Relative contribution of clinicopathological variables, genomic markers, transcriptomic subtyping and microenvironment features for outcome prediction in stage II/III colorectal cancer.

BACKGROUND: It remains unknown to what extent consensus molecular subtype (CMS) groups and immune-stromal infiltration patterns improve our ability to predict outcomes over tumor-node-metastasis (TNM) staging and microsatellite instability (MSI) status in early-stage colorectal cancer (CRC). PATIENTS AND METHODS: We carried out a comprehensive retrospective biomarker analysis of prognostic markers in adjuvant chemotherapy-untreated (N = 1656) and treated (N = 980), stage II (N = 1799) and III (N = 837) CRCs. We defined CMS scores and estimated CD8+ cytotoxic lymphocytes (CytoLym) and cancer-associated fibroblasts (CAF) infiltration scores from bulk tumor tissue transcriptomes (CMSclassifier and MCPcounter R packages); constructed a stratified multivariable Cox model for disease-free survival (DFS); and calculated the relative proportion of explained variation by each marker (clinicopathological [ClinPath], genomics [Gen: MSI, BRAF and KRAS mutations], CMS scores [CMS] and microenvironment cells [MicroCells: CytoLym+CAF]). RESULTS: In multivariable models, only ClinPath and MicroCells remained significant prognostic factors, with both CytoLym and CAF infiltration scores improving survival prediction beyond other markers. The explained variation for DFS models of ClinPath, MicroCells, Gen markers and CMS4 scores was 77%, 14%, 5.3% and 3.7%, respectively, in stage II; and 55.9%, 35.1%, 4.1% and 0.9%, respectively, in stage III. Patients whose tumors were CytoLym high/CAF low had better DFS than other strata [HR=0.71 (0.6-0.9); P = 0.004]. Microsatellite stable tumors had the strongest signal for improved outcomes with CytoLym high scores (interaction P = 0.04) and the poor prognosis linked to high CAF scores was limited to stage III disease (interaction P = 0.04). CONCLUSIONS: Our results confirm that tumor microenvironment infiltration patterns represent potent determinants of the risk for distant dissemination in early-stage CRC. Multivariable models suggest that the prognostic value of MSI and CMS groups is largely explained by CytoLym and CAF infiltration patterns.

Prediction of overall survival in stage II and III colon cancer beyond TNM system: a retrospective, pooled biomarker study.

Background: TNM staging alone does not accurately predict outcome in colon cancer (CC) patients who may be eligible for adjuvant chemotherapy. It is unknown to what extent the molecular markers microsatellite instability (MSI) and mutations in BRAF or KRAS improve prognostic estimation in multivariable models that include detailed clinicopathological annotation. Patients and methods: After imputation of missing at random data, a subset of patients accrued in phase 3 trials with adjuvant chemotherapy (n = 3016)-N0147 (NCT00079274) and PETACC3 (NCT00026273)-was aggregated to construct multivariable Cox models for 5-year overall survival that were subsequently validated internally in the remaining clinical trial samples (n = 1499), and also externally in different population cohorts of chemotherapy-treated (n = 949) or -untreated (n = 1080) CC patients, and an additional series without treatment annotation (n = 782). Results: TNM staging, MSI and BRAFV600E mutation status remained independent prognostic factors in multivariable models across clinical trials cohorts and observational studies. Concordance indices increased from 0.61-0.68 in the TNM alone model to 0.63-0.71 in models with added molecular markers, 0.65-0.73 with clinicopathological features and 0.66-0.74 with all covariates. In validation cohorts with complete annotation, the integrated time-dependent AUC rose from 0.64 for the TNM alone model to 0.67 for models that included clinicopathological features, with or without molecular markers. In patient cohorts that received adjuvant chemotherapy, the relative proportion of variance explained (R2) by TNM, clinicopathological features and molecular markers was on an average 65%, 25% and 10%, respectively. Conclusions: Incorporation of MSI, BRAFV600E and KRAS mutation status to overall survival models with TNM staging improves the ability to precisely prognosticate in stage II and III CC patients, but only modestly increases prediction accuracy in multivariable models that include clinicopathological features, particularly in chemotherapy-treated patients.

The effect of auditory stimulation on the tensor tympani in patients following stapedectomy.

CONCLUSION: We believe that a tensor tympani reflex, in response to loud sound, is present in a minority of people, although its functional significance with regard to sound transmission is questionable. The absence of startle response in our stapedectomized subjects makes us question whether the tensor component of the startle response is large enough to be identified by manometry. OBJECTIVES: This study was organized to examine reflex activity to stimulation by loud sound or by startle in the tensor tympani. Although many previous studies have been carried out, results have been contradictory, and methodological flaws have rendered the interpretations questionable. PATIENTS AND METHODS: Stapedectomized patients were invited to take part in the study. Thirteen patients underwent testing by extratympanic manometry, using a standard tympanometer. Responses were observed during repeated stimuli with loud sound at 500, 1000, 2000 and 4000 Hz to the ipsilateral and contralateral ears, and with an air jet against the closed eye. A control group was also studied for the startle test. RESULTS: Three clear ipsilateral (23%) and two clear contralateral (14%) responses to auditory stimuli were seen in the 13 patients. We found responses to startle stimuli in none of our study group and eight (42%) of control ears.

In vivo neutrophil emigration in response to interleukin-1 and tumor necrosis factor-alpha.

The migration of polymorphonuclear leukocytes (PMN) in response to recombinant interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF), C5a, and f-met-leu-phe-lys (FMLPL) in vivo was studied using a mouse subcutaneous sponge implantation model. In this model sponges were implanted in C3H/OUJ mice, and 2 days later they were injected with the test sample. After varying times, sponges were removed and digested with collagenase, and total cell counts and differentials were enumerated. IL-1 was found to stimulate a significant influx of PMN, which peaked at 6 hr and declined to near baseline levels by 24 hr. This response was dose-dependent, with the greatest response observed when 5 units of IL-1 were injected. When the IL-1 concentration was increased to 10 U, the total number of PMN migrating into the sponge was decreased, compared with that observed with 5 U of IL-1. The overall number of PMN migrating into the sponge 6 hr after injecting 5 U of IL-1 averaged 269% of the number of PMN migrating randomly into the sponge. No difference in the total number of macrophages or lymphocytes in control or IL-1-injected sponges was observed in this time frame. Heat treatment of the IL-1 at 90 degrees C for 30 min ablated the response. Similar studies with TNF and C5a showed that both of these agents also stimulated an influx of PMN that peaked 6 hr postinjection. In contrast, FMLPL did not stimulate a PMN response. When IL-1 and TNF were injected simultaneously, an additive response was observed. These data indicate that IL-1, TNF, and C5a can all stimulate a PMN response in vivo and support the hypothesis that these substances are actively involved in the mobilization of PMN to inflammatory sites in vivo.

Migration of neutrophils from lung to tracheobronchial lymph node.

Translocation of particulate antigen deposited in the lung to tracheobronchial lymph nodes (TBLN) is important in the induction of pulmonary immune responses. We have previously shown that alveolar macrophages can contribute to particle translocation to TBLN, but whether neutrophils can also contribute to this process is not known. To determine if neutrophils can carry particles to the TBLN, dog neutrophils were elicited by instillation of red or green fluorescent microspheres into individual lung lobes. Autologous neutrophils that had phagocytized fluorescent microspheres were then instilled into an unexposed lobe of the same dog's lung. After 24 hr, the TBLN of instilled dogs had numerous neutrophils, 99% of which contained either red or green fluorescent microspheres but not both. Use of the two different colored microspheres as labels precluded the possibility that neutrophils had phagocytized the microspheres in the TBLN. In a second experiment, dogs were depleted of peripheral blood neutrophils by injections of hydroxyurea. Hydroxyurea-treated and normal dogs were instilled with fluorescent microspheres and killed after 40 h. Hydroxyurea treatment reduced neutrophil accumulation in the lung by 79% and reduced particle translocation to the TBLN by 80%. Results of these experiments indicate that neutrophils are similar to pulmonary alveolar macrophages (PAM) in their ability to phagocytize particles in the lung and then migrate to the TBLN.

Substance P activates NF-kappaB independent of elevations in intracellular calcium in murine macrophages and dendritic cells.

Professional antigen presenting cells, such as macrophages, can be activated by intracellular calcium-dependent as well as calcium-independent mechanisms, depending upon the stimulus used. In this report, we addressed the mechanism of substance P-induced intracellular signalling in murine macrophages and dendritic cells. While no increases in intracellular calcium concentration were detected in macrophages or dendritic cells using sensitive fluorimetric techniques, substance P did induce rapid enhanced activation of NF-kappaB, a transcriptional activator known to regulate pro-inflammatory cytokines. These data provide an important mechanism by which substance P may augment the production of pro-inflammatory molecules.

Postsynaptic fall in intracellular pH and increase in surface pH caused by efflux of formate and acetate anions through GABA-gated channels in crayfish muscle fibres.

H(+)-selective microelectrodes and a two- or three-microelectrode voltage clamp were used to examine the influence of weak-acid, carboxylate anions on the actions of GABA on postsynaptic intracellular pH, surface pH and on membrane potential in fibres of the crayfish leg opener muscle. Substitution of 30 mM Cl- by formate or acetate promoted a GABA-induced decrease in intracellular pH, which was coupled to an increase in surface pH and to a depolarization. Such effects were not seen in the presence of an equivalent amount of lactate, methanesulphonate or glucuronate. Both the GABA-induced depolarization and the fall in internal pH promoted by formate and acetate were blocked by picrotoxin, and the fall in pH was reversibly inhibited by a K(+)-induced depolarization. The rate of the fall in intracellular pH produced by GABA (0.2 mM) was about 0.02 pH units/min in the presence of formate and 0.03 pH units/min in the presence of acetate. Under steady-state conditions, both 30 mM formate and acetate (but not lactate) induced a positive shift in the reversal potential of GABA-activated current, which was accounted for by a relative permeability vs Cl- of formate and acetate of 0.5 and 0.15, respectively. The conductance sequence of the anions was identical to the permeability sequence, i.e. Cl- greater than formate greater than acetate greater than lactate approximately equal to 0. This sequence is strictly correlated to the Stokes diameter of the anions. The relative permeabilities of the anions indicate that the effective diameter of the GABA-gated channel is about 0.5 nm. The fact that the GABA-induced acidosis was slower in the presence of formate than in the presence of acetate suggests that, in the former case, the rate-limiting step in the fall in internal pH is the entry of non-dissociated formic acid. All the above results are consistent with a scheme where GABA induces a channel-mediated efflux of permeant weak-acid anions, which gives rise to an inward (depolarizing) current and to an intracellular acidosis. A comparison of the permeability properties of crayfish and vertebrate GABA-gated channels suggests that effects similar to those seen in this work are likely to occur in mammalian and other vertebrate neurons in the presence of permeant weak-acid anions.

Stabilisation of medically refractory ventricular arrhythmia by intra-aortic balloon counterpulsation.

OBJECTIVE: To review the efficacy of intra-aortic balloon counterpulsation (IABCP) in medically refractory ventricular arrhythmia. DESIGN: Retrospective analysis of the outcome of patients with ventricular arrhythmia treated with IABCP after transfer between 1992 and 1997. SETTING: Tertiary cardiac referral centre. PATIENTS: 21 patients (mean age 58 years) who underwent IABCP for control of ventricular arrhythmia. All had significant left ventricular impairment (mean ejection fraction 28.6%); 18 had coronary artery disease. RESULTS: Before IABCP, 10 patients had incessant monomorphic ventricular tachycardia and 11 had paroxysmal ventricular tachycardia and/or ventricular fibrillation (VT/VF). IABCP resulted in suppression of ventricular arrhythmia in 18 patients, of whom 13 were weaned from IABCP. After stabilisation of ventricular arrhythmia, 10 patients were maintained on medical treatment alone and one underwent endocardial resection. IABCP was maintained until cardiac transplantation in five patients. One patient had a fatal arrest before discharge and one died from progressive heart failure. IABCP failed to control ventricular arrhythmia in three patients and was subsequently discontinued. A cardiac assist device was employed in one of these until cardiac transplantation; the other two were eventually stabilised on medical treatment. Nineteen patients were discharged from hospital. Overall survival was 95% at mean follow up of 25.7 months. CONCLUSIONS: IABCP can be an effective means of controlling refractory ventricular arrhythmia, allowing time for the institution of more definitive treatment.

Influence of clinical and angiographic factors on development of collateral channels.

BACKGROUND: The degree of coronary collateralization is believed to be related to several clinical and angiographic factors. The duration and frequency of angina may be important factors in determining development of collateral channels. OBJECTIVE: To assess these factors for a consecutive series of patients suspected to have coronary artery disease. METHODS: Patients without at least one stenosis of < 50% and patients who had previously undergone bypass surgery were excluded from our study. Severity of stenosis was quantified by digital analysis, antegrade flow in terms of TIMI grade, and collaterals using the Rentrop classification. RESULTS: We reviewed 106 patients [mean age 61 years (range 35-84), 77.6% men]. Of these, 22 (21%) had presented with an acute coronary syndrome on this admission, whilst 46 patients (43%) had previously had an acute coronary syndrome. Collaterals were more likely in patients with stenoses of > 90% (Spearman correlation 0.65, P < 0.001) in patients with lower than normal TIMI flow grade (Spearman correlation 0.86, P < 0.01) and were related to regions of hypokinesis (Spearman correlation 0.35, P < 0.01). Significant collaterals were present in 14 patients (13%) despite their having TIMI grade II/III flow. Two of these patients had grade 2/3 collaterals with TIMI grade II/III antegrade flow. Degree of collateralization was not related to chronicity and frequency of symptoms, age, risk factors for atherosclerosis and nature of presentation (i.e. acute or stable symptoms). CONCLUSION: The likelihood of coronary collateralization cannot be prospectively predicted from clinical history alone, but appears to be largely a function of severity of stenosis and level of antegrade flow. A few patients develop high-grade collateral channels despite the presence of good antegrade flow.

Enumeration of lymphocyte subpopulations in bronchoalveolar lavage of monkeys using an immunoenzymatic staining technique.

Percentages of T-helper (OKT4), T-suppressor (OKTB), and B (B1) lymphocytes were determined in peripheral blood and bronchoalveolar lavage (BAL) from six cynomolgus monkeys using an alkaline phosphatase based immunoenzymatic staining technique. The percent of each lymphocyte subset in lung lavage fluids were 40 +/- 9%, 26 +/- 7% and 11 +/- 4% for OKT4, OKT8 and B1, respectively. This cell distribution is similar to that obtained from normal human BAL samples using fluorescence techniques to evaluate binding. Values for peripheral blood lymphocyte subsets were not statistically different from BAL. This immunoenzymatic technique avoids the necessity for cell separation procedures which are used to alleviate problems with alveolar macrophage autofluorescence that can be encountered in fluorescence based assays of BAL samples. This technique also can be used by laboratories interested in lymphocyte characterization, but not equipped for fluorescence microscopy or flow cytometry.

Isolation of sublethally injured campylobacters from poultry and water sources.

Campylobacters are the most common cause of bacterial food poisoning in the UK and are prominent in many other countries worldwide. Sources that have acted as vehicles of infection in many outbreaks include milk, water and poultry, and these may contain campylobacter cells that are sublethally injured and not detected by routine laboratory techniques. Current culture media contain antibiotics to suppress competing flora, and injured campylobacter cells are more sensitive to the antibiotics used and may not grow in these selective media. Therefore, the use of selective broth as the primary culture medium may reduce the isolation rate from samples containing injured cells. To examine this, various experiments were carried out using naturally contaminated river water and fresh chicken samples. In these samples, campylobacter cells showed varying degrees of sublethal injury, with the greatest found in water. A delay of four to eight hours before adding antibiotics to broth significantly increased isolation rate, compared with direct culture in selective broth. With chicken, however, significantly better results were obtained with selective broth as the primary medium.

A study of the corrosion products of mild steel in high ionic strength brines.

The corrosion layer on steel surfaces that formed after exposure to waste isolation pilot plant (WIPP) brines under anoxic conditions was characterized for chemical composition, thickness and phase composition. The chemical composition of the corrosion layer was determined both by X-ray photoelectron spectroscopy (XPS) and by chemical analysis of acid solutions used to remove the corrosion layer. Atomic force microscopic (AFM) images indicated that the brine-corroded surface layer shows extensive granulation along the contours of the steel surface that is characteristic of sharp polishing marks. The corrosion layer seemed to be porous and could be dissolved and detached in dilute hydrochloric acid. The corrosion layer appears to be composed of iron oxides with some ionic substitutions from the brines. The 77 K Mössbauer spectrum recorded for iron powder leached under similar conditions indicated the corrosion layer was comprised principally of green rust.

Direct observation of weight counselling in primary care: alignment with clinical guidelines.

Primary care physicians provide care to a disproportionate number of overweight and obese patients and are uniquely positioned to help patients manage their weight in the context of a continuity relationship. The US National Heart, Lung and Blood Institute (NHLBI) developed evidence-based guidelines for the effective and efficient care of overweight/obese patients, but little is known about the use of these guidelines in practice. To determine the content of weight discussions and assess the elements of the NHLBI guidelines that were accomplished, office visits of 544 adult, overweight/obese patients to 28 primary care physicians were observed and audio recorded. Associations between type of weight management discussion and patient, physician and visit characteristics were examined. Fifty per cent (n = 270) of visits included weight discussions; 47% and 38% included use of at least one NHLBI assessment or treatment element during discussions about weight, respectively. Only 35% (n = 193) of discussions included an assessment and treatment strategy; none included all NHLBI-recommended elements. Overall, adherence to guidelines was poor, particularly with regard to reporting body mass index to the patient, measuring waist circumference and setting realistic weight loss goals. Weight discussions did not clearly vary by the patient, physician or visit characteristics examined. These findings suggest opportunities to develop and further tailor resources for improved physician training in patient weight management communication and treatment techniques that are both consistent with current standards for effective, evidence-based care and efficient enough for routine use during busy primary care visits.

Flexibility within the middle ears of vertebrates.

INTRODUCTION AND AIMS: Tympanic middle ears have evolved multiple times independently among vertebrates, and share common features. We review flexibility within tympanic middle ears and consider its physiological and clinical implications. COMPARATIVE ANATOMY: The chain of conducting elements is flexible: even the 'single ossicle' ears of most non-mammalian tetrapods are functionally 'double ossicle' ears due to mobile articulations between the stapes and extrastapes; there may also be bending within individual elements. SIMPLE MODELS: Simple models suggest that flexibility will generally reduce the transmission of sound energy through the middle ear, although in certain theoretical situations flexibility within or between conducting elements might improve transmission. The most obvious role of middle-ear flexibility is to protect the inner ear from high-amplitude displacements. CLINICAL IMPLICATIONS: Inter-ossicular joint dysfunction is associated with a number of pathologies in humans. We examine attempts to improve prosthesis design by incorporating flexible components.

Monitoring the intracellular store Ca2+ concentration in agonist-stimulated, intact human platelets by using Fluo-5N.

BACKGROUND: Most Ca(2+) signaling research in platelets has relied solely on monitoring the cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). Changes in [Ca(2+)](cyt) constitute the net effect of Ca(2+) fluxes into the cytosol across the plasma membrane (PM) and from intracellular stores, and Ca(2+) sequestration into the stores and Ca(2+) removal across the PM. This makes interpretation of the effects of pharmacologic or genetic interventions on Ca(2+) signaling difficult and subject to error. OBJECTIVES: To validate the use of the low-affinity Ca(2+) indicator Fluo-5N to monitor the concentration of Ca(2+) in the intracellular stores ([Ca(2+)](st)) of human platelets as a first step in developing assays for a systems-level analysis of platelet Ca(2+) signaling. METHODS: Fluo-5N-loaded and Fura-2-loaded human platelets were used to observe the effects of agonist stimulation and other manipulations on [Ca(2+)](cyt) and [Ca(2+)](st). RESULTS: Fluo-5N fluorescence changed appropriately in response to compounds that induce passive depletion of intracellular Ca(2+) stores and to physiologic agonists. Ca(2+) reuptake inhibitors and blockers of Ca(2+) release channels had the expected effects on Fura-2 and Fluo-5N fluorescence. Agonist-evoked Ca(2+) release was reversed by Ca(2+) addition to the medium, and required intact Ca(2+) reuptake mechanisms. Store refilling was observed in the presence of sarcoplasmic/endoplasmic reticulum Ca(2+) -ATPase (SERCA) inhibitors and ionomycin, suggesting the presence of a non-SERCA Ca(2+) reuptake mechanism. Evidence for a role for Ca(2+) -induced Ca(2+) release in agonist-evoked responses was obtained. CONCLUSIONS: Our data provide a validation of the use of Fluo-5N as a method for monitoring changes in [Ca(2+)](st) in human platelets.

Phorbol ester-evoked Ca2+ signaling in human platelets is via autocrine activation of P(2X1) receptors, not a novel non-capacitative Ca2+ entry.

SUMMARY BACKGROUND: Platelets are reported to possess a protein kinase C (PKC)-dependent non-capacitative Ca(2+)entry (NCCE) pathway. The phorbol ester, phorbol, 12-myristate, 13-acetate (PMA) has been suggested to stimulate platelet NCCE. Recently we demonstrated important roles in store-operated Ca(2+)entry in human platelets for Na(+)/Ca(2+) exchangers (NCXs) and autocrine signaling between platelets after dense granule secretion. As PMA evokes dense granule secretion, we have investigated the role of NCXs and autocrine signaling in PMA-evoked Ca(2+)entry. OBJECTIVES: To investigate the roles of NCXs and dense granule secretion in PMA-evoked Ca(2+)signaling in human platelets. METHODS: Fura-2- or sodium-binding benzofuran isophthalate (SBFI)-loaded platelets were used to monitor cytosolic Ca(2+)or Na(+) concentrations. Dense granule secretion was monitored as ATP release using luciferin-luciferase. RESULTS: The NCX inhibitors KB-R7943 or SN-6, and removal of extracellular Na(+), significantly reduced PMA-evoked Ca(2+)entry. PMA-evoked dense granule secretion was almost abolished by pretreatment with the PKC inhibitor Ro-31-8220 and significantly slowed by KB-R7943. The P(2X1) antagonists Ro-0437626 or MRS-2159, or desensitization of P(2X1) receptors by prior treatment with alpha,beta-Methylene-ATP or omitting apyrase from the medium, reduced PMA-evoked Ca(2+)entry. Ro-0437626 or chelation of extracellular Ca(2+) slowed but did not abolish PMA-evoked ATP release, indicating that PMA-evoked dense granule secretion does not require P(2X1) receptor activation but is accelerated by P(2X1)-mediated Ca(2+)entry. The presence of NCX3 in human platelets was demonstrated by Western blotting. CONCLUSION: PMA-evoked Ca(2+)entry results from an NCX3-dependent dense granule secretion and subsequent P(2X1) receptor activation by secreted ATP, rather than activation of a novel NCCE pathway.

Antecedent moisture and seasonal precipitation influence the response of canopy-scale carbon and water exchange to rainfall pulses in a semi-arid grassland.

The influences of prior monsoon-season drought (PMSD) and the seasonal timing of episodic rainfall ('pulses') on carbon and water exchange in water-limited ecosystems are poorly quantified. *In the present study, we estimated net ecosystem exchange of CO(2) (NEE) and evapotranspiration (ET) before, and for 15 d following, experimental irrigation in a semi-arid grassland during June and August 2003. Rainout shelters near Tucson, Arizona, USA, were positioned on contrasting soils (clay and sand) and planted with native (Heteropogon contortus) or non-native invasive (Eragrostis lehmanniana) C4 bunchgrasses. Plots received increased ('wet') or decreased ('dry') monsoon-season (July-September) rainfall during 2002 and 2003. Following a June 2003 39-mm pulse, species treatments had similar NEE and ET dynamics including 15-d integrated NEE (NEE(pulse)). Contrary to predictions, PMSD increased net C uptake during June in plots of both species. Greater flux rates after an August 2003 39-mm pulse reflected biotic activity associated with the North American Monsoon. Furthermore, August NEE(pulse) and ecosystem pulse-use efficiency (PUE(e) = NEE(pulse)/ET(pulse)) was greatest in Heteropogon plots. PMSD and rainfall seasonal timing may interact with bunchgrass invasions to alter NEE and ET dynamics with consequences for PUE(e) in water-limited ecosystems.

The interpretation of current-clamp recordings in the cell-attached patch-clamp configuration.

In these experiments we have investigated the feasibility and accuracy of recording steady-state and dynamic changes in transmembrane potential noninvasively across an intact cell-attached patch using the current-clamp mode of a conventional patch-clamp amplifier. Using an equivalent circuit mimicking simultaneous whole-cell voltage-clamp and cell-attached current-clamp recordings we have defined both mathematically and experimentally the relationship between the membrane patch resistance, the seal resistance, and the fraction of the whole-cell potential recorded across an intact membrane patch. This analysis revealed a steep increase in the accuracy of recording of steady-state membrane potential as the seal/membrane ratio increases from 0. The recording accuracy approaches 100% as the seal/membrane ratio approaches infinity. Membrane potential measurements across intact cell-attached patches in rat basophilic leukemia cells and rat megakaryocytes revealed a surprisingly high degree of accuracy and demonstrated the ability of this noninvasive technique to follow dynamic changes in potential in nonexcitable cells.