Standard and real-time multiplex PCR methods for detection of trimethoprim resistance dfr genes in large collections of bacteria.

Two multiplex PCR (mPCR) methods were developed to screen large collections of trimethoprim-resistant Escherichia coli isolates for the most prevalent resistance determinants. Five common integron-carried genes (dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17) were selected as PCR targets. Primers and conditions for standard mPCRs and real-time mPCRs were selected and tested. Two protocols using essentially the same primer pairs were established. The standard mPCR protocol also included an internal control targeting the E. coli 16S rRNA gene. Both protocols proved to be sensitive and specific for detection of the five selected genes. Screening of three different collections of clinical urinary and blood isolates (n = 368) with the two multiplex methods revealed that the five dfr genes accounted for 75-86% of trimethoprim resistance. The standard mPCR is useful and accessible for most laboratories, while the real-time mPCR requires additional equipment and expensive reagents, but is very convenient for high-throughput screening of large collections of bacterial isolates.

Antibodies from systemic lupus erythematosus (SLE) sera define differential release of autoantigens from cell lines undergoing apoptosis.

SLE is an autoimmune disease characterized by a wide range of anti-cellular and anti-nuclear autoantibodies. Many of these antigens are exposed or altered during apoptosis when the nucleus is dismantled in a controlled manner by caspases. We used Western blotting techniques to demonstrate that autoantibodies in SLE sera recognize antigens released during apoptosis. Reproducible bands, not seen in the untreated cells, with the characteristics of histones were seen when staining apoptotic cell lysates with SLE sera. Normal sera recognized some of these bands but much less strongly. Different triggers of apoptosis did not produce marked differences in the antigens recognized. We also compared different cell lines (Jurkat and U937) and found that the staining differed for one autoantigen in particular. The differential release of autoantigens by apoptotic cells may have relevance to the variety of autoantibodies seen in SLE.

Potentiation of cholinergic activity with pyridino[1,2-a]imidazo[5,4-b]indole: in vitro studies.

1. Effects of a novel imidazoindole derivative on cholinergic function were studied in isolated tissue preparations. 2. The compound demonstrated a dose-dependent (10(-11)-10(-9) potentiation (20-60%) of acetylcholine induced tension in guinea pig ileal tissue. 3. Increases in the size of end-plate potentials and nerve evoked muscle twitches were observed in frog nerve-skeletal muscle preparations. 4. Cholinesterase activity was not inhibited. 5. The results suggest that the compound has actions at the post-synaptic muscarinic receptor complex in smooth muscle and causes pre-synaptic increases in ACh release at the neuromuscular junction.

Vaso-occlusive crisis episodes in sickle cell disease.

Seventeen adult males and females with Hb-SS, Hb-SC (1) and Hb-S Thal (1) hemoglobinopathies were continuously studied for 3 years. Various hematological and biochemical parameters were measured in the venous blood of the subjects for blood gases, CBC profile, blood chemistry (SMA-18), fibrinogen, alpha-HBD and myoglobin levels, percent sickling, blood viscosity, oxygen affinity of whole blood, osmofragility of red blood cells and calcium and zinc contents in plasma and in RBC. The results were compared between those subjects who encountered more frequent vaso-occlusive crisis episodes (frequent sicklers) and those with fewer crisis episodes (infrequent sicklers), along with parameters between crisis and non-crisis states of frequent sicklers. Our studies showed that percent sickling, P50 for O2, CBC profile, PO2, serum calcium, ALP, LDH, alpha-HBD level, zinc and calcium levels in plasma and in RBC varied between crisis and non-crisis states of frequent sicklers and also between frequent sicklers and infrequent sicklers. A logical explanation of such variations may help in understanding the etiology of vaso-occlusive crisis episodes in sickle cell disease.

Effects of dietary selenium and vitamin E on plasma lipoprotein cholesterol levels in male rats.

Male Fischer-344 rats fed a diet deficient in both vitamin E and selenium (Se) for 20 weeks had higher levels of low-density lipoprotein cholesterol than age-matched rats fed an identical diet but supplemented with these micronutrients. The rats supplemented with both vitamin E and Se were switched to a diet deficient in both these micronutrients at week 20. These rats eventually developed elevated levels of low-density lipoprotein cholesterol compared to age-matched rats either continuously maintained on the diet supplemented with vitamin E and Se or rats switched (at week 20) from the vitamin E-and Se-deficient diet to a diet supplemented with both these micronutrients. In a second experiment, we found that Se deficiency alone was sufficient to significantly elevate low-density lipoprotein cholesterol. The basal diet used in these experiments had a very low cholesterol content and the observed alterations in lipoprotein cholesterol levels are likely to reflect alterations in the metabolism of endogenously synthesized cholesterol.