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The role of hormones in the pathogenesis of tendinopathy is not well recognised, even though the use of anabolic steroids is correlated with a higher incidence of spontaneous tendon ruptures. The aim of this study was to investigate the effects of dihydrotestosterone (DHT) on human tenocyte cultures from the intact supraspinatus tendon of male subjects. Cultured human tenocytes were seeded into culture plates at a density of 5 x 10(4) cells per well and incubated for 24 h. Then, 10(-9) M-10(-7) M DHT or Dulbecco's modified Eagle's medium (DMEM) only (control) was added to the culture plate wells. Cell morphology assessment and cell proliferation tests were performed 48, 72 and 96 h after DHT treatment. DHT-treated tenocytes showed an increased proliferation rate at DHT concentration higher than 10(-8) M. Differences in cell numbers between control and DHT-treated cells were statistically significant (P < 0.05) after 48 and 72 h of treatment with DHT concentrations of 10(-8) and 10(-7) M. The tenocytes treated with DHT (10(-8) and 10(-7) M) became more flattened and polygonal compared to control cells that maintained their fibroblast-like appearance during the experiment at each observation time. In conclusion, in vitro, progressive increasing concentration of DHT at doses greater than 10(-8) M had direct effects on male human tenocytes, increasing cell number after 48 and 72 h of treatment, and leading to a dedifferentiated phenotype after 48 h of treatment. This effect can be important during tendon-healing and repair, when active proliferation is required. Our results represent preliminary evidence for a possible correlation between testosterone abuse and shoulder tendinopathy.

Bilirubin-IX-alpha (BR) is an endogenous molecule with a strong antioxidant feature due to its ability to scavenge free radicals. In this paper, we demonstrated that BR, at concentrations close to those found within the cell (0.1-2.5 microM), acted as a denitrosylating agent and increased the release of nitric oxide from S-nitrosoglutathione (GSNO) and S-nitrosocysteine (SNOC) (2.5 microM). The complexation of BR with saturating concentrations of human serum albumin (HSA, 2.5 microM) did not further increase nitric oxide release from GSNO and SNOC. At concentrations similar to those reached in plasma (5-20 microM), BR denitrosylated S-nitroso-HSA (2.5 microM), the main circulating S-nitrosothiol, and this effect was potentiated by the complexation of BR with saturating HSA (20 microM). Furthermore, the product(s) of the reaction between nitric oxide and BR were identified. Ultraviolet and mass spectrometry analysis revealed that nitric oxide binds to BR forming a N-nitroso derivative (BR-nitric oxide) with extinction coefficients of 1.393 mM(-1)cm(-1) and 2.254 mM(-1)cm(-1) in methanol and NaOH, respectively. The formation of BR-nitric oxide did not occur only in a reconstituted system, but was confirmed in rat fibroblasts exposed to pro-oxidant stimuli. These results provided novel insights on the antioxidant characteristic of BR through its interaction with nitric oxide, a gaseous neurotransmitter with a well-known dual effect, namely neuroprotective under physiological conditions or neurotoxic if produced in excess, and proposed BR-nitric oxide as a new biomarker of oxidative/nitrosative stress.

Study Design: Human osteoblast cultures were exposed to a very low intensity static magnetic fields (SMF) to investigate its effects on osteoblast growth and differentiation., Objective: Analysis of the effects of periprosthetic SMF on the growth and differentiation of human osteoblast cell cultures in vitro., Summary of Background Data: The effects of pulsed electromagnetic fields (PEMF) on cell proliferation, especially in human osteoblast-like cells is well described, whereas few data are available on the effects of SMF on osteoblast cell culture. We previously demonstrated that the proliferation of human osteoblast cultures is reduced when cells are exposed to a continuous low intensity SMF comparable to the one that occurs around metal devices (Ti spinal implant) because of the generation of electric currents between the screw (Ti6Al4V) and the rod (Ti)., Methods: Primary osteoblastic cells were isolated from a human femoral head. Osteoblast cultures were exposed to SMF and alkaline phosphatase activity was evaluated in the osteoblast cell cultures at different time points. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate mRNA expression levels of osteocalcin, Runx2, and collagen I genes., Results: The SMF-treated cells showed a progressive increase in the alkaline phosphatase activity which, however, remained always lower than the one observed in the control group at each observation time (72 hours, 7 and 14 days). RT-PCR demonstrated that Runx2 and collagen I mRNA were downregulated following SMF stimulation, whereas no change in osteocalcin mRNA was observed., Conclusion: Continuous low-intensity electromagnetic field comparable to the one that generates around metal devices because of the generation of corrosion currents inhibits osteoblasts differentiation pattern and might contribute at least in part to a decrease in periprosthetic bone formation occurring in vivo.

Objectives: Cervical and vulvar cancers develop through well-defined precursor lesions but their exact pathogenesis is still unknown. The dystroglycan complex is a transmembrane glycoprotein that forms a continuous link from the extracellular matrix to the actin cytoskeleton. Deregulated expression of dystroglycan has been reported in human malignancies and related to tumor differentiation and aggressiveness. In this study, expression of dystroglycan was evaluated in the multistep cervical and vulvar tumorigenesis., Methods: Expression of the dystroglycan complex was evaluated by immunostaining in lesions representing different stages of vulvar and cervical tumorigenesis using a monoclonal antibody which recognizes carbohydratic epitopes on the alpha-dystroglycan subunit., Results: alpha-dystroglycan was constantly detected in normal cervical epithelium with a mean percentage of positive cells higher than 80%. A progressive significant reduction in the mean percentage of positive cells was observed in low (67%) and high grade SIL (14%) and in invasive carcinomas (2.6%) of the cervix. In cancers, no differences were observed in terms of percentage of positive cells when cases were stratified according with either tumor grade or stage. A progressive significant reduction in the mean percentage of positive cells was also observed from normal vulvar epithelium (90%) to VIN1 (66%), VIN2 (28%) and invasive vulvar carcinomas (22%). No significant decrease in the alpha-dystroglycan staining was observed in squamous cell hyperplasia lesions (85%) while lichen sclerosus displayed a percentage of positive cells (47%) significantly lower than normal epithelium., Conclusions: Detection of alpha-dystroglycan is frequently lost in human cervical and vulvar tumorigenesis and further studies are warranted to verify whether evaluation of this molecule might serve as marker of risk progression of preneoplastic lesions and to better understand its significance in terms of cancer development.

Fusidic acid (FA) and helvolic acid (HA) belong to a small family of naturally occurring steroidal antibiotics known as fusidanes. FA was studied for its ability to alter the biochemical properties supported by elongation factor 2 isolated from the archaeon Sulfolobus solfataricus (SsEF-2). Both poly(Phe) synthesis and ribosome-dependent GTPase (GTPase(r)) were progressively impaired by increasing concentrations of FA up to 1 mM, whereas no effect was measured in the intrinsic GTPase of SsEF-2 triggered by ethylene glycol in the presence of barium chloride (GTPase(g)). The highest antibiotic concentration caused inhibition of either poly(Phe) synthesis or GTPase(r) only slightly above 50%. A greater response of SsEF-2 was observed when HA was used instead of FA. HA caused even a weak impairment of GTPase(g). A mutated form of SsEF-2 carrying the L452R substitution exhibited an increased sensitivity to fusidane inhibition in either poly(Phe) synthesis or GTPase(r). Furthermore, both FA and HA were able to cause impairment of GTPase(g). The antibiotic concentrations leading to 50% inhibition (IC(50)) indicate that increased fusidane responsiveness due to the use of HA or the L452R amino acid replacement is mutually independent. However, their combined effect decreased the IC(50) up to 0.1 mM. Despite the difficulties in reaching complete inhibition of the translocation process in S. solfataricus, these findings suggest that fusidane sensibility is partially maintained in the archaeon S. solfataricus. Therefore, it is likely that SsEF-2 harbors the structural requirements for forming complexes with fusidane antibiotics. This hypothesis is further evidenced by the observed low level of impairment of GTPase(g), a finding suggesting a weak direct interaction between the archaeal factor and fusidanes even in the absence of the ribosome. However, the ribosome remains essential for the sensitivity of SsEF-2 toward fusidane antibiotics.

Valine 114 in the D(109)AAILVVA sequence of elongation factor 1alpha from the archaeon Sulfolobus solfataricus (SsEF-1alpha) was substituted with an acidic (V114E), basic (V114K), or cavity-forming (V114A) residue, and the effects on the biochemical properties of the factor were investigated. This sequence is well-conserved among most of eukaryal and eubacterial counterparts, and in the three-dimensional structure of SsEF-1alpha, V114 is located in a hydrophobic pocket near the first GDP-binding consensus sequence G(13)XXXXGK[T,S] [Vitagliano, L., Masullo, M., Sica, F., Zagari, A., and Bocchini, V. (2001) EMBO J. 20, 5305-5311]. These mutants displayed functions absent in the wild-type factor. In fact, although they exhibited a rate in poly(Phe) incorporation almost identical to that of SsEF-1alpha, V114K and V114A exhibited an affinity for GDP and GTP higher and a capability to bind heterologous aa-tRNA stronger than that elicited by SsEF-1alpha but similar to that of eubacterial EF-Tu. V114E instead displayed not only a weaker binding capability for aa-tRNA but also a lower affinity for GDP. The intrinsic GTPase activity of V114E was drastically reduced compared to those of SsEF-1alpha, V114K, and V114A. Interestingly, the decreased intrinsic GTPase activity of V114E was partially restored by kirromycin, an effect already observed for the G13A mutant of SsEF-1alpha [Masullo, M., Cantiello, P., de Paola, B., Catanzano, F., Arcari, P., and Bocchini, V. (2002) Biochemistry 41, 628-633]. Finally, the V114A substitution showed only a marginal effect on both the thermostability and thermophilicity of SsEF-1alpha, whereas V114K and V114E replacements strongly destabilized the molecule.

The G13A substitution in the G13XXXXGK[T,S] consensus sequence of the elongation factor 1 alpha from the archaeon Sulfolobus solfataricus (SsEF-1 alpha) was introduced in order to study the reasons for selective differences found in the homologous consensus element AXXXXGK[T,S] of the other elongation factor EF-2 or EF-G. In a previous work, it was shown that the main effect of the A26G mutation was the activation of the intrinsic GTPase of SsEF-2 [De Vendittis, E., Adinolfi, B. S., Amatruda, M. R., Raimo, G., Masullo, M., and Bocchini, V. (1994) Eur. J. Biochem. 262, 600-605]. In this work, we found that, compared to the wild-type factor (SsEF-1 alpha wt), G13ASsEF-1 alpha shows (i) a reduced rate of [(3)H]Phe polymerization that was probably due to its reduced ability to form a ternary complex with heterologous aa-tRNA and (ii) a reduced intrinsic GTPase activity that was stimulated by high concentrations of NaCl (GTPase(Na)) [Masullo, M., De Vendittis, E., and Bocchini, V. (1994) J. Biol. Chem. 269, 20376-20379]. In addition, G13ASsEF-1 alpha showed an increased affinity for GDP and GTP. Surprisingly, the decreased intrinsic GTPase(Na) of G13ASsEF-1 alpha can be partially restored by kirromycin, an effect not found for SsEF-1 alpha wt. The temperature inducing a 50% denaturation of G13ASsEF-1 alpha was somewhat lower (-5 degrees C) than that of SsEF-1 alpha wt, and the decrease in its thermophilicity was slightly more accentuated (-10 degrees C). These results indicate that the nature of the residue in position 13 is important for the functional and physical properties of SsEF-1 alpha.

A recombinant chimeric elongation factor containing the region of EF-1 alpha from Sulfolobus solfataricus harboring the site for GDP and GTP binding and GTP hydrolysis (SsG) and domains M and C of Escherichia coli EF-Tu (EcMC) was studied. SsG-EcMC did not sustain poly(Phe) synthesis in either S. solfataricus or E. coli assay system. This was probably due to the inability of the chimera to interact with aa-tRNA. The three-dimensional modeling of SsG-EcMC indicated only small structural differences compared to the Thermus aquaticus EF-Tu in the ternary complex with aa-tRNA and GppNHp, which did not account for the observed inability to interact with aa-tRNA. The addition of the nucleotide exchange factor SsEF-1 beta was not required for poly(Phe) synthesis since the chimera was already able to exchange [(3)H]GDP for GTP at very high rate even at 0 degrees C. Compared to that of SsEF-1 alpha, the affinity of the chimera for guanine nucleotides was increased and the k(cat) of the intrinsic GTPase was 2-fold higher. The heat stability of SsG-EcMC was 3 and 13 degrees C lower than that displayed by SsG and SsEF-1alpha, respectively, but 30 degrees C higher than that of EcEF-Tu. This pattern remained almost the same if the melting curves of the proteins being investigated were considered instead. The chimeric elongation factor was more thermophilic than SsG and SsEF-1 alpha up to 70 degrees C; at higher temperatures, inactivation occurred.

Guanidine-induced denaturation of Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli, Sbetagly, was investigated at pH 6.5 and 25 degreesC by means of circular dichroism and fluorescence measurements. The process proved reversible when the protein concentration was lower than 0.01 mg mL-1. Moreover, the transition curves determined by fluorescence did not coincide with those determined by circular dichroism, and the GuHCl concentration corresponding at half-completion of the transition increased on raising the protein concentration in the range 0.001-0.1 mg mL-1. Gel filtration chromatography experiments showed that, in the range 2-4 M GuHCl, there was an equilibrium among tetrameric, dimeric, and monomeric species. These findings, unequivocally, indicated that the guanidine-induced denaturation of Sbetagly was not a two-state transition with concomitant unfolding and dissociation of the four subunits. A mechanism involving a dimeric intermediate species was proposed and was able to fit the experimental fluorescence intensity transition profiles, allowing the estimation of the total denaturation Gibbs energy change at 25 degreesC and pH 6.5. This figure, when normalized for the number of residues, showed that, at room temperature, Sbetagly has a stability similar to that of mesophilic proteins.