The use of real-time PCR and fluorogenic probes for rapid and accurate genotyping of newborn mice.

Real-time PCR and fluorogenic probes were combined in a simple, rapid and sensitive method to genotype murine breeding stocks and their progeny for a point mutation. DNA from tail biopsies of newborn mice was mixed with amplification primers and fluorogenic hybridization probes in a PCR mixture. The primers were designed to amplify a region of the Fas-Ligand gene including the site for the gld natural point mutation. The fluorogenic hybridization probes overlaid this target sequence and were used to detect amplification of the PCR fragment as well as determine the presence of the point mutation using fluorescence resonance energy transfer (FRET). Both mutated and wild-type forms of the gene fragment were amplified as detected with real-time PCR. Melting curve profiles completed on each amplified sample revealed the genotype for each mouse. These genotypes were confirmed by sequencing the amplified fragments. These results suggest real-time spectrofluorometric PCR techniques incorporating FRET-based hybridization probes may be used for rapid, sensitive, inexpensive and reliable genotyping.

Transfer across the human gut of environmental technetium in lobsters (Homarus gammarus L.) from the Irish Sea.

Few data are available on the uptake by the human gut of the element technetium. Of current radiological interest in connection with discharges of technetium-99 in liquid discharges from BNFL, Sellafield, is uptake from European lobsters (Homarus gammarus), whose edible parts are known to concentrate technetium. In this study, a group of eight adult volunteers (six males and two females) ate samples of edible flesh from lobsters caught off the west Cumbrian coast and provided 24 h samples of urine and faeces for analysis. Detection of uptake from the gut by difference between intake and faecal measurements proved insensitive, suggesting a low value of the gut transfer factor (f1 value) of up to 0.1 with a maximum (two standard deviations) level of about 0.3. In urine, technetium was detectable at a relatively low level compared with the intakes, consistent with a low absorption across the gut. Values for f1 were derived with the aid of literature data for excretion following intravenous administration of technetium-95 m as pertechnetate, and gave averaged data for f1 in the range 0.046 to 0.23. These results are in broad conformity with those derived from the faecal measurements, and suggest a lower value than the 0.5 used by ICRP.

Rapid and specific detection of the Mycobacterium tuberculosis complex using fluorogenic probes andreal-time PCR.

A rapid and sensitive strategy for the specific identification of Mycobacterium tuberculosis (TB) was designed and evaluated using crude mycobacterial lysates. The speed of real-time polymerase chain reaction (PCR) was combined with the sensitivity of fluorogenic probes to confirm the presence of mycobacteria as well as specifically identify the presence of members of the mycobacteria tuberculosis complex (MTC) in a single-tube assay. Oligonucleotides were designed to amplify the internal transcribed spacer (ITS) from several mycobacterial species. Specific fluorogenic probes were included in the PCR reaction for the identification of TB as well as Mycobacterium bovia and Mycobacterium africanum in bacterial lysates. The combination of TB-specific fluorogenic probes with real-time PCR formed an approach determined to be fast (less than 40 min), sensitive (less than 800 copies of DNA) and reliable for the specific detection of the MTC. Our data demonstrate the use of real-time PCR and fluorogenic probes in a rapid and sensitive assay to distinguish members of the MTC from other mycobacterial species.

Conservative management of spontaneous rupture of Kock orthotopic ileal reservoir.

Reports of spontaneous rupture of continent urinary diversions are rare. Management of the reported cases has been solely by exploratory laparotomy with primary repair of the defect. We describe 2 cases of spontaneous rupture of an ileal reservoir successfully managed nonoperatively.

Cholesteryl ester hydrolase activity in adrenal homogenates from normal and essential fatty acid-deficient female rats.

Cholesteryl ester hydrolase was assayed in adrenal homogenates from mature female rats fed a control (corn oil-containing) or essential fatty acid (EFA)-deficient diet. Cholesteryl ester of 16:0, 18:0, 18:1, 18:2(n-6), 20:4(n-6) and 22:4(n-6) were used as substrates. In control rats, the unsaturated esters were hydrolyzed more rapidly than the saturated esters and cholesteryl arachidonate was the preferred substrate of the six investigated; cholesteryl oleate elicited the highest activity in the deficient group. Polyunsaturated esters were hydrolyzed at a significantly lower rate by homogenates from EFA-deficient rats than by those from control animals. The esters of 18:1, 18:2(n-6) and 20:4(n-6) were hydrolyzed more extensively in relation to their concentrations in adrenal tissue than were cholesteryl esters of 16:0, 18:0 and 22:4(n-6). This difference was more pronounced in control than in EFA-deficient rats. No simple relationship of adrenal cholesteryl ester hydrolase activity to ester fatty acid structure or to nutritional essentiality was evident.

Urologic manifestations of chronic granulomatous disease of infancy.

Chronic granulomatous disease of infancy is a rare hereditary disease that may have urologic manifestations. We describe 2 brothers who had intense cystitis and typical x-ray findings characteristic of this disease. The etiology, incidence and treatment of chronic granulomatous disease of infancy are discussed.

The prognostic value of cell surface antigens in low grade, non-invasive, transitional cell carcinoma of the bladder.

Tumors in 23 patients who presented with a low grade, non-invasive transitional cell carcinoma of the bladder were studied for blood group antigens A, B or O on the cell surface. Of 14 patients without cell surface antigens initially 13 suffered an invasive tumor subsequently and 1 had diffuse carcinoma in situ. Of 9 patients with cell surface antigens initially 8 did not have an invasive recurrence during a 5 or more-year followup and 1 did. The presence or absence of blood group cell surface antigen on a low grade, non-invasive transitional cell carcinoma of the bladder would seem to have value in predicting future recurrence with muscle invasion.

Cholesteryl ester depletion from the ovaries of superovulated female rats fed a normal or essential fatty acid deficient diet.

The cholesteryl ester content of the ovaries was determined in rats diets containing corn oil or hydrogenated coconut oil (essential fatty acid (EFA) deficient) and subjected to superovulation by injection of luteinizing hormone and follicle-stimulating hormone. Superovulation increased ovarian weight; the effect was greater in animals fed corn oil. Superovulation significantly decreased total ovarian cholesteryl ester concentration in animals fed corn oil, with disproportionately large decreases occurring in the esters of 20:1, 20:2, 22:5w6, and 22:6w3. Significant decreases were observed in these esters when the data were expressed on a unit mass of tissue basis or in relation to total ovarian mass. In superovulated, EFA-deficient rats, esters of 18:1, 20:1, 22:5w6, and 22:6w3 were significantly lower per unit mass of tissue but this was due, in all cases except that of 22:6w3, to the increased mass of ovarian tissue; there was no decrease in total esters per ovary weight during superovulation of deficient rats. The pattern and degree of selective changes in ovarian cholesteryl esters during superovulation were different from those previously reported for adrenal esters of stressed rats.

The family as a system in hospital-based social work.

Caring for disabled family members at home can create stress that may exacerbate existing dysfunctions in the family. By changing the orientation of hospital-based social work from "disabled family member as burden" to "family unit as an ongoing system," the authors have succeeded in helping dysfunctional, disunited families become functional family systems.

The circadian cycles of plasma corticosterone and adrenal cholesteryl esters in the normal and EFA-deficient female rat.

UNLABELLED: In female rats subjected to a 12 hr light-12 hr darkness schedule and fed a semipurified diet containing 10% corn oil, plasma corticosterone concentration showed a monophasic circadian cycle with minimum and maximum concentrations at the start of the light and dark periods, respectively. Adrenal total cholesteryl ester concentration was inversely related to plasma corticosterone, as were those of several of the individual esters; changes in cholesteryl ester concentration appeared to follow rather than precede changes in plasma corticosterone. There was preferential depletion of the cholesteryl esters of 18:1, 18:2omega6, and 20:4omega6 during glucocorticoid secretion. [ ABBREVIATIONS: EFA, essential fatty acid (s);X:YomegaZ, fatty acid with X carbon atoms and Y olefinic bonds with the terminal double bond Z carbon atoms from the methyl group.] In female rats fed hydrogenated coconut oil (EFA-deficient), a monophasic cycle for plasma corticosterone was also observed, but the peak was much broader than that recorded for rats fed corn oil, although minima and maxima occurred at similar times for the two groups. No significant cycle of adrenal total cholesteryl esters was evident in the deficient rats, but the 20:3omega9 and 22:3omega9 esters did decrease significantly during the period of high plasma corticosterone concentration. Preferential net decreases in adrenal cholesteryl esters during corticosteroidogenesis were more apparent in normal than in EFA-deficient rats.

Oral contraceptive steroids: effects on oral tolerance tests of glucose, galactose, fructose and ribose.

PIP: The effects of oral contraceptives (OCs) on oral tolerance tests of glucose, galactose, fructose, and ribose were studied in 11-week-old female rats. 1 group was fed ad libitum a basal grain diet containing norethynodrel and mestranol while a control group was pair-fed with the experimental rats and received only the basal grain diet. Oral sugar tolerance tests were performed after 1 and 4 weeks of steroid treatment. After 1 week of treatment, oral sugar loading indicated that there was no tolerance impairment based on blood serum glucose levels. However, after 4 weeks of treatment, rats administered glucose had impaired tolerances (p less than .01). Blood serum glucose levels were higher (p less than .05) for treated than for control rats after force-feeding of ribose. No impairment or increase in blood glucose levels was seen after administering fructose or galactose. Urine samples were obtained from the animals 2 weeks after the initiation of treatment to determine the quantity of urinary sugars after oral loading of the 4 sugars. Quantities of urinary sugar for the 2 groups were similar for samples obtained for 6 hours immediately after the sugar loading. During the subsequent 18 hours the treated rats had significantly greater quantities of urinary glucose than control rats when they were orally loaded with either glucose (p less than .05) or ribose (p less than .01). 8 pairs of the rats were continued on their respective diets for another 28 weeks, when the quantity of carbon dioxide exhaled subsequent to injections of glucose-u-carbon-14 was appreciably higher for pair-fed control rats compared with those fed the 2 steroids. The metabolism of injected ribose to carbon dioxide was faster for treated rats.^ieng