Ordered stereom structure in sea urchin tubercles: High capability for energy dissipation.

Tubercles in sea urchin shells serve as a base on the test plates connecting the spine; these undergo compressive or impact stress from the spines. As the volume fraction of the ordered stereom structure in a tubercle increases, the compressive load-displacement curves are gradually characterized by the typical behavior of ceramic foams. Although this ordered stereom structure only exhibits an average porosity of 50.6%, it also exhibits high fracture resistance and energy dissipation capacity. Such remarkable behavior of the ordered stereom structure is attributed to its unique hierarchical microstructure. Specifically, at the macroscale, the stereom structure is periodic. It has uniformly distributed pores that are typically round, which can effectively reduce the stress concentration around the pores, and the ordered arrangement of the trabeculae along the axial direction of the tubercle bears the most compressive stress. The trabeculae present a bottleneck shape with a specific dimension, ensuring the best fracture resistance with a relatively higher porosity. Furthermore, crack deflection in the trabeculae changes the local fracture mode of the mineral, thereby increasing the crack surface area. STATEMENT OF SIGNIFICANCE: The connecting bases of the spines in sea urchin shell, known as tubercle, effectively undergo the compressive stress or impact stress from the spines. An ordered stereom structure is found in the tubercle, and it shows an excellent fracture resistance and energy dissipation capacity. Such a fantastic behavior of the ordered stereom structure mainly takes advantage of its unique hierarchical microstructure. The stereom structure presents a periodic structure on macroscale, the trabeculae show a bottleneck shape with a specific dimension to guarantee the best fracture resistance with a relatively higher porosity, and the soft fillers among CaCO3 nanoparticles in a trabecula cause consecutive crack deflections.

Kinking and cracking behavior in nacre under stepwise compressive loading.

The damage evolution of nacre under compressive loading has not been well understood, despite numerous investigations on its compressive behavior. In the present work, quasi-in-situ loading-unloading-reloading stepwise compressive tests were performed on nacre in Pinctada maxima shell, which exhibits a distinctive gradient feature with the thickness of platelets decreasing from the external to internal parts. In the loading direction parallel to the platelets, multiple microcracks and kink bands can absorb much deformation energy, leading to a graceful failure. Kinking only occurs at the final stage of fracture process, and it thus has no obvious influence on the strength of nacre, but contributes to a much larger strain. In the loading direction perpendicular to the platelets, nacre exhibits concurrently much higher compressive strength and fracture strain, as the damage can be effectively restricted. This is attributed to the presence of gradient structure, which disperses the stress concentration in front of the crack tip, and arouses the toughening mechanisms including damage localization and crack deflection. The findings obtained in this study are expected to provide fundamental insights into the design of bio-inspired advanced engineering materials.

The measurement of ouabain binding and some related properties of digitonin-treated (Na+,K+)-ATPase.

1. Digitonin treated membrane preparations purified from dog kidney lose their (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, but the K+-phosphatase and Na+-dependent ADP-ATP exchange activities survive and remain ouabain-sensitive. Because the enzyme preparations consist largely of pure (Na+,K+)-ATPase, these effects of digitonin must be intrinsic to the Na+ pump. 2. Concomitant with these enzymatic changes, digitonin treatment alters the sensitivity of the phosphatase and exchange activities to ouabain. 3. Attempts to measure ouabain binding by the usual centrifugation or filtration methods proved unsuccessful. A filtration method involving a double 0.01 mum filter and omitting water washes is necessary to demonstrate ouabain binding. Under these conditions, ouabain binding capacity appears to be unchanged in the presence of digitonin, but the apparent dissociation constant is doubled. 4. Ouabain binding is rendered more reversible by digitonin treatment, since washing filters with water removes a large fraction of bound ouabain without affecting the retention of exchange activity. 5. The double filter method traps essentially all of the ADP-ATP exchange activity on the filter. However, a large and somewhat variable proportion of the K+-phosphatase activity passes through the filter. Sodium dodecyl sulfate polyacrylamide gel analysis of the filtrate shows that a small amount of filtrable protein catalyzed this phosphatase activity at greatly increased turnover rates. Both subunits of the (Na+, K+)-ATPase are present in this latter protein fraction.

Production of Haemophilus influenzae type-b porin in Escherichia coli and its folding into the trimeric form.

The P2 protein from pathogenic Haemophilus influenzae type b (Hib) functions as a bacterial porin and is one of several immunogenic outer membrane proteins. The P2 gene was expressed in Escherichia coli and the recombinant P2 protein (re-P2) purified to facilitate functional and immunologic studies. P2 was obtained from Hib strain Eagan using PCR and the pET vectors (17b and 11a) were used to produce re-P2 at levels exceeding 30% of the total E. coli proteins. Since previous reports had indicated that P2 was toxic to E. coli, steps were taken to control the toxicity. The plasmid was stabilized by tightly controlling the synthesis of re-P2 prior to induction. Subsequent to induction, re-P2 was sequestered into inclusion bodies rather than to membrane compartments. The refolding of the denatured re-P2 into the trimeric form involved high salt and calcium ions. re-P2 was then purified to homogeneity using gel-filtration and ion-exchange chromatography.

An anti-p24 monoclonal antibody shows cross-reactivity with multiple HIV-1 proteins.

We produced three murine monoclonal antibodies (mAbs) against the HIV-1 proteins. These three mAbs, namely CA-1, CA-2, CA-4, were IgG1 and all reacted with p24 on the HIV-1 Western blot. One of the mAbs, CA-4, also recognized p13, p21, p28, p29, p32, p39, p47, p55 on the Biotech/Du Pont HIV-1 Western blot strips and p21, p24, p28, p29, p39, p47, p55, p68, p80, p96; p110 on the Bio-Rad strips. CA-4 did not react with H-9 cell lysate nor with other retroviral antigens such as HTLV-1 or HIV-2 proteins. The binding of CA-4 to HIV-1 proteins was not blocked by deglycosylation. All three mAbs reacted with recombinant DNA derived capsid protein (p24) of HIV-1. These results suggest that many proteins in the HIV-1 Western blot contain antigenic epitope(s) similar to that of p24.

Agreements among traditional Chinese medicine practitioners in the diagnosis and treatment of irritable bowel syndrome.

BACKGROUND: Traditional Chinese Medicine was frequently used by patients with irritable bowel syndrome. AIM: To evaluate the agreement on diagnoses and prescription of irritable bowel syndrome among Traditional Chinese Medicine practitioners. METHODS: Consecutive irritable bowel syndrome patients were interviewed independently by four Traditional Chinese Medicine practitioners. The study was divided into three phases: (i) blinded individual assessment, (ii) discussion to achieve consensus on diagnosis and treatment, (iii) individual assessment based on consensual diagnostic criteria. Patients with other causes of diarrhoea were recruited as controls in phase (iii). Percentage agreement and kappa-value in diagnosis, treatment principle and regime were determined. RESULTS: Thirty-nine irritable bowel syndrome patients were assessed in phase (i) whereas 65 irritable bowel syndrome patients and 17 non-irritable bowel syndrome controls were studied in phase (iii). The mean agreement rates in diagnosis, treatment principle and regimen were: 57, 58 and 52% for phase (i) and 80, 81 and 80% for phase (iii) (P = 0.002). Accordingly, there was significant improvement in the mean kappa-values in diagnosis (0.11-0.34, P = 0.015) and treatment principle (0.16-0.37, P = 0.002) but not in treatment regime. CONCLUSIONS: Variations in diagnosis and treatment principles do exist among Traditional Chinese Medicine practitioners. Concordant diagnosis can be reached by mutual understanding and converging opinion among Traditional Chinese Medicine practitioners.

Development and performance evaluation of an electromagnetic-type shock wave generator for lipolysis.

This study aims at the design and development of electromagnetic-type intermittent shock wave generation in a liquid. The shock wave generated is focused at a focal point through an acoustic lens. This hardware device mainly consists of a full-wave bridge rectifier, 6 capacitors, a spark gap, and a flat coil. A metal disk is mounted in a liquid-filled tube and is placed in close proximity to the flat coil. Due to the repulsive force existing between the coil and disk shock waves are generated, while an eddy current is induced in the metal disk. Some components and materials associated with the device are also described. By increasing the capacitance content to enhance electric energy level, a highly focused pressure can be achieved at the focal point through an acoustic lens in order to lyse fat tissue. Focused pressures were measured at the focal point and its vicinity for different operation voltages. The designed shock wave generator with an energy intensity of 0.0016 mJ/mm(2) (at 4 kV) and 2000 firings or higher energy intensities with 1000 firings is found to be able to disrupt pig fat tissue.

Raf activation by Ras and promotion of cellular metastasis require phosphorylation of prohibitin in the raft domain of the plasma membrane.

Prohibitin (PHB) is indispensable for Ras-induced Raf-1 activation, cell migration and growth; however, the exact role of PHB in the molecular pathogenesis of cancer metastasis remains largely unexamined. Here, we found a positive correlation between plasma membrane-associated PHB and the clinical stages of cancer. The level of PHB phosphorylated at threonine 258 (T258) and tyrosine 259 (Y259) in human cancer-cell membranes correlated with the invasiveness of cancer cells. Overexpression of phosphorylated PHB (phospho-PHB) in the lipid-raft domain of the cell membrane enhanced cell migration/invasion through PI3K/Akt and Raf-1/ERK activation. It also enhanced epithelial-mesenchymal transition, matrix metalloproteinase-2 activity and invasiveness of cancer cells in vitro. Immunoprecipitation analysis demonstrated that phospho-PHB associated with Raf-1, Akt and Ras in the membrane and was essential for the activation of Raf-1 signaling by Ras. Mice implanted with cancer cells stably overexpressing PHB in the plasma membrane showed enlarged cervical tumors, enhanced metastasis and shorter survival time compared with mice implanted with cancer cells without PHB overexpression. Dephosphorylation of PHB at T258 by site-directed mutagenesis diminished the in vitro and in vivo effects of PHB. These results suggest that increase in phospho-PHB T258 in the raft domain of the plasma membrane has a role in the Ras-driven activation of PI3K/Akt and Raf-1/ERK-signaling cascades and results in the promotion of cancer metastasis.

Treatment of diarrhea-predominant irritable bowel syndrome with traditional Chinese herbal medicine: a randomized placebo-controlled trial.

BACKGROUND: As there is no effective treatment for irritable bowel syndrome (IBS), many patients turn to traditional Chinese medicine (TCM) for possible cure. We investigated the therapeutic efficacy of an ancient herbal Chinese formula in patients with diarrhea-predominant IBS. METHODS: This was a randomized double-blinded placebo-controlled trial. Chinese IBS patients with predominant diarrhea symptoms that fulfilled Rome II criteria were recruited. The diagnosis was verified by a TCM herbalist using TCM criteria. Eligible patients were randomized to receive a standard preparation of TCM extracts that contained 11 herbs or placebo with similar appearance and taste for 8 wk after a 2-wk run-in period. Patients were followed up for an additional 8 wk post-treatment. Primary outcome was patient's global symptom assessment. Other outcome measures included individual IBS symptom scores and health-related quality of life (short form 36). RESULTS: One hundred nineteen patients were randomized: 60 to receive TCM and 59 to receive placebo. There was no significant difference in the proportion of patients with global symptom improvement between the TCM and placebo groups at week 8 (35% vs 44.1%, p = 0.38) and at week 16 (31.7% vs 33.9%, p = 0.62). Moreover, there was no difference in individual symptom scores and the quality-of-life assessment between the two groups at all time points. BACKGROUND: The use of this herbal formulation for diarrhea-predominant IBS did not lead to global symptom improvement. Further controlled clinical studies may be necessary to characterize the role of TCM in the management of IBS.

Visualization of interleukin-2-like molecules in MPP(+)-lesioned rat brain.

The tissue distribution of interleukin-2 (IL-2) in normal and 1-methyl-4-phenyl-pyridinium (MPP+)-lesioned brains of rats was investigated. Intrastriatal administration of MPP+ caused visible damage in the vicinity of the injected region two weeks after injection. Autoradiography of the tissue section with anti-IL-2 antibodies plus trace amounts of radiolabeled IL-2 showed that the antibodies treatment elicited a selective radiolabeling of the brain tissues localized at the MPP(+)-lesioned region but not at normal cryo-sliced sections. Addition of radiolabeled IL-2 alone or normal rabbit immunoglobulins did not show any labeling effect. These autoradiographic imaging results suggest that there is an accumulation of cells bearing IL-2-like molecules at the MPP(+)-induced lesion sites.

Hydrophobic clustering in acid-denatured IL-2 and fluorescence of a Trp NH-pi H-bond.

The single tryptophan at position 121 of human interleukin-2 (IL-2) can form an NH-pi hydrogen bond with Phe 117 involving the indole nitrogen and the benzene aromatic ring. At pH 5.5, this type of aromatic interaction results in a fluorescence quantum yield three-fold lower than that of a fully solvent exposed tryptophan. At pH 2.1, IL-2 forms a compact denatured state with twice the emission intensity of the native protein. Global analysis of time-resolved fluorescence emission at multiple emission wavelengths shows that native and acid-denatured IL-2 can be described by four decay components. The fractional amplitudes of the shortest sub-nanosecond lifetimes are higher in the native state, suggesting rapid quenching due to the NH-pi hydrogen bond. In the denatured state, longer lifetimes have greater fractional amplitudes, indicating a smaller population of hydrogen-bonded species. Electrostatic-dipolar relaxation of the tryptophan microenvironment upon excitation is greater in the native-state of IL-2 than the acid-denatured state. This suggests that acid-denaturation sequesters Trp 121 from polar residues, while maintaining an interaction with Phe 117. This is consistent with the model of secondary structure preservation and hydrophobic clustering in molten-globule intermediates.

Intracellular form of streptococcal proteinase: a clue to a novel mechanism of secretion.

The intracellular form of streptococcal proteinase has been isolated and compared with its extracellular form. As shown by double-immunodiffusion studies and radiosequence analysis, the intracellular proteinase was identical to that of the extracellular proteinase. However, the unusual mixed disulfide, protein-S-SR, shown to be present in the extracellular proteinase, was missing in the intracellular proteinase. Protease activity is dependent upon the free sulfhydryl group of the proteinase. Thus, the intracellular proteinase was enzymatically active, while the extracellular proteinase requires activation by exposure to a reducing agent. Because this appears to be the only difference between the intracellular and extracellular protease, it is proposed that the modification of the protein-SH to form protein-S-SR is a process that is intimately related to the mechanism of secretion of the proteinase into the culture fluid by streptococci.

Analysis of IL-2 functional structure by multiple cysteine substitutions.

IL-2 has three cysteine residues. The cysteines at positions 58 and 105 of active IL-2 form an intramolecular disulfide bond while that at position 125 remains as a free form. To evaluate the importance of correct disulfide bond, mutant proteins (muteins) that have triple and double substitutions of cysteines with alanines, namely A58/105/125 and A58/125, were made by polymerase chain reaction method respectively. Thymidine incorporation assay on CTLL-2 cells showed that although these two muteins were only 0.5-2.0% as potent as that of wild type IL-2, they were 50-200 fold more active than A58, a mutein that has substitution of cysteine at position 58 with alanine. Binding inhibition study showed that the relative affinity of muteins A58/125 and A58/105/125 for high affinity IL-2 receptors was 5-25 fold higher than that of A58. These results suggest that the dramatic decrease in the activity of mutein A58 may result from the formation of an incorrect disulfide bond between the cysteines at positions 105 and 125.

Targeting shock waves in human tissue for extracorporeal shock wave therapy.

Extracorporeal Shock Wave Therapy is a relatively new alternative method to surgery for the treatment of many bone and muscle disorders. Currently, targeting of shock waves in the human body is done using either ultrasound or x-ray imaging. Many studies have shown controversial treatment results with conclusions that criticize ultrasound targeting. Here it is shown that targeting of the shock waves inside the body is crucial to the result of a treatment. The two different coupling methods of the shock wave generator onto the patient's body are used with gelatin phantoms to give insight into the actual treatments. As a conclusion, x-ray imaging or inline ultrasound with direct coupling should be preferred. In addition, the path of the shock waves should be vertical to the bone surfaces in order to preserve a focal region in the treated area.

Studies on Limulus amoebocyte. Isolation and identification of a membrane-bound protein activator of cyclic nucleotide phosphodiesterase from Limulus amoebocyte.

A protein isolated from Limulus polyphemus amoebocyte activates the hydrolysis of cyclic AMP by phosphodiesterase. The protein activator, like calmodulin, requires Ca2+ for its activity and is antagonized by calmodulin-modulating protein from bovine brain. 2-Chloro-10-(3-aminopropyl)-phenothiazine, a compound known to bind calmodulin, also inhibits the effect of the protein activator. This Limulus protein activator is an acidic protein with high percentage of glutamate and aspartate; it contains trimethyllysine, a characteristic amino acid found in all calmodulin. It is different from calmodulin isolated from other species, however, in its molecular weight (4 to 5 times greater), amino acid composition, antigenicity, and binding ability on 2-chloro-10-(3-aminopropyl)-phenothiazine affinity column chromatography. The amino acid composition, gel electrophoresis pattern, and molecular weight of this protein activator are indistinguishable from endotoxin-binding protein which we isolated previously by other independent methods. Immunologic studies demonstrate that these two proteins are essentially identical. The endotoxin-binding protein thus has the dual functions of binding endotoxin, and showing calmodulin-like activity. It may play an important role in degranulation of Limulus amoebocytes which is induced by minute amounts of gram-negative bacterial endotoxin.

Studies on Limulus amoebocyte lysate. III. Purification of an endotoxin-binding protein from Limulus amoebocyte membranes.

A protein that has been isolated from Limulus polyphemus amoebocyte membranes binds endotoxin. The protein was purified by two independent methods, organic solvent extraction and affinity chromatography, both followed by gel filtration. Immunologic studies confirm that the protein is a component of amoebocyte membranes. Although without enzymatic activity, the binding protein enhances Limulus lysate gelation. As a membrane-associated endotoxin binding "protein," it may be involved in Limulus lysate coagulation, which is initiated by minute amounts of Gram-negative bacterial endotoxin. The protein has an apparent molecular weight of 80,000.

Folding and purification of insoluble (inclusion body) proteins from Escherichia coli.

Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion bodies. To obtain the native (i.e., correctly folded) and hence active form of the protein from such aggregates, four steps are usually followed: (1) the cells are lysed and the are aggregates, (2) the cell wall and outer membrane components of the aggregates are removed, (3) the aggregates are solubilized (or extracted) with strong protein denaturants, and (4) the solubilized, denatured proteins are folded with concomitant oxidation of reduced cysteine residues into the correct disulfide bonds to obtain the native protein. This unit features three different approaches to the final step of protein folding and purification. In the first, guanidine HCl is used as the denaturant, after which the solubilized protein is folded (before purification) in an "oxido-shuffling" buffer system to increase the rate of protein oxidation. In the second, acetic acid is used to solubilize the protein which is then partially purified by gel filtration before folding, and then the protein is folded and oxidized by simple dialyzed against water. A Support Protocol is included for rapidly determining the amount of folded protein that contains the correct disulfide linkage pattern. Finally, folding and purification of a fusion protein is described using metal-chelate affinity chromatography.

Glutathione regulates interleukin-2 activity on cytotoxic T-cells.

In this study, we examined whether and how the cellular activity of interleukin-2 (IL-2) is affected by glutathione (GSH), an important tripeptide existing in most cells. Cell culture and thymidine incorporation assay showed that addition of GSH enhanced the effect of IL-2 on the proliferation and thymidine incorporation of IL-2-dependent cytotoxic T-cells such as CTLL-2 and CT-4R. Treatment of the cells with GSH resulted in a 2-fold increase in the amount of IL-2 bound to the cells and a rapid internalization of the bound IL-2. In addition, the degradation of IL-2 in the cells was enhanced by GSH treatment. These effects of GSH were accompanied by an increase in the intracellular GSH level. L-Buthionine-(S,R)-sulfoximine, an inhibitor of de novo GSH synthesis, blunted the increase of intracellular GSH level and modulated the effect of GSH on IL-2 activity. These results suggest that GSH regulates the binding, internalization, degradation, and T-cell proliferative activity of IL-2; alterations of cellular GSH concentration may thus affect the growth and replication of IL-2-sensitive cytotoxic T-cells.

Regulation by glutathione of interleukin-4 activity on cytotoxic T cells.

We have previously shown that cellular glutathione (GSH) regulates the T-cell proliferative activity of interleukin-2 (IL-2). Here, we examined whether and how GSH affects the activity of interleukin-4 (IL-4) on murine cytotoxic T cells. CT.4R, a T-cell line that is responsive to both IL-4 and IL-2, was used as a model. Although GSH alone had little effect on the thymidine incorporation of CT.4R cells, it enhanced the response of CT.4R to IL-4 and increased the level of thymidine incorporation up to more than 60-fold in a concentration-dependent manner. GSH affected the binding of IL-4 to cellular receptors. Scatchard plot analysis showed that GSH treatment did not change the dissociation constant significantly; however, it increased the receptor number from 1173 +/- 126 to 2112 +/- 492 molecules per cell. Internalization and degradation studies of IL-4 showed that the amount of IL-4 internalized and degraded in the GSH-treated cells was about twofold higher than those in the cells without GSH treatment. These results suggest that GSH regulates the binding, internalization, degradation and T-cell proliferative activity of IL-4; alteration of cellular GSH levels may thus affect the growth and replication of cytotoxic T cells through growth stimulating cytokines such as IL-2 and IL-4.