Cefoperazone/sulbactam-induced hemolytic anemia.

Drug-induced hemolytic anemia (DIHA) is a rare complication of drug therapy and usually underdiagnosed. Cefoperazone/sulbactam is a compound prepared from the third generation of cephalosporin and ß-lactamase inhibitor. There are limited data of DIHA induced from cefoperazone/sulbactam. A 93-year-old female patient, who had an operation on the biliary tract 3 months ago, was admitted to our hospital with an abdominal infection. After cefoperazone/sulbactam was given as anti-infection treatment, the patient developed hemolytic anemia on the third day. Cefoperazone/sulbactam was discontinued and replaced with meropenem. Subsequently the level of red blood cells, hemoglobin, and hematocrit returned to normal. Clinicians should pay attention to monitoring the possible adverse reactions during the use of cefoperazone/sulbactam and should be aware of the occurrence of DIHA, so as to give timely treatment.

[Evaluation of papilledema and visual improvement in patients with idiopathic intracranial hypertension after venous sinus stenting].

Objective: To evaluate the improvement of papilledema and visual acuities in patients with idiopathic intracranial hypertension (IIH) after venous sinus stenting. Methods: The clinical data of 8 IIH patients who met the inclusion criteria underwent venous sinus stenting between January 2013 and December 2016 at Department of Neurosurgery, Tianjin Huanhu Hospital were analyzed retrospectively. There were 6 females and 3 males,aged (32.9±14.4)years (range:19 to 57 years).The thickness of the retinal nerve fiber layer (RNFL) was measured by optical coherence tomography. Fundus,visual acuity and visual field examination were performed before and after operation. If pressure gradient ≥10 mmHg(1 mmHg=0.133 kPa) across the venous stenosis was indicated by intraoperative pressure measurement,the patient would be treated with venous sinus stenting. Intracranial pressure was measured by lumbar puncture 3 to 7 days after operation. RNFL thickness and eye examination were detected 6 months after surgery. CT venogram was used to observe the sinus venous conditions. Paired t test was used to compare the data before and after surgery. Results: All the 8 patients underwent venous sinus stenting successfully. The mean pressure gradient across the venous stenosis was reduced from (24±9.2) mmHg to (2.6±2.0) mmHg (t=8.02,P<0.01). Intracranial pressure decreased from preoperative (41.4±12.7) cmH2O(1 cmH2O=0.098 kPa) to postoperative (12.9±3.3) cmH2O (t=7.08, P<0.01). The RNFL thickness decreased from (275.3±68.3)µm to (131.4±31.8)µm(t=5.80,P<0.05) 6 months after surgery and the baseline visual acuity was improved from(M(QR))0.24 (0.25) to 0.65 (0.23)(Z=-2.52,P<0.05).Papilledema was significantly improved in 6 patients,and no significant change in 2 patients. CT venogram indicated adjacent stent restenosis in 1 patient. Conclusion: Venous sinus stenting can effectively improve papilledema and visual acuity caused by IIH.

The value of detecting pepsinogen and gastrin-17 levels in serum for pre-cancerous lesion screening in gastric cancer.

The aim of the study was to estimate the value of detecting pepsinogen (PG) I, PGII, and gastrin-17 (G-17) levels in serum for chronic atrophic gastritis (CAG) screening and to determine the clinical applicability of combined measurement of serum G-17, pepsinogens (PGI, PGII) and PGI/PGII ratio (PGR) as a screening test for CAG. The PGI, PGII, and G-17 levels were detected by ELISA in 68 patients with CAG and 86 healthy volunteers who underwent gastroscopy for gastroduodenal diseases at Taizhou Municipal Hospital between January 2016 and December 2016. Concentrations of all measured serum markers were lower in patients with CAG in comparison to healthy volunteers and achieved statistical significance (P<0.01) in PGI (93.25 vs 126.98) and PGR (12.67 vs 17.09). Receiver operating characteristic (ROC) curve analysis revealed the optimal cut-off values for PGI, PGII, PGR, and G-17 at 98.10 µg/l, 6.92 ng/l, 15.77 and 1.94 pmol/l, with sensitivities of 72.10%, 58.10%, 61.60%, and 59.30% and specificities of 61.8%, 51.50%, 77.90%, and 55.90%, respectively. The areas under the curve (AUCs) of PGI, PGR, and G-17 were 0.728, 0.726, and 0.556, respectively. The increase of AUC was observed only in PGR and G-17 combination (0.741) with increased sensitivity (69.10% vs 61.60%) of screening for CAG, whereas the specificity was reduced (72.10% vs 77.90%) in comparison to PGR alone. Combination of serum indicators can raise the diagnosis accuracy of CAG in some respects. However, further research including a larger sample size is necessary in order to accurately determine the sensitivity and specificity of combined detection of serum indicators.

[Collecting system percutaneous access using MR-assisted guidance: first human phantom experience].

Objective: Precise renal puncture is an essential but challenging step for successful percutaneous nephrolithotomy. We evaluated the efficiency of a novel real-time navigation system using Mixed Reality(MR) technology for human phantom kidney puncture. Methods: One human kidney phantom underwent MR-assisted percutaneous collecting system puncture. Two punctures were performed by each of 6 surgeons in the randomize selected upper, middle or lower calyces. Outcome measurements were the number of attempts for renal puncture, the time needed to evaluate the trajectory and to perform percutaneous puncture. Results: A total of 12 punctures were performed successfully using MR-Assisted Guidance. Median evaluation time and renal puncture time for the selected calyces was 13 (range 11 to 19) and 19 seconds (range 15 to 44), respectively. One or Two attempts were needed to achieve a successful renal puncture for all of the surgeons. Conclusions: The proposed MR-assisted guidance solution for renal collecting system puncture proved to be accurate, simple and quick. The inherent limitations of traditional X - ray and ultrasonic technology can be overcome.

[Colorectal resection combined with simultaneous radiofrequency ablation in the treatment of synchronous colorectal liver metastases: a retrospective analysis].

Objective: To compare the efficacy and safety of colorectal resection combined with simultaneous radiofrequency ablation (RFA) in the treatment of synchronous colorectal liver metastases(SCRLM). Methods: This retrospective study involved the patients admitted between January 1st 2010 and September 1st 2013. A total of 20 patients who underwent colorectal resection combined with simultaneous RFA of SCRLM were enrolled. Those patients (RFA group) were matched with 20 patients (Resection group) who underwent simultaneous resections of colorectal cancer and SCRLM based on the propensity scores. Perioperative parameters and survival outcomes were compared between the two groups. Results: The RFA and Resection groups were comparable in demographics, cancer characteristics and chemotherapy treatment (all P>0.05). The estimated blood loss and intraoperative blood transfusions in the RFA group were significantly lower than those in the resection group [150.00(100.00-200.00) vs 200.00(112.50-650.00), 1 vs 7, all P>0.05]. The postoperative stay in the RFA group was significantly shorter than that in laparoscopic group [8.50(8.00-10.75) vs 11.00(8.25-14.25), P=0.043]. There was no significant difference in postoperative complications (P>0.05). The 3-year disease free survival rate was 14.00% in the RFA group, and 31.20% in the Resection group (P=0.047). However, the 3-year overall rates were similar between the two groups (P>0.05). Conclusions: Compared with simultaneous resections of colorectal cancer and SCRLM resections, colorectal resection combined with simultaneous RFA of SCRLM was associated with less surgical blood loss and shorter hospitalization. Although inferior to simultaneous resections of colorectal cancer and SCRLM resections in survival outcomes, this approach extends the capability of delivering potentially curative treatment for colorectal cancer patients with unresectable SCRLM.

Miniaturized power limiter metasurface based on Fano-type resonance and Babinet principle.

In this work, we present a miniaturize power limiter, a device with size smaller than that required by the working frequency, made of coupled self-complementary electric inductive-capacitive (CELC) resonator and original electric inductive-capacitive (ELC) structure. We also make use of Babinet principle to ensure both CELC and ELC are resonating at the same frequency. The CELC structure is loaded with a Schottky diode to achieve the effect of a nonlinear power limiter. The constructive interference of CELC and ELC structure produces a new Fano-type resonance peak at a lower frequency. The Fano peak is sharp and able to concentrate electric field at a region between the inner and outer metallic patch of the metastructure, hence enhancing the nonlinear properties of the loaded diode. The Fano peak enhances the maximum isolation of the power limiter due to the local field enhancement at where the diode is loaded. Numerical simulation and experiment are conducted in the S-band frequency to verify the power limiting effect of the device designed and to discuss the formation of Fano peak. The power limiter designed has a maximum isolation of 8.4 dB and a 3-dB isolation bandwidth of 6%.

[Analysis of the influence of gender on the clinicopathological features and prognosis of pancreatic cystic neoplasms].

Objective: To compare the clinicopathological features and prognosis of pancreatic cystic neoplasms (PCN) between male and female patients. Methods: A total of 325 patients with histological confirmed PCN from January 2010 to October 2015 were enrolled. The clinicopathological features, laboratory examination, imaging features, treatment and prognosis were analyzed. Results: Of the 325 patients diagnosed as PCN, 104 were male and 221 were female. The average age of male patients was significantly higher than that of female patients (55.0±12.7 vs 47.9±13.4 years, P<0.001), however, the average tumor size of male patients was smaller than that of female patients (3.9±2.7 vs 4.9±3.0 cm, P=0.004). The location of PCN in male patients was predominantly located at pancreatic head and neck, and majority of male patients were mucinous cystic neoplasms. 1-year, 3-year and 5-year survival rates of patients with PCN were 98.5%, 92.6%, and 87.5%, respectively. 1-year, 3-year and 5-year survival rates of male patients were 97.8%, 84.9%, and 77.8%, however, those of female patients were 98.8%, 96.5%, and 92.2%, respectively. Female patients had better prognosis than male patients (χ2=5.543, P=0.019). Elevated CA19-9 (χ2=3.843, P=0.050), perineuronal invasion (χ2=6.250, P=0.012) and lymph node metastasis (χ2=4.529, P=0.033) were important prognostic factors for malignant mucinous cystic neoplasm. Conclusions: Male patients had low incidence of PCN, and were more common for mucinous cystic neoplasm. The long-term outcome of malignant PCN was poor. Even with complete resection, male patients still had worse prognosis. Close follow-up is recommended especially for male patients.

[Significance of three-dimensional reconstruction as a method of preoperative planning of laparoscopic radiofrequency ablation].

OBJECTIVE: To discuss the significance of three-dimensional reconstruction as a method of preoperative planning of laparoscopic radiofrequency ablation(LRFA). METHODS: Thirty-two cases of LRFA admitted from January 2014 to December 2015 in Department of Hepatobiliary Surgery, Chinese People's Liberation Army General Hospital were analyzed(3D-LRFA group). Three-dimensional(3D) reconstruction were taken as a method of preoperative planning in 3D-LRFA group.Other 64 LRFA cases were paired over the same period without three-dimensional reconstruction before the operation (LRFA group). Hepatobiliary system contrast enhanced CT scan of 3D-RFA patients were taken by multi-slice spiral computed tomography(MSCT), and the DICOM data were processed by IQQA(®)-Liver and IQQA(®)-guide to make 3D reconstruction.Using 3D reconstruction model, diameter and scope of tumor were measured, suitable size (length and radiofrequency length) and number of RFA electrode were chosen, scope and effect of radiofrequency were simulated, reasonable needle track(s) was planed, position and angle of laparoscopic ultrasound (LUS) probe was designed and LUS image was simulated.Data of operation and recovery were collected and analyzed. Data between two sets of measurement data were compared with t test or rank sum test, and count data with χ(2) test or Fisher exact probability test.Tumor recurrence rate was analyzed with the Kaplan-Meier survival curve and Log-rank (Mantel-Cox) test. RESULTS: Compared with LRFA group ((216.8±66.2) minutes, (389.1±183.4) s), 3D-LRFA group ((173.3±59.4) minutes, (242.2±90.8) s) has shorter operation time(t=-3.138, P=0.002) and shorter mean puncture time(t=-2.340, P=0.021). There was no significant difference of blood loss(P=0.170), ablation rate (P=0.871) and incidence of complications(P=1.000). Compared with LRFA group ((6.3±3.9)days, (330±102)U/L, (167±64)ng/L), 3D-LRFA group ((4.3±3.1) days, (285±102) U/L, (139±43) ng/L) had shorter post-operative stay(t=-2.527, P=0.016), less post-operation ALT changes (t=-2.038, P=0.048) and post-operative TNF-α changes(t=-2.233, P=0.027). Disease-free survival between two groups was significantly different (χ(2)=4.049, P=0.046). Disease-free survival of 12 months survival rates were 77.6% and 65.7% in 3D-LRFA group and LRFA group, respectively.The median disease-free survival was 16.0 months in LRFA group and over 24.0 months in 3D-LRFA group. CONCLUSIONS: Three-dimensional model of liver reconstruction based on image information is a powerful tool in liver surgery planning.It helps to simulate tumor location and vital tubular structure, make plan for interventional treatment, and therefore mean puncture time and operation time is shortened, influence on liver function is reduced, hospital stay is decreased and DFS is prolonged.

[Analysis of 113 cases with mucinous cystic neoplasms in a single institutional study].

OBJECTIVE: To describe the clinicopathological characteristics and prognostic factors of mucinous cystic neoplasm(MCN). METHODS: One hundred and thirteen patients with MCN comfirmed by histological examination admitted in People's Liberation Army General Hospital from January 1994 to June 2015 were analyzed, including 21 male and 92 female patients with mean age of (50.2±14.5) years. Forty-eight patients had incidental discovery of their cystic neoplasm for another reason, 65 patients were symptomatic, and abdominal pain or distension was main clinical symptom. Among 113 patients with MCN, 75 cases were MCN with dysplasia, and 38 cases were MCN with invasive carcinoma. Wilcoxon test and χ(2) test, et al were used to analyzed the data, respectively. RESULTS: MCN was mostly located in body and tail of pancreas.Sex, mean age, clinical symptom, tumor size, and CA19-9 showed significant difference between MCN with dysplasia and MCN with invasive carcinoma (all P<0.05). The presence of a solid component (χ(2)=32.460, P=0.000)and main pancreatic duct dilation(χ(2)= 5.729, P=0.022) were significantly associated with malignancy. Fifty-eight patients with dysplasia were followed up, only one patient occurred tumor recurrence. Thirty-one patients with malignancy were followed up, among which there were 22 patients dead, 1-, 3-, 5-year survival of MCN with malignancy was 76.9%, 56.5%, 36.6%, respectively. Lymphatic metastasis and tumor recurrence were important prognostic factors of MCN with malignancy(both P<0.05). CONCLUSIONS: MCN is most affected by old female with no specific symptom, most tumors are located at the body and tail of the pancreas.MCN with dysplasia have excellent prognosis underwent surgery. Even with complete resection, the long-term survival of MCN with malignancy is not satisfied.

TNF-αG-308A polymorphism is associated with insulin resistance: a meta-analysis.

Tumor necrosis factor-α (TNF-α) promoter polymorphisms has been reported to be associated with obesity and insulin resistance and gained widespread attention. However, results obtained so far are quite conf||licting. We therefore performed a meta-analysis to address this issue, basing on 17 studies from electronic databases (MEDLINE and EMBASE). No evidence of significant effect of TNF-αG-308A polymorphism on body mass index (BMI) or obesity risk was detected (BMI: WMDRE = 0.05, 95%CI: -0.62 to 0.73; risk of obesity: ORFE = 1.09, 95%CI: 0.87 to 1.35). G-308A variant was significantly associated with increased insulin levels in the overall (SMDFE = 0.12, 95%CI: 0.03 to 0.20) and obese subgroup analysis (SMDFE = 0.16, 95%CI: 0.03 to 0.29). In total, no significant result was observed for the association between TNF-α G-308A variant and HOMA-IR index. Nevertheless, subgroup analysis showed G-308A polymorphism was significantly associated with increased HOMA-IR in Caucasians (WMDFE = 0.49, 95%CI: 0.03 to 0.94). Our results indicate that TNF-αG-308A polymorphism has a significant effect on insulin resistance. However, it is unlikely that G-308A variant contributes to obesity.

Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis.

DNA damage induces apoptosis through a signalling pathway that can be suppressed by the BCL-2 protein, but the mechanism by which DNA damage does this is unknown. Here, using yeast two-hybrid and co-immunoprecipitation studies, we show that RAD9, a human protein involved in the control of a cell-cycle checkpoint, interacts with the anti-apoptotic Bcl-2-family proteins BCL-2 and BCL-x L, but not with the pro-apoptotic BAX and BAD. When overexpressed in mammalian cells, RAD9 induces apoptosis that can be blocked by BCL-2 or BCL-x L. Conversely, antisense RAD9 RNA suppresses cell death induced by methyl methanesulphonate. These findings indicate that RAD9 may have a new role in regulating apoptosis after DNA damage, in addition to its previously described checkpoint-control and other radioresistance-promoting functions.

Effect of mutations in a simian virus 40 PolyA signal enhancer on green fluorescent protein reporter gene expression.

Our previous studies have shown that tandem Alu repeats inhibit green fluorescent protein (GFP) gene expression when inserted downstream of the GFP gene in the pEGFP-C1 vector. We found that the 22R sequence (5'-GTGAAAAAAATGCTTTATTTGT-3') from the antisense PolyA (240 bp polyadenylation signal) of simian virus 40, eliminated repression of GFP gene expression when inserted between the GFP gene and the Alu repeats. The 22R sequence contains an imperfect palindrome; based on RNA structure software prediction, it forms an unstable stem-loop structure, including a loop, a first stem, a bulge, and a second stem. Analysis of mutations of the loop length of the 22R sequence showed that the three-nucleotide loop (wild-type, 22R) induced much stronger GFP expression than did other loop lengths. Two mutations, 4TMI (A7→T, A17→T) and 5AMI (A6→T, T18→A), which caused the base type changes in the bulge and in the second stem in the 22R sequence, induced stronger GFP gene expression than 22R itself. Mutation of the bulge base (A17→T), leading to complete complementation of the stem, caused weaker GFP gene expression. Sequences without a palindrome (7pieA, 5'-GTGAAAAAAATG CAAAAAAAGT-3', 7pieT, 5'-GTGTTTTTTTTGCTTTTTTTGT-3') did not activate GFP gene expression. We conclude that an imperfect palindrome affects and can increase GFP gene expression.

The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis.

According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1beta converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (DeltaPsim). In contrast, cytosolic CPP32/ Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the DeltaPsim is fully disrupted. Transfection with the cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the DeltaPsim collapse and subsequent apoptosis. Cytosols from anti-Fas-treated human lymphoma cells accumulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified ICE suffices to cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their DeltaPsim. In addition, ICE-treated mitochondria release an apoptosis-inducing factor (AIF) that induces apoptotic changes (chromatin condensation and oligonucleosomal DNA fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator) that can be inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.

Endothelial nitric oxide synthase gene polymorphisms and essential hypertension in Han Chinese.

We examined the effect of polymorphisms in the endothelial nitric oxide synthase gene on the risk for essential hypertension in a Han Chinese population through a meta-analysis of data from 15 studies. Associations between increased risk for essential hypertension and 4b/a were obtained in a dominant model and allele contrast (aa + ab vs bb: odds ratio (OR)(FE) = 1.26, 95% confidence interval (CI) = 1.10-1.44; a vs b allele: OR(FE) = 1.23, 95%CI: 1.09-1.40). Four studies with sample sizes over 500 produced similar results. No evidence of publication bias was found. Also, no significant heterogeneity was observed among these studies. When we examined the G894T polymorphism, we found a marginally significant association for allele contrast and the recessive model when all the eligible studies were pooled together. However, there was no evidence for a significant association after the exclusion of two studies deviating from Hardy-Weinberg equilibrium in the control group. Heterogeneity among studies was observed. Results of cumulative and recursive cumulative meta-analysis indicated that more studies are needed to objectively determine the effects of these two polymorphisms.

[Diagnosis and treatment planning of traumatic dental injury].

Traumatic dental injury (TDI), one of the common dental diseases, is defined as acute injuries to tooth hard tissues, dental pulp and/or periodontal tissues caused by sudden forces. Based on the Guidelines for the Management of TDI by International Association of Dental Traumatology (2012) and the Recommended Guidelines of the American Association of Endodontists for the Treatment of TDI (2013) for the permanent teeth, and combined with the authors' clinical experiences, the contemporary classification and clinical perspective of TDI were introduced in detail. Dental clinicians should understand and master the international guidelines for the management of TDI, including proper diagnosis, standardized treatments and regular follow-up, so as to obtain the favorable outcomes.

[Clinical observation of three-dimensional printing donor tooth model in peri-operative period of autotransplantation of tooth].

Objective: To prepare a three-dimensional (3D) printing donor tooth model and to observe its application in the peri-operative period. Methods: In part one, 192 cases (2017.9-2019.8) from Department of Oral & Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University ï¼»107 males and 85 females, age (34.2±10.7) yearsï¼½ which need autotransplantation of teeth (ATT) were collected. Whether the donor teeth can be completely extracted was predicted through clinical and imaging examination (first prediction). The second prediction was supplemented by the three-dimensional printing model of the donor teeth. Each of the prediction was compared with the actual results and the coincidence rate was calculated. In part two, 64 cases (2017.9-2019.8) from Department of Oral & Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University ï¼»28 males, 36 females, age (30.2±8.3) yearsï¼½ which need ATT were randomly divided into the model group and the donor group. The time of alveolar fossa preparation, time of donor tooth in vitro, times of trial implantation and time of pulptomy and root canal location were recorded respectively. Results: In part one, the coincidence rate between the second prediction and the actual results ï¼»97.4%(187/192)ï¼½ was significantly higher than that of the first prediction ï¼»93.2%(179/192)ï¼½ (P<0.05). In part two, the preparation time of the alveolar fossa in the maxillary and mandibular were (18.8±4.6) and (22.7±3.4) min, the time of the teeth in vitro were (3.0±0.6) and (2.1±0.6) min, the times of trial implantation were (1.3±0.8) and (1.0±0.9), and the time of pulpotomy and root canal location were (4.3±0.6) and (4.0±0.5) min. All values in the model groups were better than those in the donor group (P<0.05). Conclusions: The 3D printing model is accurate. It can be used in autogenous tooth transplantation to shorten the preparation time of alveolar fossa and time of donor tooth in vitro, and reduce the times of trial implantation of donor teeth, and to help to improve the prediction accuracy of complete extraction of donor teeth and the time of pulpotomy and root canal location.

R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism.

The Bcl-2 protein is an important regulator of programmed cell death, but the biochemical mechanism by which this protein prevents apoptosis remains enigmatic. Recently, Bcl-2 has been reported to physically interact with a member of the Ras superfamily of small GTPases, p23-R-Ras. To examine the functional significance of R-Ras for regulation of cell death pathways, the IL-3-dependent cells 32D.3 and FL5.12 were stably transfected with expression plasmids encoding an activated form (38 Glycine-->Valine) of R-Ras protein. R-Ras(38V)-producing 32D.3 and FL5.12 cells experienced increased rates of apoptotic cell death relative to control transfected cells when deprived of IL-3. Analysis of several independent clones of transfected 32D.3 cells revealed a correlation between higher levels of R-Ras protein and faster rates of cell death upon withdrawal of IL-3 from cultures. 32D.3 cells cotransfected with R-Ras(38V) and Bcl-2 exhibited prolonged cell survival in the absence of IL-3, equivalent to 32D.3 cells transfected with Bcl-2 expression plasmids alone. R-Ras(38V) also increased rates of cell death in serum-deprived NIH-3T3 cells, and Bcl-2 again abrogated most of this effect. The ratio of GTP and GDP bound to R-Ras(38V) was not significantly different in control 32D.3 cells vs those that overexpressed Bcl-2, indicating that Bcl-2 does not abrogate R-Ras-mediated effects on cell death by altering R-Ras GDP/GTP regulation. Moreover, purified Bcl-2 protein had no effect on the GTPase activity of recombinant wild-type R-Ras in vitro. When expressed in Sf9 cells using recombinant baculoviruses, R-Ras(38V) bound to and induced activation of Raf-1 kinase irrespective of whether Bcl-2 was coproduced in these cells, suggesting that Bcl-2 does not nullify R-Ras effects by interfering with R-Ras-mediated activation of Raf-1 kinase. Taken together, these findings suggest that R-Ras enhances the activity of a cell death pathway in growth factor-deprived cells and imply that Bcl-2 acts downstream of R-Ras to promote cell survival.

Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner.

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.

Rapid increase in clusters of presynaptic proteins at onset of long-lasting potentiation.

A change in the efficiency of synaptic communication between neurons is thought to underlie learning. Consistent with recent studies of such changes, we find that long-lasting potentiation of synaptic transmission between cultured hippocampal neurons is accompanied by an increase in the number of clusters of postsynaptic glutamate receptors containing the subunit GluR1. In addition, potentiation is accompanied by a rapid and long-lasting increase in the number of clusters of the presynaptic protein synaptophysin and the number of sites at which synaptophysin and GluR1 are colocalized. These results suggest that potentiation involves rapid coordinate changes in the distribution of proteins in the presynaptic neuron as well as the postsynaptic neuron.