An explanation for the decrease in cell lysis in a rotating tube with increasing ultrasound intensity

Previous observations indicate that for in vitro mammalian cells insonated in rotating test tube the amount of cell lysis initially increases to some maximum and then decreases with further increase in ultrasound exposure. The results of the present investigation support the postulate that the reduction in cell lysis with increase in ultrasound intensity is related to the development of an ultrasonically induced "cloud" of bubbles in the fluid between the transducer and test tube; these bubbles mitigate against acoustic transmission thus reducing cell lysis in the insonated test tube.     

Preparation of primary monolayer cultures of mouse pancreatic epithelial cells

Summary We describe a technique using normal and diabetic (dbdb) mice to establish primary pancreatic cultures that spread and assume a characteristic epithelial morphology. These cultures contain 4 to 7% beta cells, secrete insulin in response to stimuli for 10 to 14 d, contain few fibroblasts, and have a cell viability that is greater than 95%. The cells attach firmly to glass cover slips and are ideal for the study of insulin secretory granules or contractile proteins using indirect immunofluorescence.     

Use of buccal cells collected in mouthwash as a source of DNA for clinical testing

CONTEXT: To maximize the participation rate in population genetic studies, alternatives to invasive whole blood collection are increasing. One such alternative is buccal epithelial cell collection, which, in contrast to venipuncture and finger sticks, is painless. Buccal cells, if collected and purified efficiently, offer an acceptable source for DNA to be used in research and clinical applications. OBJECTIVE: To develop a noninvasive sampling method for collecting cells for routine DNA testing in a clinical laboratory setting. DESIGN: Five factors were used to evaluate several brands of mouthwash: (1) compatibility with the DNA purification chemistry, (2) DNA yield, (3) DNA quality, (4) DNA stability at room temperature, and (5) mouthwash taste. Next, an optimization study was undertaken to maximize DNA yield. Finally, a validation study was undertaken with the optimized protocol to test a panel of 14 donors for DNA yield and performance and to test for the stability of DNA held in mouthwash. SETTING: Industrial research and development laboratory. RESULTS: Of 5 mouthwashes tested, Scope brand mouthwash received the highest overall ranking. The addition of proteinase K and glycogen to the protocol significantly enhanced DNA yields, with a test panel (n = 14) giving a range of 12 to 60 microg of DNA per donor. In a 4-week room temperature stability study, the DNA in mouthwash samples was found to be stable for at least 2 weeks. CONCLUSION: A clinically validated DNA purification chemistry was adapted to a noninvasive specimen collection method. This method used a commercially available mouthwash, Scope, to collect buccal epithelial cells for the preparation of high-quality DNA in high yield.     

The release of adenosine triphosphate from frog skeletal muscle in vitro

1. Active frog sartorius muscle in vitro liberates a substance into the bathing solution which has a pronounced stimulatory action on the frog heart.2. The stimulatory effect is not due to an increase in the K(+) concentration of the bathing solution, nor is it due to the liberation of catecholamines.3. In a molecular sieve chromatography procedure the stimulatory substance can be eluted in a single fraction which shows a maximum absorption of U.V. light at a wave-length of 265 nm, indicative of the presence of substances containing a purine ring.4. Low concentrations (10(-7)-10(-8) g/ml.) of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and uridine triphosphate (UTP) have a marked stimulatory action on the frog heart. The action of ATP and ADP on the heart is qualitatively very similar to that of the muscle bathing solution, while the action of UTP is distinctly different. The triphosphates of inosine, cytidine and guanosine stimulate the heart when in high concentration only. Adenosine and adenosine monophosphate do not stimulate the heart.5. Incubation of the muscle bathing solution and of solutions of ATP with the enzyme apyrase for the same time produces a similar marked reduction in the stimulatory action of both on the heart. Apyrase catalyses the break-down of nucleotide triphosphates to monophosphates.6. The elution behaviour of the stimulatory substance determined by molecular sieve chromatography is the same as that for ATP.7. The muscle bathing solution causes light to be emitted from firefly lantern extract, the pattern of light emission being similar to that produced by nucleotide triphosphates.8. The concentrations of ATP having the same quantitative action on the frog heart and on firefly extract as a given muscle bathing solution are almost identical, whereas the matching concentrations of ADP and UTP in the two methods of assay are widely different.9. It is concluded that ATP is released from active frog skeletal muscle in vitro. This release may play an important part in the reactive hyperaemia of muscular exercise since ATP has a powerful vasodilator action.     

Effects of methacholine and uridine 5'-triphosphate on tracheal mucus rheology in mice

We compared the action of methacholine (MCh) and uridine 5'-triphosphate (UTP) with and without pretreatment with the chloride channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) on the transepithelial potential difference (PD), the mucus collection rate (MCR), and tracheal mucus rheology using anesthetized C57BL/6 mice. The cystic fibrosis transmembrane conductance regulator (CFTR) blocker 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) was also used as a pretreatment for MCh. After collecting baseline mucus for 1.5 h, mucus secretion was stimulated by instilling 5 microl of 10(-2) M MCh or UTP around the upper trachea. There was a significant increase in PD after MCh or UTP stimulation (-21.3+/-2.0 mV MCh versus -14.1+/-1.6 mV control; -25.4+/-2.5 mV UTP versus -19.2+/-1.9 mV control). When UTP administration was preceded by DIDS, PD shifted from -15.2+/-2.9 to -12.0+/-2.2 mV. When MCh was preceded by DIDS or by NPPB, there was no change in PD. There was a significant decrease in mucus rigidity index, logG*, with MCh (2.54+/-0.09 versus 2.99+/-0.14 for control), similar to that previously reported in other species. With UTP, 14 of 16 mice responded in terms of PD becoming more negative, and of these, there was a significant difference in logG* after UTP administration (2.29 +/-0.10 versus 2.57+/-0.10 for control), whereas there was no change in logG* with DIDS administration before UTP. When DIDS administration preceded MCh, there was a diminished but still significant decrease in logG* from control, whereas there was no change in logG* when NPPB was preadministered. The control mucus collection rate was 0.19+/-0.09 mg/h, whereas after MCh stimulation, it increased to 2.83+/-0.78 mg/h. No significant difference was measured in the MCR after either UTP or DIDS+UTP stimulation. DIDS+MCh and NPPB+MCh both resulted in significant increases in MCR, but of a much smaller magnitude than that for MCh alone. We conclude that hypersecretion owing to UTP in C57BL/6 mice is less vigorous than with MCh, reflecting the limited population of Ca(2+)-dependent Cl(-) channels stimulated by UTP P(2) receptors. The action of MCh on tracheal mucus secretion in mice appears to involve both CFTR- and non-CFTR-dependent chloride channels.     

Individual and cultural-diversity competency: focus on the therapist

The Competencies Conference: Future Directions in Education and Credentialing in Professional Psychology was held in Arizona in November 2002. One of the workshops, Individual and Cultural Differences (ICD), focused on racism, homophobia, and ageism. The consensus was that self-awareness and knowledge about the three "isms" are critical components in the education and training of psychologists. This article, authored by four of the workshop attendees, is a review of the current research and theoretical literature. Implications that address both content and context in graduate programs and training sites are presented. This is one of a series of articles published in this issue of the Journal of Clinical Psychology. Several other articles that resulted from the Competencies Conference will appear in Professional Psychology: Research and Practice and The Counseling Psychologist.     

Reciprocal translocation, 4q-; 21p+, giving rise to Down''s syndrome.

A reciprocal translocation is described, t(4;21)(q27;p11), which occurs in a balanced carrier mother and her Down's syndrome child, 47,XX,t(4q-;21p+),+21. A review is presented of Down's syndrome associated with reciprocal translations involving chromosome No. 21.     

Fetal hydrops.

Seventy-two fetuses or neonates with non-immune hydrops were examined between 1983 and 1988. The commonest association was chromosome abnormality; 11 fetuses had a 45,X karyotype and 11 autosomal trisomy. Chromosome abnormality was suspected in a further 20 on necropsy findings but chromosome culture was not possible or unsuccessful. In 11 cases there was histological evidence of infection; seven babies had major structural anomalies and six affected fetuses were twins. In six (8%) the cause of hydrops was not determined compared with eight (16%) of cases examined between 1976 and 1982. Hydrops was diagnosed more frequently while the fetus was alive, before 20 weeks' gestation, and associated with chromosome anomaly than found previously.     

Identification and properties of pathways for K+ transport in guinea-pig and rat alveolar epithelial type II cells.

86Rb+ was used to study potassium uptake and efflux in type II pneumocytes freshly isolated from adult guinea-pig and rat lung. Both species exhibited a substantial ouabain-sensitive component of potassium influx. In rats, most of the ouabain-resistant influx was abolished by bumetanide and removal of extracellular chloride elicited no further effect. In contrast, only a proportion of the ouabain-insensitive uptake was inhibitable by bumetanide in guinea-pigs and this species showed an additional component of influx, which was chloride dependent and which was reduced by either the K(+)-H(+)-ATPase inhibitor, omeprazole, or by the stilbene derivative, 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS). The chloride-dependent component was also apparent in efflux experiments in guinea-pigs, but was absent in rats. Ouabain-insensitive ATPase activity was assayed in highly purified apical membranes from guinea-pig type II pneumocytes. This activity was inhibitable by omeprazole (apparent inhibition constant, Ki, was approximately 40 microM), was potassium dependent (apparent activation constant, Ka, was approximately 200 microM) and was doubled by the addition of nigericin. While potassium transport in rat type II cells is adequately accounted for by Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- cotransport, our data suggest the additional presence of K(+)-Cl- cotransport and K(+)-H(+)-ATPase in guinea-pig type II pneumocytes. A model of how alveolar subphase acidification may occur is proposed.     

A feasibility study to investigate the use of thin-plate splines to account for prostate deformation

Image registration is an important step in the radiotherapy treatment planning process. It provides a method of fusing different types of diagnostic imaging information. One such application is to combine magnetic resonance spectroscopic images (MRSI) of the prostate with anatomical MRI and/or computed tomography images that are routinely used in the radiation treatment planning of prostate cancer. MRSI provides in vivo information related to the underlying metabolic activity of tissues, and can be related to the presence of cancer. However, the inflated endorectal coil required during MRS imaging poses a potential problem by deforming the prostate when it is filled with approximately 100 cm3 of air during image acquisition. This pushes the prostate superiorly/anteriorly, deforming the prostate and consequently the spectroscopic imaging data in a nonlinear manner. In this application, the coil-deformed MRS images are warped back to a non-deformed state, using a single data set. A nonlinear warping algorithm is presented to achieve this. Results indicate that the algorithm attains an accuracy of 97% (4 cm3 difference) when reproducing the total prostate volume compared to a Radiation Oncologist defined prostate volume. This difference is slightly smaller than the measured intra-operator variance of +/-1.5 cm3 (deflated coil) and the measured algorithm variance of +/-1.0 cm3. Additionally, intraprostatic nodules were used to assess the accuracy of the warping algorithm in regions inside the prostate. While choosing anatomical tie points along the external prostate surface, analysis of the nodules revealed the algorithm accuracy reduced to 63-93%.