Coexisting non-functioning pituitary macroadenoma and sellar-suprasellar lipoma: A case report and literature review

@#Intracranial lipomas are benign tumors that may occasionally be found in the suprasellar cistern while pituitary adenomas are far more common brain tumors. Pituitary adenomas may rarely coexist with other intracranial tumors in the sellar-suprasellar region. We share a unique case of a patient with coexisting non-functioning pituitary adenoma and sellar-suprasellar lipoma presenting with blurring of vision. We report a 55-year-old male presenting with a two-year history of blurring of vision with findings of a 2.7 x 3.0 x 3.2 cm homogeneously enhancing lobulated isointense mass on the sellar-suprasellar region. Hormonal workups revealed low cortisol and mildly elevated prolactin. He initially underwent endonasal transsphenoidal excision of the tumor which revealed to be a lipoma on histopathology. Due to minimal improvement of vision from the subtotal excision, he underwent repeat surgery through the transcranial approach which in turn showed a pituitary adenoma. The co-occurrence of two sellar-suprasellar tumors with different histology is rare, as most of the evidence is based on only a handful of case series. Intracranial lipomas result from persistence and abnormal differentiation of the meninx primitiva during the development of the subarachnoid cisterns. On the other hand, pituitary tumorigenesis is still largely unclear but appears to involve multiple tumor suppressor genes, oncogenes, cell cycle deregulation factors, and miRNAs. Given the differing pathogenesis of each tumor type, the coexistence may only be coincidental. The best surgical approach in this situation is unknown but the focus is on complete excision of the adenoma.

Manufacturing and Functional Characterization of Bioengineered Liver Grafts for Extracorporeal Liver Assistance in Acute Liver Failure.

Acute Liver Failure (ALF) is a life-threatening illness characterized by the rapid onset of abnormal liver biochemistries, coagulopathy, and the development of hepatic encephalopathy. Extracorporeal bioengineered liver (BEL) grafts could offer a bridge therapy to transplant or recovery. The present study describes the manufacture of clinical scale BELs created from decellularized porcine-derived liver extracellular matrix seeded entirely with human cells: human umbilical vein endothelial cells (HUVECs) and primary human liver cells (PHLCs). Decellularized scaffolds seeded entirely with human cells were shown to adhere to stringent sterility and safety guidelines and demonstrated increased functionality when compared to grafts seeded with primary porcine liver cells (PPLCs). BELs with PHLCs were able to clear more ammonia than PPLCs and demonstrated lower perfusion pressures during patency testing. Additionally, to determine the full therapeutic potential of BELs seeded with PHLCs, longer culture periods were assessed to address the logistical constraints associated with manufacturing and transporting a product to a patient. The fully humanized BELs were able to retain their function after cold storage simulating a product transport period. Therefore, this study demonstrates the manufacture of bioengineered liver grafts and their potential in the clinical setting as a treatment for ALF.

Exophytic and fungating papillary thyroid carcinoma: A rare and complex presentation of a well-differentiated malignancy

@#Papillary thyroid carcinoma is the most common well-differentiated thyroid malignancy accounting for more than 80 to 90% of all thyroid tumors. It has an overall excellent prognosis owing to advances in screening via imaging and ultrasound-guided fine-needle aspiration biopsy, which have facilitated early detection, diagnosis, and surgical treatment followed by adjuvant radioactive iodine therapy. Exceptionally rare cases of papillary thyroid tumors may present with enormous growth due to delayed consultation and, thus, late diagnosis, posing a challenge to definitive management, quality of life, overall survival, and prognosis. We report a case of a 35-year-old woman who presented with a 4-year history of a bleeding exophytic and fungating anterior neck mass. Computed tomography showed a fungating mass arising from the left thyroid lobe that measured 14.1 x 14.0 x 11.1 cm with areas of necrosis and hemorrhage, left internal jugular vein thrombus formation, and compression of the left internal carotid artery. The mass causes a displacement of the trachea to the right side and multiple bilateral cervical lymphadenopathies. The patient was fully aware, and she consented to undergo wide excision, total thyroidectomy, neck dissection, and pectoralis major muscle flap reconstruction. However, she went into arrest intraoperatively attributed to massive pulmonary embolism. Papillary thyroid cancer is well known for its excellent prognosis. However, outcomes may not be favorable and can even be fatal in advanced and extensive cases. Although fungating papillary cancers are rare, they remain more common in the developing countries, where early detection and access to healthcare remains limited. They also represent a big challenge to surgeons. Even if the outcome was not good, we opted to report this case as there were many learning points. If only patients with good and excellent outcomes are reported in the literature, it will overestimate the treatment success of these complex cases.

Functional characterization of a bioengineered liver after heterotopic implantation in pigs.

Organ bioengineering offers a promising solution to the persistent shortage of donor organs. However, the progression of this technology toward clinical use has been hindered by the challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions to concurrently support the health and phenotypic stability of diverse cell lineages. We previously demonstrated the ability to functionally reendothelialize the vasculature of a clinically scaled decellularized liver scaffold with human umbilical vein endothelial cells (HUVECs) and to sustain continuous perfusion in a large animal recovery model. We now report a method for seeding and engrafting primary porcine hepatocytes into a bioengineered liver (BEL) scaffold previously reendothelialized with HUVECs. The resulting BELs were competent for albumin production, ammonia detoxification and urea synthesis, indicating the presence of a functional hepatocyte compartment. BELs additionally slowed ammonia accumulation during in vivo perfusion in a porcine model of surgically induced acute liver failure. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers. Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells.

Quality of nutritional care assessment among critically-ill patients in a tertiary government hospital

@#<p style="text-align: justify;"><strong>Background and Objectives:</strong> Malnutrition is prevalent both at baseline admission and because of hospitalization. It is aggravated by adverse hospital practices and results in poor outcomes, reduced quality of life, and higher treatment costs. Improving quality of care involves nutritional intervention as a low-risk, cost-effective strategy which guides providers in improving practices systems-wise. This study aims to assess the quality of nutritional care and the nutritional status of critically- ill patients admitted in a low-resource setting.</p><p style="text-align: justify;"><strong>Materials and Methods:</strong> This is a mixed methods study among adults admitted in intensive care units (ICUs) of a tertiary government hospital. Anthropometric and biochemical indicators were obtained through chart review. The degree of malnutrition was assessed using the Subjective Global Assessment. Quality indicators under Donabedian domains were assessed and compared to current standards. The length of ICU stay and mortality rate were recorded. Dietary prescription and provision practices of healthcare providers were supplemented by a focus group discussion (FGD). Factors causing provision interruptions were also identified.</p><p style="text-align: justify;"><strong>Results and Discussion:</strong> Sixty-four ICU admissions were included. Staff-to-patient ratio was not ideal. Under process-related factors, out of 49% with actual anthropometric documentations (rest were estimates), 24% had normal body mass indices (BMI), 17% were underweight, and the rest were either overweight or obese. The baseline ICU malnutrition rate was 69%. Malnutrition screening, and assessment of risk and biochemical indicators were not done routinely. Majority (92%) had baseline dietary prescription but only 69% had specific energy and macronutrient breakdown, all done through predictive weight-based equations. Nutritional supplies arrived within 8 hours in 65% of patients. Feeding was initiated within 24-28 hours in 94% of patients. Commercial formula was the preferred type of enteral nutrition (EN). Total duration on nothing-by-mouth (NPO) (hours) throughout ICU stay was significant. Supportive measures to improve gastro-intestinal (GI) tolerance were not standardized. Common factors in delaying feeding initiation were hemodynamic instability, fasting for procedures and GI bleeding. Throughout the ICU stay, fasting for procedures, hemodynamic instability and mechanical ventilation (MV)-related factors were common. ICU mortality rate was 19% and average length of ICU stay was 5 days.</p><p style="text-align: justify;"><strong>Conclusion:</strong> Malnutrition is still prevalent in our ICUs and is affected by suboptimal healthcare practices. Staff - to-patient ratios, malnutrition risk screening and assessment, dietary referrals, documentation and minimizing interruptions in nutritional care provision needs improvement. A system review and establishment of a nutrition team is imperative.</p>

Host Gene Expression Is Regulated by Two Types of Noncoding RNAs Transcribed from the Epstein-Barr Virus BamHI A Rightward Transcript Region.

UNLABELLED: In Epstein-Barr virus-infected epithelial cancers, the alternatively spliced BamHI A rightward transcripts (BARTs) are the most abundant viral polyadenylated RNA. The BART introns form the template for the production of 44 microRNAs (miRNAs), and the spliced and polyadenylated exons form nuclear non-protein-coding RNAs. Analysis of host cell transcription by RNA-seq during latency in AGS cells identified a large number of reproducibly changed genes. Genes that were downregulated were enriched for BART miRNA targets. Bioinformatics analysis predicted activation of the myc pathway and downregulation of XBP1 as likely mediators of the host transcriptional changes. Effects on XBP1 activity were not detected in these cells; however, myc activation was confirmed through use of a myc-responsive luciferase reporter. To identify potential regulatory properties of the spliced, polyadenylated BART RNAs, a full-length cDNA clone of one of the BART isoforms was obtained and expressed in the Epstein-Barr virus (EBV)-negative AGS cells. The BART cDNA transcript remained primarily nuclear yet induced considerable and consistent changes in cellular transcription, as profiled by RNA-seq. These transcriptional changes significantly overlapped the transcriptional changes induced during latent EBV infection of these same cells, where the BARTs are exclusively nuclear and do not encode proteins. These data suggest that the nuclear BART RNAs are functional long noncoding RNAs (lncRNAs). The abundant expression of multiple forms of noncoding RNAs that contribute to growth regulation without expression of immunogenic proteins would be an important mechanism for viral oncogenesis in the presence of a functional immune system. IMPORTANCE: Infection with Epstein-Barr virus (EBV) is nearly ubiquitous in the human population; however, it does contribute to the formation of multiple types of cancer. In immunocompromised patients, EBV causes multiple types of lymphomas by expressing viral oncogenes that promote growth and survival of infected B lymphocytes. EBV-positive gastric carcinoma does not require immune suppression, and the viral oncoproteins that are frequent targets for an immunological response are not expressed. This study demonstrates using transcriptional analysis that the expression of various classes of viral non-protein-coding RNAs likely contribute to the considerable changes in the host transcriptional profile in the AGS gastric cancer cell line. This is the first report to show that the highly expressed polyadenylated BamHI A rightward transcripts (BART) viral transcript in gastric carcinoma is in fact a functional viral long noncoding RNA. These studies provide new insight into how EBV can promote transformation in the absence of viral protein expression.

Targets of drugs are generally, and targets of drugs having side effects are specifically good spreaders of human interactome perturbations.

Network-based methods are playing an increasingly important role in drug design. Our main question in this paper was whether the efficiency of drug target proteins to spread perturbations in the human interactome is larger if the binding drugs have side effects, as compared to those which have no reported side effects. Our results showed that in general, drug targets were better spreaders of perturbations than non-target proteins, and in particular, targets of drugs with side effects were also better spreaders of perturbations than targets of drugs having no reported side effects in human protein-protein interaction networks. Colorectal cancer-related proteins were good spreaders and had a high centrality, while type 2 diabetes-related proteins showed an average spreading efficiency and had an average centrality in the human interactome. Moreover, the interactome-distance between drug targets and disease-related proteins was higher in diabetes than in colorectal cancer. Our results may help a better understanding of the network position and dynamics of drug targets and disease-related proteins, and may contribute to develop additional, network-based tests to increase the potential safety of drug candidates.

Changes in expression induced by Epstein-Barr Virus LMP1-CTAR1: potential role of bcl3.

UNLABELLED: The Epstein-Barr virus protein latent membrane protein 1 (LMP1) has two NF-κB activating domains within its intracellular carboxy terminus (carboxy-terminal activating region 1 [CTAR1] and CTAR2). LMP1-CTAR1 is required for B-lymphocyte transformation, is capable of transforming rodent fibroblasts, and uniquely activates phosphoinositol (PI3) kinase, the noncanonical NF-κB pathway, and expression of the epidermal growth factor receptor (EGFR). In this study, the effects of LMP1-CTAR1 on cellular gene expression were determined by high-throughput sequencing. Additionally, the binding of bcl3 was determined using chromatin immunoprecipitation (ChIP) and sequencing. LMP1-CTAR1 induced few changes in transcription with more genes showing decreased expression. Ingenuity pathway analysis indicated significant enrichment for genes involved in cancer and cellular movement, survival, growth, and proliferation pathways. ChIP in combination with high-throughput sequencing (ChIP-Seq) identified bcl3 binding for more than 2,000 genes in LMP1-CTAR1-expressing cells with more than 90% of the peaks at genes detected within the probable promoter region. Only a small subset of the genes with significant changes in expression had corresponding peaks in the bcl3 ChIP. However, both NFKB2 and PI3 kinase were identified in the bcl3 ChIP. Additionally, many of the predicted upstream regulators for the changes in expression were identified in the bcl3 ChIP. Analysis of the proteins in the NF-κB pathway revealed many changes identified by the high-throughput RNA sequencing (RNA-Seq) and bcl3 ChIP that would likely activate noncanonical NF-κB signaling and possibly inhibit canonical NF-κB signaling. These findings suggest that the two LMP1 signaling domains modulate their combined activity and that the bcl3 transcription factor is likely responsible for some of the unique effects of CTAR1 on cellular expression. IMPORTANCE: The Epstein-Barr virus protein latent membrane protein 1 (LMP1) has potent effects on cell growth. LMP1 has two regions, carboxy-terminal activating region 1 (CTAR1) and CTAR2, that distinctly activate NF-κB, a transcription factor complex involved in activation of important host genes. In this study, analysis of the effects on cellular gene expression revealed that CTAR1 significantly affected cellular expression in part through effects on a specific form of NF-κB. The data suggest that LMP1 can activate a distinct subset of host gene expression through its CTAR1 domain which in combination with other signaling effects induced by the CTAR2 domain likely affects cell movement, survival, and growth.

Cancer stem cells display extremely large evolvability: alternating plastic and rigid networks as a potential Mechanism: network models, novel therapeutic target strategies, and the contributions of hypoxia, inflammation and cellular senescence.

Cancer is increasingly perceived as a systems-level, network phenomenon. The major trend of malignant transformation can be described as a two-phase process, where an initial increase of network plasticity is followed by a decrease of plasticity at late stages of tumor development. The fluctuating intensity of stress factors, like hypoxia, inflammation and the either cooperative or hostile interactions of tumor inter-cellular networks, all increase the adaptation potential of cancer cells. This may lead to the bypass of cellular senescence, and to the development of cancer stem cells. We propose that the central tenet of cancer stem cell definition lies exactly in the indefinability of cancer stem cells. Actual properties of cancer stem cells depend on the individual "stress-history" of the given tumor. Cancer stem cells are characterized by an extremely large evolvability (i.e. a capacity to generate heritable phenotypic variation), which corresponds well with the defining hallmarks of cancer stem cells: the possession of the capacity to self-renew and to repeatedly re-build the heterogeneous lineages of cancer cells that comprise a tumor in new environments. Cancer stem cells represent a cell population, which is adapted to adapt. We argue that the high evolvability of cancer stem cells is helped by their repeated transitions between plastic (proliferative, symmetrically dividing) and rigid (quiescent, asymmetrically dividing, often more invasive) phenotypes having plastic and rigid networks. Thus, cancer stem cells reverse and replay cancer development multiple times. We describe network models potentially explaining cancer stem cell-like behavior. Finally, we propose novel strategies including combination therapies and multi-target drugs to overcome the Nietzschean dilemma of cancer stem cell targeting: "what does not kill me makes me stronger".

[A case of thyreotoxic hypokalaemic periodic paralysis in a white male]. / Thyreotoxische hypokaliëmische periodieke paralyse bij een blanke man.

BACKGROUND: Thyreotoxic hypokalaemic periodic paralysis (THPP) is a rare and potentially life-threatening syndrome. It principally affects men of East-Asian origin and has rarely been described in a white person. CASE DESCRIPTION: A 34-year-old Dutch man, suffering from Graves' disease, presented with weakness in his lower limbs. Laboratory investigation showed severe hypokalaemia (1.8 mmol/l) and increased creatinine kinase levels. An electrocardiogram showed atrial fibrillation with a prolonged QTc-interval. The patient was admitted, cardiac rhythm was monitored, and he received potassium supplements. Laboratory investigation of thyroid function showed thyrotoxicosis. The patient was treated with propranolol and thiamazol. At follow-up, thyroid function, potassium levels and muscle strength had normalized. CONCLUSION: Hypokalaemia due to thyrotoxicosis should be considered in cases of unexplained paralysis. The treatment of THPP consists of treating for hyperthyroidism plus propranolol. Since the hypokalaemia is self-limiting, potassium supplementation is only necessary in cases of rhythm disturbances or cardiac-conduction disturbances. Despite adequate treatment, there is a risk of recurrence. Regular monitoring is indicated until euthyroidism is achieved.

Expression profile of microRNAs in Epstein-Barr virus-infected AGS gastric carcinoma cells.

Latent infection with Epstein-Barr virus (EBV) is responsible for multiple types of malignancies, including 10% of all gastric carcinomas. The microRNA (miRNA) expression in several EBV-infected AGS gastric carcinoma cell lines was determined. Infected cells expressed the viral BamHI A rightward transcript (BART) miRNAs at high levels and had consistently decreased expression of a small fraction of cellular miRNAs with specific downregulation of tumor suppressor miRNAs. These changes likely reflect expression of the viral noncoding RNAs and not latent protein expression.

Infection of Epstein-Barr virus in a gastric carcinoma cell line induces anchorage independence and global changes in gene expression.

Latent infection of EBV is linked to the development of multiple cancers that have distinct patterns of expression of viral proteins and microRNAs (miRNAs). In this study, we show that in vitro infection of a gastric epithelial cell line with EBV alters growth properties and induces growth in soft agar. The infected cells have high levels of expression of a large cluster of viral miRNAs, [the BamHI A rightward transcript (BART) miRNAs] and limited viral protein expression. Expression profile microarray analysis of this cell line revealed a large number of changes in cellular expression, with decreased expression of many genes. Inhibition of the trace-expressed levels of the viral oncoprotein, latent membrane protein 1, did not affect growth or alter the pattern of cellular expression. The expression changes are highly enriched for genes involved in cell motility and transformation pathways, suggesting these changes are important for the altered growth phenotype. Importantly, the transcripts decreased by microarray are significantly enriched in both experimentally and bioinformatically predicted BART miRNA targets. The absence of viral protein expression and the enrichment for viral miRNA targets in the modulated cell genes suggest that the BART miRNAs are major contributors to the transformed growth properties of the EBV-infected cells. The ability to affect cell growth through miRNA expression without viral protein expression would be a major factor in the development of cancer in individuals with functional immune systems.

The role of miRNAs and EBV BARTs in NPC.

The BamHI A rightward transcripts are a set of alternatively splicing transcripts produced by Epstein-Barr Virus that are highly expressed in nasopharyngeal carcinoma. These transcripts contain several open reading frames as well as precursors for twenty-two miRNAs. Although the putative proteins corresponding to these open reading frames have not been detected, several studies have identified properties that are interesting and potentially significant with respect to cellular transformation. The miRNAs, however, are very abundant in all nasopharyngeal carcinomas and several potentially significant functions have been identified for some of the miRNAs. This article will focus on the nature of this complicated set of transcripts and the evidence that they contribute to the development of nasopharyngeal carcinoma.

The Epstein-Barr Virus BART microRNAs target the pro-apoptotic protein Bim.

In Epstein-Barr Virus infected epithelial cancers, the alternatively spliced BamHI A rightward transcripts (BARTs) are abundantly expressed and are the template for two large clusters of miRNAs. This study indicates that both of these clusters independently can inhibit apoptosis in response to etoposide in an epithelial cell line. The Bcl-2 interacting mediator of cell death (Bim) was identified using gene expression microarrays and bioinformatic analysis indicated multiple potential binding sites for several BART miRNAs in the Bim 3'UTR. Bim protein was reduced by Cluster I and the individual expression of several miRNAs, while mRNA levels were unaffected. In reporter assays, the Bim 3' untranslated region (UTR) was inhibited by both clusters but not by any individual miRNAs. These results are consistent with the BART miRNAs downregulating Bim post-transcriptionally in part through the 3'UTR and suggest that there are miRNA recognition sites within other areas of the Bim mRNA.

Human tumor virus utilizes exosomes for intercellular communication.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is expressed in multiple human malignancies and has potent effects on cell growth. It has been detected in exosomes and shown to inhibit immune function. Exosomes are small secreted cellular vesicles that contain proteins, mRNAs, and microRNAs (miRNAs). When produced by malignant cells, they can promote angiogenesis, cell proliferation, tumor-cell invasion, and immune evasion. In this study, exosomes released from nasopharyngeal carcinoma (NPC) cells harboring latent EBV were shown to contain LMP1, signal transduction molecules, and virus-encoded miRNAs. Exposure to these NPC exosomes activated the ERK and AKT signaling pathways in the recipient cells. Interestingly, NPC exosomes also contained viral miRNAs, several of which were enriched in comparison with their intracellular levels. LMP1 induces expression of the EGF receptor in an EBV-negative epithelial cell line, and exosomes produced by these cells also contain high levels of EGF receptor in exosomes. These findings suggest that the effects of EBV and LMP1 on cellular expression also modulate exosome content and properties. The exosomes may manipulate the tumor microenvironment to influence the growth of neighboring cells through the intercellular transfer of LMP1, signaling molecules, and viral miRNAs.

Susceptibility of Arcobacter butzleri to human blood serum / Susceptibilidade de Arcobacter butzleri ao soro humano

A susceptibilidade de 10 amostras de Arcobacter butzleri ao soro humano foi estudada. A maior atividade bactericida foi encontrada no soro humano normal, com taxas de sobrevivência bacterianainversamente proporcionais à diluição do soro. As maiores taxas de sobrevivência foram obtidas com o soro inativado pelo calor. As taxas de sobrevivência decresceram com a adição de soro fresco ao inativado. O soro com valores reduzidos de gamaglobulinas e valores normais de complemento mostrou ativo efeito bactericida. Os resultados demonstraram que A. butzleri é altamente susceptível ao efeito bactericida do soro humano, sugerindo que pode ser capaz de ativar diretamente o complemento pela via alternativa.

Epstein-Barr virus BART microRNAs are produced from a large intron prior to splicing.

Latent Epstein-Barr virus (EBV) infection is associated with several lymphoproliferative disorders, including posttransplant lymphoma, Hodgkin's disease, and Burkitt's lymphoma, as well as nasopharyngeal carcinoma (NPC). Twenty-nine microRNAs (miRNAs) have been identified that are transcribed during latent infection from three clusters in the EBV genome. Two of the three clusters of miRNAs are made from the BamHI A rightward transcripts (BARTs), a set of alternatively spliced transcripts that are highly abundant in NPC but have not been shown to produce a detectable protein. This study indicates that while the BART miRNAs are located in the first four introns of the transcripts, processing of the pre-miRNAs from the primary transcript occurs prior to completion of the splicing reaction. Additionally, production of the BART miRNAs correlates with accumulation of a spliced mRNA in which exon 1 is joined directly to exon 3, suggesting that this form of the transcript may favor production of miRNAs. Sequence variations and processing of pre-miRNAs to the mature form also may account for various differences in miRNA abundance. Importantly, residual intronic pieces that result from processing of the pre-miRNAs were detected in the nucleus. The predicted structures of these pieces suggest there is a bias or temporal pattern to the production of the individual pre-miRNAs. These findings indicate that multiple factors contribute to the production of the BART miRNAs and to the apparent differences in abundance between the individual miRNAs of the cluster.

Nucleocytoplasmic trafficking of G2/M regulators in yeast.

Nucleocytoplasmic shuttling is prevalent among many cell cycle regulators controlling the G2/M transition. Shuttling of cyclin/cyclin-dependent kinase (CDK) complexes is thought to provide access to substrates stably located in either compartment. Because cyclin/CDK shuttles between cellular compartments, an upstream regulator that is fixed in one compartment could in principle affect the entire cyclin/CDK pool. Alternatively, the regulators themselves may need to shuttle to effectively regulate their moving target. Here, we identify localization motifs in the budding yeast Swe1p (Wee1) and Mih1p (Cdc25) cell cycle regulators. Replacement of endogenous Swe1p or Mih1p with mutants impaired in nuclear import or export revealed that the nuclear pools of Swe1p and Mih1p were more effective in CDK regulation than were the cytoplasmic pools. Nevertheless, shuttling of cyclin/CDK complexes was sufficiently rapid to coordinate nuclear and cytoplasmic events even when Swe1p or Mih1p were restricted to one compartment. Additionally, we found that Swe1p nuclear export was important for its degradation. Because Swe1p degradation is regulated by cytoskeletal stress, shuttling of Swe1p between nucleus and cytoplasm serves to couple cytoplasmic stress to nuclear cyclin/CDK inhibition.

Differential susceptibility of yeast S and M phase CDK complexes to inhibitory tyrosine phosphorylation.

BACKGROUND: Several checkpoint pathways employ Wee1-mediated inhibitory tyrosine phosphorylation of cyclin-dependent kinases (CDKs) to restrain cell-cycle progression. Whereas in vertebrates this strategy can delay both DNA replication and mitosis, in yeast cells only mitosis is delayed. This is particularly surprising because yeasts, unlike vertebrates, employ a single family of cyclins (B type) and the same CDK to promote both S phase and mitosis. The G2-specific arrest could be explained in two fundamentally different ways: tyrosine phosphorylation of cyclin/CDK complexes could leave sufficient residual activity to promote S phase, or S phase-promoting cyclin/CDK complexes could somehow be protected from checkpoint-induced tyrosine phosphorylation. RESULTS: We demonstrate that in Saccharomyces cerevisiae, several cyclin/CDK complexes are protected from inhibitory tyrosine phosphorylation, allowing Clb5,6p to promote DNA replication and Clb3,4p to promote spindle assembly, even under checkpoint-inducing conditions that block nuclear division. In vivo, S phase-promoting Clb5p/Cdc28p complexes were phosphorylated more slowly and dephosphorylated more effectively than were mitosis-promoting Clb2p/Cdc28p complexes. Moreover, we show that the CDK inhibitor (CKI) Sic1p protects bound Clb5p/Cdc28p complexes from tyrosine phosphorylation, allowing the accumulation of unphosphorylated complexes that are unleashed when Sic1p is degraded to promote S phase. The vertebrate CKI p27(Kip1) similarly protects Cyclin A/Cdk2 complexes from Wee1, suggesting that the antagonism between CKIs and Wee1 is evolutionarily conserved. CONCLUSIONS: In yeast cells, the combination of CKI binding and preferential phosphorylation/dephosphorylation of different B cyclin/CDK complexes renders S phase progression immune from checkpoints acting via CDK tyrosine phosphorylation.