Expression of proteins FGFR3, PI3K, AKT, p21Waf1/Cip1 and cyclins D1 and D3 in patients with T1 bladder tumours: clinical implications and prognostic significance. / Expresión de las proteínas FGFR3, PI3K, AKT, p21Waf1/Cip1 y ciclinas D1 y D3 en pacientes con tumores de vejiga T1: implicaciones clínicas y significado pronóstico.

OBJECTIVE: To determine the differential protein expression of biomarkers FGFR3, PI3K (subunits PI3Kp110α, PI3KClassIII, PI3Kp85), AKT, p21Waf1/Cip1 and cyclins D1 and D3 in T1 bladder cancer versus healthy tissue and to study their potential role as early recurrence markers. MATERIAL AND METHOD: This is a prospective study that employed a total of 67 tissue samples (55 cases of T1 bladder tumours that underwent transurethral resection and 12 cases of adjacent healthy mucosa). The protein expression levels were assessed using Western blot, and the means and percentages were compared using Student's t-test and the chi-squared test. The survival analysis was conducted using the Kaplan-Meier method and the log-rank test. RESULTS: Greater protein expression was detected for FGFR3, PI3Kp110α, PI3KClassIII, cyclins D1 and D3 and p21Waf1/Cip1 in the tumour tissue than in the healthy mucosa. However, these differences were not significant for PI3Kp85 and AKT. We observed statistically significant correlations between early recurrence and PI3Kp110α, PI3KClassIII, PI3Kp85 and AKT (P=.003, P=.045, P=.050 and P=.028, respectively), between the tumour type (primary vs. recurrence) and cyclin D3 (P=.001), between the tumour size and FGFR3 (P=.035) and between multifocality and cyclin D1 (P=.039). The survival analysis selected FGFR3 (P=.024), PI3Kp110α (P=.014), PI3KClassIII (P=.042) and AKT (P=.008) as markers of early-recurrence-free survival. CONCLUSIONS: There is an increase in protein expression levels in bladder tumour tissue. The overexpression of FGFR3, PI3Kp110α, PI3KClassIII and AKT is associated with increased early-recurrence-free survival for patients with T1 bladder tumours.

Purification and properties of beta-galactosidase from Aspergillus nidulans.

Beta-Galactosidase from mycelial extract of Aspergillus nidulans has been purified by substrate affinity chromatography and used to obtain anti-beta-galactosidase polyclonal antibodies. A. nidulans growing in lactose as carbon source synthesizes one active form of beta-galactosidase which seems to be a multimeric enzyme of 450 kDa composed of monomers with 120 and 97 kDa. Although the enzyme was not released to the culture medium, some enzymatic activity was detected in a cell-wall extract, thus suggesting that it can be an extracellular enzyme. Beta-Galactosidase of A. nidulans is a very unstable enzyme with an optimum pH value of 7.5 and an optimum temperature of 30 degrees C. It was only active against beta-galactoside substrates like lactose and p-nitrophenyl-beta-D-galactoside (PNPG).

Study of a hydrocarbon-utilizing and emulsifier-producing Acinetobacter calcoaceticus strain isolated from heating oil.

Twenty bacterial strains were isolated from a sample of contaminated heating oil and screened for their ability to use petroleum and several common fuels as the sole source of carbon and energy. One of the isolates, named MM5, was able to grow on petroleum derivatives and brought about an emulsification of those compounds. Gas chromatography studies showed that strain MM5 was able to degrade hydrocarbons of heating oil. MM5 has been tentatively identified as a strain of Acinetobacter calcoaceticus. The fine structure of MM5 was examined by transmission electron microscopy. Incubation in the presence of hydrocarbon substrates resulted in the development of intracellular electron-transparent inclusions. These structures were absent in the non-hydrocarbon cultures studied.

Membrane protein molecules of transmissible gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of the virion.

The binding domains of four monoclonal antibodies (MAbs) specific for the M protein of the PUR46-MAD strain of transmissible gastroenteritis coronavirus (TGEV) have been located in the 46 carboxy-terminal amino acids of the protein by studying the binding of MAbs to recombinant M protein fragments. Immunoelectron microscopy using these MAbs demonstrated that in a significant proportion of the M protein molecules, the carboxy terminus is exposed on the external surface both in purified viruses and in nascent TGEV virions that recently exited infected swine testis cells. The same MAbs specifically neutralized the infectivity of the PUR46-MAD strain, indicating that the C-terminal domain of M protein is exposed on infectious viruses. This topology of TGEV M protein probably coexists with the structure currently described for the M protein of coronaviruses, which consists of an exposed amino terminus and an intravirion carboxy-terminal domain. The presence of a detectable number of M protein molecules with their carboxy termini exposed on the surface of the virion has relevance for viral function, since it has been shown that the carboxy terminus of M protein is immunodominant and that antibodies specific for this domain both neutralize TGEV and mediate the complement-dependent lysis of TGEV-infected cells.

Ultrastructure of human astrovirus serotype 2.

The ultrastructure of human astrovirus serotype 2 (H-Ast2) grown in cell culture was analysed by electron microscopy of thin sections and negatively stained preparation. Infected LLCMK2 cells, as visualized in thin sections, contained cytoplasmic aggregates of dense or hollow-cored particles that aggregated in quasicrystalline arrays and were specifically labelled using a rabbit polyclonal anti-Ast2 antiserum. H-Ast2 particles from the supernatant of infected LLCMK2 cells in thin sections after flat- embedding were similar in size to intracellular virions. In negatively stained preparations, these virus particles had an external diameter of 41 nm and exhibited a well defined layer of surface spikes. Pentagonal and hexagonal contours were occasionally visible, and probably correspond to the projections of icosahedral structures. Star-like morphologies and particles with surface triangular hollows were seen in dark areas of the preparations only after a short treatment of the viruses of pH 10. Incubation of the viruses at pH 10.5 induced a rapid disassembly of the virus particles. The finding that the particles with icosahedral geometry and surface spikes are fully infective allows an alternative morphological model to the traditional one for astroviruses to be proposed.

Purification and molecular properties of an alpha-galactosidase synthesized and secreted by Aspergillus nidulans.

alpha-Galactosidases from mycelial extract and culture filtrate of Aspergillus nidulans have been purified to homogeneity and utilised to obtain polyclonal antibodies anti-alpha-galactosidase. The enzymatic characteristics and the cross reactivity of the antibodies suggest that alpha-galactosidases isolated from the two sources were the same enzyme. Thus, A. nidulans synthesized and secreted only one enzymatic form of alpha-galactosidase which is a multimeric enzyme of 370 kDa composed of four monomers of 87 kDa and a pI of 6.3. The optimum temperature of activity was 50 degrees C and the optimum pH 4-5. The enzyme was stable over a wide range of pH but quite unstable to temperature. alpha-Galactosidase of A. nidulans is a very specific enzyme, it is active only on p-nitrophenyl-alpha-D-galactoside (PNPG), melibiose and raffinose. When PNPG was utilised as substrate melibiose, raffinose, galactose and glucose were competitive inhibitors of the activity.