TSGA10 is a novel candidate gene associated with acephalic spermatozoa.

Acephalic spermatozoa is a rare teratozoospermia associated with male infertility. However, the pathogenesis of this disorder remains unclear. Here, we report a 27 years old infertile male from a consanguineous family, who presented with 99% headless sperm in his ejaculate. Electron microscopic and immunofluorescence analysis suggested breakage at the midpiece of the patient's sperm cells. Subsequent whole-exome sequencing analysis identified a homozygous deletion within TSGA10 (c.211delG; p.A71Hfs*12), which resulted in the production of truncated TSGA10 protein. TSGA10 is a testis-specific protein that localized to the midpiece in the spermatozoa of a normal control; however, immunostaining failed to detect TSGA10 protein in the patient's sperm. Western blot analysis also showed complete absence of TSGA10 protein in the patient. One cycle of in vitro fertilization-assisted reproduction was conducted, but pregnancy was not achieved after embryo transfer, possibly due to poor embryo quality. Therefore, we speculate that the presence of rare sequence variants within TSGA10 may be associated with acephalic spermatozoa in humans.

DNAH6 is a novel candidate gene associated with sperm head anomaly.

Globozoospermia and acephalic spermatozoa are two rare sperm head anomalies associated with male infertility. Combination of the two phenotypes in the same patient is extremely rare, so the underlying pathogenesis of this disorder remains unclear. Here, we report a 35-year-old infertile male, who presented with 30% of sperm-lacked heads and 69% of sperm round-headed or small-headed with neck thickening in his ejaculate. Subsequent whole-exome sequencing (WES) analysis identified compound heterozygous variants within the DNAH6 gene. DNAH6 is a testis-specific-expressed protein that was localised to the neck region in the spermatozoa of normal control; however, immunofluorescent staining failed to detect DNAH6 protein in the patient's spermatozoa. Quantitative real-time PCR analysis also showed the complete absence of DNAH6 mRNA in the patient's spermatozoa. Moreover, two cycles of in vitro fertilisation (IVF)-assisted reproduction were carried out, but pregnancy was not achieved after embryo transfer. Therefore, rare sequence variants in DNAH6 might be susceptibility risks for human sperm head anomaly.

Headless spermatozoa in infertile men.

Spermatozoa morphology, an important parameter in a semen specimen's potential fertility evaluation, is a significant factor for in vitro fertilisation in assisted reproductive technology. Eleven sterile men with headless spermatozoa, a type of human teratozoospermia, are presented. Their ejaculates' headless spermatozoa percentages were high with rare normal spermatozoa forms. Additionally, abnormal morphology (e.g. round-headed or microcephalic spermatozoa) was also found. Spermatozoa motility was somewhat affected, potentially because of the missing mitochondrial sheath at the sperm tail base. Patients who underwent assisted reproductive technology treatment experienced adverse pregnancy outcomes. Work types and corresponding environments seemed irrelevant, but specific family history may have prompted its genetic origin. Computer-assisted semen analysis systems easily mistake headless spermatozoa as oligozoospermia because of nonrecognition of the loose head. However, morphological testing, especially with an electronic microscope, clearly identifies abnormal spermatozoa. Future exploration requires more methods investigating the frequency and percentage of this morphological abnormality in different populations with varied fertility levels. Such research would estimate the probable correlation of the abnormality with other semen parameters and examine the potential developmental or genetic origins. During clinical work, medical staff should detect these cases, avoid misdiagnosis and provide proper consultation about diagnosis and assisted reproductive technology treatment.

Abnormal expression of PTEN and PIK3CA in pemetrexed-resistant human pancreatic cancer cell line Patu8988.

The aim of this study was to investigate the expression of PTEN and PIK3CA in the pemetrexed-resistant human pancreatic cancer cell line Patu8988, and to evaluate their effects on the biological behavior of pancreatic cancer cells. PTEN and PIK3CA gene and protein expressions were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot, respectively, in a pemetrexed-resistant pancreatic cancer cell line and in the parent strain of the pancreatic cancer cells. The discrepancies between the two types of cell lines were detected by a transwell test. RT-PCR and western blot analyses revealed that PTEN and PIK3CA were overexpressed in the pemetrexed-resistant pancreatic cancer cell line. PTEN and PIK3CA were shown to be upregulated by 89 and 76% (western blot), respectively, in the pemetrexed-resistant cell line, compared to the normal pancreatic cancer cell line. The migratory and invasive abilities of the pemetrexed-resistant pancreatic cancer cell were significantly reduced compared to those of the parent strain (P < 0.05; transwell assay). Both PTEN and PIK3CA expression was abnormally enhanced in the pemetrexed-resistant cell line Patu8988; the co-existence of high levels of PTEN and PIK3CA in the pemetrexed-resistant pancreatic cancer line cells induced a significant decrease in their migratory and invasive capacities. This suggested that the mechanism of pemetrexed resistant may be affected by PTEN and PIK3CA, and that these may alter the biological behavior of cancer cells.

Genetic variations in MOV10 and CACNB2 are associated with hypertension in a Chinese Han population.

Human hypertension is a complex, multifactorial disease. Multiple variants associated with hypertension have been identified in the large numbers of genome-wide association studies, meta-analysis, and case-control studies. The present study investigated the association between the single nucleotide polymorphisms (SNPs) of five candidate genes and the susceptibility and prognosis of hypertension in a Chinese Han population. A hospital-based case-control study in a Chinese Han population was carried out, including 500 hypertension patients and 506 healthy controls. The five SNP markers were detected using the Sequenom MassArray(®) iPLEX System. The association of genotypes with susceptibility to hypertension was analyzed using odds ratio, with 95% confidence interval and logistic regression. All five variants conformed to Hardy-Weinberg proportions in the controls. No significant differences were noted in the genotype distributions for AGTR1, PRRC2A, and CALCA polymorphisms in patients with hypertension (N = 500) and healthy controls (N = 506). SNP rs2932538, a variant in MOV10, was found to be significantly associated with an increased risk of hypertension. However, SNP rs4373814, a variant in CACNB2, showed a relevant association with a decreased risk of hypertension. In conclusion, the results of our case-control study confirmed the significant association of the SNP rs2932538 in MOV10 and SNP rs4373814 in CACNB2 with an increased risk of hypertension in a Chinese Han population, suggesting that the SNP rs2932538 may be a poor prognostic indicator for hypertension, while SNP rs4373814 may be a good prognostic indicator for hypertension in the same region. However, our findings need to be replicated in larger epidemiological and functional studies.

[Comparison of 1-year outcomes between Roux-en-Y gastric bypass and sleeve gastrectomy with Roux-en-Y duodenojejunal bypass in the patients of obesity with type 2 diabetes mellitus].

Objective: To compare the 1-year effects of Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy with Roux-en-Y duodenal bypass (SG+RYDJB) on weight loss, remission of diabetes, and postoperative complications in patients with obesity and type 2 diabetes. Methods: A single-center retrospective cohort study was conducted at the First Affiliated Hospital of Nanjing Medical University from January 2020 to December 2020. Sixty-four patients with type 2 diabetes and body mass index (BMI) of 27.5-40.0 kg/m2 were included in this study and divided into the RYGB group (n=34) and the SG+RYDJB group (n=30). In both procedures, the biliopancreatic branch was measured 100 cm distal to the Treitz ligament, and the food branch was measured 100 cm distal to the gastric or duodenojejunal anastomosis. Patients were followed up by telephone or WeChat, a free messaging and calling app at 1, 3, 6, and 12 months postoperatively to determine their weight loss and remission of diabetes. The primary outcomes were the weight loss and reduction in blood glucose concentrations at 1 year after surgery and postoperative complications. Other postoperative changes, including body weight, BMI, percentage of total weight loss (%TWL), percentage of excess weight loss (%EWL), glycated hemoglobin A1c (HbA1c), and fasting blood glucose at 1 year after surgery were also assessed. Results: There were no significant differences in baseline data between the two groups (all P>0.05). No conversion to open surgery or death occurred in either group. Operation time was longer in the SG+RYDJB than the RYGB group (137.8±22.1 minutes vs. 80.0±24.9 minutes, t=9.779, P<0.001) and the incidence of perioperative complications was higher in the SG+RYDJB than the RYGB group (20% [6/30] vs. 2.9% [1/34], χ2=4.761, P=0.029). However, the postoperative hospital stay was similar between the two groups [3.0 (3.0, 4.3) days vs. 3.0 (4.0, 6.0) days, U=641.500, P=0.071]. Perioperative complications comprised small gastric pouch anastomotic leakage in one patient in the RYGB group and leakage (three patients) and bleeding (two patients with gastrointestinal bleeding and one with trocar site bleeding) in the SG+RYDJB group. Long-term complications were as follows. The incidence of anemia was significantly higher in the RYGB than the SG+RYDJB group (26.5% [9/34] vs. 3.3% [1/30], χ2=6.472, P=0.011). However, there were no significant differences in incidences of postoperative reflux, dumping syndrome, alopecia, diarrhea, constipation or foul-smelling flatus between the two groups (all P>0.05). Compared with 1 year before surgery, the body weights and fasting plasma glucose concentrations of patients in the SG+RYDJB and RYGB group (72.4±10.6 kg vs. 98.5±14.2 kg, respectively; 68.2±10.0 kg vs. 91.9±14.8 kg, respectively), BMI (25.2±2.9 kg/m2 vs. 34.3±4.2 kg/m2, respectively; 24.3±2.4 kg/m2 vs. 32.7±3.7 kg/m2, respectively) (5.5±1.6 vs. 10.6±3.3, respectively; 5.8±2.1 vs. 9.0±3.4, respectively); HbA1c (5.7±0.8 vs. 9.7±1.2, respectively; 9.1±1.9 vs. 5.9±0.9, respectively) were significantly lower at 1 year after surgery (all P<0.05). However, the % TWL (26.5%±6.0% vs. 25.6%±4.4%, t=0.663, P=0.510) and % EWL (109.1%±38.2% vs. 109.4%±40.3%, t=-0.026, P=0.026), rate of complete remission of diabetes at 1 year (80.0% [24/30] vs. 82.4% [28/34], χ2=0.058, P=0.810] did not differ significantly between the two groups (all P>0.05). Conclusions: Although SG+RYDJB surgery compared with RYGB is more difficult to perform, it can achieve similar weight loss and remission of diabetes and is associated with a lower incidence of anemia because of the preservation of the pylorus.

[Analysis of early severe postoperative complications and risk factors in 4255 patients who underwent bariatric and metabolic surgery in a single cente].

Objective: To analyze the incidence of early severe complications following bariatric and metabolic surgery and the experience of their diagnosis, treatment, and risk factors. Methods: In this retrospective observational study, the clinical data of 4255 patients who underwent bariatric and metabolic surgery between May 2010 and May 2022 in the Department of Bariatric and Metabolic Surgery of the First Affiliated Hospital of Nanjing Medical University were retrospectively collected. Among these patients, 1125 were male and 3130 were female. The mean age and body mass index (BMI) of the patients at the time of operation were 31.3±4.5 years and 36.5±6.4 kg/m2, respectively. Regarding surgical type, 2397 patients underwent sleeve gastrectomy (SG), 489 underwent Roux-en-Y gastric bypass (RYGB), 1028 underwent sleeve gastrectomy plus jejunojejunal bypass (SG+JJB), and 341 underwent single anastomosis duodenal switch (SADS). The inclusion criteria were patients (1) with a Clavien-Dindo grade of ≥III; (2) who were undergoing SG, RYGB, SG-JJB, or SADS; and (3) who had complete clinical data. The exclusion criteria were patients (1) undergoing revisional surgery and (2) other operations during the bariatric and metabolic surgery. The Clavien-Dindo classification was used to analyze the incidence of early severe postoperative complications and their prognosis. Early severe postoperative complications were defined as Clavien-Dindo ≥ III complications within 30 days after surgery. Meanwhile, multivariate logistic regression model was used to identify risk factors of the complications. Results: Summary of early severe complications following bariatric and metabolic surgery: (1) of the 4255 patients, 22 (12 male and 10 female) exhibited early severe complications (0.52%). The mean age and BMI of these patients were 41.1±9.9 years and 36.9±8.2 kg/m2, respectively. Preoperatively, 7 patients had hypertension, 10 had type 2 diabetes mellitus, 1 had respiratory failure, and 1 had heart failure. The severe complications included 9 patients (0.21%) with grade IIIa, 11 (0.26%) with grade IIIb, 1 (0.02%) with grade IVa, and 1 (0.02%) with grade V complications. The incidences of severe postoperative complications in the different surgical procedures were 0.17% for SG (4/2397), 0.61% for RYGB (3/489), 0.58% for SG+JJB (6/1028), and 2.64% for SADS (9/341). The common severe complications were leakage (0.28%, 12 patients), bleeding (0.14%, 6 patients), and obstruction (0.05%, 2 patients). (2) Management of complications: Grade IIIa complications (including eight patients with leakage and one with severe inflammation) were treated with antibiotics, nasogastric and nutritional tube placements, and CT-guided drainage. For grade IIIa complications, five patients with bleeding were treated with reoperation, and all the patients recovered; four patients with leakage were treated with reoperation, wherein three were converted to RYGB and one patient underwent resuturing of the leakage site; two patients with obstruction were treated with adhesiolysis. The patient with grade IVa complication (including respiratory failure complicated with acid aspiration) was treated in the ICU. For the grade V complication, bleeding in a patient with SG+JJB was treated with reoperation, which confirmed the bleeding of short gastric vessels. Unfortunately, the patient died. (3) Risk factor analysis of early severe complications: univariate analysis detected that sex, age, type 2 diabetes mellitus, operation time, and surgical type were associated with postoperative complications (P<0.05). However, multivariate analysis indicated that an age of ≥31.3 years (odds ratio [OR] = 5.423, 95% confidence interval [CI]: 1.004-29.278, P=0.049) and surgical type (SADS: OR = 19.758, 95%CI: 5.803-67.282, P<0.001; RYGB: OR = 9.752, 95%CI: 2.456-38.723, P=0.001; SG+JJB: OR = 5.706, 95%CI: 1.966- 16.559, P=0.001) were independent risk factors of early severe complications following bariatric and metabolic surgery. Conclusion: Bariatric and metabolic surgery is safe. Its common postoperative complications include leakage, bleeding, and obstruction, which require early detection, diagnosis, and treatment to improve treatment outcomes. Age and surgical type are independent risk factors of early severe complications following bariatric and metabolic surgery.

Salt-responsive outer membrane proteins of Vibrio anguillarum serotype O1 as revealed by comparative proteome analysis.

AIMS: Vibrio anguillarum is a universal marine pathogen causing vibriosis. Vibrio anguillarum encounters different osmolarity conditions between seawater and hosts, and its outer membrane proteins (OMPs) play a crucial role in the adaptation to changes of the surroundings. In the present study, proteomic approaches were applied to investigate the salt-responsive OMPs of V. anguillarum. METHODS AND RESULTS: Lower salinity (0.85% NaCl) is more suitable for growth, survival and swimming motility of the bacterium. Comparative two-dimensional electrophoresis (2-DE) analysis reveals six differentially expressed protein spots among three different salinities, which were successfully identified as OmpU, maltoporin, flagellin B, Omp26La, Omp26La and OmpW respectively. CONCLUSIONS: OmpW and OmpU were highly expressed at 3.5% salinity, suggesting their role in the efficient efflux of NaCl. Maltoporin was downregulated in higher salinity, indicating that higher osmolarity inhibits carbohydrate transport and bacterial growth. Omp26La, the homologue of OmpV, functions as a salt-responsive protein in lower salinity. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report describing salt stress-responsive proteins of V. anguillarum using proteomic approaches. Our results provide a useful strategy for delineating the osmoregulatory mechanism of the marine pathogens.

Effects of polyamines on the thermal stability and formation kinetics of DNA duplexes with abnormal structure.

The effects of ions (i.e. Na+, Mg2+ and polyamines including spermidine and spermine) on the stability of various DNA oligonucleotides in solution were studied. These synthetic DNA molecules contained sequences that mimic various cellular DNA structures, such as duplexes, bulged loops, hairpins and/or mismatched base pairs. Melting temperature curves obtained from the ultraviolet spectroscopic experiments indicated that the effectiveness of the stabilization of cations on the duplex formation follows the order of spermine > spermidine > Mg2+ > Na+ > Tris-HCl buffer alone at pH 7.3. Circular dichroism spectra showed that salts and polyamines did not change the secondary structures of those DNA molecules under study. Surface plasmon resonance (SPR) observations suggested that the rates of duplex formation are independent of the kind of cations used or the structure of the duplexes. However, the rate constants of DNA duplex dissociation decrease in the same order when those cations are involved. The enhancement of the duplex stability by polyamines, especially spermine, can compensate for the instability caused by abnormal structures (e.g. bulged loops, hairpins or mismatches). The effects can be so great as to make the abnormal DNAs as stable as the perfect duplex, both kinetically and thermodynamically. Our results may suggest that the interconversion of various DNA structures can be accomplished readily in the presence of polyamine. This may be relevant in understanding the role of DNA polymorphism in cells.

Nucleotide sequence of a full-length cDNA encoding a common precursor of platelet aggregation inhibitor and hemorrhagic protein from Calloselasma rhodostoma venom.

The nucleotide sequence of a full-length cDNA encoding the common precursor of a platelet aggregation inhibitor, rhodostomin and a hemorrhagic protein from Calloselasma rhodostoma snake venom is presented. The 1.98-kb cDNA contains an open reading frame encoding 478 amino acid residues. The complete structure of the precursor protein encoded by the cDNA is elucidated.

A simple and rapid method for purification of oligodeoxyribonucleoside methylphosphonates.

An alternative dimethoxytrityl-on (dmt-on) method is described to purify hydrophobic oligodeoxyribonucleoside methyl-phosphonates (OM) with a phosphodiester linkage at the 5' end, instead of the conventional dmt-off method using a DEAE ion-exchange column. This method is modified from the reverse-phase method for purification of normal oligonucleotides.

Inhibition of alpha-fetoprotein production in a hepatoma cell line by antisense oligonucleotide analogues.

alpha-Fetoprotein (AFP) is a fetal protein which is absent in adult serum. However, the AFP gene is expressed in some neoplastic cells. According to the literature, AFP may play a role in accelerating the growth of cancer cells. In this report, 15 meric antisense oligonucleotide analogues (phosphorothioates and methylphosphonates) and their chimeric forms, which were complementary to different regions of AFP mRNA, were synthesized, and their physical characteristics such as stability, melting temperature, and toxicity were compared. They were examined as to their inhibitor effects on the translation of AFP mRNA in a AFP-producing hepatoma cell line, HuH-7. We found that chimeric oligomers with methylphosphonate or phosphorothioate linkages at both the 5' and 3' ends were more effective than prototypic oligomers. Inhibition of 72% was achieved with a chimeric oligomer against the translational initiation region, at a concentration of 25 microM. No suppressive effect of the oligomers was observed on cell viability or albumin production, indicating the specificity of the inhibition.

A method for screening antisense oligodeoxyribonucleotides effective for mRNA translation-arrest.

A transcription and translation coupled reticulocyte lysate system was established for rapid screening of antisense oligodeoxyribonucleotides (ODNs) to determine which are most effective for mRNA translation-arrest. A plasmid containing the target cDNA under the control of the T7 (or SP6) promoter was added to the lysate system in the presence of the T7 (or SP6) RNA polymerase, RNase H, and the antisense ODN under test. Transcription and translation were accomplished in a one-tube reaction. Translation-arrest caused by antisense ODN was evaluated in terms of the amounts of de novo-synthesized, [35S]-methionine or [35S]cysteine labeled target protein measured by gel electrophoresis and autoradiography. The properties of this system and optimal reaction conditions for use in antisense ODN screening were determined. Our method is simpler and more rapid than other in vitro screening methods.

Antisense oligodeoxynucleotides of IGF-II selectively inhibit growth of human hepatoma cells overproducing IGF-II.

Insulin-like growth factor II (IGF-II) is expressed in many developing embryonic tissues and is involved in mammalian growth and development. After birth, serum IGF-II is mainly produced by liver cells. Many reports have indicated that IGF-II is overexpressed in some hepatocellular carcinoma (HCC) tissue. These findings imply the possible importance of this growth factor in carcinogenesis. We screened four human HCC cell lines and three rat HCC cell lines and found that HuH-7 and HepG2 cells produced fivefold more intracellular IGF-II than the other cell lines. Experimental data indicate that IGF-II functions through the intracrine mode for HuH-7 cells. To study whether the overexpression of IGF-II is significant for the growth of HCC or only a consequence of HCC development, we used antisense oligodeoxynucleotides (ATON) to arrest the translation of IGF-II mRNA, and then measured the effects on cell growth. We found that the production of IGF-II was suppressed by ATON, and the decrease of IGF-II resulted in growth inhibition of HuH-7 and HepG2. ATON had no effect on the other tested cell lines, which produced lower levels of IGF-II. The growth inhibition was mainly attributed to a decrease of cell proliferative activity. The results indicate that the IGF-II-overproducing cell lines do depend on IGF-II for growth, and ATON of IGF-II can selectively inhibit the growth of these cells. ATON may be a potential therapeutic agent for this type of HCC in vivo.

Regulation of cyclooxygenase and 12-lipoxygenase catalysis by phospholipid hydroperoxide glutathione peroxidase in A431 cells.

Regulation of arachidonate metabolism in human epidermoid carcinoma A431 cells by phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cytosolic glutathione peroxidase (GPx1) was studied. In order to study the effect of reduced glutathione (GSH) on the catalysis regulation of these oxygenation enzymes, diethyl maleate was used to deplete the intracellular GSH. In the presence of 13-hydroperoxyoctadecadienoic acid, the enzymatic catalysis of cyclooxygenase and 12-lipoxygenase was significantly increased in the GSH-depleted cells. In terms of the inhibitory effect on 12-lipoxygenase, PHGPx was more sensitive to GSH concentrations than GPx1. Inhibition of PHGPx activity by the treatment of cells with antisense oligonucleotide of PHGPx mRNA increased the enzymatic catalysis of both cyclooxygenase and 12-lipoxygenase. In conclusion, the results indicate that catalysis of cyclooxygenase and 12-lipoxygenase in A431 cells was regulated by redox-reaction, and PHGPx seems to play an important role in the controlling of these reactions.

Scleral outfolding for macular translocation.

PURPOSE: To report experimental study and clinical observation of scleral outfolding for macular translocation. METHODS: In six human cadaver eyes, circumferential mattress sutures to create sclera infoldings were compared with radial-interrupted mattress sutures to create scleral outfoldings. In a 75-year-old man with macular degeneration and choroidal neovascular membrane, radial-interrupted mattress sutures were used for macular translocation. RESULTS: In the human cadaver eyes, circumferential mattress sutures for scleral infolding created an average decrease in corresponding internal anteroposterior retinal contour of 0.36 mm. Radial-interrupted mattress sutures for scleral outfoldings created an average decrease in the corresponding internal anteroposterior retinal contour of 4. 61 mm. The statistical significance of the difference between infoldings of the sclera versus outfoldings of the sclera had a P value of.0001. CONCLUSIONS: Initial experimental and clinical study suggests that radial-interrupted mattress sutures may generate more shortening of the internal scleral surface and greater macular translocation than circumferential mattress sutures. Additional studies are needed to evaluate the long-term effects of radial-interrupted mattress sutures and macular translocation for treatment of subfoveal choroidal neovascularization secondary to age-related macular degeneration.

Interaction between 3-(p-tolylamino)-1,5-azulenequinone and the deoxyguanosine residue in various oligonucleotides upon photolysis.

Eight single-stranded oligodeoxyribonucleotides 32P-labeled at the 5'-end were synthesized; they were annealed with the complementary oligodeoxyribonucleotides to form the corresponding double-stranded helices. These duplexes possessed standard Watson-Crick base pairs, locally perturbed sites of a base mismatch, or a bulge. Further, 5'-32P-labeled oligodeoxyribonucleotides with a hairpin loop were also synthesized. Cleavage of these single- and double-stranded oligodexyribonucleotides selectively at the deoxyguanosine residue was accomplished by use of 3-(p-tolylamino)-1,5-azulenequinone 1 upon irradiation with 350 nm UV light. The single strands were cleaved more efficiently than the double-helices. For the helices containing a deoxyguanosine residue at a bulge, at a hairpin loop or toward the end, the cleaving efficiency was increased. Computation results indicate that two possibilities exist for agent 1 to form two "Watson-Crick type" hydrogen bonds with guanine in single-stranded oligodeoxyribonucleotides; yet, only one possibility exists in duplexes.

Effect of selective cytosine methylation and hydration on the conformations of DNA triple helices containing a TTTT loop structure by FT-IR spectroscopy.

5-Methylcytosines have been introduced into triplex-forming-oligonucleotides and shown to extend the pH range over which a triplex forms with a homopurine-homopyrimidine tract of duplex DNA. As a host strand, an oligodeoxypyrimidine with a base sequence of 5'-d(TC)3T4(CT)3 ([CC]) was designed to form a hairpin triplex with a 5'-d-A(GA)2G ([AG6]) purine strand at acidic pH (Tsay, et al., (1995) J. Biomol. Str. Dyn., 13, 1235-1245). We here present results obtained by FT-IR spectroscopy concerning the conformation of the hairpin triplex as a function of the selective substitution of cytosines by 5-methylcytosines in the host strand. Namely, cytosines are substituted by 5-methylcytosines in either the 3'-pyrimidine portion ([CM]) or the 5'-pyrimidine portion ([MC]) or in both ([MM]) of the host strand. The acidic-induced transitions of the equimolar mixtures of the purine target with either of the four pyrimidine oligomers gives rise to different apparent pK values, i.e., [MM].[AG6] (6.2) > [MC].[AG6] (6.0) > [CM].[AG6] (5.7) > [CC].[AG6] (5.2) > single-stranded oligopyrimidines (4.6 +/- 0.2), indicating that cytosine methylation expands the pH range compatible with the hairpin triplex formation regardless of whether the substitution is in the 5'-pyrimidine (Hoogsteen) portion or in the 3'-pyrimidine (Watson-Crick) portion. Thermal denaturation profiles indicated that all the triplexes denatured in a monophasic manner in the pH range of 4.0 to 7.0, and that cytosine methylations in any position of the 16-base pyrimidine oligomer increase the stability of the hairpin triplex DNA. IR spectra recorded in D2O and H2O solutions revealed that cytosine methylation does not significantly influence the conformation of triplex DNA in solution, i.e., all the four triplexes accept a similar sugar conformation, and predominately take on a S-type sugar pucker with a relative proportion of two S-type sugars for one N-type. Furthermore, we also investigated the effect of relative humidity (RH) on the conformation of triplex MC.AG6 in hydrated films, and found that the conformational change induced by the decrease of RH, from predominant S-type to primary N-type sugar pucker, might first occur in the purine strand at 86% RH.

Hydrated water molecules of pyrimidine/purine/pyrimidine DNA triple helices as revealed by FT-IR spectroscopy: a role of cytosine methylation.

Hydrated water molecules of pyrimidine/purine/pyrimidine DNA hairpin triplex was studied by a comparison of triplex (CC.AG6) formed by a host oligodeoxypyrimidine of 5'-d(TC)3T4(CT)3(CC) with a target hexadeoxypurine 5'-d(AG)3(AG6) strand and by triplexes (MM.AG6, MC.AG6, and CM.AG6) formed by oligonucleotides with the exact sequences as above except 5-methylcytosine replaced all (MM), 5' end half (MC), and 3' end half (CM) cytosine bases in CC via FT-IR spectroscopy in hydrated film. Results revealed that: (i) all these triplexes have a similar hydration pattern, in which water molecules probably bound in the N7 sites of adenines and guanines in the Crick-Hoogsteen groove, and to the methyl group of thymidines in the Watson-Hoogsteen groove. There are also some bound water molecules found at the O2 sites of thymines in both Watson-Crick and Crick-Hoogsteen grooves. (ii) In the CC.AG6 triplex the S-type sugars are always dominant in all hydrated states, whereas in MM.AG6 triplex the relative population of the N-type sugars is very close to that of the S-type between 86% and 66% of humidity. Furthermore, the sugar conformation in two partially modified triplexes (CM.AG6, and MC.AG6) are dominant by the N-type at lower humidity. This phenomenon might reflect that the degree of bound water varies among the binding sites of bases. (iii) The effect of introducing a methyl group on cytosine is to generate a spine of hydrophobic region in MM (MC and MC). The enlarging hydrophobic area not only increase the stability in solution, and also the stability in sodium hydrated films of the pyrimidine/purine/pyrimidine hairpin triplexes.

A study of triplex formatin of 5'-d-T-(C-T-)2C-(T-)4C-(T-C-)2T with 5'-d-A-(G-A-)2G A hairpin triplex with three T.A.T and three C+.G.C bases triads.

The UV mixing titration, gel electrophoresis, and CD measurements indicate that oligomers with a basic sequence of 5'-d-T-(C-T-)2C-(T-)4C-(T-C-)2T form a hairpin type triplex with the target 5'-d-A-(G-A-)2G. The stability, measured UV melting temperatures, were studied in aqueous solution as functions of mC (5-methylcytidine) replacement of C, pH (4 to 7), and salt concentrations (up to 1 M). The order of stability is 5'-d-A-(G-A-)2G + 5'-d-T-(C-T-)2C-(T-)4C-(T-C-)2T < 5'-d-A-(G-A-)2G + 5'-d-T-(mC-T-)2mC-(T-)4C-(T-C-)2T approximately 5'-d-A-(G-A-)2G + 5'-d-T-(C-T-)2C-(T-)4 mC-(T-mC-)2T < 5'-d-A-(G-A-)2G + 5'-d-T-(mC-T-)2mC-(T-)4mC-(T-mC-)2T at pH 4. These results indicate that (a) a stable triplex is formed with three T.A.T and three C+.G.C base triads and (b) mC is more effective than C to stablize the triplex formation in acidic condition. Thus, this provides a simple system for further studies of triplex.