N-Acetyl β-d-glucosaminidase and α-l-fucosidase activities in relation to glycosylated hemoglobin levels and to retinopathy in diabetes

N-Acetyl β-d-glucosaminidase and α-l-fucosidase were determined in human sera from 25 control subjects, in 23 diabetic patients without retinopathy and in 22 diabetic patients with retinopathy.The results show significantly higher N-acetyl β-d-glucosaminidase activity in diabetic patients independently of the development of retinopathy and also independently of the length of diabetes. No correlation was found between either serum enzymes and serum glucose concentration and glycosylated hemoglobin (HbA,).     

Electrophoretic forms of human liver alpha-L-fucosidase and their relationship to fucosidosis (mucopolysaccharidosis F)

1. Human liver α-L-fucosidase was studied by starch gel electrophoresis and isoelectric focusing both before and after treatment with neuraminidase. These studies revealed the presence of 5–6 bands on starch gels and six isoelectric forms with pI's of 6.9, 6.5, 6.1, 5.9, 5.7 and 5.5. The activity of the two most acidic forms was reduced after neuraminidase treatment.2. Liver tissue from a patient with fucosidosis had approximately 4% of normal α-l-fucosidase activity. Starch gel electrophoresis revealed that all electrophoretic forms of the enzyme appear to be deficient.3. Apparent Michaelis constants (K_m 's) determined for both the normal and fucosidotic liver α-l-fucosidase for 4-methylumbelliferyl-α-l-fucopyranoside were found to be 0.085 mM and 0.058 mM, respectively.4. The pH activity profile of normal and fucosidotic liver α-l-fucosidase were very similar with major pH optima at 5.4 and 5.3, respectively.     

Cystic fibrosis serum α-l-fucosidase: Confirmation of normal activity levels and normal kinetic and isoelectric focusing properties

Comparable α-l-fucosidase activity was found for normal and cystic fibrosis sera, 7.8 ± 3.3 and 9.8 ± 4.7 nmol/min/ml, respectively. Isoelectric focusing of normal and cystic fibrosis sera α-l-fucosidase revealed similar isoelectric profiles with most activity between isoelectric points of 4.7–5.4. Kinetic analysis of normal and cystic fibrosis sera α-l-fucosidase revealed identical apparent Michaelis constants for the 4-methylumbelliferyl substrate (51 ± 6 μmol/l and 50 ± 8 μmol/1, respectively), similar pH optima curves (both have broad optima between pH 4.8 and 6.5) and identical thermostability curves at three different preincubation temperatures (37°, 45° and 55°C).     

Human alpha-L-fucosidases

Two forms of α-l-fucosidase from various human organs, designated by us as α-l-fucosidase I and α-l-fucosidase II, were separated by means of gel filtration on Sephadex G-200.The pH optimum for α-l-fucosidase I and α-lα-l-fucosidase II was found to be 4.5–5.0 and 5.5, respectively. Both forms differed in thermostability, pH-stability and possess strict substrate specificities.The data on the presence of fucosidases in human amniotic fluid and placenta are of interest since these organs could be used as sources for study of these enzymes in certain hereditary diseases.     

A sensitive fluorometric assay for alpha-KL-fucosidase.

A sensitive fluorometric assay for α-l-fucosidase applicable to serum or plasma is described. Some normal individuals have been found to have an extremely low level of α-l-fucosidase. This low activity enzyme has an altered pH versus activity profile but a K_m of the same order of magnitude as the high activity enzyme.     

Further evidence of human αa-l-fucosidase polymorphism

The results of a comparative study of multiple forms of α-l-fucosidase from human kidney, liver, placenta and blood serum provide evidence for α-l-fucosidase polymorphism. On the basis of the data obtained the existence of certain phenotypic groups of α-l-fucosidase, dissimilar both in the quantitative and qualitative composition of the enzyme's multiple forms, was revealed. Some possible reasons for α-l-fucosidase polymorphism are discussed. It is suggested that the data on α-l-fucosidase polymorphism may be used in diagnosis and in the isolation and preparation of individual enzyme forms.     

Fluorescent assay of alpha-L-fucosidase

Human α-l-fucosidase can be assayed using the fluorigenic substrate 4-methylumbelliferyl α-l-fucoside. This substrate is hydrolysed more readily than the p-nitrophenyl derivative by the human liver enzyme but the serum enzyme shows approximately the same activity towards the latter substrate. The pH profiles are similar with both substrates with a slight shift in the maxima and both iso-enzymic forms of α-l-fucosidase are detected by either substrate. The greatly increased sensitivity of assay by the fluorescence method favours its use in diagnostic studies on fucosidosis.     

Alpha-L-fucosidase and alpha-D-mannosidase activity in the white blood cells in the disease and carrier state of fucosidosis

α-l-Fucosidase and α-d-mannosidase activity in white blood cells from a patient with fucosidosis and her family members were studied.α-l-Fucosidase activity of the patient was completely lacking, whereas the α-l-mannosidase activity was increased and relative thermostability of this enzyme was also observed. Apparent K_m value of α-d-mannosidase was similar in the patient and the control. When the ratio of α-l-fucosidase to α-d-mannosidase activity was calculated, the value of the parents was intermediate between that of the patient and the control.     

Accumulation of boron-10 (10B) in cell cultures exposed to mercaptododecaborate (Na2H(11)10B12SH) used for the neutron capture therapy of brain tumors.

Toxicity of mercaptoundecahydro-closo-dodecaborate (MHB, Na2H(11)10B12SH) and accumulation of MHB-derived 10B were studied in E7 neuroblastoma, C6 glioma, HeLa cells and embryonic lung LEP 19 fibroblasts in culture in exponential and stationary phases of growth (2- and 7-day-old cultures, respectively). The pilot study of acute toxicity, performed on C6 glioma cells, showed good tolerance of the drug up to 1000 micrograms/ml (4.8 x 10(-3) M), when cell growth slowed and a small part of the population was lethally damaged (8.3%, 20-h incubation interval). The changes became more extensive and appeared sooner (toward 5 h) at 2000 micrograms MHB/ml (9.5 x 10(-3) M). None of the four cell lines used was found to be affected in gross morphology or growth by 200 micrograms MHB/ml within a 5-day culture interval. When exposed to this dose for 4 h, the amount of 10B accumulated in cell lines at the exponential growth phase ranged from 0.51 to 4.4 ng/micrograms protein; in the stationary cultures of the corresponding cell phenotype, the 10B values were 3 to 10 times lower (0.12-1.2 ng/micrograms protein). Irrespective of the growth phase, the values achieved in C6 glioma cells were several times higher than in the other cell lines. Furthermore, in the glioma cells, particularly in the exponential phase of growth, accumulation of 10B proceeded against the marked concentration gradient. The data provide a new indication for the use of MHB for boron neutron capture therapy of brain tumors.     

Isozymes of human alpha-L-fucosidase detectable by starch gel electrophoresis

Methods are described for the electrophoresis in starch-gel of human α-l-fucosidase and for the detection of the enzyme using the fluorogenic substrate 4-methylumbelliferyl-α-l-fucopyranoside. The electrophoretic pattern of the enzyme was examined in leucocytes, serum, cultured fibroblasts and long-term lymphoid cell lines. The enzyme from cultured lymphoid cell lines was found to consist of up to 6 clearly resolved electrophoretic isozymes plus a diffuse, more anodal region of lower staining intensity. The enzyme from leucocytes and cultured skin fibroblasts was less clearly resolved, but these cell types appeared to have components corresponding in mobility to the isozymes of lymphoid lines. In contrast, the enzyme from serum (or plasma) showed only a diffuse region of activity, with an anodal mobility slightly greater than that of the most anodal lymphoid line isozymes. Evidence is presented which indicates that the electrophoretic heterogeneity of α-l-fucosidase is due in part to the binding of sialic acid to the primary gene product. None of the isozymes was detectable in lymphoid cell lines, serum or cultured fibroblasts from a patient with fucosidosis, an inborn error of metabolism.     

RELEASE OF ENDOTOXIN IN THE FORM OF CELL WALL BLEBS DURING IN VITRO GROWTH OF NEISSERIA MENINGITIDIS

Multiple cell wall blebs were observed on the surface of three strains of N. meningitidis taken from log phase cultures. The blebs originated as evaginations of the outer layer of the cell wall. Bleb production was noted on both defined or complex media either as broth or a solid medium. The addition of 10% normal bovine serum to the various media did not affect the production and release of these surface blebs. However, as broth cultures progressed into the stationary phase of growth, the blebs disappeared from the surface of the cells. Blebs were present in substantial quantities in culture supernatant fluids and on cell surfaces and were readily isolated by ultracentrifugation. Analysis for 2-keto-3-deoxyoctonate in cultures revealed that 18% of the total endotoxin of log phase cultures was present in blebs from the cell wall.     

Cell cycle-associated changes in receptors for IgE during growth and differentiation of a rat basophilic leukemia cell line

The rat basophilic leukemia cell line (RBL-1) showed an inverse relationship between growth rate and expression of receptor activity for IgE. After prolonged exponential growth, the number of receptors per cell stabilized at 4-6 times 10-5. Cells in stationary cultures, which are arrested in the G1 phase of the cell cycle, continued to accumulate up to 0.9-1.7 times 10-6 receptors/cell with no increase in volume. Upon resuspension in fresh medium at low density, these cells were shown to lose up to 70% of the receptor activity within 4 h. Assessment of cultures synchronized by double thymidine block and cells fractionated by centrifugation of a Ficoll gradient indicated that the RBL-1 cells acquire receptors in the G1 phase of the cell cycle. No accumulation of active receptors occurred during the S and G2 phases, though the average cell volume increased. Cell division resulted in a drop in number of receptors per cell while the number of cell-bound receptors in the culture remained unchanged. This indicates that during mitosis receptors were simply distributed to daughter cells.     

α-l-Fucosidase m cystic fibrosis: analysis of skin fibroblasts and liver

The lysosomal enzyme α-l-fucosidase has been examined by thin layer gel and column isoelectric focusing in skin fibroblasts and liver from patients with cystic fibrosis and controls. All three common phenotypes of the enzyme were observed in both control and CF fibroblasts. When individuals of the same α-l-fucosidase phenotype were compared, no major differences between the isozyme profiles of cystic fibrosis patients and controls were detected in either fibroblasts or liver tissue.     

Cobalamin R binder as a possible model molecule for glycoprotein study in cystic fibrosis

The isoprotein pattern of semi-purified R binder (an acidic glycoprotein which binds cobalamin) from saliva and sera of 8 cystic fibrosis patients was compared to that of R binder from samples of 5 healthy children. In cases of cystic fibrosis, the mean isoelectric point of salivary R binder was increased from 3.78 up to 4.34 and its microheterogeneity was reduced. These significant physicochemical modifications were not observed with R binder from cystic fibrosis sera and they did not correlate with the β-galactosidase, α-mannosidase, α-l-fucosidase nor neuraminidase activity of saliva. We propose the R binder as a model molecule to study the glycoprotein metabolism in cystic fibrosis since it contains 30–40% carbohydrate, is easily complexed with cyano[⁵⁷Co]cobalamin and is present in most tissues and fluids of the human organism.     

Enzymic determination of urinary free l-fucose

Conditions necessary for the precise measurement of free l-fucose in urine by an enzymic method using l-fucose dehydrogenase have been studied. The normal urinary levels of l-fucose were 16.4 ± 9.1 μg/ml in children (22.2 ±6.5 μg/mg of creatinine) and 17.7 ± 8.5 μg/ml in adults (16.6 ± 5.7 μg/mg of creatinine). There was a close correlation between the concentration of free l-fucose and that of creatinine. A close correlation was also found between the concentration of free l-fucose and α-l-fucosidase activity. Thus, it was suggested that the free l-fucose in urine reflected the metabolism of l-fucose or l-fucose-containing glycoconjugates.In a preliminary screening test, several urine samples which showed high concentrations of free l-fucose, namely cases of “fucosuria” were found.     

Lysosomal enzyme variations in thirteen cell strains cultured from one amniotic fluid

Fifteen primary amniotic fluid cultures were established from a single sample of amniotic fluid. Three different methods were used to set up these cultures which yielded 13 cell strains. Nine lysosomal enzymes (acid phosphatase, β-glucuronidase, β-galactosidase, α-galactosidase, α-glucosidase, α-mannosidase, α-arabinosidase, $\text{N-}\text{acetyl}\text{-ß-}\text{d}\text{-}\text{glucosaminidase}$ and arylsulphatase A) were assayed in these 13 cell strains. The coefficients of variation of these enzyme levels were less than the coefficients for enzyme levels in cell strains grown from different samples of amniotic fluid but greater than those for the combined culture and assay system used. No assay values were found which could have suggested a possible enzyme deficiency disease.     

Nationwide survey of the development of drug resistance in the pediatric field in 2007 and 2010: drug sensitivity of Haemophilus influenzae in Japan (second report)

The Drug-Resistant Pathogen Surveillance Group in Pediatric Infectious Disease conducted national surveillance for Haemophilus influenzae in 2007 (phase 3) and 2010 (phase 4), following the previous surveillance conducted from 2000 to 2001 (phase 1) and in 2004 (phase 2). We examined the antimicrobial susceptibility for H. influenzae derived from clinical specimens of pediatric patients collected nationwide from 27 institutions during phases 3 (386 strains) and 4 (484 strains). The frequency of β-lactamase-nonproducing ampicillin (ABPC)-resistant (BLNAR) strains, which rapidly increased from 11.4 % in phase 1 to 43.4 % in phase 2, has gradually decreased from 38.3 % in phase 3 to 37.8 % in phase 4. In contrast, On the other hand, the frequency of β-lactamase-producing strains, which continuously decreased from 8.3 % in phase 1 to 4.4 % in phase 3, has increased to 8.7 % in phase 4. Prevalence of β-lactamase-producing clavulanic acid/amoxicillin-resistant (BLPACR) strains, especially, has increased from 1.6 % in phase 3 to 4.8 % in phase 4. The oral antimicrobial agents with the lowest MIC₉₀ were levofloxacin in both phases, and tosufloxacin in phase 4 (≤0.063 μg/ml), whereas for intravenous use the corresponding agent was tazobactam/piperacillin in both phases (0.125 μg/ml). There was no increase in the MIC₉₀ of most β-lactams between phase 3 and phase 4. In relationship to sex, age, presence of siblings, attendance at a daycare center, siblings’ attendance at a daycare center, and prior administration of antimicrobial agents within 1 month, the frequency of β-lactamase-nonproducing ABPC-intermediately resistant (BLNAI) strains + BLNAR strains was high (P = 0.005) in cases with prior administration of antimicrobial agents in phase 3.     

Penicillin-binding proteins are regulated by rpoS during transitions in growth states of Escherichia coli.

Attention has been recently focused on the role of the rpoS (formerly katF) gene product as a regulator during the transition from the exponential growth phase to the stationary phase as well as during nutritional starvation. It has been demonstrated that RpoS is an alternate sigma factor which would bind to promoters of genes induced at these times. It was previously noted that rpoS mutants do not undergo a transition to short rods during entry into the stationary phase. Because of their well-established role in morphogenesis, we investigated the status of the penicillin-binding proteins (PBPs) in Escherichia coli wild-type and isogenic rpoS mutants. Samples from cultures of E. coli ZK126 and ZK1000 (rpoS::kan) were taken in the midlogarithmic, early stationary, and late (24 h) stationary phases. The increase in PBP 6 seen upon entry of the wild-type strain into the stationary phase was not observed with the rpoS::kan cells, even after 24 h. There was also a marked decrease of PBP 3 in wild-type stationary-phase cells; PBP 3 has a known influence on morphogenesis. This decrease in PBP 3 was found to be markedly affected by the disruption of rpoS. Similar observations were made after prolonged starvation of the two strains for either glucose or a required amino acid. Inasmuch as PBPs are involved in peptidoglycan synthesis, we also examined two properties of peptidoglycan, autolysis and cross-linkage, that might be altered by the PBP differences. However, neither of these properties, which are known to undergo changes in the stationary phase, appeared to be influenced by the status of RpoS.     

Macrolide Resistance in Staphylococcus aureus: Induction of Macrolide-Resistant Protein Synthesis

Induction of resistance to macrolide-, lincosamide-, and streptogramin B-type antibiotics in Staphylococcus aureus was studied by monitoring the appearance of erythromycin A (EM)-resistant [¹⁴C]leucine incorporation. Examination of the induction process revealed saturation kinetics and a time course much like that reported for penicillinase in gram-positive bacteria. Induction kinetics in exponentially growing cells were sigmoidal and appeared to reach a maximum and constant rate when growth reached stationary phase. Since the induction of EM-resistant colony-forming ability was complete within 60 min, ribosome modification cannot be limited to a fraction of the population and must occur in essentially every cell. However, EM-resistant growth was expressed in cells where less than half the [¹⁴C]leucine-incorporating activity was resistant to EM. This suggests that resistance requires that only a threshold level of ribosome modification be exceeded and that, once exceeded, resistance is dominant to sensitivity.