Isolation and characterization of a viable adenovirus mutant defective in nuclear transport of the DNA-binding protein.

The isolation and characterization of an adenovirus mutant, Ad5dl802r1, containing two independent deletions in the 72-kilodalton (kDa) DNA-binding protein (DBP) gene is described. The two deletions remove amino acids 23 through 105 of DBP, resulting in the production of a 50-kDa product. Expression of this truncated DBP was delayed 12 to 24 h compared with that of the 72-kDa protein produced by wild-type adenovirus type 5. The DBP was located primarily in the cytoplasm of infected cells, whereas the wild-type product was predominantly nuclear. Therefore, DBP appears to contain a nuclear localization signal within the deleted region. Ad5dl802r1 DNA synthesis, viral late gene expression, and virus production were all delayed 12 to 24 h and were approximately 10-fold lower than with wild-type adenovirus type 5. These phenotypic properties can be accounted for by the delay in synthesis and the inefficient accumulation of the 50-kDa DBP within the nucleus of infected cells. The truncated DBP also lacks the majority of amino acids which are phosphorylated in the normal protein. The loss of these phosphorylation sites does not appear to seriously impair the ability of the protein to carry out its functions.     

AAT1, a gene encoding a mitochondrial aspartate aminotransferase in Saccharomyces cerevisiae.

We have isolated a gene, AAT1, encoding an aspartate aminotransferase (AspAT) from a Saccharomyces cerevisiae genomic library. AAT1 encodes a 451 amino acid protein with a predicted molecular weight of 51,687, which is likely to be the yeast mitochondrial AspAT. Sequence comparison of this yeast AspAT with AspATs from other organisms shows a high degree of homology in regions previously shown to be important for catalysis. However, the yeast mitochondrial AspAT contains four obvious insertions with respect to all other known AspATs, suggesting that the AAT1-encoded protein represents a distinct AspAT.     

Clinical, metabolic, and genetic aspects of cytochrome C oxidase deficiency in Saguenay-Lac-Saint-Jean.

Thirty-four children with lactic acidosis and Leigh encephalopathy due to cytochrome C oxidase (COX) deficiency distributed in 28 families have recently been identified in northeastern Quebec, particularly in the Saguenay-Lac-Saint-Jean (SLSJ) region. The segregation analysis was consistent with an autosomal recessive mode of inheritance. The incidence was estimated at 1/2,063 live births between 1979 and 1990, and the carrier rate was estimated at 1/23 inhabitants in SLSJ. In SLSJ, the places of origin of the COX-deficient children and their parents did not show a clustered nonuniform distribution. The genealogical reconstruction of 54 obligate carriers identified 26 ancestors common to all of them. Twenty-two were 17th-century Europeans, suggesting that the COX-deficient gene was introduced in the French-Canadian population by early settlers. These results support the hypothesis of a founder effect for COX deficiency in northeastern Quebec. Clinical findings are reported for 15 of these COX-deficient patients, age 6 mo to 11 years. Moderate developmental delay, hypotonia, ataxia, strabismus, and mild facial dysmorphism were frequent. Eleven children died in episodes of fulminant metabolic acidosis. The patients had elevated blood and cerebrospinal fluid lactate levels, decreased blood bicarbonate levels, and normal blood pH. Leigh disease and microvesicular steatosis of the liver were present in all affected patients for whom postmortem examination was performed. This biochemically uniform group of patients showed a wide range of clinical severity.     

Modulation of glycolysis induction in primary cultures of rabbit kidney proximal tubule cells: the role of shaking,glucose and insulin.

We have assessed the impact of increasing oxygen availability on cellular phenotype expression of rabbit proximal tubule cells in primary culture developed with variable glucose and/or insulin contents. To mitigate hypoxia at the cell/medium interface, cells were shaken for the whole culture duration and their expressed phenotype was compared with those expressed by static cultures. O₂ and CO₂ tensions were kept constant in the incubator atmosphere. Glycolysis and gluconeogenesis pathways, detoxication system, and mitochondrial, apical and basolateral membrane marker enzyme activities were assessed. This study showed that the induction of glycolysis which appear in primary cultures of proximal tubule cells may be partially prevented by continuously shaking the cultures. This effect was more marked in the presence of glucose, suggesting better substrate oxidation in shaken cultures.     

Antibody-forming cell response to virus challenge in mice immunized with DNA encoding the influenza virus hemagglutinin.

Immunization of mice with DNA encoding the influenza virus hemagglutinin (HA) affords complete protection against lethal influenza virus infection and the means to investigate the mechanisms of B-cell responsiveness to virus challenge. Using a single-cell enzyme-linked immunospot assay, we sought to determine the localization of HA-specific antibody-forming cells (AFCs) during the development of humoral immunity in mice given HA DNA vaccine by gene gun. At 33 days postvaccination, populations of AFCs were maintained in the spleen and bone marrow. In response to lethal challenge with influenza virus, the AFCs became localized at the site of antigenic challenge, i.e., within the draining lymph nodes of the lung compartment. Immunoglobulin G (IgG)- and IgA-producing AFCs were detected in lymph nodes of the upper and lower respiratory tracts, underscoring their importance in clearing virus from the lungs. Response to challenge required competent CD4+ T cells, without which no AFCs were generated, even those producing IgM. By contrast, in mice vaccinated with an HA-containing subunit vaccine, fewer AFCs were generated in response to challenge, and these animals were less capable of resisting infection. Our findings demonstrate the comparable localization of AFCs in response to challenge in mice vaccinated with either HA DNA or live virus. Moreover, the former strategy generates both IgG- and IgA-producing plasma cells.     

Conditions used with a continuous cultivation system to screen for d-hydantoinase-producing microorganisms

The continuous cultivation technique has been used to screen for microorganisms producing d-hydantoinase, a biocatalyst involved in the production of optically active amino acids. Pseudomonas putida strain DSM 84 was used as a model hydantoinase producer to establish selective culture conditions through the addition of various pyrimidines, dihydropyrimidines, hydantoins and 5-monosubstituted hydantoins. Thymine induced more activity than all cyclic amides tested. Addition of thymine as a non-metabolised inducer at a concentration of 0.05 g l^–1 in a continuous culture of P. putida stimulated hydantoinase production up to 80 times the basal level. Using continuous culture conditions established with the model strain, a different strain of P. putida having hydantoinase activity was isolated from commercial mixed cultures of microorganisms. DNA fingerprinting revealed that this new isolate was distinct from strain DSM 84. When used as a probe, the d-hydantoinase gene of strain DSM 84 hybridized with the DNA of the new P. putida isolate.     

Transgenic and knockout rodents: Novel insights into mechanisms of body weight regulation

Transgenic animals that over- or underexpress a protein of interest have been used to study obesity development, prevention, and susceptibility to diet-induced obesity such as a high-fat diet. Several transgenic models are resistant to diet-induced obesity including those that overexpress the insulin-sensitive glucose transporter, GLUT4, in adipose tissue only. In this animal there is increased adipose tissue mass but the animal maintains its insulin sensitivity. The overexpression of lipoprotein lipase (LPL) in skeletal muscle and the elimination of a protein kinase A subunit both resulted in lean and obesity resistant animals. By directing the production of the diphtheria toxin A chain to adipose tissue only the resulting animals not only had less adipose tissue mass but were resistant to MSG-induced obesity. Conversely, transgenic models with decreased brown adipose tissue or its function have all resulted in obese animals, highlighting the importance of thermoregulation in body weight maintenance. The use of transgenic technology in the field of obesity has emphasized the regional differences among fat pads as well as the dissimilarity between genders in fuel metabolism. Several transgenic models have separated obesity from insulin resistance allowing the importance of each state to be studied individually. Results using transgenic animals have re-emphasized that obesity is a polygenic disease.     

Vasomotor instability preceding tilt-induced syncope: does respiration play a role?

Lipsitz, Lewis A., Raymond Morin, Margaret Gagnon, DanKiely, and Aharon Medina. Vasomotor instability precedingtilt-induced syncope: does respiration play a role? J. Appl. Physiol. 83(2): 383-390, 1997.This studyaimed to determine whether alterations in cardiovascular dynamicsbefore syncope are related to changes in spontaneous respiration.Fifty-two healthy subjects underwent continuous heart rate (HR),arterial blood pressure (BP), and respiratory measurements during10-min periods of spontaneous and paced breathing (0.25 Hz) in thesupine and 60° head-up tilt positions. Data were evaluated by powerspectrum and transfer function analyses. During tilt, 27 subjectsdeveloped syncope or presyncope and 25 remained asymptomatic. Subjectswith tilt-induced syncope had significantly greater increases inlow-frequency (0.04-0.15 Hz) systolic BP, diastolic BP, and HRpower during tilt than the asymptomatic subjects(P  0.01). This difference waspresent during spontaneous but not paced breathing. However, averagetidal volume, respiratory rate, minute ventilation, proportion ofbreaths below 0.15 Hz, and low-frequency respiratory power during tilt did not differ between syncopal and nonsyncopal subjects. Transfer magnitudes between low-frequency respiration and BP, and between BP andinterbeat interval, were also similar between groups. Thus vasomotorinstability before syncope is not related to alterations in respirationor the cardiovagal baroreflex but may reflect oscillating centralsympathetic outflow to the vasculature.

     

Incorporation of radioactive proline and fucose in gastric mucosal biopsies. Correlation with histological findings

Human gastric mucosal biopsies incorporate in vitro radioactive proline and fucose into macromolecular glycoproteins (mucin). Differences were found between the incorporation pattern of antral and fundic mucosae according to their pathology, confirmed by histology. Antral mucosae with abnormal histology showed a significantly higher incorporation of proline than normal samples. Fucose incorporation was also increased. Similar results were found with fundic biopsies. The ratio of total fucose to total proline incorporated into mucin secreted during incubation also increased significantly in these samples. Biochemical analyses on the other hand showed no significant change. The results suggest a breakdown in the processing, storage and secretory processes of mucin-type glycoproteins in pathological mucosae, but not in their biosynthesis. The mucosal samples could be classified as A or B types according to their proline and fucose incorporation: A mucosae have a lower proline incorporation than B mucosae (greater than 380 cpm/micrograms DNA for the antrum and greater than 200 cpm/micrograms DNA for the fundus). These results confirm the possibility of studying abnormal mucus secretion using gastric biopsy samples.