Metabolic responses of striped bass (Morone saxatilis) to temperature acclimation. I. Alterations in carbon sources for hepatic energy metabolism.

The relative contribution of carbohydrate and lipid energy metabolism in liver tissue of temperature-acclimated striped bass was examined in vitro. Respirometry experiments were conducted to assess the role of various endogenous foodstuffs in providing reduced two-carbon fragments for aerobic metabolism. Liver composition was measured as a reflection of foodstuff flux and storage. Catabolism of 14C-labeled substrates to 14CO2 was monitored to estimate the tissue capacity for utilization of various classes of compounds. When measured at the temperature of acclimation, oxygen uptake (VO2) by liver slices shows near perfect compensation between 15 and 25 degrees C, whereas a 2.75-fold increase is found between 5 and 15 degrees C. Respiratory quotients (R.Q.) near unity are found at 5 degrees C and decrease to 0.85 and 0.82 at 15 and 25 degrees C, respectively. Inhibition of VO2 by iodoacetic acid, a glycolytic inhibitor, is near 60% at 5 degrees C and decreases to 30-40% at 15 and 25 degrees C. Evolution of 14CO2 from 14C-labeled glucoses and palmitate confirms a conservation of liver tissue capacity for carbohydrate utilization between acclimation temperatures of 5 and 15 degrees C, and an increasing capacity for utilization of fatty acids as temperature increases. Ratios of 14CO2 from 14C-6-and 14C-1-labeled glucoses indicate a relative increase in the participation of the pentose shunt in carbohydrate metabolism with cold acclimation. The results are consistent with an increasing reliance on carbohydrates for energy metabolism in the cold, whereas lipid substrates are utilized more at warm temperatures. These changes may be adaptive in partitioning energy reserves for seasonal activities of migration and reproduction.

Two compartment kinetic model with multiple artificial capillary units.

A two compartment in-vitro model was designed to simulate human pharmacokinetics and to expose bacterial cultures to changing drug concentrations, thereby avoiding limitations of conventional antibiotic testing at constant drug levels. Serially placed bacterial compartments, representing extravascular infection sites, interface with a central compartment through artificial capillaries. Drug concentrations within the culture chambers closely mimic interstitial concentrations in vivo. Simultaneous first order elimination kinetics of two drugs with different half-lives were simulated to study antibacterial effects of drug combinations. This in-vitro model is an efficient tool for optimal dosage regimen design and the study of synergistic/antagonistic effects of antibiotic combinations.

Efficacy of intermittent versus continuous administration of netilmicin in a two-compartment in vitro model.

Several aminoglycoside dosage regimens were studied in a kinetic in vitro model. Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus were exposed in serially placed artificial capillary units to netilmicin concentrations that changed based on human two-compartment pharmacokinetics. The same total dose per 24 h was administered as a continuous infusion (3.7 micrograms/ml) or in 1-h infusions given every 24 (24 micrograms/ml) or 8 h (8 micrograms/ml). The once daily administration showed the best response in terms of either faster killing of E. coli, K. pneumoniae, and S. aureus or greater reduction of the inocula of P. aeruginosa. After 28 h of treatment, however, all regimens reduced the nonpseudomonads by more than 99.99%, whereas all three P. aeruginosa strains regrew to greater than 10(8) CFU/ml due to selection of resistant subpopulations. In contrast to the bactericidal effect of the first dose, no killing occurred after subsequent doses if the ratio of peak drug concentration to MIC was low (less than or equal to 6). These results support the concept of administering high doses of aminoglycosides once every 24 h.

Comparison of Mycobacterium 23S rRNA sequences by high-temperature reverse transcription and PCR.

We describe a modified rRNA sequence analysis method which we used to determine the phylogenetic relationships among 58 species belonging to the genus Mycobacterium. We combined the sensitivity of the reverse transcriptase PCR for amplifying nanogram amounts of template rRNA material with the elevated extension temperatures used for the thermostable DNA polymerase from Thermus thermophilus. A 70 degrees C reverse transcription extension step permitted improved read-through of highly structured rRNA templates from members of the genus Mycobacterium, which have G+C contents of 66 to 71 mol%. The nucleic acid sequences of the amplified material were then determined by performing thermal cycle sequencing with alpha-33P-labeled primers, again with extension at 70 degrees C. Nonspecifically terminated bands were chased by using terminal deoxynucleotidyl transferase. Our method had a template requirement of nanogram amounts or less of purified RNA or 2,000 CFU of intact cells and had sufficient sensitivity so that lyophils obtained from the American Type Culture Collection could be used as source material. Sequences from a 250-nucleotide stretch of the 23S rRNA were aligned, and phylogenetic trees were evaluated by using the De Soete distance treeing algorithm and Rhodococcus bronchialis as the outgroup. Our 23S rRNA trees were compared with previously published 16S rRNA trees, including the comprehensive trees developed by the University of Illinois Ribosomal Database Project, and included 15 species not evaluated previously. Most of the groups were in general agreement and were consistent with relationships determined on the basis of biochemical characteristics, but some new relationships were also observed.

Use of an in-vitro kinetic model to study antibiotic combinations.

A two compartment pharmacokinetic model was used to study combinations of piperacillin with N-formimidoyl thienamycin or amikacin, and azlocillin with netilmicin against strains of Pseudomonas aeruginosa. Antibiotic antagonism seen with in-vitro static tests of piperacillin and thienamycin did not occur with the kinetic model. Piperacillin plus amikacin showed enhanced activity, and azlocillin prevented bacterial regrowth seen with netilmicin alone during multiple dosing experiments at high bacterial inocula. This model is useful in the study of antibiotic combinations.

Impact of netilmicin regimens on the activities of ceftazidime-netilmicin combinations against Pseudomonas aeruginosa in an in vitro pharmacokinetic model.

The antibacterial activities of ceftazidime and netilmicin were studied in a two-compartment in vitro model. Pseudomonas aeruginosa cultures were exposed to changing drug concentrations that mimic human pharmacokinetics. Netilmicin alone reduced the numbers of organisms in cultures of the susceptible strains by more than 99% within 4 h; however, regrowth occurred after 8 h. Although ceftazidime alone killed more slowly than netilmicin, only one of the five strains regrew within 28 h. When both drugs were combined, rapid initial killing occurred without subsequent regrowth. Studied after 24 h in combination with ceftazidime, netilmicin was as effective when given as a single daily dose as when administered in three daily doses that provided 50% more aminoglycoside per day. Decreased bacterial susceptibility was seen after ceftazidime exposure for one strain and after netilmicin exposure for all originally netilmicin-susceptible strains. No such reduction in susceptibility was observed during exposure to the combination. The results of standard in vitro checkerboard tests for synergism were predictive of the initial (4 to 8 h) but not the final (24 to 28 h) assessment of drug interaction in the pharmacokinetic model.

Comparative study with enoxacin and netilmicin in a pharmacodynamic model to determine importance of ratio of antibiotic peak concentration to MIC for bactericidal activity and emergence of resistance.

An in vitro pharmacokinetic model was used to study the comparative antibacterial activities of multiple-dose regimens of enoxacin and netilmicin. Strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus were exposed to changing drug concentrations, mimicking human two-compartment pharmacokinetics. Oral administration was simulated for the quinolone, and intravenous administration was simulated for the aminoglycoside. Similar ratios of peak concentration to MIC resulted in similar changes in bacterial concentrations over time with both compounds. Following the initial dose, a rapid bactericidal effect occurred, with a greater than 99% reduction of the bacterial counts within 4 h at peak concentrations more than three times the MIC. However, bacterial regrowth occurred within 24 h unless the peak concentration/MIC ratio exceeded 8:1 (P less than 0.01). For the regrowing bacteria, MICs were four- to eightfold higher, and little or no bactericidal effect occurred following the second and subsequent doses. These data demonstrate the equally potent bactericidal activity of orally administered enoxacin and intravenously administered netilmicin. Selection of resistant subpopulations was similar with each drug. The peak concentration/MIC ratio may be an important parameter in the clinical use of quinolone and aminoglycoside antibiotics.

Cerebellar T-cell lymphoma: an unusual primary intracranial neoplasm.

Primary T-cell lymphoma within the central nervous system is extremely rare. Imaging characteristics appear indistinguishable from the more common B-cell lymphoma. A case of such a primary tumor is discussed and the MRI and CT findings presented.

Detection of rRNA from four respiratory pathogens using an automated Q beta replicase assay.

Ribosomal RNA targets from Mycobacterium avium complex (23S), Mycoplasma pneumoniae (16S), Pneumocystis carinii (18S) and Legionella pneumophila (16S) were detected in four separate assays on a model automated Q-beta amplification instrument. Sandwich hybridization, reversible target capture, detector probe amplification and fluorescent signal detection occurred in closed, disposable packs at 38 degrees C. Packs were injected with 0.5 ml samples in 3.06 M guanidine thiocyanate. Ten samples per run were read after 7 h, requiring only 4 min loading time. Synthetic RNA transcripts and purified, natural RNAs from up to four different strains per assay were diluted to 10(6) or fewer molecules per sample (approximately 100 cells for prokaryotes, 10 cells for Pneumocystis). All analytes were detected at 10(6) targets. The limits of detection were found at 10(5) to 10(4). Discrimination against competitor RNA was tested using up to 10(9) molecules (1000 X excess) of appropriate test strains. Samples containing either zero targets or 10(7) competitors produced negative results in 95 to 100% of the samples, depending on the assay. Closely related Legionella and Mycoplasma species cross-reacted at high challenge levels of 10(9) molecules as a result of sequence similarities in the target regions. These results demonstrate the utility and versatility of an automated, high sensitivity, closed system for amplified analysis of direct-from-sample testing of respiratory pathogens.