Endothelial follicle stimulating hormone receptor in primary kidney cancer correlates with subsequent response to sunitinib

Sunitinib is an anti-angiogenic receptor tyrosine kinase inhibitor used to treat advanced metastatic renal cell carcinoma and other types of cancer. Sutent is effective in only approximately 70% of clear cell renal cell carcinoma (CCRCC) patients, has significant adverse side effects and no method is available to predict which patients will not respond. Our purpose was to explore the possibility of introducing an effective prediction method based on a marker of the tumour vasculature, the follicle stimulating hormone receptor (FSHR). Fifty patients diagnosed with advanced metastatic CCRCC have been subjected to surgery for removal of the primary tumour and were subsequently treated with sunitinib. After three months of therapy the patients were categorized as 'responsive', 'stable' or 'non-responsive' based on the RECIST guidelines. The blood vessel density and the percentage of FSHR-positive vessels were determined by immunofluorescence on sections from the primary tumours removed by surgery, prior to the sunitinib treatment. The percentage of FSHR-stained vessels was on average fivefold higher for the patients who responded to the treatment in comparison with the stable group and almost eightfold higher than in the non-responsive group. The percentage allowed the detection of responders with 87-100% sensitivity and specificity. No significant differences were detected in the total density of vessels among the three groups. The data suggest that FSHR expression levels in the blood vessels of CCRCC primary tumours can be used to predict, with high sensitivity and specificity, the patients who will respond to sunitinib therapy.     

Alternating access mechanism in the POT family of oligopeptide transporters

Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton‐dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3‐Å resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge‐like movement within the C‐terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide‐binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.     

Photoinactivation of the photosynthetic electron transport chain by accumulation of over-saturating light pulses given to dark adapted pea leaves

The effect of cumulative over-saturating pulses (OSP) of white light (1 s, >10 000 μmol photons m⁻² s⁻¹), applied every 20 min on pea leaves, was investigated during a complete diurnal cycle of 24 h. In dark-adapted leaves, this treatment leads to a progressive decline of the optimum Photosystem II (PS II) quantum yield. Continuous low background light (except far-red light) had a protective effect against this OSP-induced photoinactivation. The lack of far-red effect could be due to its absorption mainly in PS I and not in PS II, but could be also due to the general low absorption in this wavelength region. The photoinactivation was enhanced in leaves that had been previously infiltrated with chloramphenicol. The quantum yield of CO₂ assimilation, but not its maximal capacity, was inhibited by the OSP treatment. The most spectacular effects observed, in addition to an irreversible quenching of Fm, was a strong inhibition of Q_A ⁻ reoxidation revealed by a large increase in the Fs level and consequently by a decrease of ΔF/Fm′. Under such conditions, we observed that the electron flow deduced from ΔF/Fm′ underestimated the real electron flow to CO₂. Time-resolved Chlorophyll a fluorescence measurements showed that the reduced capacity of Q_A ⁻ reoxidation in OSP treated leaves was accompanied by the appearance of a 4.7 ns component attributed to PS II charge recombination. We suggest that a modification at the Q_B site may influence the redox potential of Q_A/Q_A ⁻, facilitating the reversion of the primary charge separation. In addition, a 1.2 ns fluorescence component accumulated, which appeared to be responsible for the underestimation of PS II electron flow. The observed photoinactivation seemed to be different from the photoinhibition often described in the literature, which occurs under continuous light. This revised version was published online in June 2006 with corrections to the Cover Date.     

Authentic fibroblast matrix in dermal equivalents normalises epidermal histogenesis and dermoepidermal junction in organotypic co-culture

Besides medical application as composite skin grafts, in vitro constructed skin equivalents (SEs) or organotypic co-cultures represent valuable tools for cutaneous biology. Major drawbacks of conventional models, employing collagen hydrogels as dermal equivalents (DEs), are a rather poor stability and limited life span, restricting studies to early phases of skin regeneration. Here we present an improved stabilised in vitro model actually providing the basis for skin-like homeostasis. Keratinocytes were grown on dermal equivalents (DEs) reinforced by modified hyaluronic acid fibres (Hyalograft-3D) and colonised with skin fibroblasts, producing genuine dermis-type matrix. These SEs developed a superior epidermal architecture with regular differentiation and ultrastructure, which occurred also faster than in SEs based on collagen-DEs. Critical aspects of differentiation, still unbalanced in early stages, were perfectly re-normalised, most strikingly the co-expression of keratins K1/K10 and downregulation of regeneration-associated keratins such as K16. The restriction of integrin and K15 distribution as well as keratinocyte proliferation to the basal layer underlined the restored tissue polarity, while the drop of growth rates towards physiological levels implied finally accomplishment of homeostasis. This correlated to faster basement membrane (BM) formation and ultrastructurally defined dermo-epidermal junction including abundant anchoring fibrils for strong tissue connection. Whereas the fibroblasts in the scaffold initially secreted a typical provisional regenerative matrix (fibronectin, tenascin), with time collagens of mature dermis (type I and III) were accumulating giving rise to an in vivo-like matrix with regularly organised bundles of striated collagen fibrils. In contrast to the more catabolic state in conventional DEs, the de novo reconstruction of genuine dermal tissue seemed to be a key element for maintaining prolonged normal keratinocyte proliferation (followed up to 8 wks), fulfilling the criteria of tissue-homeostasis, and possibly providing a stem cell niche.     

Receptor-mediated transcytosis of follicle-stimulating hormone through the rat testicular microvasculature

Accumulated data suggest that endothelial cells express specific receptors for several peptide and (glyco)protein hormones that may transport hormones across the cell to be delivered to the interstitial fluid and tissue target cells. Surprisingly, very little information is available on the actual endothelial organelles involved in this cellular process. In the present study the transfer of follicle-stimulating hormone (FSH) through the endothelial barrier of rat testes was examined by analysing the binding and transport of gold-tagged recombinant human (rh)FSH under various conditions using electron microscopy. At 4 degrees C the probe bound specifically to the luminal surface of the endothelial cells without internalization. The use of 125I-rhFSH, which allows precise quantitation of the binding, confirmed the specificity of hormone interaction with the testicular microvasculature. At 37 degrees C the hormone was internalized via coated pits and vesicles into an extensive subluminal tubulo-vesicular compartment and was transported across the endothelium via a system of tubules and vesicles. Moreover, monoclonal antibodies against the FSH receptor ectodomain coupled to colloidal gold followed the same route. In contrast, a non specific, fluid-phase uptake via caveolae was observed for a major plasma protein - rat serum albumin and a fluid-phase tracer - peroxidase. These results suggest that FSH transcytosis across the testicular endothelial barrier is receptor-mediated and involves luminal uptake via coated pits/vesicles, sorting at the level of luminal early endosomes, and transcellular transport through transcytotic tubulo-vesicular organelles. Similar receptor-mediated pathways are likely to be involved in the physiological functioning of a number of other protein and peptide hormones that must translocate specifically from blood to the target cells.     

Ni(II) coordination compounds based on mixed phthalate and aromatic amine ligands: synthesis, crystal structures and magnetic properties

Three new coordination compounds, [Ni(Pht)(Py)₂(H₂O)₃] (1), [Ni(Pht)(β- Pic)₂(H₂O)₃] · H₂O (2) and [Ni(Pht)(1-MeIm)₂(H₂O)₃] (3) (where Pht²⁻ = dianion of o-phthalic acid; Py = pyridine, β-Pic = 3-methylpyridine, 1-MeIm = 1-methylimidazole), have been synthesized and characterized by IR spectroscopy and thermogravimetric analysis. Crystallographic studies 1-3 reveal that each Ni(II) center has a distorted octahedral geometry being coordinated by two nitrogen atoms of aromatic amines, one oxygen atom from a carboxylate group of a phthalate ligand and three water molecules. Pht²⁻ anions act as monodentate ligands, while the remaining uncoordinated carboxylate oxygen atoms participate in the formation of hydrogen bonding. The uncoordinated oxygen atoms form hydrogen bonds with the coordinated water molecules from adjacent complexes creating a centrosymmetric dimer unit. Further, these dimer units are connected by O-H?O hydrogen bonds in double-chains. Depending on the nature of aromatic amines, the arrangement of these double-chains differs. The double-chains are held together only by van der Waals interactions in 1. In contrast, in 2 these chains form layers by π-π interactions between antiparallel molecules of β-Pic as well as by π-π interactions between β-Pic and Pht aromatic rings. In complex 3, the double-chains are knitted together via C-H?O hydrogen bonds between the methyl group of 1-MeIm and the coordinated carboxylate oxygen atom of Pht, as well as π-π contacts involving antiparallel 1-MeIm cycles. The thermal dependence of the magnetic susceptibilities for compounds 1 and 2 shows a weak antiferromagnetic interaction between the two Ni²⁺ ions of the hydrogen bonded dimers. For compound 3, a ferromagnetic interaction could be observed. Modeling the experimental data with MAGPACK resulted in: g = 2.22, |D| = 4.11 cm⁻¹ and J = −0.29 cm⁻¹ for compound 1, g = 2.215, |D| = 3.85 cm⁻¹ and J = −0.1 cm⁻¹ for compound 2 and g = 2.23, |D| = 4.6 cm⁻¹ and J = 0.22 cm⁻¹ for compound 3.     

Measuring the relative information in allele-sharing linkage studies

In the context of allele-sharing methods, this article investigates ways of measuring the information in the marker data relative to the amount of information that would have been available if the identity-by-descent (IBD) process were known. Such measures are needed to decide whether new markers can substantially modify the evidence for excess sharing. We propose new measures that take advantage of the properties of the exponential model introduced by Kong and Cox (1997, American Journal of Human Genetics61, 1179-1188). These measures are related to Fisher Information and hence are also efficiency measures. Large-sample and small-sample properties of the new and previously proposed measures of information are examined.     

A Chromosome 4 Satellite I DNA Isolated from SV40-Transformed Human Cells

Analysis of cellular DNA insert isolated from a free replicativeplasmid rescued from human cells transformed with an SV40 vectorplasmid revealed the presence of two arrays of repetitive DNAarranged in tandem. One sequence was homologous to the consensussequence of the human satellite DNA and the adjoining sequencewas a satellite DNA sequence which consisted of repetitive unitsof 42 base pairs (bp) and was designated HR42. The degree ofhomology between repetitive units was about 92%. By Southernanalysis the HR42 sequence was detected in HHW416, a somaticcell hybrid containing human chromosome 4, but not in HDm-5,the somatic cell hybrid which has human chromosome 14. By fluorescencein situ hybridization this repetitive DNA was assigned uniquelyto the centromeric region of human chromosome 4. These resultsshow that HR42 belongs to a subfamily of satellite I DNA specificfor human chromosome 4.     

MORPHOMETRIC DATA ON THE ENDOTHELIUM OF BLOOD CAPILLARIES

Local differentiations within the endothelium of both muscular (diaphragm, myocardium) and visceral (pancreas, jejunal villi) capillaries have been studied in rats on sectioned and freeze-cleaved preparations. Four distinct parts have been recognized in the endothelial cells of all these vessels on the basis of subcellular components present in each part and on the basis of variations in the local frequency of plasmalemmal vesicles: (a) the parajunctional zone, (b) the peripheral zone, (c) the organelle region, and (d) the nuclear region. Our data indicate that ~16, ~7.0, and 8.5% of the endothelial cytoplasmic volume (in the peripheral zone) is accounted for by vesicles, their content, and their membranes, respectively. The average density of vesicular openings per µm² is 78 in diaphragm, 89 in myocardium, 25 in pancreas, and 10 in jejunal mucosa capillaries. The frequency of fenestrae is 1.7 times as high in jejunal (26/µm²) as in pancreatic capillaries (15/µm²), the corresponding fractional areas being ~9.5 and ~6%, respectively, of the endothelial surface. Intercellular spaces occupy a relatively small area (~0.08 to 0.2%) of the inner endothelial surface.     

NGS combined with phylogenetic analysis to detect HIV-1 dual infection in Romanian people who inject drugs

Dual HIV infections are possible and likely in people who inject drugs (PWID). Thirty-eight newly diagnosed patients, 19 PWID and 19 heterosexually HIV infected were analyzed. V2V3 loop of HIV-1 env gene was sequenced on the NGS platform 454 GSJunior (Roche). HIV-1 dual/multiple infections were identified in five PWID. For three of these patients, the reconstructed variants belonged to pure F1 subtype and CRF14_BG strains according to phylogenetic analysis. New recombinant forms between these parental strains were identified in two PWID samples. NGS data can provide, with the help of phylogenetic analysis, important insights about the intra-host sub-population structure.