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PubMed2002

Expression and purification of aspartate beta-semialdehyde dehydrogenase from infectious microorganisms.

l-Aspartate-beta-semialdehyde dehydrogenase (ASA DH) lies at the first branch point in the aspartate metabolic pathway that leads to the formation of the amino acids lysine, isoleucine, methionine, and threonine in most plants, bacteria, and fungi. Since the aspartate pathway is not found in humans, but is necessary for bacterial cell wall biosynthesis, the enzymes in this pathway are potential targets for the development of new antibiotics. The asd gene that encodes for ASA DH has been obtained from several infectious organisms and ligated into a pET expression vector. ASA DHs from Haemophilus influenza, Pseudomonas aeruginosa, and Vibrio cholerae were expressed as soluble proteins in Escherichia coli, while ASA DH from Helicobacter pylori was obtained primarily as inclusion bodies. The V. cholerae genome contains two asd genes. Both enzymes have been expressed and purified, and each displays significant ASA DH activity. The purification of highly active ASA DH from each of these organisms has been achieved for the first time, in greater than 95% purity and high overall yield. Kinetic parameters have been determined for each purified enzyme, and the values have been compared to those of E. coli ASA DH.

Animals↗
PubMed2001

Hepatitis C virus structural proteins reside in the endoplasmic reticulum as well as in the intermediate compartment/cis-Golgi complex region of stably transfected cells.

The intracellular localization of hepatitis C virus structural proteins was analyzed by confocal immunofluorescence microscopy, cell fractionation, and immunoelectron microscopy in stably transfected cells that do not overexpress the viral proteins. The results strongly suggest that at steady state the structural proteins reside not only in the endoplasmic reticulum but also in the intermediate compartment and cis-Golgi complex region. By analogy with other viral systems, this finding raises the possibility that the intermediate compartment and cis-Golgi complex play a role in the assembly and budding of hepatitis C virus.

Calcium-Binding Proteins↗
PubMed1992

Phospholipid metabolism in rat intestinal mucosa after oral administration of lysophospholipids.

The present results indicate that PS, a phospholipid contained in small amounts in the human diet and not included in plasma lipoproteins, may be used to influence phospholipid metabolism in intestinal mucosal cells. Since PS influx into absorptive cells occurs after its hydrolysis to lysoPS, this metabolite may be used to increase the absorption of this phospholipid. These data show that lysoPS, after diffusion into intestinal cells, is sequentially converted into PS and PE (which make up a minor fraction of the lipids present in lipoproteins). As expected, lysoPS given together with radiolabeled unsaturated fatty acids was unable to promote their transfer into plasma lipoproteins. In this respect lysoPS differed from lysoPC, the latter increasing the appearance of dietary fatty acids in plasma. When given together, lysoPS decreased the lysoPC-induced transfer of unsaturated fatty acids into plasma. This effect required addition of triglycerides to the lipid mixture. In attempting to explain this triacylglycerol-dependent inhibition by lysoPS, we found that this phospholipid increased the incorporation of glycerol into mucosal cell PC. In contrast, lysoPC was inhibitory. Furthermore, in the presence of labeled inositol, lysoPC (but not lysoPS) promoted the appearance of labeled phosphatidylinositol. The data thus suggest that the two lysophospholipids differ in promoting the two main pathways of PC synthesis in the intestinal cells. While lysoPC favors PC synthesis by reacylation, lysoPS enhances the CDP-choline pathway of PC synthesis.

Administration, Oral↗
PubMed1992

TEM cytochemical study of the localization of phospholipids in interphase chromatin in rat hepatocytes.

The electron microscopy cytochemical detection of phospholipids in well-defined areas in the interphase nuclei of hepatocytes has been obtained by the acid haematein test, modified for electron microscopy and by the phospholipase A2-colloidal gold method. The specificity of both methods were controlled by enzymatic digestion with phospholipase. The main intra-nuclear localization of phospholipids is at the border between the condensed and dispersed chromatin, where non-ribosomal RNA is also revealed by RNase-gold labelling. Phospholipids are detected, too, over the clusters of interchromatin granules and in the fibrillar component of the nucleolus.

Animals↗
PubMed1995

Spinal mobility and posture: changes during growth with postural defects, structural scoliosis and spinal osteochondrosis.

A number of case studies were followed for 10 years. Children suffering from postural defects, structural scoliosis and spinal osteochondrosis were separated from a normal group. Spinal mobility and posture were compared with the mobility of normal, healthy boys and girls aged 5-14 years. In postural defects curves similar to the physiologic one were observed. The range of motion was not physiologic in scoliosis. In this disease, rotation-flexion thoracic hypermobility with decreased lumbar flexion ability was found. A physiologic trend was found in spinal osteochondrosis, although sometimes the rotational mobility of the spine was reduced. An extremely high increase of thoracic kyphosis was found only in children aged 14 years.

Adolescent↗
PubMed1995

A kluyveromyces lactis gene homologue to AAC2 complements the Saccaromyces cerevisiae op1 mutation.

A mutation (op1) in the Saccharomyces cerevisiae AAC2 gene, which codes for the most abundant ADP/ATP carrier isoform, results in lack of mitochondrial-dependent growth and in an as yet unexplained petite-negative phenotype. A gene from the petite-negative yeast Kluyveromyces lactis has been isolated by complementing in multicopy the op1 mutation of S. cerevisiae. This gene, designated KIAAC, can complement the petite-negative phenotype of op1 as well as its inability to grow on nonfermentable carbon sources. KIAAC contains a 915-base pair open reading frame coding for a protein of 305 amino acids which shows a high degree of identity to AAC2. The K. lactis ADP/ATP carrier also shares identity with other known ADP/ATP carrier sequences. In particular, the degree of identity of KIAAC is higher with the Neurospora crassa carrier (80.1%) than with AAC1 (76.6%). The nucleotide sequence upstream of the KIAAC coding region was found to contain a long DNA segment with no coding potential, but presenting features of highly regulated promoter sequences.

Amino Acid Sequence↗
PubMed1982

Application of the Salmonella mutagenicity assay and determination of polycyclic aromatic hydrocarbons in workplaces exposed to petroleum pitch and petroleum coke.

Workplaces of an Italian carbon electrode factory, exposed to petroleum pitch and petroleum coke, were studied using a coupled chemical and biological approach to evaluate occupational mutagenic/carcinogenic hazards. Analytical procedures for the determination of polycyclic aromatic hydrocarbons (PAH) and Salmonella/microsome mutagenicity tests (with TA98 and TA100 strains) were performed on both industrial ingredients (pitch and coke) and airborne particulate matter of the working environment, after fractionating by sequential Soxhlet extractions with four organic solvents of increasing polarity (benzene, chloroform, methanol and acetone). The results showed: (a) the presence of extraordinarily high PAH (carcinogenic and non-carcinogenic) contents in the benzene extracts of petroleum pitch (3.6 wt% of total PAH) and of airborne particulate samples (up to 0.35 wt% of total PAH), in correlation with very high indirect (after metabolic activation) mutagenic responses of benzene extracts with strain TA98; (b) very high indirect mutagenic responses in the other extracts of the airborne particulate samples (especially with strain TA98); (c) the production during the processing at high temperatures of directly acting mutagens (without metabolic activation) which were absent in the starting materials and their release in the air of workplaces. The comparison of chemical analytical and mutagenicity data has proved to be an interesting approach for better defining the relative health hazards due to occupational exposure to potentially mutagenic/carcinogenic petroleum products.

Air Pollutants↗
PubMed1984

Mutagenic/carcinogenic hazards in a cold-rolling steel plant exposed to mineral oils: environmental monitoring phase.

A survey was carried out in a cold-rolling steel plant exposed to mineral oils to study the mutagenic/carcinogenic hazards, following a coupled environmental/biological monitoring. The present paper deals only with the environmental phase by determining polycyclic aromatic hydrocarbons (PAH) and applying a mutagenicity test (Ames test), and includes the following steps: (a) work process and environment study; (b) sampling and analysing mineral oils before and after use (PAH determination and mutagenicity analysis); (c) sampling and analysing oil mist (TWA determination, PAH determination and mutagenicity analysis). The results showed that: (1) both unused and used (recycled) mineral oils contained only trace amounts of PAH and were not mutagenic; (2) the TWA concentrations of oil mists were lower than the TLV (less than 5 mg/m3); (3) oil mists contained only trace amounts of PAH and were not mutagenic. The authors suggest that these results could be due to the moderate temperature of the oil during the work process and that there is a relationship between low PAH content and absence of mutagenicity in the oils and air samples.

Air Pollutants, Occupational↗
PubMed1981

Induction by glucose of an antimycin-insensitive, azide-sensitive respiration in the yeast Kluyveromyces lactis.

Increasing the glucose concentration from 0.1 to 10% in exponentially growing cultures of Kluyveromyces lactis CBS 2359 does not repress the antimycin-sensitive respiration (QO2 of 80 microliter O2 . h-1 . mg-1 dry weight) but raises the antimycin-insensitive respiration from 3 to 12 microliter O2 . h-2 . mg-1 dry weight. Antimycin A inhibits the growth of K. lactis on a variety of substrates with the exception of glucose at concentrations equal to or higher than 1% where substantial antimycin-insensitive respiratory rates are induced. It can be concluded that a minimal antimycin-insensitive QO2 is necessary for cellular growth when the normal respiratory pathway is not functional. The antimycin-insensitive respiration elicited by growth in high glucose concentrations is poorly inhibited by hydroxamate and is inhibited by 50% by 90 microM azide or 1 mM cyanide. These concentrations are much higher than those necessary to inhibit cytochrome c oxidase which is not involved in the antimycin-insensitive respiration as was demonstrated by spectral measurements. A pigment absorbing at 555 nm is specifically reduced after addition of glucose to antimycin-inhibited cells. The same pigment is reoxidized by further addition of high concentrations of sodium azide indicating its participation in the antimycin-insensitive, azide-sensitive respiration.

Antimycin A↗
PubMed1983

Respiratory pathways in Hansenula saturnus.

Hansenula saturnus is a petite-negative yeast species which displays a different pattern of respiration depending on the age of the cultures. The respiration is sensitive to antimycin A (AA) in the early exponential phase, is sensitive to the simultaneous addition of AA and salicylhydroxamic acid (SHAM) in the middle exponential phase and is sensitive to SHAM in the late exponential and stationary phase. The three respiratory activities are all associated to the mitochondrial fraction. The presence of AA in the growth medium determines the induction of the AA + SHAM-insensitive respiration which is 50% inhibited by 5 mM azide. On the contrary, the presence of erythromycin in the growth medium, which inhibits mitochondrial protein synthesis in this yeast species and the synthesis of cytochromes aa3 and b, totally prevents the appearance of AA + SHAM-insensitive respiration. Moreover, the antibiotic affects cell viability, suggesting a role of the mitochondrial protein synthesis in the cell cycle of H. saturnus.

Antimycin A↗
PubMed1986

The respiratory activities of four Hansenula species.

The respiratory activities and the cytochrome spectra from four species belonging to the genus Hansenula have been analysed. The results obtained and described in this paper show that H. glucozyma possesses only the primary, antimycin A-sensitive respiration, H. anomala and H. californica possess primary and secondary (salicylhydroxamate-sensitive) respirations, whereas H. saturnus possesses three respiratory activities (AA-sensitive, SHAM-sensitive, and AA + SHAM-insensitive). The respiratory activity of H. glucozyma is glucose-repressible, whereas the activities of the other species are not. In addition, antimycin A (AA) and erythromycin (ERY) in the culture media differently inhibit the growth of the four species and regulate the respiratory pathways in the species analysed.

Antimycin A↗
PubMed1993

Lower blepharoplasty: partially joined skin and muscle flaps.

As a method of performing a lower blepharoplasty, the author proposes a partially joined skin and muscle flap. This procedure allows one to resect separately different amounts of skin and muscle for appropriate correction of the lower-lid changes that require an aesthetic blepharoplasty. It also promotes muscle raising with consequent skin flap lifting. This technique resects a large amount of skin in order to achieve a smoother lower eyelid.

Eyelids↗
PubMed1985

Antimycin A- and hydroxamate-insensitive respiration in yeasts.

In this paper evidence is presented for the mitochondrial localization of the antimycin A (AA) + salicylhydroxamate (SHAM)-insensitive respiration of the yeasts Kluyveromyces lactis, Endomycopsis capsularis and Hansenula saturnus. Such a respiration, which can be sustained by NADH and NADPH but not by succinate, is inhibited by high concentrations of azide. AA + SHAM-insensitive respiration is not phosphorylating and its postulated physiological role is to oxidize NADH.

Antifungal Agents↗
PubMed1975

Metabolic DNA in the hepatocyte nuclei in newborn rats.

The behaviour in time of labelled nuclear DNA in the hepatocytes of newborn rats was studied using autoradiographic and biochemical techniques in two groups of experiments. In the first group H-3-thymidine was injected to the mothers at the 16th day of pregnancy and the amount of labelled DNA was evaluated in the newborns after delivery. In the second group H-3-thymidine was injected to the newborns two hours after birth and the labelled DNA was studied at the same time intervals as the first group. The amount of labelled thymidine incorporated into the first group of animals remains constant for the first three days of life, thereafter a reduction in specific activity of DNA is observed concomitant with an increase of the percentage of labelled nuclei and a decrease of the number of grains per nucleus. These results show that mitotic divisions, which are absent during the first three days of life, take place between the third and sixth days of life. The decrease of the specific activity is therefore due to dilution and not to loss of labelled DNA. In the second group of experiments the DNA labelled with H-3-thymidine shows a decrease by about 30--40% per day during the first three days of life accompanied by a decrease in the number of grains per nucleus without changes in the percentage of labelled nuclei. These data show that DNA synthesized during the first day after birth is metabolically unstable, unlike that synthesized during foetal life.

Animals↗
PubMed1981

Control of antibiotic therapy in paediatric patients. II. Appropriateness of antibiotic choice in selected diseases.

The adequacy of antibiotic choice and the importance of the physician's knowledge of antibiotic use in causing errors in prescribing were investigated. A prospective three-month study was conducted in nine Italian pediatric hospital wards, involving every patient admitted to hospital for otitis, pneumonia or pharyngotonsillitis. The suspected aetiology and the antibiotic prescribed were recorded on a special form by the physician in charge. Each choice of antimicrobial agent was judged as adequate, justifiable or not justified. Out of 314 prescriptions 56.1% were assessed as adequate, 4.1% as justifiable and 39.8% as not justified. Analysis of the suspected bacteria, and of the correlation between the presumed aetiological agent and the prescribed antibiotic, demonstrates that inadequate knowledge of the physician plays a major role in producing a high percentage of unjustified prescriptions.

Anti-Bacterial Agents↗