Accuracy and repeatability of Roentgen stereophotogrammetric analysis (RSA) for measuring knee laxity in longitudinal studies

Roentgen stereophotogrammetric analysis (RSA) can be used to assess temporal changes in anterior-posterior (A-P) knee laxity. However, the accuracy and precision of RSA is dependent on many factors and should be independently evaluated for a particular application. The objective of this study was to evaluate the use of RSA for measuring A-P knee laxity. The specific aims were to assess the variation or "noise" inherent to RSA, to determine the reproducibility of RSA for repeated A-P laxity testing, and to assess the accuracy of these measurements. Two experiments were performed. The first experiment utilized three rigid models of the tibiofemoral joint to assess the noise and to compare digitization errors of two independent examiners. No differences were found in the kinematic outputs of the RSA due to examiner, repeated trials, or the model used. In a second experiment, A-P laxity values between the A-P shear load limits of +/-60 N of five cadaver goat knees were measured to assess the error associated with repeated testing. The RSA laxity values were also compared to those obtained from a custom designed linkage system. The mean A-P laxity values with the knee 30 degrees, 60 degrees, and 90 degrees of flexion for the ACL-intact goat knee (+/-95% confidence interval) were 0.8 (+/-0.25), 0.9 (+/-0.29), and 0.4 (+/-0.22) mm, respectively. In the ACL-deficient knee, the A-P laxity values increased by an order of magnitude to 8.8 (+/-1.39), 7.6 (+/-1.32), and 3.1 (+/-1.20)mm, respectively. No significant differences were found between the A-P laxity values measured by RSA and the independent measurement technique. A highly significant linear relationship (r(2)=0.83) was also found between these techniques. This study suggests that the RSA method is an accurate and precise means to measure A-P knee laxity for repeated testing over time.     

Effects of fasting and exogenous melatonin on annual rhythms in the blue fox (Alopex lagopus)

The arctic fox (Alopex lagopus) is a winter-active inhabitant of the high arctic with extreme fluctuations in photoperiod and food availability. The blue fox is a semi-domesticated variant of the wild arctic fox reared for the fur industry. In this study, 48 blue foxes were followed for a year in order to determine the effects of exogenous melatonin and wintertime food deprivation on their reproductive and thyroid axes. Half of the animals were treated with continuous-release melatonin capsules in July 2002, and in November-January, the animals were divided into three groups and either fed continuously or fasted for one or two 22-day periods. Food deprivation decreased the plasma triiodothyronine and thyroxine concentrations probably in order to preserve energy due to a decreased metabolic rate. The same was observed in the plasma testosterone levels of the males but not in the plasma estradiol concentrations of the females. Exogenous melatonin advanced the autumn moult and seasonal changes in the voluntary food intake. It also advanced the onset of the testosterone peak in the males. The plasma estradiol levels of the females were unaffected, but the progesterone levels peaked more steeply in the sham-operated females. Melatonin exerted a strong influence not only on the reproductive axis of the males but also on the seasonal food intake. The species seemed quite resistant to periodic involuntary food deprivation.     

Sequence-selective cleavage of oligoribonucleotides by 3d transition metal complexes of 1,5,9-triazacyclododecane-functionalized 2'-O-methyl oligoribonucleotides

2'-O-Methyl oligoribonucleotides bearing a 3'-[2,6-dioxo-3,7-diaza-10-(1,5,9-triazacyclododec-3-yl)decyl phospate conjugate group have been shown to cleave in slight excess of Zn(2+) ions complementary oligoribonucleotides at the 5'-side of the last base-paired nucleotide. The cleavage obeys first-order kinetics and exhibits turnover. The acceleration compared to the monomeric Zn(2+) 1,5,9-triazacyclododecane chelate is more than 100-fold. In addition, 2'-O-methyl oligoribonucleotides having the 1,5,9-triazacyclododec-3-yl group tethered to the anomeric carbon of an intrachain 2-deoxy-beta-d-erythro-pentofuranosyl group via a 2-oxo-3-azahexyl, 2,6-dioxo-3,7-diazadecyl, or 2,9-dioxo-3,10-diazatridecyl linker have been studied as cleaving agents. These cleave as zinc chelates a tri- and pentaadenyl bulge opposite to the conjugate group approximately 50 times as fast as the monomeric chelate and show turnover. The cleavage rate is rather insensitive to the length of linker. Interestingly, a triuridyl bulge remains virtually intact in striking contrast to a triadenyl bulge. Evidently binding of the zinc chelate to a uracil base prevents its catalytic action. Replacement of Zn(2+) with Cu(2+) or Ni(2+) retards the cleaving activity of all the cleaving agents tested.     

Preparation of azacrown-functionalized 2'-O-methyl oligoribonucleotides, potential artificial RNases

An improved synthesis for 3-(3-aminopropyl)- and 3-(3-mercaptopropyl)-1,5,9-triazacyclododecane has been developed and alternative methods for their conjugation to oligonucleotides have been described. Accordingly, the 3-aminopropyl azacrown and its N-(3-aminopropanoyl)-3-aminopropyl analogue have been tethered to the 3'-terminus of a 2'-O-methyloligoribonucleotide by aminolytic cleavage of the thioester linker utilized for the chain assembly. Studies on a monomeric model compound verify that the reaction proceeds solely by the attack of the primary amino group. 5'-Conjugation has been achieved by introducing a 2-benzylthio-2-oxoethyl group to the 5'-terminus as a phosphoramidite reagent and cleaving the thioester bond with the 3-aminopropyl azacrown. For intrachain conjugation, a phosphoramidite reagent derived from 1-deoxy-1-(2-benzylthio-2-oxoethyl)-beta-d-erythro-pentofuranose has been inserted in a desired position within the chain and subjected to on-support aminolysis with the 3-aminopropyl azacrown or its N-(3-aminopropanoyl)-3-aminopropyl and N-(6-aminohexanoyl)-3-aminopropyl analogues. The 3-mercaptopropyl-derivatized azacrown has been tetherd by a disulfide bond to a 3'-(3-mercaptoalkyl)phosphate-tailed oligonucleotide. The 3'- and intrachain-tethered conjugates have been shown to cleave as their Zn(II) chelate complementary oligoribonucleotide sequences.     

Detection, analysis and interactions of plasma ghrelin, leptin and growth hormone in the mink (Mustela vison)

The aim of this study was to obtain basic knowledge of the plasma concentrations and interactions of weight regulatory hormones in juvenile minks (Mustela vison). Ghrelin, leptin, and growth hormone (GH) levels were validated and determined by radioimmunoassay methods from the plasma of 30 female and 30 male minks. The female minks had higher plasma ghrelin and GH levels than the males. The plasma ghrelin concentrations of the females correlated positively with their body masses (BMs). The plasma leptin levels did not differ between sexes, but there was a positive correlation between the plasma leptin concentrations and BMs in the male minks. When the data from the male and female minks were combined, the correlation between the leptin levels and the BMs was still clear, but this was not observed in the females alone. In the male minks, the plasma GH levels correlated positively with the BMs and with the plasma leptin concentrations. However, there was no correlation between the plasma ghrelin and GH or leptin concentrations. The hormone concentrations were quite similar to earlier measurements in other carnivores.     

Adaptations of the raccoon dog (Nyctereutes procyonoides) to wintering--effects of restricted feeding or periodic fasting on lipids, sex steroids and reproduction

The raccoon dog (Nyctereutes procyonoides) is an omnivorous canid with autumnal hyperphagia and fattening followed by mid-winter passivity and fasting in boreal latitudes with seasonal snow cover. The effects of two different feeding levels (400 or 200 kcal/animal/d) or fasting (5-week fasting+1-week feeding+3-week fasting) on plasma lipids, sex steroids and reproductive success of farm-bred raccoon dogs (n=60 females and 24 males) were studied in winter. The body masses, body mass indices (BMIs) and levels of plasma triacylglycerols (TG), total cholesterol and low- and high-density lipoprotein cholesterol did not differ between the fed and the restrictively fed animals. During fasting, the plasma TG concentrations increased and the BMIs decreased, indicating the release of fatty acids from adipose tissue. After the fasting periods, the levels of plasma cholesterol and high-density lipoprotein cholesterol increased, whereas the TG levels decreased indicating the rebuilding of energy reserves. The fact that the different wintertime feeding regimes had no impact on the plasma glucose, total protein, cortisol, estradiol, progesterone or testosterone levels, or on the reproductive success, indicates versatile adaptive capacity in the species.     

Thermoregulatory adaptations of the overwintering captive raccoon dog (Nyctereutes procyonoides) in boreal climate

The raccoon dog (Nyctereutes procyonoides) is a nocturnal canid thought to utilise passive wintering strategy in the boreal climate. To record the deep body temperature (T(b)), 12 farmed raccoon dogs were implanted with intra-abdominal T(b) loggers on November 26, 2003. Between December 3, 2003 and January 27, 2004 half of the animals were fasted for 8 weeks. The amplitude of the diurnal T(b) oscillations increased due to fasting. However, the mean diurnal T(b) was lower in the fasted animals only during two occasions. Unlike observed previously in other species, not only did the raccoon dogs experience hypothermia between 0600 and 1000 hr but also hyperthermia between noon and 1800 hr. The fasted animals were as active as the fed animals measured after 42-43 days of fasting and there was a significant cross-correlation between physical activity and T(b). The nocturnal period of hypothermia is probably an adaptation to save energy during food deprivation. The diurnal hyperthermia could be explained by the opportunistic foraging behaviour of the species. Opposite to the established assumptions, the raccoon dog does not seem to enter winter sleep on fur farms. In the future it is important to determine if true winter sleep occurs in nature in the species.     

Endocrinologic adaptations to wintertime fasting in the male American mink (Mustela vison)

The aim of this study was to investigate the endocrine response to wintertime starvation in the male American mink (Mustela vison) fasted for 16 hrs, 2 days, 3 days, 5 days, or 7 days (n =10 per group). After 2 days of fasting, the plasma leptin concentrations decreased, along with the triiodothyronine, testosterone, and progesterone levels, and the blood monocyte counts. Leptin also seems to trigger the response to fasting in mustelids by inducing immunosuppression and downregulation of the reproductive and thyroid axes. The dramatic increase in the peptide YY concentrations after 3 days of fasting may be required to suppress gastrointestinal processes during food scarcity. The plasma insulin levels decreased, and those of glucagon increased after 5 days of fasting in association with efficient glucose sparing and lipid mobilization. Body energy stores cannot be wasted for growth during nutritional scarcity and, thus, the growth hormone levels of the minks decreased after 5 days of fasting. The plasma noradrenaline and cortisol concentrations also decreased after 3 and 7 days without food, respectively. The plasma ghrelin, adiponectin, resistin, thyroxine, adrenaline, or estradiol levels did not respond to fasting. The endocrine response to food deprivation is remarkably similar in divergent mammalian orders, indicating that the hormonal signals enhancing survival during nutritional scarcity must be evolutionarily old and well conserved.     

Concentrations of Tryptoline and Methtryptoline in Rat Brain

Combined gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry have been used to identify and quantify tryptoline, methtryptoline, 5-hydroxytryptoline, and 5-hydroxymethtryptoline as their heptafluorobutyryl derivatives in extracts of rat brain. Tryptoline and methtryptoline were identified on the basis of their retention times and mass spectral characteristics: they were reliably detected in brain tissue extracts without interference from artifactual formation; their whole brain concentrations ranged between 0.2 and 3 ng/g; and they had a similar neuroanatomical distribution, with the highest concentrations in the cerebellum and the cortex. Smaller quantities of 5-hydroxytryptoline and 5-hydroxymethtryptoline were also identified on the basis of their retention times and mass spectral characteristics. However, the significance of this finding is unclear, because these two compounds were accompanied by larger quantities of their tetradeuterated analogues formed from tetradeuterated-5-hydroxytryptamine added at the time of tissue homogenization; this result suggests that formation of 5-hydroxytryptoline and 5-hydroxymethtryptoline occurred during tissue homogenization, sample preparation, or both.     

Nupharopumiline,a new quinolizine alkaloid from Nuphar pumila

A new quinolizine alkaloid, (+)-nupharopumiline, has been isolated from the rhizomes of Nuphar pumila. It has been shown by spectroscopic and chemi