Detection of M. tuberculosis using DNA chips combined with an image analysis system.

OBJECTIVE: To develop a packaged DNA chip assay (the DR. MTBC Screen assay) for direct detection of the Mycobacterium tuberculosis complex. DESIGN: We described a DNA chip assay based on the IS6110 gene that can be used for the detection of M. tuberculosis complex. Probes were spotted onto the polystyrene strips in the wells of 96-well microtitre plates and used for hybridisation with biotin-labelled amplicon to yield a pattern of visualised positive spots. The plate image was scanned, analysed and interpreted automatically. RESULTS: The results corresponded well with those obtained by conventional culture as well as clinical diagnosis, with sensitivity and specificity rates of respectively 83.8% and 94.2%, and 84.6% and 96.3%. CONCLUSION: We conclude that the DR. MTBC Screen assay can detect M. tuberculosis complex rapidly in respiratory specimens, readily adapts to routine work and provides a flexible choice to meet different cost-effectiveness and automation needs in TB-endemic countries. The cost for reagents is around US$10 per sample.

Apoptosis and host restriction of vaccinia virus in RK13 cells.

Poxviruses express a number of host range (hr) genes that control virus growth in distinctive cell types. Inactivation of hr gene expression in several reported cases has led to apoptosis of virus-infected cells. In RK13 cells, the K1L gene serves as a hr gene for vaccinia virus. We therefore investigated the effect of K1L expression in apoptosis of RK13 cells. In contrast to other hr genes, no significant increase of apoptosis was detected in RK13 cells infected with a K1L- mutant virus. Also, expression of a CHO hr gene CP77 rescues K1L- mutant virus in RK13 cells with little effect on apoptosis. We then set out an experimental approach to investigate the relationship between apoptosis and host restriction in CHO and RK13 cells. A recombinant vaccinia virus expressing a human bcl-2 gene, bcl2-VV, was constructed. Expression of bcl-2 suppressed apoptosis of virus-infected CHO cells as expected. However, bcl-2 expression did not allow virus growth in CHO cells, suggesting apoptosis suppression is not sufficient to rescue host restriction. Moreover, infection of bcl-2VV in RK13 cells induced significant apoptosis with no reduction on virus production, indicating that apoptosis does not contribute to host restriction. In consideration of this data, we conclude that host restriction of vaccinia virus in CHO and RK13 cells is mediated by a pathway distinct from apoptosis.

Isolation and characterization of a Chinese hamster ovary mutant cell line with altered sensitivity to vaccinia virus killing.

The Chinese hamster ovary (CHO) cell line is nonpermissive for vaccinia virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by vaccinia virus. A vaccinia virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with vaccinia virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the virus life cycle and cell fate. We found that in parental CHO cells, vaccinia virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since vaccinia virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma virus and myxoma virus killing. On the other hand, VV5-4 remains sensitive to cowpox virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with vaccinia virus. We concluded that the cellular pathway, which blocks viral DNA replication and allows VV5-4 to survive, is independent of apoptosis. This mutant also provides evidence that an inductive signal for apoptosis upon vaccinia virus infection occurs prior to viral DNA replication.

Orion--shaping a new accreditation process.

In January, the Joint Commission on Accreditation of Healthcare Organizations announced an Action Plan in response to criticisms leveled by the American Hospital Association. A key element of the plan is the Orion Project. Already in the planning stages, it was designed to test various accreditation process improvements. Here, two of JCAHO's leaders describe what Orion is and how it will work.

Isolation of a monoclonal antibody which blocks vaccinia virus infection.

We have isolated a monoclonal antibody, B2, that neutralizes vaccinia virus infection. B2 reacts with a trypsin-sensitive cell surface epitope. B2 does not neutralize infection of herpes simplex virus, suggesting that the B2-reactive epitope is specifically involved in vaccinia virus entry. A survey of 12 different cell lines reveals a correlation between B2 reactivity and susceptibility to vaccinia virus infection. In addition, B2 interferes with vaccinia virus adsorption to target cells. Taken together, the B2-reactive epitope is part of a receptor that appears important for vaccinia virus entry.

Investigation of 2.1-microm lasing properties of Ho:Tm:Cr:YAG crystals under flash-lamp pumping at various operating conditions.

Flash-lamp-pumped normal-mode and Q-switched 2.1-microm laser operations of Ho:Tm:Cr:YAG crystals have been evaluated under a wide variety of experimental conditions in order to determine an optimum lasing condition and to characterize the laser outputs. Q-switched laser-output energies equal to or in some cases more than the normal-mode laser energies were obtained in the form of a strong single spike by optimizing the opening time of a lithium niobate Q switch. The increase of the normal-mode laser slope efficiency was observed with the increase of the Tm concentration from 2.5 to 4.5 at. % at operating temperatures from 120 K to near room temperature. Laser transitions were observed only at 2.098 and 2.091 microm under various conditions. The 2.091-microm laser transition appeared to be dominant at high-temperature operations with low-reflective-output couplers and to have an energy-level assignment from 5313 cm(-1) to 534 cm(-1) or (and) from 5313 cm(-1) to 536 cm(-1).

Uptake of cefadroxil derivatives in rat intestinal brush-border membrane vesicles.

Uptake of cefadroxil and its two acetyl-derivatives, N-acetyl- and O-acetyl-cefadroxil, into the brush-border membrane vesicles (BBMV) was measured at [pH]o = 5.5, 7.4 and [pH]i = 7.4. Both acetyl-derivatives showed a significantly slower uptake than cefadroxil at [pH]o = 5.5 and 7.4. Cefadroxil and the two derivatives showed a higher uptake rate in the presence of an inward H+ gradient ([pH]o = 5.5, [pH]i = 7.4). At [pH]o = 5.5, uptake of cefadroxil into BBMV was inhibited by N-acetyl-, O-acetyl-, N-BOC-, and N-BOC-O-acetyl-cefadroxil, but not by cephalothin and cefuroxime. At [pH]o = 7.4, no inhibition of cefadroxil uptake was evident for any inhibitors. There were two different transporters responsible for the uptake of cefadroxil at pH 5.5 and 7.4. One is the H(+)-coupled dipeptide transport system, and the other is the neutral pH-preferring system. The alpha-amino group may be essential for the transport of cefadroxil by both transport systems. Although the phenolic group in the side chain is not an essential functional group of beta-lactam antibiotics, an additional derivation on the phenolic group of cefadroxil also inhibited both the H(+)-coupled dipeptide transport system and the neutral pH-preferring transport system.

Concentration-dependent exsorption of quinidine in the rat intestine.

During intravenous infusion, the luminal concentration of quinidine was higher than the plasma concentration. The intestinal clearance (CLi) of the drug was measured by dividing the rate of appearance of the drug in the intestinal luminal perfusate by the plasma concentration. The CLi of quinidine was therefore much higher than the rate of luminal perfusion. Over the infusion dose range of 0.1-2 mg h-1, the CLi of quinidine decreased with increasing plasma concentration of quinidine. Adding quinidine into the luminal perfusate had little effect on the CLi of quinidine. Co-administration of quinidine with other agents intravenously did not alter the CLi of salicylic acid and urea, while the same treatment decreased the CLi of theophylline and S-disopyramide. In-vitro experiments on brush-border membrane vesicles showed that quinidine decreased the rate of Na+ uptake and H+ efflux. The inhibition was significant at quinidine concentrations above 20 microM. Quinidine was a more potent inhibitor than amiloride. At quinidine infusion rates less than 2 mg h-1, quinidine concentration in plasma or in the luminal perfusate was at the lower limit of the inhibitory concentration. Microclimate pH at the intestinal surface was also measured. At mid-jejunum, the microclimate pH increased 0.3 pH units by infusing 2 mg h-1 of quinidine, while the microclimate pH at most other measuring sites was not significantly altered by quinidine infusion. It was concluded that quinidine is exsorbed from blood into the intestinal lumen by a carrier-mediated pathway in addition to the passive diffusion. At high plasma concentration, quinidine exsorption becomes saturated.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of theophylline on the intestinal clearance of drugs in rats.

Intestinal exsorption of salicylic acid, urea and quinidine was measured during the perfusion of the rat intestinal lumen with Tyrode solution. The intestinal clearance (CLi) of the three compounds was measured by dividing the rate of appearance in the intestinal luminal perfusate by the plasma concentration of the compound. Co-administration of theophylline (0.2 mg h-1) with the test agents increased the CLi of salicylic acid, did not alter the CLi of urea, but decreased the CLi of quinidine. The effect of theophylline on the CLi of quinidine was enhanced with increasing dose. Theophylline was found to increase microclimate-pH at the intestinal surface, but the magnitude of delta pH alone could not explain the effect of theophylline on the CLi of quinidine. The data, together with previous observations, suggest that the intestinal exsorption of drugs was affected by the microclimate pH and by the unstirred water layer. Theophylline affects CLi of salicylic acid and quinidine partly by increasing the microclimate pH of the intestine. Theophylline may also affect quinidine CLi by inhibiting the carrier-mediated pathway.

Pulsed injection control of a titanium-doped sapphire laser.

Injection control of a tunable Ti:sapphire laser using a narrow-bandwidth pulsed dye laser operating at a wavelength removed from the peak of the Ti:sapphire-laser gain curve is reported. The free-running Ti:sapphire laser had broadband laser emission from 750 to 790 nm. Injection at 727 nm resulted in essentially complete energy extraction at that wavelength in a 2.5-pm bandwidth matching the injection source.

High-resolution spectral measurement of the HNO(3) 5.9-microm band using a tunable diode laser.

High resolution spectra in the region of the v(2) band of nitric acid have been obtained for selected portions of the HNO(3) spectrum using tunable diode laser techniques. Continuous spectra are presented from 1718.97 cm(-1) to 1729.57 cm(-1), with a spectral resolution

High resolution spectral measurement of the HNO(3) 11.3-microm band using tunable diode lasers.

High resolution spectra in the region of the 2nu(9) band of nitric acid have been obtained for selected portions of the HNO(3) spectrum using tunable diode laser techniques. Continuous spectra are presented from 891.25 cm(-1) to 898.77 cm(-1), with a spectral resolution

Measurement of HCI absorption coefficients with a DF laser.

Absorption coefficients in the fundamental P-branch of HCl at several DF laser transitions from 2439.02 cm(-1) to 2862.87 cm(-1) have been measured experimentally. The 2-1 P(3) DF laser transition has been shown to overlap the P(6) HCl(37) absorption line within the halfwidth of an atmospherically broadened line. The absorption coefficient k was measured to be 5.64 +/- 0.28 (atm-cm)cm(-1) for a 0.27% mixture of HCl in N(2) at a total pressure of 760 Torr. A theoretical and experimental comparison of the pressure dependence of k showed that the 2-1 P(3) DF transition lies 1.32 +/- 0.15 GHz from the center of the P(6) HCl absorption line. This line separation is in good agreement with published positions for this DF laser transition and the HCl absorption line. At least four other DF laser transitions in the spectral interval measured are reported showing measurable absorption (>/=10%) for a 10.2% mixture of HCl in N(2) across a 1-m pathlength. Applications of these results to differential absorption lidar (DIAL) and to heterodyne detection are also discussed.