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Biomedical subjects

B L Jones

Publications and source records attributed to B L Jones.

At least 19 recordsLinked to original sources
PubMed2001

Study of metallopeptidase isozymes from malted barley (Hordeum vulgare cv. Morex).

It has been reported that germinated barley contains peptidases that are sensitive to metal-chelating agents; however, none of these enzymes have been isolated, nor have their roles in germinated barley been investigated. Anion-exchange chromatography and chromatofocusing have been used to isolate a group of peptidases from barley (Hordeum vulgare cv. Morex) green malt that are sensitive to metal-chelating agents. Their activities were studied using one- and two-dimensional polyacrylamide gel electrophoresis. When analyzed on two-dimensional PAGE gels that contained gelatin as substrate, the enzymes separated into three major and approximately six minor activity spots with acidic pI values. The enzymes were optimally active against the gelatin substrate at pH 8.0 and were completely inhibited by 1,10-phenanthroline and EDTA, indicating that they belonged to the metallopeptidase class (EC 3.4.24.x). After the enzymes were inhibited with EDTA, the activities were recovered in the presence of low concentrations of metal ions. The hydrolysis of gelatin substrate was also impaired by the presence of reducing agents. The metallopeptidases readily digested, in vitro, the barley prolamine D hordein, indicating that they may be involved in degrading storage proteins during barley germination.

Anions↗
PubMed2001

Interactions of malt and barley (Hordeum vulgare L.) endoproteinases with their endogenous inhibitors.

For producing worts that are optimal for beer production, some, but not all, of the barley proteins must be degraded during malting and mashing. This protein hydrolysis is controlled by endoproteinases, and, in turn, is partially regulated by the presence of low-molecular-weight (LMW) proteinaceous inhibitors. This paper reports studies of the interactions between the proteinases and inhibitors and an "affinity" method for concentrating the inhibitors. The malt inhibitors (I) and proteinases (E) quickly formed strong (E-I) complexes when dissolved together, and all of the I was complexed. Heating at 100 degrees C, but not 70 degrees C, dissociated the complex, even though the enzyme activities were destroyed at 70 degrees C. The released I readily recomplexed with fresh E. Barley, however, contained insufficient E to complex all of its I complement. The E-I complex was treated with salts, detergents, and reducing agents to release active E molecules, but none disrupted the complex. By removing the LMW proteins from a malt E-I extract and dissociating the complex by heating, the concentration of I molecules was greatly increased. This "affinity" method can thus be used to concentrate the I molecules for further purification.

Beer↗
PubMed2000

Quantitative study of the formation of endoproteolytic activities during malting and their stabilities to kilning.

The proteinases of germinating barley (Hordeum vulgare L.) hydrolyze storage proteins into amino acids and small peptides that can be used by the growing plant or, during brewing, by yeast. They are critical for the malting and brewing processes because several aspects of brewing are affected by the amounts of protein, peptide, and amino acids that are in the wort. This study was carried out to quantitatively measure when endoproteinases form in green malt and whether they are inactivated at the high temperatures that occur during malt kilning. Little endoproteolytic activity was present in ungerminated barley, but the activities began forming 1 day into the "germination" phase of malting, and they were nearly maximal by the third germination day. Quantitative studies with azogelatin "in solution" assays showed that the green malt endoproteolytic activities were not inactivated under commercial kilning conditions that use temperatures as high as 85 degrees C but that some actually increased during the final kilning step. Qualitative (2-D, IEF x PAGE) analyses, which allow the study of individual proteases, showed that some of the enzymes were affected by heating at 68 and 85 degrees C, during the final stages of kilning. These changes obviously did not, however, decrease the overall proteolytic activity.

Electrophoresis, Polyacrylamide Gel↗
PubMed2000

Purification and partial characterization of a second cysteine proteinase inhibitor from ungerminated barley (Hordeum vulgare L.).

It was previously shown that ungerminated barley contains inhibitors that suppress the activities of green malt cysteine proteinases. This paper reports the purification and partial characterization of a second barley cysteine endoproteinase inhibitor, a protein called lipid transfer protein 2 (LTP2). The chromatographically purified inhibitor had a molecular mass of 7112. The amino acid composition and sequence data of the purified inhibitor indicated that it was a protein whose gene, but not the protein itself, was isolated earlier from barley aleurone tissue. The purified protein inhibited the activities of electrophoretically separated green malt cysteine proteinases but not the activities of the serine- or metalloproteinases. The purified LTP2 inhibited the same proteases as the LTP1 that was characterized previously but was present in the mature seed in much smaller amounts. Neither LTP1 nor LTP2 has been proven to transport lipids in vivo, and it seems possible that both serve to keep cysteine endoproteinases that are synthesized during barley seed development inactive until the plant needs them. The small amount of LTP2 in the seed made it impossible to determine whether it, like LTP1, is involved in beer foam formation. Because of its proteinase-inhibiting ability and its resistance to heat inactivation, some of the LTP2 may persist in beer.

Amino Acid Sequence↗
PubMed2000

Control of an outbreak of respiratory syncytial virus infection in immunocompromised adults.

Respiratory syncytial virus (RSV) is increasingly recognized as an important pathogen in immunocompromised adults, particularly those receiving bone marrow transplants, and, given the ease with which it spreads, represents a significant nosocomial problem. We describe an outbreak of RSV infection involving eight patients on a haematology/oncology ward which was controlled by early screening of patients and staff. Positive patients were cohort nursed on a separate ward and basic infection control measures including use of gowns and gloves were enforced. Children under age 12 were denied ward access. All patients with lower respiratory tract infection, and bone marrow transplant recipients with upper respiratory symptoms, were treated with nebulized ribavirin. There were no deaths. We conclude that awareness of the risk of RSV infection in immunocompromised patients coupled with rapid diagnosis and treatment, screening of symptomatic patients and staff, cohort nursing of cases and basic infection control procedures can prevent spread of RSV infection and reduce morbidity.

Adult↗
PubMed2000

An outbreak of Serratia marcescens in two neonatal intensive care units.

Outbreaks of infection in neonatal intensive care units (NICUs) due to Serratia marcescens are well recognized. In some outbreaks no point source has been found, whereas in others cross-infection has been associated with contaminated ventilator equipment, disinfectants, hands and breast pumps. We report an outbreak due to S. marcescens that involved two geographically distinct NICUs. The outbreak occurred over a six week period; 17 babies were colonized, 12 at Glasgow Royal Maternity Hospital (GRMH) and five at the Queen Mothers Hospital (QMH). At GRMH three babies developed septicaemia, of whom two died. The outbreak isolates were of the same serotype and phage type and were indistinguishable on the basis of restriction fragment length polymorphism analysis. During the outbreak, two babies shown consistently to be negative on screening, were transferred between the two units. In addition, two members of medical staff attended both units. In QMH no means of cross infection was identified. However, in GRMH the outbreak strain of S. marcescens was isolated from a laryngoscope blade and a sample of expressed breast milk.

Breast Feeding↗
PubMed2000

Learning disabilities: diagnostic considerations from an educational perspective.

This article reviews the current definitions of specific learning disability (LD). It describes the multiple causes among the specific learning disability (SLD) population, citing cognitive, neuroanatomical, cellular, and genetic research trends. Emphasis is placed on assessment components and processes, including typically used academic, cognitive, and neuropsychological measures and newly introduced dynamic or domain-based approaches. Researchers and practitioners now acknowledge that SLD represents a life-long condition, manifesting differently at each developmental stage. The impact of LD on adult adjustment and independence is described as the newest challenge in the field of LD research.

Adult↗
PubMed1999

Separation and characterization of barley (Hordeum vulgare L.) hordeins by free zone capillary electrophoresis.

Extraction conditions, separation conditions, and capillary rinsing protocols were optimized for the separation of barley hordeins by free zone capillary electrophoresis. Stable hordein extracts were obtained with a single 5 min extraction after the albumins and globulins were removed. Hordeins had to be reduced for optimal resolution. Optimum separation conditions for hordein separations were 100 mM phosphate-glycine buffer containing 20% acetonitrile and 0.05% hydroxypropylmethylcellulose. The addition of zwitterionic sulfobetaine detergents containing hydrocarbon tails of eight and ten carbons slightly improved the resolution of the separations, but not enough to warrant their use on a routine basis. The migration positions of the hordein subclasses were determined by two- dimensional reversed-phase high-performance liquid chromatography x free zone capillary electrophoresis mapping. The hordein subclasses formed clusters similar to those of wheat gliadins. Separation-to-separation repeatability was good, with migration time relative standard deviations < 1% for a 15-run period. For routine discrimination of cultivars, a 2 min post-separation rinse with 500 mM acetic acid was necessary to prevent protein build-up on the capillary walls. An example of successfully differentiating barley cultivars using this technique is shown.

Chromatography, High Pressure Liquid↗
PubMed1999

Conversion of acetaldehyde-protein adduct epitopes from a nonreduced to a reduced phenotype by antigen processing cells.

Many investigators have suggested that an immune reaction to acetaldehyde-protein adducts may be involved in the development and/or progression of alcohol liver disease. The most often reported acetaldehyde adduct is the reduced adduct prepared in vitro in the presence of strong reducing agents. However, the production of this adduct in vivo has been difficult to prove. Nevertheless, the detection of serum antibodies to this reduced adduct following alcohol exposure in animals and humans has been used to support the formation of this adduct in vivo. We have recently observed that when acetaldehyde-protein adducts prepared under nonreducing conditions are used to immunize animals, antibody to the reduced protein adduct is detected. Therefore, it was the purpose of this study to demonstrate that nonreduced (NR) adduct epitopes can be modified by intact cells to express reduced (R) adduct epitopes. This was accomplished using the monoclonal antibody RT1.1 that has been previously characterized by this laboratory and has been shown to recognize only R and not NR acetaldehyde adducts. In these studies, Balb/c mice were injected intraperitoneally (500 microg/animal) with either keyhole limpet hemocyanin (KLH)-NR or KLH-R adducted proteins. Immunization with KLH-NR produced significant amounts of antibodies that recognized both NR and R epitopes. In contrast, immunization with KLH-R produced antibodies to only R and not NR epitopes. Isolated peritoneal macrophages from nonimmunized mice were incubated in vitro with either KLH-NR, KLH-R, or unmodified KLH proteins, and the cell surface expression of the reduced epitope (RT1.1) and the activated macrophage marker (MAC-3) determined by double immunofluorescent staining. Activated macrophages incubated with KLH-NR expressed the R adduct on 11.5% of the cells, compared with 3.8% following incubation with unmodified KLH, and 19.4% following incubation with KLH-R. These data suggest that the NR adduct and/or the carrier protein are modified by peritoneal macrophages in vivo and present an epitope that is detected as a reduced adduct (RT1.1 positive). These observations may explain the presence of circulating antibodies to the reduced adduct that has been reported in human and animal studies.

Acetaldehyde↗
PubMed1998

Simplified endoproteinase assays using gelatin or azogelatin.

Endoproteinase assays with gels containing incorporated gelatin have shown that gelatin is an exceptional substrate for studying enzymes from different sources. However, due to its solubility in trichloroacetic acid, gelatin is not suited to "in-solution" assays carried out in the classic manner (by reading the absorbance of supernatants of hydrolysis reactions after the substrate has been precipitated with trichloroacetic acid). In this paper we demonstrate that gelatin can be used for such analyses by using isopropanol as precipitating reagent. Alternatively, azogelatin, which is precipitated by trichloroacetic acid, can be used. Azogelatin also serves as a very good substrate. One problem with using gelatin (or any nonderivatized protein) as substrate for measuring the activity of crude enzyme preparations is that protein contaminants in the enzyme preparation are hydrolyzed. The resulting peptides are impossible to differentiate from those released from the gelatin substrate. This problem is obviated when azogelatin is used, since its peptides are detected at 440 nm, where nonderivatized peptides do not absorb. Unlike some azo-derivatized proteins, azogelatin is soluble from pH 3.0 to 9.0. This, together with the fact that it is hydrolyzed by many different endoproteinases, makes it suitable for many applications.

Electrophoresis, Polyacrylamide Gel↗
PubMed1997

Subdural empyema due to Enterococcus faecalis.

Central nervous system infections due to Enterococcus species are uncommon. We report the first case of subdural empyema due to Enterococcus faecalis. Following partial treatment of a middle ear infection due to Enterococcus species and mixed coliforms, the patient developed signs of meningeal involvement. A lumbar puncture showed a raised polymorph cell count, but was sterile on culture; broad-spectrum antimicrobial therapy with cefotaxime, flucloxacillin and metronidazole was commenced. Following development of focal neurological signs, a CT scan revealed a subdural collection. Drainage and culture of the pus yielded a pure growth of Enterococcus faecalis. This case demonstrates the need to remain aware of the ability of the Enterococcus to cause serious infections and to direct specific antimicrobial therapy accordingly.

Adolescent↗
PubMed1996

Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley.

Proteolytic enzymes hydrolyze cereal seed storage proteins into small peptides and amino acids, which are very important for seed germination and the malting process. A cysteine-class endopeptidase was purified from 4-d-germinated barley (Hordeum vulgare L. cv. Morex). Four purification steps were used, carboxymethyl cellulose cation-exchange chromatography, chromatofocusing, size-exclusion chromatography, and electroelution from a polyacrylamide gel. The endopeptidase was most active at pH 4.5. It's isoelectric point (pI) was 4.4, as determined by isoelectric focusing, and it's SDS-PAGE molecular size was 31 kDa. The enzyme specifically hydrolyzed peptide bonds when the S2 site contained relatively large hydrophobic amino acids. The N-terminal amino acid sequence residues (1-9) of the 31-kDa endopeptidase had high homology to those of the EP-A and EP-B cysteine proteinases reported previously. The 31-kDa endopeptidase had a hydrolytic specificity similar to that of the Morex green malt 30-kDa endopeptidase we characterized previously, and also reacted with the antibody raised against the purified 30-kDa proteinase, but the two had different mobilities on non-denaturing PAGE. The hydrolytic specificities of both 30- and 31-kDa endopeptidases are such that both would very quickly cleave hordein (barley storage) proteins to small glutamine- and proline-rich peptides that could be quickly degraded to amino acids by barley exopeptidases.

Antimicrobial Cationic Peptides↗
PubMed1996

Effect of 12 atmospheres helium-oxygen on the response of mice to convulsant drugs.

The mechanism by which 12 atm abs of a helium-oxygen gas mixture (heliox) antagonizes behavioral effects of ethanol is unknown. Although the threshold for pressure-reversal of general anesthesia and expression of the high pressure neurologic syndrome (HPNS) is well above 12 atm abs in mice, the ethanol antagonism by 12 atm abs heliox could result from similar underlying excitatory effects. To investigate this possibility, the behavior of water-injected control mice and the latency to convulsions in drug-injected mice were determined in 1 atm abs air and 12 atm abs heliox. Four convulsant drugs were tested: picrotoxin (2 mg/kg), dl-allylglycine (300 mg/kg), isoniazid (300 mg/kg), and l-methionine-dl-sulfoximine (170 mg/kg). Responses were videotaped to observe behavior and to measure latency to the onset of myoclonus and clonus. Results indicated no observable excitatory effects of 12 atm abs in control mice. The latency to myoclonus was significantly reduced by pressure in allylglycine-treated mice but not in mice treated with the other convulsants. Latency to clonus was not significantly altered by pressure, relative to latency at 1 atm abs heliox, for any drug tested. In conclusion, the present findings indicate that exposure to 12 atm abs heliox is not proconvulsant and, thus, the findings do not support the hypothesis that 12 atm abs heliox antagonizes ethanol indirectly via an increase in central nervous system excitability.

Allylglycine↗
PubMed1995

Linkage of vancomycin and high-level gentamicin resistance genes on the same plasmid in a clinical isolate of Enterococcus faecalis.

A transferable 55-MDa plasmid which encoded resistance to both vancomycin and high concentrations of gentamicin was identified in a clinical isolate of Enterococcus faecalis. The plasmid hybridized with probes for the vanB and aac6'aph2" resistance genes. This is the first report of linkage of glycopeptide and high-level aminoglycoside resistance genes in an Enterococcus sp.

Base Sequence↗
PubMed1995

Aeromonas infections and their treatment.

With advances in the identification and molecular taxonomy of Aeromonas spp., these organisms, which are widely distributed in the environment, are increasingly being recognised as human pathogens. Clinical infections include gastroenteritis, skin and soft tissue infections and bacteraemia. Antibiotic resistance poses a potential problem in the antimicrobial therapy of infections cased by Aeromonas spp. While most strains are susceptible to chloramphenicol, ciprofloxacin, co-trimoxazole and the aminoglycosides, the activity of amoxycillin/clavulanate and the acylureidopenicillins is inconsistent. Addition of a beta-lactamase inhibitor does not significantly enhance the activity of the acylureidopenicillins. Aztreonam and the carbapenems, imipenem and meropenem remain highly active. Although resistance to the first and second generation cephalosporins is variable, more than 90% of Aeromonas spp. are susceptible to the third generation agents. Of potential significance is the identification of chromosomally-encoded inducible beta-lactamases, associated with resistance to extended spectrum penicillins, cephalosporins, monobactams and carbapenems, in clinical isolates of Aeromonas spp. Two distinct enzymes are produced: the A1 enzyme, a serine beta-lactamase behaving as a group 1 cephalosporinase, and the A2 enzyme, a metallo beta-lactamase which hydrolyses a wide range of beta-lactam agents including the carbapenems. The clinical relevance of these enzymes in Aeromonas spp. is unclear.

Aeromonas↗
PubMed1994

Use of monoclonal antibodies to study the structure and function of eukaryotic protein synthesis initiation factor eIF-2B.

The eukaryotic protein synthesis initiation factor, eIF-2B, is a multimeric protein of five different subunits termed alpha, beta, gamma, delta and epsilon, which facilitates recycling of a further factor, eIF-2, and is an important control point in the initiation process. In order to investigate the structure and function of eIF-2B, monoclonal antibodies have been prepared to the beta, delta and epsilon subunits of the factor from rabbit reticulocytes. All three antibodies are active in Western blotting, ELISA and immunoprecipitation. The anti-epsilon antibody inhibits both the guanine nucleotide exchange activity of eIF-2B and protein synthesis in the rabbit reticulocyte lysate at the level of initiation. The other two antibodies do not inhibit either guanine nucleotide exchange or protein synthesis. The monoclonal antibodies and a polyclonal anti-(rabbit reticulocyte eIF-2B) serum were used to investigate the subunit size and the antigenic structure of eIF-2B from a variety of rabbit tissues and from a variety of mammalian species. eIF-2B from all rabbit tissues tested was indistinguishable from that prepared from rabbit reticulocytes. Quantitative studies showed substantial variation in the relative concentrations of eIF-2 and eIF-2B between different rabbit tissues. Marked variation in both the sizes of the subunits and their reaction with the antibodies was observed between eIF-2B from rabbit, rat, guinea pig and man.

Animals↗
PubMed1994

Morphine-6-glucuronide: a potent stimulator of locomotor activity in mice.

The present study tested the hypothesis that morphine glucuronides have stimulant properties by studying their effects on locomotor activity in mice. Drug-naive C57BL/6J male mice were injected with saline, morphine, morphine-6-glucuronide (M6G) or morphine-3-glucuronide (M3G). In some experiments, mice were injected with saline or naloxone 5 min prior to drug treatment. Injection of 40 mg/kg morphine or M6G, but not M3G, significantly increased activity versus saline. The extent of activation induced by M6G was markedly higher than for morphine. Subsequent dose-response studies across a somewhat lower dose range using equimolar doses of morphine and M6G (3-80 mumoles/kg) found that both drugs significantly increased locomotor activity beginning at 20 mumoles/kg. M6G increased locomotor activity from 1.3 to 2.1 times more than for equimolar doses of morphine. Pretreatment with naloxone (10 mg/kg) completely abolished the locomotor stimulation induced by 32 mumoles/kg morphine and M6G. These findings present evidence that M6G is an active metabolite of morphine which has behaviorally stimulating effects and may play an important role in mediating the reinforcing properties of morphine in humans.

Animals↗