An Outbreak of Aseptic Meningitis due to Echo 25 Virus

In a widespread outbreak of aseptic meningitis during the summer and early fall of 1961 in Ontario, Echo type 25 virus was found to be the predominant virus type isolated from affected patients. Recovery of Echo 25 virus in several cases from the CSF of patients strongly suggests an etiological relationship between this virus type and the illness. The causative role of the virus was confirmed by demonstration of significant immunological response in patients found to be infected with the virus. The presence of antibodies to Echo 25 virus in about 70% of meningitis patients, from whom no virus was isolated, indicates that the infection in the population of the affected areas was much more prevalent than the number of successful virus isolations would suggest. It is recommended that Echo 25 virus should be added to the series of Echo virus types capable of causing outbreaks of aseptic meningitis.     

Viral Infections of Toronto Children During 1965: I. Enteroviral Disease

Enteroviruses were isolated from feces and/or cerebrospinal fluid of 29 of 43 Toronto children who contracted aseptic meningitis, pleurodynia, abdominal pain or febrile upsets between June and October, 1965. Coxsackie A9 virus was the dominant agent in aseptic meningitis and Coxsackie B1 virus in pleurodynia and other syndromes. Sero-logical evidence of recent Coxsackie B1 and Echo 6 infection was obtained in two additional patients with aseptic meningitis who did not yield virus, and elevated Coxsackie B1 antibody titres were found in one patient with pericarditis. A newborn infant died with myocarditis due to Coxsackie B1 virus following infection of the mother during the immediate antenatal period. Paired sera collected only two to four days apart from patients with enteroviral syndromes or mumps meningoencephalitis frequently showed four-fold or greater increases of antibody levels.     

Sporadic Occurrence of Echo Virus Types 27 and 31 Associated with Aseptic Meningitis in Ontario

The sporadic occurrence of Echo virus types 27 and 31 in association with aseptic meningitis during the enterovirus seasons in Ontario in 1959 to 1962 is reported.The etiological significance of both of these isolates was verified by demonstration of type-specific serological response in meningitis patients. Echo 27, which up to now has not been encountered in association with human illness, could be added to the list of viral agents capable of causing aseptic meningitis. Isolation of Echo 31 from cerebrospinal fluid provides definite proof of the etiological significance of this virus in cases of aseptic meningitis.     

Enteroviral Syndromes in Toronto, 1964

Virological or serological investigations of 72 children in Toronto and environs, who were hospitalized between January and October 1964 with a variety of syndromes, revealed evidence of enteroviral infection in 29 subjects. Coxsackie B2 was the dominant enterovirus, being isolated from feces and/or cerebrospinal fluid (CSF) of three children with aseptic meningitis, three with pleurodynia, one with myalgia and one with pericarditis; four additional patients showed rising antibody titres to this virus. Coxsackie B1 virus, which has not been isolated in Toronto since 1950, was recovered from feces of three patients with pleurodynia, CSF of one patient with myalgia, and peritoneal fluid of a child with primary peritonitis; one patient with pericarditis showed a rising antibody titre to Coxsackie B1 virus. Coxsackie B3, B4 and Echo 23 viruses were associated with one case each of pleurodynia. Coxsackie B5 virus infected five patients with aseptic meningitis, and one each with pericarditis and myocarditis.     

Aseptic Meningitis due to Frater Type Virus in Ontario

During the summer and fall of 1959 and 1960 a virus was isolated on 14 occasions from the stool or cerebrospinal fluid or both of 12 patients with a clinical picture of non-paralytic poliomyelitis or aseptic meningitis. The patients were from eight different localities in Ontario. The isolated virus was not neutralized by antisera to any of the known enteroviruses, reoviruses or adenoviruses, nor did antiserum to the isolate neutralize any of these viruses. Antiserum to Frater virus, however, did neutralize this isolate and in turn was itself neutralized by antiserum to this virus. Frater virus was isolated in Scotland from cases of aseptic meningitis during the same period in 1959 and 1960. In Ontario this virus was not encountered before 1959. Isolation of the virus from cerebrospinal fluid and demonstration of immunological response in the patients establish its etiological significance. Biological characteristics indicate that it belongs to the Echo group.     

Mumps Meningoencephalitis,Toronto, 1963

Between January and June 1963, 45 children were hospitalized with mumps meningoencephalitis. Of 39 patients with laboratory evidence of mumps infection, 24 had parotitis and 15 showed no salivary gland involvement. Cerebrospinal fluids from 18 of 40 patients yielded mumps virus by inoculation of rhesus monkey kidney cultures; 33 subjects, including 12 of the 18 virus excretors, showed rising or elevated levels of mumps antihemagglutinin during convalescence. Between May 1959 and June 1963, mumps virus was recovered from cerebrospinal fluids of 50 of 126 cases of mumps meningoencephalitis; virus isolation rates were highest during the peak incidence of mumps meningoencephalitis in winter and early spring.Mumps vaccine (inactivated) was administered to 34 parents with no history of mumps, shortly after their children developed mumps. Mumps occurred in three of 17 parents without prevaccination mumps antihemagglutinins, and in two others, but in none of 15 who had prevaccination antibodies.     

Mumps and Enteroviral Meningitis in Toronto, 1966

Of 52 children admitted to hospital for apparently typical mumps meningitis in 1966, 50 had their cerebrospinal fluid (CSF) examined. In only 17 was the mumps virus isolated from the CSF. Mumps antihemagglutinin conversions or increments were detected in 32 subjects including 10 whose CSF yielded virus. Antibody conversions were found in 16 patients and fourfold increments in another nine whose serum pairs were collected only one to four days apart. Initial sera from 20 patients were obtained three days or less after the onset of meningitis. Antibody increments were frequently noted about one day after defervescence and clinical improvement. Interferon was detected in CSF from two of eight patients, both of whom yielded virus. Enteroviruses were isolated from CSF and/or feces in seven of 15 cases of aseptic meningitis which occurred between July and October. Six patients including three virus excretors showed enteroviral neutralizing antibody increments during convalescence. The dominant enteroviral serotype was coxsackievirus A9.     

Interferon in the Cerebrospinal Fluid of Children with Central Nervous System Disorders

Interferon was detected in the cerebrospinal fluid (CSF) of 26 of 51 children with aseptic meningitis, two of 44 with bacterial meningitis, and four of 118 with miscellaneous conditions including encephalitis, convulsive disorders and leukemia with neurological involvement. The geometric mean titre of interferon in mumps meningitis was seven to eight times higher than that in enteroviral meningitis; however, levels of interferon were not related to the concentration of leukocytes in CSF from these patients. Interferon titres were relatively greater at the height of the febrile response in children with mumps meningitis or enteroviral meningitis. There was no association between the presence of interferon in the CSF and the isolation of mumps virus or an enterovirus from the same specimen. Patients frequently developed homologous antibody one to three days after signs of aseptic meningitis, obscuring the relationship of interferon production to clinical improvement.     

Isolation of Enteric Viruses in Ontario During 1960-1962

During the past three years at the Central Laboratory, Ontario Department of Health, 681 isolations were made in tissue culture from 6822 specimens submitted for virus studies by physicians and hospitals from all over Ontario. Nearly 74% of the isolates were enteroviruses, approximately 5% adenoviruses and about 1% reoviruses. The remaining 20% are still to be identified.Although the bulk of isolations was made during the same three-month period (August, September and October) of each year, the predominant virus types varied from year to year. Poliovirus 1 was most commonly encountered in 1960, Coxsackie B5 in 1961 and Echo 9 in 1962.Among other types isolated in smaller numbers were Coxsackie A1, 9 and 10, Coxsackie B1, 2, 3 and 4, Echo 1, 2, 5, 6, 7, 8, 11, 14, 17, 18 and 19, Reovirus 1, 2 and 3, Adenovirus 1, 2, 3, 4, 5, 7 and 16, as well as Frater-type virus. Most of these types were isolated for the first time in Ontario and represent additions to the existing list of viruses known to occur in this province.     

A Plaque Method for Rubella Virus Assay

A method has been described in which suitable dilutions of rubella virus will induce the formation, in a monolayer of green monkey kidney cells, of islets of infected cells which were protected from the effects of Echo 11 challenge virus. The number of islets or “negative” plaques was proportional to the dilution of rubella virus inoculated on to the monolayer. Using this method, it was observed that bentonite adsorption increased the plaque assay values of rubella virus pools. This suggested that rubella virus interference may be mediated by an interferon-like principle.     

Glutamate, glutamine, aspartate, asparagine, glucose and ketone-body metabolism in chick intestinal brush-border cells

1. Suspensions of isolated chick jejunal columnar absorptive (brush-border) cells respired on endogenous substrates at a rate 40% higher than that shown by rat brush-border cells. 2. Added d-glucose (5 or 10mm), l-glutamine (2.5mm) and l-glutamate (2.5mm) were the only individual substrates which stimulated respiration by chick cells; l-aspartate (2.5 or 6.7mm), glutamate (6.7mm), glutamine (6.7mm), l-alanine (1 or 10mm), pyruvate (1 or 2mm), l-lactate (5 or 10mm), butyrate (10mm) and oleate (1mm) did not stimulate chick cell respiration; l-asparagine (6.7mm) inhibited slightly; glucose (5mm) stimulated more than did 10mm-glucose. 3. Acetoacetate (10mm) and d-3-hydroxybutyrate (10mm) were rapidly consumed but, in contrast to rat brush-border cells, did not stimulate respiration. 4. Glucose (10mm) was consumed more slowly than 5mm-glucose; the dominant product of glucose metabolism during vigorous respiration was lactate; the proportion of glucose converted to lactate was greater with 10mm- than with 5mm-glucose. 5. Glutamate and aspartate consumption rates decreased, and alanine and glutamine consumption rates increased when their initial concentrations were raised from 2.5 to 6.7 or 10mm. 6. The metabolic fate of glucose was little affected by concomitant metabolism of any one of aspartate, glutamate or glutamine except for an increased production of alanine; the glucose-stimulated respiration rate was unaffected by concomitant metabolism of these individual amino acids. 7. Chick cells produced very little alanine from aspartate and, in contrast to rat cells, likewise produced very little alanine from glutamate or glutamine; in chick cells alanine appeared to be predominantly a product of transmination of pyruvate derived from glucose metabolism. 8. In chick cells, glutamate and glutamine were formed from aspartate (2.5 or 6.7mm); aspartate and glutamine were formed from glutamate (2.5mm) but only aspartate from 6.7mm-glutamate; glutamate was the dominant product formed from glutamine (6.7mm) but aspartate only was formed from 2.5mm-glutamine. 9. Chick brush-border cells can thus both catabolize and synthesize glutamine; glutamine synthesis is always diminished by concomitant metabolism of glucose, presumably by allosteric inhibition of glutamine synthetase by alanine. 10. Proline was formed from glutamine (2.5mm) but not from glutamine (2.5mm)+glucose (5mm) and not from 2.5mm-glutamate; ornithine was formed from glutamine (2.5mm)+glucose (5.0mm) but not from glutamine alone; serine was formed from glutamine (2.5mm)+glucose (5mm) and from these two substrates plus aspartate (2.5mm). 11. Total intracellular adenine nucleotides (22μmol/g dry wt.) remained unchanged during incubation of chick cells with glucose. 12. Intracellular glutathione (0.7–0.8mm) was depleted by 40% during incubation of respiring chick cells without added substrates for 75min at 37°C; partial restoration of the lost glutathione was achieved by incubating cells with l-glutamate+l-cysteine+glycine.     

Multiple Sclerosis—Current Etiological Concepts

An animal model for acute multiple sclerosis (ms) is experimental allergic encephalomyelitis (eae). eae is produced by intradermal injection of a protein component of central nervous system (cns) myelin. Ultrastructural studies of eae and of a peripheral nerve analog, experimental allergic neuritis (ean), have revealed an orderly sequence of cellular events leading to the destruction and removal of myelin with sparing of axons (primary demyelination). Acute ms has not been studied electron microscopically, but the ultrastructural similarities between ean and a case of acute Landry-Guillain-Barré syndrome, a primary demyelinating disease of the peripheral nervous system, suggest that a similar sequence of events might be found in acute ms. While the pathological findings support a cellmediated or delayed hypersensitivity response, there is also evidence for the pathogenetic role of circulating antibodies. Among such evidence is included the finding that sera from animals with eae and humans with acute ms rapidly produce a reversible block of complex (polysynaptic) electrical activity when applied to cns tissue cultures, which suggests a possible mechanism for transient symptoms in ms. Epidemiological and other studies link ms with a viral cause, although no direct evidence that ms is caused by a virus exists. Viral and immunological mechanisms are not mutually exclusive in considering pathogenetic possibilities for ms, for it can be postulated that a viral infection of the central nervous system acts as a triggering agent for a series of immune responses, including production of a bioelectric blocking antibody and demyelination mediated by sensitized cells, the combination of which ultimately produces the total clinical picture of ms.     

Emission of Hydrogen Sulfide by Leaf Tissue in Response to l-Cysteine

Leaf discs and detached leaves exposed to l-cysteine emitted a volatile sulfur compound which was proven by gas chromatography to be H₂S. This phenomenon was demonstrated in all nine species tested (Cucumis sativus, Cucurbita pepo, Nicotiana tabacum, Coleus blumei, Beta vulgaris, Phaseolus vulgaris, Medicago sativa, Hordeum vulgare, and Gossypium hirsutum). The emission of volatile sulfur by cucumber leaves occurred in the dark at a similar rate to that in the light. The emission of leaf discs reached the maximal rate, more than 40 picomoles per minute per square centimeter, 2 to 4 hours after starting exposure to l-cysteine; then it decreased. In the case of detached leaves, the maximum occurred 5 to 10 h after starting exposure. The average emission rate of H₂S during the first 4 hours from leaf discs of cucurbits in response to 10 millimolar l-cysteine, was usually more than 40 picomoles per minute per square centimeter, i.e. 0.24 micromoles per hour per square decimeter. Leaf discs exposed to 1 millimolar l-cysteine emitted only 2% as much as did the discs exposed to 10 millimolar l-cysteine. The emission from leaf discs and from detached leaves lasted for at least 5 and 15 hours, respectively. However, several hours after the maximal emission, injury of the leaves, manifested as chlorosis, was evident. H₂S emission was a specific consequence of exposure to l-cysteine; neither d-cysteine nor l-cystine elicited H₂S emission. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate dependent enzymes, inhibited the emission. In a cell free system from cucumber leaves, H₂S formation and its release occurred in response to l-cysteine. Feeding experiments with [³⁵S]l-cysteine showed that most of the sulfur in H₂S was derived from sulfur in the l-cysteine supplied and that the H₂S emitted for 9 hours accounted for 7 to 10% of l-cysteine taken up. ³⁵S-labeled SO₃²⁻ and SO₄²⁻ were found in the tissue extract in addition to internal soluble S²⁻. These findings suggest the existence of a sulfur cycle which converts l-cysteine to SO₄²⁻ through cysteine desulfhydration.     

Seed Dormancy in Red Rice : VIII. Embryo Acidification during Dormancy-Breaking and Subsequent Germination

Exposure of dehulled, dormant red rice (Oryza sativa) seeds to dormancy-breaking treatments (10 mm sodium nitrite, 20 mm propionic acid, 30 mm methyl propionate, 40 mm propionaldehyde, or 70 mmn-propanol) induced tissue pH acidification during chemical contact at least 12 h before visible germination. During chemical contact, the onset of embryo acidification occurred before or coincident with the chemical contact interval necessary for subsequent germination. Upon seed transfer to H₂O following chemical contact, embryo pH also decreased coincident with visible germination. During this period, the percentage of germination and embryo pH were closely linked irrespective of the dormancy-breaking compound used. Therefore, tissue acidification during the breaking of seed dormancy and the germination process may be analogous to similar tissue pH changes associated with the termination of developmental arrest in other multicellular systems, such as brine shrimp cysts and nematode larvae.     

Substrate Specificity of a Mannose-6-Phosphate Isomerase from Bacillus subtilis and Its Application in the Production of l-Ribose

The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co²⁺. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the d and l forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for l-ribulose among all pentoses and hexoses. Thus, l-ribose, as a potential starting material for many l-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter l-ribulose by mannose-6-phosphate isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter⁻¹ h⁻¹.l-Ribose is a potential starting material for the synthesis of many l-nucleoside-based pharmaceutical compounds, and it is not abundant in nature (5, 19). l-Ribose has been produced mainly by chemical synthesis from l-arabinose, l-xylose, d-glucose, d-galactose, d-ribose, or d-mannono-1,4-lactone (2, 17, 23). Biological l-ribose manufacture has been investigated using ribitol or l-ribulose. Recently, l-ribose was produced from ribitol by a recombinant Escherichia coli containing an NAD-dependent mannitol-1-dehydrogenase (MDH) with a 55% conversion yield when 100 g/liter ribitol was used in a 72-h fermentation (18). However, the volumetric productivity of l-ribose in the fermentation is 28-fold lower than that of the chemical method synthesized from l-arabinose (8). l-Ribulose has been biochemically converted from l-ribose using an l-ribose isomerase from an Acinetobacter sp. (9), an l-arabinose isomerase mutant from Escherichia coli (4), a d-xylose isomerase mutant from Actinoplanes missouriensis (14), and a d-lyxose isomerase from Cohnella laeviribosi (3), indicating that l-ribose can be produced from l-ribulose by these enzymes. However, the enzymatic production of l-ribulose is slow, and the enzymatic production of l-ribose from l-ribulose has been not reported.Sugar phosphate isomerases, such as ribose-5-phosphate isomerase, glucose-6-phosphate isomerase, and galactose-6-phosphate isomerase, work as general aldose-ketose isomerases and are useful tools for producing rare sugars, because they convert the substrate sugar phosphates and the substrate sugars without phosphate to have a similar configuration (11, 12, 21, 22). l-Ribose isomerase from an Acinetobacter sp. (9) and d-lyxose isomerase from C. laeviribosi (3) had activity with l-ribose, d-lyxose, and d-mannose. Thus, we can apply mannose-6-phosphate (EC 5.3.1.8) isomerase to the production of l-ribose, because there are no sugar phosphate isomerases relating to l-ribose and d-lyxose. The production of the expensive sugar l-ribose (bulk price, $1,000/kg) from the rare sugar l-ribulose by mannose-6-phosphate isomerase may prove to be a valuable industrial process, because we have produced l-ribulose from the cheap sugar l-arabinose (bulk price, $50/kg) using the l-arabinose isomerase from Geobacillus thermodenitrificans (20) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Schematic representation for the production of l-ribulose from l-arabinose by G. thermodenitrificans l-arabinose isomerase and the production of l-ribose from l-ribulose by B. subtilis mannose-6-phosphate isomerase.In this study, the gene encoding mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in E. coli. The substrate specificity of the recombinant enzyme for various aldoses and ketoses was investigated, and l-ribulose exhibited the highest activity among all pentoses and hexoses. Therefore, mannose-6-phosphate isomerase was applied to the production of l-ribose from l-ribulose.     

Studies of cardiac muscle with a high permeability to calcium produced by treatment with ethylenediaminetetraacetic acid

Thin strips of frog ventricle were isolated and bathed for 15 min in a solution containing 140 mM KCl, 5 mM Na₂ATP, 3 mM EDTA, and 10 mM Tris buffer at pH 7.0. The muscle was then exposed to contracture solutions containing 140 mM KCl, 5 mM Na₂ATP, 1 mM MgCl₂, 10 mM Tris, 3 mM EGTA, and CaCl₂ in amounts to produce concentrations of free calcium from 10^-4.8 M to 10^-9 M. The muscles developed some tension at approximately 10^-8 M, and maximum tension was achieved in 10^-5 M Ca⁺⁺. They relaxed in Ca⁺⁺ concentrations less than 10^-8 M. The development of tension by the EDTA-treated muscles was normalized by comparison with twitch tension at a stimulation rate of 9 per min before exposure to EDTA. In 10^-5 M Ca⁺⁺ tension was always several times the twitch tension and was greater than the contracture tension of a frog ventricular strip in KCl low Na-Ringer. Tension equal to half-maximum was produced at approximately 10^-6.2 M Ca⁺⁺. Intracellular recording of membrane potential indicated that after EDTA treatment the resting potential of cells in Ringer solution with 10^-5 M Ca or less was between 5 and 20 mv. Contracture solutions did not produce tension without prior treatment with EDTA. The high permeability of the membrane produced by EDTA was reversed and the normal resting and action potentials restored in 1 mM Ca-Ringer. Similar studies of EDTA-treated rabbit right ventricular papillary muscle produced a similar tension vs. Ca⁺⁺ concentration relation, and the high permeability state reversed with exposure to normal Krebs solution.     

Accumulation of d-Glucose from Pentoses by Metabolically Engineered Escherichia coli

Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.     

Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.l-Arabinose isomerase (l-AI) is an enzyme that mediates in vivo isomerization between l-arabinose and l-ribulose as well as in vitro isomerization of d-galactose and d-tagatose (20). l-Ribulose (l-erythro-pentulose) is a rare and expensive ketopentose sugar (1) that can be used as a precursor for the production of other rare sugars of high market value, such as l-ribose. Despite being a common metabolic intermediate in different organisms, l-ribulose is scarce in nature. The market for rare and unnatural sugars has been growing, especially in the sweetener and pharmaceutical industries. For example, several modified nucleosides derived from l-sugars have been shown to act as potent antiviral agents and are also useful in antigen therapy. Derivatives of rare sugars have also been used as agents against hepatitis B virus and human immunodeficiency virus (2, 22).For these reasons, interest in the enzymology of rare sugars has also been increasing. Various forms of l-AI from a variety of organisms have been characterized, and some have shown potential for industrial use. Several highly thermotolerant enzyme forms from Thermotoga maritima (12), Thermotoga neapolitana (10), Bacillus stearothermophilus (18), Thermoanaerobacter mathranii (9), and Lactobacillus plantarum (5) have been characterized previously. All of these reported l-AIs tend to have broad specificity, although a few l-AIs with high degrees of substrate specificity for l-arabinose have also been documented.The enzyme properties of l-AIs have been examined by engineering several forms by error-prone PCR and site-directed mutagenesis. Galactose conversion was reportedly enhanced 20% following site-directed introduction of a double mutation (C450S-N475K) into l-AI (16). Error-prone PCR manipulation of l-AI from Geobacillus stearothermophilus resulted in a shift in temperature specificity from 60 to 65°C and increased isomerization activity (11). All of these previously reported mutational studies have been aimed at improving enzymatic properties for industrial application. However, even though the three-dimensional (3D) structure of Escherichia coli l-AI has been determined previously (15), few new structural studies have been performed to decipher the reaction mechanism of this enzyme. Rhimi et al. (19) have reported an important role for D308, F329, E351, and H446 in catalysis, as indicated by findings from site-directed mutagenesis. Nonetheless, detailed analysis of the important molecular determinants controlling the catalytic activities of the l-AIs is still lacking.Previously, we have reported the cloning and characterization of a novel l-AI from Bacillus licheniformis (17). This enzyme can be distinguished from other l-AIs by its wide pH range, high degree of substrate specificity for l-arabinose, and extremely high turnover rate. In the present paper, we report the identification of an important amino acid residue responsible for the catalytic efficiency of l-AIs, as determined by a systematic screening process composed of sequence alignment and molecular dynamics (MD) simulation, followed by site-directed mutagenesis. Using the crystal structure of E. coli l-AI as a template, we have built a 3D model of B. licheniformis l-AI. Analysis of the 3D model of B. licheniformis l-AI docked with l-arabinose, followed by a systematic screening process, showed that Y333 interacted with the substrate, suggesting that this residue in B. licheniformis l-AI may be essential for catalysis. We further characterized the role of Y333 in B. licheniformis l-AI binding of and catalytic efficiency for l-arabinose.     

Loss of Hydrogen from Carbon 5 of d-Glucose during Conversion of d-[5-H,6-C]Glucose to l-Ascorbic Acid in Pelargonium crispum (L.) L'Hér

Conversion of d-[5-³H,6-¹⁴C]glucose to l-ascorbic acid in detached apices of Pelargonium crispum (L.) L'Hér cv Prince Rupert (lemon geranium) was accompanied by complete loss of tritium in the product. Chemical degradation of d-glucose which was recovered from the labeled apices yielded d-glyceric acid (corresponding to carbons 4, 5, and 6 of glucose) with a ³H:¹⁴C ratio of 4 to be compared with 9, the ratio in d-[5-³H,6-¹⁴C]glucose initially. Conversion of d-[6-³H,6-¹⁴C]glucose in the same tissue was accompanied by retention of tritium in l-ascorbic acid with a ³H:¹⁴C ratio comparable to that of compounds from the hexose pool. Results indicate that during l-ascorbic acid biosynthesis from glucose in Pelargonium crispum hydrogen at carbon 5 undergoes exchange with the medium, suggesting an epimerization at this carbon atom.