Lack of p53 function promotes radiation-induced mitotic catastrophe in mouse embryonic fibroblast cells

Background  

We have demonstrated that in some human cancer cells both chronic mild heat and ionizing radiation exposures induce a transient block in S and G2 phases of the cell cycle. During this delay, cyclin B1 protein accumulates to supranormal levels, cyclin B1-dependent kinase is activated, and abrogation of the G2/M checkpoint control occurs resulting in mitotic catastrophe (MC).     

Relative mRNA expression and immunolocalization for transforming growth factor-beta (TGF-β) and their effect on in vitro development of caprine preantral follicles

This study aimed to evaluate the immunolocalization and messenger RNA (mRNA) expression for transforming growth factor-beta (TGF-β) and its receptors (TGF-βRI and RII), as well as mRNA expression for P450 aromatase and FSH receptor in caprine preantral follicles. The effects of TGF-β, FSH alone, or in association on the in vitro follicular development were also assessed. Immunohistochemical analyses showed the expression of TGF-β and its receptors in oocytes of all follicle stages and granulosa cells of primary and secondary follicles. mRNA for TGF-β receptors and for FSH receptor (FSHR) was present in preantral follicles as well as in oocytes and granulosa cells of antral follicles. Isolated secondary follicles were cultured in α-minimum essential medium (MEM) alone or supplemented with either FSH (100 ng/ml), TGF-β (10 ng/ml), or TGF-β + FSH for 18 d. TGF-β increased significantly oocyte diameter when compared to FSH alone and control. After 18 d of culture, all groups showed a significant reduction in P450 aromatase and FSHR mRNA levels in comparison to fresh control. In contrast, treatment with FSH significantly increased the mRNA expression for TGF-β in comparison to fresh control and other treatments. In conclusion, the findings showed that TGF-β and its receptors are present in caprine ovarian follicles. Furthermore, they showed a positive effect on oocyte growth in vitro.     

赤红壤早稻田甲烷排放通量及其影响因素

用封闭箱法对广东省赤红壤早稻田CH4排放通量进行了观测。结果表明,CH4排放有明显的季节变化规律,3个排放高峰分别出现在水稻分蘖末期、孕穗抽穗期和乳熟期,平均通量为5.7mg.m-2.h-1。在测定期内,CH4排放与5和10cm土壤温度呈显著正相关,与土壤Eh呈显著负相关,与土壤pH值、水层深浅相关不明显。     

In vivo and in vitro processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps

Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the reserve protein phaseolin. The polypeptides are synthesized on membrane-bound polysomes, pass through the endoplasmic reticulum (ER) and accumulate in protein bodies. For a study of the biosynthesis and processing of phaseolin, developing cotyledons were labeled with radioactive amino acids, glucosamine and mannose, and isolated fractions (polysomal RNA, polysomes, and rough ER) were used for in vitro protein synthesis. Newly synthesized phaseolin present in the ER of developing cotyledons can be fractioned into four glycopolypeptides by SDS PAGE. In vitro synthesis with polysomal RNA results in the formation of two polypeptides by polysome run-off shows that glycosylation is a co-translational event. The two unglycosylated polypeptides formed by polysome run-off are slightly smaller than the two polypeptides formed by in vitro translation of isolated RNA, indicating that a signal peptide may be present on these polypeptides. Run-off synthesis with rough ER produces a pattern of four polypeptides similar to the one obtained by in vivo labeling. The two abundant glycopolypeptides formed by polysome run-off. This result indicates the existence of a second glycosylation event for the abundant polypeptides. Inhibition of glycosylation by Triton X-100 during chain-completion with rough ER was used to show that these two glycosylation steps normally occur sequentially. Both glycosylation steps are inhibited by tunicamycin. Analysis of carhohydrate to protein ratios of the different polypeptides and of trypsin digests of polypeptides labeled with [(3)H]glucosamine confirmed the conclusion that some glycosylated polypeptides contain two oligosaccharide chains, while others contain only one. An analysis of tryptic peptide maps shows that each of the unglycosylated polypeptides is the precursor for one glycosylated polypeptide with one oligosaccharide chain and one with two oligosaccharide chains.     

Calcium supplementation increases bone mass in GH-deficient prepubertal children during GH replacement

BACKGROUND/AIMS: Since GH plays an important role in bone mineralization, and several studies demonstrated the positive influence of a higher calcium intake on bone mass, we studied the effect of calcium supplementation in GHD children during GH therapy. METHODS: 28 prepubertal GHD children, 5.0-9.9 years old, were assigned to two groups: group A (n = 14; 7 females) treated with GH, and group B (n = 14; 7 females) treated with GH + calcium gluconolactate and carbonate (1 g calcium/day per os). Auxological parameters, total bone mineral content (TBMC) and density (TBMD), leg BMC and BMD, lumbar BMD, fat mass (FM) and lean tissue mass (LTM), blood 25-hydroxyvitamin D (25-OHD), parathyroid hormone (PTH), osteocalcin (OC) and urinary N-terminal telopeptide of type I collagen (NTx) were determined at the start of therapy and after 1 and 2 years of treatment. RESULTS: During the 2 years of the study, TBMC, TBMD, leg BMC and BMD (but not lumbar BMD) increased in both groups of patients, however after 2 years of treatment they were significantly higher in the calcium-supplemented group B than in group A (p < 0.05, for all parameters). At the start of therapy, in both groups of patients percentage FM was higher and total and leg LTM lower than in controls (p < 0.05 for each parameter). Thereafter, FM decreased and LTM increased and after 2 years they were both different from baseline (p < 0.05). After 2 years of treatment, leg BMC and BMD were more positively correlated with regional leg LTM in patients of group B (r = 0.834 and r = 0.827, respectively; p < 0.001) than in patients of group A (r = 0.617 and r = 0.637, respectively; p < 0.05). 25-OHD and PTH levels were in the normal range in all patients at the start and during treatment. OC levels were lower and urinary NTx levels higher in patients than in controls (p < 0.05 for both parameters), either at the start and after 1 year of treatment. After 2 years of treatment, OC levels were significantly higher than at the start of the study (p < 0.05) in both groups of patients, but they were higher in group B than in group A (p < 0.05); on the contrary, urinary Ntx levels were lower in group B than in group A (p < 0.05). CONCLUSION: In GHD children, treated with GH, calcium supplementation improved bone mass; it may aid in reaching better peak bone mass and in protecting weight-bearing bones, usually completed in childhood to maximum levels, from risk of osteoporosis and fractures later in life.     

Cloning, Sequencing, and Characterization of a Heat- and Alkali-Stable Type I Pullulanase from Anaerobranca gottschalkii

The gene encoding a type I pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima. The pullulanase gene from A. gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70°C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in E. coli Tuner(DE3)(pLysS), the pullulanase gene from A. gottschalkii, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg′). The N-terminal sequence of purified rPulAg′ was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E. coli. Interestingly, most of the physicochemical properties of rPulAg′ were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, β-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect. In addition, rPulAg′ was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The pullulanase from A. gottschalkii is the first thermoalkalistable type I pullulanase that has been described.     

In situ fluorescent nick translation procedure for plant chromosomes

Pectinase and cellulase, which are used to macerate plant material, always show traces of DNase activities that result in DNA nicking. Moreover, the DNA polymerase I usually applied in the in situ nick translation techniques shows both 5' to 3' and 3' to 5' exonuclease activities. As a result, significant nonspecific labeling appears in control preparations that are not digested by a restriction endonuclease. Our procedure includes blocking nonspecific nick labeling before incubation with restriction enzymes (HpaII and HaeIII). This is achieved by incorporation of ddGTP into DNA by the Taq polymerase which lacks 3' to 5' exonuclease activity. This method gives satisfactory results because it eliminates nonspecific nick translation signals that are present after applying the methods described for animal material.     

Strain persistence of invasive Candida albicans in chronic hyperplastic candidosis that underwent malignant change

Objectives: The aim of this study was to assess persistence and tissue invasion of Candida albicans strains isolated from a 65 year‐old patient with chronic hyperplastic candidosis (CHC), that subsequently developed into squamous cell carcinoma (SCC). Materials and Methods: C. albicans (n=7) were recovered from the oral cavity of the patient over seven years. Confirmation of CHC and SCC in this patient was achieved by histopathological examination of incisional biopsy tissue. DNA fingerprinting was performed on the seven isolates from the CHC patient together with a further eight isolates from patients with normal oral mucosa (n=2), chronic atrophic candidosis (n=1), SCC (n=1) and CHC (n=4). Genotyping involved the use of inter‐repeat PCR using the eukaryotic repeat primer 1251. Characterisation of the tissue invasive abilities of the isolates was achieved by infecting a commercially available reconstituted human oral epithelium (RHE; SkinEthic, Nice, France). After 24 h. C. albicans tissue invasion was assessed by histopathological examination. Results: DNA fingerprinting demonstrated strain persistence of C. albicans in the CHC patient over a seven year period despite provision of systemic antifungal therapy. The strain of C. albicans isolated from this patient was categorised as a high invader within the RHE compared to other isolates. Conclusions: Candidal strain persistence was evident in a patient with CHC over seven years. This persistence may be due to incomplete eradication from the oral cavity following antifungal therapy or subsequent recolonisation from other body sites or separate exogenous sources. The demonstration of enhanced in vitro tissue invasion by this particular strain may, in part, explain the progression to carcinoma.     

Further phenotypic delineation of subtelomeric (terminal) 4q deletion with emphasis on intracranial and reproductive anatomy

Hypoplastic left heart syndrome(HLHS) refers to the abnormal development of the left-sided cardiac structures, resulting in obstruction to blood flow from the left ventricular outflow tract. In addition, the syndrome includes underdevelopment of the left ventricle, aorta, and aortic arch, as well as mitral atresia or stenosis. HLHS has been reported to occur in approximately 0.016 to 0.036% of all live births. Newborn infants with the condition generally are born at full term and initially appear healthy. As the arterial duct closes, the systemic perfusion becomes decreased, resulting in hypoxemia, acidosis, and shock. Usually, no heart murmur, or a non-specific heart murmur, may be detected. The second heart sound is loud and single because of aortic atresia. Often the liver is enlarged secondary to congestive heart failure. The embryologic cause of the disease, as in the case of most congenital cardiac defects, is not fully known. The most useful diagnostic modality is the echocardiogram. The syndrome can be diagnosed by fetal echocardiography between 18 and 22 weeks of gestation. Differential diagnosis includes other left-sided obstructive lesions where the systemic circulation is dependent on ductal flow (critical aortic stenosis, coarctation of the aorta, interrupted aortic arch). Children with the syndrome require surgery as neonates, as they have duct-dependent systemic circulation. Currently, there are two major modalities, primary cardiac transplantation or a series of staged functionally univentricular palliations. The treatment chosen is dependent on the preference of the institution, its experience, and also preference. Although survival following initial surgical intervention has improved significantly over the last 20 years, significant mortality and morbidity are present for both surgical strategies. As a result pediatric cardiologists continue to be challenged by discussions with families regarding initial decision relative to treatment, and long-term prognosis as information on long-term survival and quality of life for those born with the syndrome is limited.