Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

Increased yield of total RNA from fine-needle aspirates for use in expression microarray analysis

Fine-needle aspirate samples hold the potential for gaining valuable insight into the molecular details and prognostic indicators for certain types of cancer in a limited volume of relatively pure tumor cells. Although limited, such clinical samples can be used with high efficiency when analyzed in conjunction with gene-dense expression microarrays. For this reason, it is essential to retrieve as much high-quality genetic material as possible from each fine-needle aspirate sample. We have conducted a study to improve the efficiency of extracting high quality total RNA to use in microarray analysis from single ex vivo fine-needle aspirate samples of 11 breast cancers added to RNAlater RNA Stabilization Reagent immediately upon collection. Approximately half the total RNA from fine-needle aspirate samples of breast cancers was isolated from the supernatant, and that RNA had similar quality and gene expression profile to the RNA that was isolated from the corresponding cell pellet. We recommend that the supernatant not be discarded when extracting RNA from fine-needle aspirate samples stored in RNAlater.     

Implications of axillary sentinel lymph node biopsy in immediate autologous breast reconstruction

For patients with invasive breast cancer, if the results of an axillary sentinel node biopsy are determined to be positive after permanent pathologic examination, the current recommendation is to perform a complete axillary node dissection. Subsequent axillary surgery may compromise the blood supply to an immediate autologous breast reconstruction. The purpose of this study was to determine which clinicopathologic factors in clinically node-negative breast cancer patients may be associated with an increased risk of positive axillary nodes. Identification of these factors will allow surgeons to modify their approach to immediate autologous breast reconstruction in these high-risk patients. The relationship between presenting clinicopathologic characteristics and the incidence of axillary metastases was analyzed by chi-square test and multivariate analysis in 167 patients with invasive breast cancer and a clinically negative axilla who underwent modified radical mastectomy with an immediate free transverse rectus abdominis musculocutaneous (TRAM) flap reconstruction. Axillary nodal metastases were found in 35 percent of clinically node-negative breast cancer patients. Multivariate analysis showed that patient age of 50 years or younger (p = 0.019), T2 tumor stage or greater (p = 0.031), and presence of lymphovascular invasion on the initial biopsy specimen (p < 0.001) were independent predictors of axillary metastases in clinically node-negative patients. Based on these results, the authors propose an algorithm for decision making in clinically node-negative breast cancer patients who desire autologous breast reconstruction and sentinel lymph node biopsy. Options for immediate autologous breast reconstruction in patients undergoing mastectomy and axillary sentinel lymph node biopsy that may minimize the risk of vascular damage on reoperation include the use of the internal mammary artery and vein as recipient vessels for a free TRAM flap or a pedicled TRAM flap. If an axillary-based blood supply is used, the authors are considering the use of cadaveric dermis to isolate the pedicle of the flap away from the remaining axillary contents. New developments in breast cancer diagnosis and treatment necessitate a team approach, with increased communication between the breast surgeon and the plastic surgeon in planning surgery for these patients.     

Fasting protects against the side effects of irinotecan but preserves its anti-tumor effect in Apc15lox mutant mice

Irinotecan is a widely used topoisomerase-I-inhibitor with a very narrow therapeutic window because of its severe toxicity. In the current study we have examined the effects of fasting prior to irinotecan treatment on toxicity and anti-tumor activity. FabplCre;Apc^15lox/+ mice, which spontaneously develop intestinal tumors, of 27 weeks of age were randomized into 3-day fasted and ad libitum fed groups, followed by treatment with a flat-fixed high dose of irinotecan or vehicle. Side-effects were recorded until 11 days after the start of the experiment. Tumor size, and markers for cell-cycle activity, proliferation, angiogenesis, and senescence were measured. Fasted mice were protected against the side-effects of irinotecan treatment. Ad libitum fed mice developed visible signs of discomfort including weight loss, lower activity, ruffled coat, hunched-back posture, diarrhea, and leukopenia. Irinotecan reduced tumor size in fasted and ad libitum fed groups similarly compared to untreated controls (2.4 ± 0.67 mm and 2.4 ± 0.82 mm versus 3.0 ± 1.05 mm and 2.8 ± 1.08 mm respectively, P < 0.001). Immunohistochemical analysis showed reduced proliferation, a reduced number of vascular endothelial cells, and increased levels of senescence in tumors of both irinotecan treated groups. In conclusion, 3 days of fasting protects against the toxic side-effects of irinotecan in a clinically relevant mouse model of spontaneously developing colorectal cancer without affecting its anti-tumor activity. These results support fasting as a powerful way to improve treatment of colorectal carcinoma patients.     

Localization of the McLeod locus (XK) within Xp21 by deletion analysis.

The McLeod phenotype is an X-linked, recessive disorder in which the red blood cells demonstrate acanthocytic morphology and weakened antigenicity in the Kell blood group system. The phenotype is associated with a reduction of in vivo red cell survival, but the permanent hemolytic state is usually compensated by erythropoietic hyperplasia. The McLeod phenotype is accompanied by either a subclinical myopathy and elevated creatine kinase (CK) or X-linked chronic granulomatous disease (CGD). Seven males with the McLeod red-blood-cell phenotype and associated myopathy but not CGD, one male with the McLeod phenotype associated with CGD, and two males known to possess large deletions of the Duchenne muscular dystrophy (DMD) locus were studied. DNA isolated from each patient was screened for the presence or absence of various cloned sequences located in the Xp21 region of the human X chromosome. Two of the seven males who have only the McLeod phenotype and are cousins exhibit deletions for four Xp21 cloned fragments but are not deleted for any portion of either the CGD or the DMD loci. Comparison of the cloned segments absent from these two McLeod cousins with those absent from the two DMD boys and the CGD/McLeod patient leads to the submapping of various cloned DNA segments within the Xp21 region. The results place the locus for the McLeod phenotype within a 500-kb interval distal from the CGD locus toward the DMD locus.     

Ouabain-insensitive salt and water movements in duck red cells. I. Kinetics of cation transport under hypertonic conditions

Duck red cells in hypertonic media experience rapid osmotic shrinkage followed by gradual reswelling back toward their original volume. This uptake of salt and water is self limiting and demands a specific ionic composition of the external solution. Although ouabain (10(-4)M) alters the pattern of cation accumulation from predominantly potassium to sodium, it does not affect the rate of the reaction, or the total amount of salt or water taken up. To study the response without the complications of active Na-K transport, ouabain was added to most incubations. All water accumulated by the cells can be accounted for by net salt uptake. Specific external cation requirements for reswelling include: sufficient sodium (more than 23 mM), and elevated potassium (more than 7 mM). In the absence of external potassium cells lose potassium without gaining sodium and continue to shrink instead of reswelling. Adding rubidium to the potassium- free solution promotes an even greater loss of cell potassium, yet causes swelling due to a net uptake of sodium and rubidium followed by chloride. The diuretic furosemide (10(-3)M) inhibits net sodium uptake which depends on potassium (or rubidium), as well as inhibits net sodium uptake which depends on sodium. As a result, cell volume is stabilized in the presence of this drug by inhibition of shrinkage, at low, and of swelling at high external potassium. The response has a high apparent energy of activation (15-20 kcal/mol). We propose that net salt and water movements in hypertonic solutions containing ouabain are mediated by direct coupling or cis-interaction, between sodium and potassium so that the uphill movement of one is driven by the downhill movement of the other in the same direction.     

Nucleotide variation in the triosephosphate isomerase (Tpi) locus of Drosophila melanogaster and Drosophila simulans

DNA sequence variation in a 1.1-kb region including the coding portion of the Tpi locus was examined in 25 homozygous third-chromosome lines of Drosophila melanogaster, nine lines of Drosophila simulans, and one line of Drosophila yakuba. Our data show that the widespread allozyme polymorphism observed in cosmopolitan D. melanogaster is due to a glutamic acid substitution occurring in a phylogenetically conserved lysine that has been identified as part of the "hinged-lid" active site of the enzyme. This observation suggests that the replacement polymorphism may have important functional consequences. One replacement polymorphism was also observed in D. simulans, although its functional relevance is more difficult to assess, since it affects a site that is not strongly conserved. This amino acid change in D. simulans is associated with a single lineage possessing seven unique silent substitutions, which may be indicative of balancing selection or population subdivision. The absence of fixed amino acid differences between D. melanogaster and D. simulans and only a single difference with D. yakuba suggests that triose phosphate isomerase is under strong functional constraint. Silent variation is slightly higher for D. melanogaster than for D. simulans. Finally, we outline the general lack of evidence for old balanced polymorphisms at allozyme loci in D. melanogaster.