Morphological and biochemical studies of human beta-mannosidosis: identification of a novel beta-mannosidase gene mutation.

BACKGROUND: There are seven well-known lysosomal storage diseases that produce angiokeratoma corporis diffusum clinically. beta-Mannosidosis (MANB1; OMIM248510), first reported in humans in 1986, is a rare hereditary lysosomal storage disease caused by a deficiency of the enzyme beta-mannosidase. Since then, 13 cases of beta-mannosidase deficiency in ten families have been described. A human beta-mannosidase mutation has been reported only by Alkhayat et al. in 1998. OBJECTIVES: To clarify its pathogenesis we did electron microscopic, biochemical and molecular biological investigations of a Japanese patient with beta-mannosidosis. METHODS: Ultrastructural analyses, enzyme assays, cell culture and mRNA and genomic DNA were sequenced to find mutations in the beta-mannosidase gene. RESULTS: Electron microscopy of skin biopsy specimens from the patient showed cytoplasmic vacuolation of lysosomes in blood and lymph vessels, endothelial cells, fibroblasts, secretory portions of eccrine sweat glands, neural cells and basal keratinocytes in the epidermis. This vacuolation was also observed in cultured keratinocytes and fibroblasts. Assays of seven enzyme activities in plasma and cultured skin fibroblasts showed a marked decrease of beta-mannosidase activity. Sequencing the beta-mannosidase cDNA revealed a four-base (ATAA) insertion between exons 7 and 8, resulting in a frameshift at codon 321 and termination at codon 325. Analysis of the patient's genomic DNA revealed a novel homozygous A(+1)-->G splice site mutation in intron 7. CONCLUSIONS: To our knowledge, this is the first case of beta-mannosidosis reported in Japan and the second report in which a gene mutation is identified. The biological importance of beta-mannose moieties in glycoproteins in basal keratinocytes is suggested.

Effects of locoweed (Oxytropis sericea) on reproduction in cows with a history of locoweed consumption.

Locoweed (Oxytropis sericea) was fed to 4 open cycling cows that had repeatedly consumed locoweed in previous grazing trails. They received locoweed at 20% of their diet for 30 d (0.68-0.76 mg swainsonine/kg/d). Locoweed induced an immediate rise in serum swainsonine (the locoweed toxin) and a concomitant drop in serum alpha-mannosidase activity in all cows accompanied by abnormal estrus behavior, increased estrous cycle lengths and failure to conceive. Serum progesterone (P4) profiles demonstrated that estrous cycles lengthened from an average of 19 d before locoweed feeding to an average of 34 d (range 24-43 d) while on locoweed. After locoweed feeding ceased, normal estrous cycles returned within an average of 14 d (range 7-25 d). Two of the 4 cows conceived on their first post-locoweed estrus at 7 and 25 d. The third cow bred twice at 13 and 31 d after lowoweed feeding stopped, and the fourth cow bred 3 times at 11, 31 and 52 d before conception occurred. Pregnancies in all 4 cows progressed normally to 7 mo gestation when locoweed was again fed at 20% of the diet for 40 d (gestation days 213 and 253) to 2/4 cows, 1 of which aborted 10 d after lowoweed feeding stopped (263 days gestation). The other cow fed lowoweed calved normally as did the 2 pregnancy control cows. Serum P4 and estradiol (E2) profiles during pregnancy appeared normal before, during and after locoweed feeding except in the cow that aborted, whose P4 declined and E2 increased prematurely. The general trend of serum prolactin was similar in locoweed-fed and control cows.

Characterization of a soluble class I alpha-mannosidase in human serum.

Class I alpha-mannosidases are thought to exist exclusively as integral membrane proteins that play intracellulary an essential role in the N-glycan biosynthesis. Using [3H]Man9GlcNAc2 as a substrate, we were able to identify a soluble alpha-mannosidase in human serum that trims the substrate Man9GlcNAc2 to Man(5-8)GlcNAc2 with Man6GlcNAc2 being the major product. This serum mannosidase is Ca2+-dependent, sensitive to 1-deoxymannojirimycin but insensitive to the class II inhibitor swainsonine and, hence, belongs to class I mannosidases. The enzymatic properties of the serum class I mannosidase are similar to that of the membrane bound class I mannosidases Golgi-mannosidase IA and IB and Man9-mannosidase.

The pathogenesis and toxicokinetics of locoweed (Astragalus and Oxytropis spp.) poisoning in livestock.

Locoweed poisoning is a chronic disease that develops in livestock grazing for several weeks on certain Astragalus and Oxytropis spp. that contain the locoweed toxin, swainsonine. The purpose of this review is to present recent research on swainsonine toxicokinetics and locoweed-induced clinical and histologic lesions. Swainsonine inhibits cellular mannosidases resulting in lysosomal storage disease similar to genetic mannosidosis. Diagnosis of clinical poisoning is generally made by documenting exposure, identifying the neurologic signs, and analyzing serum for alpha-mannosidase activity and swainsonine. All tissues of poisoned animals contained swainsonine, and the clearance rates from most tissues was about 20 hours (T1/2 half life). The liver and kidney had longer rate of about 60 hours (T1/2). This suggests that poisoned animals should be allowed a 28-day withdrawal to insure complete swainsonine clearance. Poisoning results in vacuolation of most tissues that is most obvious in neurons and epithelial cells. Most of these histologic lesions resolved shortly after poisoning is discontinued; however, some neurologic changes are irreversible and permanent.

Plasma tumor necrosis factor-a (TNF-a) levels in Gaucher disease.

Tumor necrosis factor-a (TNF-a) levels were measured in the plasma of patients with different types of Gaucher disease (GD) and patients with other lysosomal storage diseases. The highest TNF-a levels were observed in the most severe neuronopathic type of GD, exceeding those found in healthy individuals as well as patients with other lysosomal disorders. Type I GD cases showed a wide range of TNF-a levels ranging from normal to 2.5 x the highest control value. TNF-a is a pleiotropic cytokine produced mainly by activated macrophages. Our data suggest that it may play a role in the pathophysiology of GD disease.

[Value of serum glycosidase spectrum in the diagnosis of hepatocellular carcinoma].

Activities of alpha-L-fucosidase (alpha-Fucase), N-acetyl-beta-D-glucosaminidase (beta-GlcNA-case), N-acetyl-beta-D-galactosaminidase (beta-GalNAcase) and alpha-mannosidase (alpha-Manase) in sera of normal adults, patients with hepatocellular carcinoma (HCC), benign liver diseases and non-liver diseases were determined by microquantitative spectrophotometry. The results showed that the activity of the four serum glycosidases in patients with HCC was significantly higher than that in normal adults. When the maximum 95% confidence limit was used as the positive line, the positivities of alpha-Fucase, beta-GlcNAcase, beta-GalNAcase and alpha-Manase for the diagnosis of HCC were 66.80%, 37.29%, 32.20% and 18.64%, respectively. There was a close relationship among the four glycosidases without being related to serum alpha-fetoprotein (AFP). Some patients with benign liver diseases and non-liver diseases also had elevated glycosidase activity. However, the increase in several glycosidase activities was mainly found in HCC patients. Hepatitis patients with increased activities of more than one glycosidase were always accompanied with elevated SGPT or other abnormal liver functions. Therefore, serum glycosidase spectrum is useful for the diagnosis and differential diagnosis of HCC, especially in AFP negative HCC patients.

[Neutral alpha-D-mannosidase activity in human granulocytes]. / Aktivnost' neitral'noi al'fa-D-mannozidazy v granulotsitakh cheloveka.

The presence of neutral soluble alpha-D-mannosidase activity was shown in human granulocytes. For detection of the enzyme different methods were used: addition of stabilizing agents; sorption of acid alpha-D-mannosidase on concanavalin A-sepharose; inhibition of acid alpha-D-mannosidase; determination of neutral alpha-D-mannosidase in granulocytes of patients with inherited defect of acid alpha-D-mannosidase (mannosidosis). The specific activity of neutral alpha-D-mannosidase in granulocytes of donors calculated in nmol/min/mg of protein was near to the activity in lymphocytes. However the activity in granulocytes calculated in nmol/min/10(8) of cells was approximately 3 times lower than that in lymphocytes. The activity of neutral alpha-D-mannosidase in immature myeloid cells of a patient with chronic myeloid leukaemia was 10 times higher than in natural granulocytes of the same patient. This high activity may be in connection with the process of cell differentiation or the result of malignant transformation.

[Acid alpha-D-mannosidase of human leukocytes in the norm and during chronic myeloid leukemia]. / Kislaia al'fa-D-mannozidaza leikotsitov cheloveka v norme i pri khronicheskom mieloleikoze.

The activity and properties of acid alpha-mannosidase were studied in normal granulocytes and in two types of myeloid cells from patients with chronic myeloid leukemia. The activity of the enzyme in leukemic cells was 2-fold higher than that in normal granulocytes and in morphologically matured myeloid cells. Two latter types of cells did not differ in alpha-mannosidase activity. Kinetic properties, thermo- and pH stability of alpha-mannosidase from normal and leukemic cells were similar. alpha-mannosidase in leukemic and normal cells existed in two forms (A and B), which were easily separated on DEAE-cellulose column. These two forms differed in molecular mass (300 and 290 kD, respectively) and in the degree of sialylation. The quantitative ratios of A and B forms in normal and leukemic cells were different. In normal granulocytes and in mature cells from patients this ratio was 0.60 and 0.67, respectively. In leukemic cells the ratio was found to be 1.31. Thus, in leukemic cells form A of alpha-mannosidase predominanted, whereas in normal cells the predominance of form B was observed. It was suggested therefore that in leukemic cells the enhanced synthesis of alpha-mannosidase occurred in parallel with the accumulation of the B form. This accumulation was assumed as the cause of enhanced activity of the enzyme in immature leukemic cells.

beta-D-mannosidase in human polymorphonuclear leukocytes and lymphocytes: a comparative study.

All lymphocytes and polymorphonuclear leukocytes (PMNL) beta-D-mannosidase activities are adsorbed on DEAE-Trisacryl column at pH 7.0. Only one form is eluted with a 0.15 M linear gradient. The two enzymes isolated from either type of cells exhibit similar properties. The chromatographic profiles of beta-D-mannosidase from leukemic lymphocytes (chronic lymphoïd leukemia and hairy cells leukemia) differ from the normal ones by the presence of a more acidic minor form.

Biochemical improvement after treatment by bone marrow transplantation in I-cell disease.

The first case of successful bone marrow transplantation (BMT) in a patient with I-cell disease is reported. A 8-month-old girl with I-cell disease (N-acetylglucosaminylphosphotransferase deficiency) has had successful reconstitution with bone marrow from her HLA-MLC-matched brother who has heterozygous level of the transferase activity. The following biochemical and clinical improvements have occurred: the transferase in peripheral lymphocytes increased to donor's level, and lymphocytic alpha-neuraminidase, beta-galactosidase and alpha-mannosidase increased to normal levels. Plasma acid hydrolase activities, which had been 10 to 60 times higher in the patient than normal control levels, have slowly but steadily decreased from one month after the graft. Such decreases were observed in the activities of alpha-mannosidase, N-acetyl-beta-glucosaminidase, alpha-fucosidase, arylsulfatase A and acidic beta-galactosidase. There was also a marked decrease of vacuolated peripheral lymphocyte after the BMT. Three-months after the engraftment, hepatomegaly gradually decreased in size, corneal clouding has not progressed, and tight skin seems to have improved.

[Acid glycosidases in leukocytes of patients with chronic and acute myeloid leukemias]. / Kislye glikozidazy leikotsitov bol'nykh khronicheskimi i ostrymi mieloidnymi leikozami.

Activities of five acid glycosidases were examined in leukocytes of 30 healthy persons, 17 patients with chronic myeloid leukemia (CML) and 7 patients with acute myeloid leukemia (AML). In leukocytes of the patients with CML activities of alpha-D-mannosidase, N-acetyl-beta-D-hexosaminidase and beta-D-glucurodase were significantly higher (p less than 0.01) and those of alpha-D-galactosidase and alpha-D-glucosidase were somewhat higher (p less than 0.05) as compared with control leukocytes. Activities of all five glycosidases in leukocytes of patients with AML were within the normal limits. Glycosidase activities were also examined in leukocytes of three groups of patients with CML at different stages of the disease: chronic, progressive and blast crisis. All the enrymes exhibited the highest activity in the first group of patients; the activities of these enxymes in the second group were lower and those in the third group were close to normal. When CML leukocytes were fractionated using the Ficoll-verografin method, the activities of the enzymes in the interface fraction (unmatured cells) were higher than those in the bottom fraction (matured granulyocytes). These data suggest that the increased glycosidase activity in CML cells is due to the presence of the population of unmatured granulocytes which are distinct from blast cells.

[Activity of enzymes of xenobiotic metabolism in the liver of rats with vitamin A deficiency and mycotoxicosis T-2]. / Aktivnost' fermentov metabolizma ksenobiotikov v pecheni krys pri nedostatochnosti vitamina A i mikotoksikoze T-2.

Keeping male rats within a month on a ration deficient in vitamin A led to a distinct decrease in content of cytochrome P-450, in activities of carboxylase, epoxide hydrolase, aniline hydrolase and to a slight inhibition of UDP-glucuronosyl transferase in live tissue. At the same time, activity of glutathione transferase and content of reduced glutathione in liver tissue were increased. After administration of the epoxide-containing T-2 mycotoxin into rats within 10 days at a dose of 0.54 mg/kg activity of the enzymes catalyzing metabolism of xenobiotics was inhibited in the animals maintained on the complete half-synthetic ration, except of epoxide hydrolase and glutathione transferase, activity of which was elevated. The administration of T-2 toxin under conditions of deficiency in vitamin A caused especially distinct inhibition of the enzymes involved in the 1 phase of xenobiotic metabolism but it was accompanied by only slight increase in T-2 toxicosis. The enzymes participating in conjugation of xenobiotics as well as epoxide hydrolase appear to play major roles in detoxication of T-2 mycotoxin.

[Isoenzymes of blast cells in malignant hemopathies: present state and prospectives]. / Isoenzymes des cellules blastiques au cours des hémopathies malignes: actualité et prospective.

Numerous isoenzymes are used as markers in the course of malignant haemopathies. These are notably the isoenzymes of lactic dehydrogenase, hexosaminidase, esterases, acid phosphatases, and thymidine kinase. Their study already permits a finer and more rigorous classification of leukaemias and makes it possible to entertain serious hopes in at least three spheres: a better choice of treatment, surveillance of therapeutic efficacy and of remissions, and the development of new modes of therapeutic action by means of selective inhibitors.

Plasma and submandibular saliva lysosomal enzymes in cystic fibrosis.

This study determined in a blind fashion the activity levels and thermostability properties of two lysosomal hydrolytic enzymes, acid phosphatase and alpha-mannosidase, in plasma samples from 25 cystic fibrosis (CF) patients and 25 age- and sex-matched normal controls. Mean alpha-mannosidase activity (3.2 +/- 1.0 mU/ml) and acid phosphatase activities (6.5 +/- 2.9 mU/ml) in CF patients were not significantly different from those found in normal individuals (2.8 +/- 0.7 and 7.6 +/- 3.4 mU/ml, respectively). Using stringent conditions no differences in thermostability properties of these enzymes were found between plasma from CF patients as compared to that of normal controls. When activity levels of these enzymes and of four additional hydrolytic enzymes, alpha-L-fucosidase, alpha-galactosidase, alpha-glucosidase and beta-galactosidase, were determined in submandibular saliva, no significant differences in enzyme levels between CF and age- and sex-matched controls were noted nor were thermostability differences found. Our data do not support the concept that altered properties of these enzymes are useful as markers for detection of CF homozygotes and heterozygotes, nor the hypothesis that the defect underlying this disease is a deficiency of post-translational modification of glycoproteins leading to their mis-compartmentalization and qualitative alteration.

Serum alpha-mannosidase in patients with alcoholic liver disease.

Sera from 9 persons with either biopsy-proven alcoholic liver disease or a history of chronic, excessive ethanol consumption were analyzed for their content of various hydrolases. Compared to controls, significant elevations in the following enzyme activities were seen in sera from the patient population: acid phosphatase (2.0-fold), beta-glucuronidase (2.1-fold), hexosaminidase (1.4-fold), and alpha-L-fucosidase (2.3-fold). In addition, alpha-mannosidase activity, previously reported to be unchanged in cases of hepatic cirrhosis [Reglero et al., Clinica chim. Acta 130: 155-158], (1980) was found to be significantly increased (p less than 0.001) when assays were performed at acid (pH 4.5) or intermediate (pH 5.5) hydrogen ion concentrations. Fractionation of sera on DEAE-Sephadex columns showed that the increase in alpha-mannosidase activity in the serum of patients with alcoholic liver disease was due to increases in the level of at least one 'acid alpha-mannosidase' and two intermediate pH optimum alpha-mannosidases. The general increase in the activity of a group of glycosidases is consistent with a hypothesis involving decreased clearance of glycoproteins from the blood of persons with hepatic cirrhosis.

Frequency of an allele for low activity of alpha-L-fucosidase in sera: possible increase in epithelial ovarian cancer patients.

Sera from a group of patients with ovarian cancer had a statistically significant deficiency of alpha-L-fucosidase activity compared with sera from healthy females or female patients with cervical or breast cancer. Mixing experiments did not identify an inhibitor of alpha-L-fucosidase activity in the sera of ovarian cancer patients. Decreased activity of alpha-L-fucosidases was not associated with stage of disease, tumor burden, histologic type, or grade of differentiation. Unlike alpha-L-fucosidase, beta-man-nosidase and beta-N-acetylglucosamindase in sera of ovarian cancer patients were not deficient in activity. Examination of population data of healthy females and of pedigrees of ovarian cancer patients suggested that the quantitative activity alpha-L-fucosidase in serum was genetically determined. Of 60 healthy females, 4 had low enzyme activity (less than 100 U alpha-L-fucosidase/ml serum), 26 had intermediate activity (100-274 U alpha-L-fucosidase/ml), and 30 had high activity (275 U/ml), whereas of 44 ovarian cancer patients, 11 had low, 29 had intermediate, and 4 had high activity. Application of the Hardy-Weinberg law to these data revealed that low enzyme activity in sera was three times more prevalent in the ovarian cancer group, the allele for this low enzyme activity being two times more common. These observations suggested that deficiency of alpha-L-fucosidase activity in sera of females may be a hereditary condition associated with increased risk for development of ovarian cancer.

Heat inactivation of plasma hydrolases as a means of discriminating between the genotypes in cystic fibrosis: the experience in Israel.

The ability to discriminate between cystic fibrosis (CF) patients, carriers and control subjects, by determining the degree of hear inactivation of certain plasma enzymes has recently been reported. Material from Israel on 39 patients, 68 carriers and 65 control subjects, was tested under the originally described conditions with concordant results and distinct separation of the three genotypes. No carriers were detected in the 65 control subjects, perhaps because of the dilutional effect of those ethnic groups in Israel among whom the frequency of the CF gene is known to be rare. Biochemically, no evidence of heterogeneity of CF was found among the various ethnic groups.