[The safety and efficacy of combined hepatic artery resection in treatment of hilar cholangiocarcinoma: a meta-analysis].

Objective: To evaluate the safety and effectiveness of combined hepatic artery resection for the treatment of hilar cholangiocarcinoma. Methods: We searched Pubmed, The Cochrane Library, Embase, Web of Science, China Knowledge Network, Wanfang Data Resource System, Vip-Chinese Sci-tech Journal System Database, and China Biomedical Literature Database, and collected the randomized controlled studies or retrospective studies on the safety and efficacy of combined hepatic artery resection and non-hepatic artery resection in the treatment of hilar cholangiocarcinoma. The search period is from January 1, 2006 to December 31, 2019. Review Manager 5.3 software was used to analyze the extracted data indicators. Results: A total of 14 articles were collected, and a total of 2 374 patients with hilar cholangiocarcinoma were included in the study. Meta-analysis results showed that the perioperative mortality in the hepatic artery resection (HAR) group was higher than that of the control group (OR=1.70, 95%CI=0.02-2.90, P=0.05), and the total postoperative morbidity rate was higher than that of the control group (OR=1.28, 95%CI= 0.93-1.76, P=0.13), both of which were not statistically significant compared with the control group. Subgroup analysis showed that the incidence of liver failure (OR=1.15, 95%CI= 0.73-1.82, P=0.54), biliary fistula (OR=1.20, 95%CI= 0.78-1.84, P=0.40), and abdominal infection in the two groups (OR=0.98, 95%CI= 0.53-1.83, P=0.95) was without significant difference. The R0 resection rate of the HAR group was higher than that of the control group, and the difference was not statistically significant (OR=1.08, 95%CI=0.66-1.75, P=0.77). The rates of lymph node metastasis in the HAR group were higher than that in the control group (OR= 2.48, 95%CI= 1.05-5.84, P=0.04). One-year(OR=0.48, 95%CI= 0.32-0.72, P=0.000 5), 3-year (OR= 0.51, 95%CI=0.36-0.72, P=0.000 1), and 5-year (OR=0.50, 95%CI=0.35-0.70, P<0.000 1) survival rates of HAR group were lower than those of the control group. The survival rates of patients in HAR group treated with combined chemotherapy drugs after operation were significantly improved (OR= 7.33, P=0.02). Conclusions: The safety of combined HAR treatment for hilar cholangiocarcinoma is acceptable, but poor postoperative survival may be related to the high lymph node metastasis rate. Therefore, it is still necessary to be cautious in carrying out this operation. Combined with adjuvant chemotherapy after surgery may improve survival.

[Mental health survey of medical staff in a tertiary infectious disease hospital for COVID-19].

Objective: To investigate the mental health of clinical first-line medical staff in COVID-19 epidemic and provide theoretical basis for psychological intervention. Methods: The mental health status of the first-line medical staff was investigated by Self-rating Anxiety Scale(SAS) and Post-Traumatic Stress Disorder Self- rating Scale (PTSD-SS). From February 7 to 14, 2020, 246 medical staff participated in the treatment of COVID-19 were investigated using cluster sampling, and received 230 responses, with a recovery rate of 93.5%. Results: The incidence of anxiety in medical staff was 23.04% (53/230) , and the score of SAS was(42.91±10.89). Among them, the incidence of severe anxiety, moderate anxiety and mild anxiety were 2.17%(5/230) , 4.78%(11/230) and 16.09%(37/230) , respectively. The incidence of anxiety in female medical staff was higher than that in male [25.67%(48/187) vs 11.63%(5/43) , Z=-2.008, P=0.045], the score of SAS in female medical staff was higher than that in male [(43.78±11.12) vs (39.14±9.01) , t=-2.548, P=0.012]. The incidence of anxiety in nurses was higher than that in doctors[26.88% (43/160) vs 14.29% (10/70) , Z=-2.066, P=0.039], and the score of SAS in nurses was higher than that in doctors [ (44.84±10.42) vs (38.50±10.72) , t=-4.207, P<0.001]. The incidence of stress disorder in medical staff was 27.39% (63/230) , and the score of PTSD-SS was (42.92±17.88) . The score of PTSD-SS in female medical staff was higher than that in male[ (44.30±18.42) vs (36.91±13.95) , t=-2.472, P=0.014]. Conclusion: In COVID-19 epidemic , the incidence of anxiety and stress disorder is high among medical staff. Medical institutions should strengthen the training of psychological skills of medical staff. Special attention should be paid to the mental health of female nurses.

[The effect of estrogen level on Budd Chiari syndrome related hepatocellular carcinoma].

Objective: To investigate the effect of estrogen level on Budd Chiari syndrome related hepatocellular carcinoma. Methods: Immunohistochemical method was used to detect estrogen receptor-α and estrogen receptor-ß expression in 38 cases of Budd Chiari syndrome related hepatocellular carcinoma and 50 cases of HBV related hepatocellular carcinoma.Hepatoma cells of Budd Chiari syndrome related hepatocellular carcinoma were exposed to different concentrations of Estrogen for 48 hours. Tetrazolium bromide (MTT) colorimetry was used to analyze cell proliferation activities; cell cycle was analyzed by flow cytometry (FCM); cell apoptosis was analyzed by flow cytometry (FCM) and Casepase-3 activity was measured after induced by adriamycin(ADM). Results: The positive rate of estrogen receptor-α expression in the tissues of Budd Chiari syndrome related hepatocellular carcinoma was 71.05%, which was higher than that (32%)in HBV related hepatocellular carcinoma tissue evidently (P<0.01). The positive rate of estrogen receptor-ß expression in the tissues of Budd Chiari syndrome related hepatocellular carcinoma was 68.4%, which was higher than that (26%)in HBV related hepatocellular carcinoma tissue evidently (P<0.01). With the concentrations of estrogen increasing, MTT Assays showed that estrogen level increased the cell proliferation activities of Budd Chiari syndrome related hepatocellular carcinoma. The number of cells at stage S and G2/M were significantly increased and cells at stage G0/G1 were reduced with the increasing concentrations of estrogen. After being incubated under the different concentrations of estrogen for 48 h, the apoptosis rates decreased gradually and the Casepase-3 activity was significantly reduced with the increasing concentration of estrogen. Conclusions: Estrogenreceptor expression may have an important influence on hepatocellular carcinoma cell biology difference between Budd Chiari syndrome related hepatocellular carcinoma and HBV related hepatocellular carcinoma. Estrogen level can promote cell proliferation and cell cycle, and inhibit the apoptosis of hepatoma cells of Budd Chiari syndrome related hepatocellular carcinoma in vitro, and these effects were increased with the increasing of estrogen level.

GluR1 protects hypoxic ischemic brain damage via activating Akt signaling pathway in neonatal rats.

OBJECTIVE: To investigate the role of glutamic acid receptor 1 (GluR1) in hypoxic-ischemic brain damage (HIBD) in neonatal rats and its underlying mechanism. MATERIALS AND METHODS: 7-day-old neonatal rats received right common carotid artery (CCA) ligation for the establishment of HIBD. After the operation, rats were sacrificed at different time points (0, 4, 6, 12, 24, 48, and 72 h), respectively. Meanwhile, rats in Sham group underwent similar procedures without ligation. Lentivirus-GLUR1-shRNA (LV-GLUR1 shRNA group) was constructed and then transfected into the right lateral ventricles of rats to inhibit GluR1 in vivo. Rats received LV-control injection were selected in the control group (LV-control group). After injection of Lentivirus-GLUR1-shRNA, CCA ligation was performed in rats for HIBD construction. Western blot was performed to detect the protein levels of GLUR1, Akt, p-Akt, and vascular endothelial growth factor (VEGF) in brain tissues. Cell apoptosis was measured by TUNEL staining assay. RESULTS: After hypoxic ischemia (HI), GLUR1 expression increased gradually and reached a peak at 24 h. Meanwhile, p-Akt expression increased immediately and then gradually decreased. 24 h later, p-Akt expression increased again and peaked at 48 h. VEGF expression increased at 4 h after HI and reached a peak at 12 h. The expression levels of GLUR1, p-Akt, and VEGF in the brain tissues derived from rats transfected with LV-GLUR1 shRNA significantly decreased at both 4 h and 24 h after HI. In addition, results indicated that cell apoptosis was enhanced after LV-GLUR1 shRNA administration, suggesting the role of GLUR1 in protecting against HIBD. CONCLUSIONS: GLUR1 exhibits a remarkable protective role in HIBD, which may be related to the activation of the Akt signaling pathway and the upregulation of VEGF after HI.

Clinical application of kelp micro gelation (KMG) in partial splenic embolization.

OBJECTIVE: We aimed to compare and analyze the long-term efficacy and safety between kelp micro gelation (KMG) and gelfoam particles in partial splenic embolization (PSE). PATIENTS AND METHODS: This study retrospectively included 65 liver cirrhosis patients with comorbidity of hypersplenism who were admitted in Nanfang Hospital from July 2008 to May 2012. Among the included patients, 33 patients were in KMG-PSE group, and 32 cases were included in Gelfoam-PSE group. According to the Child-Pugh criteria, all cases were divided into grade A, B, and C, respectively. All eligible subjects received CT or MR examination and laboratory examination. RESULTS: Our results showed that both KMG and gelfoam particles could substantially improve the short-term efficacy of thrombocytopenia leukopenia. However, the efficacy of KMG is superior to that of gelfoam. Due to the characteristic of KMG as a permanent agent, KMG may result in an obvious pain in the spleen after PSE, especially in patients with megalosplenia. KMG was more expensive than gelfoam particles. The complication rate in patents with great embolization was much higher than that with less embolization. CONCLUSIONS: The efficacy of KMG is superior to that of gelfoam in both short-term and long-term. To effectively control the occurrence of severe complication, the embolization should be controlled less than 70% regardless of the embolization agents.

[Occupational exposure investigation and protective measures in a tertiary infectious disease hospital].

Objective: To investigate the cause of occupational exposure among 136 nurses in a tertiary infectious disease hospital, and puts forward the prevention strategy. Methods: A total of 136 nurses exposed to occupational exposure between 2014 and 2016 were included in the study. Analysis was conducted from the years of work of nurses, exposure routes, and the pathogens. Results: The nurses suffer from the highest risk of occupational exposures (73.91%) .Nurses working for less than 5 years and interns are most likely to suffer occupational exposure (45.59% and 35.29% respectively) . Occupational exposure was mainly caused by needle injuries, in which infusion was the main route of occupational exposure (36.76%) . The improper treatment of needle pulling after infusion is the main link of needle puncture (36.76%) . Occupational exposure pathogens were mainly HBV (63.24%) . Conclusion: Nursing staff is the high-risk group of occupational exposure. Irregular operation, lack of awareness of protection, improper disposal after the needle withdrawal and poor safety assessment of the operating environment are the main causes of occupational exposure. It is suggested to strengthen the training of occupational safety and protection, enhance clinical nurses occupational safety protection consciousness, standardize medical operation, so as to prevent the occurrence of occupational exposure.

Distribution and quantitative analysis of CIDEa and CIDEc in broiler chickens: accounting for differential fat deposition between strains.

1. Differences in the expression of CIDEa and CIDEc in 20 different tissues were examined. Both CIDEa and CIDEc mRNA transcripts were predominantly but variably expressed in white adipose tissue (WAT) but were also expressed at moderate levels in the kidney and liver and at lower levels in the ovary. Interestingly, among WAT types, both CIDEa and CIDEc were expressed at the lowest levels in heart coronary WAT. 2. To better understand the roles of CIDEa and CIDEc in the fat deposition of broiler chickens, the differences in lipid droplet (LD) size and mRNA levels of CIDEa and CIDEc between lean-type and fat-type broiler chicken lines were studied. LD sizes were larger in fat-type broiler lines, and CIDEa and CIDEc mRNA levels in white adipose, kidney and liver tissues were significantly higher in fat-type broiler lines than in their lean counterparts. 3. Developmental expression patterns of CIDEa and CIDEc mRNA were analysed in different tissue types (WAT, liver and kidney) in Arbor Acres broiler chickens, and CIDEa and CIDEc mRNA expression levels increased during sequential developmental stages, achieving peak expression levels at week 6. 4. These observations suggest that the functions of CIDEa and CIDEc reflect inherent characteristics of lipid metabolism that contribute to the differences in fat deposition between strains. The results in this study contribute to a more robust understanding of the tissue distribution and expression patterns of CIDEa and CIDEc mRNA and facilitate further research concerning the molecular mechanism underlying fat deposition in broiler chickens.

[Chorionic villus cell culture and karyotype analysis in 1 983 cases of spontaneous miscarriage].

Objective: To investigate the relationship between spontaneous miscarriage and embryonic chromosome abnormalities, and to evaluate the clinical application of karyotype analysis by chorionic villus cell culture. Methods: The chorionic villus karyotype of 1 983 cases of miscarriage from January 2010 to July 2016 in Guangzhou Women and Children's Mecical Center were analyzed retrospectively. The miscarried chorionic villi were obtained by curettage under sterilized condition. The chromosome specimens were prepared after chorionic villus cell culture. Karyotype analysis was performed by G-banding technique. Results: In the 1 983 samples, successful karyotype analysis was performed in 1 770 cases, with the successful rate of 89.98%. Chromosomal abnormalities were found in 1 038 cases (58.64%, 1 038/1 770). Chromosomal structural abnormalities were found in 37 cases. The numeral abnormalities were more common than structural abnormalities, and most of the numeral abnormalities were aneupoidies. In turn, they were trisomy 16, 45,X, trisomy 22, trisomy 2, trisomy 21, trisomy 15. The most common structural abnormality was balanced translocation, including Robersonian translocation. Female embryoes accounted for 61.02% (1 080/1 770) miscarriages and for 57.4%(596/1 770) of chromosomal abnormalities, while male embroyes acoounted for 61.02% (1 080/1 770) , 57.4% (596/1 770) respectively. The proportion of female embryoes was higher than male embryoes. The median age of the patients was 30 years old (16-46 years old) . As the maternal age increased, the proportion chromosomal abnormalities increased. The incidence of chromosomal abnormalities in the advanced age group (≥35 years) was 68.38% (240/351) , which was significantly higher than that in the younger group (56.24%, 798/1 419; χ(2)=17.10, P<0.01). Conclusions: Embryonic chromosomal abnormalities are the most common cause of early spontaneous miscarriage. The abnormalities centralize in some karyotypes. There is certain relationship between maternal age and the incidence of miscarriage, as well as the embryonic gender. Chorionic villus cell culture and karyotype analysis are helpful in finding the cause of miscarriage and counsel the patients.

Cisplatin plus norcantharidin alter the expression of TGF-ß1/Smads signaling pathway in hepatocellular carcinoma.

PURPOSE: To investigate the effects of cisplatin plus norcantharidin on transforming growth factor (TGF)-ß1/Smads signaling pathway in hepatocellular carcinoma cells. METHODS: Hepatocellular carcinoma cells (Hep3B) were divided into four groups: control group, cisplatin 2.0 µg/ml group, norcantharidin 10 µg/ml group, and cisplatin 2.0 µg/ml plus norcantharidin 10 µg/ml group. All cells were incubated for 24 hours. Cells proliferation was assessed using cell counting kit-8. Relative mRNA expression of TGF-ß1, Smad4 and Smad7 were assessed by quantitative RT-PCR. Protein expression of TGF-ß1 and Smad4 were investigated by western blotting. RESULTS: Cisplatin, norcantharidin and cisplatin plus norcantharidin significantly inhibited the proliferation of cells, significantly attenuated both the mRNA and protein expression of TGF-ß1 and Smad7, and significantly up-regulated the mRNA and protein expression of Smad4 in Hep3B (all p < 0.05), and cisplatin plus norcantharidin exhibited powerful effects than cisplatin and norcantharidin. CONCLUSIONS: Cisplatin, norcantharidin and cisplatin plus norcantharidin can significantly alter the expression of TGF-ß1/Smads signaling pathway and inhibit the proliferation of Hep3B cells. Cisplatin plus norcantharidin exhibited powerful effects than cisplatin and norcantharidin (Fig. 4, Ref. 23).

Bioinformatic identification of IGF1 as a hub gene in hepatocellular carcinoma (HCC) and in-vitro analysis of the chemosensitizing effect of miR-379 via suppressing the IGF1/IGF1R signaling pathway.

OBJECTIVE: We investigated the interactions among the dysregulated genes in hepatocellular carcinoma (HCC) and identified the hub genes in the protein-protein interaction (PPI) network. Also, we also investigated the regulative effect of miR-379 on the IGF1/IGF1R signaling pathway and chemoresistance in HCC. MATERIALS AND METHODS: Raw data of a microarray that compared transcriptional gene profile between 3-paired HCC tissue samples and adjacent normal tissues were downloaded from Expression Atlas (E-GEOD-33006). The raw data was reanalyzed to identify the significantly dysregulated genes, which were further used for PPI network and KEGG pathway analysis. The regulative effect of miR-379 on IGF1R expression was studied by dual luciferase assay and Western blotting. The functional role of miR-379 in chemosensitivity of HCC cells was studied by drug sensitivity and flow cytometric assay. RESULTS: IGF1 is a hub gene that is mostly upregulated in HCC and it is an important player in the p53 signaling pathway. Knockdown of IGF1R significantly decreased IC50 of 5-FU, paclitaxel (PTX) and Doxorubicin (DOX) in Huh7 and HepG2 cells. MiR-379 could directly bind to the 3'UTR of IGF1R and suppress IGF1R expression. MiR-379 overexpression sensitized Huh7 and HepG2 cells to 5-FU, PTX and DOX and also enhanced DOX-induced cell apoptosis. CONCLUSIONS: IGF1 is a hub gene in HCC and is also one of the most upregulated genes in HCC tissues compared to normal tissues. It is involved in the p53 signaling pathway regulation. MiR-379 can sensitize HCC cells to chemotherapeutic reagents via targeting IGF1R and suppressing its expression.

[Soft tissue sarcoma of larynx: a case report].

The clinical manifestation was gradually hoarseness for half a year, with aggravation of dyspnea in three days. Physical examination and laryngoscopy showed laryngeal neoplasm, the glottis was not exposed, pedicle, no adhesion with the surrounding. CT scan showed glottic soft-tissue, density, unclear boundary. Enhanced CT showed anterior commissure thickening and tumor invasion of supraglottic region. Preoperative biopsy results: fibroblasts and fibrocyte were arranged in a crisscross pattern. Postoperative pathology showed laryngeal soft tissue sarcoma.

Proline partially overcomes excess molybdenum toxicity in cabbage seedlings grown in vitro.

In vitro grown cabbage (Brassica oleracea var. capitata) seedlings exposed to excess molybdenum (Mo) ions exhibited severely reduced plant growth at the cotyledonary stage. Adding 80 mM proline (Pro) to the Mo-treated medium could help 50% seedlings to overcome the toxicity and grow true leaves. Under excess Mo stress, seedlings accumulated blue/purple anthocyanin in their cotyledons and hypocotyls. The anthocyanin content under Mo with 40 mM Pro was 4-fold higher than the control medium, MS with 40 mM Pro. The presence of Pro in the excess-Mo condition reduced chlorophyll a, whereas the chlorophyll b content was much higher than the control media of MS with and without Pro. Moreover, supplementing various concentrations of Pro into the Mo-stressed condition promoted the seedlings with higher antioxidant enzyme activities of superoxide dismutase, ascorbate peroxidate, and catalase. In addition, genes in the anthocyanin biosynthesis and accumulation pathways, phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), flavonone 3-hydroxylase (F3H), leucoanthocyanidin dioxygenase (LDOX), and glutathione-S-transferase (GST), were all upregulated. Our study indicated that, under excess Mo stress, the antioxidant activity of cabbage seedlings was induced in an attempt to protect plants from the Mo-induced toxicity and exacerbated growth. Pro, on the other hand, functioned in producing higher antioxidant enzyme activity to partially help recover plant growth.

MicroRNA-217, down-regulated in clear cell renal cell carcinoma and associated with lower survival, suppresses cell proliferation and migration.

Aberrantly expressed microRNAs (miRNAs) are frequently correlated with a variety of human cancers, including clear cell renal cell carcinoma (ccRCC). In this study, we determined the expression patterns of miR-217 in ccRCC, and tested its effect on cancer cell proliferation and migration. The expression levels of miR-217 were determined in 54 ccRCC samples using Real-Time qPCR. 786-O and ACHN cells were transfected with miR-217 mimics or miRNA mimics control. Cell proliferation and migration were evaluated by MTT assay and scratch-wound assay, respectively. We found that miR-217 was down-regulated in ccRCC compared to paired normal tissue. Lower miR-217 expression levels were associated with higher tumor grade and stage. All patients with high miR-217 expression survived 5 years, while with low miR-217 expression, only 40% survived. Cell proliferation inhibition and decreased motility were observed in cells transfected with the miR-217 mimics. In conclusion, miR-217 plays a tumor suppressor role in ccRCC.

Distinct behavioural and network correlates of two interneuron types in prefrontal cortex.

Neurons in the prefrontal cortex exhibit diverse behavioural correlates, an observation that has been attributed to cell-type diversity. To link identified neuron types with network and behavioural functions, we recorded from the two largest genetically defined inhibitory interneuron classes, the perisomatically targeting parvalbumin (PV) and the dendritically targeting somatostatin (SOM) neurons in anterior cingulate cortex of mice performing a reward foraging task. Here we show that PV and a subtype of SOM neurons form functionally homogeneous populations showing a double dissociation between both their inhibitory effects and behavioural correlates. Out of several events pertaining to behaviour, a subtype of SOM neurons selectively responded at reward approach, whereas PV neurons responded at reward leaving and encoded preceding stay duration. These behavioural correlates of PV and SOM neurons defined a behavioural epoch and a decision variable important for foraging (whether to stay or to leave), a crucial function attributed to the anterior cingulate cortex. Furthermore, PV neurons could fire in millisecond synchrony, exerting fast and powerful inhibition on principal cell firing, whereas the inhibitory effect of SOM neurons on firing output was weak and more variable, consistent with the idea that they respectively control the outputs of, and inputs to, principal neurons. These results suggest a connection between the circuit-level function of different interneuron types in regulating the flow of information and the behavioural functions served by the cortical circuits. Moreover, these observations bolster the hope that functional response diversity during behaviour can in part be explained by cell-type diversity.

A ZnO nanorod inorganic/organic heterostructure light-emitting diode emitting at 342 nm.

An inorganic/organic heterostructure light-emitting diode consisting of the hole-transporting layer N, N'-di(naphth-2-yl)- N, N'-diphenylbenzidine (NPB) and n-type ZnO nanorods fabricated by hydrothermal decomposition is reported. Poly(methyl methacrylate) was used to form a smooth surface on top of ZnO nanorod array with ZnO nanorod tops exposed for subsequent NPB deposition. An unusual ultraviolet emission at 342 nm was observed in the electroluminescence spectrum. Compared to band gap energy of ZnO (3.37 eV), the excitonic emission is blue-shifted and broadened. The mechanism of the blue shift is discussed in terms of the energy band diagram of the heterostructure.

MEMS-based flow cytometry: microfluidics-based cell identification system by fluorescent imaging.

This study utilizes MEMS technology to realize a novel low-cost microfluidics-based biochip system for flow-type cell handling. Powered by vacuum pump, the microfluidic driving system enables cells to move in order one by one in the biochip by an effect of sheath flow prefocus. Then, cells are guided to a fluorescent inspection region where two detection tasks such as cell image identification and cell counting are conducted. Currently, the glass-based biochip has been manufactured and all the related devices have been well set up in our laboratory. With this proposed prototype system, typical results about cell separation of yeast cell and PC-3 cell are available and their separated images are also presented, respectively.

The tumor cells in nodular lymphocyte-predominant Hodgkin disease are clonally related to the large cell lymphoma occurring in the same individual. Direct demonstration by single cell analysis.

Large cell lymphoma (LCL) sometimes occurs concurrently or subsequently in patients with nodular lymphocyte-predominant Hodgkin disease (NLPHD). Although there is evidence of a clonal relationship between LCL and NLPHD, there has been no direct demonstration that the lymphocytic and histiocytic (L&H) cells in NLPHD are related to the tumor cells in LCL. We identified 2 cases of NLPHD with an associated LCL. Single L&H cells, the Reed-Sternberg cell variants in NLPHD, were isolated from immunostained tissue sections by micromanipulation, and the immunoglobulin heavy chain gene (IgH) complementarity determining region (CDR) III of the cells was amplified by the polymerase chain reaction (PCR). The products were compared with those obtained from microdissected LCL cells using polyacrylamide gel electrophoresis and nucleotide sequencing. The IgH CDRIII sequences from the L&H cells were related to each other, but also showed nucleotide substitutions, consistent with a germinal center origin. The sequences from the L&H cells also were related to those from the corresponding LCL cells. We have provided direct evidence through sequence analysis of the IgH CDRIII that the L&H cells are clonally related to the corresponding LCL arising in 2 cases of NLPHD.

Phosphorylation of synthetic peptides by a CDPK and plant SNF1-related protein kinase. Influence of proline and basic amino acid residues at selected positions.

Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be inactivated by phosphorylation of Ser-158 by calmodulin-like domain protein kinases (CDPKs) or SNF1-related protein kinases (SnRK1) in vitro. While the phosphorylation site sequence is relatively conserved, most of the deduced sequences of SPS from dicot species surrounding the Ser-158 regulatory phosphorylation site contain a Pro residue at P-4 (where P is the phosphorylated Ser); spinach is the exception and contains an Arg at P-4. We show that a Pro at P-4 selectively inhibits phosphorylation of the peptide by a CDPK relative to a SnRK1. The presence of a Pro at P-4, by allowing a tight turn in the peptide substrate, may interfere with proper binding of residues at P-5 and beyond. Both kinases had greater activity with peptides having basic residues at P-6 and P+5 (in addition to the known requirement for an Arg at P-3/P-4), and when the residue at P-6 was a His, the pH optimum for phosphorylation of the peptide was acid shifted. The results are used to predict proteins that may be selectively phosphorylated by SnRK1s (as opposed to CDPKs), such as SPS in dicot species, or may be phosphorylated in a pH-dependent manner.

Identification of a novel phosphorylation motif for CDPKs: phosphorylation of synthetic peptides lacking basic residues at P-3/P-4.

The Ca(2+)-dependent protein kinases (CDPKs) are members of a large subfamily of protein kinases in plants that have been implicated in the control of numerous aspects of plant growth and development. One known substrate of the CDPKs is the ER-located ACA2 calcium pump, which is regulated by phosphorylation of Ser(45). In the present study, a synthetic peptide based on the known regulatory phosphorylation site (RRFRFTANLS(45)KRYEA) was efficiently phosphorylated in vitro by CDPKs but not a plant SNF1-related protein kinase. Phosphorylation of the Ser(45)-ACA2 peptide was surprising because the sequence lacks basic residues at P-3/P-4 (relative to the phosphorylated Ser at position P) that are considered to be essential recognition elements for CDPKs. We demonstrate that phosphorylation of the Ser(45)-ACA2 peptide is dependent on the cluster of basic residues found N-terminal (P-6 to P-9) as well as C-terminal (P + 1/P + 2) to the phosphorylated Ser. The results establish a new general phosphorylation motif for CDPKs: [Basic-Basic-X-Basic]-phi-X(4)-S/T-X-Basic (where phi is a hydrophobic residue). The motif predicts a number of new phosphorylation sites in plant proteins. Evidence is presented that the novel motif may explain the phosphorylation by CDPKs of Ser271 in the aquaporin PM28A.