Fusion and expansion of vitellogenin vesicles during Caenorhabditis elegans intestinal senescence.

Some of the most conspicuous aging phenotypes of C. elegans are related to post-reproductive production of vitellogenins (Vtg), which form yolk protein (YP) complexes after processing and lipid loading. Vtg/YP levels show huge increases with age, and inhibition of this extends lifespan, but how subcellular and organism-wide distribution of these proteins changes with age has not been systematically explored. Here, this has been done to understand how vitellogenesis promotes aging. The age-associated changes of intestinal vitellogenin vesicles (VVs), pseudocoelomic yolk patches (PYPs), and gonadal yolk organelles (YOs) have been characterized by immuno-electron microscopy. We find that from reproductive adult day 2 (AD 2) to post-reproductive AD 6 and AD 9, intestinal VVs expand from 0.2 to 3-4 µm in diameter or by >3000 times in volume, PYPs increase by >3 times in YP concentration and volume, while YOs in oocytes shrink slightly from 0.5 to 0.4 µm in diameter or by 49% in volume. In AD 6 and AD 9 worms, mislocalized YOs found in the hypodermis, uterine cells, and the somatic gonadal sheath can reach a size of 10 µm across in the former two tissues. This remarkable size increase of VVs and that of mislocalized YOs in post-reproductive worms are accompanied by extensive fusion between these Vtg/YP-containing vesicular structures in somatic cells. In contrast, no fusion is seen between YOs in oocytes. We propose that in addition to the continued production of Vtg, excessive fusion between VVs and mislocalized YOs in the soma worsen the aging pathologies seen in C. elegans.

Layer III pyramidal cells in the prefrontal cortex reveal morphological changes in subjects with depression, schizophrenia, and suicide.

Brodmann Area 46 (BA46) has long been regarded as a hotspot of disease pathology in individuals with schizophrenia (SCH) and major depressive disorder (MDD). Pyramidal neurons in layer III of the Brodmann Area 46 (BA46) project to other cortical regions and play a fundamental role in corticocortical and thalamocortical circuits. The AutoCUTS-LM pipeline was used to study the 3-dimensional structural morphology and spatial organization of pyramidal cells. Using quantitative light microscopy, we used stereology to calculate the entire volume of layer III in BA46 and the total number and density of pyramidal cells. Volume tensors estimated by the planar rotator quantified the volume, shape, and nucleus displacement of pyramidal cells. All of these assessments were carried out in four groups of subjects: controls (C, n = 10), SCH (n = 10), MDD (n = 8), and suicide subjects with a history of depression (SU, n = 11). SCH subjects had a significantly lower somal volume, total number, and density of pyramidal neurons when compared to C and tended to show a volume reduction in layer III of BA46. When comparing MDD subjects with C, the measured parameters were inclined to follow SCH, although there was only a significant reduction in pyramidal total cell number. While no morphometric differences were observed between SU and MDD, SU had a significantly higher total number of pyramidal cells and nucleus displacement than SCH. Finally, no differences in the spatial organization of pyramidal cells were found among groups. These results suggest that despite significant morphological alterations in layer III of BA46, which may impair prefrontal connections in people with SCH and MDD, the spatial organization of pyramidal cells remains the same across the four groups and suggests no defects in neuronal migration. The increased understanding of pyramidal cell biology may provide the cellular basis for symptoms and neuroimaging observations in SCH and MDD patients.

DeepContact: High-throughput quantification of membrane contact sites based on electron microscopy imaging.

Membrane contact site (MCS)-mediated organelle interactions play essential roles in the cell. Quantitative analysis of MCSs reveals vital clues for cellular responses under various physiological and pathological conditions. However, an efficient tool is lacking. Here, we developed DeepContact, a deep-learning protocol for optimizing organelle segmentation and contact analysis based on label-free EM. DeepContact presents high efficiency and flexibility in interactive visualizations, accommodating new morphologies of organelles and recognizing contacts in versatile width ranges, which enables statistical analysis of various types of MCSs in multiple systems. DeepContact profiled previously unidentified coordinative rearrangements of MCS types in cultured cells with combined nutritional conditions. DeepContact also unveiled a subtle wave of ER-mitochondrial entanglement in Sertoli cells during the seminiferous epithelial cycle, indicating its potential in bridging MCS dynamics to physiological and pathological processes.

Dietary S. maltophilia induces supersized lipid droplets by enhancing lipogenesis and ER-LD contacts in C. elegans.

Dietary and symbiotic bacteria can exert powerful influence on metazoan lipid metabolism. Recent studies have emerged that microbiota have a role in animal obesity and related health disorders, but the mechanisms by which bacteria influence lipid storage in their host are unknown. To reduce the complexity of the relationship between gut microbiota and the host, Caenorhabditis elegans (C. elegans) has been chosen as a model organism to study interspecies interaction. Here, we demonstrate that feeding C. elegans with an opportunistic pathogenic bacterium Stenotrophomonas maltophilia (S. maltophilia) retards growth and promotes excessive neutral lipid storage. Gene expression analysis reveals that dietary S. maltophilia induces a lipogenic transcriptional response that includes the SREBP ortholog SBP-1, and fatty acid desaturases FAT-6 and FAT-7. Live imaging and ultrastructural analysis suggest that excess neutral lipid is stored in greatly expanded lipid droplets (LDs), as a result of enhanced endoplasmic reticulum (ER)-LD interaction. We also report that loss of function mutations in dpy-9 in C. elegans confers resistance to S. maltophilia. Dietary S. maltophilia induces supersized LDs by enhancing lipogenesis and ER-LD contacts in C. elegans. This work delineates a new model for understanding microbial regulation of metazoan physiology.

Cellular 3D-reconstruction and analysis in the human cerebral cortex using automatic serial sections.

Techniques involving three-dimensional (3D) tissue structure reconstruction and analysis provide a better understanding of changes in molecules and function. We have developed AutoCUTS-LM, an automated system that allows the latest advances in 3D tissue reconstruction and cellular analysis developments using light microscopy on various tissues, including archived tissue. The workflow in this paper involved advanced tissue sampling methods of the human cerebral cortex, an automated serial section collection system, digital tissue library, cell detection using convolution neural network, 3D cell reconstruction, and advanced analysis. Our results demonstrated the detailed structure of pyramidal cells (number, volume, diameter, sphericity and orientation) and their 3D spatial organization are arranged in a columnar structure. The pipeline of these combined techniques provides a detailed analysis of tissues and cells in biology and pathology.

Three-dimensional reconstruction of Picea wilsonii Mast. pollen grains using automated electron microscopy.

Three-dimensional electron microscopy (3D-EM) has attracted considerable attention because of its ability to provide detailed information with respect to developmental analysis. However, large-scale high-resolution 3D reconstruction of biological samples remains challenging. Herein, we present a 3D view of a Picea wilsonii Mast. pollen grain with 100 nm axial and 38.57 nm lateral resolution using AutoCUTS-SEM (automatic collector of ultrathin sections-scanning electron microscopy). We established a library of 3,127 100 nm thick serial sections of pollen grains for preservation and observation, demonstrating that the protocol can be used to analyze large-volume samples. After obtaining the SEM images, we reconstructed an entire pollen grain comprising 734 serial sections. The images produced by 3D reconstruction clearly revealed the main components of the P. wilsonii pollen grain, i.e., two sacci and pollen corpus, tube cell, generative cell, and two degenerated prothallial cells, and their internal organization. In addition, we performed a quantitative analysis of the different pollen grain cells, including sacci, and found that there were 202 connections within a saccus SEM image. Thus, for the first time, this study provided a global 3D view of the entire pollen grain, which will be useful for analyzing pollen development and growth.

Atlastin-1 regulates morphology and function of endoplasmic reticulum in dendrites.

Endoplasmic reticulum (ER) is characterized by interconnected tubules and sheets. Neuronal ER adopts specific morphology in axons, dendrites and soma. Here we study mechanisms underlying ER morphogenesis in a C. elegans sensory neuron PVD. In PVD soma and dendrite branch points, ER tubules connect to form networks. ER tubules fill primary dendrites but only extend to some but not all dendritic branches. We find that the Atlastin-1 ortholog, atln-1 is required for neuronal ER morphology. In atln-1 mutants with impaired GTPase activity, ER networks in soma and dendrite branch points are reduced and replaced by tubules, and ER tubules retracted from high-order dendritic branches, causing destabilized microtubule in these branches. The abnormal ER morphology likely causes defects in mitochondria fission at dendritic branch points. Mutant alleles of Atlastin-1 found in Hereditary Spastic Paraplegia (HSP) patients show similar ER phenotypes, suggesting that neuronal ER impairment contributes to HSP disease pathogenesis.

Toxic effect of acrylamide on the development of hippocampal neurons of weaning rats.

Although numerous studies have examined the neurotoxicity of acrylamide in adult animals, the effects on neuronal development in the embryonic and lactational periods are largely unknown. Thus, we examined the toxicity of acrylamide on neuronal development in the hippocampus of fetal rats during pregnancy. Sprague-Dawley rats were mated with male rats at a 1:1 ratio. Rats were administered 0, 5, 10 or 20 mg/kg acrylamide intragastrically from embryonic days 6-21. The gait scores were examined in pregnant rats in each group to analyze maternal toxicity. Eight weaning rats from each group were also euthanized on postnatal day 21 for follow-up studies. Nissl staining was used to observe histological change in the hippocampus. Immunohistochemistry was conducted to observe the condition of neurites, including dendrites and axons. Western blot assay was used to measure the expression levels of the specific nerve axon membrane protein, growth associated protein 43, and the presynaptic vesicle membrane specific protein, synaptophysin. The gait scores of gravid rats significantly increased, suggesting that acrylamide induced maternal motor dysfunction. The number of neurons, as well as expression of growth associated protein 43 and synaptophysin, was reduced with increasing acrylamide dose in postnatal day 21 weaning rats. These data suggest that acrylamide exerts dose-dependent toxic effects on the growth and development of hippocampal neurons of weaning rats.

Large scale three-dimensional reconstruction of an entire Caenorhabditis elegans larva using AutoCUTS-SEM.

Large scale, high resolution three dimensional (3D) ultrastructural reconstruction of cells and tissues has become increasingly important to our understanding of complex biological systems. There have been a few partial 3D ultra-structures of Caenorhabditis elegans (C. elegans) reported, however 3D reconstruction of a whole nematode has never been achieved. Here, we independently developed a technique called automatic collector of ultrathin sections scanning electron microscopy and using this methodology, generated a 3D reconstruction of an entire C. elegans larva with 100 nm axial and 15 nm lateral resolution. Compared to the current available ATUM (automated tape-collecting ultramicrotome) technique, our work provides another alternative complete solution that can be applied to obtain large scale 3D ultrastructure of tissues. Our workflow includes an automated hardware system for high throughput serial section collection, a software package for automatic SEM imaging, and an image reconstruction program. These combined techniques can now be used together to rapidly provide access to understand the anatomy of the whole nematodes.

Association between glutathione S-transferase P1 Ile (105) Val gene polymorphism and chronic obstructive pulmonary disease: A meta-analysis based on seventeen case-control studies.

INTRODUCTION: Previous studies have shown that glutathione S-transferase P1 (GSTP1) was associated with chronic obstructive pulmonary disease (COPD). However, the association between GSTP1 Ile (105) Val gene polymorphism and COPD remains controversial. To drive a more precise estimation, we performed a meta-analysis based on published case-control studies. METHODS: An electronic search of PubMed, EMBASE, Cochrane library, Web of Science and China Knowledge Resource Integrated (CNKI) Database for papers on GSTP1 Ile (105) Val gene polymorphism and COPD risk was performed. The pooled odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association in the homozygote model, heterozygote model, dominant model, recessive model and an additive mode. Statistical heterogeneity, test of publication bias and sensitivity analysis was performed. The software STATA (Version 13.0) was used data analysis. RESULTS: Overall, seventeen studies with 1892 cases and 2012 controls were included in this meta-analysis. The GSTP1 Ile (105) Val polymorphism showed pooled odds ratios for the homozygote comparison (OR = 1.501, 95%CI [0.862, 2.614]), heterozygote comparison (OR = 0.924, 95%CI [0.733, 1.165]), dominant model (OR = 1.003, 95%CI [0.756, 1.331]), recessive model (OR = 1.510, 95%CI [0.934, 2.439]), and an additive model (OR = 1.072, 95%CI [0.822, 1.398]). CONCLUSIONS: In conclusion, the current meta-analysis, based on the most updated information, showed no significant association between GSTP1 Ile (105) Val gene polymorphism and COPD risk in any genetic models. The results of subgroup analysis also showed no significant association between GSTP1 Ile (105) Val gene polymorphism and COPD risk in Asian population and Caucasian population. Further studies involving large populations and careful control with age, sex, ethnicity, and cigarette smoking are greatly needed.

Wt1 dictates the fate of fetal and adult Leydig cells during development in the mouse testis.

Wilms' tumor 1 (Wt1) is a tumor suppressor gene encoding ∼24 zinc finger transcription factors. In the mammalian testis, Wt1 is expressed mostly by Sertoli cells (SCs) involved in testis development, spermatogenesis, and adult Leydig cell (ALC) steroidogenesis. Global knockout (KO) of Wt1 is lethal in mice due to defects in embryogenesis. Herein, we showed that Wt1 is involved in regulating fetal Leydig cell (FLC) degeneration and ALC differentiation during testicular development. Using Wt1(-/flox);Amh-Cre mice that specifically deleted Wt1 in the SC vs. age-matched wild-type (WT) controls, FLC-like-clusters were found in Wt1-deficient testes that remained mitotically active from postnatal day 1 (P1) to P56, and no ALC was detected at these ages. Leydig cells in mutant adult testes displayed morphological features of FLC. Also, FLC-like cells in adult mutant testes had reduced expression in ALC-associated genes Ptgds, Sult1e1, Vcam1, Hsd11b1, Hsd3b6, and Hsd17b3 but high expression of FLC-associated genes Thbs2 and Hsd3b1. Whereas serum LH and testosterone level in mutant mice were not different from controls, intratesticular testosterone level was significantly reduced. Deletion of Wt1 gene also perturbed the expression of steroidogenic enzymes Star, P450c17, Hsd3b6, Hsd3b1, Hsd17b1, and Hsd17b3. FLCs in adult mutant testes failed to convert androstenedione to testosterone due to a lack of Hsd17b3, and this defect was rescued by coculturing with fetal SCs. In summary, FLC-like cells in mutant testes are putative FLCs that remain mitotically active in adult mice, illustrating that Wt1 dictates the fate of FLC and ALC during postnatal testis development.

Atg7 is required for acrosome biogenesis during spermatogenesis in mice.

The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in the process of fertilization. The molecular mechanism underlying the biogenesis of this lysosome-related organelle (LRO) is still largely unknown. Here, we show that germ cell-specific Atg7-knockout mice were infertile due to a defect in acrosome biogenesis and displayed a phenotype similar to human globozoospermia; this reproductive defect was successfully rescued by intracytoplasmic sperm injections. Furthermore, the depletion of Atg7 in germ cells did not affect the early stages of development of germ cells, but at later stages of spermatogenesis, the proacrosomal vesicles failed to fuse into a single acrosomal vesicle during the Golgi phase, which finally resulted in irregular or nearly round-headed spermatozoa. Autophagic flux was disrupted in Atg7-depleted germ cells, finally leading to the failure of LC3 conjugation to Golgi apparatus-derived vesicles. In addition, Atg7 partially regulated another globozoospermia-related protein, Golgi-associated PDZ- and coiled-coil motif-containing protein (GOPC), during acrosome biogenesis. Finally, the injection of either autophagy or lysosome inhibitors into testis resulted in a similar phenotype to that of germ cell-specific Atg7-knockout mice. Altogether, our results uncover a new role for Atg7 in the biogenesis of the acrosome, and we provide evidence to support the autolysosome origination hypothesis for the acrosome.

Wnt/ß-catenin signaling regulates follicular development by modulating the expression of Foxo3a signaling components.

Wnt signaling is an evolutionarily conserved pathway that regulates cell proliferation, differentiation and apoptosis. To investigate the possible role of Wnt signaling in the regulation of ovarian follicular development, secondary follicles were isolated and cultured in vitro in the presence or absence of its activator (LiCl or Wnt3a) or inhibitor (IWR-1). We have demonstrated that activation of ß-catenin signals by activators dramatically suppressed follicular development by increasing granulosa cell apoptosis and inhibiting follicle steroidogenesis. In contrast, inhibition of Wnt signaling by IWR-1 was observed with better developed follicles and increased steroidogenesis. Further studies have shown that the transcription factor Forkhead box O3a (Foxo3a) and its downstream target molecules were modulated by the activators or the inhibitor. These findings provide evidence that Wnt signaling might negatively regulate follicular development potentially through Foxo3a signaling components.

Wt1 deficiency causes undifferentiated spermatogonia accumulation and meiotic progression disruption in neonatal mice.

Spermatogenesis is a complex process involving the regulation of multiple cell types. As the only somatic cell type in the seminiferous tubules, Sertoli cells are essential for spermatogenesis throughout the spermatogenic cycle. The Wilms tumor gene, Wt1, is specifically expressed in the Sertoli cells of the mouse testes. In this study, we demonstrated that Wt1 is required for germ cell differentiation in the developing mouse testes. At 10 days post partum, Wt1-deficient testes exhibited clear meiotic arrest and undifferentiated spermatogonia accumulation in the seminiferous tubules. In addition, the expression of claudin11, a marker and indispensable component of Sertoli cell integrity, was impaired in Wt1(-/flox); Cre-ER(TM) testes. This observation was confirmed in in vitro testis cultures. However, the basal membrane of the seminiferous tubules in Wt1-deficient testes was not affected. Based on these findings, we propose that Sertoli cells' status is affected in Wt1-deficient mice, resulting in spermatogenesis failure.

Expression of the proto-oncogene Pokemon in colorectal cancer--inhibitory effects of an siRNA.

OBJECTIVE: This study aimed to investigate expression of the proto-oncogene POK erythroid myeloid ontogenic factor (Pokemon) in colorectal cancer (CRC), and assess inhibitory effects of a small interference RNA (siRNA) expression vector in SW480 and SW620 cells. METHODS: Semi-quantitative reverse transcription-polymerase chain reaction (PCR) and immunohistochemistry were performed to determine mRNA and protein expression levels of Pokemon in CRC tissues. Indirect immunofluorescence staining was applied to investigate the location of Pokemon in SW480 and SW620 cells. The siRNA expression vectors that were constructed to express a short hairpin RNA against Pokemon were transfected to the SW480 and SW620 cells with a liposome. Expression levels of Pokemon mRNA and protein were examined by real-time quantitative-fluorescent PCR and western blot analysis. The effects of Pokemon silencing on proliferation of SW480 and SW620 cells were evaluated with reference to growth curves with MTT assays. RESULTS: The mRNA expression level of Pokemon in tumor tissues (0.845 ± 0.344) was significantly higher than that in adjacent tumor specimens (0.321 ± 0.197). The positive expression ratio of Pokemon protein in CRC (87.0%) was significantly higher than that in the adjacent tissues (19.6%). Strong fluorescence staining of Pokemon protein was observed in the cytoplasm of the SW480 and SW620 cells. The inhibition ratios of Pokemon mRNA and protein in the SW480 cells were 83.1% and 73.5% at 48 and 72 h, respectively, compared with those of the negative control cells with the siRNA. In the SW620 cells, the inhibition ratios of Pokemon mRNA and protein were 76.3% and 68.7% at 48 and 72 h, respectively. MTT showed that Pokemon gene silencing inhibited the proliferation of SW480 and SW620 cells. CONCLUSION: Overexpression of Pokemon in CRC may have a function in carcinogenesis and progression. siRNA expression vectors could effectively inhibit mRNA and protein expression of Pokemon in SW480 and SW620 cells, thereby reducing malignant cell proliferation.

The Heat-Induced Reversible Change in the Blood-Testis Barrier (BTB) Is Regulated by the Androgen Receptor (AR) via the Partitioning-Defective Protein (Par) Polarity Complex in the Mouse.

Scrotal hypothermia is essential for normal spermatogenesis, and temporal heat stress causes a reversible disruption of the blood-testis barrier (BTB). Previous studies have shown that AR expression in primary monkey Sertoli cells (SCs) was dramatically reduced after temporary heat treatment. However, the mechanisms underlying the heat-induced reversible disruption of the BTB, including whether it is directly regulated by the AR, remain largely unknown. In this study, we demonstrated that the AR acts upstream to regulate the heat-induced reversible change in the BTB in mice. When the AR was overexpressed in SCs using an adenovirus, the heat stress-induced down-regulation of BTB-associated proteins (Zonula occludens-1 (ZO-1), N-Cadherin, E-Cadherin, α-Catenin, and ß-Catenin) was partially rescued. AR knockdown by RNAi or treatment with flutamide (an AR antagonist) in SCs inhibited the recovery of BTB-associated protein expression after 43°C heat treatment for 30 min. The results of an in vivo AR antagonist injection experiment further showed that the recovery of BTB permeability induced by temporal heat stress was regulated by the AR. Furthermore, we observed that the co-localization and interactions of partitioning-defective protein (Par) 6-Par3-aPKC-Cdc42 polarity complex components were disrupted in both AR-knockdown and heat-induced SCs. AR overexpression in SCs prevented the disruption of these protein-protein interactions after heat treatment. AR knockdown or treatment with flutamide in SCs inhibited the restoration of these protein-protein interactions after heat treatment compared with heat treatment alone. Together, these results demonstrate that the AR plays a crucial role in the heat-induced reversible change in BTB via the Par polarity complex.

Expression of inhibin-alpha is regulated synergistically by Wilms' tumor gene 1 (Wt1) and steroidogenic factor-1 (Sf1) in sertoli cells.

Wt1 encodes a zinc finger nuclear transcriptional factor, which is specifically expressed in testicular Sertoli cells and knockdown of Wt1 in Sertoli cells causes male mice subfertility. However, the underlying mechanism is still unclear. In this study, we found that expression of inhibin-α is significantly reduced in Wt1-deficient Sertoli cells. Luciferase assays using the inhibin-α promoter indicated that the inhibin-α promoter is transactivated by the Wt1 A, and B isoforms (-KTS), but not the C, and D isoforms (+KTS). Analysis of the Wt1 responsive element of the inhibin-α promoter region using site-directed mutagenesis showed that the nucleotides between -58 and -49 are essential for Wt1-dependent transactivation of the inhibin-α promoter. ChIP assays indicated that Wt1 directly interacts with the inhibin-α promoter. In addition, the inhibin-α promoter is activated synergistically by Wt1 and Sf1. Mutation of the ligand binding domain (LBD) of Sf1 (residues 235-238) completely abolished the synergistic action between Wt1 and Sf1, but did not affect the physical interaction between these two proteins, suggesting that other factor(s) may also be involved in the regulation of inhibin-α in Sertoli cells. Further studies demonstrated that ß-catenin enhances the synergistic activation of Wt1 and Sf1 on the inhibin-α promoter. Given the fact that inhibin-α, a subunit of inhibin, is known to be involved in the regulation of spermatogenesis and testicular steroidogenesis, this study reveals a new regulatory mechanism of inhibin-α in Sertoli cells and also sheds light on the physiological functions of Wt1 in gonad development and spermatogenesis.

Molecular basis of cryptorchidism-induced infertility.

Artificial cryptorchidism or local testicular heat treatment can induce reversible oligospermia or azoospermia in monkeys and rats via germ cell apoptosis. Local warming of monkey testes in water at 43°C for 2 consecutive days (30 min per day) decreased the number of sperm in the semen by up to 80% on d 28, and the effect was completely reversed on d 144. Germ cells rely heavily on Sertoli cells for structural and nutritional support. Specialized junctions that play a pivotal role in spermatogenesis occur at sites of Sertoli-Sertoli and Sertoli-germ cell contact in the seminiferous epithelium. We demonstrated that expression of tight junction (TJ)-associated molecules, such as occludin and zonula occludens-1 (ZO-1), were greatly reduced 24-48 h after heat treatment, while the permeability of the blood-testis barrier (BTB) was simultaneously increased, but recovered 10 d later. These results indicate a reversible disruption of the BTB associated with transient inductions of transforming growth factor (TGF) ß2 and ß3 expression, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase activation, and concomitant loss of occludin and ZO-1. This suggests that expression of TJ-associated molecules and the BTB was reversibly perturbed by mild testicular hyperthermia, and that the heat-induced induction of TGF-ß might be involved in downregulating TJ-associated proteins, leading to cell junction reduction. This review discusses the changes in total gene expression patterns after experimental cryptorchidism in adult mouse testes, and the cloning of several novel, physiologically significant spermatogenesis-specific genes.

[Effects of mechanical force on expressions of intercellular adhesion molecule-1 in cultured human alveolar type 2 cells].

OBJECTIVE: To explore the expression of intercellular adhesion molecule-1 (ICAM-1) in cultured human alveolar type 2 cells (A549) stimulated by mechanical force in vitro. METHODS: Cells were divided into 3 groups: a tensile stress group, a compressive stress group and a control group. The four-point bending system was used to stimulate A549 cells. The cells were stimulated by tensile stress or compressive stress respectively at the same magnitude of 1000 microstrain for 6 h. Sham cells in control group were not subjected to mechanical loading. The protein level and mRNA level of ICAM-1 were measured by Western blot and RT-PCR. Then an inhibitor was added to further explore the possible mechanism. The cells were divided into a tensile stress+inhibitor group, a compressive stress + inhibitor group and a control group. The cells were pretreated with PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK) for 60 min, and then stimulated respectively by tensile stress or compressive stress at the same magnitude of 1000 microstrain for 6 h or were not subjected to mechanical loading. ICAM-1 protein and mRNA concentrations were determined by Western blot and RT-PCR, respectively. The data were analyzed by one-way ANOVA and Student-Newman-Keuls were used to compare 2 means. RESULTS: The expression of ICAM-1 protein in the tensile stress group (1.16+/-0.07) or the compressive stress group (1.05+/-0.02) were significantly higher than that of the control group (0.78+/-0.07, F=3.31, P<0.05), and the expression of ICAM-1 mRNA in the tensile stress group (1.42+/-0.05) or the compressive stress group (1.27+/-0.05) were also significantly higher than that of the control group (0.13+/-0.04, F=23.1, P<0.01). After pretreated with PD98059 for 60 min, the expression of ICAM-1 protein in the tensile stress group (1.62+/-0.10) was significantly higher than that of the control group (0.50+/-0.03, q=3.75, P<0.05), while there was no significant difference between the compressive stress group (0.60+/-0.03, q=0.32, P>0.05) and the control group. At the transcription level, the expression of ICAM-1 in the tensile stress group (1.57+/-0.03) was significantly higher than that of the control group (0.35+/-0.29, q=3.51, P<0.05), while there was no significant difference between the compressive stress group (0.46+/-0.03, q=0.32, P>0.05) and the control group. CONCLUSIONS: Mechanical forces upregulate the expression of ICAM-1 in A549 cells. PD98059 partly inhibits the upregulation of ICAM-1 induced by mechanical forces. ERK pathway may be partly involved in signal transduction of mechanical force induced expression of ICAM-1 in A549 cells.

[The clinical diagnosis value of fibro-optic bronchoscope examination combined with tumor marker determination to lung cancer].

OBJECTIVE: To investigate the diagnosis value of fibro-optic-bronchoscope combined with tumor maker determination to lung cancer. METHODS: By fibro-optic bronchoscope (FB) and electrochemiluminescence (ECL) examinations, 98 cases with lung cancer and 88 cases with benign lung disease were studied for calculating the detectable sensitivity and specificity to lung cancer, then further for evaluating the clinical value of FB examination combined with detection of tumor marker in serum/pleural fluid of patients with lung cancer. Results In patients with lung cancer, the serum levels of CEA, CA125 and CYFRA21-1 were (46.34 +/- 18.28) ng/mL, (83.34 +/- 33.26) U/mL and (25.67 +/- 10.32) ng/mL respectively, which were higher than those in patients with benign lung diseases. The serum levels of above three tumor markers in patients with lung cancer all were significantly higher than those in patients with benign lung diseases (P < 0.05). In the 36 specimens of pleural fluid, three tumor markers were higher than those in the corresponding serum samples. The detectable sensitivity of each tumor marker in pleural fluid was higher than that in serum. The sensitivity, specificity and overall accuracy of CEA in lung cancer were 37.5%, 87.5% and 63.6% respectively, of CA125 were 67.7%, 40.9% and 54.9%; of CYFRA21-1 were 56.3%, 81.8% and 68.5%; of FB were 60.4%, 100.0% and 79.4% respectively. The sensitivity, specificity or overall accuracy of fibro-optic bronchoscope combined with tumor marker (TM) examination to diagnosis of lung cancer was 90.6%, 92.0% or 91.3% respectively. CONCLUSION: The FB examination is valuable in diagnosing lung cancer, and by combined with TM determination, can further improve the accuracy to diagnosis of lung cancer.