Gene transfer to the rhesus monkey brain using SV40-derived vectors is durable and safe.

Gene transfer to central nervous system (CNS) has been approached using various vectors. Recombinant SV40-derived vectors (rSV40s) transduce human neurons and microglia effectively in vitro and in rodent brains in vivo, so we tested rSV40s gene transfer to rhesus monkey CNS in vivo, to characterize the distribution, duration and safety of such gene delivery. We used rSV40s carrying HIV-1 RevM10 with a carboxyl-terminal AU1 epitope tag as a marker, and others with the antioxidant enzymes, Cu/Zn superoxide dismutase and glutathione peroxidase. Vectors were injected stereotaxically into the caudate nucleus. Transgene expression was studied at 1 and 6 months by immunostaining serial brain sections. After intraparenchymal administration, numerous transgene-expressing cells were seen, with a longitudinal extent of 20 mm. In neurons and, more rarely, microglial cells, transgene expression remained strong throughout the 6-month study period. Astrocytes and oligodendroglia were not transduced. No evidence of inflammation or tissue damage was observed. SV40-derived vectors may thus be useful for long-term gene expression in the monkey brain and, potentially, in the human brain.

Fluctuations during growth of the plasma membrane H(+)-ATPase activity of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The plasma membrane H(+)-ATPase activity was determined under various growth conditions using the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Under early batch-growth conditions in a rich medium, the budding yeast S. cerevisiae ATPase specific activity increased 2- to 3-fold during exponential growth. During late exponential growth, a peak of ATPase activity, followed by a sudden decrease, was observed and termed "growth-arrest control". The growth arrest phenomenon of S. cerevisiae could not be related to the acidification of the culture medium or to glucose exhaustion in the medium or to variation of glucose activation of the H(+)-ATPase. Addition of ammonium to a proline minimum medium also stimulated transiently the ATPase activity of S. cerevisiae. Specific activity of the fission yeast S. pombe ATPase did not show a similar profile and steadily increased to reach a plateau in stationary growth. Under synchronous mitotic growth conditions, the ATPase activity of S. cerevisiae increased during the cell division cycle according to the "peak" type cycle, while that of S. pombe was of the "step" type.

Alcohol acetyltransferases and the significance of ester synthesis in yeast.

This paper reviews our current knowledge of yeast alcohol acyltransferases. Much of this information has been gathered over the past 10 years through the application of powerful yeast molecular biology techniques. Evidence from gene disruption and expression analysis of members of the alcohol acyltransferase (ATF) gene family indicates that different ester synthases are involved in the synthesis of esters during alcoholic fermentation. The natural physiological rationale behind these enzyme activities remains unclear. However, it is believed that these enzymes may be involved in very different functions, including cellular fatty acid homeostasis and detoxification mechanisms. Insights into the regulation of yeast ester synthesis by oxygen and unsaturated fatty acids have contributed to our understanding of the general mechanisms of gene regulation. In particular, control mechanisms that underpin the oxygen-mediated regulation of ATF1 gene transcription appear to be closely linked to those involved in the regulation of fatty acid metabolism. Data pertaining to the regulation of ATF1 gene transcription have been integrated into a working model for future research.

Osteopenia, bone fragility and reflex sympathetic dystrophy syndrome in a man with ureterosigmoidostomy.

A 68-year-old man is presented with a reflex sympathetic dystrophy syndrome (RSDS) of the right ankle diagnosed by radiography, magnetic resonance imaging and bone scintiscan. Investigations, including blood tests and bone biopsy, revealed a diagnosis of metabolic acidosis and osteomalacia. These appeared to result from a ureterosigmoidostomy performed 9 years previously for a transitional carcinoma of the bladder. Correction of the metabolic acidosis coincided with improvement in ankle pain. RSDS may be the initial presentation of osteomalacia, which in turn may be caused by the metabolic acidosis resulting from a ureterosigmoidostomy.

Amino acid replacements at seven different histidines in the yeast plasma membrane H(+)-ATPase reveal critical positions at His285 and His701.

The plasma membrane H(+)-ATPase (Pmal) from Saccharomyces cerevisiae contains 14 histidine residues. The seven most highly conserved of these were selected as targets for oligonucleotide-directed, site-specific mutagenesis. Substitutions at H240, H488, H614, H686, and H914 with a variety of amino acid residues had no effect either on cell viability or on temperature and pH growth sensitivity. In contrast, substitutions at H701, located in the putative fifth membrane-spanning region, with Asp, Gln, or Arg were dominant lethal, indicating that H701 is essential for H(+)-ATPase activity. The mutations H285Q and H285R, but not H285E, located in the hydrophilic beta-stranded domain, were tolerated in normal growth conditions. Growth of H285Q mutants was sensitive to acid pH, indicating impaired in vivo ATPase activity. The H285Q and H285R mutants showed increased in vitro ATPase-specific activity, increased vanadate resistance, increased proton competition of vanadate sensitivity, accelerated ATP hydrolysis rates at a substrate concentration much lower than the Km, and slightly uncoupled proton pumping. The most reasonable hypothesis which would take into account these observations is that H285 would not be involved in the H+ transport process but rather in the E2 to E1 transition step of the ATP hydrolysis catalytic cycle.

Functional complementation of a null mutation of the yeast Saccharomyces cerevisiae plasma membrane H(+)-ATPase by a plant H(+)-ATPase gene.

In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The presence within an organ of several isoforms prevents a detailed enzymatic characterization of individual H(+)-ATPases. We therefore used the yeast Saccharomyces cerevisiae as a heterologous host for the expression of PMA2, an H(+)-ATPase isoform of Nicotiana plumbaginifolia. Yeast transformed by the plant pma2 was still able to grow under conditions where the yeast ATPase gene (PMA1) was either repressed or deleted. The transformed yeast strain was resistant to hygromycin, and its growth was prevented when the medium pH was lowered to 5.0. The N. plumbaginifolia PMA2 expressed in S. cerevisiae has unusual low Km for ATP (23 microM) and high pH optimum (6.8). Electron microscopic examination revealed PMA2 in internal structures of the karmellae type which proliferated when cell growth was arrested, either at a nonpermissive pH or at the stationary phase in a minimal medium. Under the latter conditions, subcellular fractionation on sucrose gradients revealed, in addition to the expected plant PMA2 peak linked to the plasma membrane fraction, low density peak containing PMA2 and KAR2, an endoplasmic reticulum marker. These observations suggest that the partial internal accumulation of PMA2 occurs in membranes derived from the endoplasmic reticulum and largely depends on growth conditions.

[The self-aspirating syringe in local anesthesia]. / La seringue auto-aspirante en anesthésie locale.

Dentists routinely inject local anesthetic solutions in their daily practice. However, it is important not to inject intravascularly. Consequently, manufacturers offer two types of dental syringes: one with a mechanical aspirating device (harpoon) and the other with a self-aspirating mechanism. We have verified the efficacy of a self-aspirating syringe as presented on the market and another self-aspirating syringe which we lightly modified.

Short-chain and medium-chain aliphatic-ester synthesis in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the enzymes which catalyse the synthesis of ethyl acetate, ethyl n-hexanoate and isoamyl acetate were partly resolved from a fraction containing slowly sedimenting lipoproteins released during cell disruption with glass beads. Solubilization with detergents and fractionation by affinity chromatography have demonstrated the presence of at least three, and probably four, ester synthases which differ in their catalytic properties. Isoamyl-acetate synthase was solubilized and extensively purified to apparent homogeneity by successive chromatographies on various columns. On the basis of its specific activity in cell-free extracts, the enzyme was purified 19,000-fold with a 5% activity yield. As judged by SDS/PAGE, it consists of a single polypeptide chain with a molecular mass of 57 +/- 3 kDa and its apparent pI is 5.5. The enzyme acetylates isoamyl alcohol, ethanol and 12-DL-hydroxystearic acid from acetyl-CoA but is unable to use n-hexanoyl-CoA as a cosubstrate. This enzyme, defined as an acetyl-CoA: O-alcohol acetyltransferase, could be the product of one of the anaerobically induced genes in S. cerevisiae.

Purification and complete sequence of a small proteolipid associated with the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

The purified plasma membrane H(+)-ATPase of Schizosaccharomyces pombe and Saccharomyces cerevisiae display, in addition to the catalytic subunit of 100 kDa, a highly mobile component, soluble in chloroform/methanol. Chloroform/methanol extraction of S. cerevisiae plasma membranes led to isolation of a low molecular weight proteolipid identical to that present in purified H(+)-ATPase. NH2-terminal amino acid sequencing revealed a 38-residue polypeptide with a calculated molecular mass of 4250 Da. The polypeptide lacks the first two NH2-terminal amino acids as compared with the deduced sequence of the PMP1 gene (for plasma membrane proteolipid) isolated by hybridization with an oligonucleotide probe corresponding to an internal amino acid sequence of the proteolipid. The polypeptide is predicted to contain an NH2-terminal transmembrane segment followed by a very basic hydrophilic domain.

The yeast plasma membrane H(+)-ATPase. An essential change of conformation triggered by H+.

The plasma membrane of Schizosaccharomyces pombe contains an H(+)-ATPase similar to the cation transport ATPases of other eukaryotic organisms. The fluorescence excitation and emission spectra of the purified H(+)-ATPase are characteristic of tryptophan residues. pH reduction from 7.5 to 5.7 produces a 4% decrease in fluorescence intensity, while a further reduction to pH 5.0 leads to an increase of fluorescence. A close correlation is observed between the pH dependence of the intrinsic fluorescence and the pH dependence of (i) ATPase activity, (ii) the fluorescence of Tb-formycin triphosphate bound to the active site, and (iii) inhibition by vanadate of ATPase activity. It is proposed that the effect of pH on intrinsic fluorescence reveals the existence of an H+ induced conformational change of the H(+)-ATPase similar to the E1----E2 transition of the other plasma membrane cation transport ATPases.

[Nonfunctioning nesidioblastoma of the pancreas in children. Apropos of a case]. / Le nésidioblastome non sécrétant du pancréas de l'enfant. A propos d'une observation.

A new case of nonfunctional nesidioblastoma in a 13-year-old girl is reported. The diagnosis was made with imaging, including radiographs of the abdomen, ultrasound and computed tomography. Complete resection of the pancreatic tumor was possible. The patient has no symptoms 8 months later.

[Loss of digital substance in rheumatic diseases. Apropos of 43 cases]. / Pertes de substances digitales dans les maladies rhumatismales. A propos de 43 observations.

The authors briefly summarize 16 cases involving digital necrosis observed in the course of rheumatic disorders (3 carpal tunnel syndromes, 2 cases of panarteritis, 2 of rheumatoid arthritis, 2 of lupus erythematosus, 2 of scleroderma, 2 of dermatomyositis, 1 of adult form of Still's disease, 1 of gout, and 1 case of cryoglobulinemia). In addition the case reports of 129 patients with connective tissue disease were reviewed (74 cases of disseminated lupus erythematosus, 43 of scleroderma, and 12 mixed connective tissue disorders). Soft tissue destruction of digits was observed in 15, 39 and 25% of these patients respectively, and true tissue necrosis of varying extent in 7, 9 and 0% of these cases. The incidence and pathogenic mechanisms responsible for these disorders and lesions were discussed.

New modes of administration of salmon calcitonin in Paget's disease. Nasal spray and suppository.

In volunteers the activity of various doses of a nasal spray and of a suppository of salmon calcitonin was compared to a placebo and to the parenteral route of administration. Both new modes of administration were found to be active on the kidney (and the suppository was found to affect bone turnover as well). The parenteral route proved more effective, but the nasal and/or rectal routes were devoid of systemic side effects and had minimal local intolerance. The nasal spray was used at 200 units daily in 15 patients with Paget's disease, and at 400 units daily in another nine patients, both trials lasting one year. The two regimens proved active on the parameters of bone turnover and the higher dose was more effective than the lower one. Similarly, a 300 unit suppository was given to another 12 patients. This trial is still being completed at this time. At the third month of therapy, the parameters of bone turnover were significantly depressed. Both new modes of therapy were able to improve the focal bone balance of the osteolytic lesions monitored on sequential roentgenograms. Systemic side effects were absent and local side effects were minimal. Only one patient interrupted the nasal spray therapy, and no one interrupted the suppository therapy.

Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex.

The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H2O by D2O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex.

A single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe.

A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma-32P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors (Ulaszewski, S., Coddington, A., and Goffeau, A. (1986) Curr. Genet. 10, 359-364).

Cutaneous mucinosis associated with multiple frozen joints and bony heterotopic deposits around the hips.

A 43-year-old man suffered from migrating then additive arthralgias associated with macroscopic and microscopic cutaneous lesions compatible at first sight with reticular erythematous mucinosis. After nine months, the cutaneous picture evolved into a papular mucinosis. The patient developed "multiple frozen joints" with heterotopic bony deposits around the hips. To our knowledge, such articular problems have not yet been described in this condition. Severe systemic complications prompted the authors to treat the patient with plasma exchange and melphalan.

Decrease of the plasma membrane H+-ATPase activity during late exponential growth of Saccharomyces cerevisiae.

During the last cell division of exponential growth, the H+-ATPase activity from the yeast plasma membrane decreases by a factor of two to three. This "arrest growth control" of ATPase activity is not accompanied by modification of the sensitivity to vanadate.