Choosing highly specific primers for the polymerase chain reaction using the octomer frequency disparity method: application to Chlamydia trachomatis.

The eight-nucleotide sequence (octomer) at the 3' end of PCR primers is important to PCR specificity. We describe a correlation between the specificity of PCR primers used with human DNA and the frequency of the 3' octomer in a human database. We therefore applied a methodology (OFD) based on octomer frequency disparity to identify 16 PCR targets in the chromosome of the intracellular bacterium, Chlamydia trachomatis (Ct). In addition, the 16 sets of primers were tested with a standard procedure. All the primer pairs were highly specific for Ct and did not lead to non-specific amplification when used with human DNA. This work shows that the choice of specific PCR primers is possible using a method based on the statistical representativeness of octomers in genomes.

Antiviral activity of phorbol myristate acetate and possible relationships with interferon action.

Signal-induced turnover of membrane phospholipids represents a fundamental transducing mechanism that induces a signal cascade resulting in mobilization of calcium, activation of protein kinase C by diacylglycerol, release of arachidonic acid and stimulation of cyclic GMP production. In this pathway tumor-promoting phorbol esters such as phorbol myristate acetate (PMA) may substitute for diacylglycerol. The interferon-like antiviral effect of PMA described here suggests that the inositol phospholipid-diacylglycerol-protein kinase C signal-transducing mechanism may be involved in interferon action.

Inositol phospholipid turnover and protein kinase C translocation are stimulated by poly(I).poly(C) in human amnion cells (UAC).

Polyinosinic-polycytidylic acid, a potent inducer of inducer of interferon (IFN) production and activator of some IFN-induced enzymes, inhibits [3H]uridine incorporation into the RNA of vesicular stomatitis virus even in the absence of IFN synthesis, transiently triggers the breakdown of inositol phospholipids and activates the translocation of protein kinase C. Since IFNs also have similar activities these results suggest that IFN induction and IFN function are realised through common biochemical pathways.

Interferon (IFN)-like antiviral effect is induced by unspecific cross-linking of cell surface receptors.

Treatment of human amniotic cells (UAC) with Cytodex 1 (DEAE-dextran) results in the development of an antiviral state of the cells, as proven by studying (i) the cytopathic effect and (ii) [3H]uridine incorporation into the RNA of vesicular stomatitis virus (VSV) after VSV infection. The same treatment transiently triggers the breakdown of inositol phospholipids and activates the translocation of protein kinase C (PKC). On the basis of these data it can be suggested that cross-linking of cell surface receptors by a solid carrier bearing covalently bound positive charges may result in IFN-like effects.

Phospholipase C and phospholipase A2 are involved in the antiviral activity of human interferon-alpha.

Treatment of human amniotic cells (UAC) with human interferon-alpha (Hu-IFN alpha) or phorbol myristate acetate (PMA) resulted in translocation of protein kinase C (PK-C) activity from the cytosol fraction to that of the membranes. Analysis of 32P incorporation into phospholipid fractions and studies of alterations in fatty acid content for the major phospholipids of IFN-treated cells suggest that phospholipases C and A2 are activated by Hu-IFN alpha. Addition of neomycin (an inhibitor of phospholipase C), as well as mepacrine (an inhibitor of phospholipase A2) to IFN-treated cells inhibited the antiviral activity of Hu-IFN alpha in the vesicular stomatitis virus (VSV)-UAC system used. These observations indicate that (i) activation of PK-C and (ii) diacylglycerol formation, arachidonic acid and/or lysophosphatidylcholine release are important steps in the mechanism of action of IFN.

Ethanol-induced signal transducing mechanism associated with a transient antiviral state in human amniotic cells.

Short-term effects of ethanol on human amnion cells were investigated by studying the cellular signaling processes and the replication of vesicular stomatitis virus. Treatment of human amniotic cells with ethanol transiently triggers the breakdown of inositol phospholipids, stimulates intracellular [Ca2+]i mobilization and activates the translocation of protein kinase C. Activation of this signal transduction mechanism is associated with the development of an antiviral state, as proven by studying 3H-uridine incorporation into the RNA of vesicular stomatitis virus. Induction of the antiviral state in human amniotic cells correlates with the solubility of the alcohols in the lipid membrane of the cells.

Ultrastructural changes of human amniotic cells induced by natural human interferon-alpha.

Human interferon-alpha (Hu-IFN alpha) and phorbol myristate acetate (PMA), a direct activator of protein kinase C (PK-C), induce the translocation of protein kinase C from the cytosol to the membrane fraction. By the use of transmission (TEM) and scanning (SEM) electron microscopy we have shown that treatment of human amniotic cells (UAC) with Hu-IFN alpha resulted in profound changes in the shape, volume and ultrastructure of the cells. Most treated cells had enlarged nuclei with marginal condensation of chromatin. Nucleolar segregation, disintegration and clumping of nucleolar components were also observed. The number of interdigitating cell processes decreased and the cell surface microvilli became shortened. Similar ultrastructural alterations were induced by PMA also. All these functional and morphological data strongly support the hypothesis that protein kinase C is a key factor in IFN-mediated cell reactions.

Photo-refractive keratectomy (PRK): threat or millennium for military pilots?

The development of the excimer laser, which is capable of correcting myopia without leaving obvious scars as does radial keratotomy, makes it certain that this promising but very new modality is something the military aviation community will be facing in the immediate future. Methods of detection are available, but are expensive and time-consuming. Should military aviation permit or even sponsor a group of PRK student pilots in order to observe them closely, and then perhaps use this new modality in place of contact lenses or even spectacles?

The fate of eyewear in aircraft ejections.

Ejection from jet aircraft has been exhaustively studied from many perspectives; e.g., causes of ejection, types and causes of ejection injuries, etc. Curiously, no reports exist describing the fate of eyewear in ejections. Many pilots wear required corrective lenses during flight operations, and many wear tinted lenses. What happens to these during ejection? What injuries are a result of the eyewear? What factors can be identified that influence retention of the eyewear and severity of related injury? Do contact lenses provide significant advantages? There were 48 ejections occurring between 1977 and 1990 that involved corrective or tinted lens use that were retrospectively examined using Naval Safety Center records and personal questionnaires. There were five contact lens wearers included. Retention rates were calculated as functions of several variables. Although 37 of 46 lost all lenses, each instance of retention occurred with visor down, oxygen mask on, helmet properly secured, and at lower ejection speeds. Related injuries were minor and occurred in only 20%. The utility and need for enforcement of standard operating procedures (i.e., mask on, helmet secured, and visor down) was clearly demonstrated. Only 19 of 46 clearly met all 3 criteria. Contact lens users were too few to allow meaningful conclusions.

Comparison of bifocal and progressive addition lenses on aviator target detection performance.

The objective of this project was to determine if the type of presbyopic correction worn by aviators, conventional bifocal versus progressive addition lenses (PAL's), differentially affects aviator visual search performance. Experienced aviators, most with tactical fighter aircraft experience, searched for high-contrast targets under simulated dawn/dust (mesopic) lighting conditions (approximately 3.0 cd/m2) while wearing either a standard bifocal (ST-25) or PAL spectacle correction. Latency of locating high-contrast targets under these viewing conditions was differentially affected by the type of presbyopic correction used. Specifically, compared to a standard bifocal (ST-25), a PAL correction (Varilux Infinity) significantly lowered the time needed to locate static targets at a cockpit instrument viewing distance (83 cm). Accuracy of target location responses was not affected by the type of correction used. In addition, 7 months post-experiment, 7 of the 12 participants (58%) indicated that they used their PAL correction exclusively when flying the T-39 Sabre Liner. Three subjects (25%) used their PAL correction intermittently (primarily at night) when flying and two subjects preferred not to use the PAL's. These results suggest that relative to bifocals, speed of responding to static targets at intermediate viewing distances may be improved by wearing PAL's, and that subjects were able to adapt to PAL lenses quickly in a laboratory setting, using them later in a functional aviation environment.

[Effect of the medium composition on the synthesis of protease by Aspergillus sp]. / Influencia de la composición del medio en al síntesis de proteasa por Aspergillus sp.

The main regulatory mechanisms controlling the synthesis of neural protease in batch cultures of a strain of Aspergillus were studied. Protease was inducible in this strain. No activity appeared during the trophophase when the organism was grown in a minimal medium, unless an inducer was added. Gelatine had the best induction effect among all the tested substances. The growth kinetics and enzyme production in a culture medium containing gelatine as the sole carbon and nitrogen source was determined. In spite of these results, protease activity was clearly detectable during the idiophase in absence of an external inducer. Furthermore, the addition of gelatine to cultures during the idiophase resulted in a decrease of the enzyme activity. This behaviour could be due to internal autoinduction generated by accelerated protein turnover of the nitrogen starved cells. The addition of gelatine probably reduced the protein starvation. Protease synthesis was sensitive to repression by rapidly assimilable carbon sources such as glucose and glycerol. Ammonia also caused an important repression, while nitrate and amino nitrogen had no effect. It was concluded that the synthesis of this enzyme is controlled by induction, catabolite repression, and ammonia repression, and that the enzyme production period could be adjusted by adequate formulation of the culture medium.

Dissimilatory nitrate reduction by liquid membrane encapsulated cell-free extracts and whole cells of Micrococcus denitrificans.

The combination of Sephadex G-15 and ion-exchange resin columns allows one-step desalting and separation of cellodextrins using water as the sole eluent. The column apparatus described in this paper has the potential of producing up to 3 g cellodextrins in one day. In addition, the columns described are stable and do not require repacking or regeneration after each run. Hence the potential exists for scaling up this system for even greater production of cellodextrins if need be.