[Phosphatase and tensin homolog deleted on chromosome ten deficiency sensitizes tumor cells to lithium chloride treatment].

Objective: To identify the therapeutic efficacy of lithium chloride (LiCl) on phosphatase and tensin homolog deleted on chromosome ten (PTEN)-deficient tumors. Methods: First, the Catalogue of Somatic Mutations in Cancer for mutation spectrum of human endometrial carcinoma samples was analyzed. Second, the relationship between PTEN abundance and LiCl inhibition of endometrial cancer cell lines using Pten(+/+) and Pten(-/-) mouse embryonic fibroblast (MEF) lines was investigated. Moreover, potential alterations of mammalian target of rapamycin (mTOR) signaling pathway after treatment with LiCl were checked.Last,LiCl's efficacy on PTEN null tumors was studied. Results: PTEN was mutated in 39% of endometrial carcinomas. LiCl preferentially inhibited the proliferation of PTEN-deficient endometrial carcinoma cells and MEFs. Furthermore, LiCl blocked PTEN-deficient tumor development. Mechanistically, LiCl down-regulated mTOR signaling. Conclusions: PTEN is the most frequently mutated gene in endometrial carcinoma.By targeting mTOR signaling pathway,LiCl is a promising regimen for the treatment of tumors with PTEN deficiency.

[Metabolomics analysis identifies serum glycerophospholipid expression: a comparison between men and women with asthma].

Objective: To determine whether asthma has a different metabolic glycerophospholipid profile in serum between women and men with asthma. Methods: Fifty-one outpatients with asthma (17 men, 34 women) were enrolled from Peking University Third Hospital from Jan 2015 to Dec 2015. Clinical data such as gender, age, body mass index, pulmonary function were recorded. Glycerophospholipid profile were measured in serum using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics analysis. Projections to latent structures-discriminate analysis (PLS-DA) was used to compare the differences of glycerophospholipid level between men and women. Receiver Operating Characteristic (ROC) Curve was established from men and women. Results: Significantly different glycerophospholipid level were confirmed quantitatively between women and men. Levels of phosphatidylethanolamine (PE), phosphatidylcholine acetal phospholipid (PCP), lysophosphatidyl ethanolamine (LPE), alkyl phosphatidylethanolamine [PE(O)] were significantly higher in women relative to men [PE36: 2, PCP32: 1, LPE18: 0, and PE(O40: 7) was 0.050 (0.037, 0.079) vs 0.043 (0.000, 0.071), 0.057 (0.035, 0.727) vs 0.034 (0.000, 0.057), 0.233 (0.129, 0.390) vs 0.126 (0.075, 0.212), 0.007(0.000, 0.041) vs 0.000 (0.000, 0.000), respectively, all P<0.05). Levels of lysophosphatidylcholine (LPC), lysophosphatidylserine (LPS), and lysophosphatidylphosphatidylcholine [LPC(O)] were significantly lower in women compared to men [Levels of LPS22: 6, LPS20: 4, and LPS18: 1 were 0.000(0.000, 0.003) vs 0.009(0.000, 0.012), 0.015(0.010, 0.026) vs 0.047(0.022, 0.081), 0.008(0.003, 0.179) vs 0.020(0.008, 0.040), respectively, all P<0.05]. Area Under ROC Curve (AUC) of LPS (LPS20: 4) was 0.814. Conclusions: Glycerophospholipid levels in serum are significantly different between women and men asthmatic patients. LPS may contribute to gender based differences in asthmatics.

[Airway inflammation and small airway wall remodeling in neutrophilic asthma].

OBJECTIVE: To investigate the distribution of airway inflammation phenotype in patients with bronchial asthma (asthma), and to analyze clinical characteristics, inflammatory cytokines, pulmonary small vessels remodeling and small airway wall remodeling in patients with neutrophilic asthma. METHODS: Sixty-three patients with asthma were enrolled from January 2015 to December 2015 in Peking University Third Hospital. Clinical data including gender, age, body mass index (BMI), pulmonary function tests (PFTs), asthma control test (ACT) were recorded. All the patients underwent sputum induction. The cellular composition of the sputum was evaluatedand the concentration of active MMP-9 in the sputum tested. Blood routine tests were done and the concentration of IgE, periostin, and TGF-beta1 levels were measured in serum by enzyme-linked immunosorbent assay (ELISA). Small airway wall remodeling was measured in computed tomography (CT) scans, as the luminal diameter, luminal area, wall thickness and wall area % adjusted by body surface area (BSA) at the end of the 6th generation airway, in which the inner diameter was less than 2 mm. Small vascular alterations were measured by cross-sectional area (CSA), and the total vessel CSA < 5 mm2 was calculated using imaging software. RESULTS: The distributions of airway inflammatory phenotypes of the asthmatic patients were as follows: neutrophilic asthma (34.9%, 22/63), eosinophilic asthma (34.9%, 22/63), mixed granulocytic asthma (23.8%, 15/63), and paucigranulocytic asthma (6.3%, 4/63). The neutrophilic subtype patients had a significantly higher active MMP-9 level in sputum compared with the eosinophilic phenotypepatuents, as 179.1 (74.3, 395.5) vs. 50.5 (9.7, 225.8), P<0.05. Sputum neutrophil count was negatively correlated with FEV1%pred (r=-0.304,P<0.05), and positively correlated with active MMP-9 level in sputum (r=-0.304, P<0.05), and positive correlation trend with airway wall thickness (r=0.533, P=0.06). There was a significantly negative correlation of active MMP-9 level in sputum with FEV1%pred (r=-0.281, P<0.05), in positive correlation with small airway wall area (%)(r=0.612, P<0.05), and inpositive correlation trend with airway wall thickness (r=0.612, P=0.06). Neutrophils count in peripheral blood was positively correlated with neutrophil counts in sputum. CONCLUSION: Neutrophil count in airway is related to lung function in asthmatic patients. Neutrophils may accelerate small airway wall remodeling through the release of active MMP-9. Neutrophil count in peripheral blood is related to neutrophils count in sputum, which may be used as a substitute for evaluating inflammatory phenotype.

Effects of guanidinoacetic acid on growth performance, creatine and energy metabolism, and carcass characteristics in growing-finishing pigs.

The aim of this study was to investigate the effects of dietary supplementation with guanidinoacetic acid (GAA) on the growth performance, creatine and energy metabolism, and carcass characteristics in growing-finishing pigs. In Exp. 1, Duroc × Landrace × Yorkshire pigs (n = 180, 33.61 ± 3.91 kg average BW) were blocked by weight and sex, and allotted to 5 treatments with 6 replicates (3 gilts and 3 barrows per replicate pen). Diets were corn-soybean meal-basal diets supplemented with 0, 300, 600, 900, and 1,200 mg/kg of GAA and fed to the pigs for 98 d. From days 1 to 98, G:F increased (linear, P < 0.05) with increasing addition of dietary GAA. Using a broken-line model, the optimum level of dietary GAA was 300 mg/kg during the overall experimental period (days 1 to 98) to maximize G:F. Hot carcass weight, carcass length, and lean percentage showed a tendency to increase (quadratic, 0.05 < P < 0.10) with increasing addition of dietary GAA. On day 98, serum GAA and liver creatine tended to increase (linear, P = 0.10, 0.07) as dietary GAA increased. In addition, serum ATP on day 98 increased linearly (linear, P < 0.01), and muscle ATP and adenosine monophosphate increased quadratically (quadratic, P = 0.05) with incremental GAA supplementation. In Exp. 2, Duroc × Landrace × Yorkshire pigs (n = 180, 53.19 ± 5.63 kg average BW) were blocked by weight and sex, and allotted to 5 treatments with 6 replicates (3 gilts and 3 barrows per replicate pen). Diets were corn-soybean meal-basal diets supplemented with 0, 150, 300, 600, and 1,200 mg/kg of GAA for 35 d. As dietary GAA increased, final BW, ADG, and G:F increased quadratically (quadratic, P < 0.01), and 300 mg/kg of GAA maximized ADG and final BW (P < 0.05).The results indicate that dietary GAA could increase the creatine and ATP load in the tissues of pigs and accordingly improve growth performance. Dietary supplementation with 300 mg/kg of GAA was suitable to maximize the growth performance of growing-finishing pigs.

Surface nanotopography-induced favorable modulation of bioactivity and osteoconductive potential of anodized 3D printed Ti-6Al-4V alloy mesh structure.

The objective of the study described here is to fundamentally elucidate the biological response of 3D printed Ti-6Al-4V alloy mesh structures that were surface modified to introduce titania nanotubes with an average pore size of ∼80 nm via an electrochemical anodization process from the perspective of enhancing bioactivity. The bioactivity of the mesh structures were analyzed through immersion test in simulated body fluid, which confirmed the nucleation and growth of fine globular nanoscale apatite on the nanoporous titania-modified (anodized) mesh structure surface, and agglomerated apatite with fine flakes of apatite crystals on as-fabricated mesh structure surface, that were rich in calcium and phosphorous. The cellular activity of bioactive anodized mesh structure was explored in terms of cell-material interactions involving adhesion, proliferation, synthesis of extracellular and intracellular proteins, differentiation, and mineralization. Cells adhered with a sheet-like morphology on as-fabricated mesh structure, whereas, on anodized mesh structure, numerous filopodia-like cellular extensions interacting with nanotube pores were observed. The formation of a bioactive nanoscale apatite, cell-nanotube interactions as imaged via electron microscopy, higher expression of proteins (actin, vinculin, fibronectin, and alkaline phosphatase (ALP)), and calcium content points toward the determining role of anodized mesh structure in modulating osteoblasts functions. The unique combination of nanoporous bioactive titania and interconnected porous architecture of anodized titanium alloy mesh structure provided a multimodal roughness surface ranging from nano to micro to macroscale, which helps in attaining strong primary and secondary fixation of the implant device along with the pathway for supply of nutrients and oxygen to cells and tissue.

The effect of saikosaponin D on doxorubicin pharmacokinetics and its MDR reversal in MCF-7/adr cell xenografts.

OBJECTIVE: Multidrug resistance (MDR) is a major cause of chemotherapy failure in the treatment of cancer patients. This study aimed to determine whether saikosaponin D (SSd) can enhance the efficacy of the anticancer drug doxorubicin (Dox) both in vitro and in vivo and whether SSd can alter Dox pharmacokinetics in the serum of mice. MATERIALS AND METHODS: MCF-7/adr cells were used to investigate the effect of SSd on reversing MDR. Cell viability was assessed by MTT assay. Pharmacokinetic tests were used to evaluate the effects of SSd on serum Dox disposition. An MCF-7/adr cell xenograft model was established to investigate the effect of SSd on reversing MDR in vivo. Tumor growth and weights were measured. Immunohistochemistry staining was used to detect the expression of P-gp (P-glycoprotein), an ATP-dependent efflux pump that mediates MDR in xenograft tumor tissues. RESULTS: SSd could effectively reverse MDR in MCF-7/adr cells in vitro and had no cytotoxic effects on human amniotic epithelial cells (hAEC). There was no significant difference between the Dox pharmacokinetic parameters obtained in the mice that received Dox only and Dox combined with SSd, indicating that SSd did not alter the pharmacokinetic profiles of Dox. Furthermore, the combination of Dox and SSd had a stronger anticancer effect than Dox alone or SSd alone by inhibiting tumor growth and P-gp expression. CONCLUSIONS: Our results suggest that SSd could effectively reverse MDR in vitro and in vivo and could be a potential MDR reversal agent for P-gp-mediated MDR in breast cancer therapy.

Histone acetyltransferase PCAF accelerates apoptosis by repressing a GLI1/BCL2/BAX axis in hepatocellular carcinoma.

P300/CBP-associated factor (PCAF), a histone acetyltransferase (HAT), has been found to regulate numerous cell signaling pathways controlling cell fate by acetylating both histone and non-histone proteins. We previously reported that PCAF upregulates cell apoptosis by inactivating Serine/Threonine Protein Kinase 1 (AKT1) signaling and consequently inhibits hepatocellular carcinoma (HCC) cell growth. Here, we show that PCAF can directly acetylate cytoplasmic GLI1 protein at lysine 518, preventing its nuclear translocation and promoter occupancy, and consequently suppressing Hedgehog (Hh) signaling in HCC. Further, our results show that GLI1 can increase Bcl-2 expression and downregulate BAX. Interestingly, forced expression of PCAF reduced Bcl-2 expression, upregulated BAX and repressed cell apoptosis. Further, we provide evidence that knockdown of GLI1 abrogates the inhibitory effect of PCAF on the growth of HCC in vitro. PCAF was also found to sensitize HCC cells to 5-fluorouracil (5-FU) treatment by regulating GLI1/Bcl-2/BAX axis-dependent apoptosis. In vivo experiments also confirmed the regulatory effect of PCAF on the GLI1/Bcl-2/BAX axis and its synergistic antitumor effects with 5-FU. Gene expression microarray studies showed that PCAF was downregulated in HCC tissues compared with adjacent liver tissues and that PCAF expression was significantly associated with longer overall survival and recurrence-free survival after surgery. Together, these results show that PCAF can induce cell apoptosis by modulating a GLI1/Bcl-2/BAX axis that in turn suppresses HCC progression, and suggest that 5-FU may exert a stronger anti-tumor effect in patients with PCAF expression in HCC tumors.

Compounds from Chinese herbal medicines as reversal agents for P-glycoprotein-mediated multidrug resistance in tumours.

Multidrug resistance (MDR) is a major obstacle to successful cancer chemotherapy. One of the main underlying mechanisms of this resistance is the over-expression of P-glycoprotein (P-gp), an ATP-dependent transmembrane transporter protein encoded by the MDR1 gene. P-gp might transport anti-cancer drugs out of cancer cells and decrease effective intracellular drug concentrations. An effective approach to overcome MDR is to inhibit the function of P-gp or its expression on the surface of cancer cells. Thus, application of MDR reversal agents can be seen as a potentially important means by which to overcome the clinical drug resistance of tumour cells and improve the efficacy of chemotherapy. Recently, research efforts worldwide have focused on reversal mechanisms for MDR and on the identification of reversal agents. Chinese scholars have performed a great deal of exploratory work by screening for efficacy and low toxicity in drug resistance reversal compounds. These compounds may provide more lead compounds with greater activity, leading to the development of more effective therapies for MDR cancer cells. In this review, the function and efficiency of novel compounds derived from traditional Chinese medicines are described.

Near-zero anomalous dispersion Ge11.5As24Se64.5 glass nanowires for correlated photon pair generation: design and analysis.

We show that highly nonlinear chalcogenide glass nanowire waveguides with near-zero anomalous dispersion should be capable of generating correlated photon-pairs by spontaneous four-wave mixing at frequencies detuned by over 17 THz from the pump where Raman noise is absent. In this region we predict a photon pair correlation of >100, a figure of merit >10 and brightness of ~8×10(8) pairs/s over a bandwidth of >15 THz in nanowires with group velocity dispersion of <5 ps∙km(-1) nm(-1). We present designs for double-clad Ge(11.5)As(24)Se(64.5) glass nanowires with realistic tolerance to fabrication errors that achieve near-zero anomalous dispersion at a 1420 nm pump wavelength. This structure has a fabrication tolerance of 80-170 nm in the waveguide width and utilizes a SiO(2)/Al(2)O(3) layer deposited by atomic layer deposition to compensate the fabrication errors in the film thickness.

Rare structural variation of synapse and neurotransmission genes in autism.

Autism spectrum disorders (ASDs) comprise a constellation of highly heritable neuropsychiatric disorders. Genome-wide studies of autistic individuals have implicated numerous minor risk alleles but few common variants, suggesting a complex genetic model with many contributing loci. To assess commonality of biological function among rare risk alleles, we compared functional knowledge of genes overlapping inherited structural variants in idiopathic ASD subjects relative to healthy controls. In this study we show that biological processes associated with synapse function and neurotransmission are significantly enriched, with replication, in ASD subjects versus controls. Analysis of phenotypes observed for mouse models of copy-variant genes established significant and replicated enrichment of observable phenotypes consistent with ASD behaviors. Most functional terms retained significance after excluding previously reported ASD loci. These results implicate several new variants that involve synaptic function and glutamatergic signaling processes as important contributors of ASD pathophysiology and suggest a sizable pool of additional potential ASD risk loci.

Next-generation sequencing: the solution for high-resolution, unambiguous human leukocyte antigen typing.

Human leukocyte antigen (HLA) typing has been a challenge for more than 50 years. Current methods (Sanger sequencing, sequence-specific primers [SSP], sequence-specific oligonucleotide probes [SSOP]) continue to generate ambiguities that are time-consuming and expensive to resolve. However, next-generation sequencing (NGS) overcomes ambiguity through the combination of clonal amplification, which provides on-phase sequence and a high level of parallelism, whereby millions of sequencing reads are produced enabling an expansion of the HLA regions sequenced. We explored HLA typing using NGS through a three-step process. First, HLA-A, -B, -C, -DRB1, and -DQB1 were amplified with long-range PCR. Subsequently, amplicons were sequenced using the 454 GS-FLX platform. Finally, sequencing data were analyzed with Assign-NG software. In a single experiment, four individual samples and two mixtures were sequenced producing >75 Mb of sequence from >300,000 individual sequence reads (average length, 244 b). The reads were aligned and covered 100% of the regions amplified. Allele assignment was 100% concordant with the known HLA alleles of our samples. Our results suggest this method can be a useful tool for complete genomic characterization of new HLA alleles and for completion of sequence for existing, partially sequenced alleles. NGS can provide complete, unambiguous, high-resolution HLA typing; however, further evaluation is needed to explore the feasibility of its routine use.

Rare structural variants found in attention-deficit hyperactivity disorder are preferentially associated with neurodevelopmental genes.

Attention-deficit/hyperactivity disorder (ADHD) is a common and highly heritable disorder, but specific genetic factors underlying risk remain elusive. To assess the role of structural variation in ADHD, we identified 222 inherited copy number variations (CNVs) within 335 ADHD patients and their parents that were not detected in 2026 unrelated healthy individuals. Although no excess CNVs, either deletions or duplications, were found in the ADHD cohort relative to controls, the inherited rare CNV-associated gene set was significantly enriched for genes reported as candidates in studies of autism, schizophrenia and Tourette syndrome, including A2BP1, AUTS2, CNTNAP2 and IMMP2L. The ADHD CNV gene set was also significantly enriched for genes known to be important for psychological and neurological functions, including learning, behavior, synaptic transmission and central nervous system development. Four independent deletions were located within the protein tyrosine phosphatase gene, PTPRD, recently implicated as a candidate gene for restless legs syndrome, which frequently presents with ADHD. A deletion within the glutamate receptor gene, GRM5, was found in an affected parent and all three affected offspring whose ADHD phenotypes closely resembled those of the GRM5 null mouse. Together, these results suggest that rare inherited structural variations play an important role in ADHD development and indicate a set of putative candidate genes for further study in the etiology of ADHD.

Patients homozygous for the T435N mutation of succinyl-CoA:3-ketoacid CoA Transferase (SCOT) do not show permanent ketosis.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT; locus symbol OXCT; E.C. 2.8.3.5) is the main determinant of the ketolytic capacity of tissues. Hereditary SCOT deficiency causes episodic ketoacidosis. Permanent ketosis has been regarded as a pathognomonic feature of SCOT deficiency. There are three SCOT-deficient patients from a small region in Japan and they have not manifested permanent ketosis, even though their ketoacidotic crises were as severe as those of other SCOT-deficient patients. All three were homozygous for the T435N mutation. Transient expression analysis of wild-type and mutant cDNA showed that the T435N mutant retained significant residual SCOT activities (20% for that of the wild-type at 39.5 degrees C, 25% at 37 degrees C, and 50% at 30 degrees C). The difference of residual SCOT activities at these temperatures in expression analyses was due to differences in the level of the mutant protein. SCOT activity of the T435N protein was more vulnerable than the wild-type to heat treatment at 42 degrees C and 55 degrees C. These temperature-sensitive characteristics of the mutant protein may explain, in part, why the patients developed ketoacidotic crises during febrile illness. In SCOT-deficient patients retaining some residual activity, permanent ketosis may be absent.

Targeting of the yeast Ty5 retrotransposon to silent chromatin is mediated by interactions between integrase and Sir4p.

The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR and HML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity.

Preferential acetazolamide-induced vasodilation based on vessel size and organ: confirmation of peripheral vasodilation with use of colored microspheres.

When carbonic anhydrase activity decreases, the regional blood flow (rBF) in organs increases as hypercapnia develops. However, the effects of acetazolamide (AZ)-induced vasodilation have not been estimated with respect to vessel size and organs. The aim of this study was to determine the diameter of the capillaries in various organs that respond to inhibition of carbonic anhydrase activity by AZ. White rabbits were anesthetized with urethane and ketamine and infused with AZ. While the systolic blood pressure (SBP), pH, hemoglobin concentration, and base excess did not change, the partial pressure of arterial oxygen (PaO2) increased significantly and the partial pressure of arterial carbon dioxide (PaCO2) decreased significantly with AZ. The rBF was calculated by using 3 different sizes (15, 25, and 50 microm) of colored microspheres (CM). The rBF measured with 15 microm CM in the brain, kidneys, and liver increased in response to AZ, and the rBF in these organs was different with the different sizes of CM. However, the rBF calculated by using the different sizes of CM in the stomach and abdominal muscle did not change after the administration of AZ. The AZ-induced vasodilation occurred in all sizes of vessels in the liver, in the small and medium-sized vessels in kidneys, and in the larger capillaries in the brain.

Palladium catalysed 3-component cascade synthesis of bis(2-arylallyl) tertiary amines from aryl iodides, allene and primary amines.

A 3-component cascade synthesis of bis(2-arylallyl) tertiary amines from aryl iodide, allene and primary aliphatic amines is described; chiral amines give analogous products with no detectable racemisation; mixtures of two different aryl iodides can be utilised to give the mixed tertiary amines as the sole, or major, product; the reaction is sensitive to stereoelectronic effects which lead to mono(2-arylallyl) secondary amines.

Gene discovery using the maize genome database ZmDB.

Zea mays DataBase (ZmDB) is a repository and analysis tool for sequence, expression and phenotype data of the major crop plant maize. The data accessible in ZmDB are mostly generated in a large collaborative project of maize gene discovery, sequencing and phenotypic analysis using a transposon tagging strategy and expressed sequence tag (EST) sequencing. ESTs constitute most of the current content. Database search tools, convenient links to external databases, and novel sequence analysis programs for spliced alignment are provided and together serve as an efficient protocol for gene discovery by sequence inspection. ZmDB can be accessed at http://zmdb. iastate.edu. ZmDB also provides web-based ordering of materials generated in the project, including EST and genomic DNA clones, seeds of mutant plants and microarrays of amplified EST and genomic DNA sequences.

A single amino acid change in the yeast retrotransposon Ty5 abolishes targeting to silent chromatin.

Many retrotransposons and retroviruses are thought to select integration sites through interactions with specific chromosomal proteins. In yeast, the Ty5 retrotransposon integrates preferentially with regions bound by silent chromatin, namely the telomeres and the HMR and HML mating loci. A Ty5 mutant (M3) was identified with an approximately 20-fold decrease in targeted integration as measured by a plasmid-based targeting assay. Often chromosomal insertions generated by M3, none were located at the telomeres or silent mating loci. A single amino acid change at the boundary of integrase and reverse transcriptase is responsible for the mutant phenotype. We predict that this mutation lies within a targeting domain that mediates Ty5 target choice by interacting with a component of silent chromatin.

[Clinical analysis of surgical treatment for 86 patients with localized small-cell lung cancer].

To assess the value of surgical treatment followed by adjuvant chemotherapy for localized small-cell lung cancer (SCLC), the results of treatment in 86 SCLC patients since 1990 were reviewed. Of 86 patients, 19 patients received pneumonectomy, 53 lobectomy, 6 wedge resection and 8 segmental resection. Postoperative pathologic staging revealed 24 in stage I, 36 in stage II, and 26 in stage III. Postoperative adjuvant chemotherapy was given in 78 patients. Until the latest follow-up, 28 patients remained alive and 58 patients died. The 5-year survival rate of the 86 patients was 37%. The survival of patients in stage I was better than that of patients in stage II (P = 0.018) and stage III (P = 0.021), but the survival of patients in stage II was not significantly different from that of patients in stage III (P = 0.234). In summary, surgical treatment followed by chemotherapy improves significantly the survival of patients with localized SCLC. TNM staging is of prognostic importance.

CCAAT-box contributions to human thymidine kinase mRNA expression.

In order to examine the role of two inverted CCAAT boxes near the start of transcription of the human thymidine kinase (TK) gene, a series of constructs were prepared in which one or both CCAAT boxes were deleted or mutated. These altered promoters (1.2 kb of 5'-flanking sequence) were used to express a TK minigene containing the first two exons and introns followed by the remainder of the cDNA. RNA blots were prepared from stable cell lines of ts13 cells containing these constructs under three conditions: 1) serum deprived cells, 2) serum stimulated cells, and 3) cells that had been stimulated with serum, but were arrested in the G1 phase of the cell cycle by the temperature sensitive mutation carried by these cells. TK mRNA expression from each construct was suppressed by the temperature sensitive block to cell cycle progression. Measurement of protein expression from the various altered TK promoters indicated that both CCAAT boxes contribute to promoter strength. These experiments also suggested that the two CCAAT boxes were not equivalent and that the distal CCAAT could substitute for the proximal CCAAT, but the converse was not true.