[Hepatitis B virus down-regulates the expression of inhibin and promotes the proliferation and survival of hepatocellular carcinoma cells].

Objective: To investigate the effect and role of the hepatitis B virus (HBV) on the expression of inhibin (PHB) in the proliferation and survival of hepatocellular carcinoma (HCC) cells. Methods: The expression of PHB in 13 pairs of HBV-infected livers, normal livers and HepG2.2.15 and HepG2 cells was detected by real-time fluorescent quantitative PCR and Western blot. Liver tissues were collected from seven patients with chronic hepatitis B before and after antiviral (tenofovir) treatment, and the expression of PHB was detected by RT-PCR and Western blot. HepG2.2.15 cells were transfected with Pcmv6-AC-GFP-PHB, and control vectors were collected. DNA content was analyzed by flow cytometry. The proliferation level of each cell group was detected using the EdU cell proliferation assay. HepG2.2.15 cells transfected with Pcmv6-AC-GFP-PHB and the control vector were cultured in serum-free medium for 6 days. Apoptosis was measured at the indicated time points using fluorescence-activated cell sorting (FACS)-based Annexin-V/PI double staining. Results: Compared with normal liver tissue, the expression of PHB in HBV-infected liver tissue was down-regulated (P < 0.01). Compared with HepG2 cells, the expression of PHB in HepG2.2.15 cells was significantly decreased (P < 0.01). The expression level of PHB in liver tissue after antiviral treatment (tenofovir) was significantly higher than that before treatment (P < 0.01). Compared with the control vector, the proliferation rate of HepG2.2.15 cells transfected with Pcmv6-AC-GFP-PHB was significantly lower than that of the control vector, and the apoptosis rate of HepG2.2.15 cells transfected with the Pcmv6-AC-GFP-PHB vector was significantly higher than the control vector (P < 0.01). Conclusion: HBV down-regulates the expression of inhibin to promote the proliferation and survival of hepatocellular carcinoma cells.

[Study on long-term morphological stability of three-dimensional-printed photosensitive resin dental models].

Objective: To study the long-term morphological stability of three-dimensional (3D) printed photosensitive resin dental models under natural light and dark conditions. Methods: Eighty sets of resin dental models were made by the desktop 3D printer from one digital standard model set, and randomly divided into two groups, namely natural light group (40 sets) and dark group (40 sets). All resin models were stored in sealed bags, with 4 model sets from each group randomly collected after 1, 3, 5, 7, 14, 21, 28, 40, 60, or 90 days of storage and 3D scanned using an optical model scanner. The root-mean-square error (RMSE) was calculated to represent the mean deviation of the difference between the digital standard model and the scanned resin model. Meanwhile, three linear indexes (the width between the canines, the width between the first molars, and the arch length) of the resin dental model were measured and compared with the corresponding values of the standard model. RMSE and the linear measurements between the digital standard model and the scanned resin models were compared between the natural light group and the dark group and among models from different time points. Results: Compared with the digital standard model, the RMSE values of 96.9% (155/160) resin dental models were less than 0.1 mm within 90-day storage. Also, at the same time point, there was no significant difference in the RMSE between the natural light group and the dark group (P>0.05). 75.0% (360/480) of the absolute values of the linear differences (differences in inter-canine width, intra-molar width, and arch length between the digital standard model and the scanned resin model) were within 0.2 mm, and about 0.1% (3/480) of the linear differences were greater than 0.5 mm, and all of the linear differences were within 0.6 mm. Conclusions: 3D-printed resin dental models can be stored stably under natural light and dark conditions for a long time.

[Finite element analysis of the graft stresses after anterior cruciate ligament reconstruction].

OBJECTIVE: To explore the stress distribution characteristics of the graft after anterior cruciate ligament (ACL) reconstruction, so as to provide theoretical reference for the surgical plan of ACL reconstruction. METHODS: Based on 3D MRI and CT images, finite element models of the uninjured knee joint and knee joint after ACL reconstruction were established in this study. The uninjured knee model included femur, tibia, fibula, medial collateral ligament, lateral collateral ligament, ACL and posterior cruciate ligament. The ACL reconstruction knee model included femur, tibia, fibula, medial collateral ligament, lateral collateral ligament, ACL graft and posterior cruciate ligament. Linear elastic material properties were used for both the uninjured and ACL reconstruction models. The elastic modulus of bone tissue was set as 17 GPa and Poisson' s ratio was 0.36. The material properties of ligament tissue and graft were set as elastic modulus 390 MPa and Poisson's ratio 0.4. The femur was fixed as the boundary condition, and the tibia anterior tension of 134 N was applied as the loading condition. The stress states of the ACL of the intact joint and the ACL graft after reconstruction were solved and analyzed, including tension, pressure, shear force and von Mises stress. RESULTS: The maximum compressive stress (6.34 MPa), von Mises stress (5.9 MPa) and shear stress (1.83 MPa) of the reconstructed ACL graft were all at the anterior femoral end. It was consistent with the position of maximum compressive stress (8.77 MPa), von Mises stress (8.88 MPa) and shear stress (3.44 MPa) in the ACL of the intact knee joint. The maximum tensile stress of the graft also appeared at the femoral end, but at the posterior side, which was consistent with the position of the maximum tensile stress of ACL of the uninjured knee joint. More-over, the maximum tensile stress of the graft was only 0.88 MPa, which was less than 2.56 MPa of ACL of the uninjured knee joint. CONCLUSION: The maximum compressive stress, von Mises stress and shear stress of the ACL graft are located in the anterior femoral end, and the maximum tensile stress is located in the posterior femoral end, which is consistent with the position of the maximum tensile stress of the ACL of the uninjured knee joint. The anterior part of ACL and the graft bore higher stresses than the posterior part, which is consistent with the biomechanical characteristics of ACL.

[Anatomical and finite element analysis of anterior cruciate ligament reconstruction within biomechanical insertion].

OBJECTIVE: To provide new concepts of anterior cruciate ligament (ACL) reconstruction by anatomical gross observation of ACL tibial insertion and finite element analysis of distribution of ACL mechanical insertion. METHODS: In the anatomical study, ten fresh adult cadaveric knees were dissected, including 6 males and 4 females, all knees were generally observed through standard medial parapatellar approaches, paying attention to the close anatomical relationship of tibial insertion and anterior horn of lateral meniscus, and ACL was exposed and gradually removed from the inside. The shape of tibial insertion of ACL was observed and recorded, and anterior-posterior diameters and left-right diameters of tibial insertion were measured with vernier caliper. For the study of finite element analysis, three-dimensional thin-layer magnetic resonance imaging of normal knee joint was used to establish knee joint model. Three-dimensional reconstruction software MIMICS and finite element analysis software ANSYS were used to establish knee joint model, subsequently, clinical physical examination Lachman test and pivot-shift test were simulated to observe the force distribution of ACL tibial insertion and femoral insertion. RESULTS: The ACL tibial mechanical insertion was rather flat and long similar as an arc shape without a clear separation between anterior medial bundle (AMB) and posterolateral bundle (PLB) in gross observation. The dense fibers lies belonged to the medial intercondylar ridge and ended up anterior with the osseous landmark of anterior ridge. Its average anterior-posterior diameter was (13.8±2.0) mm, the average left-right diameter of midsubstance was (5.3±0.6) mm, and the average left-right diameter of anterior margin was (11.5±1.2) mm. The finite element analysis showed that distribution on the femoral side was oval shape mainly below the residents' ridge, while the tibial side was rather flat mainly along the medial intercondylar ridge, which was consistent with the anatomical observation. The biomechanical characteristics of ACL attachments were verified theoretically. CONCLUSION: Anatomical study and finite element analysis have confirmed the flat arc shape of ACL tibial insertion. The ideal reconstruction technique of ACL should be based on its biomechanical insertion. Based on anatomical study and biomechanical analysis, we have proposed the idea of ACL biomechanical insertion reconstruction (BIR) and established a surgical model with oval femoral tunnel and rounded-rectangle tibial tunnel.

Siglec-7 Defines a Highly Functional Natural Killer Cell Subset and Inhibits Cell-Mediated Activities.

Sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7) is an inhibitory receptor expressed on natural killer (NK) cells. In this study, we investigated the relationship between Siglec-7 expression and NK cell functions. Siglec-7 was highly expressed on NK cells and was preferentially expressed by mature NK cells from peripheral blood of healthy adults. Siglec-7(+) NK cells displayed higher levels of activating receptors CD38, CD16, DNAM1, NKp30 and NKp46, but lower levels of inhibitory receptors such as NKG2A and CD158b, compared with Siglec-7(-) NK cells. Functional tests showed that Siglec-7(+) NK cells displayed more CD107a degranulation and IFN-γ production than Siglec-7(-) NK cells. Siglec-7 inhibited NK cell functions when interacting with specific antibodies. These data suggest that Siglec-7 defines a highly functional NK cell subset and suppresses NK cell-mediated functions when cross-linked with specific antibodies.

Comparison of radiological and clinical features of temporal lobe necrosis in nasopharyngeal carcinoma patients treated with 2D radiotherapy or intensity-modulated radiotherapy.

BACKGROUND: To compare the imaging and clinical features of temporal lobe necrosis (TLN) in nasopharyngeal carcinoma (NPC) patients treated with two-dimensional radiotherapy (2D-RT) or those with intensity-modulated radiotherapy (IMRT). METHODS: We retrospectively analysed NPC patients who underwent 2D-RT (72 patients, 128 temporal lobes) or IMRT (36 patients, 50 lobes) and developed radiation-induced, MRI-confirmed TLN. RESULTS: White-matter lesions (WMLs), contrast-enhanced lesions, cysts and local mass effects were present in 128 out of 128 vs 48 out of 50 (P=0.078), 123 out of 128 vs 47 out of 50 (P=0.688), 10 out of 128 vs 1 out of 50 (P=0.185) and 57 out of 128 vs 13 out of 50 (P=0.023) temporal lobes, respectively, in the 2D-RT and IMRT groups. The WMLs were more extensive in the 2D-RT group (P<0.001). The maximum diameter of contrast-enhanced lesions was greater in the 2D-RT group (P<0.001), and these lesions tended to extend far away from the nasopharynx. The WMLs and enhancement had no impact on cyst development (both P=1). Local mass effects were always accompanied with contrast-enhanced lesions (P=0.024) but were not correlated with WMLs or cysts (P=0.523 and 0.341, respectively). There were no between-group differences in clinical features (all P-values>0.05), whereas the difference in the incidence of severe debility was of marginal significance (18.1% vs 5.6%, P=0.077). CONCLUSIONS: The IMRT-induced TLN was less extensive and milder than 2D-RT-induced TLN, but both had similar clinical features.

Free-energy-based lattice Boltzmann model for the simulation of multiphase flows with density contrast.

A free-energy-based phase-field lattice Boltzmann method is proposed in this work to simulate multiphase flows with density contrast. The present method is to improve the Zheng-Shu-Chew (ZSC) model [Zheng, Shu, and Chew, J. Comput. Phys. 218, 353 (2006)] for correct consideration of density contrast in the momentum equation. The original ZSC model uses the particle distribution function in the lattice Boltzmann equation (LBE) for the mean density and momentum, which cannot properly consider the effect of local density variation in the momentum equation. To correctly consider it, the particle distribution function in the LBE must be for the local density and momentum. However, when the LBE of such distribution function is solved, it will encounter a severe numerical instability. To overcome this difficulty, a transformation, which is similar to the one used in the Lee-Lin (LL) model [Lee and Lin, J. Comput. Phys. 206, 16 (2005)] is introduced in this work to change the particle distribution function for the local density and momentum into that for the mean density and momentum. As a result, the present model still uses the particle distribution function for the mean density and momentum, and in the meantime, considers the effect of local density variation in the LBE as a forcing term. Numerical examples demonstrate that both the present model and the LL model can correctly simulate multiphase flows with density contrast, and the present model has an obvious improvement over the ZSC model in terms of solution accuracy. In terms of computational time, the present model is less efficient than the ZSC model, but is much more efficient than the LL model.

Low expression of Mel-18 predicts poor prognosis in patients with breast cancer.

BACKGROUND: Our previous study suggested that melanoma nuclear protein 18 (Mel-18) acted as a tumor suppressor in human breast cancer. This study was designed to investigate the clinical and prognostic significance of Mel-18 in breast cancer patients. PATIENTS AND METHODS: Mel-18 was detected by immunohistochemistry in paraffin-embedded tissues from 287 breast cancer patients, of which 287 were from primary cancer sites, 63 from matched adjacent noncancerous sites, and 35 from metastatic lymph nodes. Differences in Mel-18 expression and clinical characteristics were compared by χ² test. Prognostic outcomes correlated with Mel-18 were examined using Kaplan-Meier analysis and Cox proportional hazards model. RESULTS: The decreased Mel-18 expression is incremental depending upon the magnitude of cancer progression (P < 0.001). Mel-18 was conversely correlated with the pathological classifications (P < 0.001 for T, N, and M classifications, respectively), clinical staging (P < 0.001), and progesterone receptor (P = 0.030). Furthermore, patients with higher level of Mel-18 showed prolonged overall survivals (P < 0.001). The diminished Mel-18 expression may be a risk factor for the patients' survival (P < 0.001). CONCLUSIONS: Lower Mel-18 expression is correlated with advanced clinicopathologic classifications and a poor overall survival in breast cancer patients. These findings suggest that Mel-18 may serve as a useful marker in prognostic evaluation for patients.

Pharmacokinetic and pharmacodynamic study of intratumoral injection of an adenovirus encoding endostatin in patients with advanced tumors.

Angiogenesis plays a pivotal role in tumor growth, tissue invasion and metastasis. Endostatin is an angiogenesis inhibitor and has been shown to reduce tumor growth in animal models. However, therapy with recombinant endostatin protein was hampered by its short half-life and very-low yield of bioactive protein. We performed a phase I dose-escalation clinical trial using intratumoral injection of an adenovirus containing human endostatin gene (Ad-rhE; E10A; 10(10)-10(12) virus particles (vp)) in 15 patients with advanced solid tumors. We observed intratumoral injections of E10A without dose-limiting toxicity. Most frequently reported E10A-related adverse events were transient fever and local response. Distribution studies revealed that the vector was detected in the blood, throat and injection site, but rarely in the urine and stool. An increased endostatin expression was detected using enzyme immunoassay in serum in 13 of 14 treated patients throughout the period of treatment despite the presence of neutralizing antiadenovirus antibody. Median serum basic fibroblast growth factor levels decreased from 32.4 pg ml(-1) at baseline to 24.9 pg ml(-1) after 28 days of first treatment. Thus, direct intratumoral injection of up to 10(12) vp of E10A to patients is well tolerated and further studies are necessary to define and increase clinical efficacy.

Loss of heterozygosity and its correlation with clinical outcome and Epstein-Barr virus infection in nasopharyngeal carcinoma.

Sixty-one human nasopharyngeal carcinomas (NPC) were examined by allelotype analysis for the purposes of detecting potential association between loss of heterozygosity (LOH), clinicopathological parameters and Epstein-Barr virus (EBV) infection. LOH was performed using 257 polymorphic markers on 22 chromosomes. High frequency LOH (> or = 60%) was observed on 12 chromosome arms including 1p, 2p, 2q, 3p, 3q, 5q, 9p. 9q, 11q, 13q, 14q and 17q, with the highest LOH frequency of 91% on 3p. Seventy-three loci presented LOH frequency > or = 30%; most of these loci clustered on 1p36 p34, 2p25-p24, 3p14-p21, 3p24-p26, 5q11-q14, 5q31-q33, 9p21-p23, 9q33-q34, 11q23-q25, 13q12 q14, 13q31-q33, 14q13-q11, 14q32 and 19q13. On 1p36-p34, 2p25-p24, 5q13-q11, 5q31-q33 and 19q13 are reported for the first time. LOH was correlated with specific clinicopathological parameters including tumor T-stage, N-stage, TNM-stage, tumor differentiation and serum antibody titers of IgA against virus capsid antigens (VCA) and early antigen (EA) of EBV in NPC (LOH frequency > or = 30%). Significantly high LOH frequency was observed on 9p21 (56%) and 19q13 (50%) in NPC with stage T3+T4, while significantly higher LOH frequency was observed on 12p11 (65%) in NPC with stage T1 + T2. Significantly higher LOHfrequency on 19q13 was also observed in NPC with advanced TNM-stage (III+IV). High fractional allelic loss (FAL) value and high antibody titers of EBV IgA/VCA and/or IgA/EA were significantly correlated with T3 + T4-stage, distant lymph node metastasis and advanced TNM-stage of NPC. We also found that NPC patients with high titers of IgA/VCA and IgA/EA showed high LOH frequency on 16q (48%) and 19q13 (48%). These results suggest that LOH on 9p21, l6q and 19q13 may be responsible for the aggressiveness and progression of NPC; there may be an interaction between allelic loss and EBV infection in the etiology of NPC. High frequency of LOH on 4q21 and 14q11-q12 were alsofound to be correlated with WHO type III NPC histopathology, suggesting that LOH on these regions may cause poor NPC differentiation. Our data also may be useful for the development of a NPC molecular staging system, a system which may augment the use of clinical pathological features in the diagnosis and prognosis of this disease.

Genome-wide allelotype analysis of sporadic primary nasopharyngeal carcinoma from southern China.

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in Southern China, especially in the Guangdong area. To demonstrate a comprehensive profile of loss of heterozygosity (LOH) in NPC, we applied a large panel of 382 microsatellite polymorphism markers covering all the 22 autosomes in 98 cases of sporadic primary NPC. Of the 335 informative markers, 83 loci showed high level of LOH (presence in equal to or more than 30% cases) and most of the high frequent loci were clustered to chromosome 1p36 and 1p34, 3p14-p21, 3p24-p26, 3q25-q26 and 3q27, 4q31 and 4q35, 5q15-21 and 5q32-q33, 8p22-p23, 9p21-p23 and 9q33-q34, 11p12-p14, 13q14-q13 and 13q31-q32, 14q13-q11, 14q24-q23 and 14q32. High frequency of LOH was found in chromosomes 3, 5, 9 and 11 (>/=50%), while medium frequency of LOH was found in chromosomes 1, 4, 6, 14, 17 and 19 (40-49%). Several new regions showing high frequency of LOH were found in chromosome 1p36, 3q25-q26, 3q27, 5q15-q21, 8p22-p23 and 11p12-14. The relationship between LOH and TNM stage of NPC was evaluated. Regions 6p23 (D6S289), 8p23.1 (D8S549) and 9q34.2 (D9S1826) showed higher frequency of LOH in later stages (III and IV) than in earlier stages (I and II) (P<0.05). Thus, our study provides a global view on allelic loss in the development of NPC and should shed light on the way for localization of putative tumor suppressor genes associated with the pathogenesis of NPC.

Mechanical anchoring strength of L-selectin, beta2 integrins, and CD45 to neutrophil cytoskeleton and membrane.

The strength of anchoring of transmembrane receptors to cytoskeleton and membrane is important in cell adhesion and cell migration. With micropipette suction, we applied pulling forces to human neutrophils adhering to latex beads that were coated with antibodies to CD62L (L-selectin), CD18 (beta2 integrins), or CD45. In each case, the adhesion frequency between the neutrophil and bead was low, and our Monte Carlo simulation indicates that only a single bond was probably involved in every adhesion event. When the adhesion between the neutrophil and bead was ruptured, it was very likely that receptors were extracted from neutrophil surfaces. We found that it took 1-2 s to extract an L-selectin at a force range of 25-45 pN, 1-4 s to extract a beta2 integrin at a force range of 60-130 pN, and 1-11 s to extract a CD45 at a force range of 35-85 pN. Our results strongly support the conclusion that, during neutrophil rolling, L-selectin is unbound from its ligand when the adhesion between neutrophils and endothelium is ruptured.

nm23-H1 expression in nasopharyngeal carcinoma: correlation with clinical outcome.

The expression levels of nm23-H1 mRNA and its protein in human nasopharyngeal carcinoma (NPC) were detected to clarify the relationship between nm23-H1 and metastasis and prognosis of patients with NPC. nm23-H1 mRNA expression in fresh tissues from 78 patients with NPC was investigated by in situ hybridization and RT-PCR. Routine labeling streptavidin-biotin immuno-histochemistry with the nm23-H1 murine monoclonal antibody was employed to study the expression of nm23-H1 protein in paraffin-embedded specimens from 231 patients with NPC treated in our hospital. The clinical pathologic data and results of follow-up were collected. Comparisons between expression of nm23-H1 protein or mRNA and clinical outcome were performed using the chi2 test. Multivariate prognostic analyses were performed by the Cox regression model. We found that nm23-H1-negative tumors were associated with a higher incidence of lymph-node metastasis (84.2%) than nm23-H1-positive ones (32.8%, p < 0.01). The distant metastasis and loco-regional recurrence rates in the nm23-H1-negative group were 55.8% and 31.68%, respectively but only 17.2% and 11.5%, respectively, in the nm23-H1-positive group (p < 0.01). A significant association was found between expression of nm23-H1 protein and prognosis (p < 0.01). Expression of nm23-H1 protein indicated favorable prognosis, suggesting that the absence of nm23-H1 protein expression was significantly associated with lymph-node metastasis, recurrence and distant metastasis in NPC. Expression of the nm23-H1 gene may be valuable for assessing the prognosis of NPC.

Static and dynamic lengths of neutrophil microvilli.

Containing most of the L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) on their tips, microvilli are believed to promote the initial arrest of neutrophils on endothelium. At the rolling stage following arrest, the lifetimes of the involved molecular bonds depend on the pulling force imposed by the shear stress of blood flow. With two different methods, electron microscopy and micropipette manipulation, we have obtained two comparable neutrophil microvillus lengths, both approximately 0.3 microm in average. We have found also that, under a pulling force, a microvillus can be extended (microvillus extension) or a long thin membrane cylinder (a tether) can be formed from it (tether formation). If the force is 61 pN (+/- 5 pN), a tether will be formed from the microvillus at a constant velocity, which depends linearly on the force. When the force is between 34 pN and 61 pN (transition zone), the degree of association between membrane and cytoskeleton in individual microvilli will dictate whether microvillus extension or tether formation occurs. When a microvillus is extended, it acts like a spring with a spring constant of approximately 43 pN/microm. In contrast to a rigid or nonextendible microvillus, both microvillus extension and tether formation can decrease the pulling force imposed on the adhesive bonds, and thus prolonging the persistence of the bonds at high physiological shear stresses.

The resistance to flow of individual human neutrophils in glass capillary tubes with diameters between 4.65 and 7.75 microns.

OBJECTIVE: The resistance to flow of individual human neutrophils flowing through smooth-walled glass capillary tubes at velocities ranging between 0 and 30 microns/s is measured for six different capillary tube diameters between 4.65 and 7.75 microns. METHODS: The experiments were performed with a micropipet manipulation system. The velocity of individual human neutrophils was measured under different constant pressures and compared to the velocity of the cell-free fluid in the same tube. With the aid of a theory that describes the motion of a concentric smooth-walled, sausage-shaped body in a tube, the experimental measurements are used to calculate an apparent gap width between the cell and the capillary wall. RESULTS: The maximum calculated gap width in the larger capillary tubes was on the order of 0.1 micron, whereas the minimum gap width in the smaller capillaries was about 0.015 micron. These small gap widths mean that even one neutrophil can cause a significant increase in the apparent viscosity when a cell flows through 100- to 200-micron-long capillary tubes. The neutrophils often exhibited a small static friction that tended to increase as the capillary diameter decreased. Maximum values for the adhesive force caused by the static friction were on the order of 80 pN. CONCLUSIONS: Even a single white cell entirely within a capillary can cause a significant increase in the resistance of flow.

Micropipette suction for measuring piconewton forces of adhesion and tether formation from neutrophil membranes.

A new method for measuring piconewton-scale forces that employs micropipette suction is presented here. Spherical cells or beads are used directly as force transducers, and forces as small as 10-20 pN can be imposed. When the transducer is stationary in the pipette, the force is simply the product of the suction pressure and the cross-sectional area of the pipette minus a small correction for the narrow gap that exists between the transducer and the pipette wall. When the transducer is moving along the pipette, the force on it is corrected by a factor that is proportional to the ratio of its velocity relative to its drag-free velocity. With this technique, the minimum force required to form a membrane tether from neutrophils is determined (45 pN), and the length of the microvilli on the surface of neutrophils is inferred. The strength of this technique is in its simplicity and its ability to measure forces between cells without requiring a separate theory or a calibration against an external standard and without requiring the use of a solid surface.

Intestinal cell cycle regulations. Interactions of cyclin D1, Cdk4, and p21Cip1.

OBJECTIVE: The p21Cip1 protein is a potent stoichiometric inhibitor of cyclin-dependent kinase activity, and p21Cip1 mRNA expression is localized to the nonproliferative compartment of the intestinal villus, suggesting an in vivo growth-inhibitory role in the gut. The authors determined whether nontransformed rat intestinal epithelial cells (IECs) underwent reversible cell cycle arrest by contact inhibition, and determined whether increases in the relative amount of p21 associated with cyclin D/Cdk4 protein complexes were associated with cell growth arrest. METHODS: Density arrest was achieved by prolonged culture IEC-6 in confluent conditions (5 or more days). Release from density arrest was achieved by detaching the cells from the culture plate and reseeding them at a 1:4 ratio. The DNA synthesis was estimated by [3H]-thymidine incorporation and expressed as mean plus or minus standard error of the mean (n = 4). Cyclin D1, Cdk4, and p21 mRNA and protein levels were determined by standard Northern and Western blot analyses, respectively. Cyclin D1, Cdk4, and p21 protein complex formation was analyzed by immunoprecipitating the complexes from cell lysates with an antibody to one of the constituents, followed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the precipitated complexes using antibodies to the other proteins. The kinase activity of the immunoprecipitated Cdk4 was determined using recombinant Rb as substrate. RESULTS: The IEC-6[3H]-thymidine incorporation was decreased 7.5-fold from day 1 confluence to day 7 of confluence. Twenty-four hours after release from density arrest, there was a 43-fold increase in [3H]-thymidine incorporation. Cyclin D1 and Cdk4 mRNA levels remained relatively constant during contact inhibition, whereas immunoblotting showed that the levels of cyclin D1 and Cdk4 proteins decreased by 70.9% and 68.7%, respectively, comparing day 3 with day 9 during density arrest. The levels of cyclin D1 increased 5.8-fold and Cdk4 increased by 4.4-fold by 24 hours after reseeding the day 9 density-arrested cultures, coincident with the increase in DNA synthesis. The amount of p21 associated with the cyclin D1 and Cdk4 complex in the density-arrested cells was 170% of that observed in the reseeded, proliferating cells. More important, the p21::Cdk4 ratio was 6.4-fold higher in the density-arrested (quiescent) cells as compared with rapidly proliferating cells by 24 hours after release from growth arrest. Recovery of Cdk4-dependent kinase activity occurred by 4 hours after release from growth arrest, coincident with decreased binding of p21 to the complex. CONCLUSIONS: Intestinal epithelial cells in culture can undergo density-dependent growth arrest. This process involves downregulation of cyclin D1 and Cdk4 at the level of protein expression, whereas the mRNA levels remain relatively unchanged. Further, during contact inhibition, there is more p21 associated with cyclin D1/Cdk4, which further contributes to the inhibition of the kinase complex. The authors also have shown that the process of contact inhibition is reversible, which may explain partly the ability of the intestinal epithelium to increase proliferative activity in response to injury.

Deformation and flow of membrane into tethers extracted from neuronal growth cones.

Membrane tethers are extracted at constant velocity from neuronal growth cones using a force generated by a laser tweezers trap. A thermodynamic analysis shows that as the tether is extended, energy is stored in the tether as bending and adhesion energies and in the cell body as "nonlocal" bending. It is postulated that energy is dissipated by three viscous mechanisms including membrane flow, slip between the two monolayers that form the bilayer, and slip between membrane and cytoskeleton. The analysis predicts and the experiments show a linear relation between tether force and tether velocity. Calculations based on the analytical results and the experimental measurements of a tether radius of approximately 0.2 micron and a tether force at zero velocity of approximately 8 pN give a bending modulus for the tether of 2.7 x 10(-19) N.m and an extraordinarily small "apparent surface tension" in the growth cone of 0.003 mN/m, where the apparent surface tension is the sum of the far-field, in-plane tension and the energy of adhesion. Treatments with cytochalasin B and D, ethanol, and nocodazole affect the apparent surface tension but not bending. ATP depletion affects neither, whereas large concentrations of DMSO affect both. Under conditions of flow, data are presented to show that the dominant viscous mechanism comes from the slip that occurs when the membrane flows over the cytoskeleton. ATP depletion and the treatment with DMSO cause a dramatic drop in the effective viscosity. If it is postulated that the slip between membrane and cytoskeleton occurs in a film of water, then this water film has a mean thickness of only approximately 10 A.

New preparation for oral administration of digestive enzyme. Lactase complex microcapsules.

Complex microcapsules which could protect therapeutic enzymes from inactivation in both the stomach and intestine were prepared. Thus, semipermeable microcapsules were first formed by enveloping the enzymes within spherical, ultrathin semipermeable membranes. To resist to the gastric juice, the semipermeable microcapsules were further encapsulated by enteric-soluble materials to form complex microcapsules. When the preparation passes into the intestine, the semipermeable microcapsules are released, thus the small molecules of substrates equilibrate rapidly across the semipermeable membrane to be reacted by the enveloped enzymes, and alimentary proteases remain outside. This complex microencapsulated lactase remained over 65% of its activity after 2 h simulation in gastric juice, and over 65% of its activity was retained after 6 h contact with pancreatin-containing simulated intestinal juice. By contrary, unencapsulated lactase lost immediately all of its activity under similar conditions.