Fabrication of infrared linear arrays of InAs planar avalanche photodiodes.

We report a, to the best of our knowledge, new device fabrication process for 128-pixel linear arrays of InAs planar avalanche photodiodes, utilizing selective area implantation of Beryllium ions into epitaxially-grown InAs wafers. The pixels exhibited uniform avalanche gain and responsivity. Room temperature responsivity values at 1550 and 2004 nm wavelengths are 0.49 ± 0.017 and 0.89 ± 0.024 A/W, respectively. Reverse dark current-voltage and avalanche gain measurements were carried out at different temperatures (from room temperature to 150 K). At 200 K at -15 V reverse bias, the pixels exhibited an avalanche gain of 22.5 ± 1.18 and dark current density of 0.68 ± 0.48 A/cm2.

Classification of Andrographis paniculata extracts by solvent extraction using HPLC fingerprint and chemometric analysis.

OBJECTIVE: Andrographis paniculata, widely used as an antidiabetic in Indonesian traditional medicines (jamu), contains chemical compounds whose concentration is related to its therapeutic effects. The concentration of solvents used for extraction will also affect the number of compounds extracted. Therefore, a quality control method is needed to ensure consistency in quantifying these compounds in A. paniculata to improve its therapeutic application. High-performance liquid chromatography fingerprint analysis combined with chemometrics was used to evaluate extracts from different solvent extraction treatments. The content of andrographolide, the main bioactive compound in A. paniculata, and the level of α-glucosidase inhibition activity, an indicator of its antidiabetic activity, were also determined. RESULTS: Fingerprint chromatograms of A. paniculata extracts from different treatments exhibited a similar pattern with several peaks in common, only differing in area and intensity value. The A. paniculata extracts were classified using HPLC fingerprint and principal component analysis to allow grouping according to their respective solvent extraction treatments. The highest andrographolide content and α-glucosidase inhibition activity occurred in the 50% ethanol extract and the lowest in the water extract. HPLC fingerprint analysis could be used for identifying A. paniculata extracts based on solvent extraction, thus improving quality control for their therapeutic application.

A novel and faster method of manual grading to measure choroidal thickness using optical coherence tomography.

PurposeChoroidal thickness (CT) measurements are typically obtained from manual segmentation of optical coherence tomography (OCT) B-scans. This method is time-consuming. We aimed to describe a novel and faster technique to obtain CT measurements.Patients and methodsIn a prospective cohort study of 200 healthy eyes, Spectral-Domain OCT with enhanced depth imaging were performed with the Spectralis OCT using standardised imaging protocols. The OCT scans were independently graded by reading centre-certified graders. The standard method of manual adjustment of segmentation boundaries was performed. The new method consisted of adjusting the lower segmentation line to the choroid-scleral boundary to generate the combined choroid-retina thickness, and subtracting the original retinal thickness (RT) from it to measure CT. Mean CT in the respective Early Treatment Diabetic Retinopathy Study (ETDRS) subfields was measured via the two methods, and were compared with intraclass correlation coefficients (ICC) and Bland-Altman plots.ResultsThe mean central subfield CT was 324.4 µm using the original method, compared with 328.8 µm using the new method, with a mean difference of 4.5 µm (range: -14.0 to +4.0 µm; P<0.001), and ICC for agreement of 0.9996 (P<0.001). Similar comparability was achieved for mean CT across other ETDRS subfields, with mean differences ranging from 2.4 to 3.7 µm, and ICCs ranging from 0.9993 to 0.9995 (all P<0.001).ConclusionsMean CT can be measured by subtracting the original RT from the combined choroid-retina thickness. Only one segmentation line needs to be adjusted, instead of two, reducing time required for segmentation. This method is faster and reliable.

Electrical stimulation alleviates depressive-like behaviors of rats: investigation of brain targets and potential mechanisms.

Deep brain stimulation (DBS) is a promising therapy for patients with refractory depression. However, key questions remain with regard to which brain target(s) should be used for stimulation, and which mechanisms underlie the therapeutic effects. Here, we investigated the effect of DBS, with low- and high-frequency stimulation (LFS, HFS), in different brain regions (ventromedial prefrontal cortex, vmPFC; cingulate cortex, Cg; nucleus accumbens (NAc) core or shell; lateral habenula, LHb; and ventral tegmental area) on a variety of depressive-like behaviors using rat models. In the naive animal study, we found that HFS of the Cg, vmPFC, NAc core and LHb reduced anxiety levels and increased motivation for food. In the chronic unpredictable stress model, there was a robust depressive-like behavioral phenotype. Moreover, vmPFC HFS, in a comparison of all stimulated targets, produced the most profound antidepressant effects with enhanced hedonia, reduced anxiety and decreased forced-swim immobility. In the following set of electrophysiological and histochemical experiments designed to unravel some of the underlying mechanisms, we found that vmPFC HFS evoked a specific modulation of the serotonergic neurons in the dorsal raphe nucleus (DRN), which have long been linked to mood. Finally, using a neuronal mapping approach by means of c-Fos expression, we found that vmPFC HFS modulated a brain circuit linked to the DRN and known to be involved in affect. In conclusion, HFS of the vmPFC produced the most potent antidepressant effects in naive rats and rats subjected to stress by mechanisms also including the DRN.

Buspirone induced acute and chronic changes of neural activation in the periaqueductal gray of rats.

5-HT(1A) modulation within the midbrain periaqueductal gray (PAG) is closely associated with anxiety- or panic-like behavior. Several findings have demonstrated that the properties of buspirone (a 5-HT(1A) partial agonist) would function as either anxiolytic or panicolytic in both clinical and laboratory animal research. In this study, we have investigated the neuronal activity occurring within the different regions of the PAG induced by buspirone treatment. Twenty-eight albino Wistar rats (350-400 g) were injected with either acute or chronic saline/buspirone (each, n=7), respectively. Our results show that buspirone treatment reduced locomotor activity, body weight and fecal boli, particularly in the chronic buspirone group. Two-way ANOVA revealed a significant decrease of c-Fos-immunoreactive (ir) cells expression in all regions of the rostral PAG after both acute and chronic buspirone (acute buspirone (AB) and chronic buspirone (CB), respectively) treatment. However, no effects on c-Fos-ir were detected in the caudal lateral periaqueductal gray (lPAG) and ventrolateral periaqueductal gray (vlPAG) in both the AB and CB groups, and in the dorsolateral periaqueductal gray (dlPAG) of the CB group. Interestingly, c-Fos-ir cells in the dorsomedial periaqueductal gray (dmPAG) column were reduced consistently in both the rostral and caudal PAG in both AB and CB groups. Besides, in all regions the number of c-Fos-ir cells was higher in the AB than in the CB group with exception of the rostral lPAG. In conclusion, the main anxiolytic effect of buspirone was specifically localized in all regions of the rostral PAG and in the caudal dmPAG. However, the caudal dlPAG, lPAG and vlPAG were found to be ineffective to buspirone treatment, probably due to their distinctive function in mediating higher level of anxiety in defensive behavior. This indicates that the longitudinal anatomical structure of the PAG possesses a different level of receptor sensitivity of 5-HT(1A) in the pathophysiology of anxiety and panic disorder.

Overexpression, biophysical characterization, and crystallization of ribonuclease I from Escherichia coli, a broad-specificity enzyme in the RNase T2 family.

We have constructed a strain that overproduces ribonuclease I of Escherichia coli and we have purified large quantities of the enzyme. Data from fluorescence, CD, and DSC measurements showed that it was a very stable protein. The conformation energy determined from urea and guanidine hydrochloride denaturation experiments was 11.5 kcal mol(-1) at pH 7.5. Thermal denaturation studies indicated that it had a T(m) of 64 degrees C at pH 4.0. RNase I belongs to the RNase T2/S-RNase group of endoribonucleases, but near the amino terminus it has an unusually long hydrophilic segment. Part of this was removed in the deletion construct, RNase I Delta(26-38). We have obtained crystals of both RNase I and of an enzyme-G2'p5'G complex in the P2(1) space group and oligonucleotide complexes with both wild type and mutant enzymes. The current study lays the groundwork for extensive investigation into the structure, function, and physical properties of this widely distributed group of ribonucleases.

Renin inhibitor SC-51106 complexed with human renin: discovery of a new binding site adjacent to P3.

SC-51106, a 'minimal-size' diol-based renin inhibitor lacking a P4 residue, has been co-crystallized with human renin and the structure of the complex determined by X-ray crystallography. This study defines the mode of binding of this important class of renin inhibitor, and in conjunction with molecular modeling, has led to the discovery of a new binding site adjacent to S3, which is termed the 'S3aux(iliary)' subsite.

Preliminary X-ray crystallographic studies of ribonuclease I from Escherichia coli.

Single crystals of ribonuclease I from Escherichia coli have been obtained by the vapor diffusion method. The crystals belong to the tetragonal space group P4(1)2(1)2 or its enantiomer P4(3)2(1)2 and have cell parameters a = b = 119.01 A and c = 34.40 A. There is one 27,000 dalton monomer in the asymmetric unit. The crystals diffract to beyond 3.0 A resolution.

Crystal structure analysis of amicyanin and apoamicyanin from Paracoccus denitrificans at 2.0 A and 1.8 A resolution.

The crystal structure of amicyanin, a cupredoxin isolated from Paracoccus denitrificans, has been determined by molecular replacement. The structure has been refined at 2.0 A resolution using energy-restrained least-squares procedures to a crystallographic residual of 15.7%. The copper-free protein, apoamicyanin, has also been refined to 1.8 A resolution with residual 15.5%. The protein is found to have a beta-sandwich topology with nine beta-strands forming two mixed beta-sheets. The secondary structure is very similar to that observed in the other classes of cupredoxins, such as plastocyanin and azurin. Amicyanin has approximately 20 residues at the N-terminus that have no equivalents in the other proteins; a portion of these residues forms the first beta-strand of the structure. The copper atom is located in a pocket between the beta-sheets and is found to have four coordinating ligands: two histidine nitrogens, one cysteine sulfur, and, at a longer distance, one methionine sulfur. The geometry of the copper coordination is very similar to that in the plant plastocyanins. Three of the four copper ligands are located in the loop between beta-strands eight and nine. This loop is shorter than that in the other cupredoxins, having only two residues each between the cysteine and histidine and the histidine and methionine ligands. The amicyanin and apoamicyanin structures are very similar; in particular, there is little difference in the positions of the coordinating ligands with or without copper. One of the copper ligands, a histidine, lies close to the protein surface and is surrounded on that surface by seven hydrophobic residues. This hydrophobic patch is thought to be important as an electron transfer site.

Correlation of x-ray deduced and experimental amino acid sequences of trimethylamine dehydrogenase.

The amino acid sequence of the iron-sulfur-flavoprotein, trimethylamine dehydrogenase, isolated from the bacterium W3A1 has been deduced from the x-ray diffraction pattern obtained at 2.4-A resolution. This sequence has been compared to portions of the primary sequence derived by gas-phase sequencing of isolated peptides obtained from cyanogen bromide and endoprotease Arg-C and Asp-N digestions of the purified enzyme. A consensus sequence has resulted and is comprised of 729 amino acids with Ala at both NH2- and COOH-terminal positions. The consensus sequence contains 13 cysteine residues. Approximately 80% of the sequence has been confirmed by direct sequencing with approximately 81% agreement with the x-ray deduced sequence. The calculated subunit molecular mass of the apoenzyme is 78,899 Da, in good agreement with published values of approximately 83,000. The anomalous scattering map from the native protein has also been shown to provide accurate information about the positions of most of the weak anomalous scattering centers such as sulfur or phosphorus atoms and to complement x-ray or chemical sequencing methods.

Structure and topological symmetry of the glyphosate target 5-enolpyruvylshikimate-3-phosphate synthase: a distinctive protein fold.

5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase; phosphoenolpyruvate:3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) is an enzyme on the pathway toward the synthesis of aromatic amino acids in plants, fungi, and bacteria and is the target of the broad-spectrum herbicide glyphosate. The three-dimensional structure of the enzyme from Escherichia coli has been determined by crystallographic techniques. The polypeptide backbone chain was traced by examination of an electron density map calculated at 3-A resolution. The two-domain structure has a distinctive fold and appears to be formed by 6-fold replication of a protein folding unit comprising two parallel helices and a four-stranded sheet. Each domain is formed from three of these units, which are related by an approximate threefold symmetry axis; in each domain three of the helices are completely buried by a surface formed from the three beta-sheets and solvent-accessible faces of the other three helices. The domains are related by an approximate dyad, but in the present crystals the molecule does not display pseudo-symmetry related to the symmetry of point group 32 because its approximate threefold axes are almost normal. A possible relation between the three-dimensional structure of the protein and the linear sequence of its gene will be described. The topological threefold symmetry and orientation of each of the two observed globular domains may direct the binding of substrates and inhibitors by a helix macrodipole effect and implies that the active site is located near the interdomain crossover segments. The structure also suggests a rationale for the glyphosate tolerance conferred by sequence alterations.

Preliminary crystallographic study of glycosylated recombinant human renin.

Single crystals of glycosylated recombinant human renin have been obtained using the hanging-drop vapor diffusion method with polyethylene glycol and sodium chloride as coprecipitants. The crystals belong to the cubic space group P2(1)3 with a = 143.0 A and contain two molecules of renin in the asymmetric unit. A self-rotation function study using 5.5 A data shows the orientation of a non-crystallographic 2-fold axis relating these two monomers.

Studies of crystalline trimethylamine dehydrogenase in three oxidation states and in the presence of substrate and inhibitor.

Crystals of trimethylamine dehydrogenase have been examined by difference Fourier methods at 6.0-A resolution after partial reduction by substrate and by dithionite in the presence of inhibitor. Similar studies of the inhibited oxidized enzyme and of the enzyme reduced fully by dithionite alone were also carried out. In all cases ligand binding at the active site occurred. In addition, there were small structural changes, possibly side chain movements, in the inhibited oxidized enzyme and somewhat larger changes in the partially reduced crystals. The largest changes occurred with the fully reduced enzyme. However, in no cases were subunit or domain movements observed nor were changes observed in the position of the FMN or [4Fe-4S] cofactors. Parallel studies of crystalline trimethylamine dehydrogenase were carried out by EPR spectroscopy. The results show that the electronic states of the crystalline enzyme under the conditions of the difference Fourier studies are comparable to those which occur in solution under similar conditions.

Identification of ADP in the iron-sulfur flavoprotein trimethylamine dehydrogenase.

Analysis of the 2.4-A resolution electron density map of trimethylamine dehydrogenase has revealed the unexpected presence of one molecule of ADP/subunit. This binding has been confirmed chemically. The binding site is located at the analogous position of the ADP moiety of FAD in glutathione reductase, the FAD and NADPH binding domains of which resemble two of the domains of trimethylamine dehydrogenase. Comparison of the environments of the ADP moieties in the two proteins indicates that 32 residues in 6 peptides are in equivalent positions with a root mean square deviation for C alpha positions of 1.11 A. Twelve of these amino acids are identical, based on the electron density-derived "x-ray" sequence of trimethylamine dehydrogenase. Detailed analysis of the environment of the ADP moiety indicates that most of the conserved residues are not in direct contact with the cofactor. Some of them probably represent the "fingerprint" of the beta alpha beta binding fold found in dinucleotide binding proteins, but the remaining conserved residues may indicate a closer evolutionary relationship between these two proteins.

Three-dimensional structure of flavocytochrome b2 from baker's yeast at 3.0-A resolution.

The structure of flavocytochrome b2 from baker's yeast was solved at 3.0-A resolution by the multiple isomorphous replacement method combined with solvent leveling procedures, using data collected from an area detector. The tetramer of Mr 230,000 has 4-fold symmetry. Each subunit contains a cytochrome domain consisting of the first 100 residues, a flavin-binding domain containing the next 386 residues, and an extended C-terminal tail of 25 residues. The cytochrome domain closely resembles microsomal cytochrome b5, whereas the flavin-binding domain contains a parallel beta 8/alpha 8 barrel motif similar to glycolate oxidase and trimethylamine dehydrogenase. Two of the four cytochrome domains are disordered in the crystals. The flavin ring and heme group are separated by about 16 A between their centers, and their planes are inclined by about 17 degrees to each other.

Three-dimensional structure of the iron-sulfur flavoprotein trimethylamine dehydrogenase at 2.4-A resolution.

The three-dimensional structure of trimethylamine dehydrogenase from the methylotrophic bacterium W3A1 has been determined to 2.4-A resolution. The enzyme is composed of two identical 83,000-dalton subunits, each of which is folded into three structural domains. The largest domain, at the NH2 terminus of the molecule, is folded as an eight-stranded parallel alpha/beta barrel. It contains the [4Fe-4S] and covalently bound FMN cofactors separated by about 4 A. The folding topology of the large domain and orientation of the FMN cofactor are very similar to those found in glycolate oxidase. The other two domains contain alpha/beta parallel beta sheet topologies with similar folding patterns. The topologies and spatial arrangements of these two domains are remarkably similar to the FAD- and NADPH-binding domains of glutathione reductase.

Preliminary X-ray crystallographic study of methanol dehydrogenase from Methylophilus methylotrophus.

Single crystals of methanol dehydrogenase from Methylophilus methylotrophus have been prepared by the macroseeding method. The crystals belong to the monoclinic space group C2, and have unit cell parameters a = 125.62 A, b = 63.83 A, c = 83.99 A, and beta = 93.24 degrees. There is one 62,000 Mr monomer in the asymmetric unit. The crystals diffract to beyond 2.0 A resolution.