[Analysis of efficacy and safety of a new endoscopic anastomosis clip in the treatment of defects after endoscopic full-thickness resection].

Objective: To investigate the efficacy and safety of a new endoscopic anastomosis clip in the treatment of defects after endoscopic full-thickness resection (EFTR). Methods: Retrospective cohort study. Fourteen patients [4 males and 10 females, aged (55.9±8.2) years (45-69 years)] with gastric submucosal tumors underwent EFTR at the First Affiliated Hospital of Soochow University were included from December 2018 to January 2021. Patients were divided into new anastomotic clamp group (n=6) and nylon ring combined with metal clips group (n=8). Preoperative endoscopic ultrasound examinations were required to all patients to evaluate the wound condition. The size of the defect, operation time required for wound closure, success rate of closure, postoperative gastric tube placement time, postoperative hospital stay, incidence of complications, preoperative and postoperative serological indexes were compared between the two groups. All patients were followed up after the operation, among which the general endoscopy was reviewed in the first month after the operation, and the telephone and questionnaire follow-up were used in the second, third, sixth month and one year after the operation to evaluate the therapeutic effect of the new endoscopic anastomosis clip and nylon rope combined with metal clip after the EFTR operation. Results: Both groups successfully completed EFTR and were successfully closed. There was no significant difference between the age, tumor diameter and defect diameter of the two groups (all P>0.05). Compared with the nylon ring combined with metal clip group, the operation time of the new anastomotic clip group was shortened [(5.0±1.8) minutes vs (35.6±10.2) minutes, P<0.001]. The operation time was shortened [(62.2±12.5) minutes vs (92.5±0.2) minutes, P=0.007]. Postoperative fasting time decreased [(2.8±0.8) days vs (4.9±1.1) days, P=0.002]. The hospital stay after operation was also shortened [(5.2±0.8) days vs (6.9±1.5) days, P=0.023]. The total intraoperative bleeding volume decreased [(20.00±5.48) ml vs (35.63±14.75) ml, P=0.031]. The patients in both groups received endoscopic examination 1 month after operation, and there was no delayed perforation and bleeding after operation. There was no obvious symptoms of discomfort. Conclusion: The new anastomotic clamp is suitable for the treatment of full-thickness gastric wall defects after EFTR, and shows advantages of shorter operation, less bleeding, and fewer postoperative complications.

[Association between polymorphism in notch signaling pathway and lung cancer risk].

Objective: To analyze the relationship between single nucleotide polymorphisms (SNP) of Notch signaling pathway and susceptibility to lung cancer. Methods: The present study was a hospital-based case-control study. All 1 121 patients of lung cancer diagnosed by histopathology three hospitals in Fujian and Nanjing were selected as cases from January 2006 to December 2012. At the same time, 1 121 healthy population from other departments of the hospital to visit patients or community, excluding those with tumor, chronic disease, and immediate family members of lung cancer, were enrolled in control group. A uniform questionnaire was used to collect general information. Matrix-assisted laster desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was used to identify the polymorphisms of 9 SNP (Notch3 rs3815188, Notch4 rs915894, Notch4 rs520692, DLL1 rs1033583, JAG1 rs8708, JAG2 rs9972231, HEY1 rs1046472, HEY2 rs3734637, HES2 rs11364) in 1 121 lung cancer patients and 1 121 healthy controls. The association between SNP and lung cancer was analyzed by χ(2) and logistic regression model. Results: The average age of cases and controls was (58.70±10.73) and (58.98±10.85) years old. The OR for genotype AC carriers of HEY1 rs1046472 was 0.80 (95%CI: 0.66-0.97) when comparing with genotype CC. The OR for genotype AC+AA carriers of HEY1 rs1046472 was 0.81 (95% CI: 0.67-0.98) when comparing with genotype CC. The OR for genotype AC carriers of HEY2 rs3734637 was 0.82 (95%CI: 0.67-0.99) when comparing with genotype AA. In the stratified analysis, Notch3 rs3815188, DLL1 rs1033583, JAG1 rs8708, JAG2 rs9972231, HEY1 rs1046472, HEY2 rs3734637, HES2 rs11364 were associatied with the risk of lung cancer, P were 0.041, 0.030, 0.043, 0.003, 0.004, 0.026 and 0.038, respectively.The interactions analysis done by logistic regression model showed JAG1 rs8708 and family history, JAG2 rs9972231 and BMI had interaction in the study, OR were 2.07 (95% CI:1.21-3.52) and 1.73 (95% CI:1.21-2.47), respectively. Conclusion: Notch3 rs3815188, DLL1 rs1033583, JAG1 rs8708, JAG2 rs9972231, HEY1 rs1046472, HEY2 rs3734637 and HES2 rs11364 were significantly associated with susceptibility to lung cancer.

[Clinical application of Acutegastrointestinal injury grading system assocaited with clinical severity outcome in critically ill patients: a multi-center prospective, observational study].

Objective: To investigate the feasibility of utilizing the current acute gastrointestinal injury(AGI) grading system, and explore the association of severity of AGI grade with clinical outcome in critically ill patients. Methods: The adult patients from 14 general ICUs in Zhejiang Province with an expected admission to ICU for at least 24 h were recruited, and all clinical, laboratory, and survival data were prospectively collected. The AGI grade was daily assessed based on GIsymptoms, feeding details and organ dysfunctionon the first week of admission to ICU.The intra-abdominal pressures(IAP) was measured using AbViser device. Results: Of 550 patients enrolled, mean values for age and APACHE Ⅱ score were (64.9±17.2) years and (19.5±7.4), respectively. 456 patients(82.9%) took mechanical ventilation, and 470 patients were identified for AGI. The distribution of AGI grade on the frist day of ICU admission were 50.6%(Ⅰ grade, n=238), 34.2%(Ⅱ grade, n=161), 12.4%(Ⅲ grade, n=58) and 2.8%(Ⅳ, n=13), respectively, while the distribution of the global AGI grade based on the 7-day AGI assessment of ICU admission were 24.5%(Ⅰ grade, n=115), 49.4%(Ⅱ grade, n=232), 20.6%(Ⅲ grade, n=97) and 5.5%(Ⅳ, n=26), respectively. 28- and 60-day mortality rate was 29.3%(n=161) and 32.5%(n=179), respectively. The patients with AGI had a higher 28-(31.1% vs 18.8%, P=0.025) and 60-day survival rate(34.7% vs 20.0%, P=0.01) than those with non-AGI, and also there were positive correlations between AGI grade and 28- and 60-day mortality(P<0.001). Univariate Cox regression analysis showed that age, the source of medicial admission, diabetes mellitus, coronary heart disease, the use of vasoactive drugs, serum creatinine and lactate, mechanical ventilation, APACHE Ⅱ score, the AGI grade in the first day of ICU admission and feeding intolerance within the first week of ICU stay were significantly(P≤0.02) associated with mortality. In multivariate analysis including all these variables, the source of medical admission(χ(2)=4.34, P=0.04), diabete mellitus(χ(2)=3.96, P=0.05), the use of vasoactive drugs(χ(2)=6.55, P=0.01), serum lactate(χ(2)=4.73, P=0.03), the global AGI grade in the 7-day of ICU admission(χ(2)=7.10, P=0.008), and APACHE Ⅱ score(χ(2)=12.1, P<0.001) remained independent predictors for 60-day mortality.In the further subgroup analysis including 402 patients with 7-day survival, the feeding intolerance within the first week of ICU stay could provide independent and incremental prognostic value of 60-day mortality wtih increased χ(2)value of Cox regression model(χ(2)=52.2 vs 41.9, P=0.007) . Conclusion: The AGI grading system is useful for identifying the severity of gastrointestinal dysfunction, and could be used as a strong predictor of impaired outcome. The results provide evidence to support that feeding intolerance within 7 days of admission to ICU was an independent determinant of mortality.

[Effect of gender on the prognosis of patients with non-small cell lung cancer].

Objective: To analyze the effect of gender on the prognosis of patients with non-small cell lung cancer (NSCLC). Methods: Data of 1 195 patients with NSCLC were analyzed by Chi-square, Kaplan-Meier, log-rank tests and Cox regression models. Results: Women had a longer survival than men (median overall survival 31.64 versus 22.71 months, P<0.01) in the participants of this study. Differences seen in overall survival remained the similar, after stratified by age, pathologic types, clinical stage, sizes, pleural effusion and surgery of the patients, respectively. Data from the multivariate analysis revealed that factors as smoking, clinical stage, metastatic when diagnosis was made and surgery, but not gender, were independent prognostic factors for patients with NSCLC. After adjustment for potential confounders, we found that smoking was a major confounding factor, affecting the relationship between gender and prognosis of NSCLC. Conclusion: Gender did not seem an independent prognostic factor for NSCLC patients while the survival advantages of females might be attributed to the lower prevalence of smoking in this population.

[Association between human papillomavirus infection and lung cancer].

Objective: To investigate the association between human papillomavirus (HPV) and lung cancer. Methods: We examined a series of 83 lung cancer patients with HPV DNA in both lung tumor specimens and adjacent normal specimens from Fujian province. Twenty-one of the most clinically relevant HPV types from the highly conserved L1 region of the viral genome were analyzed, using the PCR amplification and were followed by reverse hybridization with specific probes. Chi-square test of paired design was used to test the difference of HPV positive rates between lung cancer specimens and adjacent normal specimens. Chi-square test and Fisher's exact test were used to analyze the differences of HPV positive rate of tumor specimens on factors as gender, age, histological subtype, clinical stage, smoking status and alcohol consumption. Results: HPV was detected in 7 of the 83 tumor specimens and in 6 of the paired normal lung tissues. There was no significant correlation between HPV and lung cancer (P>0.999). Neither demographic characteristics nor clinical features were found with significant differences on HPV in lung cancer tissues (P>0.05). Conclusion: Our data showed that HPV was not significantly associated with the risk of lung cancer in Fujian province.

Effects and mechanisms of triacetylshikimic acid on platelet adhesion to neutrophils induced by thrombin and reperfusion after focal cerebral ischemia in rats.

AIM: To investigate the effect of triacetylshikimic acid (TSA) on the platelet adhesion to neutrophils and P-selectin expression on activated platelet membrane induced by thrombin and reperfusion after focal cerebral ischemia. METHODS: The platelet adhesion to neutrophils was evaluated by rosette assay, and P-selectin expression on platelet membrane was determined by flow cytometry. RESULTS: TSA 10 - 1000 micromol/L markedly inhibited thrombin(0.4 kU/L)-induced platelet adhesion to neutrophils. The platelet adhesion to neutrophils induced by a 21-h reperfusion after middle cerebral artery occlusion for 3 h was also inhibited in a dose-dependent manner by TSA 50 - 200 mg/kg given by ig immediately and at 60 min again after the onset of cerebral ischemia. TSA was also shown to decrease the P-selectin expression on platelet surface induced by thrombin in washed platelet and by adenosine diphosphate (ADP) 5 micromol/L in whole blood. CONCLUSION: Reperfusion after cerebral ischemia and thrombin induced platelet adhesion to neutrophils, which could be reduced by TSA probably due to its inhibition of P-selectin expression on activated platelets.

[Experimental study on protective effective of congsheng capsule in acute cerebral ischemia and improvement of cerebral blood flow and energy metabolism].

OBJECTIVE: To investigate the protective effect of Congsheng capsule (CSC) on acute cerebral ischemia in mice and rats. METHODS: Cerebral ischemia model was established by adding FeCl3 to block the blood flow of unilateral middle cerebral artery (MCA) for 30 mins. Acute incomplete cerebral ischemia was induced by ligation of bilateral carotid arteries in rats. Bilateral carotid arteries of old mice were repeatedly ischemia and reperfusion combined with caudal bloodletting to build the cerebral ischemia model. The effect of CSC on neurologic symptom, cerebral infarction size, cerebral water content, cerebral blood flow and cerebral tissue energy metabolism were observed. RESULTS: CSC 1, 3, 6 g/kg obviously reduce the size of cerebral infarction and cerebral water content, markedly improve the neurological symptoms and cerebral blood flow; 3, 6 g/kg significantly ameliorate the energy burden. CONCLUSION: CSC has anti-cerebral ischemia function through increasing blood supply of cerebral tissue and improve the energy metabolism.

Mutations of the S4-S5 linker alter activation properties of HERG potassium channels expressed in Xenopus oocytes.

1. The structural basis for the activation gate of voltage-dependent K+ channels is not known, but indirect evidence has implicated the S4-S5 linker, the cytoplasmic region between the fourth and fifth transmembrane domains of the channel subunit. We have studied the effects of mutations in the S4-S5 linker of HERG (human ether-á-go-go-related gene), a human delayed rectifier K+ channel, in Xenopus oocytes. 2. Mutation of acidic residues (D540, E544) in the S4-S5 linker of HERG channels to neutral (Ala) or basic (Lys) residues accelerated the rate of channel deactivation. Most mutations greatly accelerated the rate of activation. However, E544K HERG channels activated more slowly than wild-type HERG channels. 3. Mutation of residues in the S4-S5 linker had little or no effect on fast inactivation, consistent with independence of HERG channel activation and inactivation 4. In response to large hyperpolarizations, D540K HERG channels can reopen into a state that is distinct from the normal depolarization-induced open state. It is proposed that substitution of a negatively charged Asp with the positively charged Lys disrupts a subunit interaction that normally stabilizes the channel in a closed state at negative transmembrane potentials. 5. The results indicate that the S4-S5 linker is a crucial component of the activation gate of HERG channels.

Antagonistic effects of shikimic acid against focal cerebral ischemia injury in rats subjected to middle cerebral artery thrombosis.

AIM: To study the effects of shikimic acid (SA) on focal cerebral ischemic injury after middle cerebral artery thrombosis (MCAT). METHODS: Thrombosis was induced by FeCl3 in middle cerebral artery of rats. The influences of SA on neurologic deficit (ND), infarct size (IS), brain edema, and cerebral blood flow (CBF) in ischemic region were observed. RESULTS: SA 25 and 50 mg.kg-1 i.p. for 3 d before MCAT attenuated ND, and reduced IS by 51% and 42%; and decreased brain water content from 80.7% to 79.8% and 79.9%; and increased CBF after ischemia from 50.2% of the preischemic level to 75.5% and 73.3%, respectively. In pathologic examination, there was much less thrombosis in MCA in the rat with the pretreatment by SA 25 mg.kg-1. The extent of brain ischemia was much less than that of control. CONCLUSIONS: SA reduced focal cerebral ischemic injury induced by middle cerebral artery thrombosis.

A mutation in the pore region of HERG K+ channels expressed in Xenopus oocytes reduces rectification by shifting the voltage dependence of inactivation.

1. The effects of a mutation in the human ether-a-go-go-related gene (HERG) (Ser631 to Ala, S631A) on the voltage- and extracellular [K+] dependence of inactivation were studied in Xenopus oocytes using two microelectrode and single channel voltage-clamp techniques. 2. The voltage required for half-inactivation of S631A HERG was 102 mV more positive than for wild-type (WT)-HERG, resulting in reduced rectification of the steady-state current-voltage relationship. In contrast, the voltage dependence of channel activation was not altered by the S631A mutation. These findings indicate that inactivation of HERG channels is not linked to activation. 3. Rectification of whole-cell S631A HERG current was caused by a voltage-dependent reduction in open probability, and inward rectification of the current-voltage relationship of single channels. 4. Elevation of extracellular [K+] from 2 to 20 mM shifted the half-point for inactivation by +20 mV for WT-HERG, and +25 mV for S631A HERG. Thus, elevated [K+]o and the S631A mutation affect HERG inactivation by different mechanisms. 5. The S631A mutation altered the ion translocation rate of HERG channels. The single channel conductance (gamma) of S631A HERG was 20 pS between -40 and-100 mV, and 6.0 pS between +40 and +100 mV (120 mM extracellular K+). This compares to a gamma of 12.1 and 5.1 pS for WT-HERG channels under the same conditions.

Belladonna alkaloids-induced behavioral changes and amnesia on open-field and step-through in 18-, 28-, and 38-day-old mice.

AIM: To study the age-related changes of atropine (Atr), scopolamine (Sco), anisodine (AT3), and anisodamine (Ani) on behaviors and memories. METHODS: The behaviors and memories were measured with open-field test and step-through task. M-cholinergic receptors were determined by [3H] quinuclidinyl benzilate ([3H] QNB). RESULTS: During acquisition session (d 1) the 18-, 28-, and 38-d-old mice pretreated with Atr, Sco, and AT3 (0.02, 0.2, 2, or 20 mg.kg-1, i.p.) in open-field test showed increase in walking counts by 26%-42%, but decrease in rearing, grooming, and defecating counts for 50%-92%, 67%-100%, and 75%-100%, respectively. On recall session (d 2) the walking and rearing behaviors in the 18- and 28-d-old mice receiving Atr, Sco, and AT3 on d 1 were higher than those in the mice receiving saline. But a lower grooming behavior on d 2 was found in the mice receiving the drugs on d 1. On d 1 Ani 20 mg.kg-1 reduced the rearing behavior by 50% in 18-d-old mice and defecation by 33%-36% in 18- and 28-d-old mice. All the 4 belladonna alkaloids increased the number of avoidance-response errors and decreased the retention latencies in step-through task. Bmax of [3H] QNB binding sites in frontal cortex and hippocampus regions in the 38-d-old mice increased 7% and 23% vs in the mice of 18 d of age, respectively. CONCLUSION: 1) The effects of the belladonna alkaloids on behaviors and memories in adult mice were weaker than those in young mice. 2) The belladonna alkaloids-induced amnesia on passive avoidance-response in step-through was more sensitive than behavioral changes and amnesia on open-field. 3) According to the lowest effective doses which insulted the behaviors or memories in young mice, Sco was about 10, 100, and 1000 times more potent than Atr, AT3, and Ani, respectively.

Characterization of the cooperative function of inhibitory sequences in Ets-1.

DNA binding by the eukaryotic transcription factor Ets-1 is negatively regulated by an intramolecular mechanism. Quantitative binding assays compared the DNA-binding activities of native Ets-1, three deletion mutants, and three tryptic fragments. Ets-1 and activated Ets-1 polypeptides differed in DNA-binding affinity as much as 23-fold. Inhibition was mediated by two regions flanking the minimal DNA-binding domain. Both regions regulated affinity by enhancing dissociation of the protein-DNA complex. Three lines of evidence indicated that inhibition requires cooperative interaction between the two regions: first, the two inhibitory regions acted through a common mechanism; second, neither region functioned independently of the other; finally, mutation of the C-terminal inhibitory region altered the conformation of the N-terminal inhibitory region. In addition, partial proteolysis detected an identical altered conformation in the N-terminal inhibitory region of Ets-1 bound to DNA. This finding suggested that repression is transiently disrupted during DNA binding. These results provide evidence that the two inhibitory regions of Ets-1 are structurally, as well as functionally, coupled. In addition, conformational change is shown to be a critical component of the inhibition mechanism. A cooperative, allosteric model of autoinhibition is described. Autoinhibition of Ets-1 could be relieved by either protein partner(s) or posttranslational modifications.

Tumour necrosis factor-alpha can modulate the phenotype of aortic smooth muscle cells.

In culture, rabbit aortic smooth muscle cells (SMC) from an atheroma differed phenotypically from SMC from normal media (M-SMC) in their growth rate, secretion of SMC-derived growth factor (SDGF), and metabolism of acetylated low density lipoproteins (a-LDL). The factor responsible for this in vivo phenotypic change of SMC was investigated in vitro. After preincubation of M-SMC with 0.1-10 U ml-1 of tumour necrosis factor-alpha (TNF) for 1-3 days, the cells grew faster than control cells and secreted a substantial amount of SDGF. The population doubling time and secretion of SDGF were inversely correlated. Moreover, after preincubation with TNF, the SMC metabolized [125I]a-LDL, unlike control M-SMC. These findings show that TNF can modulate the phenotype of SMC and suggest that it is important in the pathogenesis of atherosclerosis.

Metalloproteinases and tissue inhibitor of metalloproteinases in mesothelial cells. Cellular differentiation influences expression.

Mesothelial cells play a critical role in the remodeling process that follows serosal injury. Although mesothelial cells are known to synthesize a variety of extracellular matrix components including types I, III, and IV collagens, their potential to participate in matrix degradation has not been explored. We now report that human pleural and peritoneal mesothelial cells express interstitial collagenase, 72- and 92-kD gelatinases (type IV collagenases), and the counterregulatory tissue inhibitor of metalloproteinases (TIMP). Our initial characterization of the mesothelial cell metalloenzymes and TIMP has revealed: (a) they are likely identical to corresponding molecules secreted by other human cells; (b) they are secreted rather than stored in an intracellular pool; (c) a primary site of regulation occurs at a pretranslational level; (d) phorbol myristate acetate, via activation of protein kinase C, upregulates expression of collagenase, 92-kD gelatinase, and TIMP, but has no effect on expression of 72-kD gelatinase; and (e) lipopolysaccharide fails to upregulate the biosynthesis of either metalloproteinases or TIMP. Of particular interest is the observation that the state of cellular differentiation has a striking influence on the expression of metalloenzymes and TIMP, such that epitheloid cells display a more matrix-degradative phenotype (increased 92-kD gelatinase and decreased TIMP) than their fibroblastoid counterparts. We speculate that mesothelial cells directly participate in the extracellular matrix turnover that follows serosal injury via elaboration of metalloproteinases and TIMP. Additionally, the reactive cuboidal mesothelium which is characteristic of the early response to serosal injury may manifest a matrix-degenerative phenotype favoring normal repair rather than fibrosis.

[The effects of different lipoproteins on cholesterol metabolism in smooth muscle cells of rabbit aorta].

In the present experiment, M-SMC were cultured from rabbit aorta by an explant method and I-SMC cultured by the explant method from cannulated aortic intima of rabbits, and the effects of LDL, A-LDL, OX-LDL and HDL on the cholesterol metabolism in both types of cells were investigated by using 14C oleic acid as the source of cholesterol re-esteri fication in cells. The results showed that LDL enhanced cholesterol re-esterification in both types of cells and HDL had an opposite effect, A-LDL could increase CE synthesis only in I-SMC, while OX-LDL showed a complex effect on the level of CE in different cells by different concentration. The present experiment has studied the relationship between lipoprotein and cholesterol metabolism in SMC at the level of cell metabolism, and suggests that lipoproteins play a key role in AS.

Pulmonary epithelial cell urokinase-type plasminogen activator. Induction by interleukin-1 beta and tumor necrosis factor-alpha.

Diffuse alveolar damage, presenting clinically as adult respiratory distress syndrome, is characterized initially by widespread intra-alveolar fibrin deposition. Alveolar epithelial cells play a central role in the subsequent repair process. We have recently shown that alveolar epithelial cells have the capacity to promote fibrinolysis (Marshall, B. C., Sageser, D. S., Rao, N. V., Emi, M., and Hoidal, J. R. (1990) J. Biol. Chem. 265, 8198-8204) and may therefore directly participate in the extensive remodeling that follows acute lung injury. Because the tissue repair process occurs in an acute inflammatory setting, we investigated the effects of inflammatory mediators on urokinase-type plasminogen activator (u-PA) expression by pulmonary epithelial cells. We found that interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) upregulated PA activity in A549 human pulmonary epithelial cells. Biosynthetic labeling and immunoprecipitation showed that both cytokines caused marked accumulation of newly synthesized u-PA. Northern blot analyses demonstrated that both IL-1 beta and TNF-alpha induced relatively rapid accumulation of u-PA mRNA which did not require de novo protein synthesis and was substantially inhibited by glucocorticoids. Nuclear run-off transcription studies showed that both cytokines caused rapid transcriptional activation of the u-PA gene. While the effects of IL-1 beta and TNF-alpha were qualitatively similar, some differences emerged. Most notably, TNF-alpha led to a more sustained accumulation of u-PA mRNA than did IL-1 beta. In contrast to their effects on u-PA expression, IL-1 beta and TNF-alpha had minimal effect on PA inhibitor-1 expression. These effects of IL-1 beta and TNF-alpha, mediators known to play a key role in acute lung injury and inflammation, may promote lysis of alveolar fibrin by alveolar epithelium, thereby aiding in restoration of normal lung architecture.

[Comparative studies on the febrifugal analgesic and anticonvulsive activities of water extracts from cultivated and wild Saposhnikovia divaricata].

In this paper, comparative studies on main pharmacological activities and toxilogical activities of water extracts from cultivated Saposhnikovia divaricata (SD) and wild SD in Qixia county of Shandong province were performed. The results showed that the febrifugal analgesic and anticonvulsived activities of water extracts from cultivated SD and wild SD were primary same. Estimative results of LD50 showed that order of toxicity were such as firstly direct cultivated SD, secondly cuttage SD and finally wild SD. Therefore it was recognized that cultivated SD completely can replaced wild one in medical use.

Reticulocytes II: Reexamination of the in vivo survival of stress reticulocytes.

Very young reticulocytes are released into the circulation in response to the stress of anemia. These stress reticulocytes have shortened in vivo survival when transfused into normal recipients, and are generally considered to be abnormal because they have skipped a terminal cell division. We reevaluated one aspect of their abnormality: that of in vivo survival. Using methodology that accounted for all cells transfused, in vivo survival of both normal and stress reticulocytes was investigated in both normal and anemic recipients. The experiments demonstrate that: (1) survival of reticulocytes is normal only when normal reticulocytes are injected into nonanemic animals; (2) intrinsic properties of stress reticulocytes lead to their immediate removal from the circulation by normal recipients to a significantly greater extent than by anemic recipients; and (3) both stress and normal reticulocytes are removed at an accelerated rate over time by anemic recipients. Taken together, the data indicate that in the course of becoming anemic, an adaptation occurs that allows cells produced during anemia to circulate considerably longer in anemic animals than they could in normal nonanemic animals. Other studies disclosed that increased reticulocyte survival in anemic animals could not be attributed to reticuloendothelial overload, but is induced by adaptation of the spleen, decreasing its removal of stress reticulocytes.

Reticulocytes. I. Isolation and in vitro maturation of synchronized populations.

Studies of reticulocyte maturation have been limited by the inability to obtain pure populations of age-synchronized reticulocytes and by the absence of well-defined methods for the maturation of reticulocytes in vitro. Many of these problems were overcome using temporary suppression of erythropoiesis with thiamphenicol and phlebotomy resulting in a highly reproducible reticulocyte response, Percoll density gradient separation of cells yielding essentially pure populations of age-synchronized reticulocytes, and liquid culture techniques where cell lysis is minimal. The system allows reproducible study of well-defined cohorts of reticulocytes as they mature into erythrocytes. During in vitro maturation we serially monitored changes in reticulocyte count, glucose consumption, 125I-transferrin binding, fluorescein (FITC)-labeled transferrin binding, the activities of four erythrocyte enzymes (glucose-6-phosphate dehydrogenase, pyruvate kinase, phosphofructokinase, and lactate dehydrogenase) and the appearance of cells on scanning electron microscopy. These variables changed at different rates suggesting that multiple mechanisms underlie these maturational events. Transferrin binding and reticulocyte count decreased most rapidly and reached values near zero after three to four days in culture. The four enzyme activities decreased much more slowly, and only two reached pretreatment values after seven days in culture. In contrast to the findings of others, scanning electron microscopy suggested that cells do not assume the normal biconcave shape in this system. The methods described make it feasible to study the process of reticulocyte maturation in vitro. The data presented represent a first step in the study of the coordination and interrelationships of various maturational processes.

Photoaffinity labeling of beef liver monoamine oxidase-B by 4-fluoro-3-nitrophenyl azide.

4-Fluoro-3-nitrophenyl azide (FNPA) competitively inhibited beef liver monoamine oxidase-B (MAO-B) in the dark (Ki = 2.8 microM). Upon irradiation in the presence of FNPA, a concentration-dependent photoinactivation of MAO-B was observed. The kinetic analysis showed that the photoinactivation of MAO-B resulted in a decrease in Vmax but no change in Km. This result suggests that an irreversible linkage may be formed between the enzyme and the photolyzed FNPA. When [3H]FNPA was photoirradiated with the purified MAO-B, a single radioactive band associated with MAO-B was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The photo-dependent incorporation could be protected by phenylethylamine, the substrate for MAO-B, in a concentration-dependent manner. Complete tryptic-chymotryptic digestion of [3H]FNPA-labeled MAO-B resulted in three radioactive peaks on Sephadex G-25 column chromatography. With the same digestion and separation procedures, only one major radioactive peak was observed for the [3H]pargyline-labeled MAO-B, and its elution volume was different from that of [3H]FNPA-labeled peptides. These results suggest that, upon photolysis, FNPA may incorporate into a region in the active site of MAO-B which may be different from the pargyline binding site--the FAD prosthetic group of the enzyme.