First Report of Neofusicoccum nonquaesitum Causing Branch Cankers on Giant Sequoia (Sequoiadendron giganteum) in North America.

Between 2001 and 2007, samples from three California native plants showing canker symptoms were submitted to the California Department of Food and Agriculture's Plant Pest Diagnostics laboratory. Giant sequoia (Sequoiadendron giganteum) and coast redwood (Sequoia sempervirens) showed branch cankers and dieback, whereas tanoak (Lithocarpus densiflora) had bleeding bole cankers. Samples were collected from mature trees in private landscapes in El Dorado, Sacramento, and Alameda counties in California. A fungus was isolated on one-half strength acidified potato dextrose agar (APDA) from the canker margins of all three hosts. Colonies were moderately fast growing, initially white, later turning olivaceous black. Pycnidia developed singly or in small groups and contained conidia that measured 18 to 29 × 6 to 8 µm (average 21.5 × 6.8 µm). Conidia were aseptate, hyaline, and fusiform, with truncate bases. rDNA sequences of the internal transcribed spacer (ITS) region of the isolates (GenBank JQ282157 through JQ282159), amplified using primers ITS1 and ITS4 (2), were 100% identical to the holotype isolate of Neofusicoccum nonquaesitum Inderb., Trouillas, Bostock & Michailides (1) by a BLAST query (GenBank GU251163). Pathogenicity of the N. nonquaesitum isolate from giant sequoia (CDFA4) was tested on five saplings using cultures grown on APDA for 14 days. A single wound was made approximately 2 cm above the soil line on the cambium of each plant using a 3-mm cork borer. One 3-mm colonized agar plug was placed on each wound and secured with Parafilm. Plugs of APDA were placed onto wounds of five plants as controls. All plants were kept in a growth chamber at 23°C with a 12-h photoperiod. After 4 days, Parafilm was removed to reveal dark brown cankers measuring 12 to 43 mm long on the inoculated plants. Fourteen days after inoculation, cankers were black, sunken, and measured 79 to 117 mm (average 91.4 mm) long. Most of the inoculated plants were wilted with chlorotic to necrotic foliage. Mature pycnidia with cirri developed in most of the cankers. N. nonquaesitum was reisolated on APDA from all of the cankers. No symptoms developed on the control plants. The experiment was repeated once with similar results. Botryosphaeria dothidea, also in the Botryosphaeriaceae, has been reported to cause similar cankers on giant sequoia and coast redwood in California (3). However, rDNA sequencing of the ITS region of this isolate obtained from the American Type Culture Collection (ATCC 60344) (GenBank JQ284384) showed it matched the type specimen of Neofusicoccum australe (GenBank GU251219), not our isolate. To our knowledge, this is the first report of N. nonquaesitum as a pathogen of giant sequoia in North America. This study expands the host range of N. nonquaesitum from almond (Prunus dulcis), California bay (Umbellularia californica), and blueberry (Vaccinium spp.) (1) to include giant sequoia, coast redwood, and tanoak, which are economically important trees in California forests and landscapes. References: (1) P. Inderbitzin et al. Mycologia 102:1350, 2010. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (3) J. J. Worrall et al. Plant Dis. 70:757, 1986.

Evidence of neurodegeneration in brains of older adults who do not yet fulfill MCI criteria.

We sought to determine whether there are structural and metabolic changes in the brains of older adults with cognitive complaints yet who do not meet MCI criteria (i.e., preMCI). We compared the volumes of regional lobar gray matter (GM) and medial temporal lobe structures, including the hippocampus, entorhinal cortex (ERC), fusiform and parahippocampal gyri, and metabolite ratios from the posterior cingulate in individuals who had a Clinical Demetia Rating (CDR) of 0.5, but who did not meet MCI criteria (preMCI, N=17), patients with mild cognitive impairment (MCI, N=13), and cognitively normal controls (N=18). Controls had more ERC, fusiform, and frontal gray matter volume than preMCI and MCI subjects and greater parahippocampal volume and more posterior cingulate N-acetylaspartate (NAA)/myoinosotil (mI) than MCI. There were no significant differences between MCI and preMCI subjects on any of these measures. These findings suggest there are neurodegenerative changes in the brains of older adults who have cognitive complaints severe enough to qualify for CDR=0.5 yet show no deficits on formal neuropsychological testing. The results further support the hypothesis that detection of individuals with very mild forms of Alzheimer's disease (AD) may be facilitated by use of the CDR, which emphasizes changes in cognition over time within individuals rather than comparison with group norms.

Identification and characterization of a chitinase antigen from Pseudomonas aeruginosa strain 385.

A chitinase antigen has been identified in Pseudomonas aeruginosa strain 385 using sera from animals immunized with a whole-cell vaccine. The majority of the activity was shown to be in the cytoplasm, with some activity in the membrane fraction. The chitinase was not secreted into the culture medium. Purification of the enzyme was achieved by exploiting its binding to crab shell chitin. The purified enzyme had a molecular mass of 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.2. NH2-terminal amino acid sequencing revealed two sequences of M(I/L)RID and (Q/M/V)AREDAAAAM that gave an exact match to sequences in a translated putative open reading frame from the P. aeruginosa genome. The chitinase was active against chitin azure, ethylene glycol chitin, and colloidal chitin. It did not display any lysozyme activity. Using synthetic 4-methylumbelliferyl chitin substrates, it was shown to be an endochitinase. The Km and kcat for 4-nitrophenyl-beta-D-N,N'-diacetylchitobiose were 4.28 mM and 1.7 s(-1) respectively, and for 4-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, they were 0.48 mM and 0.16 s(-1) respectively. The pH optimum was determined to be pH 6.75, and 90% activity was maintained over the pH range 6.5 to 7.1. The enzyme was stable over the pH range 5 to 10 for 3 h and to temperatures up to 50 degrees C for 30 min. The chitinase bound strongly to chitin, chitin azure, colloidal chitin, lichenan, and cellulose but poorly to chitosan, xylan, and heparin. It is suggested that the chitinase functions primarily as a chitobiosidase, removing chitobiose from the nonreducing ends of chitin and chitin oligosaccharides.

Characterisation of a thermostable pepstatin-insensitive acid proteinase from a Bacillus sp.

An acid proteinase, Wai 21a, produced by a thermophilic Bacillus species (strain Wai 21a) has been purified to homogeneity by cation-exchange chromatography, phenyl-Sepharose chromatography and anion-exchange chromatography. A pI of 3.8 was determined by isoelectric focussing. The protein contained some associated carbohydrate (20 mol hexose equiv/mol proteinase). Optimal proteolytic activity was observed at pH 3.0 (at 60 degrees C). The Leu15-Tyr16 bond was the major site of hydrolysis for the oxidized B chain of insulin. Enzyme activity was not affected by inhibitors of the cysteine, metallo or serine class of proteinases. The aspartate proteinase inhibitor, pepstatin, did not inhibit enzyme activity. Inhibition of enzyme activity by 1,2-epoxy-3-(p-nitrophenoxy)-propane indicated the presence of at least one carboxyl group essential to the catalytic mechanism of the enzyme. Proteinase activity was inhibited by diazoacetyl-DL-norleucine methyl ester in a slow and non-specific manner atypical of pepstatin-sensitive aspartate proteinases. Wai 21a proteinase may be classified as member of the pepstatin-insensitive group of aspartate proteinases. The thermal stability at pH 3.0 and 60 degrees C increased 2.1-fold (t1/2, 4.5-9.7 hr) in the presence of 5 mM Ca++. An increase in both pH (3.0-4.5) and Ca++ concentration (0-30 mM) resulted in a 15-fold increase (t1/2, 15-230 min) in thermal stability at 75 degrees C. The amino acid composition of Wai 21a proteinase was found to be similar to other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the thermostable enzyme, kumamolysin.

Five human gastric aspartic proteinases: N-terminal amino acid sequences and amino acid composition.

Human pepsin A consists of 4 or more isoenzymes (designated 1, 3a, 3b and 3c) one of which, pepsin 1, contains up to 50% carbohydrate moieties. The amino-acid composition and N-terminal sequence of pepsin 1 and the other isoforms have been determined and compared with data obtained for pepsin 3b and gastricsin (pepsin C or pepsin 5). Pepsins were isolated from penta-gastrin stimulated gastric juice using repetitive chromatography on DEAE-cellulose, or high performance ion-exchange chromatography. Sequencing was performed using automated solid-phase Edman degradation with a microsequence facility. The amino-acid compositions were similar for pepsins 1, 3a, b and c and the N-terminal sequences of pepsins 1, 3a and c, reported for the first time, were shown to be identical with that for pepsin 3b (the main component of pepsin A) although residue 28 was unassigned in pepsin 1. Residue 30 in all four isoenzymes is valine and we cannot confirm reports of major pepsins with leucine in this position. For gastricsin the sequence differed from the pepsin isoenzymes and in position 24 we find pro rather than ala as was first described. These observations suggest that pepsin 1 is identical to 3b or a mixture of 3a, 3b and 3c but not gastricsin. This data supports the hypotheses that the four pepsin isoenzymes are products of the same gene(s) but have undergone varying levels of post translational modification.

Calorie sources and recovery from central nervous system ischemia.

OBJECTIVES: Glucose is the primary substrate for the energy requirements of the nervous system. Nevertheless, administration of glucose to critically ill patients with central nervous system trauma may have adverse effects on their neurologic recovery. The purpose of this study was to evaluate the effects of other sources of nonprotein calories on spinal cord lactate accumulation and on electrophysiologic recovery after a period of severe spinal cord ischemia. DESIGN: Two randomized, blinded studies were performed: one of glycolytic energy substrates (fructose, xylitol, sorbitol, glycerol) and one of ketogenic energy substrates (beta-hydroxybutyrate, acetate, butyrate). SETTING: College teaching hospital laboratory. SUBJECTS: New Zealand albino rabbits (weight 3.5 to 4.5 kg). INTERVENTIONS: After infusion of the randomly assigned treatment, temporary ischemia was produced in the lumbosacral spinal cord by occluding the abdominal aorta with a balloon catheter. MEASUREMENTS AND MAIN RESULTS: Blood concentrations of glucose, lactate, pyruvate, and ketone bodies and spinal cord dialysate concentration of lactate were measured before and after infusion of the assigned treatment, and during ischemia and during the first 2 hrs after reperfusion. Spinal somatosensory evoked potentials were recorded during ischemia to assure a similar severity of ischemia in all animals and during the first 2 hrs after reperfusion as a measure of electrophysiologic recovery. Infusion of the glycolytic nutrients xylitol and fructose increased blood glucose and lactate concentrations, and resulted in increased lactate accumulation in the spinal cord during ischemia and resulted in a significantly poorer recovery of the spinal somatosensory evoked potential than infusion of saline. Infusion of sorbitol and glycerol did not have these adverse effects in the doses administered. None of the ketogenic nutrients increased blood glucose concentration or increased lactate accumulation in the spinal cord during ischemia when compared with infusion of saline. Infusion of butyrate and acetate caused arterial hypotension and resulted in a poorer recovery of the spinal somatosensory evoked potential than saline. Infusion of beta-hydroxybutyrate did not have an adverse effect on blood pressure or on evoked potential recovery. CONCLUSIONS: Glycerol, sorbitol, and beta-hydroxybutyrate deserve further evaluation as potential nonprotein calorie sources in patients with neurologic injury. Xylitol and fructose are not suitable since these substrates resulted in hyperglycemia and increased lactate accumulation in the central nervous system, and had detrimental effects on electrophysiologic recovery after ischemia. The short-chain fatty acids (acetate and butyrate) also had adverse effects on electrophysiologic recovery after ischemia, probably because of their hypotensive effects when given intravenously, rather than from the effects of their metabolism.

Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A.

A proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac-Phe-OEt and Bz-Ala-OMe. Tur-NH(2), Trp-NH(2), Leu-pNA, and Val-pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz-ala-Tyr-NH(2) was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz-ala-OMe, 25 mM Tyr-NH(2), 70 degrees C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz-Ala-OMe and 615 mM Tyr-NH(2) at 40 degrees C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; -betaNA and -pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield.

Immobilization of a proteinase from the extremely thermophilic organism Thermus Rt41A.

An extracellular proteinase from Thermus strain Rt41A was immobilized to controlled pore glass (CPG) beads. The properties of the free and CPG-immobilized enzymes were compared using both a large (azocasein) and a small (peptidase) substrate. The specific activity of the immobilized proteinase was 5284 azoU/mg with azocasein and 144 sucU/mg for SucAAPFpNA. The percentage recovery of enzyme activity was unaffected by pore size when it was immobilized at a fixed level of activity/g of beads, whereas it increased with increasing pore size when added at a fixed level/m(2) of support. Saturation of the CPG beads was observed at 540 azoU/m(2) of 105-nm beads. Lower levels (50 azoU/m(2) of 50-nm beads) were used in characterization experiments. The pH optimum of the immobilized Rt41A proteinase was 8.0 for azocasein and 9.5 for SucAAPFpNA, compared with the free proteinase which was 10.5 for both substrates. The immobilized enzyme retained 65% of its maximum activity against azocasein at pH 12, whereas the free proteinase retained less than 10% under the same conditions. Stability at 80 degrees C increased on immobilization at all pH values between 5 and 11, the greatest increase in half-life being approximately 12-fold at pH 7.0. Temperature-activity profiles for both the free and immobilized enzymes were similar for both substrates. The stability of the immobilized proteinase, however, was higher than that of the free enzyme in the absence and presence of CaCl(2). Overall, the results show that low levels of calcium (10 muM) protect against thermal denaturation, but that high calcium or immobilization are required to protect against autolysis. (c) 1994 John Wiley & Sons, Inc.

Characterization of a chelator-resistant proteinase from Thermus strain Rt4A2.

The Thermus isolate Rt4A2 was found to produce an extracellular chelator-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtration chromatography, and weak anion-exchange chromatography. The Rt4A2 proteinase was found to have properties typical of an alkaline serine proteinase. It had a pH optimum of 9.0 and was specifically inhibited by phenylmethanesulphonyl fluoride. Its isoelectric point was greater than 10.25. Its molecular-mass was 31.6 kDa as determined by SDS/PAGE. N-terminal sequencing has shown it to have high sequence similarity with other serine proteinases from Thermus species. The proteinase hydrolysed a number of substrates including fibrin, casein, haemoglobin, collagen, albumin and the synthetic chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition was observed above an azocasein concentration of 0.05% (w/v). Esterase activity was directed mainly towards those substrates containing the aliphatic or aromatic residues of alanine, glycine, tryptophan, tyrosine and phenylalanine. Thermostability half-lives of greater than 7 days at 70 degrees C, 43 h at 80 degrees C and 90 min at 90 degrees C were found in the presence of 5 mM CaCl2. At 90 degrees C increasing the CaCl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increase in the half-life of the enzyme from 30 to 130 min. Half-lives of 19.4 min at 100 degrees C and 4.4 min at 105 degrees C were found in the presence of 50 mM CaCl2. The metal chelators EGTA and EDTA reduced the stability at higher temperatures but had no effect on the activity of the proteinase. Activity was not stimulated by common metal activators such as Ca2+, Mg2+ and Zn2+.

Some characteristics of a proteinase from a thermophilic Bacillus sp. expressed in Escherichia coli: comparison with the native enzyme and its processing in E. coli and in vitro.

Proteinase Ak.1 was produced during the stationary phase of Bacillus sp. Ak.1 cultures. It is a serine proteinase with a pI of 4.0, and the molecular mass was estimated to be 36.9 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 60 and 70 degrees C, with half-lives of 13 h and 19 min at 80 and 90 degrees C, respectively. Maximum proteolytic activity was observed at pH 7.5 with azocasein as a substrate, and the enzyme also cleaved the endoproteinase substrate Suc-Ala-Ala-Pro-Phe-NH-Np (succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanalide). Major cleavage sites of the insulin B chain were identified as Leu-15-Tyr-16, Gln-4-His-5, and Glu-13-Ala-14. The proteinase gene was cloned in Escherichia coli, and expression of the active enzyme was detected in the extracellular medium at 75 degrees C. The enzyme is expressed in E. coli as an inactive proproteinase at 37 degrees C and is converted to the mature enzyme by heating the cell-free media to 60 degrees C or above. The proproteinase was purified to homogeneity and had a pI of 4.3 and a molecular mass of 45 kDa. The NH2-terminal sequence was Ala-Ser-Asn-Asp-Gly-Val-Glu-, showing the exact signal peptide cleavage point. Heating the proenzyme resulted in the production of active proteinase with an NH2-terminal sequence identical to that of the native enzyme. The characteristics of the cloned proteinase were identical to those of the native enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in contact lens comfort related to the menstrual cycle and menopause. A review of articles.

Many women report changes in contact lens comfort during different phases of their menstrual cycle. Dryness and tearing, decreased visual acuity, swollen lids, foreign body sensations and visual coordination problems are examples of what may occur. This paper is a review of recent and older articles relating changes in contact lens comfort to modifications of the glands, conjunctiva and cornea of the eye during different phases of the menstrual cycle. Both high and low estrogen levels seem to affect the eye differently in various phases of the cycle. Some assumptions and predictions of what may happen to the eye at menopause also will be discussed.

The use of phenyl-Sepharose for the affinity purification of proteinases.

Phenyl-Sepharose is most often used as an adsorbent for hydrophobic interaction chromatography (HIC). We report on its effective use for the affinity purification of some extracellular thermostable proteinases from bacterial sources. Proteinases belonging to the serine, aspartate and metallo mechanistic classes were effectively retained by the media. Purification factors in the range of 2.9-60 and enzyme activity yields in excess of 88% were obtained. In some cases homogeneous enzyme was obtained from culture supernatants in a single step. A number of other proteinases from mammalian sources were also retained. The specificity of the enzyme/support interaction was studied. Proteinases complexed with peptide inhibitors (pepstatin and chymostatin) showed reduced binding to phenyl Sepharose indicating interaction with the active site cleft whereas modification with low molecular weight active site directed inactivators such as PMSF and DAN did not, indicating that binding may not be dependent on the catalytic site. Pepsinogen and the pro-enzyme form of the serine proteinase from the thermophilic Bacillus sp. strain Ak.1 were not retained by the media and could be resolved in an efficient manner from their active counterparts.

Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A.

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25-10.5) which can be exploited in purification by using cation-exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0-10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0-10.0 and maximum activity, in a 10-min assay, was observed at 90 degrees C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70 degrees C and the half-lives at 80 degrees C and 90 degrees C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60 degrees C and the half-life at 70 degrees C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents. Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1' amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1' position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1' sites.

Hypercalcaemia due to calcium binding by a polymeric IgA kappa-paraprotein.

Total serum calcium concentration was raised in a 63-year-old lady with multiple myeloma and markedly elevated serum IgA kappa-paraprotein concentration. Symptoms of hypercalcaemia were absent, and serum ionized calcium was normal, suggesting calcium binding by the abnormal protein. This was demonstrated directly after isolation of the paraprotein and characterization of the calcium/protein interaction. After reduction of the paraprotein with mercaptoethanol, sodium dodecyl sulphate polyacrylamide gradient gel electrophoresis revealed two bands corresponding to light and heavy chains, but under non-reducing conditions the isolated paraprotein migrated in a series of bands, possibly representing polymeric forms of the basic immunoglobulin moiety.

Glucose metabolism and acidosis in the metabolic penumbra of rat brain.

The characterization of tissue acid-base status related to the penumbral zone of increased glucose consumption surrounding a focal cerebral ischemic lesion may suggest therapeutic techniques to maximize tissue survivability from stoke. We measured local cerebral metabolic rate for glucose (1 CMRglc) and an index of brain tissue pH (pHt) concurrently and characterized their interaction in a model of focal cerebral ischemia in rats in a double-label autoradiographic study, using [14C]2-deoxyglucose and [14C]dimethyloxazolidinedione. Computer-assisted digitization and analysis permitted the simultaneous quantification of the two variables on a pixel-by-pixel basis in the same brain slices. Hemispheres ipsilateral to intravascular tamponade-induced middle cerebral artery occlusion showed areas of normal, depressed, and elevated glucose metabolic rate (as defined by an interhemispheric asymmetry index) after 2 hr of ischemia. Regions of increased 1 CMRglc showed moderate acidosis (6.87 +/- 0.05), while regions of normal glucose metabolic rate showed normal pHt (pH +/- SD = 6.98 +/- 0.05) and regions of decreased 1 CMRglc showed severe acidosis (6.69 +/- 0.11). A repeated-measures analysis of variance found these values to differ from each other at the P less than 0.0005 significance level. The finding of moderate acidosis coupled with increased 1 CRMglc in the metabolic penumbra suggests that the excess protons may result from the anaerobic dissociation of ATP synthesis and hydrolysis.

Improved separation of human pepsins from gastric juice by high-performance ion-exchange chromatography.

A simple and precise separative procedure can now be used to isolate individual pepsins from gastric juice, and by measurement of the protein absorbance at 280 nm enable their direct quantitation. This will facilitate the study of pepsin secretion, particularly in patients with peptic ulcer disease.

Heterogeneity of human pepsin 1, as shown by high-performance ion-exchange chromatography.

We have shown that pepsin 1 can be prepared in milligram quantities from human gastric juice by semi-preparative high-performance ion-exchange chromatography. Further investigation into the elution of this enzyme using linear chloride gradients have shown it to be a heterogeneous mixture, the components of which all have peptic activity, but differing specific activities. These components are changed in number and retention time by incubation with hyaluronidase and aryl sulphatase, but not by neuraminidase or acid phosphatase, implying the presence of a sulphated proteoglycan.

Effects of moderate hypoxemia and unilateral carotid ligation on cerebral glucose metabolism and acid-base balance in the rat.

We used our recently developed method for the simultaneous measurement of the local CMRglc (LCMRglc) and composite tissue pH to evaluate the response to unilateral carotid ligation and moderate hypoxia [40.1 +/- 4.8 (SD) mm Hg]. The LCMRglc and tissue pH were measured simultaneously in brain slices using [14C]2-deoxy-D-glucose and [14C]5,5-dimethyl-2,4-oxazolidinedione. The ipsilateral LCMRglc was increased significantly in the caudate-putamen and medical thalamus and was surrounded by a much more extensive zone of acidosis, as shown by significant reductions in the tissue pH, which was affected in parietal cortex, caudate-putamen, lateral septal nucleus, medial geniculate, Ammon's horn, and nucleus reticularis of substantia nigra. In regions with an elevated LCMRglc and acidosis, anaerobic glycolysis combined with ATP hydrolysis are likely to co-exist. In regions characterized by normal glucose metabolism and acidosis, we hypothesize that a direct effect of hypoxia on the sodium/hydrogen ion antiporter may lead to secondary acidosis. Disturbed acid-base balance during hypoxia may have an adverse effect on cerebral function and cause clinical symptoms.

Simultaneous double-isotope autoradiographic measurement of local cerebral glucose metabolic rate and acid-base status in rat brain.

We developed a double-isotope autoradiographic method for the simultaneous measurement of the local cerebral metabolic rate for glucose (1CMRG) and index of regional acid-base status (rABI) in single brain slices using [2-14C]deoxy-D-glucose (DG) and 5,5-dimethyl-[2-14C]oxazolidine-2,4,dione (DMO). After iv isotope administration, paper chromatography separates plasma DMO from DG activity using a methanol-methylene chloride solvent system. Initial tissue autoradiograms depict regional DMO plus DG and DG metabolite distribution. After 14 days in a well-ventilated hood, 97.5 +/- 0.5% of all DMO is lost from tissue sections by sublimation, and a second autoradiogram depicts DG plus DG metabolite distribution. Retention of brain lipids does not alter beta-particle self-absorption, avoiding problems associated with isotope extraction with solvents. Autoradiograms are digitized and converted to isotope-content images. The second autoradiogram is used for 1CMRG computation. After subtracting the second regional isotope-content value from the first, the DMO content is obtained and used to compute rABI. Application of this method to normal animals yields expected values for 1CMRG and rABI. This method is amenable to whole-slice digitization and creation of functional images of 1CMRG and ABI followed by pixel-by-pixel correlations of the two variables, making this a potentially valuable tool for the investigation of the relationships between glucose metabolism and brain acid-base balance.