Syrian refugee women's breastfeeding behaviors and use of contraceptive methods: a qualitative study.

While migration leads to massive changes in women's lives, it brings along reproductive health issues as well. Determining the issues experienced by migrant women on fertility, breastfeeding and use of contraceptive methods and providing recommended solutions are important for planning and quality of maternal and child health services to be provided to women. The aim of this study is to evaluate the breastfeeding behaviors and contraceptive use of Syrian refugee women who have given birth. The study was conducted at an obstetrics and gynecology clinic of a state hospital in Ankara, Turkey, in 2018-2019. The study used a phenomenological pattern as a qualitative research method. The sample of the study consisted of 15 married Syrian refugee women of reproductive age (from 18 to 49 years of age) who gave birth before. Data collected were grouped under five main themes as "initiation of breastfeeding," "maintenance of breastfeeding and traditional practices," "situations affecting the decision of introducing complementary feeding," "use and awareness of contraceptive method," and "having a child from individual and social perspectives." Our study determined that knowledge and use of contraceptive methods and breastfeeding by Syrian women are insufficient. It is important to overcome the barriers to access to safe motherhood, reproductive health and protective health services.

Genotoxic damage in Maras powder consumers from Kahramanmaras province of Turkey.

In the present study, chromosomal aberrations (CAs) and micronucleus (MN) levels in the lymphocytes from 60 male individuals consisting of 40 habitues of Maras powder (a kind of smokeless tobacco) and 20 unexposed subjects were determined to investigate the possible inducing effect of Maras powder. The consumers of Maras powder had no exposure to any other known mutagens or toxicants. The mean exposure period to Maras powder was 12.25 + 0.93 years (range 3-22). Data obtained from microscopic examination of the slides was analyzed by SPSS (10.0) package programme. Mean frequency of CA and MN was found to be significantly higher in Maras powder consumers as compared to controls. Similarly, there was a significant elevation in the level of aberrant cells (Ab.Cells) with CAs and binucleated cells with MN (BNMN) in habitues. Spearman's rho correlation analysis indicated a significant increase in the frequency of CA and MN with increase in both age and years of exposure in consumers. Our finding of a significant elevation of CA and MN frequencies in peripheral lymphocytes from smokeless tobacco consumers demonstrated a potential cytogenetic hazard associated with Maras powder exposure.

Tumorigenic potential and the molecular mechanism of the carcinogenic effect exerted by 2-nitroanisole.

The host-mediated in vitro/in vivo assay system was used to evaluate the tumorigenic potential of the aromatic nitro compound 2-nitroanisole (2-NA). After intraperitoneal administration of the compound, resident macrophages were recovered by peritoneal lavage from treated and untreated mice and cultured in soft agar. 2-NA was shown to be carcinogenic, and the tumorigenic potential was evaluated. Additionally, by establishment of a transformed peritoneal macrophage cell line, the underlying molecular mechanism of 2-NA's carcinogenic effect was studied.

Molecular interactions of the type 1 human immunodeficiency virus transregulatory protein Tat with N-methyl-d-aspartate receptor subunits.

We investigated the effect of type 1 human immunodeficiency virus (HIV-1) regulatory protein Tat on N-methyl-d-aspartate (NMDA) receptors expressed in Xenopus oocytes by voltage-clamp recording and its role in NMDA-mediated neurotoxicity using cultured rat hippocampal neurons. Tat (0.01-1muM) potentiated NMDA-induced currents of recombinant NMDA receptors. However, in the presence of Zn(2+), the potentiating effect of Tat was much more pronounced, indicating an additional Zn(2+)-related effect on NMDA receptors. Consistently, Tat potentiated currents of the particularly Zn(2+)-sensitive NR1/NR2A NMDA receptor with a higher efficacy, whereas currents from a Zn(2+)-insensitive mutant were only marginally augmented. In addition, chemical-modified Tat, deficient for metal binding, did not reverse Zn(2+)-mediated inhibition of NMDA responses, demonstrating that Tat disinhibits NMDA receptors from Zn(2+)-mediated antagonism by complexing the cation. We therefore investigated the interplay of Tat and Zn(2+) in NMDA-mediated neurotoxicity using cultures of rat hippocampal neurons. Zn(2+) exhibited a prominent rescuing effect when added together with the excitotoxicant NMDA, which could be reverted by the Zn(2+)-chelator tricine. Similar to tricine, Tat enhanced NMDA-mediated neurotoxicity in the presence of neuroprotective Zn(2+) concentrations. Double-staining with antibodies against Tat and the NR1 subunit of the NMDA receptor revealed partial colocalization of the immunoreactivities in membrane patches of hippocampal neurons, supporting the idea of a direct interplay between Tat and glutamatergic transmission. We therefore propose that release of Zn(2+)-mediated inhibition of NMDA receptors by HIV-1 Tat contributes to the neurotoxic effect of glutamate and may participate in the pathogenesis of AIDS-associated dementia.

Inhibition of histone-mediated gene transfer in eucaryotic cells by anti-histone IgG.

In our laboratory, the gene transfer efficiency of some lipofection reagents (lipofectine, lipofectamine, DOTAP and Dosper) and histones H3 and H4 was compared to that of DEAE-Dextran (64). The histones H3 and H4 were found to have the highest transfection efficiency of all the agents tested. In the present study we have analyzed other parameters important for gene delivery by the histones H3 and H4. We transferred the HIV-1 tat gene to Jurkat cells and measured the transactivation of HIV-1-LTR by the transactivator protein, expressed in Jurkat cells. The expression of CAT as a reporter gene hybridized to LTR was a direct measure of transactivation potential. In order to investigate whether the transfection was only due to the positive ionic character of the histones H3 and H4 we tested other histones (H1 and H2A) and polylysine in our system. Under our experimental conditions, neither polylysine, nor the histones H1 and H2A were able to promote gene transfer in Jurkat cells. The inability of these reagents to promote gene transfer was not dependent on DNA condensation; in EMSA (Electrophoretic Mobility Shift Assay) all these reagents exhibited a strong retardation of DNA. In the presence of anti-histone-IgG the transfection potential of histones H3 and H4 was diminished in a concentration - dependent manner. To investigate whether the histone antibodies inhibited the condensation of DNA by histones we carried out gel retardation assays (EMSA) in the absence and in the presence of histone antibodies. Anti-histone-IgG had no effect on the retardation of histone-DNA complexes; on the contrary, retardation was increased. This observation has led us to postulate two models for the possible mechanism by which the histones H3 and H4 catalyze gene transfer in eucaryotic cells.

Expression and purification of recombinant HIV-1 tat protein using HIV-1-tat specific monoclonal antibodies.

We expressed tat protein from HIV-1 coding AA 1-67 (strain HIV-Bru) in the inducible vector pTrc 99A in E. coli. The tat coding region was cloned in the ATG site of the expression vector. A sequence coding for 15 AA was added at the 3'region as a molecular marker. After sonification, the tat protein was routinely detected in Western blots using the Mab developed by us. Following precipitation and centrifugation the resulting pellets were dissolved and purified by three different methods: 1. immunoaffinity-chromatography using Affi-gel HZ coupled with a Mab recognizing the N-terminal sequence of HIV-1-tat; 2. ion exchange chromatography using DEAE-52 cellulose, and 3. isoelectric focusing in free solution. The resulting protein extracts obtained from the three purification protocols were checked in ELISA with the antibody. The peak fraction from all the procedures showed tat activity. No cross reaction in the presence of sera from uninfected persons was observed. The results showed that the purification of tat protein using monoclonal antibodies leads to highly purified preparations.

Inhibition of tat-mediated HIV-1-LTR transactivation and virus replication by sulfhydryl compounds with chelating properties.

D-Penicillamine, a structural analog of cysteine, has the ability to chelate metal ions and reacts with cysteine. We have shown earlier that D-Penicillamin is a potential inhibitor of tat-mediated transactivation of HIV-1-LTR (14) and possesses anti-HIV-1 activity (23). Following this approach, we evaluated the anti-tat and anti-HIV-1 activity of several sulfhydryl compounds with chelating properties. The tested compounds: N-(2-Mercapto-propionyl)-glycin (MPG), 2,3-Dimercapto-propanol (DMP) and 2,3-Dimercapto-propane-sulfonic acid (DMPS) exhibited an inhibitory effect on the tat-mediated transactivation in Jurkat cells, as well as in U937 cells. The highest inhibitory response was shown by DMP leading to about 50% inhibition of transactivation in Jurkat cels and an 80% inhibition in U937 cells. On the contrary, DMPS (30 micrograms/ml) had no inhibitory effect in U937 cells, but did exhibit a 50% inhibition of transactivation in Jurkat cells at 30 micrograms/ml. The antiviral activity of DMP and DMPS was evaluated in H9 cells. In the concentration range which is effective for antiviral effect, both the compounds were highly cytotoxic. Mercapto-propionyl-glycin, although a weak inhibitor of transactivation, was able to inhibit synctia formation to more than 90% and inhibit the viral antigene expression to about 70%. The concentration of MPG needed to achieve this antiviral effect was very high, but it had no cytotoxicity at this concentration. We suggest that a search for compounds using this approach may be useful in developing potential inhibitors of tat-mediated transactivation.

Antibody spectrum against the viral transactivator protein in patients with human immunodeficiency virus type 1 infection and Kaposi's sarcoma.

We analyzed patterns of antibody response to recombinant transactivator protein (human Immunodeficiency virus type 1 [HIV-1] tat) in serum samples from HIV-1-negative subjects (n = 60), HIV-1-infected asymptomatic patients (n = 20), HIV-1-infected patients with Kaposi's sarcoma (n = 25), and patients with Kaposi's sarcoma without HIV-1 infection. None of the healthy subjects possessed anti-tat immunoglobulin G (IgG) in their serum. All asymptomatic patients with HIV-1 infection were anti-tat IgG-positive. Epitope mapping revealed that these sera had anti-tat IgG to all the functional domains of tat protein. Histochemical studies on lymph nodes from five asymptomatic HIV-1-infected patients showed that, in all cases, tat-positive cells were present within the germinal center at the stage of follicular fragmentation containing immunoblasts and small lymphocytes. Of the 25 HIV-1-infected patients with Kaposi's sarcoma, 4 were anti-tat IgG-positive; however, the epitope analysis revealed that IgG to functional domains of tat protein--in particular to transactivating response element (TAR)-binding site--were absent. All patients with Kaposi's sarcoma without HIV-1 infection were anti-tat IgG-negative. Presence or absence of anti-tat IgG and a prevalence of different antibody profiles in different groups of patients indicated the pathophysiologic role of tat protein. Thus, a passive immunization with anti-tat IgG could be a useful strategy to influence the pathophysiologic state of the disease.

Peritoneal macrophages from 17 alpha-ethinylestradiol-treated NMRI mice secrete transformation-specific low molecular weight proteins.

Analysis of protein secretion was performed for a macrophage-like cell line, which was established from the peritoneal cells of NMRI mice treated with 17 alpha-ethinylestradiol. The protein secretion pattern was investigated by computerized analysis of high resolution two-dimensional gel electrophoresis (2-D PAGE) and compared to that of control macrophages, intraperitoneally activated by bacterial lipopolysaccharide. The transformed cells encode a number of low molecular weight proteins (10-20 kDa), which were not observed in control cells under identical experimental conditions. In conclusion the transformation of peritoneal macrophages by 17 alpha-ethinylestradiol leads to an upregulation of polypeptides, in particular of low molecular weight proteins. A high similarity between the induced low molecular weight protein secretion by macrophages of 17 alpha-ethinylestradiol-treated and that of 2,3,7,8-tetrabromodibenzo-p-dioxin-treated mice was found.

Intercellular traffic of human immunodeficiency virus type 1 transactivator protein defined by monoclonal antibodies.

Monoclonal antibodies (mAbs) directed against the amino-terminal region (N-terminal sequence 2-19) of transactivator protein (tat) of HIV-1 have been shown to inhibit intercellular transactivation mediated by the extracellular tat protein. The intracellular transactivation was not significantly affected by anti-tat mAbs. The specificity of anti-tat mAbs in abolishing the transactivating potential of extracellular tat is documented by studies with mAbs to HIV-1 reverse transcriptase, or to a human mammary cancer protein. None of these antibodies showed any inhibitory effect on intercellular transactivation. Specific interaction of anti-tat IgG with tat protein expressed in Jurkat cells is further supported by experiments on immunoblotting. Extracellular tat is responsible for signals which induce a variety of biological responses in HIV-infected cells, as well as in uninfected cells. The fact that anti-tat mAbs can abolish the intercellular traffic of tat protein offers a unique strategy in the development of vaccines against AIDS.

Detection of distinct patterns of anti-tat antibodies in HIV-infected individuals with or without Kaposi's sarcoma.

Patterns of antibody response to recombinant transactivator protein (HIV-1 tat) in serum samples from HIV-1-negative persons (n = 60), HIV-1-infected asymptomatic persons (n = 20), HIV-1-infected people with Kaposi's sarcoma (n = 25) and of people with Kaposi's sarcoma without HIV-1 infection have been analyzed. None of the healthy people had anti-tat IgG in their serum. All asymptomatic patients with HIV-1 infection were anti-tat IgG-positive. Epitope mapping revealed that these sera have anti-tat IgG to all the functional domains of tat protein. Four of the 25 HIV-1-infected patients with Kaposi's sarcoma were anti-tat IgG-positive; however, epitope analysis revealed that IgG to functional domains of tat protein, in particular to TAR-binding site, were absent. All patients with Kaposi's sarcoma without HIV-1 infection were anti-tat IgG-negative. Presence or absence of anti-tat IgG, and prevalence of different antibody profiles in different groups of patients suggest the pathophysiologic role of tat protein. Thus, a passive immunization with anti-tat IgG could be a useful strategy to influence the pathophysiologic state of the disease.

Histone-mediated transfer and expression of the HIV-1 tat gene in Jurkat cells.

We studied the gene transfer efficiency of lipofection reagents in comparison to DEAE-Dextran. DOTAP, Dosper, and Lipofectin have lower transfection efficiency; Lipofectamine has a 2.5-fold better efficiency compared with DEAE-Dextran. We report a novel and highly efficient DNA transfer system based on the DNA-binding proteins histone 3 and histone 4. We have transferred the HIV-1 tat gene and measured the transactivation of HIV-1 LTR by the transactivator protein, expressed in Jurkat cells. The HIV-1 LTR was linked to the CAT gene as a reporter. Compared to DEAE-Dextran-mediated transfection, histone-mediated transfection resulted in a sevenfold higher expression of the CAT gene. The maximum transfection efficiency mediated by histones is dependent on the relative concentration (DNA:histone ratio) and the incubation time. In a gel-retardation assay, an optimal complex formation was observed under the same conditions that allowed the highest transfection efficiency. This ability of histones to increase the delivery and transgenic expression of foreign DNA in eukaryotic cells is not simply due to the positive ionic character of the histone proteins. Polylysine, histone H1, and histone H2A were unable to mediate gene transfection in our system. Monoclonal antibodies that recognize antigenic determinant present on all five histone proteins (anti-histone, pan) were able to neutralize the transfection-enhancing potential of histone 3 and histone 4. However, anti-histone IgG enhanced the retardation of mobility of histone-DNA complexes. The results of this study allow us to conclude that histones H3 and H4 can catalyze gene transfer and gene expression in eukaryotic cells without any requirement for additional constituents. For this reason, we have termed the new gene-delivery system as histonefection.

Low molecular weight proteins secreted by peritoneal macrophages obtained from 2,3,7,8-tetrabromodibenzo-p-dioxin-treated NMRI mice.

The protein secretion patterns in a macrophage-like cell line (CBrD), established from the peritoneal cells of NMRI mice treated with the dioxin analog 2,3,7,8-tetrabromodibenzo-p-dioxin (TBrDD), were analyzed by high resolution two-dimensional gel electrophoresis (2-D PAGE), and compared to the pattern of proteins secreted by control macrophages which were intraperitoneally activated by bacterial lipopolysaccharide. The most striking alterations were observed in the low molecular range. The transformed cells encode a number of low molecular mass proteins (10-20 kDa) which were not detected in control cells under identical experimental conditions. The protein pattern with respect to isoelectric point, molecular weight, optical density (OD) and area of the spot (in mm2) has been depicted by computer analysis in relation to a standardized spot outline and the spot's background (in OD). It is concluded that the transformation of murine peritoneal macrophages by TBrDD leads to an upregulation of proteins, in particular of low-molecular-weight proteins.

Characterization of the RNA dependent DNA polymerase of bovine leukemia virus.

The reverse transcriptase-RNA dependent DNA polymerase of Bovine Leukemia Virus (BLV) was isolated and characterized. The enzyme has a molecular weight of about 80kd and the isoelectric point is 7.6. The enzyme prefers magnesium, as a divalent cation using synthetic homopolymeric template primer poly (C) oligo (dG). Monoclonal antibodies directed against reverse transcriptase of human immunodeficiency virus type I (HIV-I) did not crossreact with the isolated polymerase.

Expression of a reverse transcriptase activity in a cell line established from peritoneal macrophages of mice treated with N-methyl-N-nitrosourea.

We have established a cell line from peritoneal macrophages of mice treated intraperitoneally with N-methyl-N-nitrosourea. The present communication describes the identification of an RNA-dependent DNA-polymerase activity in a particulate fraction from supernatants of the cell culture. This activity is similar to retroviral Reverse Transcriptase (RT) based on its template specificity and ionic preference. The proof of the retrovirus-like nature of RT was obtained by ultracentrifugation of the pelleted proteins secreted in the medium on a sucrose gradient. The main RT activity was obtained in fractions of 1.14-1.16 g/ml densities, which are comparable to those of type C retroviruses. The presented data support constitutive expression of the retrovirus gene in a chemically transformed cell line.

Gene-targeted inhibition of transactivation of human immunodeficiency virus type-1 (HIV-1)-LTR by antisense oligonucleotides.

We have used an in vitro approach to study the efficiency of antisense oligonucleotides in inhibiting LTR-(HIV-1)-directed CAT expression catalyzed by tat protein, the functional protein of the transactivator gene. We selected the target sequence localized near the 5' end of the tat mRNA. The following conclusions can be drawn from the data presented here: a) Antisense oligonucleotides modified by conjugation of cholesterol at the 3' end have a severalfold higher inhibitory response, b) inhibitory response is dependent on the mode of introducing oligonucleotides, and c) the inhibition by antisense oligonucleotides is sequence specific and directed towards the targeted region. This approach could be useful for targeting functional regions of regulatory gene products and designing gene-targeted inhibitors of virus replication.

D-penicillamine inhibits transactivation of human immunodeficiency virus type-1 (HIV-1) LTR by transactivator protein.

D-Penicillamine, an amino acid analogue of cysteine, has been shown to inhibit the transactivation of HIV-1 LTR by the transactivator protein, tat protein. The transactivation was studied in Jurkat cells co-transfected with plasmids containing HIV-LTR sequences fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and HIV tat gene. The expression of CAT activity was a measure of transactivation of LTR by the tat protein. Incubation of transfected Jurkat cells with D-penicillamine led to inhibition of CAT activity. This inhibition was found to be concentration-dependent; more than 90% inhibition of chloramphenicol acetylation was seen in extracts prepared from cultures incubated with 40 micrograms/ml of D-penicillamine. Earlier experiments have shown that D-penicillamine at 40 micrograms/ml can completely inhibit HIV-1 (HTLV-III B) replication in H9 cells [(1986) Drug Res. 36, 184-186]. These results suggest that inhibition of transactivation may be the molecular mechanism involved in the inhibition of HIV-1 replication by D-penicillamine.

Serological relationship between reverse transcriptases from human T-cell lymphotropic viruses defined by monoclonal antibodies. Evidence for two forms of reverse transcriptases in the AIDS-associated virus, HTLV-III/LAV.

The immunological relationship between reverse transcriptases purified from human T-cell lymphotropic viruses (HTLV-I, HTLV-II, HTLV-III) was defined using monoclonal antibodies specific for HTLV-III reverse transcriptase, secreted by a mouse/mouse hybridoma clone (4F8) developed in our laboratory. The viral proteins from HTLV-I and HTLV-II do not bear any cross-reactive epitope to antibodies secreted by this clone. These antibodies specifically cross-react with HTLV-III reverse transcriptase. The antibodies failed to neutralize the catalytic activity of reverse transcriptase; however, after immunoprecipitation with a magnetic conjugate of goat anti-mouse IgG, the residual activity was completely inhibited. This shows that the antibodies are not directed towards the catalytic active center of the enzyme. Using an immunoblotting technique (Western blotting), we have found two cross-reactive proteins with HTLV-III lysate with molecular masses of 53 and 66 kDa. This suggests that HTLV-III possesses two reverse transcriptase activities with a common determinant recognized by the same epitope.

A study of antitemplate inhibition of mammalian, bacterial and viral DNA polymerases by 2- and 2'-substituted derivatives of polyadenylic acid.

In the present study, effects of various 2- and 2'-substituted polyadenylic acid analogs on eukaryotic, bacterial and viral DNA polymerases were investigated. The polymer containing 2'-deoxy-2'fluoroadenosine, (dAfl)n, showed a concentration dependent stimulation of (rA)n . (dT)12-catalyzed reverse transcriptase reaction from Rauscher Leukemia Virus (RLV). A similar stimulation of the (rA)n . (dT)12-catalyzed DNA polymerase-gamma reaction was also observed. However, the (rC)n . (dG)12-dependent reverse transcriptase activity was inhibited by (dAfl). The DNA polymerase-beta activity catalyzed by (dA)n . (dT)12 was also inhibited by (dAfl)n. The reported data indicate that (dAfl)n closely resembles (rA)n as a functional template. In contrast, the 2-substituted derivatives, poly(2-methylthioadenylic acid) and poly(2-ethylthioadenylic acid), are not able to discriminate between the reactions catalyzed by different templates. For example, both derivatives inhibit (rA)n . (dT)12- and (rC)n . (dG)12-catalyzed reverse transcriptase reaction to the same extent; though the methylthio derivative is a much better inhibitor than the ethylthio analog. The DNA polymerase-alpha was less sensitive to these inhibitors; whereas the bacterial DNA polymerase (Kornberg enzyme; DNA polymerase I) was completely resistant to the action of all the derivatives used in this study.