Characterization of Hprt mutations in cDNA and genomic DNA of T-cell mutants from control and 1,3-butadiene-exposed male B6C3F1 mice and F344 rats

A multiplex PCR procedure for analysis of genomic DNA mutations in the mouse hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene was developed and then used with other established methods for the coincident identification of large- and small-scale genetic alterations in the Hprt gene of mutant T-cell isolates propagated from sham- and 1,3-butadiene (BD)-exposed mice and rats. The spectra data for RT-PCR/cDNA analysis and multiplex PCR of genomic DNA from Hprt mutants were combined, and statistical analyses of the mutant fractions for the classes of mutations identified in control versus exposed animals were conducted. Under the assumption that the mutant fractions are distributed as Poisson variates, BD exposure of mice significantly increased the frequencies of (1) nearly all types of base substitutions; (2) single-base deletions and insertions; and (3) all subcategories of deletions. Significantly elevated fractions of G:C-->C:G and A:T-->T:A transversions in the Hprt gene of BD-exposed mice were consistent with the occurrence of these substitutions as the predominant ras gene mutations in multiple tumor types increased in incidence in carcinogenicity studies of BD in mice. BD exposure of rats produced significant increases in (1) base substitutions only at A:T base pairs; (2) single-base insertions; (3) complex mutations; and (4) deletions (mainly 5' partial and complete gene deletions). Future coincident analyses of large- and small-scale mutations in rodents exposed to specific BD metabolites should help identify species differences in the sources of deletion mutations and other types of mutations induced by BD exposures in mice versus rats.     

Fatal fat embolism following amphotericin B lipid complex injection

A case of amphotericin B lipid complex induced fatal fat embolism is described. A 41-year-old Caucasian man with AIDS was undergoing treatment for cryptococcal meningitis with amphotericin B. His course was complicated by renal failure necessitating a change in therapy to amphotericin B lipid complex (Abelcet). At approximately 48 h, the patient developed tachycardia, tachypnea, respiratory failure, decline in hematocrit, thrombocytopenia, and alteration in mental status. Autopsy findings included fat emboli involving heart, lungs, kidney, and brain. To our knowledge, this is the first case report of a fatal fat embolism caused by intravenous liposome drug delivery.     

An invasion-independent pathway of blood-borne metastasis: a new murine mammary tumor model

It is generally believed that active invasion by cancer cells is essential to the metastatic process. In this report, we describe a murine mammary tumor (MCH66) model of metastasis that does not require invasion into the vascular wall of both the primary tumor and the target organ, in this case, the lung. The process involves intravasation of tumor nests surrounded by sinusoidal blood vessels, followed by intravascular tumor growth in the lung, without penetration of the vascular wall during the process. Comparative studies using a nonmetastatic MCH66 clone (MCH66C8) and another highly invasive metastatic cell line (MCH416) suggested that high angiogenic activity and sinusoidal remodeling of tumor blood vessels were prerequisites for MCH66 metastasis. Differential cDNA analysis identified several genes that were overexpressed by MCH66, including genes for the angiogenesis factor pleiotrophin, and extracellular matrix-associated molecules that may modulate the microenvironment toward neovascularization. Our analyses suggest that tumor angiogenesis plays a role in the induction of invasion-independent metastasis. This model should prove useful in screening and development of new therapeutic agents for cancer metastasis.     

Acute inflammatory response to endotoxin in mice and humans

Endotoxin injection has been widely used to study the acute inflammatory response. In this study, we directly compared the inflammatory responses to endotoxin in mice and humans. Escherichia coli type O113 endotoxin was prepared under identical conditions, verified to be of equal biological potency, and used for both mice and humans. The dose of endotoxin needed to induce an interleukin-6 (IL-6) concentration in plasma of approximately 1,000 pg/ml 2 h after injection was 2 ng/kg of body weight in humans and 500 ng/kg in mice. Healthy adult volunteers were injected intravenously with endotoxin, and male C57BL/6 mice (n=4 to 12) were injected intraperitoneally with endotoxin. Physiological, hematological, and cytokine responses were determined. Endotoxin induced a rapid physiological response in humans (fever, tachycardia, and slight hypotension) but not in mice. Both mice and humans exhibited lymphopenia with a nadir at 4 h and recovery by 24 h. The levels of tumor necrosis factor (TNF) and IL-6 in plasma peaked at 2 h and returned to baseline levels by 4 to 6 h. IL-1 receptor antagonist RA and TNF soluble receptor I were upregulated in both mice and humans but were upregulated more strongly in humans. Mice produced greater levels of CXC chemokines, and both mice and humans exhibited peak production at 2 h. These studies demonstrate that although differences exist and a higher endotoxin challenge is necessary in mice, there are several similarities in the inflammatory response to endotoxin in mice and humans.     

Cross-orientation inhibition in cat is GABA mediated

Summary Visual evoked potentials (VEPs) were recorded from cat cortex (area 17) before, during and after application of the GABA blocker bicuculline (iontophoretic or topical). The stimuli comprised a test sinusoidal grating, and a mask grating oriented either parallel or orthogonal to the test. Both test and mask alternated in contrast at different temporal frequencies. VEPs were averaged in synchrony with the test contrast reversal, so the mask did not contribute directly to the averaged VEP response. Before application of bicuculline, both parallel and orthogonal masks attenuated the amplitude of VEPs and changed the phase response, but in different ways. Orthogonal masks lowered the slope of the contrast response curve without affecting extrapolated threshold, while parallel masks caused the curve to shift to the right. Orthogonal masks increased the phase advance, while parallel masks eliminated it. During application of bicuculline, neither parallel nor the orthogonal masks attenuated VEP amplitudes. The results suggest that although the mechanisms for the action of parallel and orthogonal masks are clearly distinct, both are mediated by the GABA-ergic inhibitory system. Given this evidence, measurement of VEP contrast response curves may provide a simple non-invasive technique for monitoring visual inhibition in humans.     

CD28 and CTLA4 coordinately regulate airway inflammatory cell recruitment and T-helper cell differentiation after inhaled allergen

Airway inflammation after inhaled allergen exposure requires the recruitment, activation, and differentiation of antigen-specific T cells into T helper (Th) 2 effector cells. These processes are regulated not only by antigen engagement of the T-cell receptor, but also by specific accessory molecules on the surface of the T cell. We examined how the balance of signals derived through the CD28 and cytotoxic T-lymphocyte antigen (CTLA) 4 receptors modulate the outcome of inhaled antigen exposure in a murine model of allergic airway inflammation. Mice deficient in CD28 have defective Th2 cell development and failed to develop inflammation after sensitization and inhaled challenge with ovalbumin. Prevention of B7-CTLA4 interactions in CD28-deficient mice restored lymphocyte but not eosinophil recruitment to the airway. Analysis of cytokine gene expression revealed that T cells from CD28-deficient mice failed to differentiate into Th2 cells in either the presence or absence of B7-dependent signals, and therefore did not recruit eosinophils to the airway. Thus, the processes of T-cell recruitment to the airway and T-cell differentiation have distinct requirements for signals mediated through the CD28 and CTLA4 receptors, demonstrating that these receptors are important regulatory components in the development of allergic airway inflammation.     

Abacavir once or twice daily combined with once-daily lamivudine and efavirenz for the treatment of antiretroviral-naive HIV-infected adults: results of the Ziagen Once Daily in Antiretroviral Combination Study

The long intracellular half-life of abacavir (ABC) supports its once-daily use, and this would be expected to simplify treatment if ABC could be given as part of a complete once-daily regimen. A randomized double-blind clinical trial compared the efficacy and safety of 600 mg of ABC administered once daily (n = 384) versus 300 mg of ABC administered twice daily (n = 386) in combination with 300 mg of lamivudine (3TC) and 600 mg of efavirenz (EFV) administered once daily in antiretroviral-naive patients over 48 weeks. The baseline median plasma HIV-1 RNA level was 4.89 log10 copies/mL (44% with viral load >100,000 copies/mL), and the median CD4 cell count was 262 cells/mm. ABC administered once daily was non-inferior to the twice-daily regimen, with 66% and 68% of patients in these respective treatment arms achieving a confirmed plasma HIV-1 RNA level <50 copies/mL (95% confidence interval: -8.4%, 4.9%). The ABC once-daily and twice-daily regimens were similar with respect to infrequency of virologic failure (10% vs. 8%), emergence of resistance mutations, CD4 cell increases from baseline (median, 188 vs. 200 cells/mm), safety profile, and incidence of ABC-related hypersensitivity reactions (9% vs. 7%). ABC administered once daily in combination with 3TC and EFV administered once daily was non-inferior to the ABC twice-daily dosing schedule when combined with 3TC and EFV over 48 weeks.     

Amphibian cardiac troponin I gene's organization, developmental expression, and regulatory properties are different from its mammalian homologue.

In mammals, the expression of the troponin I-slow (TnIs) isoform is predominant in the heart during embryogenesis and, shortly after birth, is replaced by the cardiac-specific isoform, TnIc; a developmental switch thought to be mediated by thyroid hormone. Whereas, in Xenopus, TnIc is expressed at the onset of heart formation and is the only TnI isoform expressed in the heart. Herein, we demonstrate that the expression patterns of these genes appear to be common within the anuran lineage and, unlike their mammalian counterparts, are not affected by thyroid hormone. To elucidate the regulatory mechanism(s) governing the expression of the amphibian TnIc gene, we characterized the TnIc gene from Rana catesbeiana and used its 5'-flanking region to drive expression of green fluorescent protein in the Xenopus transgenic system. Our results demonstrate that a 300-bp minimal promoter containing intact GATA and CArG-box elements is sufficient to drive expression of this reporter gene in a pattern that mimics, both spatially and temporally, the expression of the endogenous Xenopus TnIc gene.     

Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha

Telomere proteins protect the chromosomal terminus from nucleolytic degradation and end-to-end fusion, and they may contribute to telomere length control and the regulation of telomerase. The current studies investigate the effect of Oxytricha single-stranded telomere DNA-binding protein subunits α and β on telomerase elongation of telomeric DNA. A native agarose gel system was used to evaluate telomere DNA-binding protein complex composition, and the ability of telomerase to use these complexes as substrates was characterized. Efficient elongation occurred in the presence of the α subunit. Moreover, the α–DNA cross-linked complex was a substrate for telomerase. At higher α concentrations, two α subunits bound to the 16-nucleotide single-stranded DNA substrate and rendered it inaccessible to telomerase. The formation of this α·DNA·α complex may contribute to regulation of telomere length. The α·β·DNA ternary complex was not a substrate for telomerase. Even when telomerase was prebound to telomeric DNA, the addition of α and β inhibited elongation, suggesting that these telomere protein subunits have a greater affinity for the DNA and are able to displace telomerase. In addition, the ternary complex was not a substrate for terminal deoxynucleotidyltransferase. We conclude that the telomere protein inhibits telomerase by rendering the telomeric DNA inaccessible, thereby helping to maintain telomere length.     

Rapid diagnosis of typhoid fever through identification of Salmonella typhi within 18 hours of specimen acquisition by culture of the mononuclear cell-platelet fraction of blood.

Detection of Salmonella typhi in blood by culture of the mononuclear cell-platelet layer was compared with other methods currently used for the diagnosis of typhoid fever. Colonies of S. typhi were present in all mononuclear cell-platelet layer-positive cultures within 18 h of plating and were identified within an additional 10 min by a coagglutination technique. In contrast, identification of all positive cultures by conventional blood culture required 3 days.