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Pasteurella multocida is a pathogen of veterinary and medical importance. Here, we report the 1.85 Å resolution crystal structure of the class C acid phosphatase from this organism (denoted rPmCCAP). The structure shows that rPmCCAP exhibits the same haloacid dehalogenase fold and dimeric assembly as the class C enzyme from Haemophilus influenzae. Formation of the dimer in solution is demonstrated using analytical ultracentrifugation. The active site is devoid of a magnesium ion due to the presence of citrate in the crystallization buffer. Absence of the metal ion minimally perturbs the active site structure, which suggests that the main role of the ion is to balance the negative charge of the substrate rather than stabilize the active site structure. The crystal lattice displays unusual crystal packing involving the C-terminal polyhistidine tag mimicking the substrate. Steady-state kinetic constants are determined for the substrates NMN, 5´-AMP, 3´-AMP, 2´-AMP, and p-nitrophenyl phosphate. The highest catalytic efficiency is observed with NMN. The production of polyclonal anti-rPmCCAP antibodies is demonstrated, and these antibodies are shown to cross-react with the H. influenzae class C phosphatase. The antibodies are used to detect PmCCAP in clinical P. multocida and Mannheimia haemolytica strains cultured from infected animals.

The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD{sup +} utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5{prime},3{prime}-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5{prime}-AMP, 3{prime}-AMP, and 2{prime}-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5{prime}-nucleotides and 3{prime}-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5{prime} substrates in an anti conformation and 3{prime} substrates in a syn conformation. Finally, themore » structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.« less

Complex type N-glycosides of commercial bovine fetuin preparations from pooled fetal calf serum have been shown to contain comparable amounts of Gal4,4,4TRI (see structure A below) and Gal4,4,3TRI (structure B) as major asialo-structures. To investigate whether there is a clear genetic specificity for synthesis of these oligosaccharides, N-glycosides from two preparations of bovine fetuin, each from a single calf, were examined. Both of these structures were present in each calf, and there was only a subtle quantitative difference in the ratio of these two structures between the calves. Thus, a specific galactosyltransferase, presumably required for the biosynthesis of the Gal4,4,3TRI structure, may exist in both of these individual calves. Comparison of fetuin N-glycosides was also extended to sheep, pig, and human alpha 2-HS-glycoprotein, the human counterpart of bovine fetuin, using high-pH anion-exchange chromatography of the reducing oligosaccharides as well as HPLC of their pyridinylamino derivatives. The N-glycosides of ovine fetuin also have both Gal4,4,4TRI and Gal4,4,3TRI structures in a ratio similar to that of bovine fetuin. However, the major N-glycoside of porcine fetuin is of a fucosyl biantennary complex type structure (structure C below) and human alpha 2-HS-glycoprotein has an N-glycoside which is almost exclusively a nonfucosylated biantennary structure (structure D). This species-specific presence of N-glycosides of fetuins and comparison with N-glycosides of other glycoproteins suggest that the polypeptide sequence of a glycoprotein may affect its N-glycan structure by regulating the activity of specific glycosyltransferases. [formula: see text]

Class C nonspecific acid phosphatases are bacterial enzymes that are secreted across the cytoplasmic membrane and hydrolyze a variety of phosphomonoesters at acidic pH. These enzymes are of interest for the development of improved vaccines and clinical diagnostic methods. In one case, the category A pathogen Francisella tularensis, the class C phosphatase plays a role in bacterial fitness. Here, the cloning, expression, purification and crystallization methods for the class C acid phosphatases from F. tularensis and Pasteurella multocida are reported. Crystals of the F. tularensis enzyme diffracted to 2.0 A resolution and belonged to space group C222(1), with one enzyme molecule in the asymmetric unit. Crystals of the P. multocida enzyme diffracted to 1.85 A resolution and belonged to space group C2, with three molecules in the asymmetric unit. Diffraction patterns from crystals of the P. multocida enzyme exhibited multiple interpenetrating reciprocal-space lattices, indicating epitaxial twinning. Despite this aberrance, autoindexing was robust and the data could be satisfactorily processed to 1.85 A resolution using MOSFLM and SCALA.

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis a cid p hosphatase A (MapA) was most similar (the BLAST probability score was 10 −10 ) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis . Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity.

Lipoprotein e (P4) from Haemophilus influenzae belongs to the "DDDD" superfamily of phosphohydrolases and is the prototype of class C nonspecific acid phosphatases. P4 is also a component of a H. influenzae vaccine. We report the crystal structures of recombinant P4 in the ligand-free and tungstate-inhibited forms, which are the first structures of a class C phosphatase. P4 has a two-domain architecture consisting of a core alpha/beta domain and a smaller alpha domain. The core domain features a five-stranded beta-sheet flanked by helices on both sides that is reminiscent of the haloacid dehalogenase superfamily. The alpha domain appears to be unique and plays roles in substrate binding and dimerization. The active site is solvent accessible and located in a cleft between the two domains. The structure shows that P4 is a metalloenzyme and that magnesium is the most likely metal ion in the crystalline recombinant enzyme. The ligands of the metal ion are the carboxyl groups of the first and third Asp residues of the DDDD motif, the backbone carbonyl of the second Asp of the DDDD motif, and two water molecules. The structure of the tungstate-bound enzyme suggests that Asp64 is the nucleophile that attacks the substrate P atom. Dimerization appears to be important for catalysis because intersubunit contacts stabilize the active site. Analysis of the structural context of mutations engineered for vaccine studies shows that the most promising mutations are located in the dimer interface. This observation suggests a structure-based vaccine design strategy in which the dimer interface is disrupted in order to expose epitopes that are buried in dimeric P4.

Cloning, expression, purification and crystallization studies of a recombinant class C acid phosphatase from the Category A pathogen Bacillus anthracis are reported. Large diffraction-quality crystals were grown in the presence of HEPES and Jeffamine ED-2001 at pH 7.0. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.4, b = 90.1, c = 104.2 angstroms. The asymmetric unit is predicted to contain two protein molecules with a solvent content of 38%. Two native data sets were collected from the same crystal before and after flash-annealing. The first data set had a mosaicity of 1.6 degrees and a high-resolution limit of 1.8 angstroms. After flash-annealing, the apparent mosaicity decreased to 0.9 degrees and the high-resolution limit of usable data increased to 1.6 angstroms. This crystal form is currently being used to determine the structure of B. anthracis class C acid phosphatase with experimental phasing techniques.

Haemophilus influenzae infects the upper respiratory tract of humans and can cause infections of the middle ear, sinuses and bronchi. The virulence of the pathogen is thought to involve a group of surface-localized macromolecular components that mediate interactions at the host-pathogen interface. One of these components is lipoprotein e (P4), which is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 angstroms resolution native X-ray diffraction data set. The space group is P4(2)2(1)2, with unit-cell parameters a = 65.6, c = 101.4 angstroms, one protein molecule per asymmetric unit and 37% solvent content. This is the first report of the crystallization of a class C acid phosphatase.

Francisella tularensis is the etiologic agent of the potentially fatal human disease tularemia and is capable of survival and multiplication within professional phagocytes of the host. While the mechanisms that allow intracellular survival of the bacterium are only now beginning to be elucidated at the molecular level, previous work demonstrated that F. tularensis produces copious levels of an acid phosphatase which in crude and purified form affected the dose-dependent abrogation of the respiratory burst of stimulated neutrophils. The work presented here was undertaken to provide a source of recombinant F. tularensis acid phosphatase for detailed biochemical, biological, and structural studies. Results from this work are consistent with the ability to generate milligram amounts of recombinant enzyme whose attributes are demonstrably equivalent to those of the native enzyme. Such properties include molecular mass, broad substrate specificity, sensitivity and resistance to various inhibitors, pH optimum, and reactivity with rabbit polyclonal antibody to the native enzyme.

Francisella tularensis is a highly infectious bacterial pathogen that is classified as a Category A Pathogen by the Centers for Disease Control and Prevention. Here, we report crystallization of a recombinant form of F. tularensis AcpA, a unique and highly expressed acid phosphatase that is thought to play a role in intracellular survival by inhibiting the host respiratory burst. Three crystal forms have been obtained, with form III being the most suitable for high-resolution structure determination. Form III crystals were grown in the presence of PEG 1500 and the competitive inhibitor sodium orthovanadate (5 mM). The space group is C222(1) with unit cell parameters a = 112.1 A, b = 144.4 A, c = 123.9 A. The asymmetric unit is predicted to contain two protein molecules and 43% solvent. A 1.75-A native data set was recorded at beamline 8.3.1 of the Advanced Light Source. To our knowledge, this is the first report of high-resolution crystals of any F. tularensis protein.

To the Editor: Tularemia is a highly pathogenic zoonosis caused by the gram-negative intracellular bacterium Francisella tularensis. F. tularensis causes serious septicemia in animals, especially wild rodents and lagomorphs (rabbits and hares), and potentially fatal, multisystemic disease in humans. The human mortality rate can reach 30% in untreated persons (1). F. tularensis is listed as a category A bioterrorism agent by the Centers for Disease Control and Prevention alongside the causative agents of anthrax, plague, smallpox, botulism, and viral hemorrhagic fevers. Generally, lesions associated with septicemic tularemia include multifocal 1–2-mm, white foci of necrosis in the liver, spleen, lymph nodes, and lungs. Eimeria stiedae is the causative agent of hepatic coccidiosis, a common disease of wild rabbits (2) that can result in severe hepatic injury and death in juveniles and neonates. The gross lesion associated with hepatic coccidiosis is unique and nearly pathognomonic. Because E. stiedae causes proliferation of bile duct epithelial cells, affected livers contain multifocal, well-demarcated, linear, occasionally branching, bosselated, yellow to pearl-gray lesions that reflect the course of the biliary tree. We describe a unique case of tularemia in a rabbit co-infected with E. stiedae. This case was initially misdiagnosed as simple E. stiedae infection on the basis of the classical gross lesions of hepatic coccidiosis, which overshadowed the more subtle tularemia lesions. A juvenile wild rabbit was brought to a local veterinary clinic for postmortem examination. The owner, located in southwestern Missouri near the Arkansas–Kentucky border, raises wild-captured rabbits in a 10-acre, fenced area reserved for the training of hunting dogs. Beginning in the summer of 2009, a gradual rabbit die-off occurred, progressing to almost complete depopulation by May 2010. The liver from the dead rabbit was submitted to the University of Missouri Veterinary Medical Diagnostic Laboratory (Columbia, MO, USA). Gross examination showed the liver contained multifocal to coalescing, linear, yellow to gray nodules consistent with the classical appearance of hepatic coccidiosis. Although no gross evidence of tularemia was observed, the specimen was treated as potentially infected with tularemia because the veterinarian requested F. tularensis testing. Samples were collected and processed for bacteriologic culture, PCR, and histologic evaluation within the confines of a certified biological hood. The liver contained 2 distinct microscopic lesions. The first was severe biliary hyperplasia with numerous intraepithelial coccidia, consistent with hepatic coccidiosis, as was anticipated. The second, more surprising lesion was an acute, multifocal, necrotizing hepatitis (Figure). The differential diagnoses for acute, multifocal, necrotizing heptatitis in a rabbit include tularemia, Tyzzer disease, listeriosis, and salmonellosis. In this instance, F. tularensis was identified by bacterial culture (3) and PCR as previously described (4). No other pathogenic bacteria were isolated on culture. These results were reported to the veterinarian, the owner, and public health officials. All remaining biological specimens were immediately discarded following the University of Missouri’s select agent protocols, and further analysis was halted, preventing further typing of the isolated F. tularensis. Figure Liver from a juvenile wild rabbit with numerous oval Eimeria stiedae oocysts in the convoluted hyperplastic bile ducts (asterisks) and necrotizing hepatitis (arrow) by Francisella tularensis. Hematoxylin and eosin stain; scale bar = 200 ... According to the Centers for Disease Control and Prevention, ≈126 cases of tularemia are reported annually in the United States (5). During 2000–2008, Missouri had the highest number of reported cases (228) followed by Arkansas (149) (5). Two subspecies of F. tularensis are endemic to the United States: the highly virulent F. tularensis subsp. tularensis (type A) and the moderately virulent F. tularensis subsp. holarctica (type B). Transmission of the bacterium occurs primarily through bites from arthropods, including the dog tick (Dermacentor variabilis), the wood tick (D. andersoni), the lone star tick (Amblyomma americanum), and the deer fly (Chrysops spp.). In addition, contact with infected animals, most commonly rabbits, wild rodents, and cats, is another common route of transmission to humans (1,6). Tularemia occurs in various animal species. Lagomorphs, rodents, and sheep are most susceptible; infected animals are frequently found dead or moribund. Carnivores are less susceptible; however, feline tularemia occurs sporadically, and human infections associated with bites and scratches from infected cats have been recognized (7). In addition to arthropod bites, contact with infected dead rabbits or their tissues appears to be the most common source of human infection. A wide variety of case reports have been published describing unique incidences of rabbit–human transmission, including a lawn mower aerosolizing rabbit nests along with their occupants (8), consumption of undercooked rabbit meat (9), and contact with a “lucky” rabbit’s foot (10). The purpose of this report is to alert veterinarians, veterinary laboratory personnel, and public health officials that rabbit tularemia can be easily overlooked on gross examination in animals displaying lesions of hepatic coccidiosis, a common disease of the wild rabbit. Therefore, all rabbits submitted for postmortem examinations should be regarded as potentially infected with tularemia, particularly during seasons when vectors are active.

Haemophilus influenzae is an important respiratory tract pathogen. Toward understanding the progression of H. influenzae from commensal to pathogen, we need to understand the steps of colonization and infection, processes which must involve overcoming the normal host mucociliary clearance mechanism. A reliable method for the screening and quantitation of mucin– H. influenzae binding to allow for the assessment of the physiological variables significant to H. influenzae –mucin interactions in the normal and diseased conditions, will provide insight on how to intervene to prevent, inhibit, or treat infection. The current methods for enumeration of mucin-bound H. influenzae are labor intensive and rely on viable organisms. In this report, we present a new detection method, which reduces the number of variables, processing steps, and time involved, providing an economical, rapid, and reliable means to screen for and quantitate mucin-bound H. influenzae . Organisms are applied to mucin-coated microtiter wells for a set time; nonadherent organisms are removed with gentle rinses; wells are incubated with the phosphomonoesterase substrate p -nitrophenyl phosphate; and the absorbance, reflecting phosphatase activity of the mucin-bound organisms, is read at 410 nm in a microtiter plate reader against enzymatic activity calibration curves. All nonencapsulated and encapsulated H. influenzae tested exhibited significant acid phosphate activity within 20 min, which provided linear relationships with the numbers of organisms present. H. influenzae mucin binding characteristics obtained by this method were generally comparable to published data, and ranged from 10 3 to 10 6 organisms per well, depending on both strain of organism and type of mucin employed. This convenient, rapid and economical mucin adherence assay, will enable more extensive and comprehensive studies of the interactions of H. influenzae adhesins and specific ligands on mucin macromolecules, as well as the nonspecific means by which mucins function in preventing bacterial infection.

Reversible phosphorylation of tyrosine residues serves as a biochemical “switch” that alters the functional properties of many proteins involved in cellular signal transduction processes. Protein tyrosine phosphatases (PTPs) catalyze the removal of phosphoryl groups from tyrosine residues in target proteins thereby playing a central role in the regulation of diverse cellular processes including glucose metabolism, cell cycle control, and immune responses. Accordingly, small molecules capable of inactivating PTPs may find use as therapeutic agents and tools for the study of diverse signal transduction pathways. In the work reported here, peroxymonophosphate (2–O3POOH) was reported to be an exceptional inactivator of PTP1B, an archtypcal member of the PTP enzyme family (KI = 6.6 × 10−7 M and kinact = 0.043 s−1). In this regard, peroxymonophosphate is over 7,000 times more potent than hydrogen peroxide, the endogenous regulator of PTP1B. Inactivation of PTP1B by peroxymonophosphate is active site directed and, like that by hydrogen peroxide, is readily reversed by treatment with dithiothreitol (5 mM). Together the findings suggest that peroxymonophosphate oxidizes the active site cysteine residue of PTP1B to the sulfenic acid oxidation state. Importantly, peroxymonophosphate (100 nM) yields substantial inactivation of PTP1B even in the presence of physiologically-relevant concentrations of the biological thiol glutathione (1 mM). Collectively, these properties may make peroxymonophosphate a useful tool for probing the role of cysteine-dependent PTPs in various signal transduction pathways. Finally, it is interesting to note that peroxymonophosphate may be biosynthetically accessible via the reaction of endogenous hydrogen peroxide with phosphoryl donors. Peroxymonophosphate possesses key properties expected for the endogenous signaling molecule involved in the redox regulation of PTP activity.

Human renal epithelial cells are capable of internalizing Escherichia coli regardless of whether the bacteria are isolated from individuals with pyelonephritis or from healthy volunteers. In this study, we investigated the role of host cell tyrosine kinase activity in internalization. We found that internalization of both fecal and pyelonephritis isolates is blocked by tyrosine kinase inhibitors. We found increased intensity of two tyrosine-phosphorylated proteins, with relative mobilities of approximately 123,000 and 110,000, in Western blots of extracts from human renal epithelial cells infected with E. coli. The increased intensity of these tyrosine-phosphorylated proteins was observed only in the Triton X-100-insoluble fraction, suggesting that these proteins could be associated with the cytoskeleton. Increased tyrosine phosphorylation of these proteins upon E. coli infection was observed in both transformed and primary human renal epithelial cells and in cells infected with several different strains of E. coli isolated from the feces of healthy individuals or from the blood or urine of patients with pyelonephritis. The increased tyrosine phosphorylation of these proteins required live bacteria and was blocked by tyrosine kinase inhibition but not by protein synthesis inhibitors or cytochalasin D. These experiments establish a strong link between E. coli internalization and host cell signaling through tyrosine kinases in human kidney cells and provide evidence that specific proteins are involved in these processes.

The link between rap and mental health is the promotion of harmful, self-destructive behaviour. The issue of drugs has been central to the gangster rap phenomenon. Alcohol abuse and casual sex are similarly ubiquitous in modern rap. Arguably, however, it is not much different from other musical

Acid phosphatases (Acp) of intracellular pathogens have recently been implicated as virulence factors that enhance intracellular survival through suppression of the respiratory burst. We describe here the identification, purification, characterization, and sequencing of a novel burst-inhibiting acid phosphatase from the facultative intracellular bacterium, Francisella tularensis. Similar to other the burst-inhibiting Acps, F. tularensis Acp (AcpA) is tartrate-resistant and has broad substrate specificity. The AcpA enzyme is unique, however, in that it is easily released from the bacterial cell in soluble form, is a basic enzyme, suppresses the respiratory burst of not only fMet-Leu-Phe but also phorbol 12-myristate 13-acetate-stimulated neutrophils and does not fit into any of the three currently recognized classes of acid phosphatase. We also report the complete nucleotide sequence of the gene acpA, encoding AcpA, and the deduced primary structure of its encoded polypeptide. Comparative sequence analyses of AcpA is discussed. To our knowledge, this is the first report describing the cloning and sequencing of a burst-inhibiting acid phosphatase.

Bipolar Disorder (Oxford Psychiatry Library) By Lakshmi Yatham, Gin Malhi. Oxford University Press. 2011. £9.99 (pb). 96pp. ISBN: 9780199562305 ‘Pocketbook’ is usually a misnomer; few of these books actually fit into the average pocket. But this title really is a pocket reference in the most

In this experiment, which is intended for the introductory organic laboratory, the widely used local anesthetic Lidocaine is synthesized in two steps from 2,6-dimethylaniline. In the first step, th...

If silicone greases are used on the standard taper joints of round bottom flasks, they will eventually get inside the flask, contaminating the contents and introducing spurious peaks into the IR and NMR spectra of samples. Removal of these deposits with solvents or conventional cleaning solutions is difficult. This article describes a fast and efficient method for removing the deposits.

Substrate specificity of cathepsin C derived from rat liver, describing polymeric structure and behavior as acidic protein

Historically, the study of aminopeptidase activity in mammalian systems has been directed primarily toward tissue extracts. This work culminated in the isolation and characterization of the classical leucine aminopeptidase ( Smith and Hill, 1960 ). Little attempt has been made to characterize the aminopeptidases of normal plasma and serum, and perhaps it is for this reason that the activity has been tacitly identified with tissue leucine aminopeptidase (LAP), and commonly designated serum LAP. Behal et al. (1963) have chromatographically resolved the LAP activity of human blood into a number of aminopeptidase components. In this report, some distinguishing properties are described for a variety of aminopeptidases which can occur in blood plasma. The aminopeptidase activity of normal plasma could not be attributed to the presence of leucine aminopeptidase.

Dipeptidyl arylamidase I of bovine pituitary tissue and chloride and sulfhydryl activation of seryltyrosyl-beta-naphthylamide hydrolysis

Dipeptidyl-beta-naphthylamidase activated by chloride in extracts of rat and bovine anterior pituitary glands

An enzyme is described that was observed in extracts of rat and bovine pituitary glands. This enzyme, named dipeptidyl arylamidase II, catalyzes the cleavage of the dipeptide moiety from Lys-Ala-β-naphthylamide at pH 5.5, but not from monoamino acid arylamides. Dipeptidyl arylamidase II exhibits no metal, halide, or sulfhydryl dependences, and its specificity is restricted to substrates having an unsubstituted NH2-terminus. Lower but significant rates of hydrolysis were also exhibited at pH 5.5 on the β-naphthylamides of Arg-Ala and Leu-Ala, with only traces of activity on the arylamides of Ala-Ala, Gly-Pro, and Ser-Met. No activity was detected on the β-naphthylamides of benzylodicarbonyl-Lys-Ala, Lys-Lys, Arg-Arg, Gly-Arg, Gly-Phe, Ser-Tyr, or His-Ser. Cations were exceptionally inhibitory to the hydrolysis of Lys-Ala-β-naphthylamide by dipeptidyl arylamidase II, and the sensitivity of the enzyme increased with the size of the cation: Li+ l Na+ l K+ l Tris l puromycin aminonucleoside l puromycin. A 50 mm concentration of Tris, pH 5.5, in the assay system gave a 96% inhibition of arylamidase II activity, while puromycin at 1 mm gave a 76% inhibition. A Km of 1.1 x 10-5 m was determined for the hydrolysis of Lys-Ala-β-naphthylamide at pH 5.5 at 37°. The inhibition by cations was competitive for all cations tested, with Ki values at pH 5.5 and 37° of 1.8 x 10-3 m for Na+, 3.2 x 10-4 m for Tris, 1.9 x 10-5 m for puromycin, and 3.0 x 10-5 m for puromycin aminonucleoside. Dipeptidyl arylamidase II activity was detected in tissues other than pituitary; thyroid, spleen, and kidney were relatively rich sources, whereas liver, pancreas, and serum were poor. The hydrolysis of Lys-Ala-β-naphthylamide at pH 5.5 by all tissues tested showed the same sensitivity to Tris ions. As indicated by studies with sucrose homogenates of fresh rat pituitary glands, and with subcellular fractions prepared by differential and density gradient centrifugation, dipeptidyl arylamidase II was located, along with acid phosphatase, in pituitary lysosomes. However, unlike acid phosphatase, arylamidase II appeared to be located almost exclusively in the lysosomes, and exhibited a functional latency of about 91 to 95%. Ser-Tyr arylamidase (dipeptidyl arylamidase I) was also found to be lysosomal, whereas Arg-Arg arylamidase (dipeptidyl arylamidase III) was cytoplasmic. The aminopolypeptidase and dipeptidase activities measured on Lys- and Arg-β-naphthylamide were predominantly (90 to 95%) cytoplasmic.

SummaryTitratable ash-acidity (ash-TA) is an acid-base parameter which is defined in terms of mineral composition and may be evaluated as the sum of values for Cl, S and P minus the sum of values for Na, K, Ca and Mg. A relatively simple and essentially direct titrimetric procedure for determining ash-TA in urine, feces and foods is shown to yield values which agree satisfactorily with those calculated from the mineral composition of these materials.

The carborane C2B3H5 is metastable relative to polymerization, which occurs easily under high-energy conditions or when the electronic symmetry of the trigonal-bipyramidal structure is disturbed. Attempts to halogenate or NO2-oxidize C2B3H5 also produced only intractable nonvolatiles. However, highly covalent substitution derivatives are possible. The apparently stable B-B linked dicarborane C4B6H8 is formed during the spontaneous polymerization of C2B3H5 at 25C or in a hot-cold reactor. Any of the three carboranes C2B3H5, C2B4H6, or C2B5H7 polymerizes under high-energy conditions, such as a microwave discharge. (Author)

Dipeptides separation and identification by column and paper chromatography for elution times prediction and sequence studies

SummaryA relatively simple titrimetric method for determining total cation-forming mineral elements in urine and feces is described and demonstrated to yield dependable values. Addition of fecal minerals to an otherwise constant diet in rats is shown to effect an increased urinary excretion of total cation-forming mineral elements together with a correspondingly decreased renal “net acid” excretion. The theoretical basis for this result and for analogous results of others is discussed. It is concluded that the extent to which cation-forming mineral elements are excreted in the feces rather than in urine can considerably influence the extent of renal “net acid” excretion.Addendum: While the preceding report was in press, studies by Lennon, Lemann and Litzow(13,14) appeared, in which collective values for Na+, K+, Ca++ and Mg++ (ions derivable from cation-forming elements) in diet and feces, respectively, are calculated as a negative and positive component, respectively, of “total effective acid production”...

BackgroundSodium nitroprusside (SNP) has been reported to rapidly reduce psychotic symptoms in patients with schizophrenia. This has the potential to revolutionize treatment for schizophrenia. In this study, we tested the hypothesis that SNP leads to a reduction in psychotic symptoms and an improvement in spatial working memory (SWM) performance in patients with schizophrenia.MethodThis was a single-centre, randomized, double-blind, placebo-controlled trial performed from 27 August 2014 to 10 February 2016 (clinicaltrials.gov identifier: NCT02176044). Twenty patients with schizophrenia aged 18–60 years with a diagnosis of schizophrenia or schizoaffective disorder were recruited from psychiatric outpatient clinics in the South London and Maudsley NHS Trust, London, UK. Baseline symptoms were measured using the Positive and Negative Syndrome Scale (PANSS) and the 18-item Brief Psychiatric Rating Scale (BPRS-18), and SWM was assessed using the CANTAB computerized test. Participants received either an infusion of SNP (0.5 μg/kg per min for 4 h) or placebo and were re-assessed for symptoms and SWM performance immediately after the infusion, and 4 weeks later.ResultsSNP did not lead to any reduction in psychotic symptoms or improvement in SWM performance compared to placebo.ConclusionsAlthough this study was negative, it is possible that the beneficial effects of SNP may occur in patients with a shorter history of illness, or with more acute exacerbation of symptoms.