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Restoration by T4 ligase of DNA sequences sensitive to "flush" cleaving restriction enzyme.

Fouteen "flush"-ended segments originate from the action of the restriction endonuclease Hae III of Haemophilus aegiptius on the DNA of the colicinogenic factor ColE 1 (A. Oka and M. Takanami, Nature, 264, 191, 1976). They are joined by the T4 polynucleotide ligase. The reaction can be monitored by gel electrophoresis, electron microscopy and resistance to phosphatase of the 5'-32P labelled ends. The joined products are a random recombination of the original segments, and can be cleaved by the same Hae III endonuclease to restore the exact electrophoretic pattern of the Hae III-cut ColE 1 DNA. In a properly diluted mixture of 5'-32P segments treated with T4 ligase, the level of phosphatase resistance is very close to the frequency of circle-formation as determined by electron microscopy: thus, the joining of the "flush"-ends involves the formation of circular structures covalently closed in both strands.

Coliphages↗

Intermittent saline flushes during haemodialysis do not alleviate coagulation and clot formation in stable patients receiving reduced doses of dalteparin.

BACKGROUND: Heparin-free haemodialysis (HD) with intermittent saline flushes (ISF) in patients with bleeding risk is widely used. The aim of this study was to investigate if ISF reduce coagulation and clotting in stable patients receiving reduced doses of dalteparin. METHODS: Inclusion criteria were stable chronic HD patients >or=18 years of age and haemoglobin >or=11 g/dl. Exclusion criteria were use of warfarin and acetylsalicylic acid. Six HD sessions were evaluated per patient. Dalteparin was given as one bolus dose at start of HD (50% of the conventional dose). In HD number 1, 3 and 5, 100 ml saline solution was flushed through the filter each 30 min. In HD 2, 4 and 6, no ISF were given. Potential clotting in the bubble trap was visually observed each hour and graded on a 4-point scale: 1 = normal, 2 = fibrinous ring, 3 = clot formation and 4 = coagulated system. The dialyser was visually inspected at the end of each session: 1 = normal, 2 = a few blood stripes (affecting less than 5% of the surface fibres), 3 = many blood stripes (more than 5% of the fibres) and 4 = coagulated filter. The coagulation marker PF1+2, the platelet activation marker beta-TG and anti-FXa activity were repeatedly measured during HD. RESULTS: Six men and two women were included. In four cases (four different patients), HD was stopped due to a coagulated system, all cases on days with ISF performed. Multiple linear regression analyses with repeated measurements showed that ISF adjusted for dalteparin dose/kg significantly increased mean clot in the bubble trap, estimate (B) = 0.717, P = 0.0001 and also showed that ISF increased PF1+2, B = 0.16, P = 0.001 when adjusted for anti-FXa activity and hours of dialysis, whereas beta-TG was only borderline increased, B = 0.09, P = 0.055. CONCLUSIONS: ISF during HD does not alleviate visible clotting or intravascular coagulation activity in stable patients receiving reduced doses of dalteparin and polysulphone dialysers. Whether this applies to unstable patients with increased bleeding risk not receiving any anticoagulation remains to be shown.

Adult↗

MUC1 in secretory phase endometrium: expression in precisely dated biopsies and flushings from normal and recurrent miscarriage patients.

MUC1 is a cell-surface and secretory product of endometrial epithelium. Immunohistochemical studies carried out using two different antibodies to the mucin-type tandem repeat region of MUC1 indicate a cell-surface location in proliferative phase glands, with intracellular deposits accumulating in the early secretory phase. Commencing 3-4 days after the luteinizing hormone (LH) peak and continuing into the late secretory phase, secretory MUC1 appears in gland lumens. Uterine flushings were collected as a function of time after the LH peak and were analysed using a two-site enzyme-linked immunosorbent assay for MUC1. Low but measurable concentrations were observed up to day 7, while on days 7-13 much higher values were obtained. In women suffering from recurrent spontaneous miscarriage, the concentration of MUC1 in flushings was significantly lower than in the controls on day LH + 10. Lower values were observed on days 7 and 13. Reduced epithelial secretory function and a resultant change in uterine fluid composition are features of endometrium from recurrent miscarriage patients.

Abortion, Habitual↗

Quantification of an estrogen-dependent cat uterine protein (CUPED) in uterine flushings of estrogen- and progesterone-treated ovariectomized cats by radioimmunoassay.

We previously reported the purification of an estrogen-dependent cat uterine protein (CUPED) and the preparation of a specific anti-CUPED serum in rabbits. Here, we describe a specific radioimmunoassay for CUPED using the purified CUPED and anti-CUPED serum that was utilized to quantify CUPED in daily uterine flushings obtained from steroid-treated ovariectomized cats. The radioimmunoassay was sufficiently sensitive to measure 0.1-100 ng CUPED. CUPED levels were low in untreated ovariectomized cats, increased within one day after the onset of treatment with estradiol, and remained elevated as long as estradiol was unopposed by progesterone. The levels of CUPED decreased when progesterone was added to the treatment regimen either 7, 14, or 28 days after the initiation of estradiol treatment. The data indicate that the presence of CUPED in the uterine flushings is dependent on the presence of estradiol and the absence of progesterone, that CUPED appears in the uterine lumen within one day after the onset of treatment with estradiol, and that the levels of CUPED are sharply reduced within one day of administration of progesterone and become nondetectable after three days.

Animals↗

The fast flush test measures the dynamic response of the entire blood pressure monitoring system.

The fast flush test (FT) is the only test that allows clinicians to determine in vivo the natural frequency (fn) and damping coefficient (zeta) of an invasive blood pressure monitoring system. The underlying assumption to the validity of the FT is that it activates the whole system including the distal catheter. We devised an in vitro model of a typical invasive blood pressure monitoring system to determine whether this assumption was true. The model consisted of a conventional transducer with a flush device attached to various lengths of connecting tubing (91.4, 182.9, and 274.3 cm) terminated by four different diameter catheters (5.1 cm 14 G, 16 G, 18 G, and 20 G). A microtipped transducer catheter was inserted into the distal catheter tubing system. A FT was performed and the fn and zeta were recorded from the conventional transducer and simultaneously from the microtipped transducer catheter. Similar studies were conducted using the ROSE damping device as well as with systems including 0.1 ml of air near the conventional transducer. These studies utilized 18- and 20-G catheters with each of the three lengths of connecting tubing. All measurements of fn and zeta at the proximal conventional transducer were identical to those measurements as recorded by the distal microtipped transducer catheter. We conclude that the FT activates the whole monitoring system and that fn and zeta are the same throughout the system including the distal catheter.

Blood Pressure Monitors↗

Contrast-enhanced CT with saline flush technique using two automated injectors: how much contrast medium does it save?

PURPOSE: The purpose of this work was to investigate the volume of contrast medium saved by the saline flush technique. METHOD: Thoracic helical CT was performed by injecting 75 ml of contrast material (Ioversol 320) only (n = 25; Group A) or 75 ml of contrast medium pushed with 6 ml (n = 25; Group B), 12 ml (n = 25; Group C), 25 ml (n = 25; Group D), or 50 ml (n = 25; Group E) of saline at a rate of 2 ml/s. The aortic CT numbers were measured from 30 to 55 s after the beginning of injection. We compared the time to peak aortic enhancement (TPAE) among Groups A-E. RESULTS: The TPAEs of the Groups A-E were 42.6, 45.2, 48.6, 48.4, and 48.3 s, respectively, and there was a statistically significant difference among them (p < 0.0001, analysis of variance). Post hoc test revealed statistically significant differences in TPAE between Groups A and B, C, D, and E and between Groups B and C, D, and E but no differences among Groups C, D, and E. CONCLUSION: The saline flush technique prolongs TPAE by 6 s and saves 12 ml of contrast medium.

Adult↗

Replacement estrogen therapy for menopausal vasomotor flushes. Comparison of quinestrol and conjugated estrogens.

Quinestrol, conjugated estrogens, or placebo was used to treat 156 patients with pernicious vasomotor instability in a prospective, double-blind, randomized, multiinvestigator trial. Vasomotor flushes were severe in approximately 80% of the cases and moderate in 20%, relatively equally distributed among the various drug groups. Both qinestrol and conjugated estrogens were significantly more effective than placebo in relieving vasomotor symptoms (by chi2 analysis, P less than or equal to 0.05). Greatest improvement was seen in the group receiving the higher once weekly quinestrol dosage of 0.2 mg followed by the group on the lower quinestrol dosage of 0.1 mg once weekly and the group on conjugated estrogens, 1.25 mg daily for 21 days on and 7 days off. No significant difference in relief of vasomotor flushes was shown between the active drug groups. No drug-related complications or side reactions of significance occurred. The results indicate that once weekly quinestrol is effective in relieving the vasomotor symptoms of the menopause. Either of two once weekly quinestrol regimens is an effective as conjugated estrogens given daily in a cyclic manner and therefore offers an alternative form of exogenous estrogen therapy.

Chemical Phenomena↗

Three simple solutions for renal flush and preservation.

The harvesting of kidneys at small hospitals where standard flush solutions are not available has led to the use of several flush solutions that can be made from readily available materials. Mongrel dogs (15) had bilateral nephrectomy after receiving intravenously a mixture of Ringer's lactate, furosemide (Lasix), mannitol, and chlorpromazine (Thorazine). One kidney was perfused with iced Collins' solution and stored for 24 hours, submerged in the perfusate at 4 C. Five dogs each had the contralateral kidney perfused with a solution of either (1) Ringer's lactate, salt-poor albumin, and heparin; (2) 6% dextran 70 and Fenwall buffered heparin solution; or (3) Ringer's lactate, mannitol, and heparin, and were stored in their respective perfusate. After 24 hours' storage the kidneys were transplanted into the neck, and creatinine clearance (Crcl) was determined on days 1, 2, 3, 5, and 7. The dogs were sacrificed on day 7 and biopsy specimens were obtained from each kidney for pathologic examination. The kidneys perfused with Collins' solution had immediate function after transplantation with an average Crcl of 10.5 at 25 hours and 20.2 at seven days. The kidneys perfused with Ringer's lactate and albumin had minimal function immediately after transplantation and were anuric within 24 hours. The kidneys perfused with dextran solution and Ringer's lactate and mannitol solution had immediate function but output remained low, and by seven days the average Crcl was only 3.0 and 4.5 respectively. All solutions except the Ringer's lactate and albumin were hyperosmolar, thus helping to prevent the "no reflow" phenomenon. These results indicate that the solutions tested do not result in the adequate preservation to warrant clinical use.

Animals↗

Distant procurement of organs for clinical heart-lung transplantation using a single flush technique.

A more widespread application of combined heart-lung transplantation (HLT) for the treatment of pulmonary vascular disease has been limited by the lack of a simple and inexpensive method for preserving donor organs for transplantation from distant hospitals. Of the 12 HLT patients treated at Papworth Hospital, the last 7 involved distant procurement of donor organs. For this, a single flush hypothermic system was used, with a Ringer's solution containing albumin, mannitol, prostacyclin, and heparin. This was preceded by a prostacyclin infusion into the donor's pulmonary artery. A retrospective comparison has been made of the post-operative pulmonary function between the HLT patients with distant procurement, those with near procurement of organs, and a group of patients who underwent coronary artery bypass graft (CABG) over the same time span. The mean ischemic time for distant-procurement patients was 110 (+/- 10) min, compared with 50 (+/- 0.5) min for near procurement. During the initial 18 hr postoperatively, the alveolar-arterial oxygen gradients (A-aDO2) for both groups of HLT patients and the CABG patients were closely comparable. Two near-procurement patients died: 1 from tracheal dehiscence, and 1 from cytomegalovirus (CMV). One distant-procurement patient died of CMV. All the others recovered fully and are alive and well at home. No patient developed a classical reimplantation response, and pulmonary function in the distant-procurement group compared closely with the CABG patients. These observations suggest that a simple single flush preservation system can effectively extend the pulmonary ischemic time so facilitating distant organ procurement.

Coronary Artery Bypass↗

Glycolytic support of adenine nucleotides in rat liver flush-preserved with UW or Collins' II. Importance of donor nutritional status.

Correlation of hepatocellular adenine nucleotides in donor liver with clinical posttransplant outcome has recently been reported. Our earlier work with rats has shown that pretreatment of donors with glucose effectively retards hepatocellular ATP losses in livers preserved in Collins' II solution through potentiation of their glycolytic capacity. The primary substrate--i.e., endogenous or exogenous glucose--was not identified. The current study was undertaken to compare the relative efficacy of the University of Wisconsin (UW) solution, which is devoid of glucose, with Collins' II in the support of adenine nucleotides through anaerobic glycolysis in flush-preserved rat liver. Adult rats were either pretreated with 25% dextrose or fasted prior to liver harvesting and preservation in either UW or Collins' II. Adenine nucleotide degradation and lactate production during preservation were assessed. For a given dietary pretreatment, losses of ATP and adenylate energy charge and lactate production were similar for UW- and Collins' II-preserved livers. Donor pretreatment with dextrose resulted in significantly higher ex vivo liver ATP, energy charge, and lactate regardless of the preservation solution. Salvageable nucleotide degradates were increased significantly in UW livers, presumably through the effects of allopurinol. These results demonstrate that effective support of adenine nucleotides by glycolysis in flush-preserved liver is independent of the presence of exogenous glucose but dependent upon the nutritional status of the donor prior to liver procurement.

Adenine Nucleotides↗

Six-hour porcine liver storage without flushing or perfusion.

Livers from normal porcine donors were preserved by surface cooling only, without flushing or perfusion, for periods up to 24 hr. All recipients of livers stored for 6 hr survived until sacrifice at 7 days. In a separate, similar group, survival up to 21 days was noted. Only 2 of 6 recipients survived after 9-hr liver storage, but one of these lived for greater than 120 days. No animals survived longer than 2 days after transplantation of livers stored for 12 or 24 hr. The changes in plasma levels of aspartate aminotransferase of recipients of 6-hr surface-cooled livers were not significantly different from AST levels of recipients of livers stored in University of Wisconsin or Euro-Collins solution as observed in previous studies in this laboratory. At sacrifice after 7 days, there was no histologic evidence of damage after surface cooling. In the light of recent reports of evidence of endothelial and reticuloendothelial damage caused by flushing solutions, it is suggested that surface cooling of the liver may provide adequate preservation for 6 hr in appropriate circumstances. Further studies will be needed to confirm that this method of preservation is applicable to livers removed from brain-dead donors and that it does not cause endothelial damage.

Animals↗

Bacterial colonization of tunneled right atrial catheters in pediatric oncology: a comparison of sterile saline and bacteriostatic saline flush solutions.

In a double-blind, randomized controlled trial, children with malignant diseases had their tunneled right atrial catheters flushed with either sterile saline or bacteriostatic saline, once per week for 26 weeks. There was no significant difference in the rates of catheter colonization between the two groups, which did differ, however, in terms of the time from entry into the study to the first infective event (64 +/- 34 days vs. 146 +/- 27 days; p less than 0.001). This was strongly suggestive of a seasonal effect, as all of the colonizations in the bacteriostatic saline group were delayed until the summer months. We conclude that the use of a bacteriostatic saline flush solution for tunneled right atrial catheters is beneficial in efforts to prevent catheter colonization.

Bacterial Infections↗

Thrombocytopenia following heparin flush.

A total of 23 patients who developed heparin-induced thrombocytopenia following exposure to heparin solely due to intravascular catheter or filter flush were the subjects of prospective studies of argatroban therapy. Heparin doses were 10-13,000 U, with a mean exposure of 8+/-4 days. Following heparin-induced thrombocytopenia diagnosis and heparin cessation, 13 patients received argatroban (mean dose of 1.8+/-1.1 mg/kg/min for 5.5+/-3.9 days), achieving activated partial thromboplastin times of 63+/-23 seconds, and 10 historical control patients received no direct thrombin inhibitors. Platelet count recovered to a mean of 207+/-153 x 10(9)/L (n=12) after 5.5+/-3.9 days of argatroban therapy and to a mean of 127+/-63 x 10(9)/L (n=8) 5 days after baseline in the control group. A composite end point of death, amputation, or new thrombosis within 37 days occurred in five (38.5%) argatroban-treated patients and four (40%) controls. Death was the most common untoward outcome (approximately 30% of each group). No argatroban-treated patient and two (20%) control patients experienced new thrombosis. Major bleeding was comparable between groups. Heparin-induced thrombocytopenia can occur following minimal heparin exposure, including heparin flushes; in these patients, argatroban provides effective alternative anticoagulation as compared with historical controls.

Amputation, Surgical↗

Chlorpropamide-alcohol flushing in relation to macroangiopathy and peripheral neuropathy in non-insulin dependent diabetes.

Seventy patients with non-insulin dependent diabetes (NIDD) were studied for the chlorpropamide-alcohol flush (CPAF), first degree family history of diabetes, macroangiopathy and for peripheral neuropathy. Positive CPAF challenge tests were found in 65% of the tested subjects and in 77% if there was a family history of diabetes. Signs of macroangiopathy (loss of foot pulses) were significantly (p less than 0.05) less common in the CPAF positive than in the CPAF negative diabetics with a duration of diabetes of ten years or less. With a longer duration this difference between the two groups was reduced. Also signs of peripheral neuropathy (abnormal vibration sense) were less common (p less than 0.05) in the CPAF positive diabetics than in the CPAF negative. Previously a low prevalence of retinopathy in teh CPAF positive non-insulin dependent diabetics has been reported. We have shown that this is also true of peripheral macroangiopathy and peripheral neuropathy. Chlorpropamide-alcohol flushing seems to be related to a relative protection against late complications in diabetes and the test might be used to find patients at risk.

Aged↗

A comparison of flushing fluids for initial perfusion of kidneys for transplantation.

Four solutions for initial flushing of kidneys prior to transplantation were tested under conditions designed to resemble those of clinical cadaveric donor renal transplantation. The experimental model was the dog subjected to bilateral nephrectomy with renal autograft. Kidney grafts were subjected to 15 minutes' anoxia in vivo, 30 minutes' warm ischaemia at 37 degrees C ex vivo, and two hours' cold ischaemia before reimplantation. The four solutions used were Collins (C3), Perfudex (P), hyperosmolar citrate (HC), and a solution of bovine albumin containing dog red blood cells (BBA). Effects of the flushing fluids were compared by parameters relating to dog survival, renal function, and serum enzyme levels. With all parameters studied the best results occurred in HC perfused kidneys. Results with BBA perfusion were marginally worse, while C3 perfused kidneys were again inferior. P perfused kidneys clearly did least well. The results support the use of HC for clinical application.

Animals↗

Baobab trees (Adansonia) in Madagascar use stored water to flush new leaves but not to support stomatal opening before the rainy season.

Baobab trees (Adansonia, Bombacaceae) are widely thought to store water in their stems for use when water availability is low. We tested this hypothesis by assessing the role of stored water during the dry season in three baobab species in Madagascar. In the dry season, leaves are present only during and after leaf flush. We quantified the relative contributions of stem and soil water during this period through measures of stem water content, sap flow and stomatal conductance. Rates of sap flow at the base of the trunk were near zero, indicating that leaf flushing was almost entirely dependent on stem water. Stem water content declined by up to 12% during this period, yet stomatal conductance and branch sap flow rates remained very low. Stem water reserves were used to support new leaf growth and cuticular transpiration, but not to support stomatal opening before the rainy season. Stomatal opening coincided with the onset of sap flow at the base of the trunk and occurred only after significant rainfall.

Adansonia↗

Cyclic changes in the concentration of prostaglandin F2alpha in human uterine flushings.

Prostaglandin F2alpha (PGF2alpha) was measured by radioimmunoassay in uterine flushings obtained from 41 women at various stages of the menstrual cycle. The concentration of PGF2alpha changed significantly during the cycle, with highest values in the mid secretory phase of the cycle (mean 9.1 ng/ml of uterine flushing, range 3.9 to 16.0 ng/ml, n = 9). The still unresolved role of intrauterine PGF2alpha is discussed in relation to luteolysis, myometrial contractility and implantation.

Body Fluids↗

A study of the effectiveness of propranolol in menopausal hot flushes.

A prospective, double-blind, randomized comparison of propranolol, 40 mg three times daily, and matching placebo showed propranolol to be no more effective than placebo in controlling hot flushes in a group of 25 perimenopausal women. Other menopausal symptoms, such as insomnia and palpitations, were equally unaffected. However, a very close correlation was found between the daily atmospheric temperature and the number of flushes occurring in the group.

Adult↗