Isolation, folding and structural investigations of the amino acid transporter OEP16.

Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on a Ni-NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24 mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of α-helices. ¹5N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection.

Using ex vivo proton magnetic resonance spectroscopy to reveal associations between biochemical and biological features of meningiomas.

OBJECT: The goal in this study was to determine if proton ((1)H) MR spectroscopy can differentiate meningioma grade and is associated with interpretations of biological behavior; the study was performed using ex vivo high-resolution spectra indicating metabolic characteristics. METHODS: Sixty-eight resected tissue samples of meningiomas were examined using ex vivo (1)H MR spectroscopy. Of these meningiomas, 46 were WHO Grade I, 14 were WHO Grade II, and 8 were WHO Grade III. Fifty-nine were primary meningiomas and 9 were recurrences. Invasion of adjacent tissue (dura mater, bone, venous sinus, brain) was found in 32 cases. Thirty-nine meningiomas did not rapidly recur (as defined by expansion on MR imaging within a 5-year follow-up period), whereas rapid recurrence was confirmed in 24 meningiomas, and follow-up status was unknown in 5 cases. RESULTS: The absolute concentrations of total alanine and creatine were decreased in high-grade compared with low-grade meningiomas, as was the ratio of glycine to alanine (all p < 0.05). Additionally, alanine and the glycine/alanine ratio distinguished between primary and recurrent meningiomas (all p < 0.05). Finally, the absolute concentrations of alanine and creatine, and the glycine/alanine and choline/glutamate ratios were associated with rapid recurrence (p < 0.05). CONCLUSIONS: These data indicate that meningioma tissue can be characterized by metabolic parameters that are not typically identified by histopathological analysis alone. Creatine, glycine, and alanine may be used as markers of meningioma grade, recurrence, and the likelihood of rapid recurrence. These data validate a previous study of a separate group of Grade I meningiomas.

Identification, structural analysis and function of hyaluronan in developing fish larvae (leptocephali).

Hyaluronan (HA) has been identified as the principal glycosaminoglycan (CAG) in the highly hydrated, extracellular body matrix of the larval stage (leptocephalus) of seven species of true eels (Teleostei: Elopomorpha: Anguilliformes) and the ladyfish Elops saurus (Elopiformes), and was found as a minor GAG component in the bonefish Albula sp. (Albuliformes). Identification was based on: (1) HPLC separation of unsaturated disaccharides derived from chondroitinase ABC digests of whole-body GAG extracts; (2) 1H NMR analyses of native GAG polymers; and (3) degradation of GAG extracts by Streptomyces hyaluronan lyase. The unsaturated disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose (DeltaDi-HA) accounted for 92.4-99.8% of the total disaccharides in chondroitinase digests. Trace amounts of unsaturated disaccharides of chondroitin sulfate were also present. Two-dimensional gCOSY spectra of the native HA polymer were similar for all species. Proton assignments for the HA disaccharide repeat (GlcAbeta1-3GlcNAcbeta1-4) in D(2)O, based on gCOSY, DQF-COSY and TOCSY analyses for the eel Ahlia egmontis, were concordant with published chemical shifts for HA oligosaccharides. In addition to its presumed role in maintaining the structural integrity and hydration of the gelatinous body of the leptocephalus, HA is postulated to function as a storage polysaccharide in those species in which it is the predominant GAG.

Fluorescent in situ hybridization and ex vivo 1H magnetic resonance spectroscopic examinations of meningioma tumor tissue: is it possible to identify a clinically-aggressive subset of benign meningiomas?

OBJECTIVE: Although histologically benign, Grade I meningiomas can sometimes behave aggressively. The clinically-aggressive subset of Grade I meningiomas is typically indistinguishable from clinically-benign Grade I meningiomas in vivo. We compared molecular genetic and biochemical findings to clinical, pathological, and immunohistochemical information in a series of clinically-aggressive Grade I meningiomas with a series of clinically-benign meningiomas to identify characteristics that may be used to distinguish between these two groups. METHODS: Tumor tissue samples from 30 patients with Grade I meningiomas were harvested. Half of the sample was embedded in paraffin to be used for fluorescent in situ hybridization to examine aberrations of chromosomes 1p, 14q, and 22q; the other half was snap frozen and examined with proton magnetic resonance spectroscopy to identify concentrations of key metabolites in the tissue ex vivo. Clinical and pathological parameters were retrospectively reviewed as part of routine clinical management. These data were evaluated for potential unique associations with diagnostic significance. RESULTS: Molecular genetic and biochemical findings correlated with clinical behavior of the two Grade I meningioma groups. Specific chromosomal abnormalities correlated with the aggressive phenotype: homogeneous loss of 1p, homogeneous loss of 14q, and the presence of any of the examined chromosomal aberrations (P < 0.05). The presence of aberrations also influenced meningioma regrowth after subtotal resection. The ratio of choline to glutamate correlated with histopathological subtype (P < 0.05). The ratio of glutamine to glutamate, and the ratio of glycine to total glutamine and glutamate, and creatine correlated with recurrence. Alanine was decreased in meningiomas with chromosomal aberrations in tumors that recurred. CONCLUSION: Distinct molecular genetic and biochemical alterations differentiated clinically-aggressive Grade I meningiomas from clinically-benign Grade I meningiomas.

Synthesis of butane-like SiGe hydrides: enabling precursors for CVD of Ge-rich semiconductors.

The synthesis of butane-like (GeH(3))(2)(SiH(2))(2) (1), (GeH(3))(2)SiH(SiH(3)) (2), and (GeH(3))(2)(SiH(2)GeH(2)) (3) Si-Ge hydrides with applications in low-temperature synthesis of Ge-rich Si(1-x)Ge(x) optoelectronic alloys has been demonstrated. The compositional, vibrational, structural, and thermochemical properties of these compounds were studied by FTIR, multinuclear NMR, mass spectrometry, Rutherford backscattering, and density functional theory (DFT) simulations. The analyses indicate that the linear (GeH(3))(2)(SiH(2))(2) (1) and (GeH(3))(2)(SiH(2)GeH(2)) (3) compounds exist as a mixture of the classic normal (n) and gauche (g) conformational isomers which do not seem to interconvert at 22 degrees C. The conformational proportions in the samples were determined using a new fitting procedure, which combines calculated molecular spectra to reproduce those observed by varying the global intensity, frequency scale, and admixture coefficients of the individual conformers. The (GeH(3))(2)(SiH(2))(2) (1) species was then utilized to fabricate Si(0.50)Ge(0.50) semiconductor alloys reflecting exactly the Si/Ge content of the precursor. Device quality layers were grown via gas source MBE directly on Si(100) at unprecedented low temperatures 350-450 degrees C and display homogeneous compositional and strain profiles, low threading dislocation densities, and atomically planar surfaces. Low energy electron microscopy (LEEM) analysis has demonstrated that the precursor is highly reactive on Si(100) surfaces, with H(2) desorption kinetics comparable to those of Ge(2)H(6), despite the presence of strong Si-H bonds in the molecular structure.

Monitoring of recombinant survival motor neuron protein using fiber-optic surface plasmon resonance.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of the survival motor neuron 1 (SMN1) gene. A nearly identical copy gene exists known as SMN2, however, due to an aberrant splicing event, the SMN2 gene fails to produce sufficient full-length protein to protect against disease development in the absence of SMN1. While a number of compounds have recently been identified that can stimulate full-length survival motor neuron (SMN) expression from the nearly identical copy SMN2, one of the difficulties has been the lack of a highly reproducible and quantitative means to measure the levels of SMN protein. To develop a technique that allows the rapid and highly sensitive measurement of SMN protein, a Surface Plasmon Resonance (SPR) application has been developed. The ability to quantify unassociated SMN protein and monitor the binding of SMN with other proteins in solution using a SPR sensor in less than 15 min and at low ng mL(-1) levels in HEPES Buffer Saline (HBS) has been achieved. The detection limit for the specific binding of SMN in HBS pH 7.4 solution is 0.99 ng mL(-1) with non-specific binding accounting for approximately 30% of the signal. Quantification of SMN is based on an immunoassay performed on the gold surface of the SPR sensor. 16-mercaptohexadecanoic acid (MHA) was reacted with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) to form a pre-activated thiol (MHA-NHS). Antibodies for SMN were then coupled to the sensor with the pre-activated thiol. Sensor specificity was examined with mixtures of myoglobin (MG) and SMN. SMN sensor response decreases by more than 60% when MG was added to SMN. The decrease in sensor response can be attributed to non-specific binding of SMN to MG, verified with a sensor for MG.

Antineoplastic agents. 485. Isolation and structure of cribrostatin 6, a dark blue cancer cell growth inhibitor from the marine sponge Cribrochalina sp.

Cribrostatin 6, a dark blue cancer cell growth inhibiting (P388 ED(50) 0.3 microg/mL) constituent of the Republic of Maldives marine sponge Cribrochalina sp., has been assigned structure 3 on the basis of a combination of HRMS, high-field (500 MHz, HMBC, and GOESY experiments) (15)N, (1)H, and (13)C NMR, and X-ray crystal structure analyses. Cribrostatin 6 also was found to inhibit the growth of a number of pathogenic bacteria and fungi.