[Clinical diagnosis and treatment of multiple pulmonary nodules].

CT screening has markedly reduced the lung cancer mortality in high-risk population and increased the detection of early-stage pulmonary neoplasms, including multiple pulmonary nodules, especially those with a ground-glass appearance on CT. Multiple primary lung cancer (MPLC) constitutes a specific subtype of lung cancer with indolent biological behaviors, which is predominantly early-stage adenocarcinoma. Although MPLC progresses slowly with rare lymphatic metastasis, existence of synchronous lesions and distributed location of these nodules still pose difficulty for the management of such patients. One single operation is usually insufficient to eradicate all neoplastic lesions, whereas repeated surgical procedures bring about another dilemma: whether clinical benefits of surgical treatment outweigh loss of pulmonary function following multiple operations. Therefore, despite the anxiety for treatment among MPLC patients, whether and how to treat the patient should be assessed meticulously. Currently there is a heated discussion upon the timing of clinical intervention, operation mode and the application of local therapy in MPLC. Based on clinical experience of our multiple disciplinary team, we have summarized and commented on the evaluation, surgical treatment, non-surgical local treatment, targeted therapy and immunotherapy of MPLC in this article to provide further insight into this field.

Circle RNA circCSPP1 promotes human osteosarcoma cell proliferation and increases glucose metabolism by suppressing miR-200c maturation.

INTRODUCTION: MiR-200c plays a central role in glucose metabolism in cancer cells. However, its upstream regulators in this process are unknown. CircRNA CSPP1 (circCSPP1) was predicted to bind to premature miR-200c, an oncogenic miRNA. Therefore, we explored their interaction in osteosarcoma (OS). METHODS: Differential circCSPP1 and miR-200c expression in OS was analyzed using RT-qPCR. Glucose metabolism was analyzed by glucose uptake assay. Subcellular circCSPP1 location in OS cells was detected using cellular fractionation assay. The direct interaction between circCSPP1 and miR-200c was explored using RNA-RNA pull-down assay. The role of circCSPP1 in miR-200c maturation was investigated by analyzing both mature and premature miR-200c levels in OS cells with circCSPP1 overexpression. RESULTS: CircCSPP1 and premature miR-200c levels were increased while mature miR-200c level was decreased in OS. CircCSPP1 was detected in both the nuclear and cytoplasm fractions of OS cells. CircCSPP1 directly interacted with premature miR-200c. CircCSPP1 overexpression increased premature miR-200c level, glucose uptake, and cell proliferation, but decreased mature miR-200c level. MiR-200c overexpression suppressed the role of circCSPP1 in OS cells. CONCLUSIONS: CircCSPP1 promotes OS cell proliferation and increases glucose metabolism by suppressing miR-200c maturation.

[Joint association of sedentary behavior and physical activity on anxiety tendency among occupational population in China].

OBJECTIVE: To investigate the status and influencing factors of anxiety tendency among occupational population in China and to examine the joint association between sedentary behavior and physical activity with anxiety tendency. METHODS: The data were from the 2021 Asia Best Workplace (Chinese mainland) program. The Generalized Anxiety Tendency scale was used to assess employees' anxiety status, and Logistic regression was used to analyze the factors influencing anxiety tendency and calculate the odds ratio (OR) within different groups. The OR of sitting for each sitting-physical activity (PA) combination group and within PA strata were calculated to explore the joint association. RESULTS: A total of 11 903 workers with an average age of 32.9 years were included in this study. Among them, 3 562 workers had anxiety tendency (29.9%) and the prevalence of those under 40 years old (30.6%) was significantly higher than the other age group (26.7%). 41.0% of the respondents had the moderated to vigorous physical activity. Their average daily sitting time was 9.4 h, and the percentage of those who exceeded 8 h sitting reached 73.9% in the past week. The analysis of Logistic regression showed that smoking (OR=1.24, 95%CI: 1.23-1.39), longer sedentary time and lower physical activity level were risk factors for anxiety tendency, and longer average daily sleep time (OR=0.56, 95%CI: 0.51-0.61) was a protective factor. The joint association analysis and stratified analysis of physical activity and sedentary behavior with anxiety tendency showed that increased sedentary time combined with decreased physical activity intensity was significantly associated with increased risk of anxiety tendency (range of OR: 1.64-3.14). The threshold for sedentary time in total as a risk factor for anxiety tendency gradually decreased as physical activity intensity increased. CONCLUSION: The anxiety tendency and sedentary behavior among the occupational population should recieve more attention. Lack of physical activity and sedentary behavior are both risk factors for anxiety tendency, and strengthening the intensity of physical activity can attenuate the harmful effects of sedentary behavior on anxiety tendency.

[Analysis of relationship between distribution of pathogenic microorganisms in olfactory cleft and olfactory disorders among patients with upper respiratory inflammation during the prevention and control of COVID-19].

Objective: To explore the relationship between pathogens in the olfactory cleft area and olfactory disorders in patients with upper respiratory inflammation (URI) during the prevention and control of 2019 novel coronavirus disease (COVID-19). Methods: A total of 234 URI patients including acute upper respiratory infection, chronic rhinosinusitis (CRS), allergic rhinitis (AR) were continuously selected from September 2020 to March 2021 in Beijing Anzhen Hospital and 98 healthy adults were enrolled as controls. The secretions from the olfactory cleft of all subjects were collected with nasal swabs under nasal endoscopy. Multiple real-time fluorescent quantitative polymerase chain reaction detection method was used to detect nucleic acids of 33 types of respiratory pathogenic microorganism. Sniffin' Sticks olfactory test was performed on all patients with URI. URI patients with olfactory dysfunction were followed up for 9 (8, 10) months (M (Q1, Q3)). SPSS 20.0 software was used for statistical analysis. Results: Among the 98 controls, 9 (9.18%) were positive for pathogenic microorganisms, including 1 (1.02%) rhinovirus, 1 (1.02%) parainfluenza virus type 3, 3 (3.06%) enterovirus, 1 (1.02%) staphylococcus aureus and 3 (3.06%) Moraxella catarrhalis. Among the 234 URI patients, 111 (47.44%) had olfactory disorders and 123 (52.56%) had normal sense of smell. In the olfactory disorder group (111 cases), 38 cases (34.23%) were positive for pathogenic microorganisms, and 4 cases (3.60%) were mixed infection, including 11 cases of rhinovirus (9.91%), 5 cases of coronavirus 229E (4.50%), 2 cases of coronavirus OC43/NL63 (1.80%), 3 cases of parainfluenza virus type 1 (2.70%), 2 cases of enterovirus (1.80%), 1 case of influenza B virus type BV (0.90%), 11 cases of Staphylococcus aureus (9.91%), 7 cases of Moraxella catarrhalis (6.31%), and 1 case of Klebsiella pneumoniae (0.90%). In the normal smell group (123 cases), 18 cases (14.63%) were positive for pathogenic microorganisms, and 1 case (0.81%) was mixed infection, including 3 cases of rhinovirus (2.44%), 4 cases of coronavirus 229E (3.25%), 1 case of Influenza virus type 3 (0.81%), 3 cases of enterovirus (2.44%), 3 cases of Staphylococcus aureus (2.44%), 4 cases of Moraxella catarrhalis (3.25%), and 1 case of Klebsiella pneumoniae (0.81%). Univariate analysis between the two groups found that there were significant differences in the detection rate of pathogenic microorganisms, rhinovirus and Staphylococcus aureus between the groups (all P<0.05). The detection rate of parainfluenza virus type 1, Staphylococcus aureus, and rhinovirus were different between the patients with olfactory disorder and normal olfactory function in the three subgroups of acute upper respiratory tract infection, CRS and AR, respectively (χ2 value was 3.88, 4.53 and 4.73, respectively, all P<0.05). During the follow-up period, among the 111 patients with olfactory disorder, 71 (63.96%) patients' olfactory function returned to normal, 32 (28.83%) patients' olfactory function improved but not completely returned to normal, 8 (7.21%) patients' olfactory function did not improve. Conclusions: During the prevention and control of COVID-19, rhinovirus or Staphylococcus aureus infection or colonization of URI patients is closely related to olfactory disorders. Parainfluenza virus type 1 infection can cause relatively persistent olfactory disorders in patients with acute upper respiratory tract infection. Staphylococcus aureus and rhinovirus colonization are related to the occurrence of olfactory dysfunction in CRS and AR patients respectively.

[Analysis of factors affecting olfactory disfunctions in patients with chronic rhinosinusitis with nasal polyps].

Objective: To analyze the factors affecting olfactory disfunctions in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Methods: This was a retrospective analysis. Eighty-eight patients with CRSwNP who underwent endoscopic sinus surgery in Beijing Anzhen Hospital from 2014 to 2018 were enrolled, including 22 males and 66 females, with the age of (48.1±11.3) years old(Mean±SD). Sniffin' Sticks olfactory test, Lund-Mackay score and modified sinus CT olfactory cleft score, nasal resistance and acoustic reflex examination, blood routine and blood biochemistry test, serum specific IgE test were performed before surgery and nasal polyps of all patients were collected for eosinophil count during surgery. According to bilateral total TDI score, the patients were divided into normal olfactory function group and olfactory disfunction group. The clinical baseline data were compared between the two groups. According to the results of single factor analysis, factors which were significant different between the two groups and clinically useful indicators were further included in the multivariate Logistic regression model analysis, then a model predicting olfactory disfunction in patients with CRSwNP was initially established. P<0.05 was considered statistically significant. Results: Among 88 patients with CRSwNP, 32 (36.4%) patients were with normal olfaction and 56 (63.6%) patients were with olfactory disfunction, including 40 (45.5%) of hyposmia and 16 (18.2%) of anosmia. Tissue eosinophil count, blood eosinophil percentage and blood urea concentration had significant difference between the two groups (12.7[2.0, 52.3]/HP (M[P(25), P(75)]) vs 38.6[16.2, 87.0]/HP, 2.75[1.60, 4.80]% vs 4.35[2.50, 6.60]%, (5.56±1.15) mmol/L vs (4.98±1.33) mmol/L, all P<0.05). Modified sinus CT olfactory cleft score and Lund-Mackay score except for ostiomeatal complex score were statistically significant between the two groups (all P<0.05). Multivariate Logistic regression analysis showed that the bilateral and total olfactory cleft score and blood urea concentration were statistically significant, in addition, the bilateral and total olfactory cleft score was a risk factor (OR=2.108, 95%CI: 1.407-3.159, P<0.001) and blood urea within a certain concentration was a protective factor (OR=0.461, 95%CI: 0.240-0.884, P=0.020). Further studies found that the area under the ROC curve of the model with tissue eosinophil count, blood eosinophil percentage, bilateral and total olfactory cleft score, total inspiratory volume and blood urea concentration was 0.888 (P<0.01), which had good predictive value for olfactory disorders in CRSwNP. Conclusions: The modified sinus CT olfactory cleft score is closely related to the olfactory disorders in patients with CRSwNP. A certain degree of elevated blood urea concentration may have a protective effect on the olfactory function of patients with CRSwNP.

[Effect of endoscopic sinus surgery on olfactory function in patients with chronic rhinosinusitis with nasal polyps].

Objective:To evaluate the effect of surgery on olfactory function in patients with chronic rhinosinusitis with nasal polyps（CRSwNP） by subjective and objective olfactory tests. Method:This was a retrospective study. Forty patients with CRSwNP who underwent endoscopic sinus surgery（ESS） from 2015 to 2017 in Beijing Anzhen Hospital were enrolled. Postoperative time was 6-46 months. The patients were followed up in October 2018 and examined using Sniffin' sticks olfactory test and olfactory event-related potentials （oERP）. The polyps collected during surgery were performed to eosinophil count and percentage calculation. They were divided into eosinophilic CRSwNP（ECRS） and non-eosinophilic CRSwNP（NECRS）. The subjective and objective olfactory functions between the two groups were compared before and after surgery respectively and the paired T test was performed between the postoperative and the preoperative Sniffin' sticks olfactory test. Covariance analysis was used to adjust the effect of different postoperative time on postoperative olfactory recovery. Result:There were 21（52.5%） ECRS and 19（47.5%） NECRS patients of the 40 patients with nasal polyps. There was statistical difference in the posterior ethmoid score and the posterior olfactory cleft score of CT. According to the criterion of total TDI increased more than 5.5, olfactory function in 21（52.5%） patients had improved. In addition, there was a significant improvement in olfactory function in ECRS group either in unilateral T/TDI or bilateral T/D/TDI, but only unilateral T/TDI increased in NECRS group. There was no significant difference in Sniffin' sticks olfactory test between the two groups, but there was a statistically significant difference in the latency of oERP after surgery. Conclusion:ESS could improve olfactory function in patients with CRSwNP by more than 50%. Bilateral olfactory improvement in ECRS was better than that in NECRS, but olfactory function in postoperative ECRS was still lower than that in NECRS. oERP can more objectively and accurately reflect the severity of olfactory disorders associated with eosinophilic inflammation.

[Correlation of tissue eosinophil count and modified CT olfactory cleft score and olfactory function in patients with nasal polyps].

Objective:To investigate the effect of tissue eosinophil count on olfactory function in patients with chronic sinusitis with nasal polyps（CRSwNP）. Method:We prospectively selected 59 patients with CRSwNP. All the patients were not treated with oral or topical glucocorticoids for at least 1 month at the time of enrollment. All the nasal polyps were taken under local anesthesia in the outpatient department for eosinophil count and percentage calculation. The patients were divided into eosinophilic CRSwNP（ECRS group） and non-eosinophilic CRSwNP（non-ECRS group）. Baseline data was compared between the two groups. Spearman correlation analysis was performed on tissue eosinophil count and Lund-Mackay score, modified olfactory cleft scores, Sniffin' Sticks olfactory test, olfactory evoked potential peak latency and amplitude respectively. Result:Thirty-five of 59 patients with CRSwNP completed olfactory evoked potential test, from whom we collected stable waveforms. The ECRS group had a higher olfactory cleft area CT score, ethmoid sinus CT score and blood eosinophil count and percentage; there was a significant difference between the ECRS group and the non-ECRS group in single and bilateral odor test threshold（T value）（P=0.017）. There was no significant correlation between tissue eosinophil count and subjective olfactory VAS score（P>0.05）. Tissue eosinophil count was related to the bilateral T value in Sniffin' Sticks olfactory test（r=-0.322, P=0.013）, anterior and posterior olfactory cleft area scores（r=0.431 and 0.415, respectively, P=0.001） and olfactory evoked potential N1 latency（r=0.504, P=0.001） and P2 latency（r=0.374, P=0.020）, but not related to Lund-Mackay scores. In addition, there was a significant correlation between the unilateral T score in Sniffin' Sticks test and the unilateral olfactory evoked potential N1 latency（r=-0.505, P=0.002）. Conclusion:The increase of tissue eosinophilia was closely related to olfactory disfunctions in CRSwNP. It could be reflected by modified CT olfactory cleft score, Sniffin' Sticks olfactory test T value and oERP peak latency. In addition, T value was negatively consistent with latency of oERP N1 peak.

Correlations of MC4R and MSH2 expression with obesity in colon cancer patients.

OBJECTIVE: To investigate the correlations of MC4R and MSH2 with adult obesity, a total of 46 patients with early-stage colon cancer were treated in our hospital between February 2008 and February 2009 and were enrolled. PATIENTS AND METHODS: Venous blood was regularly drawn from subjects of the observation group and 48 healthy subjects for 6 years. Expression levels of MC4R and MSH2 genes were tested using quantitative polymerase chain reaction analyses, and the ensuing proteins were determined using enzyme-linked immunosorbent assays and Western blotting and immunohistochemical analyses. Finally, correlations with body mass index (BMI) and the presence of colon cancer were identified using multivariate analyses. RESULTS: Compared with the control group, MSH2 mRNA and protein expression increased significantly over time (p < 0.05) in patients with colon cancer. Moreover, MSH2 expression was correlated with colon cancer progression, and MC4R mRNA and protein expression increase concurrently in comparison with the control group (p < 0.05). Also, the mean BMI among patients with colon cancer was 30.8, whereas that among control subjects was only 21.4. These data indicate a relationship between BMI and colon cancer. CONCLUSIONS: Expression of MSH2 in patients with colon cancer may promote the expression of the obesity gene MC4R, potentially contributing to body weight gains.

[Phenotypic characteristics of LAP(+) CD4(+) T lymphocytes in colorectal cancer tissues].

OBJECTIVE: To analyze the phenotypic characteristics of LAP(+) CD4(+) T lymphocytes and investigate their molecular mechanisms in colorectal cancer (CRC) microenvironment. METHODS: Fifty colorectal cancer patients treated in our two hospitals from January 2014 to May 2014 were included in this study. Their tumor tissues and adjacent normal tissues, peripheral blood samples, and peripheral blood samples of 25 healthy donors (HD) were collected to isolate the lymphocytes. The different expressions of CCR7, CD45RA, Foxp3, CTLA-4, CCR4 and CCR5 in LAP(+) CD4(+) T and LAP(-)CD4(+) T lymphocytes were analyzed by flow cytometry (FCM). RESULTS: The FCM assay detected that the percentage of LAP(+) CD4(+) T cells in peripheral blood of the CRC patients were significantly higher than that of HD [(9.44±3.18)% versus (1.49±1.00)%, P<0.001]. In addition, significantly more LAP(+) CD4(+) T cells were also recruited into tumor tissue than those in the tumor-adjacent normal tissue [(11.76±3.74)% versus (3.87±1.64)%, P<0.001]. LAP(+) CD4(+) T cells in the tumor-adjacent normal tissue and peripheral blood of both HDs and CRC patients mainly displayed a central memory phenotype. However, effector memory lymphocytes were predominant in the tumor tissue.In the tumor tissue, the expression of Foxp3 in the LAP(+) CD4(+) T cells was (3.87±1.12)%, significantly lower than that in the LAP(-)CD4(+) T cells (16.70±2.61)%, (P<0.001); the expression of CTLA-4 in the LAP(+) CD4(+) T cells was (36.36±19.14)%, significantly higher than the (19.60±8.91)% in the LAP(-)CD4(+) T cells (P<0.001); the expression of CCR4 in the LAP(+) CD4(+) T cells was (37.72±11.14)%, significantly higher than the (30.06±9.14)% in the LAP(-)CD4(+) T cells (P<0.001); and the expression of CCR5 in the LAP(+) CD4(+) T cells was (18.86±7.10)%, significantly higher than the (13.92±3.31)% in the LAP(-)CD4(+) T cells (P<0.001). CONCLUSIONS: LAP(+) CD4(+) T cells with low expression of Foxp3 and high expressions of CTLA-4, CCR4 and CCR5 are tend to be enriched and accumulated in the tumor tissue. The unique phenotypic characteristics make these cells a distinct subset of lymphocytes, apparently different from the traditional CD4(+) CD25(+) Treg cells.

Application of retrograde dissection method for isolation of bone marrow cells from rat femurs and tibiae.

Currently, there is no practical and efficient method for the isolation of bone marrow cells (BMCs) from rat femurs and tibiae. Here, we attempted to develop a rapid, simple, effective, and non-contaminating method for the isolation of BMCs from rat femurs and tibiae. Rat femurs and tibiae were dissected from the ankle to the hip joint; subsequently, a three-step "locate-slide-twist" procedure was performed using scissors and forceps to remove the femurs and tibiae completely, from the surrounding musculature. The bones were flushed with phosphate-buffered saline to harvest BMCs. The femurs and tibiae were dissected in 1.8 ± 0.6 min, and the BMC suspension preparation time was 13.1 ± 2.3 min. The bone marrow cavities did not incur any fractures or injuries during the isolation. Culture of harvested BMCs for 72 h led to a significant increase in cell number from 4.4 ± 0.3 x 106 to 6.9 ± 0.7 x 10(6) (P < 0.01) with no significant decrease in viability (98.1 ± 0.6% vs 96.2 ± 1.1%; P > 0.05). Microscopic examination of the isolated BMCs after the 72-h incubation period revealed the no-microbial or muscle cell contamination. Furthermore, flow cytometry revealed that cultured BMCs (72-h culture) grew well. Here, we have reported a rapid, simple, effective, and non-contaminating method for the isolation of BMCs from rat femurs and tibiae by using retrograde dissection. This method can be used to harvest a large number of viable BMCs without the risk of contamination from muscle and connective tissues.

Molecular cloning, tissue expression pattern, and copy number variation of porcine SCUBE3.

The signal peptide CUB EGF-like domain-containing protein 3 (SCUBE3) gene is a member of SCUBE gene family and plays important roles in bone cell biology and the determination of limb bone length. In this study, the full-length transcript of porcine SCUBE3 was cloned using reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The full-length sequence of porcine SCUBE3 cDNA was 4131 base pairs and included 21 exons. The SCUBE3 gene contained a 2895-base pair open reading frame that encoded a peptide of 965 amino acids. Comparison of the deduced amino acid sequences of porcine SCUBE3 with those of human, mouse, zebrafish, and rat showed 96, 95, 73, and 95% identities, respectively. Porcine SCUBE3 mRNA expression levels were highest in the backfat, bone marrow, and cartilage tissues. Copy number variation was detected in porcine SCUBE3 and validated by real-time quantitative polymerase chain reaction. Different copy number variations were present in randomly selected individuals and may, therefore, be a good marker for identifying phenotypic traits. Our findings provide a basis for further investigation of the functions and regulatory mechanisms of SCUBE3 in pigs.

[Research of nasopharyngeal carcinoma and the applicationof epstein-barr virus immediate early protein].

Epstein-barr virus is closely related to the occurrence and development of nasopharyngeal carcinoma;EB virus immediate early protein Zta and Rta is the control factor for the virus to enter the replication and cleavage stage.At present,ELISA method is commonly used to detect serum Zta and Rta related antibodies in the clinical as one of the serological indicators for early nasopharyngeal cancer screening and the auxiliary diagnosis.

Association between polymorphisms in insulin-like growth factor-1 and risk of osteoporosis.

We conducted a case-control study to assess the relation-ship between rs35767, rs2288377, and rs5742612 insulin-like growth factor-1 (IGF-1) and osteoporosis risk in a Chinese female population. The genotypes of rs35767, rs2288377, and rs5742612 of IGF-1 were determined by polymerase chain reaction-restriction fragment length polymorphism. Patients with osteoporosis were more likely to have drinking and smoking habits and have lower bone mineral density in the L2-L4 vertebrae, femoral neck, and total hip. According to conditional regression analysis, individuals carrying the TT genotype of rs35767 had an increased risk of osteoporosis, with an adjusted odds ratio (95% confidence interval) of 2.29 (1.35-4.97). In conclusion, our results sug-gest that the TT genotype of IGF-I rs35767 was associated with an in-creased risk of osteoporosis, suggesting that this polymorphism can be used as a predictive factor for osteoporosis risk.

NO mediated increase of Fos protein and NMDA1A R mRNA expression in rat spinal cord during morphine withdrawal.

AIM: To investigate the effects of nitric oxide (NO) on activation of the rat spinal cord neurons during naloxone-precipitated morphine withdrawal. METHODS: Fos immunocytochemistry, NADPH-d histochemistry, Fos/NADPH-d double-labeling, intrathecal injection, antisense oligonucleotides (AS-ONs) techniques, and RT-PCR were used. RESULTS: Acute administration of naloxone and chronic administration of morphine did not change the expression of Fos protein and NADPH-d positive neurons, and there was no expression of Fos/NADPH-d double-labeled neurons in the spinal cord of rats. Morphine withdrawal increased the expression of Fos protein, NADPH-d positive, and Fos/NADPH-d double-labeled neurons, and they were observed in all the laminae of the rat spinal cord. Intrathecal injection of nNOS antisense oligonucleotides (nNOS-AS) inhibited the increase of Fos protein and NMDA(1A)R mRNA expression in the rat spinal cord during morphine withdrawal and decreased the scores of morphine withdrawal symptoms. The effect of nNOS-AS was greater than that of eNOS-AS. There was no effect in nNOS sense oligonucleotides (nNOS-S) group. CONCLUSION: NO mediated the increase of Fos protein and NMDA1A R mRNA expression in the rat spinal cord during morphine withdrawal.

Degenerative disorders caused by Bcl-2 deficiency prevented by loss of its BH3-only antagonist Bim.

Apoptosis is triggered when proapoptotic members of the Bcl-2 protein family bearing only the BH3 association domain bind to Bcl-2 or its homologs and block their antiapoptotic activity. To test whether loss of the BH3-only protein Bim could prevent the cellular attrition caused by Bcl-2 deficiency, we generated mice lacking both genes. Mice without Bcl-2 have a fragile lymphoid system, become runted, turn gray, and succumb to polycystic kidney disease. Concomitant absence of Bim prevented all these disorders. Indeed, loss of even one bim allele restored normal kidney development, growth, and health. These results demonstrate that Bim levels set the threshold for initiation of apoptosis in several tissues and suggest that degenerative diseases might be alleviated by blocking BH3-only proteins.

Gene structure alternative splicing, and chromosomal localization of pro-apoptotic Bcl-2 relative Bim.

Bim is a proapoptotic protein of the Bcl-2 family that shares only the short BH3 domain with other members. It has three isoforms, apparently produced by alternative splicing. The demonstration that Bim is essential for certain apoptotic responses and to prevent overproduction of hematopoietic cells suggests that it may be a tumor suppressor. We have, therefore, investigated the organization of the mouse Bim gene, delineating its promoter and splicing, and positioned the gene on both mouse and human chromosomes. Bim has six exons, but the third is a facultative intron that is spliced out in the mRNAs for the smaller isoforms (BimL and BimS), but not that encoding the largest isoform (BimEL). The 0.8-kb region 5' to exon 1, which contains a TATA-less promoter and binding sites for several transcription factors, can drive expression of a reporter gene. Mouse Bim localizes to the distal third of Chromosome (Chr) 2, near the F-G boundary, and its human counterpart to Chr 2q12 or q13. Deletions of these bands have been reported in ten tumors (eight hematopoietic), reinforcing the possibility that Bim is a tumor suppressor. These findings should help to clarify the regulation of Bim expression and to assess whether mutations involving Bim contribute to neoplastic and other diseases.

[The effect of nociception on expression of NADPH-d and fos protein at supraspinal level].

AIM: To study the distribution of NADPH-diaphorase positive neurons in periaqueductal gray matter(PAG) and dorsal raphe nucleus (DR), and the correlation between nitric oxide and peripheral nociception at supraspinal level. METHODS: 20 Sprague-Dawley rats were randomly divided into two groups. In experimental group (n = 10), formalin (5%, 0.1 ml) was injected subcutaneously into the plantar surface of the unilateral hindpaw, and in control group (n = 10), normal saline solution was used alike. The rats were perfused after 2 hours, and the midbrain were removed. Frozen seriate transverse sections were divided into three sets, all of which were processed for NADPH-d histochemistry. Subsequently, the first set that contained NADPH-diaphorase positive neurons were stained with neutral red, and the other two sets were processed for c-fos immunohistochemistry. RESULTS: NADPH-diaphorase positive neurons were densely distributed in CGLD, CGLV and DR. NADPH-d, Fos and NADPH/Fos double-labeled neurons appeared in CGLD, CGLV and DR after injection of formalin in the rat hindpaw. CONCLUSION: At supraspinal level NO may play an important role in the modulation of nociception in PAG and DR.

Gamma-radiation-induced growth arrest and apoptosis in p53-null lymphoma cells is accompanied by modest transcriptional changes in many genes.

Damage to DNA produces cell cycle arrest, apoptosis, or both. The response in cells with p53 tumor suppressor function involves transcriptional changes, but whether that holds for cells lacking active p53, as in most tumors, is not known. Better characterization of the DNA damage response in tumors lacking p53 function is relevant to cytotoxic therapy. We have explored whether gamma-irradiated p53-null mouse T lymphoma cells undergo marked changes in transcription. Their arrest in G2/M prior to apoptosis required transcription. Transcripts whose abundance altered on irradiation were sought by subtractive hybridization, and 1010 candidate clones from two oppositely enriched cDNA populations were sequenced. Hybridization revealed small (<3-fold) increases or decreases in the transcripts of more than 15 genes, including some implicated in cell cycle control (e.g., BTG, Bap1) or apoptosis (e.g., STAT1, calpain), but no marked changes like those associated with other forms of T-cell death. Moreover, the expression of some critical apoptosis regulators, such as Bcl-2 family members, did not change. Hence, the G2/M arrest and apoptosis in the irradiated p53-null lymphoma appears to involve modest expression changes for many genes, but post-transcriptional alterations may be more critical.