony growth both macrophage and granulocyte colonies were detected while following 20-25 days in culture all colonies tested revealed monocyte-macrophage morphology. To obtain higher numbers of colonies, we tested different cell sources, different CM preparations and the effect of steroid hormones on colony development. We found that the mononuclear cells obtained from cord blood (CB) or from some patients with inflammatory bowel disease yielded much higher numbers of colonies than PBM from normal individuals. Colony development from these two sources did not depend on an external source of colony stimulating factor (CSF) but was augmented as a result of CSF supplementation. CM obtained from CB mononuclear cells as well as supernatants from some human monoblastic cell lines were similar in their CSF activity to CM from normal PBM and made possible the development of macrophage/granulocyte colonies. Higher numbers of colonies were induced by including physiological concentrations of estradiol in the culture medium, in the absence of external sources of CSF. The system described above enabled the analysis of cloned macrophages and their circulating progenitor cells as well as the assay of different preparations of CSF.

The effect of estradiol on human myelomonocytic cells. II. Mechanism of enhancing activity of colony formation.

Elevated proportions of monocytes have previously been found in the blood of healthy women during the ovulation period as well as in other conditions associated with increased blood estradiol (E2). This phenomenon was explained, in part, by an augmenting effect which physiological concentrations of E2 may have on the development of granulocyte-macrophage (GM) colonies derived from normal peripheral blood mononuclear cells. To analyze this effect, we tested possible alternatives for the interaction between E2, colony-stimulating factor (CSF) and GM colony progenitor cells. E2 was found not to interact synergistically with CSF, but pre-treatment of the progenitor cells with E2 resulted in higher numbers of colonies in response to CSF. Moreover, E2 did not induce higher secretion of CSF but treatment with anti-CSF antibodies abolished the enhancing effect of E2. Based on these results, we suggest that the augmenting effect of E2 on GM colony formation is mediated by inducing the colony precursor cells to be more responsive to CSF. These findings may help to elucidate some of the complex relationships between estrogens, immune responses and hemopoiesis.

The effect of estradiol on human myelomonocytic cells. 1. Enhancement of colony formation.

Cyclic changes were observed in the content of blood monocytes during the menstrual cycle of normal women. Elevated blood monocytes were found during the ovulation period as well as in other conditions which are associated with increased blood estradiol (E2). To understand the possible association between E2 and monocytosis, we analysed the in vitro effect of E2 on the development of myelomonocytic colonies in culture. E2 in physiological concentrations was found to increase the number of colonies developed from peripheral blood mononuclear cells (PBM) of both females and males. The optimal concentration for the augmenting effect on males' PBM was lower than that for females. Mononuclear cells derived from cord blood, which yielded much higher numbers of colonies than adult PBM, also responded to the stimulatory effect of E2. Estrone and estriol were less effective than E2 in adult PBM. In contrast, progesterone, diethylstilbestrol and testosterone did not affect the number of colonies at the range of physiological concentrations tested. The anti-estrogen Tamoxifen did not inhibit the stimulatory effect of E2. The augmenting effect of E2 on monomyelocytic colony formation may explain at least in part the increase in blood monocyte content of women with high E2 as well as other phenomena of macrophage and granulocyte changes associated with the menstrual cycle.

Genetics and biochemistry of Creutzfeldt-Jakob disease in Libyan Jews.

A focus of Creutzfeldt-Jakob disease (CJD) among Jews from Libyan origin was identified in Israel 20 years ago. The incidence of the disease in this ethnic group is about 100 times more than in the worldwide population. The consumption of lightly cooked sheep brain has been invoked to explain the high incidence of CJD in this community. The discovery of mutations in the PrP gene which segregates with other familial prion diseases such as Gerstmann-Straussler syndrome (GSS) lead us to perform a molecular genetic study and compare it to an epidemiological survey among the Libyan community. The epidemiological data suggests a very high familial incidence of CJD in this population and a molecular genetic research elucidated that CJD segregates with a point mutation at codon 200 of the PrP gene resulting in the substitution of Lysine for Glutamate. This mutation was found in some 40 CJD patients of Libyan origin and was not found in one Moroccan Jew suffering from CJD. It was also absent in almost 100 healthy Libyan controls above the age of 60. This result strongly supports a genetic etiology for CJD pathogenesis in the Libyan Jewish community and disregards the previous culinary hypothesis. The disease is vertically transmitted in autosomal dominant inheritance with unknown penetrance. All our patients were heterozygote for the mutation except one homozygote patient. The course of the disease in this patient was identical to the heterozygote patients, strongly arguing that inherited CJD displays complete phenotypic dominance.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of respiratory muscle training on improvement of the internal and external thoraco-pulmonary respiratory mechanism in COPD patients.

INTRODUCTION: Chronic obstructive pulmonary disease (CPOD) is a severe, incapacitating pathology. Inspiratory and/or expiratory muscle training may favorably impact the indicators of both specific and general improvement with regard to this disease. We are hypothesizing that when combined with bronchial decluttering, this training will have a beneficial effect on lung function and quality of life in these patients. METHOD: Fourty COPD subjects classified Gold I and Gold II and aged 60.38±8.02years were divided into four groups of 10. Three of the groups were trained with the help of Threshold(®) tools used for (1) inspiratory, (2) expiratory and (3) inspiratory and expiratory purposes; their training supplemented the decluttering and lower limb muscle exercise that the 4th group concurrently received. The patients underwent 16 rehabilitation sessions over an 8-week period. The variables consisted in: (1) forced expiratory volume in 1s (FEV1) and spirometrically measured peak expiratory and inspiratory flow rates (PEFR and PIFR); (2) fatigability, dyspnea, heart rate and walking distance evaluated during the 6-minute walk test; (3) maximum inspiratory pressure and (4) maximum expiratory pressure as assessed by the Threshold(®) tools and (5) the signs of quality of life in terms of the Saint-George's respiratory questionnaire (SGRQ) score. RESULTS: Only in group 1, there was significant improvement with regard to FEV1 and PEFR. There was no PIFR modification in any of the groups. On the other hand, signs of quality of life scores along with dyspnea, fatigability and heart rate showed significant improvement in the three experimental groups, and significant improvement in maximum inspiratory pressure was observed in groups 1 and 3. DISCUSSION: When associated with decluttering techniques, diaphragmatic rehabilitation and lower limb muscle exercise along with psychological support and educational efforts, respiratory muscle training is beneficial when compared with the usual protocols in rehabilitation of COPD patients.

Determination of the electrophoretic pattern of somatic and excretory-secretory proteins of Ligula intestinalis parasite in spirlin (Alburnoides bipunctatus).

Ligula intestinalis parasite is a cestodes that causes remarkable damages to fish. It is also of prime importance in economic and hygienic aspects. SDS-PAGE and western blotting are the methods that can be used to determine the electerophoretic pattern of somatic and excretory-secretory proteins of parasites. In this study, after obtaining the plerocercoidal stage of this parasite from the spirlin (Alburnoides bipunctatus), its somatic proteins were prepared using ultrasonicae, and excretory-secretory proteins were prepared using the PBS solution. After protein assay, which included using the Bradford method and then SDS-PAGE on these two antigenic solutions, 5 protein bands of 26, 33, 38, 58, 70kDa in somatic antigens, and 7 bands of 25, 28, 33, 43, 49, 60, 70kDa in excretory-secretory antigens were observed. After western blotting on both antigens and adding the primary antibody (the sera of infected fish) and then the secondary antibody (Rabbit Anti-fish Polyclonal Antibody Conjugated from Abnova Corporation) no band was seen in excretory-secretory antigen. And only in the 55kDa band of somatic antigen, a positive response, in comparison of fish positive serum was observed.

Human papillomavirus infection in lung vs. oral squamous cell carcinomas: a polymerase chain reaction study.

The role of Human Papillomavirus (HPV) has been suspected in pathogenesis of various malignancies; however, the available data are not conclusive. This study aimed to determine and compare the frequency of HPV infection in oral and lung Squamous Cell Carcinoma (SCC) by a sensitive method. Sixty specimens of oral and lung SCC (30 cases each one) were reevaluated in Tabriz Imam Reza Centre in a 24 month period. Following genomic DNA extract, the Polymerase Chain Reaction (PCR) amplification was performed in presence of specific MY11 and MY09 primers for HPV infection. Three cervical specimens and a combination of PCR solution lacking DNA plus healthy persons' DNA samples were employed as positive and negative controls, respectively. The oral group was significantly older than the lung group (68.90 vs. 56.67 y, p < 0.001) with more males in the latter (83.3 vs. 60%; p = 0.04). Percentages of HPV infection in the oral and lung groups were comparable (20 vs. 10%, respectively; p = 0.47). Majority of patients with HPV infection were older than 60 years (88.9%) or male (88.9%). In the oral group, all these cases were well differentiated and the majority was of lower lip origin (83.3%). In the lung group, 66.7% of these specimens were moderately differentiated and the origin was bronchus in all cases. In conclusion, the rate of HPV infection in lung and oral SCC samples is rather lower than the previous reports in the literature. This rate is apparently higher in the oral than the lung SCC specimens.

Flora, life forms and chorotypes of plants of Salok protected area (North Khorassan Province Iran).

Salok protected Area is located in the 37 degrees 15' to 37 degrees 08' of the North latitude and 57 degrees 16' to 57 degrees 06' of East longitude, in West North Esfarayen in North khorassan province. In this research 52 families, 174 genera and 213 species were identified. The largest plant family is Asteraceae (34 species), Poaceae (18 species), Brassicaceae (17 species) and Fabaceae (17 species), respectively. Chief life forms are Hemicryptopytes (49.29%), Therophytes 23.47% and cryptophytes (12.67%). The most of plants chorotype with 62.91% is influenced by Irano-Touranina elements. Among 213 identified species of this region and 15 species endemic of Iran contain 0.87% of total endemic species of Iran's flora.

Expression of epidermal growth factor receptor tyrosine kinase family in fine needle aspiration and permanent specimens of invasive lobular and ductal breast cancers.

Recently, the role of HER-2/Neu gene amplification has been enthusiastically investigated in breast cancer. Determining the HER-2/Neu status could be achieved by evaluating either histologic samples or cytologic specimens obtained by Fine Needle Aspiration (FNA). This study aimed at determining the concordance of HER-2/Neu expression in FNA and histologic sections. FNA samples, as well as their corresponding histologic sections of 90 cases with breast cancer were evaluated in Tabriz Sina Teaching Center in a 13-month period of time. The immunohistochemistry was employed for determining the HER-2/Neu amplification for both methods. The concordance rate and agreement were determined between the two methods. Ninety specimens of women with a mean age of 50.93 +/- 10.64 (29-84) years were assessed. There were 84 cases with invasive ductal carcinoma and 6 cases with invasive lobular carcinoma. Lymph nodes were involved in 50 cases and there were vascular and neural involvement in 40 and 35 cases, respectively. Her-2/Neu was not detected in 27 cases (30%) with weak and strong amplifications in 47 (52.2%) and 16 (17.8%) cases of FNA specimens, respectively. Her-2/Neu was not detected in 29 cases (32.2%) with weak and strong amplifications in 42 (46.7%) and 19 (21.1%) cases of histologic specimens, respectively. The concordance rate was 70% between the two methods. The agree ment was statistically significant between the two methods, as well (kappa = 0.51, p < 0.001). HER-2/neu gene amplification can be reliably estimated by immunohistochemistry on breast cancer FNAs and a good correlation has been found between this and results on histological sections.

Expression of P27, Ki67 and P53 in squamous cell carcinoma, actinic keratosis and Bowen disease.

This study aims at evaluating the expression of P27, Ki67 and P53 in Squamous Cell Carcinoma (SCC), Actinic Keratosis (AK) and Bowen Disease (BD) specimens. In an analytic-descriptive setting, skin biopsy specimens of 45 patients were evaluated in three 15-case groups including BD, AK and SCC specimens. Fifteen normal skin biopsy specimens were obtained and used as the control group. Immunohistochemical staining was performed in all the specimens and the expression rates and patterns of Ki67, P27 and P53 were determined. The results were compared between the four groups. Ki67 was expressed in 0.8, 23.7, 12.3 and 19.3% of the cells in the normal skin, AK, BD and SCC groups, respectively. No significant difference was seen between the three pathological conditions regarding the expression rate of Ki67. P27 was positive in 23.4, 26.2, 25.9 and 4.5% of specimens in the normal skin, AK, BD and SCC groups, respectively. This rate was significantly the lowest in the SCC group. P53 expression was detected in 26.6, 41.8 and 54.6% of the assessed cells in the AK, BD and SCC groups, respectively. There was no expression of P53 in the normal skin specimens. This rate was significantly the highest again in the SCC group. Based on these results, the quantitative and qualitative (pattern of distribution) evaluation of the expressions of Ki67, P27 and P53 may be helpful in differentiating malignant and premalignant epidermal lesions, particularly in unsatisfactory or fragmented specimens.

A rare case of intramedullary cervical spinal cord meningioma and review of the literature.

STUDY DESIGN: Case report. SETTINGS: Department of Neurosurgery, Imam Hospital, Faculty of Medicine, Tabriz University of Medical Sciences, Daneshgah Street, Tabriz, East Azerbayjan, Iran. REPORT: A 21-year-old man presented with progressive paraesthesia and weakness in the four limbs, with disturbances in the vibration and positional senses. Magnetic resonance imaging revealed a cervical intramedullary tumour from the cervicomedullary junction to C3. Radical resection of the tumour was performed and histology revealed a meningioma. His clinical outcome after a 3-year follow-up is relatively good. CONCLUSION: Intramedullary cervical meningiomas are very rare and have been reported only five times before. Because of its tendency to recur after surgery and because of a possible aggressive behaviour, meticulous histopathological examination is mandatory to predict the evolution and plan the follow-up. Outcome is mainly related to the type of tumour and the complete removal.