Keratan sulphate levels in mucopolysaccharidoses and mucolipidoses.

The mucopolysaccharidoses (MPS) is characterized by accumulation of glycosaminoglycans (GAGs), and mucolipidosis (ML) by accumulation of GAGs and sphingolipids. Each type of MPS accumulates specific GAGs. The lysosomal enzymes N-acetylgalactosamine-6-sulphate sulphatase and beta-galactosidase involve the stepwise degradation of keratan sulphate (KS). Deficiency of these enzymes results in elevation of KS levels in the body fluids and in tissues, leading to MPS IV disease. In this study, we evaluated blood and urine KS levels in types of MPS and ML other than MPS IV. Eighty-five plasma samples came from MPS I (n = 18), MPS II (n = 28), MPS III (n = 20), MPS VI (n = 3), MPS VII (n = 5) and ML (n = 11) patients while 127 urine samples came from MPS I (n = 34), MPS II (n = 34), MPS III (n = 32), MPS VI (n = 7), MPS VII (n = 9) and ML (n = 11) patients. KS levels were determined using the ELISA method. Plasma KS levels varied with age in both control and patient populations. In all age groups, the mean values of plasma KS in MPS and ML patients were significantly higher than those in the age-matched controls. Plasma KS values in four newborn patients were above the mean + 2SD of the age-matched controls (mean, 41 ng/ml). Overall, 85.9% of individual values in non-type IV MPS and ML patients were above the mean + 2SD of the age-matched controls. For urine KS levels, 24.4% of individual values in patients were above the mean + 2SD of the age-matched controls. In conclusion, KS in blood is elevated in each type of non-type IV MPS examined, in contrast to the conventional understanding. This finding suggests that measurement of KS level provides a new diagnostic biomarker in a wide variety of mucopolysaccharidoses and mucolipidoses in addition to MPS IV.

Investigation of loss of heterozygosity and SNP frequencies in the RET gene in papillary thyroid carcinoma.

In both medullary carcinoma and papillary carcinoma of the thyroid, altered expression of the RET gene is implicated in tumorigenesis. Recent studies suggest that loss of heterozygosity (LOH) at the G691S SNP may be associated with tumors from patients with a history of radiation exposure. We investigated LOH for three RET SNPs (G691S, S904S, and L769L) in tumor and normal tissue from 46 patients from Ukraine and Belarus who were exposed to radioactive fallout following the Chernobyl nuclear accident and were operated for papillary thyroid carcinoma between 1995 and 2000. Normal tissue from 28 patients was heterozygous for at least one SNP; DNA from the corresponding tumor samples was also heterozygous, indicating that no LOH had taken place. To assess SNP frequencies in a radiation-associated thyroid cancer cohort, we investigated a further 68 unpaired post-Chernobyl samples. For G691S, there was considerable deviation from Hardy-Weinberg equilibrium; more detailed analysis showed that this was linked to age at onset of disease. Among younger patients, the distribution of genotypes conformed to Hardy-Weinberg equilibrium; among older patients, we observed marked deviation (p = 0.0072), with significant over-representation of the rare S allele relative to the younger groups (Fisher's exact, p = 0.0233). This suggests that SNPs in the RET oncogene may play a role in sporadic papillary thyroid carcinoma.

Biodistribution, kinetics, and efficacy of highly phosphorylated and non-phosphorylated beta-glucuronidase in the murine model of mucopolysaccharidosis VII.

Enzyme replacement therapy (ERT) has been shown to be effective at reducing the accumulation of undegraded substrates in lysosomal storage diseases. Most ERT studies have been performed with recombinant proteins that are mixtures of phosphorylated and non-phosphorylated enzyme. Because different cell types use different receptors to take up phosphorylated or non-phosphorylated enzyme, it is difficult to determine which form of enzyme contributed to the clinical response. Here we compare the uptake, distribution, and efficacy of highly phosphorylated and non-phosphorylated beta-glucuronidase (GUSB) in the MPS VII mouse. Highly phosphorylated murine GUSB was efficiently taken up by a wide range of tissues. In contrast, non-phosphorylated murine GUSB was taken up primarily by tissues of the reticuloendothelial (RE) system. Although the tissue distribution was different, the half-lives of both enzymes in any particular tissue were similar. Both preparations of enzyme were capable of preventing the accumulation of lysosomal storage in cell types they targeted. An important difference in clinical efficacy emerged in that phosphorylated GUSB was more efficient than non-phosphorylated enzyme at preventing the hearing loss associated with this disease. These data suggest that both forms of enzyme contribute to the clinical responses of ERT in MPS VII mice but that enzyme preparations containing phosphorylated GUSB are more broadly effective than non-phosphorylated enzyme.

Crystal structure of the dimeric extracellular domain of human carbonic anhydrase XII, a bitopic membrane protein overexpressed in certain cancer tumor cells.

Overexpression of the zinc enzyme carbonic anhydrase (CA; EC ) XII is observed in certain human cancers. This bitopic membrane protein contains an N-terminal extracellular catalytic domain, a membrane-spanning alpha-helix, and a small intracellular C-terminal domain. We have determined the three-dimensional structure of the extracellular catalytic domain of human CA XII by x-ray crystallographic methods at 1.55-A resolution. The structure reveals a prototypical CA fold; however, two CA XII domains associate to form an isologous dimer, an observation that is confirmed by studies of the enzyme in solution. The identification of signature GXXXG and GXXXS motifs in the transmembrane sequence that facilitate helix-helix association is additionally consistent with dimeric architecture. The dimer interface is situated so that the active site clefts of each monomer are clearly exposed on one face of the dimer, and the C termini are located together on the opposite face of the dimer to facilitate membrane interaction. The amino acid composition of the active-site cleft closely resembles that of the other CA isozymes in the immediate vicinity of the catalytic zinc ion, but differs in the region of the nearby alpha-helical "130's segment." The structure of the CA XII-acetazolamide complex is also reported at 1.50-A resolution, and prospects for the design of CA XII-specific inhibitors of possible chemotherapeutic value are discussed.

Active site mutant transgene confers tolerance to human beta-glucuronidase without affecting the phenotype of MPS VII mice.

Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is an autosomal recessive lysosomal storage disorder due to an inherited deficiency of beta-glucuronidase. A naturally occurring mouse model for this disease was discovered at The Jackson Laboratory and shown to be due to homozygosity for a 1-bp deletion in exon 10 of the gus gene. The murine model MPS VII (gus(mps/mps)) has been very well characterized and used extensively to evaluate experimental strategies for lysosomal storage diseases, including bone marrow transplantation, enzyme replacement therapy, and gene therapy. To enhance the value of this model for enzyme and gene therapy, we produced a transgenic mouse expressing the human beta-glucuronidase cDNA with an amino acid substitution at the active site nucleophile (E540A) and bred it onto the MPS VII (gus(mps/mps)) background. We demonstrate here that the mutant mice bearing the active site mutant human transgene retain the clinical, morphological, biochemical, and histopathological characteristics of the original MPS VII (gus(mps/mps)) mouse. However, they are now tolerant to immune challenge with human beta-glucuronidase. This "tolerant MPS VII mouse model" should be useful for preclinical trials evaluating the effectiveness of enzyme and/or gene therapy with the human gene products likely to be administered to human patients with MPS VII.

Expression of membrane-associated carbonic anhydrase XIV on neurons and axons in mouse and human brain.

Although long suspected from histochemical evidence for carbonic anhydrase (CA) activity on neurons and observations that CA inhibitors enhance the extracellular alkaline shifts associated with synaptic transmission, an extracellular CA in brain had not been identified. A candidate for this CA was suggested by the recent discovery of membrane CA (CA XIV) whose mRNA is expressed in mouse and human brain and in several other tissues. For immunolocalization of CA XIV in mouse and human brain, we developed two antibodies, one against a secretory form of enzymatically active recombinant mouse CA XIV, and one against a synthetic peptide corresponding to the 24 C-terminal amino acids in the human enzyme. Immunostaining for CA XIV was found on neuronal membranes and axons in both mouse and human brain. The highest expression was seen on large neuronal bodies and axons in the anterolateral part of pons and medulla oblongata. Other CA XIV-positive sites included the hippocampus, corpus callosum, cerebellar white matter and peduncles, pyramidal tract, and choroid plexus. Mouse brain also showed a positive reaction in the molecular layer of the cerebral cortex and granular cellular layer of the cerebellum. These observations make CA XIV a likely candidate for the extracellular CA postulated to have an important role in modulating excitatory synaptic transmission in brain.

Purification and kinetic analysis of recombinant CA XII, a membrane carbonic anhydrase overexpressed in certain cancers.

Carbonic anhydrase XII (CA XII) is a transmembrane glycoprotein with an active extracellular CA domain that is overexpressed on cell surfaces of certain cancers. Its expression has been linked to tumor invasiveness. To characterize its catalytic properties, we purified recombinant secretory forms of wild-type and mutant CA XIIs. The catalytic properties of these enzymes in the hydration of CO(2) were measured at steady state by stopped-flow spectrophotometry and at chemical equilibrium by the exchange of (18)O between CO(2) and water determined by mass spectrometry. The catalysis of CO(2) hydration by soluble CA XII has a maximal value of k(cat)/K(m) at 34 microM(-1) small middle dots(-1), which is similar to those of the membrane-associated CA IV and to soluble CA I. The pH profiles of this catalysis and the catalyzed hydrolysis of 4-nitrophenylacetate indicate that the pK(a) of the zinc-bound water in CA XII is 7.1. His64 in CA XII acts as a proton shuttle residue, as evidenced by the reduced rate constant for proton transfer in the mutants containing the replacements His64 --> Ala and His64 --> Arg, as well as by the selective inhibition of the proton transfer step by cupric ions in wild-type CA XII. The catalytic rate of CO(2) hydration by the soluble form of CA XII is identical with that of the membrane-bound enzyme. These observations suggest a role for CA XII in CO(2)/HCO(3)(-) homeostasis in cells in which it is normally expressed. They are also compatible with a role for CA XII in acidifying the microenvironment of cancer cells in which CA XII is overexpressed, providing a mechanism for CA XII to augment tumor invasiveness and suggesting CA XII as a potential target for chemotherapeutic agents.

Second opinions, and becoming a care manager.

After an unsuccessful period of orthodontic treatment (by another provider), state-of-the-art methods are used to gain a patient's trust. Diagnostic set-ups and digital imaging techniques help the patient feel confident enough to accept a comprehensive multidisciplinary treatment plan.

Mitochondrial carbonic anhydrase CA VB: differences in tissue distribution and pattern of evolution from those of CA VA suggest distinct physiological roles.

A cDNA for a second mouse mitochondrial carbonic anhydrase (CA) called CA VB was identified by homology to the previously characterized murine CA V, now called CA VA. The full-length cDNA encodes a 317-aa precursor that contains a 33-aa classical mitochondrial leader sequence. Comparison of products expressed from cDNAs for murine CA VB and CA VA in COS cells revealed that both expressed active CAs that localized in mitochondria, and showed comparable activities in crude extracts and in mitochondria isolated from transfected COS cells. Northern blot analyses of total RNAs from mouse tissues and Western blot analyses of mouse tissue homogenates showed differences in tissue-specific expression between CA VB and CA VA. CA VB was readily detected in most tissues, while CA VA expression was limited to liver, skeletal muscle, and kidney. The human orthologue of murine CA VB was recently reported also. Comparison of the CA domain sequence of human CA VB with that reported here shows that the CA domains of CA VB are much more highly conserved between mouse and human (95% identity) than the CA domains of mouse and human CA VAs (78% identity). Analysis of phylogenetic relationships between these and other available human and mouse CA isozyme sequences revealed that mammalian CA VB evolved much more slowly than CA VA, accepting amino acid substitutions at least 4.5 times more slowly since each evolved from its respective human-mouse ancestral gene around 90 million years ago. Both the differences in tissue distribution and the much greater evolutionary constraints on CA VB sequences suggest that CA VB and CA VA have evolved to assume different physiological roles.

Expression of a novel transmembrane carbonic anhydrase isozyme XII in normal human gut and colorectal tumors.

Carbonic anhydrase isozyme XII is a recently discovered member of the alpha-carbonic anhydrase gene family with a suggested role in von Hippel-Lindau gene-mediated carcinogenesis. Increased expression of its mRNA has been observed in renal and lung carcinomas. This paper presents the localization of CA XII in the normal human gut and in colorectal tumors. Immunohistochemistry performed using a polyclonal antibody raised against truncated CA XII revealed prominent polarized staining for CA XII in the basolateral plasma membrane of the enterocytes of the normal large intestine, the reaction being most intense in the surface epithelial cuff region. Most colorectal tumors displayed abnormal expression of CA XII; the most dramatic change was observed in the deep parts of the adenomatous mucosa, where the positive immunoreaction clearly increased along with the grade of dysplasia. Adenomas with severe dysplasia and carcinomas showed an equal, diffuse staining pattern. The results indicate region-specific regulation of CA XII expression along the cranial-caudal axis of the human gut, whereas its diffuse expression in the most malignant tumors seems to correlate with their biological behavior.

Identification of carbonic anhydrase XII as the membrane isozyme expressed in the normal human endometrial epithelium.

Although previous studies demonstrated carbonic anhydrase (CA) activity in the human endometrium, the CA isozyme(s) responsible for this activity has not been established. In this report, we provide the first evidence that the CA isozyme XII, a recently identified transmembrane isozyme that is expressed in normal kidney and greatly overexpressed in some renal cancers, is present in endometrium. We show by immunohistochemistry that CA XII is expressed in the basolateral plasma membrane of epithelial cells of normal human endometrium. Expression of CA XII in uterus was confirmed by Northern blotting. Detergent-solubilized CA XII was isolated from human endometrium by inhibitor affinity chromatography and characterized by isoelectric focusing and Western blot as a polypeptide with a pI of 6.3. The high expression of CA XII in the endometrial epithelium suggests that it may be functionally linked to the pH-dependent events in spermatozoa that precede fertilization. Its basolateral location and extracellular active site could also allow it to influence the morphological changes in endometrium that occur during the menstrual cycle.

Active site residues of human beta-glucuronidase. Evidence for Glu(540) as the nucleophile and Glu(451) as the acid-base residue.

Human beta-glucuronidase (hGUSB) is a member of family 2 glycosylhydrolases that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. Amino acid sequence and structural homology of hGUSB and Escherichia coli beta-galactosidase active sites led us to propose that residues Glu(451), Glu(540), and Tyr(504) in hGUSB are involved in catalysis, Glu(451) being the acid-base residue and Glu(540) the nucleophile. To test this hypothesis, we introduced mutations in these residues and determined their effects on enzymes expressed in COS cells and GUSB-deficient fibroblasts. The extremely low activity in cells expressing Glu(451), Glu(540), and Tyr(504) hGUSBs supported their roles in catalysis. For kinetic analysis, wild type and mutant enzymes were produced in baculovirus and purified to homogeneity by affinity chromatography. The k(cat)/K(m) values (mM(-1).s(-1)) of the E540A, E451A, and Y504A enzymes were 34,000-, 9100-, and 830-fold lower than that of wild type hGUSB, respectively. High concentrations of azide stimulated the activity of the E451A mutant enzyme, supporting the role of Glu(451) as the acid-base catalyst. We conclude that, like their homologues in E. coli beta-galactosidase, Glu(540) is the nucleophilic residue, Glu(451) the acid-base catalyst, and Tyr(504) is also important for catalysis, although its role is unclear. All three residues are located in the active site cavity previously determined by structural analysis of hGUSB.

The mannose 6-phosphate/insulin-like growth factor-II receptor is a substrate of type V transforming growth factor-beta receptor.

The type V transforming growth factor beta (TGF-beta) receptor (TbetaR-V) is a ligand-stimulated acidotropic Ser-specific protein kinase that recognizes a motif of SXE/S(P)/D. This motif is present in the cytoplasmic domain of the mannose 6-phosphate/insulin-like growth factor-II (Man-6-P/IGF-II) receptor. We have explored the possibility that the Man-6-P/IGF-II receptor is a substrate of TbetaR-V. Purified bovine Man-6-P/IGF-II receptor was phosphorylated by purified bovine TbetaR-V in the presence of [gamma-32P]ATP and MnCl2 with an apparent Km of 130 nM. TGF-beta stimulated the phosphorylation of the Man-6-P/IGF-II receptor at 0 degrees C in mouse L cells overexpressing the Man-6-P/IGF-II receptor and in wild-type mink lung epithelial (Mv1Lu cells) metabolically labeled with [32P]orthophosphate. The in vitro and in vivo phosphorylation of the Man-6-P/IGF-II receptor occurred at the putative phosphorylation sites as revealed by phosphopeptide mapping and amino acid sequence analysis. TGF-beta stimulated Man-6-P/IGF-II receptor-mediated uptake (approximately 2-fold after 12 h treatment) of exogenous beta-glucuronidase in Mv1Lu cells and type II TGF-beta receptor (TbetaR-II)-defective mutant cells (DR26 cells) but not in type I TGF-beta receptor (TbetaR-I)-defective mutant cells (R-1B cells) and human colorectal carcinoma cells (RII-37 cells) expressing TbetaR-I and TbetaR-II but lacking TbetaR-V. These results suggest the Man-6-P/IGF-II receptor serves as an in vitro and in vivo substrate of TbetaR-V and that both TbetaR-V and TbetaR-I may play a role in mediating the TGF-beta-stimulated uptake of exogenous beta-glucuronidase.

Case report: treatment for a patient with a history of TMJ disorder.

Establishing a knowledge-based protocol for the treatment of orthodontic patients who report a history of temporomandibular dysfunction can alert the practitioner to potential treatment pitfalls before they happen. While the joints can be extremely adaptive, some individuals are subject to painful and/or limited function. Others have acquired condylar positions that, if not recognized, could lead to serious alterations in the original treatment plan. Combining a thorough diagnostic protocol with a therapeutic regimen that seeks to establish a stable condylar and occlusal position-prior to initiating treatment- is essential.

Enzyme replacement therapy improves reproductive performance in mucopolysaccharidosis type VII mice but does not prevent postnatal losses.

Mice with mucopolysaccharidosis type VII (MPS VII) are devoid of beta-glucuronidase and accumulate glycosaminoglycans in lysosomes resulting in bone dysplasia, learning disabilities, and decreased mobility. MPS VII males do not breed and, while MPS VII females occasionally mate with heterozygous males, they do not maintain their young postnatally. Heterozygous matings produce less than 25% MPS VII offspring, but until now it was unclear whether this results from prenatal or postnatal losses. The administration of recombinant beta-glucuronidase from birth significantly reduces glycosaminoglycan storage in most tissues, increases life span, and improves the animal's cognitive ability and mobility. To determine whether reproductive failure is corrected by such therapy, male and female MPS VII mice were injected with enzyme at weekly intervals from birth to 5 wk of age (6xinj). Enzyme-replaced MPS VII mice bred when mated together. The 6xinj MPS VII males mated repeatedly until they were killed 135 d postinjection. All mated 6xinj MPS VII females gave birth to two litters, but maintained few of their young. Selective loss of MPS VII offspring was observed in matings between heterozygotes. Analysis of 379 preterm fetuses from heterozygous matings showed a frequency of 24.6% MPS VII pups, indicating that the decreased number of MPS VII pups produced by mating heterozygotes results from postnatal losses. The ovaries of young adult MPS VII mice have follicles and corpora lutea, and the testes generate sperm. Results suggest that the reproductive failure in MPS VII mice is related to impaired mobility and/or impaired cognitive function, and enzyme replacement restores mating capacity.

Effects of second language study of phonemic discrimination and auditory event-related potentials in adults.

This study was designed to investigate the effects of acquisition of a second language on auditory even-related brain potentials and discrimination of foreign language phonemes by 36 women (ages 18 to 47 years), and 25 men (ages 18 to 36 years) and of varying linguistic background, in response to synthetic versions of Japanese phonemes. Subjects were subsequently tested on discrimination between spoken Japanese phonemes. Analysis indicated that the men and women differed in phonological processing and in the way acquisition of the second language affected phonological processing.

Identification of Glu-540 as the catalytic nucleophile of human beta-glucuronidase using electrospray mass spectrometry.

Human beta-glucuronidase is a member of the Family 2 glycosylhydrolases that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. The enzyme is shown to catalyze glycoside bond hydrolysis with net retention of anomeric configuration, presumably via a mechanism involving a covalent glucuronyl-enzyme intermediate. Incubation of human beta-glucuronidase with 2-deoxy-2-fluoro-beta-D-glucuronyl fluoride resulted in time-dependent inactivation of the enzyme through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-glucuronyl-enzyme, as observed by electrospray mass spectrometry. Regeneration of the free enzyme by hydrolysis or transglycosylation and removal of excess inactivator demonstrated that the covalent intermediate was kinetically competent. Peptic digestion of the 2-deoxy-2-fluoro-alpha-D-glucuronyl-enzyme intermediate and subsequent analysis by liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry indicated the presence of a 2-deoxy-2-fluoro-alpha-D-glucuronyl peptide. Sequence determination of the labeled peptide by tandem mass spectrometry in the daughter ion scan mode permitted the identification of Glu-540 as the catalytic nucleophile within the sequence SEYGAET.

The role of computerized video imaging in predicting adult extraction treatment outcomes.

The purpose of this study was to evaluate the accuracy of computerized video imaging in predicting the soft tissue outcome of extracting four premolars in adults. The pretreatment and posttreatment cephalometric and facial photographic records of 31 previously treated, nongrowing patients were digitized and computer-generated cephalometric VTOs and video images were compared with the known outcomes. The results showed that both the VTOs and video images were accurate enough to be used for patient education and communication, as well as for diagnosis and treatment planning. While lay people found that the predicted video images adequately resembled the actual outcomes, orthodontists were more critical, particularly of the lower lip area where variable soft tissue responses to treatment were noted.

Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers.

We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic C-terminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30-42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43- to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using fluorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.