The novel HLA-C*01:02:89 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-C*01:02:89 differs from HLA-C*01:02:01:01 by one nucleotide in exon 2.

Systematic screening and validation of reliable reference genes for qRT-PCR analysis in Okra (Abelmoschus esculentus L.).

Quantitative real-time polymerase chain reaction (qRT-PCR) is a sensitive and widely used technique for quantifying gene expression levels, and its accuracy depends on the reference genes used for data normalization. To date, no reference gene has been reported in the nutritious and functional vegetable okra (Abelmoschus esculentus L.). Herein, 11 candidates of reference genes were selected and evaluated for their expression stability in okra in different tissues at different developmental stages by using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). Among them, eukaryotic initiation factor 4 alpha (eIF4A) and protein phosphatase 2A (PP2A) showed the highest stability, while TUA5 had the lowest stability. The combined usage of these two most stable reference genes was sufficient to normalize gene expression in okra. Then, the above results were further validated by normalizing the expression of the cellulose synthase gene CesA4. This work provides appropriate reference genes for transcript normalization in okra, which will facilitate subsequent functional gene research on this vegetable crop.

Inhibition of KIR2.1 decreases pulmonary artery smooth muscle cell proliferation and migration.

The investigation of effective therapeutic drugs for pulmonary hypertension (PH) is critical. KIR2.1 plays crucial roles in regulating cell proliferation and migration, and vascular remodeling. However, researchers have not yet clearly determined whether KIR2.1 participates in the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and its role in pulmonary vascular remodeling (PVR) also remains elusive. The present study aimed to examine whether KIR2.1 alters PASMC proliferation and migration, and participates in PVR, as well as to explore its mechanisms of action. For the in vivo experiment, a PH model was established by intraperitoneally injecting Sprague­Dawley rats monocrotaline (MCT). Hematoxylin and eosin staining revealed evidence of PVR in the rats with PH. Immunofluorescence staining and western blot analysis revealed increased levels of the KIR2.1, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) proteins in pulmonary blood vessels and lung tissues following exposure to MCT, and the TGF­ß1/SMAD2/3 signaling pathway was activated. For the in vitro experiments, the KIR2.1 inhibitor, ML133, or the TGF­ß1/SMAD2/3 signaling pathway blocker, SB431542, were used to pre­treat human PASMCs (HPASMCs) for 24 h, and the cells were then treated with platelet­derived growth factor (PDGF)­BB for 24 h. Scratch and Transwell assays revealed that PDGF­BB promoted cell proliferation and migration. Immunofluorescence staining and western blot analysis demonstrated that PDGF­BB upregulated OPN and PCNA expression, and activated the TGF­ß1/SMAD2/3 signaling pathway. ML133 reversed the proliferation and migration induced by PDGF­BB, inhibited the expression of OPN and PCNA, inhibited the TGF­ß1/SMAD2/3 signaling pathway, and reduced the proliferation and migration of HPASMCs. SB431542 pre­treatment also reduced cell proliferation and migration; however, it did not affect KIR2.1 expression. On the whole, the results of the present study demonstrate that KIR2.1 regulates the TGF­ß1/SMAD2/3 signaling pathway and the expression of OPN and PCNA proteins, thereby regulating the proliferation and migration of PASMCs and participating in PVR.

The promoting role of Cx43 on the proliferation and migration of arterial smooth muscle cells for angiotensin II-dependent hypertension.

BACKGROUND: Recent studies have shown that endothelin-1 and angiotensin II (AngII) can increase gap junctional intercellular communication (GJIC) by activating Mitogen-activated protein kinases (MAPKs) pathway. However, not only the precise interaction of AngII with Connexin43(Cx43) and the associated functions remain unclear, but also the regulatory role of Cx43 on the AngII-mediated promotion proliferation and migration of VSMCs is poorly understood. MATERIAL AND METHODS: Our research applicated pressure myography measurements, immunofluorescence and Western blot analyses to investigate the changes in physiological indicators in spontaneously hypertensive rats (SHRs) and AngII-stimulated proliferation and migration of A7r5 SMCs(Rat vascular smooth muscle cells). The aim was to elucidate the role of CX43 in hypertension induced by AngII. RESULTS: Chronic ramipril (angiotensin converting enzyme inhibitor) management for SHRs significantly attenuated blood pressure and blood vessel wall thickness, also reduced contraction rate in the cerebral artery. The cerebral artery contraction rates, mRNA and protein expression of Cx43, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) protein expression in the SHR + ramipril and SHR + ramipril + carbenoxolone (CBX, Cx43 specific blocker) groups were significantly lower than those in the SHR group. Cx43 protein expression and Ser368 phosphorylated Cx43 protein levels increased significantly in AngII-stimulated A7r5 cells. However, the levels of phosphorylated Cx43 decreased after pre-treatment with candesartan (AT1 receptor blocker), GF109203X (protein kinase C (PKC) blocker) and U0126 (mitogen-activated protein kinases/extracellular signal-regulated kinase1/2(MEK/ERK1/2)-specific blocker) in AngII-stimulated A7r5 cells. Cx43 was widely distributed in the cell membrane, nucleus, and cytoplasm of the SMCs. Furthermore, pre-treatment of the AngII- stimulated A7r5 cells with Gap26 (Cx43 blocker) significantly inhibited cell migration and decreased the expression levels of MEK1/2, ERK1/2, P-MEK1/2, and P-ERK1/2. CONCLUSION: Our research confirms that Cx43 plays an important role in the regulation of proliferation and migration of VSMCs via MEK/ERK and PKC signal pathway in AngII-dependent hypertension.

Effects of eye drops of Buddleja officinalis Maxim. extract on lacrimal gland cell apoptosis in castrated rats with dry eye.

OBJECTIVE: To explore the possible mechanism of eye drops of Buddleja officinalis extract in treating dry eye of castrated rats by analyzing the expressions of Bax and Bcl-2 proteins. METHODS: Forty-five Wistar male rats were randomly divided into sham-operated group, untreated group and eye drops of Buddleja officinalis Maxim. extract (treatment) group. The dry eye model was established with orchiectomy in the untreated group and treatment group. Rats in the treatment group were treated with eye drops of Buddleja officinalis Maxim. extract, one drop once, three times daily. Eyes of rats in the sham-operated group and untreated group were instilled with normal saline. After one-, two-, or three-month treatment, five rats in each group were scarified respectively. Then samples were taken to detect related indices. Expressions of Bax and Bcl-2 of lacrimal gland were checked by immunohistochemical method and quantity of apoptotic cells was counted. RESULTS: After one-, two- or three-month treatment, the quantities of expressions of Bax in acinar epithelial cells and glandular tube cells were significantly lower, and those of Bcl-2 were significantly higher in the treatment group than in the untreated group, and the quantities of apoptotic cells of the treatment group were significantly lower than those of the untreated group (P<0.01). CONCLUSION: The main components of extract of Buddleja officinalis Maxim. are flavonoids, which can significantly inhibit cell apoptosis in lacrimal gland.

Effects of extract of Buddleja officinalis eye drops on androgen receptors of lacrimal gland cells of castrated rats with dry eye.

AIM: To evaluate the effects of the extract of Buddleja officinalis eye drops in basic tears secretory volume, tear film stability, expression of androgen receptors (AR) in castrated rats with dry eye, and to investigate the therapeutic effects of the extract of Buddleja officinalis on dry eye caused by gonadal hormones level imbalance. METHODS: Forty-five Wistar masculinity rats were divided at random into nine groups, including normal groups (A1, A2 and A3); model groups (B1, B2 and B3); therapy groups with extract of Buddleja officinalis eye drops (C1, C2 and C3). The "1" stood for being fed for 1 month, and "2" for 2 months, and "3" for 3 months. The dry eye model was established with orchiectomy on groups B and C. Group C was treated with Buddleja officinalis extract eye drops for one month. All rats were checked with Schirmer I test (SIT) and tear film break-up time (BUT). Expression of AR was analyzed by flow cytometer (FCM). RESULTS: The SIT value of group C was significantly higher than that of group B (P<0.01) and the BUT value of group C was significantly longer than that of group B (P<0.01), which indicated the eye drop could significantly keep basic tears secretory volume and tear film stability. And the expression of AR of group C was much higher than that of group B, which showed that available composition of the eye drops maybe display androgen-like activity. CONCLUSION: The main components of the extract of Buddleja officinalis is the flavonoids that can significantly inhibit happening of dry eye of rat after androgen level lowered. Its mechanism is like androgen's and it can display androgen-like activity to keep basic tears secretory volume and tear film stability.

[Effect of C/N ratio on nitrous oxide production during denitrification with different electron acceptors].

The experiment investigated the nitrous oxide production under different C/N ratios during denitrification, taking nitrate and nitrite as electron acceptor respectively. Ethanol was selected as carbon source. The C/N ratios were 0, 1.2, 2.4, 3.5, 5.0 and 20 when nitrate was taken as electron acceptor and C/N ratios 0, 1.8, 2.4, 3.0, 4.3, 5.2, 6.6, 20.6 when electron acceptor was nitrite. The results indicated that: the optimum C/N ratio was 3.0 taking nitrite as electron acceptor and the N2O production was 0.044 mg x L(-1); the optimum C/N ratio was 5.0 taking nitrate as electron acceptor and the N2O production was 0.135 mg x L(-1) which was 3 times higher than that of nitrite as electron acceptor. Though the electron acceptor changed, the trend of N2O production was similar: when carbon source was badly insufficient, the production of N2O and denitrification rate were both quite small; the N2O production increased with the increasing of the quantity of carbon source; when the carbon source was excessive, the N2O production sharply raised. Consequently, compared to complete nitrification and denitrification, short-cut nitrification and denitrification could save 40% carbon source. Moreover, controlling C/N = 3 could reduce the production of N2O in short-cut nitrification.

[Effects of salinity concentration on N2O production during nitrification].

The experiment investigated the production and conversion rate of N2O during nitrification using saline sewage and municipal wastewater. Different kinds of sludge were used, domesticated by saline sewage influent (salinity 7.5 g/L) and municipal wastewater (salinity 0.1 g/L), respectively. The results showed that the production of N2O using saline sewage was 2.85 times higher than that using municipal wastewater. The production and conversion rate of N2O during nitrification under different salinities were also investigated. The results showed that the production of N2O was almost the same when salinity decreased from 7.5 g/L to 5.0 g/L, even 2.5 g/L. However, specific ammonia oxidation rate was increased with the decrease of salinity. The sudden increase of salinity, from 7.5 g/L to 10 g/L, resulted in the increase of N2O production and conversion rate but decrease of specific ammonia oxidation rate. Consequently, It is important to avoid the severe fluctuation of salinity resulted in the increase of N2O production and conversion rate.

[N2O emission and control in shortcut nitrification and denitrification and simultaneous nitrification and denitrification biological nitrogen removal systems].

SBR reactors were used to investigate the N2O emission in shortcut nitrification and simultaneous nitrification and denitrification (SND). Shortcut nitrification with nitrosation rate above 90% was realized by real-time control strategy. The N2O emission and variation of nitrosation rate were investigated under 4 DO levels (0.5, 1.0, 1.5, 2.0 mg/L). The results turned out that the optimal DO to maintain high nitrosation rate and minimum N2O emission was 1.5 mg/L and the N4O emission was 0.06 g per ammonium removed. The SBR filled with carbon fiber performed under low DO and pulse feeding. The SND rate was over 79% during the experiment. The N2O emission was studied under DO 0.2, 0.4, 1.0 and 1.5 mg/L. It turned out that the optimal DO was 1.0 mg/L and the N2O emission was 0.021 g per ammonium removed. Compared to the shortcut nitrification, the N2O emission of SND was 1/3 of the short-cut nitrification under optimal DO.