Cloning and expression of the gene involved in Sanfilippo B syndrome (mucopolysaccharidosis III B)

Sanfilippo B syndrome is caused by a deficiency of alpha-N- acetylglucosaminidase, a lysosomal enzyme involved in the degradation of heparan sulphate. Accumulation of the substrate in lysosomes results in degeneration of the central nervous system with progressive dementia often combined with hyperactivity and aggressive behaviour. In order to clone the deficient gene, we purified the enzyme from human placenta and obtained amino acid sequence information. Alignment of one of the CNBr generated internal peptides to sequence from the database revealed the chromosomal location of the gene in the 5' upstream flanking region of the gene for 17-beta-hydroxysteroid-dehydrogenase at 17q21.1. The available DNA sequence was used to clone the cDNA coding for alpha-N- acetylglucosaminidase and analyse its gene structure. The gene is fully contained in the 5' upstream flanking region of the gene for 17-beta- hydroxysteroid-dehydrogenase and interrupted by five introns. The cDNA clone has a length of 2575 bp and encodes a protein of 743 amino acids. Chinese hamster ovary cells transfected with the cDNA construct show alpha-N-acetylglucosaminidase activity about 17-fold over background. This will allow correction studies with NAG deficient Sanfilippo B cell lines and facilitate the development of enzyme replacement therapy for these patients.      

Haem arginate: a new stable haem compound

Intravenous administration of haem in acute hepatic porphyrias inhibits the induction of delta-aminolaevulinic acid synthase, reduces the formation of potentially harmful metabolites of porphyrin synthesis and corrects the haem deficiency. Typically, haem therapy has been given in the form of haematin--haem dissolved in alkali. Such haematin solutions are, however, extremely unstable. Thus, the rapid decomposition of this therapeutic agent may have been responsible for the ineffectiveness of treatment in some clinical states and adverse reactions may have been caused by haematin degradation products. There is, therefore, a need for a stable, effective and well-tolerated haem preparation. We have prepared certain highly soluble haem compounds of which haem arginate has proved to be the most promising. Pure haemin was isolated from HIV and hepatitis B negative human blood. The haem derivatives prepared were screened as substrates for haem oxygenase. Haem arginate and haem lysinate were found to be as good substrates as methaemalbumin. Stock solutions of haem arginate were stable for 2 years at +6 degrees C. After dilution with sterile isotonic saline the haem arginate infusion was clearly more stable than haematin solutions made in the laboratory or prepared by dissolving commercial lyophilized haematin. The antiporphyrogenic effect of haem arginate (even after storage for two years) in 2-allyl-2-isopropylacetamide-induced experimental porphyria of rats was equal to that of freshly prepared haematin. The acute oral toxicity of haem arginate was low compared with the parenterally administered drug, indicating poor oral bioavailability. The acute toxic effects after high intravenous or intraperitoneal doses were directed to the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

A reversible monoamine oxidase inhibitor, toloxatone: Structural and electronic properties

Toloxatone is a reversible MAO_A-inhibitor, marketed as antidepressant (Humoryl^®), with an original chemical structure. It differs from first generation irreversible MAOIs, known to induce covalent bonds with the enzyme active site. In order to understand the mechanism of the reversible inactivation of the MAO, as a first step, a detailed structural and electronic analysis was undertaken. An X-ray diffraction-crystallographic study showed that toloxatone is a planar molecule and brought to light hydrogen bonds and π-π interactions. MO calculations confirmed the planar structure as energetically favoured. Electronic analysis demonstrated a delocalization of both ring systems. The combined results give evidence for the potential of toloxatone to participate in reversible, long distance interactions with an appropriate partner.     

银杏内酯类合成物F抗血小板激活因子和组织胺的实验研究

目的：研究银杏内酯类化合物Ｆ（简称药物Ｆ）同时拮抗组织胺（ＨＡ）和血小板激活因子（ＰＡＦ）的作用。方法：测定离体豚鼠肺组织的收缩率以及用药物Ｆ后的致敏豚鼠肺功能的变化。结果：（１）加入药物Ｆ后离体豚鼠的气管条和肺条对ＨＡ的收缩率分别由（５０．９７±１６．００）％和（５４．２４±１２．１７）％降至（２１．６９±３．８５）％和（２２．９７±１７．７８）％（Ｐ〈０．０５）；（２）药物Ｆ保护后，离体豚鼠肺     

Boron Neutron Capture Therapy of Brain Tumors: Clinical Trials at the Finnish Facility Using Boronophenylalanine

Two clinical trials are currently running at the Finnish dedicated boron neutron capture therapy (BNCT) facility. Between May 1999 and December 2001, 18 patients with supratentorial glioblastoma were treated with boronophenylalanine (BPA)-based BNCT within a context of a prospective clinical trial (protocol P-01). All patients underwent prior surgery, but none had received conventional radiotherapy or cancer chemotherapy before BNCT. BPA-fructose was given as 2-h infusion at BPA-dosages ranging from 290 to 400mg/kg prior to neutron beam irradiation, which was given as a single fraction from two fields. The average planning target volume dose ranged from 30 to 61Gy (W), and the average normal brain dose from 3 to 6Gy (W). The treatment was generally well tolerated, and none of the patients have died during the first months following BNCT. The estimated 1-year overall survival is 61%. In another trial (protocol P-03), three patients with recurring or progressing glioblastoma following surgery and conventional cranial radiotherapy to 50–60Gy, were treated with BPA-based BNCT using the BPA dosage of 290mg/kg. The average planning target dose in these patients was 25–29Gy (W), and the average whole brain dose 2–3Gy (W). All three patients tolerated brain reirradiation with BNCT, and none died during the first three months following BNCT. We conclude that BPA-based BNCT has been relatively well tolerated both in previously irradiated and unirradiated glioblastoma patients. Efficacy comparisons with conventional photon radiation are difficult due to patient selection and confounding factors such as other treatments given, but the results support continuation of clinical research on BPA-based BNCT.     

Ace gene dosage influences the development of renovascular hypertension

1. Clinical and experimental evidence highlights the importance of the renin–angiotensin system in renovascular hypertension. Furthermore, genetic factors affecting angiotensin‐converting enzyme (ACE) could influence the development of renovascular hypertension. 2. To test the effect of small gene perturbations on the development of renovascular hypertension, mice harbouring two or three copies of the Ace gene were submitted to 4 weeks of two‐kidney, one‐clip (2K1C) hypertension. Blood pressure (BP), cardiac hypertrophy, baroreflex sensitivity and blood pressure and heart rate variability were assessed and compared between the different groups. 3. The increase in BP induced by 2K1C was higher in mice with three copies of the Ace gene compared with mice with only two copies (46 vs 23 mmHg, respectively). Moreover, there was a 3.8‐fold increase in the slope of the left ventricle mass/BP relationship in mice with three copies of the Ace gene. Micewith three copies of the Ace gene exhibited greater increases in cardiac and serum ACE activity than mice with only two copies of the gene. Both baroreflex bradycardia and tachycardia were significantly depressed in mice with three copies of the Ace gene after induction of 2K1C hypertension. The variance in basal systolic BP was greater in mice with three copies of the Ace gene after 2K1C hypertension compared with those with only two copies of the gene (106 vs 54%, respectively). In addition, the low‐frequency component of the pulse interval was higher mice with three copies of the Ace gene after 2K1C hypertension compared with those with only two (168 vs 86%, respectively). Finally, in mice with three copies of the Ace gene, renovascular hypertension induced a 6.1‐fold increase in the sympathovagal balance compared with a 3.2‐fold increase in mice with only two copies of the gene. 4. Collectively, these data provide direct evidence that small genetic disturbances in ACE levels per se have an influence on haemodynamic, cardiac mass and autonomic nervous system responses in mice under pathological perturbation.