Short-term culturing of low-grade superficial bladder transitional cell carcinomas leads to changes in the expression levels of several proteins involved in key cellular activities

Fresh, superficial transitional cell carcinomas (TCCs) of low-grade atypia (3 grade I, Ta; 6 grade II, Ta), as well as primary cultures derived from them were labeled with [35S]methionine for 16 h, between 2 and 6 days after inoculation. Whole protein extracts were subjected to IEF (isoelectric focusing) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by autoradiography. Proteins were identified by a combination of proteomic technologies that included microsequencing, mass spectrometry, 2-D PAGE immunoblotting and comparison with the bladder TCC protein database available on the internet (http://biobase.dk/cgi-bin/celis). Comparison of the IEF 2-D gel protein profiles of fresh tumors and their primary cultures showed that the overall expression profiles were strikingly similar, although differing significantly in the levels of several proteins whose rate of synthesis was differentially regulated in at least 85% of the tumor/culture pairs as a result of the short-term culturing. Most of the proteins affected by culturing were upregulated and among them we identified components of the cytoskeleton (keratin 18, gelsolin and tropomyosin 3), a molecular chaperone (hsp 28), aldose reductase, GST pi, metastasin, synuclein, the calreticulin precursor and three polypeptides of unknown identity. Only four major proteins were downregulated, and these included two fatty acid-binding proteins (FABP:FABP5 and A-FABP) which are thought to play a role in growth control, the differentiation-associated keratin 20, and the calcium-binding protein annexin V. Proteins that were differentially regulated in only some of the cultured tumors included alpha-enolase, triosphosphate isomerase, members of the 14-3-3 family, hnRNPs F and H, PGDH, hsp (heat-shock protein) 60, BIP, the interleukin-1 receptor antagonist, the nucleolar protein B23, as well as several proteins of yet unknown identity. The suitability of in vitro bladder tumor culture models to study complex biological phenomena such as malignancy and invasion is discussed.     

An evaluation of the time dependence of the anisotropy of the water diffusion tensor in acute human ischemia

We have performed MRI examinations to determine the water diffusion tensor in the brain of six patients who were admitted to the hospital within 12 h after the onset of cerebral ischemic symptoms. The examinations have been carried out immediately after admission, and thereafter at varying intervals up to 90 days post admission. Maps of the trace of the diffusion tensor, the fractional anisotropy and the lattice index, as well as maps of cerebral blood perfusion parameters, were generated to quantitatively assess the character of the water diffusion tensor in the infarcted area. In patients with significant perfusion deficits and substantial lesion volume changes, four of six cases, our measurements show a monotonic and significant decrease in the diffusion anisotropy within the ischemic lesion as a function of time. We propose that retrospective analysis of this quantity, in combination with brain tissue segmentation and cerebral perfusion maps, may be used in future studies to assess the severity of the ischemic event.     

Formation of dielectric layers and charge regulation in protein adsorption at biomimetic interfaces

Protein charge is an important parameter in the understanding of protein interactions and function. Proteins are subject to dynamic charge regulation, that is, the influence of the local environment (such as charged interfaces and biopolymers) on protein charge. Charge regulation is governed by differences in the dielectric and electrostatic environment between adsorbed protein and the free protein in bulk solution. In this work protein charge regulation is addressed experimentally by employing electrochemistry at interfaces between two immiscible electrolyte solutions (ITIES) as well as theoretically by developing a new protein adsorption model at ITIES. Electrochemistry at ITIES is shown to be particularly well suited to study protein charge regulation as the adsorbed protein experiences a different dielectric environment compared to the bulk phase and the external control of the water/oil potential difference allows systematic studies on how potential induced ion gradients affect protein charge. The theoretical model incorporates all the features of the experimental system and specifically takes into account protein charge regulation at ITIES as well as the impact of the formation of dielectric layers on the experimentally observed impedance. The model parameters include the protein charge-pH profile, bulk pH, and the overall potential difference. It is shown that the formation of a dielectric layer and the associated charge regulation are the main factors dictating the observed experimental behavior. Finally, the theoretical model is used to interpret literature results, and the consistency between the model and the relatively large data set suggests that the model may be used more generally for understanding and predicting protein adsorption.     

Characterization of oil-free and oil-loaded liquid-crystalline particles stabilized by negatively charged stabilizer citrem

The present study was designed to evaluate the effect of the negatively charged food-grade emulsifier citrem on the internal nanostructures of oil-free and oil-loaded aqueous dispersions of phytantriol (PHYT) and glyceryl monooleate (GMO). To our knowledge, this is the first report in the literature on the utilization of this charged stabilizing agent in the formation of aqueous dispersions consisting of well-ordered interiors (either inverted-type hexagonal (H(2)) phases or inverted-type microemulsion systems). Synchrotron small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM) were used to characterize the dispersed and the corresponding nondispersed phases of inverted-type nonlamellar liquid-crystalline phases and microemulsions. The results suggest a transition between different internal nanostructures of the aqueous dispersions after the addition of the stabilizer. In addition to the main function of citrem as a stabilizer that adheres to the surface of the dispersed particles, it has a significant impact on the internal nanostructures, which is governed by the following factors: (1) its penetration between the hydrophobic tails of the lipid molecules and (2) its degree of incorporation into the lipid-water interfacial area. In the presence of citrem, the formation of aqueous dispersions with functionalized hydrophilic domains by the enlargement of the hydrophilic nanochannels of the internal H(2) phase in hexosomes and the hydrophilic core of the L(2) phase in emulsified microemulsions (EMEs) could be particularly attractive for solubilizing and controlling the release of positively charged drugs.     

Complexation of tauro- and glyco-conjugated bile salts with three neutral beta-CDs studied by ACE

Complexation of the bile salts (BS) taurocholate, tauro-beta-muricholate, taurodeoxycholate, taurochenodeoxycholate, glycocholate, glycodeoxycholate, and glycochenodeoxycholate common in rat, dog, and man with natural beta-CD and the chemically modified beta-CDs 2-hydroxypropyl-beta-CD and 2-O-methyl-beta-CD was studied using mobility shift ACE. The CDs were selected due to their frequent use in preformulation and drug formulation as oral excipients for the solubilization of drug substances with low aqueous solubility. ACE was demonstrated to be a feasible and efficient technique for investigation of the interactions between BS and beta-CDs. All the investigated BS possessed affinity for the three CDs with stability constants ranging from 2x10(3) to 4x10(5) M(-) (1). The requirements and assumptions related to the use of ACE for estimating high affinity stability constants were discussed. The extent and pattern of hydroxylation significantly influenced the affinity of the glyco- and tauro-conjugated BS toward the beta-CDs (chenodeoxycholates > deoxycholates > cholates) whereas the nature of the beta-CD derivatization and BS conjugation played a minor role only. The results indicate that displacement of drug substances from beta-CD inclusion complexes is likely to occur in the small intestine where BS are present potentially influencing drug bioavailability.     

Psoriasin (S100A7): a putative urinary marker for the follow-up of patients with bladder squamous cell carcinomas

To search for bladder squamous cell carcinoma markers that are released to the urine a blind and systematic analysis of the protein profiles of fresh tumors, their secreted proteins, as well as the patient's urine was carried out using state-of-the-art proteomic technology. We review the data concerning the putative marker psoriasin (S100A7), which, alone or in combination with other biomarkers, may be valuable for the noninvasive follow-up of patients bearing these tumors.     

Quantitative perfusion imaging in carotid artery stenosis using dynamic susceptibility contrast-enhanced magnetic resonance imaging

Quantitative, multislice dynamic susceptibility contrast-enhanced MRI perfusion measurements were used to determine the patterns of cerebral blood flow (CBF), cerebral blood volume (CBV), mean transit time (MTT), and normalized first moment of the tissue deltaR2-time curve (N) in 11 subjects with carotid artery occlusion or stenosis. MTT correlated with degree of carotid stenosis, whereas a range of alterations in CBF and CBV were found presumably reflecting variables degrees of collateral flow. There was no significant correlation between MRI and SPET flow perfusion measurements, with increasing disparity between the two techniques at higher inter-hemispheric flow ratios. The effect of obtaining the arterial input function (AIF) from the middle cerebral artery (MCA) ipsilateral or contralateral to the stenosis was determined. Despite the use of an AIF from the MCA, which is distal to the circle of Willis, and hence the major sources of collateral supply, there was still some extra dispersion of the contrast agent bolus due to differences in arrival time.