Silence of ATAD2 inhibits proliferation of colorectal carcinoma via the Rb-E2F1 signaling.

OBJECTIVE: This study aims to clarify the potential function of ATAD2 (ATPase family, AAA domain containing 2) in regulating proliferative and apoptotic abilities of colorectal carcinoma (CRC). PATIENTS AND METHODS: ATAD2 levels in CRC specimens and cell lines were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Overall survival in CRC patients with high or low level of ATAD2 was assessed by Kaplan-Meier method. The correlation between ATAD2 level and clinical characteristics of CRC patients was analyzed by χ2 test. Univariable and multivariable Cox regression models were generated to illustrate potential risk factors for the overall survival of CRC. After knockdown of ATAD2 in SW620 cells, relative levels of Cyclin D1, ppRb, pRb, E2F1, Cyclin E and cleaved Caspase 3 were detected by Western blot. Regulatory effects of ATAD2 on viability, clonality, cell cycle distribution, and apoptosis in SW620 and HCT15 cells were examined by a series of functional experiments. RESULTS: Upregulated ATAD2 in CRC was correlated to tumor size, tumor node metastasis (TNM) staging, and histological classification of CRC. High level of ATAD2 predicted poor prognosis in CRC patients. Cox regression test suggested that ATAD2 level, tumor size, TNM staging and histological classification were independent factors influencing overall survival in CRC. Knockdown of ATAD2 reduced viability and clonality in SW620 and HCT15 cells. In addition, cell cycle was arrested in G1 phase and apoptosis was stimulated in CRC cells with ATAD2 knockdown. In SW620 cells transfected with ATAD2 shRNA, protein levels of Cyclin D1, ppRb, E2F1 and Cyclin E were downregulated, and cleaved Caspase 3 was upregulated. CONCLUSIONS: ATAD2 is upregulated in CRC tissues and correlated to poor prognosis of CRC patients. It exerts an anti-proliferation role in CRC by arresting cell cycle in G1/S phase and triggering apoptosis via the Rb-E2F1 signaling.

[Clinical effects of expanded super-thin perforator flaps in the shoulder, neck, and chest in reconstruction of extensive burn scars in the face].

Objective: To explore the clinical effects and key techniques of expanded super-thin perforator flaps in the shoulder, neck, and chest in reconstruction of extensive burn scars in the face. Methods: From January 2008 to November 2018, 22 patients with extensive burn scars in the face were admitted to the Department of Plastic Surgery of Dongguan Kanghua Hospital and the Department of Plastic Surgery of Dermatology Hospital of Southern Medical University, with 3 males and 19 females, aged from 4 to 48 years. There were 16 cases of type Ⅱ and 6 cases of type Ⅲ in facial scars. Before the first stage of expansion surgery, Doppler blood flow survey meter or multi-slice CT was used to locate the perforator vessels. One to four expanders with rated capacity ranged from 100 to 600 mL were placed in the patients. We gave 20% to 30% of the rated capacity of expander intro-operation and common injection with 10% to 15% of the rated capacity of expander per week post-operation until the volume reached 1.5 to 2.5 times of the rated capacity of expander during the past 3 to 4 months. At the second stage of surgery, the perforators were located again before surgery with the same method. The size of defects after the excision of facial scars ranged from 6 cm×4 cm to 18 cm×16 cm. With perforators used as nutrient vessels, narrow pedicle flaps or random flaps ranging from 6 cm×6 cm to 22 cm×18 cm were elevated as rotating or advancing to reconstruct the defects. The donor sites were sutured directly. Some of the flaps needed stage Ⅲ operation for cutting the pedicle. The survival of flaps, post-operation complications, and follow-up were assessed. Results: All flaps of 22 patients survived. All the donor sites were closed simultaneously. One patient underwent an additional surgery for 5 cm×4 cm necrosis on distal part of flap caused by subcutaneous hematoma. Two patients with epidermis blister on the flaps were healed by themselves after dressing change. Due to rapid expansion, blood capillary proliferation appeared on the central part of the flap in 3 cases, after slowing down the expansion speed properly, which had no impact on flap transfer. No ischemia or venous congestion phenomenon were observed in the other flaps. During follow-up of 5 to 48 months, the flaps of patients showed no significant bloated appearance, with good complexion and texture, and even could reproduce facial fine-grained expressions naturally. Conclusions: For the reconstruction of extensive burn scars in the face, expanded super-thin perforator flaps can not only acquire large and thin flaps with high matching degree surface skin defect, but also reproduce facial fine-grained expressions. It is a simple and safe method which conforms to the facial aesthetic standard.

Clinical analysis of Krukenberg tumours in patients with colorectal cancer-a review of 57 cases.

BACKGROUND: A Krukenberg tumour (KT) is defined as an ovarian metastasis from a gastrointestinal adenocarcinoma and suggests a terminal condition. This study aimed to identify the prognostic factors affecting the survival of patients with KTs of colorectal origin who receive cytoreductive surgery. METHODS: Medical records of patients who had received cytoreductive surgery and had been pathologically diagnosed with KT of colorectal origin in two centres were reviewed. Information about the patients' clinicopathological features and follow-up visit were collected. Factors influencing patient survival were analysed. RESULTS: Fifty-seven patients were included in this study. The median survival time was 35 months. Five-year overall survival was 25%. Patients who had recurrence 2 years after resection of the primary tumour, achieved complete cytoreduction, had metastases confined to the pelvis, had no lymph node involvement, and received systemic chemotherapy had a significantly longer median survival than those who had recurrence at the same time as resection of the primary tumour (P = 0.027), received incomplete cytoreduction (P < 0.001), had metastases beyond the pelvis (P < 0.001), had lymph node involvement (P = 0.011), and did not receive systemic chemotherapy (P = 0.006) on log-rank test. Less extensive metastatic disease, achievement of complete cytoreduction, and use of systemic chemotherapy were significantly associated with improved prognosis on multivariate analysis. CONCLUSIONS: Cytoreductive surgery may confer survival benefits in patients with KTs of colorectal origin who attain complete cytoreduction and whose metastases are confined to the pelvis and when combined with active systemic chemotherapy.

Rabbit MSTN gene polymorphisms and genetic effect analysis.

We analyzed meat samples of nine pure lines of rabbit and its 37 hybrid combinations by sequencing and single-strand conformation polymorphism techniques to explore genetic polymorphisms of all the three exon regions and part of the 5'-regulatory region of the myostatin (MSTN) gene. Thus, we detected a single nucleotide mutation (T→C) on the 476 locus of the 5'-regulatory region, but no mutation sites were detected in the exon areas. The correlation analysis showed that the mutation had some favorable genetic effects, and it resulted in increased liver weight, carcass weight, forelegs weight, back and waist weight, ham weight, and tare weight, whereas it decreased muscle drip loss and cooking loss (P < 0.05). These results suggest that the mutations in the upstream regulatory region of the MSTN gene are beneficial to the rabbit soma development, and the mutations can be used as molecular markers for the selection of the meat quality of rabbits.

Clinical significance of Mena and Her-2 expression in breast cancer.

OBJECTIVE: The aim of this study was to determine the expression patterns of Mena and Her-2 in breast cancer tissues and to explore their clinical significance and correlation with clinicopathological parameters. METHODS: The expression of Mena and Her-2 was detected in 40 breast cancer tissues and 14 normal breast tissues by immunohistochemistry, and the relationship of Mena and Her-2 expression with clinicopathological parameters was analyzed. RESULTS: Both Mena (70%) and Her-2 (40%) were more commonly expressed in breast cancer than in normal breast tissue (7.1%, 0%, respectively; p < 0.05); further, Mena and Her-2 expression in breast cancer were positively correlated (r = 0.530, p < 0.05). In comparing expression with clinicopathological parameters of tumor samples, Mena and Her-2 were both associated with axillary lymph node metastasis and TNM stage (p < 0.05), but not with patient age or pathological type. CONCLUSIONS: Mena and Her-2 are related to the malignancy degree and metastasis of breast cancer, and thus may play a coordinating role in the occurrence and progression of breast cancer.

Rapid determination of transgene copy number in tobacco by competitive PCR using a pair of SSR primers.

We developed a straightforward, rapid, and inexpensive method to determine transgene copy number in tobacco. The plasmid (pSSRCopy) used for tobacco transformation contains a simple sequence repeat (SSR) locus, PT1199, which was partially deleted in the middle, a homogenous SSR locus in tobacco K326. A 168-bp segment of the cloned PT1199 was shortened to 95 bp by deleting a 73-bp internal fragment. Using a pair of SSR primers, competitive PCR was amplified from genomic DNA from transgenic tobacco harboring pSSRCopy, and the two expected bands were found. The 168-bp band (SSR-168) corresponds to endogenous PT1199 and the 95-bp band (SSR-95) comes from the integrated pSSRCopy. A single copy of a transgene can be easily distinguished from multiple copies by comparing band densities.

Site-specific antibody of (Na(+) + K(+))-ATPase augments cardiac myocyte contraction without inactivating enzyme activity.

(Na(+) + K(+))-ATPase regulates both excitability and contractility of the heart. Little is known about the molecular basis of the enzyme that underlies its cardiac regulatory functions. Here we demonstrate that the (833)KRQPRNPKTDKLVNE(847) region, which resides in the alpha-subunit of rat (Na(+) + K(+))-ATPase, directly participates in the regulation of cardiac contraction. A site-specific antibody (SSA95) against this peptide sequence markedly increased intracellular Ca(2+) transients and contraction (EC(50) = 11.4 nM) in intact rat heart cells without inactivating the (Na(+) + K(+))-ATPase. These novel findings establish the first link between a precise structural region of the (Na(+) + K(+))-ATPase and cardiac positive inotropy.

A pyrroline derivative of mexiletine offers marked protection against ischemia/reperfusion-induced myocardial contractile dysfunction.

The efficacy and mechanism of protection of a new 2,2,5, 5-tetramethylpyrroline derivative of mexiletine, MEX-NH, against ischemia/reperfusion-induced cardiac dysfunction are reported. The MEX-NH and its nitroxide metabolite are membrane-permeable antioxidants. Studies were performed in an isolated rat heart model to measure the efficacy of MEX-NH in preventing postischemic injury. Serial measurements of contractile function and coronary flow were performed on hearts subjected to 30 min of global 37 degrees C ischemia followed by 45 min of reperfusion. Hearts were either untreated or treated with 25 microM MEX-NH or MEX for 1 min before ischemia. The hearts treated with MEX-NH showed marked recovery of left ventricular developed pressure (96.3 +/- 2.7% of preischemic value) compared with untreated (13.7 +/- 1.0%) or MEX-treated (19.9 +/- 2.7%) hearts. The cardiac sarcolemmal Na(+),K(+)-ATPase activity showed that the enzyme activity was fully restored in hearts treated with MEX-NH compared with 65 +/- 5.3% inhibition in the untreated hearts. Competitive inhibition of [(3)H]ouabain binding revealed that the MEX-NH binds at the K(+)-binding site of the enzyme. The present study establishes that the compound MEX-NH provides marked protection against ischemia/reperfusion-induced contractile dysfunction in isolated hearts. A combination of reversible inhibition of Na(+)/K(+)-ATPase activity during ischemia and site-targeted antioxidative effect upon reperfusion seems to contribute to this cardioprotection.

Nitric oxide protects nitric oxide synthase function from hydroxyl radical-induced inhibition.

The interdependent relationships among nitric oxide synthase (NOS), its coenzyme, cofactors and nitric oxide (NO(free radical) were studied using electron paramagnetic resonance spectroscopy. It was found that superoxide-dependent hydroxyl free radical (OH(free radical), derived from NOS coenzyme and cofactors, inhibits NOS activity, and that endogenous NO(free radical) generated by NOS scavenges OH(free radical) and protects NOS function. These results reveal a new role for NO(free radical) that may be important in NOS function and cellular free radical homeostasis.

Indirect neurotrophic effect of neuropeptide ZNC(C)PR on PC12 cells via peptide-stimulation of C6 cells.

AIM: To understand the mechanism of neurotrophic action of neuropeptide ZNC(C)PR and its effect on which could affect both growth and apoptosis of C6 cells. METHODS: Effects of ZNC(C)PR-treated C6 conditioned medium was observed on on culture of PC12 cells. The development of PC12 cells was determined by ratio of neurite-bearing cells in the total cells. The specific binding of ZNC(C)PR on C6 cells was determined by radioligand binding assay (RBA). RESULTS: ZNC(C)PR-treated C6 conditioned medium increased the ratio of neurite-bearing PC12 cells by 36% compared to the untreated C6 conditioned medium or to a mixture of ZNC(C)PR with the untreated C6 conditioned medium. RBA showed a specific binding site of ZNC(C)PR on C6 cells with Kd value of 2.74 nmol.L-1 and Bmax value of 19 pmol.g-1 protein. CONCLUSION: ZNC(C)PR enhanced C6 cells induced secretion of some neurotrophic factors which acted as enhancers for PC12 cells differentiation, through its specific receptor sites on the neuroglioma cell.

Nitric oxide synthase in cardiac sarcoplasmic reticulum.

NO. is a free radical that modulates heart function and metabolism. We report that a neuronal-type NO synthase (NOS) is located on cardiac sarcoplasmic reticulum (SR) membrane vesicles and that endogenous NO. produced by SR-associated NOS inhibits SR Ca2+ uptake. Ca2+-dependent biochemical conversion of L-arginine to L-citrulline was observed from isolated rabbit cardiac SR vesicles in the presence of NOS substrates and cofactors. Endogenous NO. was generated from the vesicles and detected by electron paramagnetic resonance spin-trapping measurements. Immunoelectron microscopy demonstrated labeling of cardiac SR vesicles by using anti-neuronal NOS (nNOS), but not anti-endothelial NOS (eNOS) or anti-inducible NOS (iNOS) antibodies, whereas skeletal muscle SR vesicles had no nNOS immunoreactivity. The nNOS immunoreactivity also displayed a pattern consistent with SR localization in confocal micrographs of sections of human myocardium. Western blotting demonstrated that cardiac SR NOS is larger than brain NOS (160 vs. 155 kDa). No immunodetection was observed in cardiac SR vesicles from nNOS knockout mice or with an anti-nNOS mu antibody, suggesting the possibility of a new nNOS-type isoform. 45Ca uptake by cardiac SR vesicles, catalyzed by Ca2+-ATPase, was inhibited by NO. produced endogenously from cardiac SR NOS, and 7-nitroindazole, a selective nNOS inhibitor, completely prevented this inhibition. These results suggest that a cardiac muscle nNOS isoform is located on SR of cardiac myocytes, where it may respond to intracellular Ca2+ concentration and modulate SR Ca2+ ion active transport in the heart.

Cerebral asymmetry in a selected Chinese population.

Previous studies have demonstrated anatomical differences between the two cerebral hemispheres and ethnic differences in cerebral asymmetry. This study examined asymmetry of Chinese living in Shanghai. Measurements were taken across the frontal, mid-cerebral and occipital regions from normal head computed tomography (CT) scans of 200 Chinese Shanghai residents (100 male and 100 female, aged 6-73 years, average 48.7 years). The results were compared with reported data in the literature. The following results were found: (i) In the frontal region the right side was larger than the left in 57.5% of cases, equal in 10.5% and smaller in 32% of cases; in the mid-cerebral region the right side was larger than the left in 65.5% of cases, equal in 12.5% and smaller in 22% of cases; in the occipital regions the right side was larger than the left in 34.5% of cases, equal in 8.5% and smaller in 57% of cases. The average right-left differences between the frontal, mid-cerebral and occipital regions were 0.43 mm, 0.9 mm and 0.4 mm respectively. No difference in cerebral asymmetry existed between males and females. The occipital lobes showed the greatest individual asymmetry. The distribution of cerebral asymmetry of Chinese in Shanghai showed similarity to North American Whites rather than North American Blacks, but the average right-left differences were smaller than those of Whites.

Ultrastructural localization of glycolytic enzymes on sarcoplasmic reticulum vesticles.

We have previously obtained indirect evidence that sarcoplasmic reticulum (SR) vesicles from cardiac and skeletal muscle contain the complete chain of glycolytic enzymes from aldolase to pyruvate kinase. To investigate directly whether pyruvate kinase and other glycolytic enzymes are anatomically associated with the SR, electron microscopic immunogold++ labeling studies were carried out in isolated SR vesicles using specific primary antibodies against selected glycolytic enzymes and Ca2+-ATPase, and appropriate secondary antibodies labeled with 6-nm or 12-nm gold particles. Pyruvate kinase was broadly dispersed on the cytoplasmic side of the SR membrane of both cardiac and skeletal muscle vesicles. With 6-nm gold particles, density of binding to pyruvate kinase was 2522 +/- 445 and 4171 +/- 1379 particles/microm2 for cardiac and skeletal muscle SR, respectively. Binding densities to Ca2 +/- ATPase were similar (2550 +/- 639 particles/ microm2 for cardiac SR, 3877 +/- 408 particles/microm2 for skeletal muscle SR). Immunogold labeling of ultrathin sections indicated that pyruvate kinase was attached to the SR membrane and located immediately adjacent to the Ca2+-ATPase. Aldolase and glyceraldehyde phosphate dehydrogenase were also found to be attached to the cytoplasmic side of SR vesicles and located in close proximity to Ca2+-ATPase. These results provide the first ultrastructural evidence that glycolytic enzymes are anatomically associated with SR membranes and located near the SR C2+-ATPase. The results further support the hypothesis that ATP generated by SR-associated glycolytic enzymes is coupled to SR Ca2+ active transport.

Hydroxyl radical inhibits sarcoplasmic reticulum Ca(2+)-ATPase function by direct attack on the ATP binding site.

Oxygen-derived free radicals have been reported to damage the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, potentially contributing to cellular Ca2+ overload and myocardial damage after ischemia and reperfusion. To determine whether the ATP binding site on Ca(2+)-ATPase is involved in oxygen radical injury, SR vesicles containing bound Ca(2+)-ATPase were isolated from rabbit cardiac and skeletal muscle and exposed to a hydroxyl radical (.OH)-generating system consisting of H2O2 and Fe(3+)-nitrilotriacetic acid in amounts that generate a magnitude of .OH similar to that which occurs in the reperfused heart. .OH exposure completely inhibited Ca(2+)-ATPase activity and SR 45Ca uptake for both cardiac and skeletal muscle. In contrast, when the purified vesicles were premixed with 1 mmol/L ATP before exposure to .OH, complete protection was observed: there was no loss of ATPase activity or 45Ca transport. No significant protection occurred with adenosine, sucrose, AMP, or ADP (1 mmol/L each). SDS-gel electrophoresis indicated that .OH did not damage the primary structure of the enzyme. Electron paramagnetic resonance spin-trapping experiments demonstrated that ATP did not scavenge .OH. These results suggest that .OH denatures the SR Ca(2+)-ATPase by directly attacking the ATP binding site, and occupation of the active site by ATP protects against .OH-induced loss of enzymatic activity and SR Ca2+ transport. The depletion of ATP that occurs during ischemia may enhance the toxic effect of .OH at the time of reperfusion.

Functional coupling between glycolysis and sarcoplasmic reticulum Ca2+ transport.

To investigate whether the energy derived from glycolysis is functionally coupled to Ca2+ active transport in sarcoplasmic reticulum (SR), we determined whether glycolytic enzymes were associated with SR membranes and whether metabolism through these enzymes was capable of supporting 45Ca transport. Sealed right-side-out SR vesicles were isolated by step sucrose gradient from rabbit skeletal and cardiac muscle. Intravesicular 45Ca transport was measured after the addition of glycolytic substrates and cofactors specific for each of the glycolytic reactions being studied or after the addition of exogenous ATP and was expressed as transport sensitive to the specific Ca(2+)-ATPase inhibitor thapsigargin. We found that the entire chain of glycolytic enzymes from aldolase onward, including aldolase, GAPDH, phosphoglycerate kinase (PGK), phosphoglyceromutase, enolase, and pyruvate kinase (PK), was associated with SR vesicles from both cardiac and skeletal muscle. Iodoacetic acid, an inhibitor of GAPDH, eliminated 45Ca transport supported by fructose-1,6-diphosphate, the substrate for aldolase, but transport was completely restored by phosphoenolpyruvate (the substrate for PK), indicating that both of the ATP-producing glycolytic enzymes, GAPDH/PGK and PK, were associated with the SR and functionally capable of providing ATP for the Ca2+ pump. Addition of a soluble hexokinase ATP trap eliminated 45Ca transport fueled by exogenous ATP but had markedly less effect on 45Ca transport supported by endogenously produced ATP (via glycolysis). Similarly, at very low concentrations of ATP and ADP (10 to 50 nmol/L), ATP that was produced endogenously from ADP and phosphoenolpyruvate supported 15-fold more 45Ca transport than ATP that was supplied exogenously at the same concentration. These results are consistent with functional coupling of glycolytic ATP to Ca2+ transport and support the hypothesis that ATP generated by SR-associated glycolytic enzymes may play an important role in cellular Ca2+ homeostasis by driving the SR Ca2+ pump.