Some properties of a fungal β-D-glucanase preparation

A commercial enzyme preparation, of fungal origin, contained a mixture of β-D-glucanases which were fractionated by ion-exchange chromatography to give a mixture of an endo-(1→4)- and an exo-(1→3)-β-D-glucanase. These two enzymes were then separated by molecular-sieve chromatography on Sephadex G-150. The purified exo-(1→3)-β-D-glucanase has a relatively high specificity for (1→3)-β-D-glucosidic linkages, and has no action on lichenin.     

Protein and carbohydrate moieties of a preparation of β-lactamase II

1. A crystalline preparation of beta-lactamase II has been separated into two moieties by gel filtration on a column of Sephadex G-100. 2. The first moiety consisted mainly of carbohydrate and showed virtually no beta-lactamase activity. 3. The second moiety was a protein of molecular weight 22500, which was enzymically active. 4. The protein moiety, like the original protein-carbohydrate complex, required Zn(2+) for beta-lactamase activity. It did not differ significantly from the complex in its behaviour to a number of cephalosporin substrates, but was less stable to heat than the complex. 5. About 30% of the total beta-lactamase activity was lost when the protein-carbohydrate complex was separated into the two moieties. This activity was regained when the protein and carbohydrate moieties were mixed, but the mixture did not show the heat stability of the original complex.     

The preparation of adenosine 5′-pyrophosphate by a non-enzymic method

1. A non-enzymic method for the preparation of adenosine 5′-diphosphate is described, in which the terminal phosphate of adenosine 5′-triphosphate is transferred to methanol in the presence of hydrochloric acid. The final purified product can be obtained in 60% yield. 2. Experiments with [¹⁴C]methanol showed that no methylation of the adenosine diphosphate occurs during the reaction. 3. Confirmation that the pyrophosphate moiety of the adenosine diphosphate produced was in the 5′-position was obtained by: (a) periodate oxidation; (b) treatment with apyrase and examination of the resulting adenylic acid isomer by paper chromatography. 4. The method appears to be generally applicable to the preparation of nucleoside 5′-diphosphates from the corresponding nucleoside 5′-triphosphates.     

Enzymatic preparation of d-p -trimethylsilylphenylalanine

In this paper we report on the enzymatic preparation of d-p-trimethylsilylphenylalanine (d-TMS-Phe). First, dl-5-(p-trimethylsilylphenylmethyl)hydantoin␣(dl-TMS-Phe-Hyd) was synthesized chemically and subjected to bacterial hydrolysis to obtain N-carbamoyl-d-p-trimethylsilylphenylalanine (C-d-TMS-Phe), but no strains examined showed sufficient hydantoinase activity on this compound. However, Blastobacter sp. A17p-4, which is known to produce N-carbamoyl-d-amino acid amidohydrolase (DCase), was found to be able to hydrolyze C-dl-TMS-Phe prepared chemically from the hydantoin. When C-dl-TMS-Phe was hydrolyzed with cells of Blastobacter sp. A17p-4, its optical purity was low because N-carbamoyl-l-amino acid amidohydrolase (LCase) coexisted in the cells. DCase and LCase in the cell-free extract of Blastobacter sp. A17p-4 could be separated by DEAE-Sephacel column chromatography. The optimum pH for the hydrolysis of C-dl-TMS-Phe by the partially purified DCase was 8.0 and addition of 2.5 % N,N-dimethylformamide was effective in raising the substrate concentration without inactivation of DCase. Under the optimized conditions, highly optically pure (98 % enantiomeric excess) d-TMS-Phe could be obtained from C-dl-TMS-Phe with partially purified DCase. Received: 12 July 1996 / Received revision: 11 September 1996 / Accepted: 2 November 1996     

Purification and characterization of glycyrrhizin-β-d-glucuronidase and baicalin-β-d-glucuronidase from a commercial enzyme preparation

Abstract

Five commercial enzyme preparations were screened for hydrolysis of the glucuronic acid units of glycyrrhizin (GL) and baicalin. Two preparations hydrolyzing GL to glycyrrhetic acid (GA) and four enzyme preparations hydrolyzing baicalin to baicalein were obtained. One enzyme preparation with the ability to hydrolyze both GL and baicalin, namely Rapidase Pineapple, was purified by anion exchange, cation exchange and molecular sieve chromatography. The results of purification indicated that the enzymes containing the glycyrrhizin-β-d-glucuronidase (GBDG) and baicalin-β-d-glucuronidase (BBDG) activities were distinct, with different substrate specificities, molecular weights and enzymatic characteristics. GBDB hydrolyzed GL to GA, but had no detectable activity on baicalin, and BBDG hydrolyzed baicalin to baicalein, but could not hydrolyze GL. However, both GBDG and BBDG could hydrolyze the artificial substrate p-nitrophenyl- β-d-glucuronide (pNPGA).     

Prolastin,a pharmaceutical preparation of purified human α1-antitrypsin,blocks endotoxin-mediated cytokine release

Background

α1-antitrypsin (AAT) serves primarily as an inhibitor of the elastin degrading proteases, neutrophil elastase and proteinase 3. There is ample clinical evidence that inherited severe AAT deficiency predisposes to chronic obstructive pulmonary disease. Augmentation therapy for AAT deficiency has been available for many years, but to date no sufficient data exist to demonstrate its efficacy. There is increasing evidence that AAT is able to exert effects other than protease inhibition. We investigated whether Prolastin, a preparation of purified pooled human AAT used for augmentation therapy, exhibits anti-bacterial effects.

Methods

Human monocytes and neutrophils were isolated from buffy coats or whole peripheral blood by the Ficoll-Hypaque procedure. Cells were stimulated with lipopolysaccharide (LPS) or zymosan, either alone or in combination with Prolastin, native AAT or polymerised AAT for 18 h, and analysed to determine the release of TNFα, IL-1β and IL-8. At 2-week intervals, seven subjects were submitted to a nasal challenge with sterile saline, LPS (25 μg) and LPS-Prolastin combination. The concentration of IL-8 was analysed in nasal lavages performed before, and 2, 6 and 24 h after the challenge.

Results

In vitro, Prolastin showed a concentration-dependent (0.5 to 16 mg/ml) inhibition of endotoxin-stimulated TNFα and IL-1β release from monocytes and IL-8 release from neutrophils. At 8 and 16 mg/ml the inhibitory effects of Prolastin appeared to be maximal for neutrophil IL-8 release (5.3-fold, p < 0.001 compared to zymosan treated cells) and monocyte TNFα and IL-1β release (10.7- and 7.3-fold, p < 0.001, respectively, compared to LPS treated cells). Furthermore, Prolastin (2.5 mg per nostril) significantly inhibited nasal IL-8 release in response to pure LPS challenge.

Conclusion

Our data demonstrate for the first time that Prolastin inhibits bacterial endotoxin-induced pro-inflammatory responses in vitro and in vivo, and provide scientific bases to explore new Prolastin-based therapies for individuals with inherited AAT deficiency, but also for other clinical conditions.     

The physiological effect of ingested β-N-methylamino-L-alanine on a glutamatergic synapse in an in vivo preparation

The neurotoxin, BMAA (β-N-methylamino-L-alanine), may be a risk factor for amyotrophic lateral sclerosis (ALS), Parkinson's (PD) and Alzheimer's (AD) disease. In vivo experiments have demonstrated that BMAA can cause a number of motor dysfunctions if ingested or injected, and in vitro experiments show that this toxin binds to glutamate receptors with deleterious results. Also, BMAA exists in the human food chain worldwide, and has been detected in the brains of ALS and AD patients. This paper offers the first demonstration by intracellular recording of the effect of ingested BMAA on the postsynaptic response of an identified glutamatergic cell in a living, undissected organism (Drosophila melanogaster), and correlates these observations with the specific motor dysfunctions that result from ingestion. The results suggest that BMAA acts as a glutamate agonist, causing NMDA receptor channels to remain open for prolonged periods of time, thereby damaging the cell by excitotoxicity. The effect on the postsynaptic response became apparent days before the function of the postsynaptic cell (wing beat) became affected. Severely depolarized cells were able to fully recover with the removal of BMAA from the food source, suggesting that blocking BMAA binding in the brain might be a good treatment strategy.     

Regioselective preparation of 2′, 3′-di-O-acylribonucleosides carrying lipophilic acyl groups through a lipase-catalysed alcoholysis

Candida antarctica B lipase-catalysed alcoholysis of 2, 3, 5-tri-O-hexanoyluridine (1a), 2, 3, 5-tri-O-dodecanoyluridine (1b), 2, 3, 5-tri-O-hexanoylinosine (1c) and 2, 3, 5-tri-O-dodecanoylinosine (1d) proceeded regioselectively to produce the corresponding 2, 3-di-O-acylribonucleosides 2a–d, providing a simple and efficient access to these new lipophilic compounds. Contrasting to the alcoholysis, enzymatic hydrolysis of 1a–d using different enzymes and experimental conditions did not proceed regioselectively.     

Whole-blood immunoassay for γH2AX as a radiation biodosimetry assay with minimal sample preparation

The current state of the art in high-throughput minimally invasive radiation biodosimetry involves the collection of samples in the field and analysis at a centralized facility. We have developed a simple biological immunoassay for radiation exposure that could extend this analysis out of the laboratory into the field. Such a forward placed assay would facilitate triage of a potentially exposed population. The phosphorylation and localization of the histone H2AX at double-stranded DNA breaks has already been proven to be an adequate surrogate assay for reporting DNA damage proportional to radiation dose. Here, we develop an assay for phosphorylated H2AX directed against minimally processed sample lysates. We conduct preliminary verification of H2AX phosphorylation using irradiated mouse embryo fibroblast cultures. Additional dosimetry is performed using human blood samples irradiated ex vivo. The assay reports H2AX phosphorylation in human blood samples in response to ionizing radiation over a range of 0–5 Gy in a linear fashion, without requiring filtering, enrichment, or purification of the blood sample.     

A simplified preparation of hematoporphyrin-diethers—IV

• 1.1. A relatively simple technique is described for the synthesis of hematoporphyrin ethers, from dimethyl to dioctyl, based upon reaction under prescribed conditions of temperature and time of the porphyrin with the relevant alcohol containing 20% (w/v) H₂SO₄ and subsequent hydrolysis of ester functions.
• 2.2. Yields are good and means of purification, where necessary, are described.
• 3.3. The procedures should be suitable for large scale production and are relatively inexpensive.

     

Are Ranvier's nodes artifacts by preparation?

To avoid artifacts, native nerve fibers were investigated by phase contrast light microscopy. It was shown that Ranvier's nodes cannot be seen. At the sites at which they can be produced by thermal and chemical effects, a tight joint of the tube ends of the 'internodes' inverted to the inside are to be found. Some structures of Ranvier's nodes do not exist in intact living nerves. This fact can also be proved by electron micrography.     

Lipases in the preparation of β-blockers

β-Adrenergic blocking agents are pharmaceutical products used for the treatment of hypertension and angina pectoris. These β-blockers can be prepared in optically active form by conventional crystallization procedures, by asymmetric chiral synthesis and by using stereoselective enzymes.     

Fractionation of a commercial cellulase preparation from Penicillium funiculosum and its use in the purification of a water soluble α-glucan from milled barley

A commercial enzyme preparation from a selected strain of Penicillium funiculosum has been partially purified using a single stage chromatofocusing fractionation to produce an amylase-free mixture of hydrolytic enzymes. This mixture has been used to remove the non-starch polysaccharides from aqueous extracts of laboratory milled barley. The structure of the resulting purified α-glucan fraction has been examined by gel filtration before and after enzymic debranching and by iodine staining. The mild damage caused to the starch within the barley kernel releases a low molecular weight amylopectin molecule with no detectable amylose in the product. In this respect the product is different from that produced after severe, direct grinding of the purified barley starch where some amylose appears in the water soluble extract. Although the small amount of α-glucan is not of any quantitative industrial significance in itself, it does reflect the extent and type of physical damage which is taking place in the starch granule. The model proposed to explain these results - a starch granule with a solid amylose/amylopectin core but with a number of patches of protruding amylopectin clusters - may have important implications in an industrial context.     

Interactions between a commercial preparation of Bacillus thuringiensis and β-exotoxin in the fall armyworm,Spodoptera frugiperda

A commercial preparation of Bacillus thuringiensis (Dipel) and its β-exotoxin were assayed alone and in combination against neonate (3- to 6-hr-old) fall armyworm, Spodoptera frugiperda, larvae using a microdroplet technique. Positive dosage-mortality response curves were determined for each agent. Combination bioassays were then conducted using dosages based on these curves. Presence or absence of interactions was determined by comparing mortality levels from the combination assays with expected mortality levels using single degree of freedom χ² tests. Synergism occurred in 14 of 18 combinations of B. thuringiensis and β-exotoxin tested. Four combinations, all of which contained low individual dosages, exhibited additivity of effects. Maximum deviation of observed from expected mortality occurred at the combination of LC₃₀ for B. thuringiensis plus LC₃₀ for β-exotoxin (observed = 100%, expected = 51%). The data suggested that both agents were contributing to the synergism in this system, but that B. thuringiensis was a slightly stronger synergist than β-exotoxin.     

Modified xanthan—its preparation and viscosity

Xanthan with various pyruvic acid and acetate contents has been prepared from a single commercial polysaccharide sample using optimised chemical conditions (acid and alkali hydrolysis, respectively) for removal of acetal and acyl groups. The only significant change found on analysis of the modified xanthans was loss of pyruvic acid and/or acetate; no low moleculur weight carbohydrate-containing material was released. Contrary to some previous reports, evidence is presented to show that the pyruvic acid acetal and o-acetyl contents of xanthan do not affect solution viscosity. The viscosities of native, pyruvate-free and pyruvate/acetate-free xanthan solutions (0·3% w/v) were similar at shear rates 8·8–88·3 s^?1 in both distilled water and 1% KCl. Over the concentration range 0·2-1·5%, the viscosities of native and pyruvate-free xanthan at 10 s^?1 were similar. The viscosity increase on addition of 1% KCl to salt-free xanthan solutions was independent of pyruvic acid acetal substitution. Our results suggest that xanthan samples with various pyruvic acid acetal and o-acetal contents, prepared under different fermentation conditions of Xanthomonas campestri should not normally be used for assessing the contribution of these groups to solution viscosity.     

Liquefaction of dulse (Palmaria palmata (L.) Kuntze) by a commercial enzyme preparation and a purified endo ,β-1,4-D-xylanase

Liquefaction of dry and freshPalmaria palmata by food grade enzyme preparations and a purified endo--1,4-D-xylanase was studied.The endo--1,4-D-xylanase (EC 3.2.1.8) was purified to homogeneity from a commercial food grade enzyme prepared fromAspergillus niger. It has a molecular weight of 22 500, a pI of 3.5, is inactive toward corn arabinoxylan,p-nitrophenyl--D-xylose, carboxymethyl cellulose but shows a weak activity toward microcrystalline cellulose. It hydrolyzes oat and dulse xylan equally well in seawater and deionized water essentially into xylose and xylobiose. It is stable between pH 5.5 to 9.0 and 0 to 30 °C and its activity is optimal at pH 4.5–5.5 and 40–60 °C. It has a K_m of 2.2 and 2.8 mg ml^-1 and V_max of 3600 and 3900 nkat mg^-1 of protein on oat and dulse xylan, respectively.Acetate buffer, deionized water and seawater alone extracted 62.6 to 64.5 % of the dry weight of dry dulse, but the use of commercial food grade enzyme preparations or the purified xylanase improved liquefaction to 81.2–87.1 %. Xylose and galactose were the only sugars present in the soluble extracts. Deionized and seawater extracted 58.8–52.7 and 39.1–42.2% of the dry weight of the fresh algae collected in fall and summer, respectively. Only galactose was found in the seawater extract, while some xylose with galactose were measured in the deionized water extract of the fresh autumn algal sample. Purified and crude xylanase improved liquefaction of fresh algae to 79.8–81.4 and 71.9–77.9% of the fresh dry weight (fall and summer, respectively) in deionized and seawater, respectively, and increased the xylose content of the soluble fractions. Polysaccharides in the soluble residues were composed of 1,3/1,4-linked xylose, 1-linked galactose (floridoside) and 1,4-linked glucose (cellulose) and contained essentially 1,4-linked xylose and 1,4-linked glucose in insoluble fractions obtained after enzymatic treatment.The use of xylanase-containing food grade enzyme preparations improves liquefaction ofPalmaria palmata, particularly from fresh alga. This study indicates that processing such as drying may modify markedly the solubility ofP. palmata cell wall polysaccharides, which would imply the existence of some organization and/or other components in the fresh cell wall that lower xylan solubility in seawater.     

Fat metabolism in higher plants. β-hydroxylation of fatty acids by a soluble preparation from maturing avocado mesocarp

1. An avocado supernatant fraction converted fatty acids of medium chain length (C(8)-C(12)) into a polar product. 2. The product was identified as the beta-hydroxy derivative of the substrate by g.l.c. and t.l.c. analysis. 3. For hydroxylation of the fatty acids, CoA, ATP and molecular oxygen were required. Acyl carrier protein gave some stimulation. The reaction took place with oxygen alone if acyl-CoA was the substrate. 4. Hydroxylation was maximal with decanoic acid but dodecanoic acid and octanoic acid were also very active. Acids of shorter or longer chain lengths were not hydroxylated. 5. NAD(+) concentration caused complete inhibition at 0.5mm and may be an important control mechanism for the reaction in vivo. 6. The reaction was inhibited by iodoacetamide and by bipyridyl and carbon monoxide, indicating involvement of thiol and heavy metal groups.     

A facile preparation of α-hydroxy fatty acids

A convenient, one-step synthesis of α-hydroxy long chain, very long-chain and branched chain fatty acids from non-oxygenated fatty acids is described. The procedure involves preparation of fatty acid dianion using lithium diisopropylamide and oxygenation of the dianion by molecular oxygen.