Utility of endoscopic therapy in the management of Boerhaave syndrome.

Background/aims: The optimal intervention for Boerhaave perforation has not been determined. Options include surgical repair with/without a pedicled muscle flap, T tube placement, esophageal resection or diversion, or an endoscopic approach. All management strategies require adequate drainage and nutritional support. Our aim was to evaluate outcomes following Boerhaave perforation treated with surgery, endoscopic therapy, or both. Patients and methods: We performed a 10-year review of our prospectively maintained databases of adult patients with Boerhaave perforations. We documented clinical presentation, extent of injury, primary intervention, "salvage" treatment (any treatment for persistent leak), and outcome. Results were analyzed using the Fisher's exact and Kruskal - Wallis tests. Results: Between October 2004 and October 2014, 235 patients presented with esophageal leak/fistula with 17 Boerhaave perforations. Median age was 68 years. Median length of perforation was 1.25 cm (range 0.8 - 5 cm). Four patients presented with systemic sepsis (two treated with palliative stent and two surgically). Primary endotherapy was performed for eight (50 %) and primary surgery for eight (50 %) patients. Two endotherapy patients required multiple stents. Median stent duration was 61 days (range 56 - 76). "Salvage" intervention was required in 2/8 (25 %) endotherapy patients and 1/8 (13 %) surgery patient (stent). All patients healed without resection/reconstruction. There were no deaths in the surgically treated group and two in the endotherapy group (stented with palliative intent due to poor systemic condition). Readmission within 30 days occurred in 3/6 of alive endotherapy patients (50 %) and 0/8 surgery patients. Re-intervention within 30 days was required for one endotherapy patient. Conclusion: Endoscopic repair of Boerhaave perforations can be useful in carefully selected patients without evidence of systemic sepsis. Endoscopic therapy such as stenting is particularly valuable as a "salvage" intervention. The benefits of endoscopic therapy and esophageal preservation are offset against an increased risk of readmission in patients primarily treated endoscopically.

ASCL1 and RET expression defines a clinically relevant subgroup of lung adenocarcinoma characterized by neuroendocrine differentiation.

ASCL1 is an important regulatory transcription factor in pulmonary neuroendocrine (NE) cell development, but its value as a biomarker of NE differentiation in lung adenocarcinoma (AD) and as a potential prognostic biomarker remains unclear. We examined ASCL1 expression in lung cancer samples of varied histologic subtype, clinical outcome and smoking status and compared with expression of traditional NE markers. ASCL1 mRNA expression was found almost exclusively in smokers with AD, in contrast to non-smokers and other lung cancer subtypes. ASCL1 protein expression by immunohistochemical (IHC) analysis correlated best with synaptophysin compared with chromogranin and CD56/NCAM. Analysis of a compendium of 367 microarray-based gene expression profiles in stage I lung adenocarcinomas identified significantly higher expression levels of the RET oncogene in ASCL1-positive tumors (ASCL1(+)) compared with ASCL1(-) tumors (q-value <10(-9)). High levels of RET expression in ASCL1(+) but not in ASCL1(-) tumors was associated with significantly shorter overall survival (OS) in stage 1 (P=0.007) and in all AD (P=0.037). RET protein expression by IHC had an association with OS in the context of ASCL1 expression. In silico gene set analysis and in vitro experiments by ASCL1 shRNA in AD cells with high endogenous expression of ASCL1 and RET implicated ASCL1 as a potential upstream regulator of the RET oncogene. Also, silencing ASCL1 in AD cells markedly reduced cell growth and motility. These results suggest that ASCL1 and RET expression defines a clinically relevant subgroup of ∼10% of AD characterized by NE differentiation.

Patterns of operative mortality following esophagectomy.

Esophagectomy has one of the highest mortality rates among all surgical procedures. We investigated the type and frequency of complications associated with perioperative mortality after esophagectomy. We performed a retrospective review of all perioperative deaths following esophagectomy for esophageal cancer at the Mayo Clinic, Rochester from 1993 through 2009. Of 1522 esophagectomies, perioperative mortality occurred in 45 (3.0%). The majority who died were male (82%); median age was 72 years (range 46-92). The median age-adjusted Charlson comorbidity score was 6. Twenty-three (51%) underwent neoadjuvant chemoradiotherapy. The type of esophagectomy was transthoracic in 27 patients (60%), transhiatal in eight (18%), tri-incisional in seven (16%), left thoracoabdominal in one (2%), and transabdominal in one (2%). A mean of 3.2 major complications occurred prior to death (median 2.5, range 1-8), with the most common being pulmonary complications occurring in 30 patients (67%) and anastomotic complications in 20 (44%). The primary underlying cause of death was pulmonary complications and anastomotic complications in 18 patients (40%) each, respectively, abdominal sepsis in three (7%), fatal hemorrhage in three (7%), and pulmonary embolism, stroke and multisystem organ failure in one each (2%), respectively. Patients died a median of 19 days (range 3-98) following esophagectomy. Most patients who died following esophagectomy experienced multiple serious complications rather than a single causative event. Major pulmonary and anastomotic complications were implicated in the vast majority of perioperative mortality, and should remain the focus of efforts to improve clinical outcomes.

Transcriptional targets of hepatocyte growth factor signaling and Ki-ras oncogene activation in colorectal cancer.

Both Ki-ras mutation and hepatocyte growth factor (HGF) receptor Met overexpression occur at high frequency in colon cancer. This study investigates the transcriptional changes induced by Ki-ras oncogene and HGF/Met signaling activation in colon cancer cell lines in vitro and in vivo. The model system used in these studies included the DLD-1 colon cancer cell line with a mutated Ki-ras allele, and the DKO-4 cell line generated from DLD-1, with its mutant Ki-ras allele inactivated by targeted disruption. These cell lines were transduced with cDNAs of full-length Met receptor. Microarray transcriptional profiling was conducted on cell lines stimulated with HGF, as well as on tumor xenograft tissues. Overlapping genes between in vitro and in vivo microarray data sets were selected as a subset of HGF/Met and Ki-ras oncogene-regulated targets. Using the Online Predicted Human Interaction Database, novel HGF/Met and Ki-ras regulated proteins with putative functional linkage were identified. Novel proteins identified included histone acetyltransferase 1, phosphoribosyl pyrophosphate synthetase 2, chaperonin containing TCP1, subunit 8, CSE1 chromosome segregation 1-like (yeast)/cellular apoptosis susceptibility (mammals), CCR4-NOT transcription complex, subunit 8, and cyclin H. Transcript levels for these Met-signaling targets were correlated with Met expression levels, and were significantly elevated in both primary and metastatic human colorectal cancer samples compared to normal colorectal mucosa. These genes represent novel Met and/or Ki-ras transcriptionally coregulated genes with a high degree of validation in human colorectal cancers.

Functional topology in a network of protein interactions.

MOTIVATION: The building blocks of biological networks are individual protein-protein interactions (PPIs). The cumulative PPI data set in Saccharomyces cerevisiae now exceeds 78 000. Studying the network of these interactions will provide valuable insight into the inner workings of cells. RESULTS: We performed a systematic graph theory-based analysis of this PPI network to construct computational models for describing and predicting the properties of lethal mutations and proteins participating in genetic interactions, functional groups, protein complexes and signaling pathways. Our analysis suggests that lethal mutations are not only highly connected within the network, but they also satisfy an additional property: their removal causes a disruption in network structure. We also provide evidence for the existence of alternate paths that bypass viable proteins in PPI networks, while such paths do not exist for lethal mutations. In addition, we show that distinct functional classes of proteins have differing network properties. We also demonstrate a way to extract and iteratively predict protein complexes and signaling pathways. We evaluate the power of predictions by comparing them with a random model, and assess accuracy of predictions by analyzing their overlap with MIPS database. CONCLUSIONS: Our models provide a means for understanding the complex wiring underlying cellular function, and enable us to predict essentiality, genetic interaction, function, protein complexes and cellular pathways. This analysis uncovers structure-function relationships observable in a large PPI network.

Binary tree-structured vector quantization approach to clustering and visualizing microarray data.

MOTIVATION: With the increasing number of gene expression databases, the need for more powerful analysis and visualization tools is growing. Many techniques have successfully been applied to unravel latent similarities among genes and/or experiments. Most of the current systems for microarray data analysis use statistical methods, hierarchical clustering, self-organizing maps, support vector machines, or k-means clustering to organize genes or experiments into 'meaningful' groups. Without prior explicit bias almost all of these clustering methods applied to gene expression data not only produce different results, but may also produce clusters with little or no biological relevance. Of these methods, agglomerative hierarchical clustering has been the most widely applied, although many limitations have been identified. RESULTS: Starting with a systematic comparison of the underlying theories behind clustering approaches, we have devised a technique that combines tree-structured vector quantization and partitive k-means clustering (BTSVQ). This hybrid technique has revealed clinically relevant clusters in three large publicly available data sets. In contrast to existing systems, our approach is less sensitive to data preprocessing and data normalization. In addition, the clustering results produced by the technique have strong similarities to those of self-organizing maps (SOMs). We discuss the advantages and the mathematical reasoning behind our approach.

Epstein-Barr virus serology and posttransplant lymphoproliferative disease in lung transplantation.

BACKGROUND: Posttransplant lymphoproliferative disease (PTLD) is now a widely recognized complication of lung transplantation. In the current study, we present our experience with PTLD over a 15-year period, which includes the incidence rates in 242 lung allografts and the relative risk of developing PTLD in 146 patients with known pretransplantation Epstein-Barr virus (EBV) status. METHODS: Inpatient and outpatient charts of 300 consecutive lung transplant recipients between 1984 and 1999 were retrospectively reviewed. RESULTS: Twelve cases of PTLD were observed for a total incidence rate of 5.0%. Ten of these patients had pretransplantation EBV testing, and the consequent increase in relative risk for patients who were EBV negative was 6.8-fold. The mean time between organ transplantation and tissue diagnosis of PTLD was 17.6 months. Total 1-year survival rate from the time of diagnosis for the cohort was 58%, whereas 2-year survival rate was 50%. Median survival for the six patients who died was 4.5 months. CONCLUSIONS: These data suggest that although EBV seronegativity does carry a 6.8-fold increase in the relative risk of developing PTLD, long-term survival despite the development of PTLD can be achieved, and thus EBV seronegativity by itself should not be considered a contraindication to lung transplantation.

Mining mouse microarray data.

Microarrays of mouse genes are now available from several sources, and they have so far given new insights into gene expression in embryonic development, regions of the brain and during apoptosis. Microarray data posted on the internet can be reanalyzed to study a range of questions.

AML1-like transcription factor induces serine elastase activity in ovine pulmonary artery smooth muscle cells.

In previous studies, we showed that induction of pulmonary artery (PA) smooth muscle cell (SMC) elastase activity by serum-treated elastin (STE) requires DNA transcription. We therefore used differential mRNA display to identify transcripts expressed coincident with elastase induction. Twenty-four individual transcripts were differentially expressed from a screen of approximately 2000 mRNA sequences. An mRNA with sequence homology to the human transcription factor AML1 was identified and subsequently cloned from ovine PA SMCs. Since AML1 binds to a consensus sequence in the promoter of neutrophil elastase, we pursued the possibility that AML1 is a candidate transcription factor for SMC elastase. We documented by immunohistochemistry that serum stimulation induces increased expression of AML1 in the nucleus of PA SMCs. We also showed that STE induction of elastase activity is associated with early expression of AML1 mRNA and protein and that AML1 consensus sequence DNA binding activity is increased in nuclear extracts of STE-treated cells. In addition, AML1 antisense oligonucleotides reduced serum induction of elastase activity. Our study thus provides the first functional evidence of AML1 transcriptional activity related to elastase genes and offers novel insights into the broader biological significance of AML1 in nonmyeloid cells.

Developmental expression of the collagen-binding heat-shock protein GP46 and collagen types I and IV in rat tissues.

The temporal expression of protein and mRNA encoding the collagen-binding heat-shock glycoprotein, gp46, were determined in the heart, kidney, and lung during early rat postnatal development. The steady-state levels of collagen types I and IV mRNA expression were also examined to determine if gp46 and these collagen types are co-regulated during ontogenesis. Western blot analysis using a monoclonal antibody to gp46 revealed that gp46 levels are developmentally regulated. In heart and kidney, gp46 levels were high on days 3 and 8, reduced significantly on day 25, and low to undetectable on day 69. Protein levels of gp46 in the lung exhibited a similar temporal pattern except on day 3, when very low levels of gp46 were detected. mRNA expression of gp46 during early postnatal development did not correlate with gp46 protein accumulation in these tissues, suggesting a complex pre- and post-translational regulatory scheme. In the heart, protein levels of gp46 correlated well with collagen type I alpha 1(I) mRNA expression but not with collagen type IV alpha 1(IV). In contrast, gp46 protein levels closely paralleled alpha 1(IV) expression in the kidney. Gp46 levels exhibited no apparent correlation with either alpha 1(I) or alpha 1(IV) levels in the lung. These results show that gp46 is developmentally regulated at both the protein and mRNA levels in a tissue specific manner. The relationship between gp46 and collagen alpha 1(I) and alpha 1(IV) chain mRNA expression also has been shown to be tissue specific.

Time-dependent decreases of atrial natriuretic peptide release from the isolated rat atrium: evidence for a readily releasable pool.

In vitro, atrial distension causes a rapid increase in atrial natriuretic peptide (ANP) release. This stretch-induced release, however, declines to baseline levels within minutes without significant depletion of the total hormone stores. It has been observed that the basal rate of ANP release from isolated atria also declines over time despite evidence that the tissue retains its viability. We examined this time-dependency of ANP release from isolated rat atria and some parameters that may explain the diminishing release. Mean ANP secretion was 60 pg/min for both spontaneously beating and electrically paced preparations. Although ANP secretion steadily declined over time, there was no time-dependent effect on the amplitude of intracellularly recorded action potentials. The total ANP content in atria obtained after dissection was 133 +/- 28.9 micrograms/g (n = 3) which was not significantly different from atria that were perfused for 3 h (137 +/- 21.2 micrograms/g; n = 3). Only the 28 amino acid circulating form of ANP was released. The ANP mRNA appeared to be partially degraded in atria after 30 min equilibration or after perfusion for 3 h. These results demonstrated that ANP release from isolated atrial preparations declines steadily despite the maintenance of normal electrophysiological activity. This decline was not due to significant depletion of the ANP stores suggesting that a readily releasable pool of ANP exists and represents only a small fraction of the total hormone stores. Finally, degradation of ANP mRNA implies a reduction of de novo synthesis in our preparation which suggests that the observed depletion of the releasable pool was related to a decline in newly synthesized ANP.

Gene expression of A- and B-type natriuretic peptides in response to acute ethanol ingestion.

Given that ethanol ingestion is associated with a disruption of water and electrolyte balance in addition to being a significant risk factor for cardiovascular disease, we have investigated the gene expression of ANP and BNP in response to acute doses of ethanol. Wistar rats were administered either a 5 g/kg dose of ethanol or an equivalent volume of water, and atrial and ventricular tissue samples were removed at 30, 60, and 120 min for analyses. Although no differences in ANP mRNA were observed between ethanol and water-treated rats during the time course, BNP mRNA levels in ethanol-treated rats were 43% of those present in water-treated animals in atrial tissue at 120 min. In ventricular tissue, BNP mRNA levels were reduced similarly to 38% of control. These results suggest a possible differential regulation of A- and B-type natriuretic peptides under the influence of ethanol ingestion.

ANP secretion from small cell lung cancer cell lines: a potential model of ANP release.

Although atrial distension is widely accepted as the primary stimulus for atrial natriuretic peptide (ANP) release, a number of agonists are also known to induce its secretion. The mechanisms underlying these processes are not well understood. Studies of this nature are hampered by the inherent difficulty in culturing homogeneous populations of cardiac myocytes in sufficient quantities to perform molecular investigations. For this reason, we have examined the possibility of using other cell types as a model of ANP release. It has been reported that a number of tumor samples from small cell lung cancer (SCLC) patients express the ANP gene. Characterization of a large number of cell lines derived from SCLC tumor samples indicated that two of these cell lines, OS-A and SHP-77, secrete ANP at rates of approximately 10(-20) g.cell-1.min-1. This is a sufficient quantity to facilitate secretion studies using a perifusion system. We have demonstrated that ANP is released through regulated secretory pathways, as the Ca2+ ionophore A-23187, arginine vasopressin (AVP), and the sodium ionophore, monensin, were capable of modifying secretion rates. High-pressure liquid chromatography (HPLC) analysis indicated that the primary secretory product is ANP-(99-126), the circulating form of this hormone. Intracellularly, both ANP-(99-126) and ANP-(1-126) were present, suggesting the synthesis and appropriate cleavage of pro-ANP-(1-126). Because both of these cell lines have doubling times in the range of 3-5 days, they could serve as a rapidly proliferating and easily maintainable supply of homogeneous tissue for release studies.(ABSTRACT TRUNCATED AT 250 WORDS)

The antidiuretic effect of pneumadin requires a functional arginine vasopressin system.

Pneumadin is an antidiuretic decapeptide, recently isolated from rat and human lung. Bolus intravenous injection of 5 nmol of pneumadin into water-loaded rats caused a rapid and significant antidiuresis and a reduction in Na+ and Cl- excretion. Pneumadin administration did not alter mean arterial pressure, right atrial pressure, heart rate or haematocrit. Bolus intravenous injection of 20 nmol of pneumadin into non-water-loaded rats caused a significant increase in arginine vasopressin (AVP) within 10 min. Pneumadin administration also increased circulating atrial natriuretic peptide (ANP) but did not alter aldosterone or plasma renin activity levels. Injection of pneumadin into water-loaded Brattleboro rats, which genetically lack circulating AVP, did not change urine flow, confirming that the pneumadin induced antidiuresis is AVP dependent. Radioactive pneumadin was cleared from the circulation with a t1/2 beta of 480.3 s. Radioactive pneumadin, isolated from plasma, eluted at an altered position on reverse phase HPLC, which indicated that the peptide was modified in vivo. This modification was also observed when synthetic pneumadin was incubated in rat plasma in vitro. Purification and sequencing of the modified synthetic peptide indicated that the modification is not a proteolytic cleavage. These results indicate that pneumadin injected into the rat caused an antidiuresis by altering circulating AVP levels.

Radioimmunoassay for rat B-type natriuretic peptide (BNP-45).

Rat BNP-45 is the main circulating form of BNP in rat plasma. To understand the role of BNP in physiological and pathophysiological conditions, a specific radioimmunoassay (RIA) for the quantitative determination of the peptide in plasma and tissues is necessary. An assay using rBNP-45 as the standard in conjunction with antisera directed against this peptide has not been described in the literature, though some investigators have reported values ranging from 0.73-2.0 pmol/L using either BNP-26 or BNP-32 as the standard peptide. Unfortunately, these forms of BNP do not exist in rat plasma. In our studies, we have developed a specific RIA for rBNP-45 using rBNP-45 as the standard peptide and Tyro-rBNP-45 as the radioligand. We have used two specific antisera for assay purposes; one against rBNP-45, and the second to a peptide composed of the first 20 amino acids of rBNP-45 (rBNP[1-20]). The recovery of various amounts of rBNP-45 added to control plasma was 50-80% depending on the method of extraction and purification. The interassay and intraassay coefficients of variation were 12% and 6% respectively. Values obtained were similar for blood sampled by either cardiac puncture, decapitation, or aortic puncture. The method was used to measure rBNP-45 in the plasma of normal (WKY) and Spontaneously Hypertensive (SHR) rats. The values obtained were 5.46 +/- 0.43 and 19.6 +/- 2.36 pmol/L respectively. The rat atrial natriuretic peptide (ANP[99-126]) values in the same extracts were 23.2 +/- 0.45 and 51.6 +/- 3.16 pmol/L.

Chronic ethanol ingestion modifies the renin-aldosterone axis independent of alterations in the regulation of atrial natriuretic peptide.

Using an animal model, we have investigated the effects of chronic ethanol ingestion on the regulation of atrial natriuretic peptide (ANP) synthesis and release. Male Sprague-Dawley rats were maintained for 6 weeks on a liquid diet of ethanol (up to 20% v/v) as part of a 2% solution of calf milk replacer. Weight-matched controls received an equal volume of ethanol-free solution, and normal animals drank ad libitum. All animals received rat chow throughout the experiment. This model produced physiologically relevant levels of blood ethanol, as concentrations at the time of sacrifice were 171.98 +/- 39.26 mg/dl. Plasma renin activity was significantly elevated in response to ethanol treatment, whereas circulating aldosterone concentration was reduced. No alterations in the plasma or atrial tissue levels of ANP were evident, although we did observe a significant increase in the ventricular tissue levels of ANP from 45.1 to 71.8 ng/g as a consequence of ethanol treatment. Levels of both atrial and ventricular ANP mRNA were not different between alcohol-treated and liquid-restricted control animals, although both groups showed significant increases in the amount of transcript in comparison with rats drinking ad libitum. No significant increases in either arterial blood pressure or heart/body weight ratio were observed for ethanol-treated rats. These results suggest that modifications in the renin-aldosterone axis can occur independently of alterations in the regulation of ANP under the influence of chronic ethanol ingestion.

Chronic ethanol treatment increases the circulating plasma levels of B-type natriuretic peptide (BNP-45) in the rat.

Chronic ethanol ingestion is associated with a number of cardiovascular disorders, including stroke, heart failure, and hypertension. Given that the regulation of A-type natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) is known to be altered in both congestive heart failure and essential hypertension, we have investigated the regulation of BNP under the influence of ethanol ingestion. Sprague-Dawley rats were given ethanol in drinking fluid for a 6-week period, while a weight-matched liquid-restricted group received an equivalent volume of ethanol-free solution. Plasma BNP levels were increased in ethanol-treated animals relative to both liquid-restricted and normal control groups. No changes in cardiac BNP gene expression were observed, but an increased trend in atrial tissue BNP levels was evident. No changes in either the mRNA, tissue, or plasma levels of ANP were evident. These results suggest a differential regulation of natriuretic peptides under the influence of ethanol, and implicate chronic ethanol ingestion as a further clinical condition under which the plasma levels of a natriuretic peptide may be elevated.

Acute ethanol ingestion modifies the circulating plasma levels of atrial natriuretic peptide.

Since ethanol ingestion is associated with a disruption of water and electrolyte balance in a variety of species, we sought to evaluate the regulatory control of atrial natriuretic peptide (ANP) in response to acute doses of ethanol. Male Sprague-Dawley rats were administered a 5-g/kg dose of ethanol (40% w/v) via a gastric tube, while control animals received an equivalent volume of water. Expressed as a percentage of control, plasma ANP levels were 39.0%, 28.5%, and 23.6% in the ethanol-treated animals at 30, 60, and 120 min postintubation, respectively. Ethanol-treated animals displayed blood alcohol concentrations of 89.0, 137.6, and 214.1 mg/dl at the same time periods. After 120 min, plasma renin activity was elevated from 8.7 to 20.3 ng/ml/h in conjunction with an increase in the levels of circulating aldosterone from 16.3 to 42.5 ng/dl and an increase in plasma vasopressin from 2.2 to 3.6 pg/ml. Levels of atrial ANP mRNA remained consistent over the time course of the experiment, and no changes in the amount of ventricular ANP transcript were observed. Tissue ANP levels were similar between ethanol-treated and water-loaded control animals. In vitro experiments using cultured cardiac myocytes suggest that ethanol exposure may not directly affect ANP secretion. We propose that acute ethanol treatment may inhibit atrial distension and subsequently modify the control of ANP release under volume loading conditions.

Differential expression and activity of p34cdc2 in cultured aortic adventitial fibroblasts derived from spontaneously hypertensive and Wistar-Kyoto rats.

OBJECTIVE: The present investigation was undertaken to determine whether p34cdc2, a cell-cycle regulatory kinase, is involved in the manifestation of the altered proliferation evident in fibroblasts isolated from spontaneously hypertensive rats (SHR). DESIGN: Experiments were performed on quiescent aortic adventitial fibroblasts stimulated to re-enter the cell cycle in order to examine the timing of cell cycle-related events. METHODS: The cell-cycle phase was determined by flow cytometry and was related to the cellular content and kinase activity of p34cdc2. RESULTS: SHR fibroblasts displayed a heightened basal level of p34cdc2 at quiescence relative to Wistar-Kyoto (WKY) rat cells. Both SHR and WKY fibroblasts showed a cell cycle-dependent increase in p34cdc2 content, beginning in S phase. However, the SHR adventitial fibroblasts exited G0-G1 several hours earlier than the WKY fibroblasts as indicated by the time of initiation of DNA synthesis and increase in activity of p34cdc2. CONCLUSIONS: SHR aortic adventitial fibroblasts appear to have a heightened proliferative capacity relative to WKY fibroblasts, which is evident in a quicker exit from G0 and faster transition to DNA synthesis, followed by the earlier activation of p34cdc2.