A novel HYLS1 homozygous mutation in living siblings with Joubert syndrome.

The p.Asp211Gly homozygous HYLS1 mutation is so far known to cause only hydrolethalus syndrome, a lethal malformation syndrome. We report living sibling patients with a homozygous no-stop mutation in exon 4 of HYLS1, NM_145014.2:c.900A>C (p.Ter300TyrextTer11) in the second decade of life. The proband has Joubert syndrome (JS). The younger brother also has JS and an enlarged posterior fossa that was initially diagnosed as Dandy-Walker malformation. The present mutation is unique as it affects the stop codon. The product protein HYLS1 plays an essential role in the formation of the primary cilium. This report provides insight into the spectrum of disorders involving midline brain defects closely related to cilium dysfunction or ciliopathy.

Mannitol facilitates neurotrophic factor up-regulation and behavioural recovery in neonatal hypoxic-ischaemic rats with human umbilical cord blood grafts.

We recently demonstrated that blood-brain barrier permeabilization using mannitol enhances the therapeutic efficacy of systemically administered human umbilical cord blood (HUCB) by facilitating the entry of neurotrophic factors from the periphery into the adult stroke brain. Here, we examined whether the same blood-brain barrier manipulation approach increases the therapeutic effects of intravenously delivered HUCB in a neonatal hypoxic-ischaemic (HI) injury model. Seven-day-old Sprague-Dawley rats were subjected to unilateral HI injury and then at day 7 after the insult, animals intravenously received vehicle alone, mannitol alone, HUCB cells (15k mononuclear fraction) alone or a combination of mannitol and HUCB cells. Behavioural tests at post-transplantation days 7 and 14 showed that HI animals that received HUCB cells alone or when combined with mannitol were significantly less impaired in motor asymmetry and motor coordination compared with those that received vehicle alone or mannitol alone. Brain tissues from a separate animal cohort from the four treatment conditions were processed for enzyme-linked immunosorbent assay at day 3 post-transplantation, and revealed elevated levels of GDNF, NGF and BDNF in those that received HUCB cells alone or when combined with mannitol compared with those that received vehicle or mannitol alone, with the combined HUCB cells and mannitol exhibiting the most robust neurotropic factor up-regulation. Histological assays revealed only sporadic detection of HUCB cells, suggesting that the trophic factor-mediated mechanism, rather than cell replacement per se, principally contributed to the behavioural improvement. These findings extend the utility of blood-brain barrier permeabilization in facilitating cell therapy for treating neonatal HI injury.

Lack of exercise, via hindlimb suspension, impedes endogenous neurogenesis.

Bedridden patients who receive good physical rehabilitation are able to exhibit clinical improvement. Accumulating evidence demonstrates that exercise increases endogenous neurogenesis and may even protect against central nervous system (CNS) disorders. Here, we explored the effects of lack of exercise on neurogenesis in rats by employing a routine hindlimb suspension (HS) model over a 2-week period, which consists of elevating their tails, thereby raising their hindlimbs above the ground and unloading the weights in these extremities. In addition, the effects of exercise and recovery time with normal caging after HS were also explored. BrdU (50 mg/kg, i.p.) was injected every 8 h over the last 4 days of each paradigm to label proliferative cells. Immunohistochemical results revealed that HS significantly reduced the number of BrdU/Doublecortin double-positive cells in the subventricular zone and dentate gyrus. Exercise and recovery time significantly improved atrophy of the soleus muscle, but did not attenuate the HS-induced decrement in BrdU/Dcx-positive cells. A separate cohort of animals was exposed to the same HS paradigm and enzyme-linked immunosorbent assay (ELISA) of neurotrophic factors was performed on brain tissue samples harvested at the end of the HS period, as well as plasma samples from all animals. ELISA results revealed that HS reduced the levels of brain-derived neurotrophic factor in the hippocampus and vascular endothelial growth factor plasma levels. This study revealed that lack of exercise reduced neurogenesis with downregulation of neurotrophic factors. The use of the HS model in conjunction with CNS disease models should further elucidate the role of exercise in neurogenesis and neurotrophic factors in neurologic disorders.

Overexpression of D2/D3 receptors increases efficacy of ropinirole in chronically 6-OHDA-lesioned Parkinsonian rats.

Ropinirole, which is a non-ergot dopamine agonist derivative, exerts therapeutic benefits in Parkinson's disease (PD). Based on recent studies implicating dopamine receptors 2 and 3 (D2R and D3R) as possible targets of ropinirole, we over-expressed these dopamine receptor genes in the dopamine-denervated striatum of rodents to reveal whether their over-expression modulated ropinirole activity. Adult Sprague-Dawley rats initially received unilateral 6-hydroxydopamine lesion of the medial forebrain bundle. At 1 month after surgery, successfully lesioned animals (3 or less forelimb akinesia score, and 8 or more apomorphine-induced rotations/min over 1 h) were randomly assigned to intrastriatal injection (ipsilateral to the lesion) of blank lentiviral vector, D2R, D3R or both genes. At about 5 months post-lesion, ropinirole (0.2 mg/kg, i.p.) was administered daily for 9 consecutive days. The subtherapeutic dose of ropinirole improved the use of previously akinetic forelimb and produced robust circling behavior in lesioned animals with striatal over-expression of both D2R and D3R compared to lesioned animals that received blank vector. In contrast, the subtherapeutic dose of ropinirole generated only modest motor effects in lesioned animals with sole over-expression of D2R or D3R. Western immunoblot and autoradiographic assays showed enhanced D2R and D3R protein levels coupled with normalized D2R and D3R binding in the ventral striatum of lesioned animals with lentiviral over-expression of both D2R and D3R relative to vehicle-treated lesioned animals. Immunohistochemical analyses showed that D2R and D3R GFP fluorescent cells colocalized with enkephalin and substance P immunoreactive medium spiny neurons. These data support the use of the subtherapeutic dose of ropinirole in a chronic model of PD.

Multiple genes, including a member of the AAA family, are essential for degradation of unassembled subunit 2 of cytochrome c oxidase in yeast mitochondria.

Cytochrome c oxidase consists of three mitochondrion- and several nucleus-encoded subunits. We previously found that in a mutant of Saccharomyces cerevisiae lacking nucleus-encoded subunit 4 of this enzyme (CoxIV), subunits 2 and 3 (CoxII and CoxIII), both encoded by the mitochondrial DNA, were unstable and rapidly degraded in mitochondria, presumably because the subunits cannot assemble normally. To analyze the molecular machinery involved in this proteolytic pathway, we obtained four mutants defective in the degradation of unassembled CoxII (osd mutants) by screening CoxIV-deficient cells for the accumulation of CoxII. All of the mutants were recessive and were classified into three different complementation groups. Tetrad analyses revealed that the phenotype of each mutant was caused by a single nuclear mutation. These results suggest strongly that at least three nuclear genes (the OSD genes) are required for this degradation system. Interestingly, degradation of CoxIII was not affected in the mutants, implying that the two subunits are degraded by distinct pathways. We also cloned the OSD1 gene by complementation of the temperature sensitivity of osd1-1 mutants with a COXIV+ genetic background on a nonfermentable glycerol medium. We found it to encode a member of a family (the AAA family) of putative ATPases, which proved to be identical to recently described YME1 and YTA11. Immunological analyses revealed that Osd1 protein is localized to the mitochondrial inner membrane. Disruption of the predicted ATP-binding cassette by site-directed mutagenesis eliminated biological activities, thereby underscoring the importance of ATP for function.

Purification and characterization of neutrophil chemotactic factors of Streptococcus sanguis.

Two neutrophil chemotactic factors were isolated from the culture filtrates of Streptococcus sanguis ATCC 10556 and were chemically characterized as N-terminal blocked peptides of low molecular weight. One of the factors consisted of proline, valine, methionine, isoleucine and leucine and the other of methionine, isoleucine, leucine and phenylalanine. In both factors, methionine was detected as the sole N-terminal amino acid, but the amino group was blocked. The removal of N-terminal methionine yielded several N-terminal amino acids, suggesting that S. sanguis produced several N-terminal blocked methionyl peptides, all of which could be chemotactically active.

Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis.

A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.

Cloning and expression in Escherichia coli of cDNA encoding house dust mite allergen Der f 3, serine protease from Dermatophagoides farinae.

Der f 3 is one of the allergens produced by house dust mite Dermatophagoides farinae showing serine protease activity. Based on its amino acid sequence, a cDNA clone encoding Der f 3 was isolated from a cDNA library of D. farinae. Sequencing analysis of the clone revealed the presence of an open reading frame of 780 bp, which encodes a mature protein of 232 amino acids with 27 amino acids of pre-pro sequence at the N-terminus. When proDer f 3 was produced in Escherichia coli as a fused protein with glutathione-S-transferase, the fused protein was accumulated as inclusion bodies. The protein purified with 8 M urea and glutathione-affinity column chromatography, however, did not show protease activity. When an arginine residue was introduced at the C-terminus of the pro-region in place of threonine, removal of the pro-region to produce an active mature protease was observed. The specificity and the activity of this recombinant protease were almost the same as those of native Der f 3.

Synthesis of the vasoconstrictor peptide endothelin in kidney cells.

The expression plasmid containing human prepro-endothelin cDNA was constructed and introduced into COS-7 cells. Mature endthelin, consisting of 21 amino acid residues, was secreted into the culture medium of the transfected cells and was also synthesized by non-transfected COS-7 cells. Normal kidney cells derived from other species also synthesized and secreted endothelin. Partial characterization of endothelins produced by kidney cells suggested that existence of new types of endothelin. This is the first report of the vasoconstrictor peptide endothelin being synthesized in kidney cells.

Synthesis of ala-pro-gly-[Ile3, Val5]angiotensin II isolated from the skin of the australian frog Crinia georgiana.

Ala-Pro-Gly-[Ile3, Val5]angiotensin II was synthesized by Merrifield's solid-phase procedure. The peptide was purified by chromatography on successive columns of anion-exchange resin, Sephadex G-25 and SP-Sephadex C-25; its homogeneity was determined by degradation with alpha-chymotrypsin, ionophoresis, thin-layer chromatography, and high-pressure liquid chromatography (HPLC). The dansyl derivative of this angiotensin has the same chromatographic behavior (TLC) as the dansyl undecapeptide, "Crinia angiotensin II", isolated from the skin of the Australian frog Crinia georgiana. The pressor activity of the synthetic undecapeptide (in rats anesthetized with sodium amytal, followed by treatment with a solution of hexamethonium chloride containing polyvinylpyrrolidone, and vagotomy) was 90.6 +/- 4.99% (n = 26, 7 rats) of that of [Ile5]angiotensin II (human angiotensin II).

Purification and characterization of an aminopeptidase from sperm of the sea urchin, Strongylocentrotus intermedius. Ca2(+)-dependent substrate specificity as a novel feature of the enzyme.

An aminopeptidase showing broad substrate specificity was purified to electrophoretic homogeneity from spermatozoa of the sea urchin, Strongylocentrotus intermedius. It is a single chain protein (Mr = 110,000) with an isoelectric point of 5.2 and shows the highest activity in a pH range between 7.0 and 7.5. Ni2+, Cu2+, Zn2+, and Hg2+, as well as 1,10-phenanthroline and p-chloromercuribenzoate, inhibit the enzyme irrespective of the substrates used, but Ca2+, Mn2+, Mg2+, and Co2+ modified the activity differently depending on the nature of the substrate. The effect of Ca2+ was most marked; it stimulated the activity toward some 4-methylcoumaryl-7-amide (MCA) substrates (for example leucine MCA), whereas it depressed the activity toward some other substrates such as arginine-MCA and lysine-MCA in a competitive manner. The rate of enzymatic hydrolysis determined for a mixture of leucine-MCA and arginine-MCA, in respect to the release of their common product (7-amino-4-methylcoumarin), was in good agreement with the value calculated on the assumption that these two substrates compete with each other for a single active site of the enzyme. Furthermore, the enzyme showed an identical Ki value for each of the competitive inhibitors examined, irrespective of the type of substrate. Ca2+ also influenced the activities toward various peptide substrates in a dual way similar to that observed on the MCA substrates. These results indicate that the sea urchin sperm aminopeptidase has an active site that alters its substrate preference depending on the Ca2+ concentration of the reaction medium.

ATP-dependent proteolysis in yeast mitochondria.

An ATP-dependent proteolysis system in yeast mitochondria was characterized by examining the hydrolysis of mitochondrial translation products in isolated mitochondria. Degradation of [35S]methionine-labeled polypeptides synthesized in isolated yeast mitochondria was activated by exogenously added ATP. ADP, GTP, and CTP substituted for ATP to some extent, but nonhydrolyzable ATP analogues did not. Adenosine-5'-O-(3'-thio-triphosphate) effectively competed with ATP as activator. Carboxyatractyloside, an inhibitor of adenine nucleotide translocation across the mitochondrial inner membrane, and the metal chelator o-phenanthroline inhibited the ATP-dependent proteolysis. The latter inhibition was abolished by subsequent addition of Mn2+ or Co2+ but not Ca2+ or Zn2+. Hemin inhibited the ATP-dependent proteolysis of mitochondrial translation products with a half-maximum inhibition at 12 microM. Analysis by SDS-polyacrylamide gel electrophoresis showed that [35S]methionine-labeled polypeptides were rapidly degraded into low-molecular-weight species. Submitochondrial particles retained the ATP-dependent proteolytic activity and had the same spectrum of inhibitors as intact mitochondria except for a reduced effect of carboxyatractyloside. These results indicate that yeast mitochondria contain an ATP-dependent and hemin-sensitive proteolysis system which is associated with the inner membrane and can hydrolyze mitochondrial translation products, and that a chelator-sensitive protease is probably involved in this system.

Divalent metal ion-dependent mitochondrial degradation of unassembled subunits 2 and 3 of cytochrome c oxidase.

Intracellular protein degradation plays an important role in maintaining the stoichiometry of the different subunits of an oligomeric enzyme. In a Saccharomyces cerevisiae mutant defective in cytochrome c oxidase subunit 4 encoded in nuclear DNA, mitochondrial-encoded subunits 2 and 3 cannot assemble normally [Dowhan et al. (1985) EMBO J. 4, 179-184]. In this study, we show that those unassembled forms of subunits 2 and 3 in this strain are eliminated rapidly by degradation. Reduction of the intracellular ATP level by inhibiting the glycolytic pathway, or inhibition of the entry of ATP into mitochondria by bongkrekic acid, both of which are expected to reduce the intramitochondrial ATP level in respiratory-deficient cells such as WD1, significantly suppressed the degradation, suggesting that the degradation requires intramitochondrial ATP. The degradation was also inhibited by o-phenanthroline, a membrane-permeable metal chelator, and this inhibitory effect was suppressed by addition of an excess amount of Co2+, Mn2+, or Zn2+, but not by Ca2+ or Mg2+, suggesting a novel metal-dependence of the degradation of unassembled Cox II and Cox III which has not been reported previously for mitochondrial metabolic protein degradation systems. A potential advantage of using this strain for identifying the factor(s) involved by a genetical approach is discussed.

Differences between homogeneous spermidine synthases isolated from rat and pig liver.

Spermidine synthase was purified to homogeneity from rat and pig liver by a method modified from a previously reported one using DEAE-Sepharose, S-adenosyl(5')-3-thiopropylamine-Sepharose affinity chromatography, Sephacryl S-300 gel filtration and polyacrylamide gel electrophoresis. No apparent difference between the two enzymes was observed in specific activity, molecular weight (74,000), or subunit composition (two subunits). However, significant differences were observed in their pI values, which were 5.16 for the pig enzyme and 5.34 for the rat enzyme, and their peptide maps. Amino acid compositions of the two enzymes were closely related, but differed significantly in some amino acids. In addition, the rat enzyme was more sensitive to inhibition by S-adenosyl-1,8-diamino-3-thiooctane than the pig enzyme.

Cerulenin resistance in a cerulenin-producing fungus. III. Studies on active-site peptides of fatty acid synthetase from Cephalosporium caerulens.

Active-site peptides of acetyl transferase, condensing enzyme and acyl carrier protein in the neighborhood of the prosthetic group, 4'-phosphopantetheine, of Cephalosporium caerulens fatty acid synthetase were investigated. The enzyme was reacted with [14C]acetyl-CoA or [14C]iodoacetamide. 14C-Labeled enzyme was digested with pepsin, trypsin or both. 14C-Labeled peptides were isolated by several purification procedures. The amino acid sequence of the active site of condensing enzyme was determined to be Tyr-Gln-Val-Glu-Ser-Cys-Pro-Ile-Leu-Glu-Gly-Lys and that of acetyl transferase was Phe-Ser-Gly-Ala-Thr-Gly-His-Ser-Gln-Gly. The amino acid composition around the 4'-phosphopantetheine-carrying serine was determined to be Asx2, Thr, Ser, Glx3, Gly2, Ala, Ile, Leu3, and Lys. When these active-site peptides were compared with those of Saccharomyces cerevisiae synthetase, a high degree of homology was observed in the active-site peptides of the acetyl transferase and acyl carrier protein domains. However, that of the condensing enzyme domain gave lower homology. These findings may support the assumption that the low reactivity of cerulenin with C. caerulens synthetase is a consequence of the structure of the condensing enzyme domain.

Wasp venom peptides; wasp kinins, new cytotrophic peptide families and their physico-chemical properties.

In addition to wasp kinins, the wasp venom contains a series of hydrophobic peptides, mastoparans and chemotactic peptides as major peptidergic components. The first major component in the venom is mastoparam. The peptides in the mastoparan family are tetradecapeptide amides which cause degranulation of the mast cells to release histamine from the cells, and act on the adrenal chromaffin cells to release catecholamines and adenylic acids. Some mastoparans cause hemolysis and serotonin release from the platelets. The new cytotrophic peptides as the second major components are tridecapeptide amides possessing chemotactic activity for polymorphonuclear leucocytes and monocytes. Some of the peptides in this family also cause histamine release from the mast cells. Mastoparan takes a random coil structure in aqueous solution but changes its conformation to alpha-helix in methanolic solution or in the presence of lysophosphatidyl choline. This fact is confirmed also by the transferred nuclear overhauser effect by NMR analysis. The similar phenomenon was observed in the family of chemotactic peptides. The helical conformation of these peptides are amphipathic structure in which all of side chains of the hydrophobic amino acids are located on one side of the axis, and those of the basic or the hydrophilic amino acid residues are on an opposite side. Mastoparan enhances the membrane conductivity of the lipid bilayer when the peptide is investigated by the black lipid membrane experiment. This indicates that the peptide may be assembled in the membrane by changing its conformation and, for some reason, enhances the ion transfer through the membrane. These properties of the peptide may reveal various activities on the cell membrane.

Molecular determinants of binding of a wasp toxin (PMTXs) and its analogs in the Na+ channels proteins.

The structural specificity of alpha-PMTX, a novel peptide toxin derived from wasp venom has been studied on the neuromuscular synapse in the walking leg of the lobster. alpha-PMTX is known to induce repetitive action potentials in the presynaptic axon due to sodium channel inactivation. We synthesized 29 analogs of alpha-PMTX by substituting one or two amino acids and compared threshold concentrations of these mutant toxins for inducing repetitive action potentials. In 13 amino acid residues of alpha-PMTX, Arg-1, Lys-3 and Lys-12 regulate the toxic activity because substitution of these basic amino acid residues with other amino acid residues greatly changed the potency. Determining the structure-activity relationships of PMTXs will help clarifying the molecular mechanism of sodium channel inactivation.

Alpha-pompilidotoxin (alpha-PMTX), a novel neurotoxin from the venom of a solitary wasp, facilitates transmission in the crustacean neuromuscular synapse.

A new neurotoxin, named alpha-pompilidotoxin (alpha-PMTX) has been found in the venom of the solitary wasp Anoplius safnariensis. In the neuromuscular synapse of the lobster walking leg preparation, alpha-PMTX (10-100 micro/M) caused great enhancement of both the excitatory and inhibitory postsynaptic potentials. Recordings of the excitatory post synaptic currents (EPSCs) at the synaptic sites showed that alpha-PMTX reversibly and dose-dependently potentiates EPSCs. Alpha-PMTX may act primarily on the presynaptic membrane but the mode of action of the toxin is clearly different from other known facilitatory neurotoxins, such as alpha-latrotoxin, apamin or charybdotoxin. This novel toxin will serve as a useful tool in the research field of neuroscience.

Multiple cranial arteriovenous malformations in a child with eventual blindness in the affected eye.

PURPOSE: To report a case of multiple cranial arteriovenous malformations involving the orbit and retina. METHOD: Case report. We treated a 7-year-old girl who was diagnosed with a left submaxillary, a left retinal, a left orbital, and a middle subdural arteriovenous malformation. RESULTS: Enlargement of the arteriovenous malformations, except for the retinal arteriovenous malformation, was observed. After external carotid artery embolizations and radiation therapy for uncontrolled oral cavity bleeding, loss of light perception in the affected eye occurred, but no marked changes occurred in the retinal arteriovenous malformation. CONCLUSION: This rare case suggests that the clinical finding of a stable retinal arteriovenous malformation may be associated with enlargement of arteriovenous malformation lesions at other sites.

Corneal endothelial changes in schizophrenic patients with long-term administration of major tranquilizers.

PURPOSE: To examine corneal endothelial changes in schizophrenic patients who underwent long-term administration of major tranquilizers. METHODS: We performed slit-lamp examination and endothelial specular microscopy on 100 eyes of 50 schizophrenic patients (range, 31 to 68 years old; mean, 54 years) who underwent long-term (12 to 44 years) treatment with major tranquilizers. We also studied 50 eyes of 25 patients (range, 31 to 65 years old; mean, 53 years) with no history of corneal disease, as a control group of similar age. Mean cell density, coefficient of variation, and percentage of hexagonal cells were calculated and statistically compared between patients and controls using an unpaired t-test. RESULTS: Slit-lamp examination disclosed pigmentation of the cornea in nine eyes of five patients and pigmentation of the lens in 25 eyes (25%) of 35 patients. Corneal pigmentary changes were seen only in patients with lenticular changes. No eyes showed corneal edema. In contrast, no corneal abnormalities were seen in any control eye. Specular microscopic analysis showed mean cell density of 3,484.4 +/- 462.6 cells/mm2, coefficient of variation of 0.31 +/- 0.06 and percentage of hexagonal cells to be 60.2% +/- 7.5% in the patient group, and 3,291.3 +/- 384.4 cells/mm2, 0.32 +/- 0.07, and 60.6% +/- 7.0%, respectively, in the control subjects. There were no statistically significant differences between patient and control eyes in these three factors. The nine eyes with corneal pigmentation showed no significant differences in these three factors as compared with the control subjects. CONCLUSIONS: These results indicate that long-term treatment with major tranquilizers is not associated with morphometric abnormalities of the corneal endothelium.