Evaluation of haploidization efficiency in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.) through anther culture

The production of large numbers of haploids is the crucial point of the dihaploidisation process. Although in vitro haploid plants were successfully produced by irradiated pollen technique in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.), the frequency is still insufficient for using in a large-scale breeding programme. Thus, the present study was conducted to determine the efficacy of the anther culture technique on the production of in vitro haploids in the aforementioned species for which there have been no successful reports concerning by androgenesis. The anthers at uninucleate microspore stage were collected at different florescence times and then cultured on a solid MS medium supplemented by different combinations of 2,4-D (2,4-dichlorophenoxyacetic acid), BAP (6-benzylaminopurine), KN (kinetin) with the constant addition of NAA (naphthalene acetic acid) to induce callogenesis, embryogenesis and plantlet initiation. The combination of PGR, genotype and anther collection time played an important role in the androgenic response. The highest response was obtained from 57S?21 and G9 lines with the combination of 2.0 or 4.0 mg/l BAP?+?0.05 mg/l NAA (E6 medium) at the first anther collection time. Plantlets were regenerated and rooted on MS medium supplemented by 0.01 mg/l IAA. In total, 74 plants were recovered and propagated with micro-cuttings. The ploidy analyses revealed that 35 plants (47.3?%) were haploid (n?=?20), and the others (52.7?%) were diploid (2n?=?40).     

The Effects Of Short Time Right Ventricular Pacing On Left Atrial Mechanical Functions

Objectives

Left atrium (LA) plays an important role in left ventricular filling. It is well known that right ventricular apical pacing has unfavorable effects on ventricular systolic and diastolic performance. The aim of this study is to evaluate the LA mechanical functions with 2D echocardiography in patients with a permanent pacemaker after short time ventricular pacing.

Design

Echocardiographic examination was performed in 38 patients (mean age 63.0± 10.9, 18 female) with dual chamber pacemakers or defibrillators (< 20% ventricular pacing within previous 6 months, all of them on sinus rhythm) before and after 4 hours > 90% ventricular pacing at 70 beats per minute in DDD mode with an optimal AV interval. Left atrial volumes (LAV) including at the time of mitral valve opening (Vmax), at closure (Vmin), and at the onset of atrial systole (Volp) were measured. The passive emptying, conduit, active emptying and total emptying volume, stroke volumes were also calculated.

Results

No significant differences were noted at baseline and after pacing for absolute Vmax, Volp, passive emptying, conduit, active emptying, total emptying volumes as well as the volumes indexed to body surface area (p >0.05).

Conclusions

Short - time RV pacing seems to have no acute effects on left atrial mechanical functions.     

The critical role of S-lactoylglutathione formation during methylglyoxal detoxification in Escherichia coli

Survival of exposure to methylglyoxal (MG) in Gram-negative pathogens is largely dependent upon the operation of the glutathione-dependent glyoxalase system, consisting of two enzymes, GlxI (gloA) and GlxII (gloB). In addition, the activation of the KefGB potassium efflux system is maintained closed by glutathione (GSH) and is activated by S-lactoylGSH (SLG), the intermediate formed by GlxI and destroyed by GlxII. Escherichia coli mutants lacking GlxI are known to be extremely sensitive to MG. In this study we demonstrate that a ΔgloB mutant is as tolerant of MG as the parent, despite having the same degree of inhibition of MG detoxification as a ΔgloA strain. Increased expression of GlxII from a multicopy plasmid sensitizes E. coli to MG. Measurement of SLG pools, KefGB activity and cytoplasmic pH shows these parameters to be linked and to be very sensitive to changes in the activity of GlxI and GlxII. The SLG pool determines the activity of KefGB and the degree of acidification of the cytoplasm, which is a major determinant of the sensitivity to electrophiles. The data are discussed in terms of how cell fate is determined by the relative abundance of the enzymes and KefGB.     

Production of in vitro haploid plants from in situ induced haploid embryos in winter squash (<Emphasis Type="Italic">Cucurbita maxima</Emphasis> Duchesne ex Lam.) via irradiated pollen

The influence of pollen irradiation on the production of in vitro haploid plants from in situ induced haploid embryos was investigated in winter squash (Cucurbita maxima Duchesne ex Lam.). Pollen were irradiated at different gamma-ray doses (50, 100, 200 and 300 Gray) and durations (9, 11, 15, 21, and 28 July). Production of in vitro haploid plantlets was influenced by irradiation dose, irradiation duration, genotype, and embryo type and embryo stage. Embryos were only obtained from lower irradiation doses (50 Gray and 100 Gray) and earlier irradiation durations (9, 11, and 15 July). The greatest embryo number per fruit was procured from “G14” and “55SI06” genotypes at 50 Gray gamma-ray dose. Necrotic embryos were higher than normal embryos at delayed harvest times (5 and 6 weeks after the pollination). The convenient harvest time for embryo rescue was observed about 4 weeks (between 25 and 30 days) after pollination. All cotyledon and amorphous embryos had only diploid plants while late-torpedo, arrow-tip, and pro-cotyledon embryos produced 33.3, 50.0, and 66.7% haploid plant. The frequency of haploid plantlets was 0.11, 1.17, 10.96 and 0.28 per 100 seeds, 100 embryos, 100 plantlets and a fruit at 50 Gray gamma-ray dose, respectively.     

Anaesthesia and the acute phase protein response in children undergoing circumcision

Concentrations of acute phase proteins (CRP: C-reactive protein, albumin) change during surgery. We investigated the acute phase response to circumcision and the effects of anaesthesia on this response. The children were divided into four groups; group 1 (intratracheal general anaesthesia, n=40), group 2 (general anaesthesia with mask, n=20), group 3 (ketamine, n=20), group 4 (local anaesthesia, n=35). Blood samples were obtained, 24 hours before circumcision, after premedication, and 24 hours after circumcision. CRP and albumin before circumcision were comparable for all groups. There was no increase in CRP, and albumin remained steady throughout the study. No difference was observed among the groups, and related to anaesthesia. No responsiveness may be explained with the size of injured tissue or anatomical and histological type of preputium.     

Regulation of somatostatin-14 and gastrin I binding sites in rat gastrointestinal mucosa by ulcerogenic dose of cysteamine

A single duodenal ulcerogenic dose of cysteamine administered into rats induced time-dependent depletion of immunoreactive somatostatin in the gastric corporeal, antral, and duodenal mucosa with a parallel increase (up-regulation) of somatostatin binding sites. The concentration of somatostatin binding sites returned to the control level in the corporeal mucosa when measured at 24 hrs; however, in the duodenal mucosa there was only a partial return to the control level. Somatostatin binding sites in the antral mucosa did not return to control level even after 24 hrs. Except for the duodenum mucosal immunoreactive gastrin level was unaffected by cysteamine administration, but corporeal mucosal gastrin I binding sites were diminished (down-regulation) after 24 hrs.     

Somatostatin as a mediator of the effect of neurotensin on pentagastrin-stimulated acid secretion in rats

Neurotensin and somatostatin have both been shown to inhibit gastric acid secretion, but no interaction between these peptides has been demonstrated. To determine whether somatostatin might be a mediator of neurotensin's effect on pentagastrin-stimulated gastric acid secretion, we performed the following three experiments. First, we collected 0.2-ml samples of portal venous blood as frequently as every 5 min, and we confirmed a significant release of somatostatin-like immunoreactivity into portal venous blood during neurotensin-induced inhibition of acid secretion. This release of somatostatin-like immunoreactivity and inhibition of acid secretion were only seen in pentobarbital-anesthetized rats, but no sustained release of somatostatin-like immunoreactivity or inhibition of acid secretion occurred in urethane-anesthetized animals. In the second experiment, we analyzed portal plasma by high pressure liquid chromatography, and found that portal somatostatin-like immunoreactivity in blood collected during neurotensin infusion was composed of a single peak corresponding to somatostatin-14. In the third experiment, we found that infusion of antibody to somatostatin prevented neurotensin from inhibiting pentagastrin-stimulated acid secretion. Taken together, these data show that somatostatin, possibly from the stomach itself, is a necessary mediator of neurotensin's inhibitory effect in pentobarbital-anesthetized rats.     

N‐acetyl‐L‐cysteine inhibits bleomycin induced apoptosis in malignant testicular germ cell tumors

Antioxidants may prevent apoptosis of cancer cells via inhibiting reactive oxygen species (ROS). However, to date no study has been carried out to elucidate the effects of strong antioxidant N‐acetylcysteine (NAC) on Bleomycin induced apoptosis in human testicular cancer (NTERA‐2, NT2) cells. For this reason, we studied the effects of Bleomycin and NAC alone and in combination on apoptotic signaling pathways in NT2 cell line. We determined the cytotoxic effect of bleomycin on NT2 cells and measured apoptosis markers such as Caspase‐3, ‐8, ‐9 activities and Bcl‐2, Bax, Cyt‐c, Annexin V‐FTIC and PI levels in NT2 cells incubated with different agents for 24 h. Early apoptosis was determined using FACS assay. We found half of the lethal dose (LD₅₀) of Bleomycin on NT2 cell viability as 400, 100, and 20 µg/ml after incubations for 24, 48, and 72 h, respectively. Incubation with bleomycin (LD₅₀) and H₂O₂ for 24 h increased Caspase‐3, ‐8, ‐9 activities, Cyt‐c and Bax levels and decreased Bcl‐2 levels. The concurrent incubation of NT2 cells with bleomycin/H₂O₂ and NAC (5 mM) for 24 h abolished bleomycin/H₂O₂‐dependent increases in Caspase‐3, ‐8, ‐9 activities, Bax and Cyt‐c levels and bleomycin/H₂O₂‐dependent decrease in Bcl‐2 level. Our results indicate that bleomycin/H₂O₂ induce apoptosis in NT2 cells by activating mitochondrial pathway of apoptosis, while NAC diminishes bleomycin/H₂O₂ induced apoptosis. We conclude that NAC has antagonistic effects on Bleomycin‐induced apoptosis in NT2 cells and causes resistance to apoptosis which is not a desired effect in eliminating cancer cells. J. Cell. Biochem. 114: 1685–1694, 2013. © 2013 Wiley Periodicals, Inc.     

Investigation of the effect of naringenin on oxidative stress‐related alterations in testis of hydrogen peroxide‐administered rats

Testis tissue is prone to oxidation because its plasma membrane contains many polyunsaturated fatty acids. Naringenin is a plant‐derived natural flavonoid. We investigated the possible ameliorative role of naringenin on the hydrogen peroxide (H₂O₂)‐induced testicular damage in Wistar rats. Animals received 12 mg/kg H₂O₂ by intraperitoneal injection, and 50 mg/kg naringenin via orogastric gavage for 4 weeks. In the H₂O₂ group, the testis malondialdehyde level increased, while the amount of reduced glutathione, glutathione transferase activities, and the testis weight decreased. There were severe testicular damages in the H₂O₂ group otherwise their grade were less in the naringenin + H₂O₂ group. However, the serum testosterone concentrations decreased in both the H₂O₂ and the naringenin + H₂O₂ groups. The testicular zinc and calcium levels reduced in the H₂O₂‐treated rats. In conclusion, the administration of H₂O₂ caused oxidative stress in the testes and naringenin supplementation decreased the H₂O₂‐induced effects, except for changes in testosterone levels.     

A peptide epitope derived from the cancer testis antigen HOM-MEL-40/SSX2 capable of inducing CD4+ and CD8+ T-cell as well as B-cell responses

Background

Antigen-derived HLA class I-restricted peptides can generate specific CD8⁺ T-cell responses in vivo and are therefore often used as vaccines for patients with cancer. However, only occasional objective clinical responses have been reported suggesting the necessity of CD4⁺ T-cell help and possibly antibodies for the induction of an effective anti-tumor immunity in vivo. The SSX2 gene encodes the cancer testis antigen (CTA) HOM-MEL-40/SSX2, which is frequently expressed in a wide spectrum of cancers. Both humoral and cellular immune responses against SSX2 have been described making SSX2 an attractive candidate for vaccine trials.

Methods

SYFPEITHI algorithm was used to predict five pentadecamer peptides with a high binding probability for six selected HLA-DRB1 subtypes (*0101, *0301, *0401, *0701, *1101, *1501) which are prevalent in the Caucasian population.

Results

Using peripheral blood cells of 13 cancer patients and 5 healthy controls, the HOM-MEL-40/SSX2-derived peptide p101-111 was identified as an epitope with dual immunogenicity for both CD4⁺ helper and cytotoxic CD8⁺ T cells. This epitope also reacted with anti-SSX2 antibodies in the serum of a patient with breast cancer. Most remarkably, SSX2/p101-111 simultaneously induced specific CD8, CD4, and antibody responses in vitro.

Conclusions

p101-111 is the first CTA-derived peptide which induces CD4⁺, CD8⁺, and B-cell responses in vitro. This triple-immunogenic peptide represents an attractive vaccine candidate for the induction of effective anti-tumor immunity.