Quantitative PCR: Procedures and precisions

We develop a multitype branching-process model for the Polymerase Chain Reaction (PCR). We apply the model to a comparison of three methods for estimating the initial number of molecules of target present in a PCR. These three methods are: one which uses a coamplified, internal control; one which uses an external control series; and one which uses simple extrapolation of log outputvs time (no control). We identify assumptions for each method which permit mathematical analysis of bias and precision. All three methods perform well if: (1) replication efficiencies are stable among reactions; (2) other method-specific conditions on efficiencies are met; and (3) product accumulates exponentially throughout the range where it is observed. When replication efficiencies vary among reactions but other optimal conditions for each method hold, the no-control and external-control methods lose precision relative to the internal control method, but they may still perform satisfactorily for many applications. The internal control method continues to perform well even if accumulation of product plateaus. This method depends, however, on a condition we call equivalence of replication efficiencies, the attainability of which in practice remains to be proven.     

Diseases and parasites of red foxes, gray foxes, and coyotes from commercial sources selling to fox-chasing enclosures.

Fifty-six red foxes (Vulpes vulpes), 18 gray foxes (Urocyon cinereoargenteus), and 13 coyotes (Canis latrans) obtained by the South Carolina Wildlife and Marine Resources Department during an investigation of suspected illegal wildlife translocation were examined for diseases and parasites. Red foxes and coyotes were confiscated from an animal dealer based in Ohio (USA), and gray foxes were purchased from an animal dealer in Indiana (USA). Emphasis was placed on detection of pathogens representing potential health risks to native wildlife, domestic animals, or humans. All animals were negative for rabies; however, 15 gray foxes were incubating canine distemper at necropsy. Serologic tests disclosed antibodies to canine parvovirus, canine distemper virus, canine adenovirus, canine coronavirus, canine herpesvirus, and canine parainfluenza virus in one or more host species. Twenty-three species of parasites (two protozoans, three trematodes, four cestodes, eleven nematodes, and three arthropods) were found, including species with substantial pathogenic capabilities. Echinococcus multilocularis, a recognized human pathogen not enzootic in the southeastern United States, was found in red foxes. Based on this information, we conclude that the increasingly common practice of wild canid translocation for stocking fox-chasing enclosures poses potential health risks to indigenous wildlife, domestic animals, and humans and, therefore, is biologically hazardous.     

Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Triglyceride interesterification by lipases. 3. Alcoholysis of pure triglycerides

Lipase-catalyzed alcoholysis of triolein dissolved in ethanol or isopropanol for the formation of ethyl and isopropyl esters was investigated. Of 16 lipases screened, Amano lipase from P. fluorescens was selected for investigation of the effects of basic reaction conditions on alcoholysis yields. Ethanolysis yields were only slightly affected by water additions to immobilized lipase preparations. Isopropyl ester yields decreased with water addition. Good operational stability was observed over 17 days. Changes in initial triolein concentration in the range 5–50 mM had very little effect on ester yields. The ionic strength of the phosphate buffer used in lipase immobilization affected ethanolysis and isopropanolysis yields in opposite ways. The highest ethanolysis yields were obtained with lipases immobilized from 250 mM buffer, while isopropyl ester yields were highest with lipases immobilized from water. In addition, the quantities and isomers of monoglyceride intermediates in ethanolysis were affected by the immobilization buffer strength. Larger quantities of 2-monoglycerides were formed in ethanolysis reactions with lipase preparations immobilized from water.     

The thermal and energetic significance of clustering in the speckled mousebird,Colius striatus

Summary The effect of clustering behaviour on metabolism, body temperature, thermal conductance and evaporative water loss was investigated in speckled mousebirds at temperatures between 5 and 36°C. Within the thermal neutral zone (approximately 30–35 °C) basal metabolic rate of clusters of two birds (32.5 J·g^-1·h^-1) and four birds (28.5 J·g^-1·h^-1) was significantly lower by about 11% and 22%, respectively, than that of individuals (36.4 J·g^-1·h^-1). Similarly, below the lower critical temperature, the metabolism of clusters of two and four birds was about 14% and 31% lower, respectively, than for individual birds as a result of significantly lower total thermal conductance in clustered birds. Body temperature ranged from about 36 to 41°C and was positively correlated with ambient temperature in both individuals and clusters, but was less variable in clusters. Total evaporative water loss was similar in individuals and clusters and averaged 5–6% of body weight per day below 30°C in individuals and below 25°C in clusters. Above these temperatures total evaporative water loss increased and mousebirds could dissipate between 80 and 90% of their metabolic heat production at ambient temperatures between 36 and 39°C. Mousebirds not only clustered to sleep between sunset and sunrise but were also observed to cluster during the day, even at high ambient temperature. Whereas clustering at night and during cold, wet weather serves a thermoregulatory function, in that it allows the brrds to maintain body temperature at a reduced metabolic cost, clustering during the day is probably related to maintenance of social bonds within the flock.Abbreviations BMR basal metabolic rate - bw body weight - C _totab total thermal conductance - EWI evaporative water loss - M metabolism - RH relative humidity - T _a ambient temperature - T _b body temperature - T _ch chamber temperature - T _cl cluster temperature - TEWL total evaporative water loss - LCT lower critical temperature - TNZ thermal neutral zone     

Fetuin: an acute phase protein in cattle

Summary Fetuin is a plasma protein present in high concentrations during fetal development in animals of the order Artiodactyla. Its role is not known. The human homologue of fetuin — ₂HS glycoprotein — has been shown to be a negative acute phase protein in adult plasma. In the present study, the concentration of fetuin was measured in the serum of healthy cattle (Bovis bovis) and in animals with various injuries and inflammatory disorders. The levels were decreased by 30% in pregnancy but increased up to 10-fold in some trauma cases. A significant negative correlation between the concentrations of fetuin and albumin has also been found. Thus, fetuin appears to be a positive acute phase protein in cattle.Abbreviations ₂HS ₂HS glycoprotein - AP acute phase     

Structure-function studies of human cholesteryl ester transfer protein by linker insertion scanning mutagenesis

Human plasma cholesteryl ester transfer protein (CETP) enhances transfer and exchange of cholesteryl ester (CE) and triglyceride (TG) between high-density lipoprotein and other lipoproteins. To define regions responsible for the neutral lipid transfer activities at the molecular level, a total of 27 linker insertion mutants at 18 different sites along the CETP molecule were prepared and transiently expressed in a mammalian cell line (COS). The inserted linkers were small (usually 6 bp) and did not interrupt the translational reading frame of the CETP cDNA. Although secretion of each mutant protein was less than that of wild-type CETP, the majority of the mutants had normal cholesteryl ester transfer activity (transfer activity per nanogram of CETP in media). However, insertional alterations in three regions severely impaired CE transfer activity: (1) in the region of amino acids 48-53; (2) at amino acid 165; and (3) in the region of amino acids 373-379. Although the impaired activities could also be a result of globally incorrect folding of these CETP mutants, hydrophobicity analysis and secondary structure predictions tended to exclude this possibility for most of the insertion sites at which insertions resulted in inactivation. The insertion at amino acid 379 occurs immediately after a triplet of lysine residues, suggesting that this region might be involved in an essential step in the mechanism of CE and TG transfer, such as the binding of CETP to phosphatidylcholine molecules in the lipoprotein surface. Effects on TG transfer activity were generally similar to those on CE transfer activity, suggesting a similar structural requirement for both neutral lipid transfer activities.     

Occurrence of 28- and 27-Carbon ecdysteroids and sterols in developing worker pupae of the leaf-cutting ant acromyrmex octospinosus (reich) (hymenoptera,formicidae: Attini)

The major ecdysteroids in large worker pupae of the leaf-cutting ant Acromyrmex octospinosus were characterized at the peak ecdysteroid concentration by using high-performance liquid chromatography, enzyme immunoassay, and mass spectrometry. In decreasing amounts, they were determined to be makisterone A, an unidentified C₂₈ ecdysteroid bearing a molecular weight of 494, 20-hydroxyecdysone (ratio of 1 to 6 as compared to makisterone A), and putative but negligible ecdysone. The presence of both C₂₈ and C₂₇ ecdysteroids is discussed in relation to the content of 4-desmethylsterols determined by gas chromatography and mass spectrometry to be ergosta-5,7,24 (28)-trien-3β-ol, ergosterol, ergosta-5,7-dien-3β-ol and ergosta-7,24(28)-dien-3β-ol for the main sterols, and with a small amount of cholesterol.