Fluorescence in situ hybridization (FISH): DNA probe production and hybridization criteria

We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA sequences with accuracy and sensitivity for application in both basic research and clinical diagnosis. Due to the frequent use of FISH in many laboratories, it is important to apply the most convenient and reproducible approach. This review describes some of the most recent techniques, and includes versatile, effective and simple methods of probe production and fluorescence in situ hybridization. We also describe methods for the production of region-specific and chromosome-specific DNA probes and hybridization techniques for the visualization of these probes.     

The genetic effects of environmental lead

Distribution and metabolism of the thyroid hormone 3,5, 3'-l-triiodothyronine (T3) were studied in several ways to gain insights into these processes in the warm water fish tilapia Oreochromis mossambicus. Trace doses of 125I-labeled T3 (T*3)1 were injected intraarterially, extraarterially, or intraperitoneally in freshwater-reared male tilapia to explore plasma clearance kinetic responses to these different input modalities. Multicompartmental analysis of the plasma clearance data indicated a kinetic distribution of T*3 much like that reported for the rat and human, with about 2% of total body T*3 in plasma, 5% in rapidly exchanging tissues such as kidney and liver, and 93% in slowly exchanging tissues such as muscle. However, plasma clearance rates (PCR, 5.37 mL/h . 100 g body wt) and plasma appearance rates (PAR3 = PCR x [T3] plasma = 36.3 ng/h . 100 g body wt) were quite different than these indices in rat and human and 5 to 50 times larger than values reported for rainbow trout. On a whole-body basis, normalized for body weight, the tilapia we studied produced and accumulated much more T3 than rat, human, or rainbow trout. Enzymatic and chromatographic analyses of the plasma clearance data samples indicated substantial production of labeled glucuronide, but not sulfate, conjugates of iodothyronines (TiG) of unknown origin appearing in plasma. The TiG appeared beginning a few hours postinjection, peaked at 6 hours, and yielded a predicted steady-state TiG level of 8.3% of the T3 level in plasma. In contrast, in published studies, no conjugates were detected in rainbow trout plasma from 2 to 24 h after iv injection of T*3, T*4, or reverse-T*3, although conjugates of all were present in bile. To our knowledge, although T3 and T4 sulfate conjugates are present in the sera of several mammals, this is the first quantification of iodothyronine glucuronides reported in blood of any species under normal conditions. This might have physiological significance for the tilapia, with T3G providing a reversible storage form of T3 in blood, as has been suggested for sulfate conjugates of T3 and T4 in blood of several mammals.     

Extended-spectrum beta-lactamase-producing Klebsiella pneumoniae strains causing nosocomial outbreaks of infection in the United Kingdom

Representative isolates from 10 distinct extended-spectrum beta-lactamase-producing strains of Klebsiella pneumoniae that caused hospital outbreaks in the United Kingdom from 1991 to 1994 were examined for relationships between their enzymes and plasmids. The beta-lactamases were identified by a combination of isoelectric focusing and gene sequencing. SHV-2 beta-lactamase was produced by isolates from four outbreaks, SHV-5 was involved in three, and SHV-4, TEM-15, and TEM-26 were involved in one outbreak each. All of the extended-spectrum beta-lactamases were encoded by self-transmissible plasmids, with sizes ranging from about 70 to 160 kb. No similarities between the restriction digest patterns of the extended-spectrum beta-lactamase-encoding plasmids were detected, except to some extent between those that produced TEM-15 and TEM-26. Thus, outbreaks of hospital infection with these organisms in the United Kingdom from 1991 to 1994 involved distinct organisms and resistance plasmids and appeared to be unrelated.     

Aorto-iliac thrombosis in a foal

A six-day-old Missouri foxtrotter colt was examined because it had had diarrhoea since it was 24 hours old. A diagnosis of colitis, septicaemia, and disruption of the arterial blood flow to the pelvic limbs was made on the basis of clinical and laboratory findings. Despite intensive medical therapy, the foal died 13 hours after being examined. Postmortem examination revealed diffuse fibrinous enteritis with lymphoid necrosis, multifocal fibrinonecrotic typhlocolitis, disseminated intravascular coagulation, and a large occluding thrombus at the aortic termination. The results of bacteriological culturing supported the diagnosis of septicaemia leading to activation of the clotting cascade, disseminated intravascular coagulation, aorto-iliac thrombosis and infarction of the pelvic limbs.     

High-performance liquid chromatography determination of mycophenolic acid and its glucuronide metabolite in human plasma

Two HPLC-UV assays are reported here: one is a rapid assay for mycophenolic acid (MPA) and the other is a simultaneous assay for MPA and its metabolite mycophenolic acid glucuronide (MPAG). For both methods, plasma samples (500 microl) with added internal standard were acidified and extracted using C18 solid-phase extraction cartridges. Chromatographic separation was achieved on a C18 Novapak column using a mobile phase consisting of methanol-0.05% orthophosphoric acid (40:60, v/v) for the rapid MPA assay and 30:70 for the simultaneous MPA and MPAG assay. The assays were linear over the ranges 0.1 to 50.0 mg/l for MPA and 2.8 to 225.8 mg/l for MPAG. Mean absolute recovery for all analytes was >99%. These methods are suitable for therapeutic drug monitoring and pharmacokinetic studies.     

The influence of advanced age on the outcome of assisted reproduction

This study investigates within-subject variations and associations of salivary viscosities and flow rates in a test panel of healthy adults. After several practice sessions, unstimulated and stimulated whole saliva samples were collected 5 times daily (at 0800, 1100, 1400, 1700, and 2000 h) from 30 university students. There was a significant within-subject variation in viscosity and flow rate of unstimulated saliva (P<0.001). Intra-item correlations were significantly different for salivary flow rates (r= 0.82 for unstimulated, r= 0.88 for stimulated, P< 0.001) and viscosity of unstimulated saliva (r= 0.54, P< 0.05), but viscosity of stimulated saliva was different in this respect. Our results indicate that there is a significant within-subject variation in viscosity of unstimulated saliva.     

Preweaning growth traits for Senepol, Hereford, and reciprocal crossbred calves and feedlot performance and carcass characteristics of steers

We conducted a multiyear study in two phases to determine preweaning performance traits of Senepol (S x S), Hereford (H x H), and reciprocal (S x H and H x S) F1 crossbred calves and feedlot performance and carcass characteristics of steers. In Phase I, from 1985 to 1989, data from S x S (n = 194), H x H (n = 383), and S x H (n = 120) calves were used. Numbers of S x S cows were increased during Phase I so that data from H x S (n = 74) calves could be included in Phase II (1990 to 1992) in addition to S x S (n = 118), H x H (n = 130), and S x H (n = 56) calves. Also during Phase II, feedlot performance and carcass characteristics were determined for S x S (n = 30), H x H (n = 26), H x S (n = 36), and S x H (n = 26) steers. In Phase I, S x S calves had heavier (P < .01) birth weights and heavier (P < .01) 205-d adjusted weaning weights than H x H calves. Birth weights of S x H calves were heavier (P < .01) than the mean of the purebred calves, but 205-d adjusted weaning weights did not differ (P > .10). In phase II, direct heterosis was 3.5% for birth weight (P < .05) and 5.1% for 205-d adjusted weaning weight (P < .01). Senepol maternal breed effects were 1.9 kg for birth weight (P < .10) and 37.9 kg for 205-d adjusted weaning weight (P < .01). Levels of direct heterosis, Senepol maternal breed effects, and Hereford direct breed effects were significant for most feedlot performance traits of steer calves that were fed to a common end point. Breeds did not differ (P > .10) for USDA yield and quality grades, and direct heterosis was not significant for Warner-Bratzler shear force. These results demonstrate significant levels of heterosis in preweaning performance between S x S and H x H calves and in feedlot performance of steers. Levels of heterosis were smaller and nonsignificant for most carcass traits including meat tenderness, which did not differ between S x S and H x H steers in this study.     

The use of controlled-release oxycodone for the treatment of chronic cancer pain: a randomized, double-blind study

1. We have studied the effects of muscarinic cholinoceptor agonists and subtype-preferring antagonists on the isometric contraction of smooth muscle strips from dog prostate. 2. Acetylcholine and carbachol induced contraction of prostate strips from the peripheral zone, ('the capsule'). Bethanechol contracted the tissue but not at lower doses. McN-A-343 and oxotremorine-M showed the same effects. 3. Blocking alpha- and beta-adrenoceptors with phentolamine and propranolol, respectively, did not modify carbachol-induced contractions. 4. The nicotinic receptor blocker, hexamethonium (10(-6)-10(-4) M) did not affect the contractile response evoked by a single dose of carbachol (10(-5) M), whilst the muscarinic receptor antagonist, atropine (10(-11)-10(-9) M), inhibited it in a competitive manner. 5. The muscarinic M1 (pirenzepine), M2 [AF-DX 116, himbacine (M2/M4) and methoctramine], M3 (HHSID and f-F-HHSID), and putative M4 (tropicamide) antagonists reduced significantly the carbachol-induced contractions. The pIC50 values were: atropine (10.01) > himbacine (8.3) > methoctramine (7.85) > AF-DX 116 (7.60) > HHSID (7.21) > p-F-HHSID (7.10) > pirenzepine (7.30) > tropicamide (7.00). 6. The antagonist profile indicates that an predominant M2 receptor subtype could mediate the muscarinic contraction in the canine prostate.