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.

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.

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.

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.

Sex differences in the middle ear of the bullfrog (Rana catesbeiana).

Impedance transformation by the middle ear of terrestrial vertebrates is considered to be a function of the ossicular lever apparatus, which increases force and decreases velocity at the stapes footplate relative to the tympanic membrane, and the ratio of tympanic membrane area to stapes footplate area (the area ratio), which increases pressure at the stapes. A recent vibrometric study has found that the velocity ratio between tympanic membrane and stapes footplate is higher in male bullfrogs (Rana catesbeiana) than in females. Although the tympanic membrane area is known to be larger in male bullfrogs, the area ratio has apparently never been compared between the sexes. The hypothesis that a lower area ratio in males helps to compensate for the higher velocity ratio was investigated by comparing tympanic membrane, stapes footplate and operculum areas in frogs of both sexes. Contrary to expectation, the difference in stapes footplate area between males and females is very small, and area ratios are considerably higher in males. Area ratios increase with increasing body size in both sexes. Operculum area does not differ between the sexes. The apparent difference in impedance transformation between male and female frogs might be necessary due to different inner ear impedances, or might reflect serious inadequacies in the conventional models of middle ear function.

Depolarisation-evoked Ca2+ waves in the non-excitable rat megakaryocyte.

1. A combination of patch clamp, confocal microscopy and immunohistochemistry was used to examine the spatial properties of Ca2+ signalling in the rat megakaryocyte, a non-excitable cell type in which membrane potential can markedly modulate agonist-evoked Ca2+ release. 2. Intracellular calcium ion concentration ([Ca2+]i) increases, stimulated by both ADP and depolarisation, frequently originated from a peripheral locus and spread as a wave throughout the cell. Spatially restricted [Ca2+]i increases, consistent with elementary Ca2+ release events, were occasionally observed prior to ADP-evoked waves. 3. ADP- and depolarisation-evoked Ca2+ waves travelled approximately twice as fast around the periphery of the cell compared to across its radius, leading to a curvilinear wavefront. There was no significant difference between wave velocities generated by the two stimuli. 4. Immunohistochemical staining of type III IP3 receptors, the endoplasmic reticulum-specific protein GRP78/BiP and calreticulin indicated a major peripheral location of the cellular Ca2+ stores which probably accounts for the accelerated wave velocity at the cell periphery. 5. These data demonstrate that [Ca2+]i increases, stimulated by depolarisation or the agonist ADP, have indistinguishable spatial properties, providing evidence that similar underlying mechanisms are responsible for their generation.

Voltage-dependent Ca2+ release in rat megakaryocytes requires functional IP3 receptors.

Using simultaneous whole-cell patch-clamp and fluorescence measurements of [Ca2+]i in rat megakaryocytes we have investigated the requirement for functional inositol 1,4,5-trisphosphate (IP3) receptors in Ca2+ release induced by membrane depolarization during agonist stimulation. Voltage-dependent Ca2+ release was observed during application of the IP3-generating agonists U46619 (a thromboxane A2 analogue) and ADP. Furthermore, voltage-dependent Ca2+ release was observed in the absence of exogenous agonist following sensitization of IP3 receptors with thimerosal. Depolarization-induced Ca2+ release was not detected during depletion of intracellular Ca2+ stores by thapsigargin. Thus, depletion of stores alone is not sufficient to confer voltage dependence upon the Ca2+ release mechanism. Block of IP3 receptors by carbacyclin-stimulated elevations in cAMP, uncaging of cAMP or exposure to a high concentration of caffeine reversibly abolished Ca2+ increases stimulated by both ADP and depolarization. The cAMP-dependent block was prevented by a peptide inhibitor of protein kinase A, indicating that an alteration of adenylate cyclase activity leading to modulation of protein kinase A activity does not underlie the control of Ca2+ release by voltage. These results are consistent with the requirement for functional IP3 receptors for voltage control of Ca2+ release from intracellular stores during inositol lipid signalling. The data also indicate the involvement of a voltage sensor downstream of surface membrane receptors in the depolarization-evoked Ca2+ response.

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.

Investigation of Head Injury Mechanisms Using Neutral Density Technology and High-Speed Biplanar X-ray.

The principal focus of this study was the measurement of relative brain motion with respect to the skull using a high-speed, biplanar x-ray system and neutral density targets (NDTs). A suspension fixture was used for testing of inverted, perfused, human cadaver heads. Each specimen was subjected to multiple tests, either struck at rest using a 152-mm-diameter padded impactor face, or stopped against an angled surface from steady-state motion. The impacts were to the frontal and occipital regions. An array of multiple NDTs was implanted in a double-column scheme of 5 and 6 targets, with 10 mm between targets in each column and 80 mm between columns. These columns were implanted in the temporoparietal and occipitoparietal regions. The impacts produced peak resultant accelerations of 10 to 150 g, and peak angular accelerations between 1000 and 8000 rad/s(2). For all but one test, the peak angular speeds ranged from 17 to 22 rad/s. The relative 3D displacements between the skull and the NDTs were analyzed. The localized motions of the brain generally followed loop or figure eight patterns, with peak displacements on the order of +/- 5 mm. These results can be used to further finite-element modeling efforts.

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.

A novel role for membrane potential in the modulation of intracellular Ca2+ oscillations in rat megakaryocytes.

1. The effect of membrane potential (Vm) on ADP-evoked [Ca2+]i oscillations was investigated in rat megakaryocytes, a non-excitable cell type recently shown to exhibit depolarisation-evoked Ca2+ release from intracellular stores during metabotropic purinoceptor stimulation. 2. Hyperpolarising voltage steps caused a transient fall in [Ca2+]i and either abolished Ca2+ oscillations or reduced the oscillation amplitude. These effects were observed in both the presence and absence of extracellular Ca2+ and also in Na+-free saline solutions, suggesting that hyperpolarisation leads to a reduction in the level of ADP-dependent Ca2+ release without a requirement for altered transmembrane Ca2+ fluxes. 3. In the presence of Ca2+ oscillations, depolarising voltage steps transiently enhanced the amplitude of Ca2+ oscillations. Following run-down of Ca2+ oscillations, depolarisation briefly restimulated oscillations. 4. Simultaneous [Ca2+]i and current-clamp recordings showed that Ca2+ and Vm oscillate in synchrony, with an average fluctuation of approximately 30-40 mV, due to activation and inactivation of Ca2+-dependent K+ channels. Application of a physiological oscillating Vm waveform to non-oscillating cells under voltage clamp stimulated [Ca2+]i oscillations. 5. Analysis of the relationship between [Ca2+]i and Vm showed a threshold for activation of hyperpolarisation at about 250-300 nM. The implications of this threshold in the interaction between Vm and Ca2+ release during oscillations are discussed. 6. We conclude that the ability of voltage to control release of endosomal Ca2+ in ADP-stimulated megakaryocytes is bipolar in nature. Our data suggest that Vm changes are active components of the feedback/feedforward mechanisms contributing to the generation of Ca2+ oscillations.

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.

Clinical mental health training within a multidisciplinary school-based health clinic.

A model is presented to illustrate a clinical mental health training program within a multidisciplinary School-Based Health Clinic (SBHC). In collaboration with schools of education, medicine, nursing, and social work, a multidisciplinary training and treatment program was established that provided unique opportunities for clinical training. An ecological/public health model was utilized as the conceptual framework for clinical mental health training, treatment, and research. Preliminary clinical outcome data suggest that out of a sample of 381 patients, 15% of those utilizing the SBHC's mental health services were being treated for substance abuse disorders. This finding supports current research that has demonstrated that the SBHCs are providing access to students who are in the most serious need to mental health services. Process data suggest that the SBHC is an effective site for multidisciplinary clinical mental health training.

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.

Transitional changes in membrane potential and intracellular [Ca2+] in rat basophilic leukemia cells.

Using whole-cell current-clamp measurements we have found that thapsigargin-mediated activation of store-regulated Ca2+ entry in rat basophilic leukemia cells is accompanied by complex changes in membrane potential. These changes consisted of: (i) an initial slow, small depolarization, (ii) a transitional change in potential to a depolarized value and (iii) transitional changes between a hyperpolarized and a depolarized potential. These complex changes in potential can be explained by the interaction between the endogenous inwardly rectifying K+ conductance and the generation of a small inward current. To investigate the possible influence of these changes of potential on [Ca2+]i, single cell measurements of fura2 fluorescence were undertaken alone or in combination with current-clamp measurements. Thapsigargin-mediated activation of the store-regulated Ca2+ entry pathway was accompanied by a marked increase of [Ca2+]i. During this increase, transient, abrupt declines in [Ca2+]i were detected in approximately 60% of the cells investigated. These changes of [Ca2+]i are consistent with the observed changes of membrane potential recorded under current-clamp.