Heat-shock protein 70-2 (HSP70-2) expression in bladder urothelial carcinoma is associated with tumour progression and promotes migration and invasion

Purpose: Testis specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, is essential for the growth of spermatocytes and cancer cells. We investigated the association of HSP70-2 expression with clinical behaviour and progression of urothelial carcinoma of bladder. Experimental design: We assessed the HSP70-2 expression by RT-PCR and HSP70-2 protein expression by immunofluorescence, flow cytometry, immunohistochemistry and Western blotting in urothelial carcinoma patient specimens and HTB-1, UMUC-3, HTB-9, HTB-2 and normal human urothelial cell lines. Further, to investigate the role of HSP70-2 in bladder tumour development, HSP70-2 was silenced in the high-grade invasive HTB-1 and UMUC-3 cells. The malignant properties of urothelial carcinoma cells were examined using colony formation, migration assay, invasion assay in vitro and tumour growth in vivo. Results: Our RT-PCR analysis and immunohistochemistry analysis revealed that HSP70-2 was expressed in both moderate to well-differentiated and high-grade invasive urothelial carcinoma cell lines studied and not in normal human urothelial cells. In consistence with these results, HSP70-2 expression was also observed in superficially invasive (70%) and muscle-invasive (90%) patient's tumours. Furthermore, HSP70-2 knockdown significantly suppressed cellular motility and invasion ability. An in vivo xenograft study showed that inhibition of HSP70-2 significantly suppressed tumour growth. Conclusions: In conclusion, our data suggest that the HSP70-2 expression is associated with early spread and progression of urothelial carcinoma of bladder cancer and that HSP70-2 can be the potential therapeutic target for bladder urothelial carcinoma. (C) 2009 Elsevier Ltd. All rights reserved.

STAT-1 expression is regulated by IGFBP-3 in malignant glioma cells and is a strong predictor of poor survival in patients with glioblastoma Laboratory investigation

Object. Insulin-like growth factor binding proteins (IGEBPs) have been implicated in the pathogenesis of glioma. In a previous study the authors demonstrated that IGFBP-3 is a novel glioblastoma biomarker associated with poor survival. Since signal transducer and activator of transcription 1 (STAT-1) has been shown to be regulated by IGFBP-3 during chondrogenesis and is a prosurvival and radioresistant molecule in different tumors, the aim in the present study was to explore the functional significance of IGFBP-3 in malignant glioma cells, to determine if STAT-1 is indeed regulated by IGFBP-3, and to study the potential of STAT-1 as a biomarker in glioblastoma. Methods. The functional significance of IGFBP-3 was investigated using the short hairpin (sh)RNA gene knockdown approach on U251MG cells. STAT-1 regulation by IGFBP-3 was tested on U251MG and U87MG cells by shRNA gene knockdown and exogenous treatment with recombinant IGFBP-3 protein. Subsequently, the expression of STAT-1 was analyzed with real-time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) in glioblastoma and control brain tissues. Survival analyses were done on a uniformly treated prospective cohort of adults with newly diagnosed glioblastoma (136 patients) using Kaplan-Meier and Cox regression models. Results. IGFBP-3 knockdown significantly impaired proliferation, motility, migration, and invasive capacity of U251MG cells in vitro (p < 0.005). Exogenous overexpression of IGFBP-3 in U251MG and U87MG cells demonstrated STAT-1 regulation. The mean transcript levels (by real-time RT-PCR) and the mean labeling index of STAT-1 (by IHC) were significantly higher in glioblastoma than in control brain tissues (p = 0.0239 and p < 0.001, respectively). Multivariate survival analysis revealed that STAT-1 protein expression (HR 1.015, p = 0.033, 95% CI 1.001-1.029) along with patient age (HR 1.025, p = 0.005, 95% CI 1.008-1.042) were significant predictors of shorter survival in patients with glioblastoma. Conclusions. IGFBP-3 influences tumor cell proliferation, migration, and invasion and regulates STAT-1 expression in malignant glioma cells. STAT-1 is overexpressed in human glioblastoma tissues and emerges as a novel prognostic biomarker.

Bimodal distribution of motility and cell fate in Dictyostelium discoideum

Pre-starvation amoebae of Dictyostelium discoideum exhibit random movements. Starved cells aggregate by directed movements (chemotaxis) towards cyclic AMP and differentiate into live spores or dead stalk cells. Many differences between presumptive spore and stalk cells precede differentiation. We have examined whether cell motility-related factors are also among them. Cell speeds and localisation of motility-related signalling molecules were monitored by live cell imaging and immunostaining (a) in nutrient medium during growth, (b) immediately following transfer to starvation medium and (c) in nutrient medium that was re-introduced after a brief period of starvation. Cells moved randomly under all three conditions but mean speeds increased following transfer from nutrient medium to starvation medium; the transition occurred within 15 min. The distribution of speeds in starvation medium was bimodal: about 20% of the cells moved significantly faster than the remaining 80%. The motility-related molecules F-actin, PTEN and PI3 kinase were distributed differently in slow and fast cells. Among starved cells, the calcium content of slower cells was lower than that of the faster cells. All differences reverted within 15 min after restoration of the nutrient medium. The slow/fast distinction was missing in Polysphondylium pallidum, a cellular slime mould that lacks the presumptive stalk and spore cell classes, and in the trishanku (triA(center dot)) mutant of D. discoideum, in which the classes exist but are unstable. The transition from growth to starvation triggers a spontaneous and reversible switch in the distribution of D. discoideum cell speeds. Cells whose calcium content is relatively low (known to be presumptive spore cells) move slower than those whose calcium levels are higher (known to be presumptive stalk cells). Slow and fast cells show different distributions of motility-related proteins. The switch is indicative of a bistable mechanism underlying cell motility.

A thermal molecular migration in the solid-state - structures of isomeric 5-amino-4-(2,6-dichlorophenyl)-1-(2-nitrophenyl)-1h-1,2,3-triazole (yellow form i) and 4-(2,6-dichlorophenyl)-5-(2-nitroanilino)-2h-1,2,3-triazole (red form-ii), c14h9cl2n5o2

Yellow form (I): Mr= 350.09, monoclinic, P2Jn, Z--4, a=9.525(1), b=14.762(1), c= 11.268(1),/t, fl= 107.82 (1) o , V= 1508.3 A 3 , Din(flotation in aqueous KI)= 1.539 (2), D x= 1.541 (2) g cm -3, #(Cu Ka, 2 = 1.5418 A) = 40.58 cm -~, F(000) = 712, T= 293 K, R = 8.8% for 2054 significant refections. Red form (II): Mr= 350.09, triclinic, Pi, Z=2, a=9.796(2), b= 10.750 (2), c= 7.421 (1)A, a= 95.29 (2), fl= 0108-2701/84/111901-05501.50 70.18 (1), y = 92-.76 (2) °, V= 731.9 A 3, Din(flotation in KI) = 1.585 (3), D x = 1.588 (3) g cm -3, ~t(Cu Ka, 2 = 1.5418/~) = 40.58 cm -1, F(000) = 356, T=293 K, R = 5.8% for 1866 significant reflections. There are no unusual bond distances or angles. The triazole and two phenyl rings are planar. On the basis of packing considerations the possibility of intermolecular interactions playing a role in the reactivity of the starting material is ruled out.

Flow through a plateau border of cellular foam

Experimentally measured average velocities through plateau borders of stationary cellular foam, when compared with those calculated with the assumption of rigid Plateau Border walls, show that the assumption of rigid walls severely underestimates the velocities. An analysis of the situation wherein plateau border walls have velocities, as decided by the surface viscosity of the system, is presented here. The plateau border is idealized as a pipe of equilateral triangular cross-section with vertices of the triangle having zero velocity. The pertinent form of Navier-Stoke's equations with inhomogeneous boundary conditions and its solution through a procedure of successive approximations is presented in dimensionless form. The solution reduces to the known solution of slow steady flow through a triangular pipe, when surface viscosity is infinite. Results indicate that the assumption of rigid plateau border walls is valid only when value of the inverse of dimensionless surface viscosity is less than 0.044. Beyond that the assumption severely underestimates the flow and the effect of nonrigidity of the wall must be considered.

Solid-State Acyl Migration In Salicylamides - An X-Ray Study Of The Ortho-Propionyl And Normal-Propionyl Derivatives

In recent years there has been an upsurge of interest in the study of organic reactions in the solid state. It is now realised that the crystalline matrix provides an extra-ordinary spatial control on the initiation and progress of these reactions. Electronic and dipolar effects which are important in solution are replaced by structural and geometric effects in solids. These 'spatial' or 'topochemical' aspects are important in understanding the mechanistic details of the reaction. In our laboratory, the thermally induced acyl migration in salicylamides from 0- to N- position in the solid state has been under study (Scheme 1). The structures of the acetyl and benzoyl derivatives (Ia,IIa, Ib and IIb) have been reported.

Thermally Induced Acyl Migration in Salicylamides. 1. Structures of the Reactants, O-Acetylsalicylamide and O-Benzoylsalicylamide

O-Acetylsalicylamide (Ia), C9H9NO3, M r =179.18, monoclinic, P2Jc, a=8.155(5), b=8.571 (2), c= 13.092 (3)A, fl=99.54 (5) ° , V= 902.4(6)A 3, Z=4, Dm=l.31, Dx=l.319gcm -3, 2(Mo Ka) = 0.71069 A,/~ = 1.08 cm -1, F(000) = 376, T = 295 K, R = 0.076 for 1604 reflections. O-Benzoylsalicylamide (Ib), C14HtlNO 3, M,=241.2, monoclinic, P2t/e, a=9.423(1), b=5.116(1), e= 26.424 (2) A, fl= 103.97 (1)% V= 1236.2 (3)/~3, Z= 4, D~ = 1.28, D x = 1.296 gcm -3, ,;L(Cu Ks) = 1.5418 A, p = 7.71 cm-', F(000) = 504, T= 295 K, R =0.050 for 2115 reflections. The dihedral angles between the amide group and the benzene ring are 39.9 ° (Ia) and 37.9 ° (Ib), whereas between the acyl group and the benzene ring they are 78.1 ° (Ia) and 93.4 ° (Ib). The differences in the packing of the two structures are brought out in terms of the observed hydrogen-bonding patterns. Based on the crystallographic results, an intramolecular mechanism for the migration of the acyl group from the O to the N position is suggested in both compounds.

Rectangular universal cellular array

A rectangular universal cellular array consisting of cells having three inputs and one output is described. This array is based on the Reed-Muller canonical expansion of a switching function. Although the total number of external input pins required in this array is the same as that of a rectangular array proposed in the literature, the number of cells is very much less.

Studies on the mechanism of follitropin action at the cellular level

With a view toward understanding better the mechanism of action of follitropin, an attempt was made using granulosa cells obtained from intact immature estrogenized rats to study in short-term incubations the effect of highly purified ovine follitropin on the binding of the hormone to the cells and the associated aromatase response. A modified radioimmunoassay procedure has been used to monitor unlabeled physiologically fully active follitropin bound to the cell. A linear relationship between the actual amount of hormone bound to the cells and the estradiol produced in vitro has been established. The amount of ovine follitropin bound that can elicit a half-maximal response in estrogen production was calculated to be 400 pg. The number of follitropin binding sites per cell was 375 and the Kd of binding was 3.03 × 10−10 Image . By the addition of ovine follitropin antiserum at different time points of a 4-h incubation period, a continual need for follitropin support for estradiol production has been established.

Pentoxifylline induces hyperactivation and acrosome reaction in spermatozoa of golden hamsters: changes in motility kinematics

Pentoxifylline (PF) is used to enhance motility of spermatozoa from infertile human subjects. We have previously shown that 0.45 mM PF improved capacitation of spermatozoa and fertilization of oocytes in vitro in hamsters. The present study was carried out to assess PF- induced changes in motility kinematics of hamster spermatozoa by a computer-aided sperm analyser (CASA) and determine the timing of onset of hyperactivation (HA) and acrosome reaction (AR) in PF-treated spermatozoa. Motility kinematics were analysed by CASA for 0-8 h in the absence or presence of 0.45 mM PF in Tyrode's medium supplemented with lactate, pyruvate and polyvinyl alcohol (TLP-PVA) or in TLP-PVA with bovine serum albumin (TALP-PVA). Conventional assessment was also made on the percentage of motility and quality of motility of spermatozoa; values were expressed as sperm motility index (SMI). Both in TALP-PVA and TLP-PVA, PF markedly increased SMI, especially the quality of motility (P < 0.02) by 2-3 h which was sustained up to 6 h. The motility kinematic data of PF-treated spermatozoa in TALP-PVA showed that average path velocity, curvilinear velocity and amplitude of lateral head displacement significantly (P < 0.05) increased as early as 2 h, with the expected decrease in straightness (STR) and linearity (LIN). Similar changes were also observed with PF-treated spermatozoa in TLP-PVA. Moreover, the percentage of hyperactivated spermatozoa in PF-treated samples was significantly (P < 0.001) higher than the untreated control at 2 h. To determine whether PF could induce AR, independent of bovine serum albumin, quantitative AR was assessed by observing the presence or absence of acrosomal cap on viable spermatozoa. PF significantly (P < 0.001) increased the percentage of AR as early as 2 h, reaching maximum at 4 h both in TALP-PVA (P < 0.05) and in TLP-PVA (P < 0.001). These results show that, in hamsters, PF induces early onset (by 2 h) of HA and AR and increases the proportion of spermatozoa undergoing physiological maturation.

Arabidopsis thaliana J-class heat shock proteins: cellular stress sensors

Plants are sessile organisms that have evolved a variety of mechanisms to maintain their cellular homeostasis under stressful environmental conditions. Survival of plants under abiotic stress conditions requires specialized group of heat shock protein machinery, belonging to Hsp70:J-protein family. These heat shock proteins are most ubiquitous types of chaperone machineries involved in diverse cellular processes including protein folding, translocation across cell membranes, and protein degradation. They play a crucial role in maintaining the protein homeostasis by reestablishing functional native conformations under environmental stress conditions, thus providing protection to the cell. J-proteins are co-chaperones of Hsp70 machine, which play a critical role by stimulating Hsp70s ATPase activity, thereby stabilizing its interaction with client proteins. Using genome-wide analysis of Arabidopsis thaliana, here we have outlined identification and systematic classification of J-protein co-chaperones which are key regulators of Hsp70s function. In comparison with Saccharomyces cerevisiae model system, a comprehensive domain structural organization, cellular localization, and functional diversity of A. thaliana J-proteins have also been summarized. Electronic supplementary material The online version of this article (doi:10.1007/s10142-009-0132-0) contains supplementary material, which is available to authorized users.

Cellular Automata Models of Traffic Behavior in Presence of Speed Breaking Structures

In this article, we study traffic flow in the presence of speed breaking structures. The speed breakers are typically used to reduce the local speed of vehicles near certain institutions such as schools and hospitals. Through a cellular automata model we study the impact of such structures on global traffic characteristics. The simulation results indicate that the presence of speed breakers could reduce the global flow under moderate global densities. However, under low and high global density traffic regime the presence of speed breakers does not have an impact on the global flow. Further the speed limit enforced by the speed breaker creates a phase distinction. For a given global density and slowdown probability, as the speed limit enforced by the speed breaker increases, the traffic moves from the reduced flow phase to maximum flow phase. This underlines the importance of proper design of these structures to avoid undesired flow restrictions.

Cellular and molecular regulation of mammalian blastocyst hatching

In mammals including humans, failure in blastocyst hatching and implantation leads to early embryonic loss and infertility. Prior to implantation, the blastocyst must hatch out of its acellular glycoprotein coat, the zona pellucida (ZP). The phenomenon of blastocyst hatching is believed to be regulated by (i) dynamic cellular components such as actin-based trophectodermal projections (TEPs), and (ii) a variety of autocrine and paracrine molecules such as growth factors, cytokines and proteases. The spatio-temporal regulation of zona lysis by blastocyst-derived cellular and molecular signaling factors is being keenly investigated. Our studies show that hamster blastocyst hatching is acelerated by growth factors such as heparin binding-epidermal growth factor and leukemia inhibitory factor and that embryo-derived, cysteine proteases including cathepsins are responsible for blastocyst hatching. Additionally, we believe that cyclooxygenase-generated prostaglandins, estradiol-17 beta mediated estrogen receptor-alpha signaling and possibly NF kappa B could be involved in peri-hatching development. Moreover, we show that TEPs are intimately involved with lysing ZP and that the TEPs potentially enrich and harbor hatching-enabling factors. These observations provide new insights into our understanding of the key cellular and molecular regulators involved in the phenomenon of mammalian blastocyst hatching, which is essential for the establishment of early pregnancy.

Inhibition of Tyrosine Phosphorylation of Sperm Flagellar Proteins, Outer Dense Fiber Protein-2 and Tektin-2, Is Associated With Impaired Motility During Capacitation of Hamster Spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.