Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

Abstract:  The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.     

Ultrasonic root-end preparation in apical surgery: a prospective randomized study.

OBJECTIVE: The purpose of this study was to evaluate the potential benefit of an ultrasonic device in apical surgery on the outcome of treatment. STUDY DESIGN: A randomized prospective design was used in a standardized treatment protocol. Patients were allocated to treatment with an ultrasonic device (P-Max Newtron) or treatment with a bur in an otherwise similar protocol. One year after treatment the results were evaluated by 2 oral and maxillofacial surgeons who were blinded for the therapy. RESULTS: Out of a total group of 399 patients who were included in the study, adequate follow-up could be obtained in 290 patients. The overall success rate in the ultrasonic group was 80.5% and in the group treated with a bur 70.9% (P = .056). In molars, the difference in success rate was significant (P = .02). CONCLUSION: The use of an ultrasonic device in apical surgery improved the outcome of treatment. In molars this effect was significant.     

Ventilation imaging of the lung: Comparison of hyperpolarized helium-3 MR imaging with Xe-133 scintigraphy

RATIONALE AND OBJECTIVES: To compare hyperpolarized helium-3 (HHe) magnetic resonance imaging (MRI) of the lung with standard Xe-133 lung ventilation scintigraphy. MATERIALS AND METHODS: We performed a retrospective review of 15 subjects who underwent HHe MRI and Xe-133 lung ventilation imaging. Coronal MRI sections were acquired after a single inhalation of HHe gas, and standard posterior planar lung ventilation scintigraphy was performed during continuous breathing of Xe-133 gas. The first breath scintigram of each patient was compared with a composite MR image composed of the sum of the individual MR images and with the individual helium-3 MR images. Ventilation defects on the two imaging modalities were compared for size, conspicuity, and concordance in presence and location. Assessment was done separately for each of four lung quadrants. RESULTS: Comparing the composite HHe MR images with Xe-133 scintigraphy, ventilation defect size, conspicuity and concordance were the same in 67% (40/60), 63% (38/60), and 62% (37/60) quadrants, respectively. Comparing the individual HHe MR image sections with the Xe-133 ventilation scan, there was concordance between the ventilation defects in 27% (16/60) of quadrants. More defects were identified on the individual HHe MR images in 62% (37/60) of quadrants. CONCLUSION: There was good agreement between composite HHe MR image and first breath Xe-133 scintigraphic images, supporting the widely held assumption that HHe MRI likely depicts first breath lung ventilation.     

Ureteral catheterization and manipulations using flexible cystoscope on patients in semiprone position

Cystoscopy using the flexible cystoscope was performed on 15 patients in the semiprone position. Ureteral catheters were placed to facilitate percutaneous nephrolithotomy in 8 patients, and indwelling ureteral stents were removed in 7. The procedure offers the urologist an additional technique in select situations.     

Effect of buffering on pharmacokinetics of ketoprofen enantiomers in man

Aims Concomitant administration of magnesium hydroxide may affect the rate or extent of absorption of non-steroidal anti-inflammatory drugs. In order to find out whether or not buffering modifies the pharmacokinetics of ketoprofen, plasma concentration-time courses resulting from oral administration of unbuffered formulations were compared with those of buffered formulations.
Methods Two groups of 12 healthy and young male subjects were included in two randomized cross-over studies and received single oral doses of ketoprofen 12.5 or 25  mg, respectively, given as tablets which were either unbuffered or buffered with magnesium hydroxide/citrate. Ketoprofen enantiomers in plasma were determined by h.p.l.c. up to 24  h post-dose.
Results Maximum plasma concentrations ( C _max ) of both the (R)- and (S)-enantiomer, observed after administration of the buffered formulations (12.5 and 25  mg), were higher compared with the unbuffered tablets by about 50–80%. The area under concentration-time data (AUC) was unaffected, and, hence, C _max/AUC was increased by buffering. Time to C _max ( t _max ) and mean residence time (MRT) tended to be or was shortened by buffering.
Conclusions It is concluded that buffering of two ketoprofen formulations with magnesium hydroxide/citrate enhanced the concentration maximum by increasing the rate of absorption and leaving AUC unaffected.     

Gastric emptying patterns of a liquid meal in newborn infants measured by epigastric impedance

Epigastric impedance was used to measure the gastric emptying patterns of a liquid non-caloric meal (5 mL water kg⁻¹) in 30 healthy newborn infants. Twenty-six mature infants were examined in the first eight days of life, and four preterm infants were examined within 6 weeks after birth. The recordings consisted of two components: the emptying signal (the DC component), and a phasic 3 cycles per minutes (CPM) signal (the AC component). In some of the infants the phasic 3 CPM signal was also seen during the fasting state.
For mature infants the median half emptying time (T₅₀) was 6.9 min. For a second meal given within one hour after the first meal the half emptying time was 5.5 min ( P  < 0.01). In preterm infants the emptying times were not significantly different from mature infants.
Day-to-day variation was low with a coefficient of variation of 17% in nine infants.
A periodic change of the impedance signal, the phasic 3 CPM signal, was observed after a meal in 24 of the infants. The median frequency was 3.0 CPM in 20 mature and 2.9 CPM in four preterm infants. In nine infants a phasic 3 CPM signal was also observed during the fasting state, with a median frequency of 2.9 CPM.
Measurement of gastric emptying pattern with epigastric impedance is a simple investigation for the evaluation of gastric emptying time and phasic activity in mature and preterm infants. However, the method is sensitive to spontaneous movements of the children, resulting in non-valid measurements in around one fourth of the infants.     

Report of the Canadian Hypertension Society Consensus Conference: 2. Nonpharmacologic management and prevention of hypertensive disorders in pregnancy

OBJECTIVE: To provide Canadian physicians with comprehensive, evidence-based guidelines for the nonpharmacologic management and prevention of gestational hypertension and pre-existing hypertension during pregnancy. OPTIONS: Lifestyle modifications, dietary or nutrient interventions, plasma volume expansion and use of prostaglandin precursors or inhibitors. OUTCOMES: In gestational hypertension, prevention of complications and death related to either its occurrence (primary or secondary prevention) or its severity (tertiary prevention). In pre-existing hypertension, prevention of superimposed gestational hypertension and intrauterine growth retardation. EVIDENCE: Articles retrieved from the pregnancy and childbirth module of the Cochrane Database of Systematic Reviews; pertinent articles published from 1966 to 1996, retrieved through a MEDLINE search; and review of original randomized trials from 1942 to 1996. If evidence was unavailable, consensus was reached by the members of the consensus panel set up by the Canadian Hypertension Society. VALUES: High priority was given to prevention of adverse maternal and neonatal outcomes in pregnancies with established hypertension and in those at high risk of gestational hypertension through the provision of effective nonpharmacologic management. BENEFITS, HARMS AND COSTS: Reduction in rate of long-term hospital admissions among women with gestational hypertension, with establishment of safe home-care blood pressure monitoring and appropriate rest. Targeting prophylactic interventions in selected high-risk groups may avoid ineffective use in the general population. Cost was not considered. RECOMMENDATION: Nonpharmacologic management should be considered for pregnant women with a systolic blood pressure of 140-150 mm Hg or a diastolic pressure of 90-99 mm Hg, or both, measured in a clinical setting. A short-term hospital stay may be required for diagnosis and for ruling out severe gestational hypertension (preeclampsia). In the latter case, the only effective treatment is delivery. Palliative management, dependent on blood pressure, gestational age and presence of associated maternal and fetal risk factors, includes close supervision, limitation of activities and some bed rest. A normal diet without salt restriction is advised. Promising preventive interventions that may reduce the incidence of gestational hypertension, especially with proteinuria, include calcium supplementation (2 g/d), fish oil supplementation and low-dose acetylsalicylic acid therapy, particularly in women at high risk for early-onset gestational hypertension. Pre-existing hypertension should be managed the same way as before pregnancy. However, additional concerns are the effects on fetal well-being and the worsening of hypertension during the second half of pregnancy. There is, as yet, no treatment that will prevent exacerbation of the condition. VALIDATION: The guidelines share the principles in consensus reports from the US and Australia on the nonpharmacologic management of hypertension in pregnancy.     

FEASIBILITY OF CONDUCTING CARDIOVASCULAR OUTCOME RESEARCH IN AUSTRALIAN GENERAL PRACTICE: RESULTS FROM THE ANBP2 PILOT STUDY

1. The present study aimed to determine the feasibility of conducting a 5 year cardiovascular outcome trial of the treatment of 6000 elderly hypertensive patients in Australian general practices. 2. General practitioners (GPs) were invited to participate by mail and personal follow-up. Patient records were reviewed to identify subjects for a blood pressure (BP) screening programme. Blood pressure was measured on three occasions and eligible subjects were included if the average BP was 160 mmHg systolic or 90 mmHg diastolic if systolic BP was 140 mmHg. 3. Seven hundred and forty-one GPs were approached and 89 were enrolled in the study (12% of mail invites and 75% of those receiving a personal contact). In 16 practices where screening was completed, 82 000 records were reviewed to identify 4% patients eligible for screening. Twenty-two per cent of eligible subjects attended screening. Of 1938 subjects screened, 180 (9%) had BP 5=160/90 mmHg. Forty-seven percent of subjects (n = 916) were receiving antihypertensive therapy and 184 (20%) were withdrawn from therapy. One hundred and sixteen (63%) of these subjects had BP return to study entry levels within 6 weeks. Fifty-seven newly diagnosed and 81 previously treated subjects were randomized (7% of the screened population). 4. Based on the high participation rate of GPs, the response rate of patients to attend a BP screening programme and the 7% randomization to screening ratio for entry into the study, the ANBP2 pilot study has demonstrated that it is feasible to recruit subjects from Australian general practices to a cardiovascular outcome trial.