Extraction of human DNA for PCR from chewed residues of betel quid using a novel "PVP/CTAB" method.

Residues of chewed betel quid (BQ) are often found on crime scenes in Taiwan and possibly some of the Southeast Asian countries. Although these residues are important biological evidences relating to the suspects, the forensic analysis of BQ evidence has been hindered by failures in extraction of human DNA for PCR analysis. Therefore, it is a prerequisite for relevant forensic casework to establish a reliable method for extracting DNA from chewed BQ residues. Three conventional methods (salt/chloroform, 5% Chelex-100 resin, and QIAamp) were first tested for extraction of human DNA from 33 mock BQ samples, which had been stored for less than two months, and 50 four-year-old forensic BQ samples. PCR amplifications from the HLA-DQA1&PM and the STR loci were then used to test the quality of the extracted DNA. For the mock samples, three observations were made. First, PCR amplification of DNA extracted by using these conventional methods had low success rate. Second, the addition of extra Taq DNA polymerase could compensate the lost enzyme activities due to putative inhibitors and, thus, increase the yield. Third, using the Centricon-100 column to remove putative inhibitors substantially improved the efficiency of PCR. However, for the four-year-old forensic BQ samples, none of the attempts for PCR were successful. In order to solve the problem in PCR analysis of DNA from old BQ samples, we developed a DNA extraction method based on the use of polyvinyl pyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB), which bind to two common classes of PCR inhibitors in plants, polyphenols, and polysaccharides, respectively. The result showed that this "PVP/CTAB" method is completely successful for the mock BQ samples, and 92% (46 out of 50) successful for the four-year-old forensic BQ samples. To our best knowledge, this is the first report of a reliable method for the extraction of human DNA for PCR from chewed BQ residues. This method should provide a useful means for forensic identification in countries where betel chewing is common.

Purification and characterization of a naturally processed hepatitis B virus peptide recognized by CD8+ cytotoxic T lymphocytes.

In vitro studies in patients with hepatitis B virus (HBV) infection have suggested that hepatocytolysis induced by CD8+ cytotoxic T lymphocytes (CTLs) is the most important effector pathway in eliminating infected cells. The recognition is implicated in the endogenously processed HBV antigens in the context of HLA class I molecules presented on the liver cell membrane. However, the naturally occurring HBV peptide antigens have not yet been demonstrated. We report here that a naturally processed peptide antigen P2 was isolated from HLA class I molecules of HBV-infected liver cell membrane. The P2 peptide exhibited the activity of sensitizing target cells for lysis by CD8+ CTLs. The P2 sequence (YVNVNMGLK) purified from liver tissue was in concordance with that encoded by the viral genome for the HBV nucleocapsid antigen or HBcAg 88-96. P2 peptide could also be isolated from the EBV-transformed B cells that were transfected by HBcAg-expressing vector. The P2 epitope, sharing the HLA-A11 binding motifs, was recognized by HLA-A11-restricted CD8+ CTLs. The data provided direct evidence that, in hepatitis B patients, antigenic peptides of HBV were processed by hepatocytes, presented with the class I MHC molecules, and recognized by CD8+ CTLs.

Fish scale-induced dermatitis.

BACKGROUND: Fish scales loosened during the process of cleaning that come in contact with human skin will adhere and "grow," creating a raised skin lesion if not washed off immediately. OBJECTIVE: The purpose of this study was to examine the process of fish scale-induced dermatitis. METHODS: Fish scales from the blue gill (Lepomis machrochirus) were placed on the intact skin of denuded Swiss Webster mice that had no prior sensitization. RESULTS: The scales physically adhered and formed a raised fold of skin within minutes after placement. Lesions were submitted for pathologic evaluation on days 2 and 7. A subacute irritant dermatitis was observed that evolved into a chronic dermatitis with hyperkeratosis. Inverted fish scales (the anatomically reversed surface) and fish epidermis did not produce such lesions on the mice. CONCLUSION: Fish scales are able to induce an irritant dermatitis. They most likely do so through initial adhesion via mucopolysaccharide secretions.

Microvascular access to the uveal tract in the rabbit eye for ocular pharmacologic studies.

Drug delivery to the eyes is quite inefficient regardless if the drugs are administered topically or intravenously. It is known that less than 1% of topically instilled drug can be absorbed into the eyes while even less of intravenously injected drugs reach the eyes. A research model has been developed in this study which allows delivery of drugs effectively to uveal and anterior structures of the eye. This experimental model allows drugs to be delivered to the eye via cannulation of and retrograde flow through the valveless vortex veins. In addition, the superficial branch of the vortex vein (draining the ciliary body and iris) or the deep branch (draining the choroid plexus of the retina) can be selectively cannulated to suit the researcher's needs. Finally, this procedure minimizes systemic drug actions which otherwise would complicate interpretation of experimental results. This model can be used for in vivo laboratory studies on (a) metabolic and physiological processes of the uveal tract in the eye, and (b) drug delivery to selective tissues of the uveal tract in the eye. Dose-response relationships of pilocarpine and timolol to lower intraocular pressure were demonstrated with this model.

Effects of dopamine antagonists injected through vortex veins on intraocular pressure.

In order to avoid the factors of the corneal barrier and tearing washout on absorption of eye drops, dopamine and antagonists were injected into rabbit eyeballs through vortex veins. Many dopamine antagonists, such as haloperidol, moperone, metoclopramide, trifluperidol, lenperone, and fluoropipamide, lowered the intraocular pressure (IOP), whereas a few of them, such as chlofluperol and trifluoperazine, raised the IOP. Furthermore, it was found that dopamine agonists also could either increase or decrease the IOP. The ocular hypertensive effect of dopamine was blocked by haloperidol as expected. However, when the hypotension-inducing dopamine agonist, bromocriptine, and antagonist, haloperidol, were combined, the IOP remained unchanged. It was concluded that dopamine agonists and antagonists act at different sites and changes in IOP resulted from a combined summary of these complex effects. As a result, dopamine agonists and antagonists, while they may both lower the IOP when given alone, can antagonize each other through different sites and produce no net change in IOP.

Effects of epinephrine on retinal and choroidal blood flow through different routes of drug administration.

Epinephrine (1, 3 and 10 micrograms/kg) was found to increase retinal and choroidal blood flow (22, 49 and 67%) when it was administered intravenously. The increase in retinal and choroidal blood flow coincided well with the increase in systemic blood pressure (44, 65 and 96%, respectively). When epinephrine (2%) was instilled to the eyes topically the retinal and choroidal blood flow decreased (15%) while systemic blood pressure remained unchanged. It is suggested that the decrease in retinal and choroidal blood flow by topical epinephrine could be the cause of epinephrine maculopathy reported previously. L-Timolol (0.25%, topically) did not affect retinal and choroidal blood flow significantly.

Stereospecific effect of naloxone hydrochloride on cyanide intoxication.

Cyanide intoxication in mice can be antagonized by the opiate antagonist, (-)naloxone HCl, alone or in combination with sodium thiosulfate and/or sodium nitrite. Potency ratios, derived from LD50 values, were compared in groups of mice pretreated with sodium nitrite (sc, 100 mg/kg), sodium thiosulfate (ip, 1 g/kg), and (-)naloxone HCl (sc, 10 mg/kg) either alone or in various combinations. These results indicate that naloxone HCl provides a significant protection against the lethal effects of potassium cyanide. The protective effect of sodium thiosulfate, but not sodium nitrite, was enhanced with (-)naloxone HCl. The combined administration of sodium nitrite and sodium thiosulfate was further enhanced with (-)naloxone HCl. This protective effect of naloxone HCl against the lethal effect of cyanide appears to be restricted to the (-)stereoisomer, as the (+)stereoisomer, the inactive opiate antagonist, is also inactive in protecting against the lethal effects of cyanide. The mechanism of antagonism is discussed.

Compensatory mutations in receptor function: a reevaluation of the role of methylation in bacterial chemotaxis.

During bacterial chemotaxis membrane receptor proteins are methylated and demethylated at glutamate residues. The generally accepted view is that these reactions play an essential role in the chemosensing mechanism. Strains may be isolated, however, that exhibit chemotaxis in the complete absence of methylation. These are readily obtained by selecting for chemotactic variants of a mutant that completely lacks the methylating enzyme. Methyltransferase activity is not restored; instead, the sensory-motor apparatus is genetically restructured to compensate for the methylation defect. Genetic and biochemical analyses show that the compensatory mutational locus is the structural gene for the demethylating enzyme. Thus, although mutants lacking either the methylating or demethylating enzymes are nonchemotactic, strains defective in both activities exhibit almost-wild-type chemotactic ability.

Effects of prostaglandins on intraocular pressure recovery rate in rabbits.

The actions of a number of prostaglandins (PGs) were studied in unanesthetized rabbits using an intraocular pressure (IOP) recovery-rate method. In topical doses of 0.1 to 10 micrograms, these compounds accelerated the rate at which IOP returned to control levels after an infusion of hypertonic saline. In general, PGE1 appeared more potent than the other PGs at these doses. Arachidonic acid also increased the IOP recovery rate. The effect of arachidonic acid was completely blocked by the cyclooxygenase inhibitor indomethacin. Recovery rate responses to arachidonic acid were increased further after pretreatment with the lipoxygenase inhibitor phenidone. When administered alone, phenidone itself accelerated IOP recovery; this action was also blocked by indomethacin. The IOP recovery rate method appears to be a useful tool for studying ocular effects of PGs and other products or inhibitors of arachidonic acid metabolism.

Dopaminergic involvement in intraocular pressure in the rabbit eye.

The involvement of dopamine in maintaining intraocular pressure (IOP) was investigated with the rabbit IOP recovery model after intravenous infusion of hypertonic saline. Dopamine facilitated the IOP recovery while reserpine did the opposite. When dopamine was administered after reserpinization, the IOP recovery was facilitated again. These results indicate that dopamine is involved in the maintenance of IOP because depletion of dopamine with reserpine resulted in an opposite effect produced by dopamine whereas administration of dopamine in reserpinized animals induced dopaminergic responses. Timolol produced similar effects as reserpine, which supports the idea that timolol reduces aqueous humor formation through elimination of dopaminergic function and reduction of blood flow in the ciliary body.