Piperlongumine increases the apoptotic effect of doxorubicin and paclitaxel in a cervical cancer cell line.

OBJECTIVE: Piperlongumine (PL) is an alkaloid derived from the edible pepper (Piper longum L) and it has been described to have various biologic activities including anticancer effects. Our aim in this study was to assess the cytotoxic role of PL on a cervical cancer cell line (HeLa) and to evaluate the effects of PL/doxorubicin and PL/paclitaxel combination therapies on apoptotic cancer cell death. MATERIAL AND METHODS: The cytotoxicity, IC50 doses by MTT assay confirmed by fluorescent imaging, and apoptotic cell rates by Annexin V staining using flow cytometry were determined for PL, doxorubicin, paclitaxel, and for their combinations. RESULTS: It was shown that the PL by itself induced the apoptosis in HeLa cells. PL in combination with doxorubicin and paclitaxel increased apoptotic cell death compared to either chemotherapeutic agent alone. CONCLUSION: We conclude that the PL inhibits cancer cell growth by inducing apoptosis and has a potential anticancer activity in cervical cancer, especially when combined with doxorubicin and paclitaxel.

Cytotoxic Antibody Detection by Means of Flow-Cytometric Cross-Match.

BACKGROUND: Complement-dependent lymphocytotoxicity (CDC-XM) and flow-cytometric (FCXM) cross-match are analyzed individually for each donor and recipient pair, because these techniques have fundamental differences for the evaluation of histocompatibility. Lately, cytotoxic flow-cytometric cross-match (cFCXM) has been developed as an alternative to both CDC-XM and FCXM techniques. We evaluated the limits of cFCXM with the use of different positive serum dilutions. METHODS: CDC-XM, FCXM, and cFCXM tests were performed with the use of commercially available negative and positive serum samples and lymphocytes from healthy donors. RESULTS: Complement-dependent cell death was successfully detected with the use of cFCXM. Complement-dependent cell death ratios in cFCXM were similar those in CDC-XM. With cFCXM, not only complement-dependent cell death but also IgG binding could be detected within a single assay. At higher concentrations of the positive serum, IgG-fluorescein isothiocyanate (FITC) mean fluorescent intensity (MFI) values detected with the use of cFCXM were less than those of conventional FCXM. Correspondingly, for dead cells, MFI values of IgG-FITC were less than those of live cells in higher positive serum concentrations in the cFCXM assay. Moreover, our results demonstrated that in cFCXM analysis, the decreasing ratio of dead cells at increasing positive serum dilutions was not in parallel with the same decrease in IgG-FITC MFI values. CONCLUSIONS: The cFCXM technique detects complement-mediated cytotoxic cell death with the additional ability to show IgG binding in the same tube and therefore may reduce the necessary bench time and workload.

Two novel mutations in the 3' untranslated region of the beta-globin gene that are associated with the mild phenotype of beta thalassemia.

INTRODUCTION: There are approximately 800 different genomic alterations of the ß-globin gene described in the human hemoglobin variant (HbVar) database. In this study, we have identified two novel putative mutations (HBB:c.*+108 A>G and HBB:c.*+132 C>T) in the 3' untranslated region (3'-UTR) of the ß-globin gene and describe their clinical implications. METHODS: Four patients from two unrelated families, all with hematological and clinical features associated with beta-thalassemia (ß-thal), and their family members were included. The molecular diagnoses of the ß-globin gene mutations were performed by direct sequencing. RESULTS: A novel mutation, HBB:c.*+108 A>G, was found in combination with the IVS-I-110 G>A (HBB:c.93-21 G>A) mutation in three siblings (two brothers and one sister) from one of the families involved in our study. Their mother was found to be a carrier for HBB:c.*+108 A>G with normal HbA2 levels. The other novel mutation, HBB:c.*+132 C>T, was found in combination with IVS-I-1 G>A (HBB:c.92 + 1G>A) in a 7-year-old boy diagnosed as ß-thal intermedia from the second family. His father and two brothers were all carriers of HBB:c.*+132 C>T with borderline HbA2 levels. CONCLUSION: Based on the observed ß-thal intermedia phenotypes and the accompanying mutations, we conclude that these novel ß-globin gene 3' UTR mutations are associated with the mild phenotype of ß-thal.

C.428_451 dup(24bp) mutation of the ARX gene detected in a Turkish family.

ARX mutations give rise to both syndromic and nonsyndromic forms of mental retardation (MR). We investigated the most common ARX mutations, c.428_451 dup(24bp) and c.333ins (GCG)7 in a series of 370 mentally retarded FMR1 (CGG)n expansion mutation negative Turkish patients using PCR amplification and high resolution MetaPhor agarose gel electrophoresis. Sequence analysis was also performed for confirmation and discrimination of the mutations. One patient representing non-syndromic X-linked MR showed an abnormal band pattern on agarose gel and sequence analysis of exon 2 of the ARX gene revealed that the patient had the c.428_451 dup(24bp) mutation. When we screened the family members, we found that his sister and mother were also carrier for the same mutation. The proband showed mild MR and subtle clinical findings like dysarthria and lack of fine motor functions. In conclusion, the patients with weak fine motor skills and positive family history for X-linked MR should be screened for the most common ARX gene mutations.

Frequencies of four genetic polymorphisms in the CYP1A2 gene in Turkish population.

Cytochrome P450 (CYP) 1A2 gene is involved in the metabolic activation of several carcinogens and altered metabolization of some clinically used drugs. We aimed to investigate the distributions of genetic polymorphisms -3860 (G/A)(CYP1A2*1C) and -2467 (T/del)(CYP1A2*1D) in the 5'-flanking region and -739 (T/G)(CYP1A2*1E) and -163(C/A)(CYP1A2*1F) in the first intron of the CYP1A2 gene in 110 unrelated healthy Turkish volunteers by PCR-RFLP technique. The frequencies of each polymorphism in Turkish population were found as 0.04, 0.92, 0.01, 0.27 for CYP1A2*1C, CYP1A2*1D, CYP1A2*1E, CYP1A2*1F, respectively. Compared with other populations, CYP1A2*1D has been found to be significantly increased in Turkish population. On the other hand, in general, the frequencies of the other polymorphisms were concordant with those in the Egyptian and Caucasian populations, and were different from those in the Japanese, Chinese and Ethiopian populations. Our results suggest that due to increased frequency of CYP1A2*1D in Turkish population, functional significance of CYP1A2*1D should be evaluated. It might be screened to determine the relationship between CYP1A2*1D and CYP1A2 related drug metabolisms in associated groups.

The effect of MDR1 (ABCB1) polymorphism on the pharmacokinetic of tacrolimus in Turkish renal transplant recipients.

There is marked interindividual variability in trough blood levels of tacrolimus (TRL) following standard dosing. TRL is a substrate for P-glycoprotein (P-gp), the product of the multidrug resistance-1 (MDR1)(ABCB1) gene. P-gp acts as a membrane efflux pump, which affects TRL absorption from the gut. Some of the single nucleotide polymorphisms (SNP) of ABCB1 gene are associated with pharmacokinetic characteristics of TRL. The objective of this study was to determine the role of ABCB1 C3435T polymorphism on TRL dose requirements, trough values and dose-adjusted trough TRL concentrations among Turkish renal transplant recipients. Renal transplant recipients receiving TRL (n=92) were genotyped for ABCB1. TRL daily doses, trough concentrations, dose-adjusted trough concentrations, demographic features, and clinical data were obtained at 1, 6, and 12 months after renal transplantation. The frequency of the ABCB1 3435 CC genotype was 30.4%, whereas 47.8% of patients were 3435 CT and 21.7% of patients were 3435 TT. TRL daily doses were significantly lower among patients with the 3435 TT genotype at months 1 and 6. At 6 and 12 months after transplantation patients who were homozygous for the ABCB1 3435 CC showed significantly lower dose-adjusted trough TRL concentrations compared with subjects of 3435 TT and CT genotypes. Knowledge of ABCB1 genotype may be useful to adjust the optimal dose of TRL in transplant patients, thereby rapidly achieving target concentrations.

Novel cytogenetic findings revealed by conventional cytogenetic and FISH analyses in leukaemia patients.

AIM: To describe novel cytogenetic findings in four leukaemia patients. METHODS: Conventional cytogenetic (CC) and fluorescence in situ hybridization (FISH) analyses were performed on bone marrow samples of four leukaemia patients. RESULTS: In this study, t(3;10)(q11;q25) and t(2;22)(p21;q11.2) were detected as novel translocations. t(8;16;21)(q22.1;q13;q22) and t(1;6;9;22)(p36.1;p21.3;q34;q11) were found as variant translocations, and these variant translocations were confirmed by Interphase-FISH and Multi-colour-FISH. CONCLUSION: Newly identified cytogenetic findings can lead us to characterize cytogenetic evolution of the haematological malignancies. Further investigations are certainly warranted to resolve the prognostic impact of these new cytogenetic abnormalities.

Molecular analysis of fragile X syndrome in Antalya Province.

BACKGROUND: Detection of the (CGG)n repeats in the FMR1 gene that cause the fragile X syndrome (FXS), has become a milestone for phenotype-genotype correlation in FXS. AIMS: To screen the FMR1 gene CGG repeats in index cases with FXS and their family members in the Antalya Province. SETTING AND DESIGN: This study was prospectively conducted between January 2000 and March 2005 in Department of Medical Biology and Genetics, Faculty of Medicine, Akdeniz University, Antalya. MATERIALS AND METHODS: A series of 132 cases from three hospitals in Antalya Province were studied. All cases were molecularly screened using non-radioactive Expand Long PCR method that was confirmed by Southern blotting. RESULTS: Seventeen out of 132 cases were found to have a full mutation, including three that were mosaic for premutations/full mutations. Of the 132 cases, eight were found to have the premutation size of the CGG repeats. The remaining 107 cases were identified as normal. CONCLUSIONS: Due to premature ovarian failure and Fragile X premutation Tremor/Ataxia Syndrome related with the premutation, the detection of the premutation will provide valuable information both for clinical follow-up and genetic counseling. In conclusion, our data suggest that expansion of CGG repeats in the FMR1 gene can be analyzed by Expand Long PCR, an efficient and non-radioactive method that can be used to monitor the expansion of premutation to full mutation, which would eventually lead to reduce the FXS prevalence.

Consanguineous marriages in the province of Antalya, Turkey.

To assess the trends in the frequency and the medical effects of consanguinity in the south coast of Turkish population using local and national data in the last 11 years. This cross-sectional study was carried out in Manavgat province, which is a major tourism center on the Mediterranean coast of Turkey. The authors studied consanguineous marriages in rural and urban population in the Mediterranean coast, Manavgat province, Turkey, via a 1500 random survey sample of married couples. There has been a significant increase in the incidence of consanguineous marriages in rural areas (40.7%) since 1989 in the southern population of Turkey. The results showed that the most frequent type of marriage was between the first cousins. It is found that there is no statistically significant difference between the consanguineous and non-consanguineous marriages in the different age groups. The results were discussed on the basis of educational status, reasons for having consanguineous marriages and the general medical effects as well as with the relation of congenital malformations. The custom of consanguineous unions in the Mediterranean population of Turkey is still extremely high, and preventive measures should be done to decrease its frequency and associated complications.

EsMlp, a muscle-LIM protein gene, is up-regulated during cold exposure in the freeze-avoiding larvae of Epiblema scudderiana.

Screening of a cDNA library identified transcripts that were up-regulated by cold (4 or -20 degrees C) exposure in larvae of the freeze-avoiding goldenrod gall moth, Epiblema scudderiana. One clone contained a full-length open reading frame encoding a protein of 94 amino acids. The gene product, with 79.1% of residues identical with the Drosophila LIM protein Mlp60A, was named EsMlp and contained a single LIM domain and consensus sequences characteristic of a LIM protein. Transcript levels rose approx twofold when larvae were shifted from 4 to -20 degrees C and approx threefold over the midwinter months compared with larvae sampled in October or April. EsMlp expression was high in larval head (possibly due to expression in pharyngeal muscles) and body wall but was not detected in fat body. Immunoblotting revealed a three- to fourfold increase in EsMlp protein in midwinter larvae (January-February) compared with November-collected animals and a further rise to eightfold higher than November values in larvae collected in April. Cold up-regulation of EsMlp and the pattern of EsMlp levels in the larvae suggest possible roles for the protein, such as in muscle maintenance over the winter or as a preparative function that could facilitate the rapid resumption of development and metamorphosis when environmental temperatures rise in the spring.

Nature and control of chloride transport in insect absorptive epithelia.

Insect epithelia most commonly absorb from KCI-rich, low Na+ fluids. This is true of the locust hindgut, which is functionally analogous to vertebrate kidney tubules. Active absorption of Cl- at the apical membrane is the predominant transport process giving rise to a large short-circuit current (Isc) after stimulation by neuropeptides (CTSH, ITP) via cAMP as second messenger. This Cl- transport is not coupled to or driven secondarily by Na+, K+, HCO3-, Ca2+, or Mg2+ movements. An apical V-type H+ ATPase acidifies the hindgut lumen but at a rate that is 10-15% of Cl-dependent Isc. The evidence to date as to whether the resulting large apical proton gradient is used to drive Cl- transport secondarily by an apical H+/Cl- symport is mixed. Thus a primary mechanism of Cl- absorption remains an alternative possibility. The complete primary structure of a large neuropeptide stimulant (ITP: 72 amino acids) of locust ileal Cl- transport has recently been deduced from its cDNA. This is the first putative insect neuropeptide hormone shown to stimulate ion transport across absorptive epithelia for which the primary sequence has been deduced.