Spectrally silent transitions in the bacteriorhodopsin photocycle.

The photocycle kinetics of bacteriorhodopsin were analyzed from 0 to 40 degrees C at 101 wavelengths (330-730 nm). The data can be satisfactorily approximated by eight exponents. The slowest component (half-time 20 ms at 20 degrees C) belongs to the 13-cis cycle. The residual seven exponentials that are sufficient to describe the all-trans photocycle indicate that at least seven intermediates of the all-trans cycle must exist, although only five spectrally distinct species (K, L, M, N, and O) have been identified. These seven exponentials and their spectra at different temperatures provide the basis for the discussion of various kinetic schemes of the relaxation. The simplest model of irreversible sequential transitions includes after the first K--> L step the quasiequilibria of L<-->M, M<-->N, and N<-->O intermediates. These quasiequilibria are controlled by rate-limiting dynamics of the protein and/or proton transfer steps outside the chromophore region. Thus there exists an apparent kinetic paradox (i.e., why is the number of exponents of relaxation (at least seven) higher than the number of distinct spectral intermediates (only five)), which can be explained by assuming that some of the transitions correspond to changes in the quasiequilibria between spectrally distinct intermediates (i.e., are spectrally silent).

Acyclovir triphosphate inhibits the diagnostic polymerase chain reaction for cytomegalovirus.

Immunocompromised patients are frequently treated with guanine analogs such as acyclovir and ganciclovir. Acyclovir triphosphate, the active intracellular metabolite of acyclovir, exerts its antiviral effect by inhibiting herpesviral DNA polymerases through premature chain termination. PCR has recently been used for early detection of cytomegalovirus. However, we and others have experienced false-negative results for cytomegalovirus-PCR in patients on both acyclovir and ganciclovir. The impact of these agents on PCR assay is unknown. In an attempt to investigate the role of guanosine analogs in these false-negative results, we exposed the DNA-PCR for murine beta-actin, a murine CMV IE gene sequence, and a human CMV IEA1 product, to phosphorylated acyclovir derivatives. Varying concentrations of acyclovir-5'-triphosphate (final: 70-6000 microM) in the reaction mix resulted in an absence of detectable product at or above 490-670 microM. Inhibition was not observed with up to 1400 microM acyclovir-monophosphate. Increasing the Taq concentration to 10 units/100 microL stopped the inhibition. Our data demonstrate that acyclovir-5'-triphosphate inhibits PCR amplification of various gene products in a concentration-dependent manner. Furthermore, this inhibition appears to be specifically directed against the Taq polymerase and can be completely reversed by higher concentrations of the enzyme. Thus, false-negative PCR results for a viral gene product in patients under prophylaxis/treatment with acyclovir could potentially be due to contamination by acyclovir triphosphate. Therefore, negative PCR results in these patients need be interpreted with caution.

Direct evidence using in situ polymerase chain reaction that the endothelial cell and T-lymphocyte harbor latent murine cytomegalovirus.

The latent viral genome, harbored indefinitely, threatens reactivation from its remote location. Although polymerase chain reaction (PCR) has detected the organs responsible for latency, it is not known whether latent cytomegalovirus (CMV) infection is maintained within organ-specific cells or ubiquitous elements such as macrophages, endothelial cells, or perhaps others. PCR lacks correlation with tissue structure. However, PCR-based in situ hybridization maintains cellular architecture while allowing the identification of the latently infected cells. Murine CMV (MCMV) nucleic acid sequences in organs of latently infected Balb/C mice were amplified by PCR incorporating digoxigenin-11-dUTP, holding the product DNA in situ (appropriate controls analyzed in parallel). Product DNA was then hybridized in situ with a biotinylated oligonucleotide probe for detection via streptavidin-alkaline phosphatase and light microscopy. Immunohistochemistry verified the positive cell types. Using this technique, we have shown directly in multiple organs of latently infected Balb/C mice including kidney (5/5), liver (5/5), and spleen (5/5) that the endothelial cell and/or T-lymphocyte harbor latent MCMV, whereas in uninfected animals, MCMV DNA was not detected. PCR-based in situ hybridization allows detection of the specific cell(s) harboring latent MCMV DNA while allowing conservation of cellular architecture.

Total anomalous origin of the coronary arteries from the pulmonary artery.

The clinicopathologic features of four patients with total anomalous origin of the coronary arteries from the pulmonary artery (TCAPA) are presented and compared with 21 previously reported cases. Patients with TCAPA usually present with left ventricular heart failure, angina, or associated cardiovascular anomalies. Of the 19 patients in whom a clinical history was available, 16 were symptomatic before three days of age. All patients died with 60% dying before two weeks of age. Longer survival was associated with additional cardiovascular anomalies that increased pulmonary arterial perfusion pressure, oxygen saturation, or both. Seventeen (68%) patients had additional cardiovascular anomalies, most commonly atrial (nine cases) or ventricular (eight cases) septal defects and tetralogy of Fallot or other variants of pulmonary atresia (four cases). Only five (22%) of 23 had noncardiovascular anomalies. The coronary arteries arose equally from either one ostium or from two, and the number of ostia was not related to either anomalous coronary artery distribution or to the presence of additional cardiovascular anomalies. Cardiomegaly was present in 56% of cases and the majority of patients had myocardial fibrosis or infarction. Embryology is reviewed and evidence is presented to support the theory of involution-persistence of coronary artery anlagen as the pathogenetic mechanism of TCAPA.

Bioceramic implants in surgically produced infrabony defects.

Tricalcium phosphate ceramic of hydroxyapatite structure with 50% porosity and 800- to 1000-mum pore diameter was implanted in surgically produced infrabony defects in dogs. The defects were evaluated histologically at different time intervals, 1, 2, 4, 8, 16, and 24 weeks. The results show that the ceramic is well tolerated by the tissue and yields no toxic reactions. Bone ingrowth into the pores and repair of the periodontium are clearly demonstrated. No significant hematological changes were observed.