Ectopic expression of glucagon-like peptide 1 for gene therapy of type II diabetes.

Glucagon-like peptide 1 (GLP-1) is a promising candidate for the treatment of type II diabetes. However, the short in vivo half-life of GLP-1 has made peptide-based treatments challenging. Gene therapy aimed at achieving continuous GLP-1 expression presents one way to circumvent the rapid turnover of GLP-1. We have created a GLP-1 minigene that can direct the secretion of active GLP-1 (amino acids 7-37). Plasmid and adenoviral expression vectors encoding the 31-amino-acid peptide linked to leader sequences required for secretion of GLP-1 yielded sustained levels of active GLP-1 that were significantly greater than endogenous levels. Systemic administration of expression vectors to animals using two diabetic rodent models, db/db mice and Zucker Diabetic Fatty (ZDF) rats, yielded elevated GLP-1 levels that lowered both the fasting and random-fed hyperglycemia present in these animals. Because the insulinotropic actions of GLP-1 are glucose dependent, no evidence of hypoglycemia was observed. Improved glucose homeostasis was demonstrated by improvements in %HbA1c (glycated hemoglobin) and in glucose tolerance tests. GLP-1-treated animals had higher circulating insulin levels and increased insulin immunostaining of pancreatic sections. GLP-1-treated ZDF rats showed diminished food intake and, in the first few weeks following vector administration, a diminished weight gain. These results demonstrate the feasibility of gene therapy for type II diabetes using GLP-1 expression vectors.

Novel cloning method for recombinant adenovirus construction in Escherichia coli.

pAd(vantage) is a rapid cloning system for generating recombinant adenoviruses. The system is based on manipulating the full-length adenovirus genome as a stable plasmid in E. coli using intron-encoded endonucleases. These intron-encoded endonucleases cut their recognition sequences, which range from 15-39 bp, with high specificity. Their unusual long homing sequence makes them rare-cutting and ideal for use as cloning sites. We report how transgenes can easily be cloned directly into the E1 region of an adenoviral plasmid, followed by transfection into a mammalian packaging cell line, to produce homogeneous recombinant viruses without the need for plaque purification.

Maturation and function of cystic fibrosis transmembrane conductance regulator variants bearing mutations in putative nucleotide-binding domains 1 and 2.

One feature of the mutations thus far found to be associated with the disease cystic fibrosis (CF) is that many of them are clustered within the first nucleotide-binding domain (NBD) of the CF transmembrane conductance regulator (CFTR). We sought to discover the molecular basis for this clustering by introducing into the two NBDs of CFTR mutations either mimicking amino acid changes associated with CF or altering residues within highly conserved motifs. Synthesis and maturation of the mutant CFTR were studied by transient expression in COS cells. The ability of the altered proteins to generate cyclic AMP-stimulated anion efflux was assessed by using 6-methoxy-N-(sulfopropyl) quinolinium (SPQ) fluorescence measurements in HeLa cells expressing mutated plasmids. The results show that (i) all CF-associated mutants, with one exception, lack functional activity as measured in the SPQ assay, (ii) mutations in NBD1 are more sensitive to the effects of the same amino acid change than are the corresponding mutations in NBD2, (iii) cells transfected with plasmids bearing CF-associated mutations commonly but not exclusively lack mature CFTR, (iv) NBD mutants lacking mature CFTR fail to activate Cl- channels, and (v) the glycosylation of CFTR, per se, is not required for CFTR function. We reason that the structure of NBD1 itself or of the surrounding domains renders it particularly sensitive to mutational changes. As a result, most NBD1 mutants, but only a few NBD2 mutants, fail to mature or lack functional activity. These findings are consistent with the observed uneven distribution of CFTR missense mutations between NBD1 and NBD2 of CF patients.

Demonstration that CFTR is a chloride channel by alteration of its anion selectivity.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) generates adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, indicating that CFTR is either a chloride channel or a chloride channel regulator. To distinguish between these possibilities, basic amino acids in the putative transmembrane domains were mutated. The sequence of anion selectivity of cAMP-regulated channels in cells containing either endogenous or recombinant CFTR was bromide greater than chloride greater than iodide greater than fluoride. Mutation of the lysines at positions 95 or 335 to acidic amino acids converted the selectivity sequence to iodide greater than bromide greater than chloride greater than fluoride. These data indicate that CFTR is a cAMP-regulated chloride channel and that lysines 95 and 335 determine anion selectivity.

Viability of Ascaris lumbricoides eggs eliminated after anti-helminthic therapy.

The viability of Ascaris lumbricoides eggs passed in the feces was evaluated after treatment of patients with one of the anti-helminthic drugs (thiabendazole, levamisole, cambendazole, pyrantel pamoate, mebendazole or praziquantel). For each drug, a group of 5 children was selected and their feces collected 24 h before treatment and 24, 48 and 72 h after drug administration, except for mebendazole, with the feces being collected throughout the period of treatment. After sedimentation, the total amount of eggs from each collection was transferred to tissue culture flasks containing 10 ml H2SO4 0,1N, with the addition of 3 drops of a miconazole solution, and incubated at 28 degrees C, individually, for 80 days. The flasks were maintained open and the culture were oxygenated daily by manual agitation. On the 80th day of culture, 20-days-old albino mice were inoculated with 3,200 embryonated eggs, per os. Larvae were recovered from their lungs and hearts, on the 8th day after infection, according to Baerman's method (Morais, 1948). Thiabendazole showed 100.0% ovicidal capacity as early as 48 h after treatment. Inhibition of embryonal development was observed when thiabendazole was used. This drug also had an effect on the eggs infectivity when inoculated into normal mice. No significant effect on embryonal development was observed for the other drugs tested.

Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.

The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered in CF chromosomes and in residues believed to play a role in its function. Examination of the various mutant proteins in COS-7 cells indicated that mature, fully glycosylated CFTR was absent from cells containing delta F508, delta 1507, K464M, F508R, and S5491 cDNA plasmids. Instead, an incompletely glycosylated version of the protein was detected. We propose that the mutant versions of CFTR are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded. Since mutations with this phenotype represent at least 70% of known CF chromosomes, we argue that the molecular basis of most cystic fibrosis is the absence of mature CFTR at the correct cellular location.

Cone visual cell degeneration in ground squirrel retina: disruption of morphology and cyclic nucleotide metabolism by lodoacetic acid.

Photoreceptor degeneration was induced in the cone-dominant retina of the ground squirrel by intracardiac injection of iodoacetic acid. Morphology was examined and cyclic nucleotide levels were determined in retinas taken at various times between 35 min and 11 days after injection. Degenerating cone cells were first detected at day 1 and all cone cells were reduced to compact dense masses by day 4. Cellular debris was removed by macrophages that entered the photoreceptor layer from the inner neuroretina. Cyclic AMP levels of dark-adapted retinas were doubled 24 hrs after injection and were reduced to approximately 50% of the dark-adapted level of control retina between days 1 and 3. The concentration of cyclic GMP was 3 to 4 times higher than normal at 4 to 5 hrs postinjection, dropped to 9% of normal at day 4, and was 2% at day 11. Since these changes were coincident with the loss of morphologic integrity of cone cells, they imply that at least 50% of cyclic AMP and most of the cyclic GMP in the cone-dominant retina of the ground squirrel is present in cone cells. The elevation of cyclic AMP and cyclic GMP levels prior to pathological morphology suggests that the iodoacetic acid-induced disruption of cyclic nucleotide metabolism may be associated with the degeneration of the cone photoreceptors.

Cyclic nucleotides of cone-dominant retinas. Reduction of cyclic AMP levels by light and by cone degeneration.

Dark-adapted retinas or whole eyes of 13-line ground squirrels (Citellus tridecemlineatus) and western fence lizards (Sceloporus occidentalis) contain higher levels of cyclic AMP than of cyclic GMP. In these cone-dominant retinas, light reduces cyclic AMP content selectively. Freezing of dark- or light-adapted retinas or eyes also reduces cyclic AMP content, with only minimal changes in cyclic GMP levels. In addition, exposure of frozen retinas of dark-adapted ground squirrel to light results in a significant decrease in cyclic AMP content. The destruction of cone visual cells of ground squirrel retina by iodoacetic acid injection decreases the cyclic nucleotide content of the dark-adapted retina. Considering the relative loss of cyclic nucleotides from cone degeneration, we estimate that the content of cyclic AMP in visual cells of ground squirrel retina is about four times greater than that of cyclic GMP.

Therapeutic efficacy of oral oxamniquine in the toxemic form of schistosomiasis mansoni: treatment of eleven individuals from two families, and experimental study.

Two families, comprising 11 individuals in the toxemic form of schistosomiasis mansoni, infected in Belo Horizonte, Brazil were treated. Parasitological cure was obtained in 5 (45%) of the patients after a single oral dose of oxamniquine (Mansil), 20 mg/kg body weight. No significant side effects were observed. To evaluate the possibility of resistance to the drug, cercariae collected from Biomphalaria glabrata infected with micracidia from eggs obtained from three of the individuals not cured were studied. Mice infected with these three strains were cured after a single dose of examniquine. It is suggested that research be continued with other therapeutic schedules and perhaps other, more potent, drugs.

Effect of X-irradiation on vitamin B12 binding capacity to intrinsic factor.

The effects of whole-body X-irradiation on vitamin B12-protein complex formation in gastric juice after oral administration of [57Co]-B12 have been studied. Two proteins with B12-binding activity have been isolated by gel filtration from gastric juice. 57Co-activity, recovered from B12-protein complex in gastric juice, is found to be about 30% less in the X-irradiated rat. In serum, vitamin B12 is mainly associated with alpha1-globulin. Radioactivity distribution in serum globulins after intraperitoneal injection of [57Co]-B12 was similar in control and X-irradiated rats.

Vitamin B12 absorption in x-irradiated rats.

The effect of whole-body exposure of rats to a sub-lethal dose (400 rad) of x-rays on the absorptive capacity of intestinal mucosal cells for vitamin B12 has been studied. The rate of absorption of vitamin B12 from the intestinal loops is decreased in x-irradiated rat. Inclusion of gastric juice from normal rat does not improve the rate. A severe interference in the absorption and retention of orally fed (57-Co)-B12, as evidenced by low serum levels, decreased organ uptake and increased excretion, is observed. However, when the vitamin is administered intraperitoneally, its uptake by organs is not affected in the irradiated animal. This suggests that the observed morphological degeneration of mucosal cells in x-irradiated rats is the main reason for the malabsorption of vitamin B12. Atrophy of intestinal cells in the protein-fasted rat accentuates the adverse effects of radiation. A sharp drop in viable intestinal flora is observed within 24 h post-irradiation, but there is an increase after 3 days.