[Bioactive compounds of Jingfang Granules against SARS-CoV-2 virus proteases 3CLpro and PLpro].

OBJECTIVE: Jingfang Granules have been recommended for the prevention and treatment of corona virus disease 2019 (COVID-19). Through chemical analysis and bioactivity evaluation, this study aims to elucidate the potential effective components of Jingfang Granules. METHODS: The inhibitory acti-vities of Jingfang Granules extract against 3-chymotrypsin-like protease (3CLpro), papain like protease (PLpro), spike protein receptor-binding domain (S-RBD) and human cyclooxygenase-2 (COX-2) were evaluated using enzyme assay. The antitussive effects were evaluated using the classical ammonia-induced cough model. The chemical constituents of Jingfang Granules were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometry (LC/MS). The 3CLpro and PLpro inhibitory activities of the major compounds were determined by enzyme assay, molecular docking, and site-directed mutagenesis. RESULTS: Jingfang Granules exhibited 3CLpro and PLpro inhibitory activities, as well as COX-2 inhibitory and antitussive activities. By investigating the MS/MS behaviors of reference standards, a total of fifty-six compounds were characterized in Jingfang Granules. Sixteen of them were unambiguously identified by comparing with reference standards. The contents of the 16 major compounds were also determined, and their total contents were 2 498.8 µg/g. Naringin, nodakenin and neohesperidin were three dominating compounds in Jingfang Granules, and their contents were 688.8, 596.4 and 578.7 µg/g, respectively. In addition, neohesperidin and naringin exhibited PLpro inhibitory activities, and the inhibition rates at 8 µmol/L were 53.5% and 46.1%, respectively. Prim-O-glucosylcimifugin showed significant inhibitory activities against 3CLpro and PLpro, and the inhibitory rates at 8 µmol/L were 76.8% and 78.2%, respectively. Molecular docking indicated that hydrogen bonds could be formed between prim-O-glucosylcimifugin and amino acid residues H163, E166, Q192, T190 of 3CLpro (binding energy, -7.7 kcal/mol) and K157, D164, R166, E167, T301 of PLpro(-7.3 kcal/mol), respectively. Site-directed mutagenesis indicated amino acid residue K157 was a key active site for the interaction between prim-O-glucosylcimifugin and PLpro. CONCLUSION: Prim-O-glucosylcimifugin, neohesperidin, and naringin as the major compounds from Jingfang Granules could inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus proteases 3CLpro and PLpro. The results are valuable for rational clinical use of Jingfang Granules.

[Advances in research of protein kinase A in parasites: a review].

Protein kinase A (PKA) is an important intracellular substance that regulates substance metabolism and biological functions, which exerts a wide range of biological effects through phosphorylation of specific serine/threonine residues in specific proteins. PKA plays an important role in the cAMP signaling pathway, and is involved in various life activities of parasites. Therefore, investigating the role of PKA in the life activities of parasites may provide insights into the development of novel anti-parasitic targets. The review mainly describes the structure and function of PKA and its role in life activities of parasites.

PAG1 stimulates proliferation and metastasis of nasopharyngeal carcinoma through downregulating PTEN.

OBJECTIVE: We aim to uncover the expression pattern and biological functions of PAG1 in the progression of nasopharyngeal carcinoma (NPC). PATIENTS AND METHODS: PAG1 levels in 28 paired NPC tissues and paracancerous tissues were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Then, the potential influences of PAG1 on proliferative, migratory and invasive abilities of SUNE2 and CNE2 cells were assessed by cell counting kit-8 (CCK-8) and transwell assay, respectively. Next, the interaction between PAG1 and its direct target gene of phosphate and tension homology deleted on chromosome ten (PTEN) was verified by Dual-Luciferase reporter gene assay. At last, rescue experiments were conducted to uncover the role of PAG1/PTEN axis in the malignant progression of NPC. RESULTS: PAG1 was highly expressed in NPC tissues and cell lines. Knockdown of PAG1 blocked NPC cells to proliferate, migrate, and invade. Dual-Luciferase reporter gene assay indicated the binding relationship between PAG1 and PTEN. In addition, both mRNA and protein levels of PTEN were negatively regulated by PAG1 in NPC cells. Notably, PTEN was responsible for PAG1-regulated malignant progression of NPC. CONCLUSIONS: PAG1 is upregulated in NPC tissues and cells and stimulates the proliferative and metastatic abilities in NPC by targeting PTEN, thus aggravating the malignant progression of NPC.

[Effect of overexpression of integrin ß2 on clinical prognosis in triple negative breast cancer].

Objective: To investigate the clinical significance of the expression of integrin ß2 (ITGB2) and tumor infiltrating dendritic cells CD80 and CD86 in triple negative breast cancer (TNBC). Methods: The expression levels of ITGB2 and MHC-1 in MDA-MB-231 cells and TNBC tissues were detected by Western bolt and RT-PCR, and the expressions of ITGB2, CD80 and CD86 in TNBC tissues were detected by immunohistochemistry. Results: 1.The expression level of ITGB2 in TNBC cell line (1.67±0.38) was higher than that in MCF-10A cell line (1.06±0.38), and the expression level of MHC-1 molecules in TNBC cell line (0.64±0.20) was lower than that in MCF-10A cell line (1.02±0.21). The difference was statistically significant (P=0.036 and 0.020, respectively). 2.The expression level of ITGB2 in TNBC tissues (1.55±0.47) was significantly higher than that in normal breast tissues (1.04±0.31). 3. Immunohistochemistry showed that the expression level of ITGB2 in TNBC tissues (3.31±2.29) was significantly higher than that in paracancerous tissues (1.20±1.19). Combined with clinicopathological data, it was suggested that the expression of ITGB2 was correlated with tumor stage (P=0.038), histological grade (P=0.022), lymph node metastasis (P=0.019), ki67 expression (P=0.000) and total survival (P=0.003). 4. Immunohistochemistry showed that the expression of CD80 and CD86 in TNBC tissues (3.07±1.57, 3.93±1.64) was significantly lower than those in paracancerous tissues (4.63±2.92 vs 5.23±2.85) (P=0.032 and 0.027). Conclusion: The high expression of ITGB2 in triple negative breast cancer affects the prognosis of patients.

MiR-507 inhibits the growth and invasion of trophoblasts by targeting CAMK4.

OBJECTIVE: To elucidate the potential influences of miR-507 and CAMK4 on the progression of preeclampsia (PE). PATIENTS AND METHODS: Placental tissues were collected from 24 PE pregnancies and 24 healthy pregnancies. The relative levels of miR-507 and CAMK4 in placental tissues were detected. In addition, expressions of apoptosis-associated genes in collected tissues were examined by both quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The influences of miR-507 and CAMK4 on proliferative and migratory abilities in HTR-8/SVneo cells were assessed by CCK-8 and transwell assay, respectively. The target relationship between miR-507 and CAMK4 was detected by Luciferase assay. RESULTS: MiR-507 was upregulated in placental tissues collected from PE pregnancies. Overexpression of miR-507 suppressed proliferative and migratory abilities, and stimulated apoptosis in HTR-8/SVneo cells. CAMK4 was the target gene of miR-507, which was downregulated in placental tissues collected from PE pregnancies and negatively correlated to miR-507 level. The knockdown of CAMK4 suppressed proliferative and migratory abilities, and stimulated apoptosis in HTR-8/SVneo cells, and these trends were abolished by silence of miR-507. CONCLUSIONS: Highly expressed miR-507 in PE pregnancies inhibits proliferative and migratory potentials, and induces apoptosis in trophoblasts by targeting CAMK4.

[Effect of high expression of type â collagen α1 chain protein on the prognosis of triple negative breast cancer].

Objective: To investigate the expression of type Ⅰ collagen α1 chain protein (COL1A1) in triple negative breast cancer (TNBC), and its relationship with clinicopathological features and prognosis of TNBC. Methods: A total of 148 TNBC specimens were collected from the Affiliated Cancer Hospital of Xinjiang Medical University from 2013 to 2015. The mRNA expression of COL1A1 was detected by fluorescence quantitative RT-PCR and the protein expression of COL1A1 was detected by Western blot. The expression of COL1A1 and α-smooth muscle actin (α-SMA) in TNBC were detected by immunohistochemistry. The relationship between the expression of COL1A1 and clinicopathological parameters and prognosis of TNBC patients was analyzed. Results: The mRNA and protein expression of COL1A1 in MDA-MB-231 cells were 1.696±0.486 and 0.550±0.088, respectively, which were higher than those in MCF-10A cells (1.020±0.231 and 0.350±0.083, P=0.032, P=0.046). The mRNA and protein expression of COL1A1 in TNBC tissues were 1.632 ±0.598 and 0.733 ±0.068, respectively, which were higher than those in paracancerous tissues (1.041±0.316 and 0.612±0.016, P=0.003, P=0.039). The high expression rates of COL1A1 and α-SMA in TNBC tissues were 35.8% and 56.7% respectively, which were higher than those in paracancerous tissues (16.7% and 30.0%, P=0.041, P=0.037). The expression of COL1A1 was correlated with tumor size, TNM stage, lymph node metastasis, vascular invasion and α-SMA expression (all P<0.05). The median survival time in COL1A1 high expression group was 64 months, which was lower than that in low expression group (73 months, P<0.05). Multivariate analysis of Cox proportional hazard regression model showed that COL1A1 expression was an independent influencing factor for the survival of TNBC patients (HR=3.952, P=0.004). Conclusion: The high expression of COL1A1 in TNBC is an independent prognostic factor of TNBC patients.

Pyruvate kinase genes in grass carp Ctenopharyngodon idella: molecular characterization, expression patterns, and effects of dietary carbohydrate levels.

To explore features of carbohydrate metabolism and evolution of carbohydrate metabolism-associated genes in herbivorous fishes, the open reading frames (ORF) of PKL, PKMa, and PKMb genes of grass carp (Ctenopharyngodon idella) were obtained, encoding 538, 528, and 532 amino acids, respectively. Comparative genomic analysis showed that adjacent PK genes were highly conserved between fish and mammals. Gene expression profiles were quite different between the three PK genes in tissues and at developmental stages. PKL, PKMa, and PKMb had the highest expression levels in the liver, heart, and muscle, respectively. During embryogenesis, high expression levels of PKMa and PKMb were detected in unfertilized and fertilized eggs. Following a non-expression period, PKMa and PKMb exhibited high expressions again after the hatching stage. In contrast, PKL transcripts could not be detected in early developmental stages, and expression levels continued to increase from the hatching stage to 144 h post hatching. After the 8-week feeding trial with 18%, 30%, and 42% dietary carbohydrate levels, the concentrations of glucose and insulin in serum, pyruvate kinase enzymes, and gene expression levels in brain, muscle, and liver tissues all increased with the increase in carbohydrate levels in the diets. Furthermore, high carbohydrate levels (30% and 42% carbohydrate diets) had a greater effect on grass carp growth. This indicated that PKL, PKMa, and PKMb genes were not only very important in catalytic enzymes, which can be up-regulated by high carbohydrate dietary conditions, but also exhibited a complex and detailed division of labor in different tissues and developmental stages.

Inhibition effect of exosomes-mediated Let-7a on the development and metastasis of triple negative breast cancer by down-regulating the expression of c-Myc.

OBJECTIVE: Triple negative breast cancer is typically characterized by high malignancy, easy recurrence and metastasis, and poor prognosis. However, effective treatment for triple negative breast cancer has not yet been identified. Our research explores the underlying mechanisms of exosomes in transporting Let-7a and regulating c-Myc gene and their roles in the development of triple negative breast cancer, and to provide new ideas for targeted therapy of triple negative breast cancer. PATIENTS AND METHODS: Based on previous studies that focused on the roles of c-Myc and Let-7a in the development of triple negative breast cancer, triple negative breast cancer cell lines have been constructed by c-Myc knockout and overexpression of Let-7a, respectively, to evaluate the effects of c-Myc, and Let-7a, as well as exosomes transmitting Let-7a on the development of triple negative breast cancer. RESULTS: Let-7a, which is mediated by exosomes, inhibited proliferation, migration, and invasion of MDA-MB-231 cells by binding on the 3'UTR region of the c-Myc gene and silencing of the c-Myc expression. CONCLUSIONS: Our study revealed the role of c-Myc, Let-7a, and exosomes in the development of triple negative breast cancer, which lay the theoretical foundation for further usage of exosomes to construct tumor killing vectors and for exploring specific targets for triple negative breast cancer.

Tissue distribution and early developmental expression patterns of aldolase A, B, and C in grass carp Ctenopharyngodon idellus.

Aldolase is a key enzyme involved in glycolysis, gluconeogenesis, and the pentose phosphate pathway. To establish the expression patterns of all three aldolase isozyme genes in different tissues and during early embryogenesis in lower vertebrates, as well as to explore the functional differences between these three isozymes, the grass carp was selected as a model owing to its relatively high glucose-metabolizing capability. Based on the cDNA sequences of the aldolase A, B, and C genes, the expression patterns of these three isozymes were analyzed in different tissues and during early embryogenesis using quantitative real-time polymerase chain reaction (qRT-PCR). Sequence analysis of cDNAs indicated that aldolase A, B, and C (GenBank accession numbers: KM192250, KM192251, and KM192252) consist of 364, 364, and 363 amino acids, respectively. The qRT-PCR results showed that the expression levels of aldolase A, B, and C were highest in the muscle, liver, and brain, respectively. Aldolase A and C exhibited similar expression patterns during embryogenesis, with high levels observed in unfertilized and fertilized eggs and at the blastocyst stage, followed by a decline and then increase after organogenesis. In contrast, aldolase B transcript was not detected during the unfertilized egg stage, and appeared only from gastrulation; the expression increased markedly during the feeding period (72 h after hatching), at which point the level was higher than those of aldolase A and C. These data suggest that the glucose content of grass carp starter feed should be adjusted according to the metabolic activity of aldolase B.

[Association between the time of sedentary behaviors and risk of type 2 diabetes].

Objective: To explore the relationship between the time of sedentary behaviors and the risk of type 2 diabetes mellitus (T2DM) among rural adults in Henan Province. Methods: A total of 1 227 adults from Houzhai village in Zhengzhou City and Qiaomiao village in Jiaozuo City in Henan Province were randomly selected to participate in the survey; each of them finished a questionnaire and accepted physical examination and test of biochemical indicators. The participants were divided into case and control groups according to whether suffering from T2DM. A Pearson linear correlation and linear regression model analysis were used to investigate the relevance between the time of sedentary behaviors and fasting plasma glucose (FPG); meanwhile, a multi factor logistic regression model was used to analyze the relationship between the time of sedentary behaviors and T2DM. Results: The average FPG level among the participants was (5.4±2.5) mmol/L, and the average time of sedentary behaviors was (234.5±156.5) min per day. The prevalence rate of T2DM in subgroups whose time of sedentary behaviors were separately 0-<2.5, 2.5-<4.5, 4.5-<6.5 and ≥6.5 h/day were 15.8% (72/457), 19.3% (73/379), 16.7% (35/210) and 20.4% (37/181), respectively. There was a linear regression relationship between the time of sedentary behaviors(x) and FPG(y), the regression equation was: y=5.081+0.001x (t=3.01, P=0.003). Logistic regression model analysis showed that participants whose time of sedentary behaviors ≥6.5 h/day had significantly higher risk of T2DM than those whose time of sedentary behaviors between 0-2.4 h/day (OR=1.77, 95%CI: 1.11-2.81) after age and gender adjusted. However, the associations only exist in males and adults ≥50 years old according to sex and age stratification. It showed that participants with sedentary time ≥6.5 h/day had significantly higher risk of T2DM than those with sedentary time between 0-<2.5 h/d, with the corresponding OR (95%CI) at 2.34 (1.21-4.52) and 2.22 (1.19-4.16), respectively. Conclusion: The prolonged sedentary time is a risk factor of type 2 diabetes. After stratification by gender and age, the correlation only found in males and adults aged ≥50 years old.

Swimming intervention mitigates HFD-induced obesity of rats through PGC-1α-irisin pathway.

OBJECTIVE: Irisin, a newly discovered myokine, can drive the browning of white adipocytes to control body weight or mitigate obesity progression through regulating energy metabolism. However, the underlying mechanisms or specific signal pathways of exercise-induced irisin on the management of obesity are still unclear. MATERIALS AND METHODS: Totally 30 rats were subjected to high fat diet (HFD) feeding for 8 weeks to establish the rat model with obesity successfully. HFD-induced obese model rats were provided with 8 weeks swimming intervention at moderate intensity for exploring the treatment of obesity through exercise intervention. In addition, another 15 rats were subjected to HFD feeding coupled with total 16 weeks swimming intervention at a moderate intensity from the beginning of the experiment, which was used for exploring the prevention of obesity through exercise intervention. Blood and gastrocnemius samples were harvested from obese rats after swimming intervention to explore its specific signal pathways through ELISA analysis and Western blotting. RESULTS: HFD feeding of rats for 8 weeks could lead to the obesity due to the disorders of lipid metabolism. Totally 8 weeks swimming intervention at moderate intensity for rats with obesity could obviously alleviate the progression of obesity and 16 weeks swimming intervention from the beginning of the experiment could significantly inhibit the development of obesity. Meanwhile, swimming intervention could result in an increased phosphorylation of AMPK and up-regulation of irisin and PGC-1α as the biomarkers of energy metabolism. CONCLUSION: Exercise intervention can activate PGC-1α-dependent irisin to induce the browning of white adipocytes, thus inhibiting or alleviating the occurrence and development of obesity.

A prospective evaluation of minimal residual disease as risk stratification for CCLG-ALL-2008 treatment protocol in pediatric B precursor acute lymphoblastic leukemia.

OBJECTIVE: The aim of this prospective study was to evaluate the cut-off value of minimal residual disease (MRD) in predicting the efficacy of CCLG-ALL-2008 or CCLG-2008 treatment protocol on pediatric B-precursor ALL (BP-ALL). PATIENTS AND METHODS: Three hundred and seventy-nine Chinese pediatric BP-ALL were enrolled in this study between Dec 2008 and Sep 2013 in two stratified cohorts. One hundred and fifty-three patients enrolled between Dec 2008 and Oct 2010 as the first cohort, and 196 patients enrolled from Nov 2010 to Sep 2013 as the second cohort. Clinical and biological characteristics and 5 years EFS, RFS, and OS were analyzed. RESULTS: Patients with E2A-PBX1 showed a favorable treatment response with a lower minimal residual disease (MRD) level (< 10-4) at the time point 1 (TP1, p = 0.039) and the highest proportion of the 5-year EFS, RFS, and OS. A high level of MRD was associated with high WBC counts, increased age, BCR-ABL1 fusion gene, MLL rearrangements and adverse karyotypes. In comparison with the first cohort, the second cohort with the MRD assay incorporated prospectively, the standard risk (SR) and the intermediate risk (IR) patients showed a better RFS, EFS, and OS while the high-risk (HR) patients displayed worse RFS, EFS, and OS than those of the first cohort, respectively. Patients with MRD level at either 10-4 or 10-3 showed a similar OS at TP1 or TP2, and patients with MRD level above 10-2 had the worst OS. CONCLUSIONS: This study indicated that the levels of MRD to be an adequate guide in risk-adapted treatment under the CCLG-ALL-2008 protocol and can be adapted to the future development of advanced clinical protocols.

Characterization of a TIR-NBS-LRR gene associated with downy mildew resistance in grape.

Grapevine downy mildew, caused by Plasmopara viticola, is a devastating disease that results in considerable economic losses as well as environmental damage through the repeated application of fungicides. The nucleotide-binding site leucine-rich repeat gene family functions in plant immunoactivity against various pathogens and pests. In this study, the 5' and 3' ends of the resistance gene homology fragment RGA5 were obtained by rapid amplification of cDNA ends. The 4282-base pair full-length cDNA was obtained using gene-specific primers, and the corresponding 1335-amino acid protein sequence contained characteristic nucleotide-binding site leucine-rich repeat domains of plant resistance proteins, including the toll-interleukin receptor type region. Expression of RGA5 during P. viticola infection and abiotic stress was investigated using quantitative real-time polymerase chain reaction. The results showed that treatment with P. viticola and 4 abiotic stimuli (salicyclic acid, methyl-jasmonate, abscisic acid, H2O2) significantly induced RGA5 within 12 days of inoculation. Therefore, RGA5 may play a critical role in protecting grapevines against P. viticola via signaling pathways involving these molecules.

A rat model designed for the continuous intraarterial infusion of cyclosporine.

BACKGROUND: Limb allotransplantation is not a life-saving treatment. However, large doses of immunosuppressive agents are needed. There is an urgent need to increase the selectivity and targeting of drugs. METHODS: We designed a rat model for intraarterial infusion of cyclosporine (CSA) based on the hindlimb replanted model to simulate the limb allotransplantation. To investigate whether intraartery infusion could improve the drug's distribution, we infused CSA 4.0 mg/kg per day continuously into either the superficial epigastric artery (IA group) or superficial epigastric vein (IV group) of Lewis rats. RESULTS: On day 10, CSA concentrations were measured in skin, muscle, and bone tissues of hindlimb. Samples were taken from different parts of the bilateral hindlimbs in the IA group and right hindlimb only in the IV group. Tissue concentrations of the perfusion side were much higher in IA group. Systemic concentrations of IA group were higher than IV group. CONCLUSIONS: These results warrant further research in our next limb allotransplantation model.

Transcriptome analysis of the grass carp (Ctenopharyngodon idella) using 454 pyrosequencing methodology for gene and marker discovery.

Total RNA isolated from the brain, muscle, liver, gonad, and intestinal tissues of grass carp was pooled to construct cDNA libraries. Using 454 pyrosequencing, a total of 738,604 high-quality reads were generated from the normalized cDNAs of the pooled individuals. Clustering and assembly of these reads produced a set of 37,086 all-unigene sequences after BLAST. Of these, 24,010 (64.74%) were annotated in the National Center for Biotechnology Information database, and 3715 simple sequence repeats and 2008 single nucleotide polymorphisms were identified in this EST dataset as potential molecular markers. This study provides new data for functional genomic and biological research on grass carp. The markers identified in this study will enrich the currently used molecular markers and facilitate marker-assisted selection in grass carp-breeding programs. These results also demonstrate that transcriptomic analysis based on 454 sequencing is a powerful tool for gene discovery and molecular marker development in non-model species.

A 66-bp deletion in growth hormone releasing hormone gene 5'-flanking region with largemouth bass recessive embryonic lethal.

Growth hormone releasing hormone (GHRH) regulates the secretion of growth hormone (GH) in the pituitary gland. A 66-bp deletion (c.-923_-858del) was detected in the 5'-flanking sequence of the largemouth bass (Micropterus salmoides) GHRH gene. In two cultured random populations of adult individuals (A: n = 170 and B: n = 150), the genotype ratios of +/+:+/- were 2.5:1 and 2.8:1 respectively. Only one -/- fish was detected. A Largemouth bass family was constructed with two heterozygous individuals (+/-) as parents. The genotype ratio of +/+:+/-:-/- in the filial generation embryos was 1:1.6:0.1 at the neurula and 1:2:0 at hatched larvae stages. This indicated that the 66-bp deletion was a recessive lethal site and that homozygous individuals (-/-) died off in embryonic development. The growth traits (body weight, body length and body depth) were measured, and the GHRH mRNA expression levels in brain tissue were detected using real-time PCR. The effects of genotype (+/-) on growth traits and GHRH mRNA expression were not significant. Although the cause of death was not clear, the results hint that the 66-bp deletion site in GHRH 5'-flanking sequence significantly affects the livability in largemouth bass embryonic development.

Isolation and characterization of a Vibrio vulnificus mutant deficient in both extracellular metalloprotease and cytolysin.

We isolated a Vibrio vulnificus mutant that was deficient in both metalloprotease and cytolysin by allelic exchange. The virulence of this mutant in mice and its cytotoxicity for HEp-2 cells were comparable to those of the wild-type strain, indicating that neither factor was essential for these properties. The cytolysin, but not the protease, seemed to be important for causing damage in the alimentary tract of the mice.

Differences in gene expression in muscle-invasive bladder cancer: a comparison of Italian and American patients.

OBJECTIVE: To seek differences in gene expression in the primary muscle-invasive bladder cancers of two cohorts of patients having different survival rates. An Italian group treated by transurethral resection of the bladder tumor (TURBT) and neo-adjuvant chemotherapy using methotrexate, vinblastine, adriamycin and cisplatin (M-VAC) followed by TURBT, partial cystectomy or radical cystectomy (75% 3-year survival) was compared to an American cohort treated by radical cystectomy (51% 3-year survival). METHODS: Immunohistochemistry was used to examine the protein expression levels of three genes that act at the G1/S cell cycle checkpoint, p53, p21/waf-1/cip1 (a downstream effector gene in the p53 pathway) and Rb, plus a major inhibitor of apoptosis, Bcl-2. RESULTS: For the bladder cancers of the Italian patient cohort, there was a significantly higher rate of p53 immunopositivity (93 vs. 63%, p = 0.002) and a significantly lower rate of Rb loss (25 vs. 54%, p = 0.009). In bivariate analysis, 72% of Italian tumors were immunopositive for both p53 and p21 (p53+/p21+) vs. 49% for the American tumors. The subset of Italian patients with p53+/p21+ tumors were more frequently disease-free (stage pT0) following chemotherapy and were less likely to fail therapy than those with p53+/p21- tumors (p = 0.0357). Loss of Rb staining was associated with a decreased 5-year survival in the Italian, but not in the American patients. CONCLUSIONS: (1) Significant differences in the expression of the p53, p21 and Rb genes were found between the 2 groups of patients. (2) Italian patients with p53+/p21+ tumors had significantly lower recurrence rates after TURBT and chemotherapy than those having p53+/p21- tumors. (3) Absence of p21 immunopositivity in the Italian tumors may identify alterations in the p53 pathway that predict poor outcome.