Behavioral and clinical pathology changes in koi carp (Cyprinus carpio) subjected to anesthesia and surgery with and without intra-operative analgesics

Fish surgery is becoming increasingly common in laboratory and clinical settings. Behavioral and physiologic consequences of surgical procedures may affect experimental results, so these effects should be defined and, if possible, ameliorated. We document behavioral and clinical pathology changes in koi carp (Cyprinus carpio) undergoing surgery with tricaine methanesulphonate (MS-222) anesthesia, with and without intraoperative administration of the opiate butorphanol (0.4 mg/kg intramuscularly) or the nonsteroidal antiinflammatory analgesic ketoprofen (2 mg/kg intramuscularly). For all fish combined, surgery resulted in reduced activity, lower position in the water column, and decreased feeding intensity at multiple time points after surgery. The butorphanol-treated group was the only one not to experience significant (P < 0.05) alterations from presurgical behaviors. Clinical pathology changes at 48 h after anesthesia and surgery included decreased hematocrit, total solids, phosphorus, total protein, albumin, globulin, potassium, and chloride and increased plasma glucose, aspartate aminotransferase, creatine kinase, and bicarbonate. The only clinical pathology difference between treatment groups was a lower increase in creatine kinase in the ketoprofen-treated group. No adverse effects of butorphanol or ketoprofen at these doses were identified. These results suggest a mild behavioral sparing effect of butorphanol and reduced muscle damage from the antiinflammatory activity of ketoprofen.     

Fractionation of plant protoplast types by iso-osmotic density gradient centrifugation

Summary A simple effective technique for the fractionation of protoplast populations is described. Protoplasts are separated by low-speed centrifugation in an iso-osmotic, discontinuous density gradient system on the basis of differences in their buoyant densities. At a constant osmolality of 660±20 mOs/kg H₂O, the gradients provide a density range from 1.017 to 1.069 g/cm³ at 20 °C which corresponds to the buoyant densities of most protoplast types studied. Characteristics of the KMC/S-density gradient system and factors affecting the fractionation were investigated. Protoplasts were isolated from various tissues and cultivars of tobacco, barley, wheat, rye, oat and maize. Their density-dependent distribution profiles in KMC/S-gradients and their average buoyant densities were determined under standardized conditions. Great differences in the buoyant densities were found between protoplasts of different tissues. Mixed populations of two types of protoplasts, differing in buoyant density by about 15–20 mg/cm³, were separated to give highly purified fractions. Factors affecting the buoyant densities of protoplasts have been investigated. Ploidy level and species differences did not significantly affect the fractionation profiles. However, an age-dependent variation in the average buoyant density of tobacco mesophyll protoplasts was observed. Fractionation of tobacco mesophyll protoplasts and their subsequent regeneration to plants demonstrates the practicability and physiological compatibility of the KMC/S-density gradient system under sterile conditions. The morphogenetic potential of protoplasts was not affected by the separation procedure or the gradient components.     

Smaller lungs in women affect exercise hyperpnea

We subjected 29 healthy young women (age: 27 ± 1 yr) with a wide range of fitness levels [maximal oxygenuptake (O_{2 max}): 57 ± 6 ml · kg¹ · min¹;35-70ml · kg¹ · min¹]to a progressive treadmill running test. Our subjects had significantly smaller lung volumes and lower maximal expiratory flow rates, irrespective of fitness level, compared with predicted values for age-and height-matched men. The higher maximal workload in highly fit(O_{2 max} > 57 ml · kg¹ · min¹,n = 14) vs. less-fit(O_{2 max} < 56 ml · kg¹ · min¹,n = 15) women caused a higher maximalventilation (E) with increased tidal volume (VT)and breathing frequency (f_b) atcomparable maximal VT/vitalcapacity (VC). More expiratory flow limitation (EFL; 22 ± 4% ofVT) was also observed duringheavy exercise in highly fit vs. less-fit women, causing higherend-expiratory and end-inspiratory lung volumes and greater usage oftheir maximum available ventilatory reserves.HeO₂ (79% He-21%O₂) vs. room air exercise trialswere compared (with screens added to equalize external apparatusresistance). HeO₂ increasedmaximal expiratory flow rates (20-38%) throughout the range ofVC, which significantly reduced EFL during heavy exercise. When EFL wasreduced with HeO₂, VT,f_b, andE (+16 ± 2 l/min) weresignificantly increased during maximal exercise. However, in theabsence of EFL (during room air exercise),HeO₂ had no effect onE. We conclude that smaller lungvolumes and maximal flow rates for women in general, and especiallyhighly fit women, caused increased prevalence of EFL during heavyexercise, a relative hyperinflation, an increased reliance onf_b, and a greater encroachment onthe ventilatory "reserve." Consequently,VT andE are mechanically constrained duringmaximal exercise in many fit women because the demand for highexpiratory flow rates encroaches on the airways' maximum flow-volumeenvelope.

     

Anaerobic utilization of alkylbenzenes and n-alkanes from crude oil in an enrichment culture of denitrifying bacteria affiliating with the beta-subclass of Proteobacteria

Denitrifying bacteria were enriched from freshwater sediment with added nitrate as electron acceptor and crude oil as the only source of organic substrates. The enrichment cultures were used as laboratory model systems for studying the degradative potential of denitrifying bacteria with respect to crude oil constituents, and the phylogenetic affiliation of denitrifiers that are selectively enriched with crude oil. The enrichment culture exhibited two distinct growth phases. During the first phase, bacteria grew homogeneously in the aqueous phase, while various C₁–C₃ alkylbenzenes, but no alkanes, were utilized from the crude oil. During the second phase, bacteria also grew that formed aggregates, adhered to the crude oil layer and emulsified the oil, while utilization of n -alkanes (C₅ to C₁₂) from the crude oil was observed. During growth, several alkylbenzoates accumulated in the aqueous phase, which were presumably formed from alkylbenzenes. Application of a newly designed, fluorescently labelled 16S rRNA-targeted oligonucleotide probe specific for the Azoarcus / Thauera group within the β-subclass of Proteobacteria revealed that the majority of the enriched denitrifiers affiliated with this phylogenetic group.     

Inhibition of peptide bond formation by pleuromutilins: the structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin

Tiamulin, a prominent member of the pleuromutilin class of antibiotics, is a potent inhibitor of protein synthesis in bacteria. Up to now the effect of pleuromutilins on the ribosome has not been determined on a molecular level. The 3.5 A structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin provides for the first time a detailed picture of its interactions with the 23S rRNA, thus explaining the molecular mechanism of the antimicrobial activity of the pleuromutilin class of antibiotics. Our results show that tiamulin is located within the peptidyl transferase center (PTC) of the 50S ribosomal subunit with its tricyclic mutilin core positioned in a tight pocket at the A-tRNA binding site. Also, the extension, which protrudes from its mutilin core, partially overlaps with the P-tRNA binding site. Thereby, tiamulin directly inhibits peptide bond formation. Comparison of the tiamulin binding site with other PTC targeting drugs, like chloramphenicol, clindamycin and streptogramins, may facilitate the design of modified or hybridized drugs that extend the applicability of this class of antibiotics.     

Laser light-scattering evidence for an altered association of beta B1-crystallin deamidated in the connecting peptide

Deamidation is a prevalent modification of crystallin proteins in the vertebrate lens. The effect of specific sites of deamidation on crystallin stability in vivo is not known. Using mass spectrometry, a previously unreported deamidation in beta B1-crystallin was identified at Gln146. Another deamidation was investigated at Asn157. It was determined that whole soluble beta B1 contained 13%-17% deamidation at Gln146 and Asn157. Static and quasi-elastic laser light scattering, circular dichroism, and heat aggregation studies were used to explore the structure and associative properties of recombinantly expressed wild-type (wt) beta B1 and the deamidated beta B1 mutants, Q146E and N157D. Dimer formation occurred for wt beta B1, Q146E, and N157D in a concentration-dependent manner, but only Q146E showed formation of higher ordered oligomers at the concentrations studied. Deamidation at Gln146, but not Asn157, led to an increased tendency of beta B1 to aggregate upon heating. We conclude that deamidation creates unique effects depending upon where the deamidation is introduced in the crystallin structure.     

Ribosomal crystallography: from poorly diffracting microcrystals to high-resolution structures

The cellular organelles translating the genetic code into proteins, the ribosomes, are large, asymmetric, flexible, and unstable ribonucleoprotein assemblies, hence they are difficult to crystallize. Despite two decades of intensive effort and thorough searches for suitable sources, so far only three crystal types have yielded high-resolution structures: two large subunits (from an archaean and from a mesophilic eubacterium) and one thermophilic small subunit. These structures have added to our understanding of decoding, have revealed dynamic aspects of the biosynthetic process, and have indicated the strategies adopted by ribosomes for interacting between themselves as well as with inhibitors, factors and substrates.