Orthosiphon stamineus (Misai Kucing) ameliorated postmenopausal osteoporosis in rat model.

OBJECTIVE: Orthosiphon stamineus (OS) or Misai Kucing (Java tea) is a popular herbal supplement from Southeast Asia for various metabolic, age-related diseases. This study investigated the potential use of OS leaf extracts to ameliorate osteoporosis in ovariectomized rats. METHODS: Fifty-six female Sprague-Dawley rats were randomly allocated into eight groups (n = 7): SHAM (healthy sham control); OVX (ovarietomized) nontreated rats (negative control); OVX + Remifemin (100 mg/kg body weight), and 2% green tea extract (positive controls); OVX + OS 50% ethanolic and aqueous extracts, both at either 150 or 300 mg/kg. After 16 weeks, the rats' bones and blood were evaluated for osteoporosis indicators (protein and mRNA expressions), micro-computed tomography for bone histomorphometry, and three-point bending test for tibia mechanical strength. RESULTS: The extracts dose-dependently and significantly (P < 0.05) improved bone strength and flexibility, bone mineral density, bone formation protein markers (P1NP), and bone histomorphometry. All extracts reduced the inflammation biomarker (interleukin-6). The extracts up-regulated osteoblastogenesis (bone morphogenetic protein-2) and collagen-1 synthesis (collagen type 1 alpha-1) mRNA expressions, and down-regulated bone resorption (TNFSF11 and nuclear factor-kappa B) mRNA expressions. Both the water and 50% ethanolic extract were effective. The effective dose is equivalent to 25 to 50 mg/kg extract for humans. CONCLUSIONS: The extract showed bone-protective and antiosteoporotic effects (improving bone strength, flexibility, bone density, and bone morphometry) by reducing inflammation and the bone resorption biomarkers, while enhancing bone formation biomarkers and collagen synthesis.

Role of both actin-myosin cross bridges and NO-cGMP pathway modulators in the contraction and relaxation of human placental stem villi.

INTRODUCTION: Human placental stem villi (PSV) present contractile properties. We studied the role of actin-myosin cross bridges (CBs) and the effects of NO-cGMP pathway modulators in the PSV contraction and relaxation. METHODS: In vitro contractile properties were investigated in 71 PSV from term human placentas studied according to their long axis. Contraction was induced by both KCl and electrical tetanic stimulation. Relaxation was induced by inhibiting the CB cycle with either 2,3-butanedione monoxime (BDM) or blebbistatin (BLE) and by activating the NO-cGMP pathway with isosorbide dinitrate (ISDN), sildenafil (SIL) or ISDN + SIL. RESULTS: PSV tension slowly increased by 140% of the basal tone after KCl exposure and by 85% after tetanus. The addition of BDM, BLE, ISDN, SIL and ISDN + SIL induced a relaxation of PSV, the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). These relaxation kinetics were particularly slow. Other relaxation parametres, i.e., maximum lengthening, -peak dT/dt, and resting tension, did not differ between these 5 subgroups. DISCUSSION AND CONCLUSION: Isolated human PSV were able to contract after both KCl exposure and tetanus. This increase in contractility was reversed by inhibiting the CB cycle with BDM or BLE and by stimulating the NO-cGMP pathway with ISDN or SIL. The association ISDN + SIL did not potentiate the relaxing processes.

Quantitative analysis of the flexibility effect of cisplatin on circular DNA.

We study the effects of cisplatin on the circular configuration of DNA using atomic force microscopy (AFM) and observe that the DNA gradually transforms to a complex configuration with an intersection and interwound structures from a circlelike structure. An algorithm is developed to extract the configuration profiles of circular DNA from AFM images and the radius of gyration is used to describe the flexibility of circular DNA. The quantitative analysis of the circular DNA demonstrates that the radius of gyration gradually decreases and two processes on the change of flexibility of circular DNA are found as the cisplatin concentration increases. Furthermore, a model is proposed and discussed to explain the mechanism for understanding the complicated interaction between DNA and cisplatin.

Surface agents' influence on the flexural strength of bilaminated ceramics

The objective of this study was to evaluate the influence of different surface agents on the flexural strength of a ceramic system. Eighty bar-shaped specimens of zirconia were divided into four groups according to the agent to be used: group Control - to be cleaned with alcohol; group VM9 - application of a fluid layer of porcelain; group Effect Bonder - application of a bonding agent; and group Coloring Liquid - application of coloring liquid. All specimens received the porcelain application by the layering technique and were then subjected to thermocycling. The four-point bending test was performed to calculate the strength values (σ, MPa) and the failure modes were classified. ANOVA did not detect significant differences among the groups. The Weibull modulus were 5 (Control, VM9 and Effect Bonder) and 6 (Coloring Liquid). The cracking of the porcelain ceramic toward the interface was the predominant failure mode. It was concluded that the surface agents tested had no effect on the flexural strength of the bilaminated ceramic specimens.

On the mechanism of action of SJ-172550 in inhibiting the interaction of MDM4 and p53.

SJ-172550 (1) was previously discovered in a biochemical high throughput screen for inhibitors of the interaction of MDMX and p53 and characterized as a reversible inhibitor (J. Biol. Chem. 2010; 285:10786). Further study of the biochemical mode of action of 1 has shown that it acts through a complicated mechanism in which the compound forms a covalent but reversible complex with MDMX and locks MDMX into a conformation that is unable to bind p53. The relative stability of this complex is influenced by many factors including the reducing potential of the media, the presence of aggregates, and other factors that influence the conformational stability of the protein. This complex mechanism of action hinders the further development of compound 1 as a selective MDMX inhibitor.

Cytotoxicity of superoxide dismutase 1 in cultured cells is linked to Zn2+ chelation.

Neurodegeneration in protein-misfolding disease is generally assigned to toxic function of small, soluble protein aggregates. Largely, these assignments are based on observations of cultured neural cells where the suspect protein material is titrated directly into the growth medium. In the present study, we use this approach to shed light on the cytotoxic action of the metalloenzyme Cu/Zn superoxide dismutase 1 (SOD1), associated with misfolding and aggregation in amyotrophic lateral sclerosis (ALS). The results show, somewhat unexpectedly, that the toxic species of SOD1 in this type of experimental setting is not an aggregate, as typically observed for proteins implicated in other neuro-degenerative diseases, but the folded and fully soluble apo protein. Moreover, we demonstrate that the toxic action of apoSOD1 relies on the protein's ability to chelate Zn(2+) ions from the growth medium. The decreased cell viability that accompanies this extraction is presumably based on disturbed Zn(2+) homeostasis. Consistently, mutations that cause global unfolding of the apoSOD1 molecule or otherwise reduce its Zn(2+) affinity abolish completely the cytotoxic response. So does the addition of surplus Zn(2+). Taken together, these observations point at a case where the toxic response of cultured cells might not be related to human pathology but stems from the intrinsic limitations of a simplified cell model. There are several ways proteins can kill cultured neural cells but all of these need not to be relevant for neurodegenerative disease.

PpGalNacT2 participating in vanadium-induced HL-60 cell differentiation.

The current study demonstrates vanadium plays the role of antitumor, and its antitumor effect is dosage-dependent. N-acetyl-galactosamine-transferase 2 (polypeptide: N-acetyl-α-galactosaminyl-transferases 2, ppGalNAc-T2) is a member of ppGalNAcTs (polypeptide: N-acetyl-α-galactosaminyl-transferases) family, which proves to play a vital role in the tumor emergence and development process. In this study, we focused on ppGalNAc-T2 and vanadium and aimed to determine whether ppGalNAc-T2 is correlated with vanadium's antitumor effect. We discovered that ppGalNAc-T2 changed with the variation of HL-60 cell growth induced by vanadium at mRNA level. Peanut agglutinin (PNA) is an exogenous lectin. PpGalNacT2 can be indirectly recognized by PNA. By means of flow cytometry and immunofluorescent staining, we found the deviation of PNA binding increased significantly at high concentration vanadium. Then we docked one of the possible compound substances of vanadium onto the body, VO(3) (molecular formula O(13)V(4), partial vanadate tetramer) and ppGalNAcT2, and simulated them via molecular dynamics, which showed that VO(3) may inhibit the activity of the enzyme by stemming conformational changes of a key loop of ppGalNAcT2. To sum up, our results suggested that ppGalNacT2 participated in vanadium induced HL-60 cell differentiation, which might be able to provide a new mechanism of vanadium's antitumor effect.

Comparison of physical and mechanical properties of microwave-polymerized acrylic resin after disinfection in sodium hypochlorite solutions

This study evaluated the color stability, surface roughness and flexural strength of a microwave-polymerized acrylic resin after immersion in sodium hypochlorite (NaOCl), simulating 20 min of disinfection daily during 180 days. Forty disk-shaped (15 x 4 mm) and 40 rectangular (65 x 10 x 3 mm) specimens were prepared with a microwave-polymerized acrylic resin (Onda-Cryl). Specimens were immersed in either 0.5 percent NaOCl, 1 percent NaOCl, Clorox/Calgon and distilled water (control). Color measurements were determined by a portable colorimeter. Three parallel lines, separated by 1.0 mm, were registered on each specimen before and after immersion procedures to analyze the surface roughness. The flexural strength was measured using a 3-point bending test in a universal testing machine with a 50 kgf load cell and a crosshead speed of 1 mm/min. Data were analyzed statistically by ANOVA and Tukey's test (?=0.05). There was no statistically significant differences (p>0.05) among the solutions for color, surface roughness and flexural strength. It may be concluded that immersion in NaOCl solutions simulating short-term daily use during 180 days did not influence the color stability, surface roughness and flexural strength of a microwave-polymerized acrylic resin.

Este estudo avaliou a estabilidade de cor, rugosidade superficial e resistência à flexão de resina acrílica polimerizada por microondas após desinfecção em hipoclorito de sódio, simulando 20 min de desinfecção diária durante 180 dias. Quarenta espécimes circulares (15 x 4 mm) e 40 retangulares (65 x 10 x 3 mm) foram preparados em resina acrílica polimerizada por microondas (Onda-Cryl). As amostras foram imersas em hipoclorito de sódio a 0,5 por cento, hipoclorito de sódio a 1 por cento, Clorox/Calgon e água destilada (controle). Medidas de cor foram determinadas por um espectrocolorímetro portátil. Três linhas paralelas, separadas por 1,0 mm, foram registradas em cada amostra antes e depois dos procedimentos de imersão para analisar a rugosidade superficial. A resistência à flexão foi medida utilizando um teste de flexão por 3 pontos em uma máquina universal de ensaios com uma célula de carga de 50 Kgf e uma velocidade de 1 mm/min. Os dados foram analisados estatisticamente por ANOVA e teste de Tukey (?=0,05). Não houve diferença estatisticamente significante (p>0,05) entre as soluções para cor, rugosidade superficial e resistência à flexão. Pode-se concluir que a imersão em soluções de hipoclorito de sódio, simulando um uso diário de curta duração durante 180 dias, não influenciou a estabilidade de cor, rugosidade superficial e resistência à flexão de resina acrílica polimerizada por microondas.

Expression of fusion IL2-B7.1(IgV+C) and effects on T lymphocytes.

The search for an effective immunotherapeutic treatment for tumors is an important area of cancer research. To prepare a more effective form of the bifunctional fusion protein IL2-B7.1(IgV+C) and analyze its effect on the stimulation of T lymphocyte proliferation, we used DNAStar 5.03 software to predict the structural diversity and biochemical character of IL2-B7.1(IgV+C). We then prepared fusion protein IL2-B7.1(IgV+C) by establishing its prokaryotic expression system, and tested its effect on the stimulation of T lymphocytes in vitro. The results indicated that IL2-B7.1(IgV+C) correctly formed a secondary structure in which both IL2 and B7.1(IgV+C) maintained their original hydrophilicity and epitopes. Western blot analysis revealed that IL2-B7.1(IgV+C) was efficiently expressed. Our analysis of CTLL-2 and T-cell proliferation showed that recombinant human (rh) IL2-B7.1(IgV+C) exerted the combined stimulating effects of both rhIL2 and rh B7.1(IgV+C) on cell proliferation, and that these effects could be blocked by adding either anti-IL2 or anti-B7.1 monoclonal antibodies. A >2-fold increase in [3H]TdR incorporation compared with that of cells treated with recombinant protein IL2, or B7.1(IgV+C) alone, revealed that rhIL2-B7.1(IgV+C) had dose-dependent synergetic effects on T-cell activation in the presence of anti-CD3 monoclonal antibody. We concluded that the augmented potency of rhIL2-B7.1(IgV+C) resulted in a stronger stimulation of T-cell proliferation than either rhB7.1(IgV+C) or rhIL2 alone.

Effect of zoledronate on bone quality in the treatment of aseptic loosening of hip arthroplasty in the dog.

Periprosthetic bone loss, which is a direct cause of aseptic loosening in total hip arthroplasty (THA), can be suppressed by bisphosphonates. It is unknown how the quality of this bone is affected in the presence of both wear debris (from implant) and bisphosphonates. The objective of this study was to evaluate the effect of zoledronate (ZLN) on bone quality in the presence of wear debris [polyethylene (PE) particles] in a canine model of uncemented THA. Thirty dogs underwent THA, and aseptic loosening was induced via implantation of PE particles packed into the femoral component. For 26 weeks until sacrifice, two groups (each n = 10) received weekly injections of ZLN (low dose 2 mug/kg, high dose 10 mug/kg) and the third group (control) received saline. Histological and radiographic examinations were performed to evaluate the degree of implant reaction. Histomorphometry (static/dynamic) was performed to evaluate bone turnover. Back-scattered electron imaging was used to quantify the newly formed bone and to evaluate the mineralization distribution. Density fractionation and X-ray diffraction were used to evaluate mineral properties, while four-point bending was used to determine mechanical properties. A dose-dependent presence of newly formed subperiosteal bone was found, which appeared to be less mineralized than the adjacent cortical bone. The high-dose ZLN group showed decreased cortical porosity and turnover and increased mineralization profile, failure strength, and modulus. We conclude that ZLN affects some of the material properties of cortical bone and allows the newly formed subperiosteal bone to remain and therefore affect the overall quality of the bone.

Increase in the conformational flexibility of beta 2-microglobulin upon copper binding: a possible role for copper in dialysis-related amyloidosis.

A key pathological event in dialysis-related amyloidosis is the fibril formation of beta(2)-microglobulin (beta 2-m). Because beta 2-m does not form fibrils in vitro, except under acidic conditions, predisposing factors that may drive fibril formation at physiological pH have been the focus of much attention. One factor that may be implicated is Cu(2+) binding, which destabilizes the native state of beta 2-m and thus stabilizes the amyloid precursor. To address the Cu(2+)-induced destabilization of beta 2-m at the atomic level, we studied changes in the conformational dynamics of beta 2-m upon Cu(2+) binding. Titration of beta 2-m with Cu(2+) monitored by heteronuclear NMR showed that three out of four histidines (His13, His31, and His51) are involved in the binding at pH 7.0. (1)H-(15)N heteronuclear NOE suggested increased backbone dynamics for the residues Val49 to Ser55, implying that the Cu(2+) binding at His51 increased the local dynamics of beta-strand D. Hydrogen/deuterium exchange of amide protons showed increased flexibility of the core residues upon Cu(2+) binding. Taken together, it is likely that Cu(2+) binding increases the pico- to nanosecond fluctuation of the beta-strand D on which His51 exists, which is propagated to the core of the molecule, thus promoting the global and slow fluctuations. This may contribute to the overall destabilization of the molecule, increasing the equilibrium population of the amyloidogenic intermediate.

Spinal flexibility increase after chymopapain injection is dose dependent: a possible alternative to anterior release in scoliosis.

STUDY DESIGN: Experimental animal study. OBJECTIVES: To investigate whether the increase in spinal flexibility after chymopapain injection is dose dependent and determine the "optimal" dosage of chymopapain to increase spinal flexibility in a rabbit model. SUMMARY OF BACKGROUND DATA: Spinal instability after chymopapain injection may result in severe back pain. However, this undesired mechanical effect in treating disc herniation may provide a safe minimally invasive approach for anterior spinal release in scoliosis correction. METHODS: A total of 138 lumbar intervertebral discs from 46 New Zealand white rabbits were randomly injected with chymopapain at 6.25, 12.5, 25, 50, 75, and 100 picokatals (pKats)/0.05 mL/disc. The rabbits were killed 1 week after the injection, and the lateral bending stiffness of the spinal segments without posterior elements was determined. RESULTS: The lateral bending spinal stiffness showed no significant change after injection of 6.25 and 12.5 pKats/0.05 mL/disc but reduced significantly following chymopapain injection of 25, 50, 75, and 100 pKats (all P < 0.05 by post hoc least significant difference tests). While the lateral bending stiffness was lowest at the 100-pKats dose, there were no significant differences between the four higher dosages. CONCLUSION: The reduction in the lateral bending spinal stiffness after chymopapain injection is dose dependent, and an optimal dosage for spinal release existed; doses greater than the optimal dosage did not result in further significant decrease in lateral bending spinal stiffness.

DNA-bending finger: artificial design of 6-zinc finger peptides with polyglycine linker and induction of DNA bending.

DNA structural changes such as bending play an important role in various biological reactions. Not only protein binding to its specific DNA sequence but also DNA bending induced by the protein is indispensable for unique gene expression. Therefore, an artificial protein that induces a DNA conformational change is interesting as a transcriptional regulator of a specific gene. We created 6-zinc finger proteins, Sp1ZF6(Gly)n (n = 4, 7, 10), by connecting two DNA binding domains of transcription factor Sp1 with flexible polyglycine peptide linkers, and their effects on DNA structure were compared with that of native 3-zinc finger Sp1(530-623). Gel electrophoretic methods revealed that Sp1ZF6(Gly)7 and Sp1ZF6(Gly)10 bind to two distal GC boxes and result in DNA bending. Evidently, the hydroxyl radical footprinting analysis demonstrated that hypersensitive cleavage was observed at the 5'-TA step in the intervening region bound by Sp1ZF6(Gly)7 or Sp1ZF6(Gly)10. The phasing assays strongly suggested that the induced DNA bending was directed toward the major groove and that Sp1ZF6(Gly)7 caused the most drastic directional change in DNA bending. Of special interest are the facts that the newly designed 6-finger peptides Sp1ZF6(Gly)7 and Sp1ZF6(Gly)10 can induce DNA bending at the intervening region of the two distal binding sites and that the linker length between two 3-zinc finger motifs has a crucial effect on the entire DNA-bending direction. Such DNA-bending fingers may be feasible for use as a gene expression regulator based on the structural change in DNA in the future.