Commensal flora triggered target anti-inflammation of alginate-curcumin micelle for ulcerative colitis treatment.

Ulcerative colitis (UC) is a chronic, idiopathic inflammatory bowel disease characterized by dysregulation of colon immune response. Curcumin (Cur) has strong anti-inflammatory activities, but the application is severely hindered by the extremely hydrophobicity and pitiful bioavailability. Alginate (Alg), a natural polysaccharide with ideal solubility and biosafety, was introduced to prepare the esterified alginate-curcumin conjugate (Alg-Cur) and constructed stable Alg-Cur micelle in physiological solutions. Compared with crystalline Cur, the target anti-inflammatory activities of Alg-Cur were systematically investigated. The results showed that Alg-Cur exerted effective anti-inflammatory effects in Raw 264.7 cells. After oral administration, 92.32 % of Alg-Cur reached colon, and the ester bonds were quickly sheared by abundant esterase produced by commensal anaerobic flora. The released Cur was quickly absorbed in-situ in monomolecular state, and effectively ameliorated the colonic inflammation and tissue damage by inhibiting the TLR4 expression in colonic epithelial cell, reducing the transcription and expression of the pro-inflammation cytokines downstream, as well as the infiltration of lymphocytes, macrophages and neutrophils. The Alg-Cur micelle effectively enhanced the hydrophilicity and bioavailability of Cur, and the commensal flora triggered Cur release showed great potential for UC treatment.

Defecation function and quality of life in patients with slow-transit constipation after colectomy.

BACKGROUND: Although total or subtotal colectomy for slow-transit constipation (STC) has been proven to be a definite treatment, the associated defecation function and quality of life (QOL) are rarely studied. AIM: To evaluate the effectiveness of surgery for STC regarding defecation function and QOL. METHODS: From March 2013 to September 2017, 30 patients undergoing surgery for STC in our department were analyzed. Preoperative, intra-operative, and postoperative 3-mo, 6-mo, 1-year, and 2-year follow-up details were recorded. Defecation function was assessed by bowel movements, abdominal pain, bloating, straining, laxative, enema use, diarrhea, and the Wexner constipation and incontinence scales. QOL was evaluated using the gastrointestinal QOL index and the 36-item short form survey. RESULTS: The majority of patients (93.1%, 27/29) stated that they benefited from the operation at the 2-year follow-up. At each time point of the follow-up, the number of bowel movements per week significantly increased compared with that of the preoperative conditions (P < 0.05). Similarly, compared with the preoperative values, a marked decline was observed in bloating, straining, laxative, and enema use at each time point of the follow-up (P < 0.05). Postoperative diarrhea could be controlled effectively and notably improved at the 2-year follow-up. The Wexner incontinence scores at 6-mo, 1-year, and 2-year were notably lower than those at the 3-mo follow-up (P < 0.05). Compared with those of the preoperative findings, the Wexner constipation scores significantly decreased following surgery (P < 0.05). Thus, it was reasonable to find that the gastrointestinal QOL index scores clearly increase (P < 0.05) and that the 36-item short form survey results displayed considerable improvements in six spheres (role physical, role emotional, physical pain, vitality, mental health, and general health) following surgery. CONCLUSION: Total or subtotal colectomy for STC is not only effective in alleviating constipation-related symptoms but also in enhancing patients' QOL.

Can integration reduce inequity in healthcare utilization? Evidence and hurdles in China.

BACKGROUND: Integration of medical insurance schemes has been prioritized as one of the key strategies to address inequity in China's health system. The first pilot attempt to integrate started in 2003 and later expanded nationwide. This study aims to assess its intended impact on inequity in inpatient service utilization and identify the main determinants contributing to its ineffectiveness. METHODS: A total of 49,365 respondents in the pilot integrated area and 77,165 respondents in the non-integration area were extracted from the Fifth National Health Services Survey. A comparative analysis was conducted between two types of areas. We calculate a concentration index (CI) and horizontal inequity index (HI) in inpatient service utilization and decompose the two indices. RESULTS: Insurance integration played a positive role in reducing inequality in inpatient service utilization to some extent. A 13.23% lower in HI, a decrease in unmet inpatient care and financial barriers to inpatient care in the pilot integrated area compared with the non-integration area; decomposition analysis showed that the Urban-Rural Residents Basic Medical Insurance, a type of integrated insurance, contributed 37.49% to reducing inequality in inpatient service utilization. However, it still could not offset the strong negative effect of income and other insurance schemes that have increased inequality. CONCLUSIONS: The earlier pilot attempt for integrating medical insurance was not enough to counteract the influence of factors which increased the inequality in inpatient service utilization. Further efforts to address the inequality should focus on widening access to financing, upgrading the risk pool, reducing gaps within and between insurance schemes, and providing broader chronic disease benefit packages. Social policies that target the needs of the poor with coordinated efforts from various levels and agencies of the government are urgently needed.

Selective Strengthening of Intracortical Excitatory Input Leads to Receptive Field Refinement during Auditory Cortical Development.

In the primary auditory cortex (A1) of rats, refinement of excitatory input to layer (L)4 neurons contributes to the sharpening of their frequency selectivity during postnatal development. L4 neurons receive both feedforward thalamocortical and recurrent intracortical inputs, but how potential developmental changes of each component can account for the sharpening of excitatory input tuning remains unclear. By combining in vivo whole-cell recording and pharmacological silencing of cortical spiking in young rats of both sexes, we examined developmental changes at three hierarchical stages: output of auditory thalamic neurons, thalamocortical input and recurrent excitatory input to an A1 L4 neuron. In the thalamus, the tonotopic map matured with an expanded range of frequency representations, while the frequency tuning of output responses was unchanged. On the other hand, the tuning shape of both thalamocortical and intracortical excitatory inputs to a L4 neuron became sharpened. In particular, the intracortical input became better tuned than thalamocortical excitation. Moreover, the weight of intracortical excitation around the optimal frequency was selectively strengthened, resulting in a dominant role of intracortical excitation in defining the total excitatory input tuning. Our modeling work further demonstrates that the frequency-selective strengthening of local recurrent excitatory connections plays a major role in the refinement of excitatory input tuning of L4 neurons.SIGNIFICANCE STATEMENT During postnatal development, sensory cortex undergoes functional refinement, through which the size of sensory receptive field is reduced. In the rat primary auditory cortex, such refinement in layer (L)4 is mainly attributed to improved selectivity of excitatory input a L4 neuron receives. In this study, we further examined three stages along the hierarchical neural pathway where excitatory input refinement might occur. We found that developmental refinement takes place at both thalamocortical and intracortical circuit levels, but not at the thalamic output level. Together with modeling results, we revealed that the optimal-frequency-selective strengthening of intracortical excitation plays a dominant role in the refinement of excitatory input tuning.

SIRT1 inhibitory compounds from the roots of Codonopsis pilosula.

Three new compounds, pilosulinene A (1), pilosulinols A (2), and B (3), along with seven known compounds, were isolated from the roots of Codonopsis pilosula cultivated in Xundian County of Yunnan Province. The structures of new compounds were established by spectroscopic methods. In particular, the presence of an aromatic ring in the structure of 1 makes it intriguing. The inhibitory activity of compounds against SIRT1 was evaluated. The results showed that 8 could inhibit Sirt1 in a dose-dependent manner.

Two New Triterpenoids from the Roots of Codonopsis pilosula.

Pseudolarolides U and V, two new triterpenoids, and four biogenetically related compounds, pseudolarolides E, F, K, and P were isolated from the roots of Codonopsis pilosula (Campanulaceae). Their structures were determined by spectroscopic data. The regulation of Sirtuin 1 (SIRT1) activity by all the isolated compounds was evaluated.

Two Novel Proline-Containing Catechin Glucoside from Water-Soluble Extract of Codonopsis pilosula.

Choushenflavonoids A (1) and B (2), two unusual proline-containing catechin glucosides, were isolated from the roots of Codonopsis pilosula cultivated in a high-altitude location of Yunnan province. Their structures were determined by spectroscopic data and chemical methods. Specifically, the absolute configuration of glucose residue in 1 and 2 was assigned by acid hydrolysis followed by derivatization and gas chromatography (GC) analysis. In addition, biological evaluation of 1 and 2 against Sirtuin 1 (SIRT1) was carried out.

Simultaneous determination and pharmacokinetic study of three flavonoid glycosides in rat plasma by LC-MS/MS after oral administration of Rubus chingii Hu extract.

A simple and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of isoquercitrin, kaempferol-3-O-rutinoside and tiliroside in rat plasma. Plasma samples were deproteinized with methanol and separated on a Hypersil Gold C18 column (2.1 × 50 mm, i.d., 3.0 µm) using gradient elution with the mobile phase of water and methanol at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed with negative ion electrospray ionization in selected reaction monitoring mode. All analytes showed good linearity over their investigated concentration ranges (r2 > 0.99). The lower limit of quantification was 1.0 ng/mL for isoquercitrin and 2.0 ng/mL for kaempferol-3-O-rutinoside and tiliroside, respectively. Intra- and inter-day precisions were <8.2% and accuracy ranged from -11.5 to 9.7%. The mean extraction recoveries of analytes and IS from rat plasma were >80.4%. The assay was successfully applied to investigate the pharmacokinetic study of the three ingredients after oral administration of Rubus chingii Hu to rats.

[Interpretation of Chinese guidelines for the diagnosis and treatment of constipation surgery (2017)].

The Chinese Society of Coloproctology (CSCP) released the updated "guidelines for the diagnosis and treatment of constipation surgery" in 2017. The major amendments include the following aspects: (1) According to defecation reflex involving parts of the classification, the cause of constipation is divided into three parts, including colorectal factors, internal and external colorectal factors. The new division is more rational, easy to make clear the location and the possible pathogenesis. (2) Constipation is divided into slow transit constipation, outlet obstructive constipation and mixed constipation. The main consideration of this division is the name of the surgery for the outlet obstructive constipation, such as the rectal prolapse and the rectocele. (3) The 6-hour observation time point is added in the colonic transit test and to make clear whether the stomach and the small intestine has slow transit. (4) Micro ecological preparations, promoting dynamic drugs and promoting secretion drugs as well as psychological treatment and acupuncture treatment are added in non-surgical treatment of constipation (5) The antegrade colonic enema is removed from surgical treatment of slow transit constipation; in the surgical treatment of internal rectal prolapse, STARR surgery is added and Delorme surgery is deleted; transrectal and transperineal approach are added in the surgical treatment of rectocele. In this paper, we compare the domestic and international constipation-related guidelines, and summarize the main revisions in the 2017 edition of "guidelines for the diagnosis and treatment of constipation surgery".

Tetramethylpyrazine Protects Against Glucocorticoid-Induced Apoptosis by Promoting Autophagy in Mesenchymal Stem Cells and Improves Bone Mass in Glucocorticoid-Induced Osteoporosis Rats.

Glucocorticoid-induced osteoporosis (GIOP) is a widespread clinical complication due to the common use of glucocorticoids. Excess glucocorticoids induce apoptosis of bone marrow-derived mesenchymal stem cells (BMSCs), which have been shown to play an increasingly important role in the pathogenesis and therapy of osteoporosis. Tetramethylpyrazine (TMP), an extract from one of the most recognized herbs in traditional Chinese medicine (Chuanxiong), has been reported to have antiapoptotic properties. In this study, we tested whether TMP protects rat BMSCs following exposure to glucocorticoids in vitro and in vivo. We treated BMSCs with different concentrations of TMP (50, 100, or 200 µM) and exposed them to 10-6 M dexamethasone (Dex) for 48 h in vitro. Our data showed that TMP inhibited Dex-induced cytotoxicity and protected BMSCs from apoptosis. Interestingly, further results demonstrated that TMP prevented apoptosis in BMSCs by promoting autophagy in an AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathway-dependent manner. In addition, calcein fluorescence double labeling and microcomputed tomography scanning indicated that 12 weeks of TMP administration augmented bone formation and protected trabecular bone mass in GIOP rats. We also discovered that first-passage BMSCs isolated from the TMP treatment group had a lower rate of apoptosis and a higher light chain 3 (LC3)-II/LC3-I ratio than the GIOP group. Our findings demonstrate for the first time that TMP can protect BMSCs from exposure to excess glucocorticoids by promoting autophagy through AMPK/mTOR pathway and might be an effective agent for the prevention and treatment of GIOP.

Identification and characterization of a phospholipid scramblase encoded by planarian Dugesia japonica.

Phospholipid scramblases (PLSCRs) are the conserved calcium-binding, type II transmembrane proteins synthesized in all eukaryotic organisms. In mammals, these proteins play essential roles in various physiological processes, especially in the immune responses. However, the existence of PLSCRs and their biological functions in planarian are still unknown at present. In this study, a new member of PLSCRs was identified in planarian Dugesia japonica (D. japonica), named DjPLSCR. The sequence analysis revealed that it contains an opening reading frame consisting of 726bp encoding a putative protein of 241 amino acids with a predicted molecular mass of ~28.7kDa and an isoelectric point of 6.21. Whole-mount in situ hybridization showed that mRNAs of DjPLSCR are predominantly expressed in adult and regenerative pharynx which is an important organ of immune system in planarians. Importantly, we found that the transcription level of DjPLSCR was significantly upregulated when planarians were stimulated with the pathogen-associated molecular patterns [polyinosinic-polycytidylic acid, lipopolysaccharide, peptidoglycan and ß-glucan], suggesting that DjPLSCR is involved in the immune response upon pathogen invasion. Our findings provide the first experimental insights into the characteristics and potential functions of PLSCR in planarians.

Linear transformation of thalamocortical input by intracortical excitation.

Neurons in thalamorecipient layers of sensory cortices integrate thalamocortical and intracortical inputs. Although we know that their functional properties can arise from the convergence of thalamic inputs, intracortical circuits could also be involved in thalamocortical transformations of sensory information. We silenced intracortical excitatory circuits with optogenetic activation of parvalbumin-positive inhibitory neurons in mouse primary visual cortex and compared visually evoked thalamocortical input with total excitation in the same layer 4 pyramidal neurons. We found that intracortical excitatory circuits preserved the orientation and direction tuning of thalamocortical excitation, with a linear amplification of thalamocortical signals of about threefold. The spatial receptive field of thalamocortical input was slightly elongated and was expanded by intracortical excitation in an approximately proportional manner. Thus, intracortical excitatory circuits faithfully reinforce the representation of thalamocortical information and may influence the size of the receptive field by recruiting additional inputs.

Histone H4 lysine 16 hypoacetylation is associated with defective DNA repair and premature senescence in Zmpste24-deficient mice.

Specific point mutations in lamin A gene have been shown to accelerate aging in humans and mice. Particularly, a de novo mutation at G608G position impairs lamin A processing to produce the mutant protein progerin, which causes the Hutchinson Gilford progeria syndrome. The premature aging phenotype of Hutchinson Gilford progeria syndrome is largely recapitulated in mice deficient for the lamin A-processing enzyme, Zmpste24. We have previously reported that Zmpste24 deficiency results in genomic instability and early cellular senescence due to the delayed recruitment of repair proteins to sites of DNA damage. Here, we further investigate the molecular mechanism underlying delayed DNA damage response and identify a histone acetylation defect in Zmpste24(-/-) mice. Specifically, histone H4 was hypoacetylated at a lysine 16 residue (H4K16), and this defect was attributed to the reduced association of a histone acetyltransferase, Mof, to the nuclear matrix. Given the reversible nature of epigenetic changes, rescue experiments performed either by Mof overexpression or by histone deacetylase inhibition promoted repair protein recruitment to DNA damage sites and substantially ameliorated aging-associated phenotypes, both in vitro and in vivo. The life span of Zmpste24(-/-) mice was also extended with the supplementation of a histone deacetylase inhibitor, sodium butyrate, to drinking water. Consistent with recent data showing age-dependent buildup of unprocessable lamin A in physiological aging, aged wild-type mice also showed hypoacetylation of H4K16. The above results shed light on how chromatin modifications regulate the DNA damage response and suggest that the reversal of epigenetic marks could make an attractive therapeutic target against laminopathy-based progeroid pathologies.

[Investigation on the relationship between the solar term of onset and syndrome types in 430 patients with acute myocardial infarction by circular statistical analysis].

OBJECTIVE: To study the relationship between the solar term of onset of acute myocardial infarction (AMI) and its syndrome types in traditional Chinese medicine (TCM). METHODS: The clinical data about 430 patients with AMI hospitalized in Foshan Hospital of TCM from February 4th 2003 (Beginning of Spring) to February 3rd 2008 (Beginning of Spring) were collected, and the solar term of onset as angle coordinate was regarded, then the peak phase of the onset solar term in each syndrome type of AMI was calculated by circular statistical analysis. RESULTS: Among 430 patients with AMI, 134 patients were considered to have qi stagnancy and blood stasis syndrome, 188 patients showed the syndrome of turbid sputum obstruction, 29 of them showed deficiency of yin-blood, and 79 showed deficiency of yang qi. The clinical manifestation of AMI was mainly asthenia syndrome (qi stagnancy and blood stasis+turbid sputum obstruction, 74.9%). According to the circular statistical analysis, the peak of the solar terms of AMI onset occurred at the Beginning of Spring in all cases (r=0.127 4, P<0.01), and standard deviation (s)=116.300 6 degree angle, showed it mainly occurred in winter and spring. As the peak of the onset of qi stagnancy and blood stasis occurred at Winter Solstice and Lesser Cold (r=0.200 5, P<0.01), its peak occurred in winter; the turbid sputum obstruction syndrome occurred at Spring Equinox (r=0.147 0, P<0.05), mainly in spring, yet the symptoms of above two peaks were generally mild. Besides, there was no significant difference in onset of the solar term in regard to onset of deficiency of yin-blood and deficiency of yang qi (both P>0.05). CONCLUSION: There is a close relationship between periodicity of the solar terms and onset of AMI. The main treatment for AMI is to expel turbid sputum, activate blood to resolve stasis and promote blood circulation to relieve pain; also the method of activating blood to resolve stasis is frequently contemplated in winter, and the method of expelling turbid sputum is the main strategy in spring.

[Prevalence of equol producer phenotype and its relations to lifestyle factors and dietary intakes among lifestyle factors and dietary intakes among healthy Chinese adults in Beijing].

OBJECTIVE: To investigate the prevalence of equol producers and the physiological range of urinary equol excretion, and also to evaluate relations between equol phenotype and lifestyle among Chinese adults in Beijing. METHODS: 100 male and 100 female adults participated in a cross-sectional study and provided twice 1d urine samples on regular diet and after 3d soy isoflavone challenge respectively. A health and demographics questionnaire, and 2d food record were completed before the urine collections. Isoflavones and their metabolites in urine were measured to determine equol phenotype by HPLC. RESULTS: The physiological range of 24h urinary equol excretion was 0-76.56 micromol/24h, and the percentage of the equol producer phenotype was 26.8% on regular diet and 60.4% after soy isofavone challenge, respectively. There was no indication that habitual consumption of soy foods is associated with the equol producer phenotype. The correlations of isoflavone intake from 2d food record with those from urinary isoflavone levels were 0.58 for total isoflavones, 0.49 for daidzein, 0.56 for genistein, and 0.50 for glycitein (P < 0.01). CONCLUSION: About one fourth of Chinese adults in Beijing were detected equol excretion in urine under the usually lifestyle. However, equol_producing potential was higher.

Preceding inhibition silences layer 6 neurons in auditory cortex.

A canonical feedforward circuit is proposed to underlie sensory cortical responses with balanced excitation and inhibition in layer 4 (L4). However, in another input layer, L6, sensory responses and the underlying synaptic circuits remain largely unclear. Here, cell-attached recordings in rat primary auditory cortex revealed that for the majority of L6 excitatory neurons, tonal stimuli did not drive spike responses, but suppressed spontaneous firings. Whole-cell recordings further revealed that the silencing resulted from tone-evoked strong inhibition arriving earlier than excitation. This pattern of inputs can be attributed to a parallel feedforward circuit with both excitatory and inhibitory inputs disynaptically relayed. In contrast, in the other neurons directly driven by thalamic input, stimuli evoked excitation preceding relatively weak inhibition, resulting in robust spike responses. Thus, the dichotomy of L6 response properties arises from two distinct patterns of excitatory-inhibitory interplay. The parallel circuit module generating preceding inhibition may provide a gating mechanism for conditional corticothalamic feedback.

[Research development on relation between equol and soy foods health effect].

Under the function of human intestinal bacteria, soybean can produce equol and O-desmethylangolensin. Among humans, 30%-55% have the bacteria capable of producing equol. Factors that influence the capacity to produce equol are not clearly established; however, gut physiology, host genetics, and diet factors are probably related to the individual difference of equol producing. Some studies on high soy-consuming populations reported an inverse association between urinary and serum equol concentrations and breast and prostate cancer risk. Furthermore, several studies of soy supplementation and bone density suggest that soy products may be more effective in maintaining bone density in equol-producing individuals. It requires further identification of the factors influencing the equol producing and the relationship between equol and human health.

[Effects of p53 inhibitor-alpha on the proliferation and apoptosis in large intestinal epithelial cells damaged by hyperthermic chemotherapy].

OBJECTIVE: To investigate the effects of p-fifty three inhibitor-alpha (PFT-alpha), a p53 inhibitor, on the proliferation and apoptosis of colon epithelial cells damaged by hyperthermic chemotherapy. METHODS: Normal epithelial cells were obtained from the mucosa at least 10 cm away from the cancer tissue in a specimen of large intestine cancer resected during operation and cultured. PFT-alpha at different concentrations was added into the culture fluid to observe its effects on the proliferation of the epithelial cells. Epithelial cell in logarithmic growth phase were inoculated in 6-well plate and divided into 3 groups: normal control (CON) group; hyperthermic chemotherapy (HTC) group, undergoing treatment of cisplatin and bath in water at 43 degrees C; and PFT-alpha + HTC group, undergoing treatment of PFT-alpha at different concentrations, cisplatin, and warm water bath. The cell apoptosis was observed by annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining and flow cytometry (FCT). The cell cycle was observed by PI staining and FCT. Western blotting was used to detect the protein expression of cyclinB1 and Cdc2, and RT-PCR was used to detect the mRNA expression of cyclinB1. RESULTS: PFT-alpha at the concentration > 60 micromol/L significantly inhibited the proliferation of the large intestine epithelial cells. The natural apoptosis rate of the large intestine epithelial cells (CON group) was 2.9% +/- 0.4%, the apoptosis rate was 27.0% +/- 2.1% in the HTC group, and the apoptosis rates of the PFT-alpha + HTC group were 14.8% +/- 1.5%, 9.7% +/- 1.2%, 6.1% +/- 1.3%, and 3.8% +/- 0.3%, on a downward trend, corresponding to the increase of PFT-alpha concentration from 0, 20, 30, to 40 micromol/L (all P < 0.05). The G(0)/G(1) phase rate of epithelial cells was higher and the S phase rate was lower significantly in the PFT-alpha + HTC group. The G(2)/M phase rate was higher since the PFT-alpha concentration reached 10 micromol/L and then increased along with the increase of the PFT-alpha concentration; the S phase rates of the PFT-alpha + HTC group with different PFT-alpha concentrations were all significantly higher than that of the HTC group (all P < 0.01), however, were still lower than that of the CON group (all P < 0.01). The protein expressions of cyclinB1 and Cdc2 in the PFT-alpha + HTC group were both significantly higher than those in the CON and HTC groups (all P < 0.01), without a significant difference between the latter 2 groups. The mRNA expression of cyclinB1 in the PFT-alpha + HTC group increased along with the increase of the PFT-alpha concentration, and there wee significant differences in the mRNA expression of cyclinB1 between the CON and PFT groups and PFT-alpha + HTC group with the PFT-alpha concentration > or = 10 micromol/L (P < 0.05 or P < 0.01). CONCLUSION: PFT-alpha dose-dependently protects the hyperthermic chemotherapy-induced damage to the large intestine epithelial cells via upregulation of protein and mRNA expression of cyclinB1, increasing the phosphorylation level of Cdc2, decreasing the cyclinB1/Cdc2 activity, and increasing the G(2)/M phase rate of the cells.

Characteristics and mechanism of enzyme secretion and increase in [Ca2+]i in Saikosaponin(I) stimulated rat pancreatic acinar cells.

AIM: This investigation was to reveal the characteristics and mechanism of enzyme secretion and increase in [Ca2+]i stimulated by saikosaponin(I) (SA(I)) in rat pancreatic acini. METHODS: Pancreatic acini were prepared from male Wistar rats. Isolated acinar cells were suspended in Eagle's MEM solution. After adding drugs, the incubation was performed at 37 degrees for a set period of time. Amylase of supernatant was assayed using starch-iodide reaction. Isolated acinar single cell was incubated with Fura-2/AM at 37 degrees, then cells were washed and resuspended in fresh solution and attached to the chamber. Cytoplasm [Ca2+]i of a single cell was expressed by fluorescence ratio F340/F380 recorded in a Nikon PI Ca2+ measurement system. RESULTS: Rate course of amylase secretion stimulated by SA(I) in rat pancreatic acini appeared in bell-like shape. The peak amplitude increased depended on SA(I) concentration. The maximum rate responded to 1 x 10(-5)mol/L SA(I) was 13.1-fold of basal and the rate decreased to basal level at 30 min. CCK-8 receptor antagonist Bt(2)-cGMP markedly inhibited amylase secretion stimulated by SA(I) and the dose-effect relationship was similar to that by CCK-8. [Ca2+]i in a single acinar cell rose to the peak at 5 min after adding 5 x 10(-6)mol/L SA(I) and was 5.1-fold of basal level. In addition, there was a secondary increase after the initial peak. GDP could inhibit both the rate of amylase secretion and rising of [Ca2+]i stimulated by SA(I) in a single pancreatic acinar cell. CONCLUSION: SA(I) is highly efficient in promoting the secretion of enzymes synthesized in rat pancreatic acini and raising intracellular [Ca2+]i. Signaling transduction pathway of SA(I) involves activating special membrane receptor and increase in cytoplasm [Ca2+]i sequentially.

CARP is a novel caspase recruitment domain containing pro-apoptotic protein.

Many CARD-containing caspase mediators interact with CARD-containing caspases and participate in activation or suppression of caspases. We cloned a novel CARD-containing protein from our EST database, named CARP. Computational characterization revealed that CARP encoded 445 amino acids with predicted MW 49.7 kDa, localized at chromosome 10p13 with 15 exons, and four putative function domains, one CARD domain (aa 160-243), one nuclear receptor-binding motif, two EF-hand motifs, and 42% alpha-helix content. Stable transfection of CARP into lung carcinoma A549 and HEK293S cells leads to 23% of the cells undergoing apoptosis, but only 3% in the cells transfected with empty control vector. The cell proliferation was significantly inhibited by 1.2-5 folds (P<0.02) in seven CARP-transfected tumor cell lines-lung carcinoma A549 and PG, melanoma WM451, prostate cancer PC-3 and PC-3M, liver cancer H7402, and bladder cancer BIU87. Our results suggest that CARP is a novel CARD-containing pro-apoptotic protein.