Measuring cellular forces using bis-aliphatic hydrazone crosslinked stress-relaxing hydrogels.

Studies focused on understanding the role of matrix biophysical signals on cells, especially those when cells are encapsulated in hydrogels that are locally remodelled, are often complicated by appropriate methods to measure differences between the bulk and local material properties. From this perspective, stress-relaxing materials that allow long-term culture of embedded cells provide an opportunity to elucidate aspects of this biophysical signalling. In particular, rheological characterization of the stress relaxation properties allows one to link a bulk material measurement to local aspects of cellular functions by quantifying the corresponding cellular forces that must be applied locally. Here, embryonic stem cell-derived motor neurons were encapsulated in a well-characterized covalently adaptable bis-aliphatic hydrazone crosslinked PEG hydrogel, and neurite outgrowth was observed over time. Using fundamental physical relationships describing classical mechanics and viscoelastic materials, we calculated the forces and energies involved in neurite extension, the results of which provide insight to the role of biophysical cues on this process.

Changes in the expression of potassium channels during mouse T cell development.

In this report we have combined the whole-cell electrophysiological recording technique with flow microfluorometry to isolate phenotypically defined thymocytes and T lymphocytes. Results obtained showed that J11d-/Lyt-2-/L3T4- cells express none or very few delayed rectifier K+ channels, whereas most other Lyt-2-/L3T4- cells, as well as typical cortical thymocytes (Lyt-2+/L3T4+), do express K+ channels. Mature (Lyt-2+/L3T4- or Lyt-2-/L3T4+) thymocytes, which are heterogeneous for J11d expression, were also found to be heterogeneous for K+ channel expression. Consistent with this finding was the observation that the cortisone-resistant subpopulation of thymocytes, which express low levels of J11d, were enriched for cells expressing low levels of K+ channels. Mature phenotype peripheral T lymphocytes expressed very low levels of K+ channels, but upon activation with Con A were found to express high levels of K+ channels. The results suggest that K+ channel expression in T cells is developmentally regulated. Increased expression of the channel is induced in response to mitogenic signals throughout the T cell lineage. Expression of the channel, therefore, serves as a useful marker in defining steps in the T cell differentiation pathway.

KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel.

The M-current regulates the subthreshold electrical excitability of many neurons, determining their firing properties and responsiveness to synaptic input. To date, however, the genes that encode subunits of this important channel have not been identified. The biophysical properties, sensitivity to pharmacological blockade, and expression pattern of the KCNQ2 and KCNQ3 potassium channels were determined. It is concluded that both these subunits contribute to the native M-current.

Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues.

HCN cation channel mRNA expression was determined in the rabbit heart and neonatal and adult rat ventricle using RNase protection assays. In the rabbit SA node, the dominant HCN transcript is HCN4, representing >81% of the total HCN message. HCN1 is also expressed, representing >18% of the total HCN mRNA. Rabbit Purkinje fibers contained almost equal amounts of HCN1 and HCN4 transcripts with low levels of HCN2, whereas rabbit ventricle contained predominantly HCN2. The SA node contained 25 times the total HCN message of Purkinje fibers and 140 times the total HCN message of ventricle. No reports of hyperpolarization-activated current (If) exist in rabbit Purkinje fibers, and we could not record If in rabbit ventricular myocytes. To investigate the possible role of isoform switching in determining the voltage dependence of If, we determined the prevalence of HCN isoforms in neonatal and adult rat ventricle. We had previously determined the threshold for activation of If to be approximately -70 mV in neonatal rat ventricle and -113 mV in adult rat ventricle. In both neonatal and adult rat ventricle, only HCN2 and HCN4 transcripts are present. The ratio of HCN2 to HCN4 is approximately 5:1 in the neonate and 13:1 in the adult. Taken together, these results suggest that different cardiac regions express different isoforms of the HCN family. The HCN1 and HCN4 isoforms are most closely associated with a depolarized threshold for If activation, whereas the HCN2 isoform is associated with a more negative activation curve.

Effects of the renin-angiotensin system on the current I(to) in epicardial and endocardial ventricular myocytes from the canine heart.

The Ca(2+)-independent portion of transient outward K(+) current (I(to)) exhibits a transmural gradient in ventricle. To investigate control mechanisms for this gradient, we studied canine epicardial and endocardial ventricular myocytes with use of the whole-cell patch-clamp technique. I(to) was larger in amplitude, had a more negative voltage threshold for activation, and had a more negative midpoint of inactivation in epicardium. Recovery from inactivation was >10-fold slower in endocardium. Incubation of epicardial myocytes with angiotensin II for 2 to 52 hours altered I(to) to resemble unincubated endocardium and reduced the amplitude of the phase 1 notch of the action potential. In contrast, incubation of endocardial myocytes with losartan for 2 to 52 hours altered I(to) to resemble unincubated epicardium and induced a phase 1 notch in the action potential. With RNase protection assays, we determined that incubations with angiotensin II or losartan did not alter mRNA levels for either Kv4.3 or Kv1.4; thus, a change in the alpha subunit for I(to) is unlikely to be responsible. To test whether posttranslational modification produced the effects of angiotensin II, we coexpressed Kv4.3 and the angiotensin II type 1a receptor in Xenopus oocytes. Incubation with angiotensin II increased the time constant for recovery from inactivation of the expressed current by 2-fold with an incubation time constant of 3.7 hours. No effect on activation or inactivation voltage dependence was observed. These results demonstrate that the properties of I(to) in endocardium and epicardium are plastic and likely under the tonic-differing influence of the renin-angiotensin system.

MinK-related peptide 1: A beta subunit for the HCN ion channel subunit family enhances expression and speeds activation.

The HCN family of ion channel subunits underlies the currents I(f) in heart and I(h) and I(q) in the nervous system. In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a beta subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a beta subunit for the cardiac delayed rectifier I(Kr), these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org.

Ischemic but not pharmacological preconditioning requires protein synthesis.

BACKGROUND: Ischemic preconditioning (IPC) and pharmacological preconditioning (PPC) have both been shown to confer cardioprotective effects. However, the role of protein synthesis in preconditioning is unclear. METHODS AND RESULTS: Isolated rabbit hearts were treated with cycloheximide (CHx, 10 micromol/L), a protein synthesis inhibitor at the translational level, before 2 cycles of IPC (5 minutes of global ischemia/5 minutes of reperfusion, n=6) or PPC by pinacidil (PIN, 10 micromol/L; n=6), an ATP-sensitive potassium channel opener. Six rabbit hearts received actinomycin D (Act D, 20 micromol/L; n=6), a protein synthesis inhibitor at the transcriptional level, before IPC. The left anterior descending coronary artery was then occluded for 60 minutes and reperfused for 120 minutes. Control hearts received no treatment before prolonged ischemia (n=6). Left ventricular pressure, action potential duration, and coronary flow were measured. Infarct size is expressed as a percentage of the area at risk. IPC (n=6) and PIN (n=8) hearts experienced reduced infarct size compared with control hearts (22+/-3% and 27+/-2% versus 46+/-3%, IPC and PIN versus control; P:<0.01). Translational blockade (CHx) reversed the IPC infarct size reduction effect (22+/-3% versus 48+/-4%, IPC versus CHx+IPC; P:<0.01) but not the effects of pinacidil (27+/-2% versus 29+/-3%, PIN versus CHx+PIN; P:=NS). Transcriptional blockade (Act D) did not abolish the IPC effect (23+/-5% versus 22+/-3%, Act D+IPC versus IPC; P:=NS). There were no significant differences in electromechanical function consequent to CHx and Act D treatment. CONCLUSIONS: These findings suggest an important role for protein synthesis in the mechanism for IPC-mediated protection at the translational level, which may be different from PPC.

Transient outward current, Ito1, is altered in cardiac memory.

BACKGROUND: Cardiac memory refers to an altered T-wave morphology induced by ventricular pacing or arrhythmias that persist for variable intervals after resumption of sinus rhythm. METHODS AND RESULTS: We induced long-term cardiac memory (LTM) in conscious dogs by pacing the ventricles at 120 bpm for 3 weeks. ECGs were recorded daily for 1 hour, during which time pacing was discontinued. At terminal study, the heart was removed and the electrophysiology of left ventricular epicardial myocytes was investigated. Control (C) and LTM ECG did not differ, except for T-wave amplitude, which decreased from 0.12+/-0.18 to -0.34+/-0.21 mV (+/-SEM, P<0.05), and T-wave vector, which shifted from -37+/-12 degrees to -143+/-4 degrees (P<0.05). Epicardial action potentials revealed loss of the notch and lengthening of duration at 20 days (both P<0.05). Calcium-insensitive transient outward current (Ito) was investigated by whole-cell patch clamp. No difference in capacitance was seen in C and LTM myocytes. Ito activated on membrane depolarization to -25+/-1 mV in C and -7+/-1 mV (P<0.05) in LTM myocytes, indicating a positive voltage shift of activation. Ito density was reduced in LTM myocytes, and a decreased mRNA level for Kv4.3 was observed. Recovery of Ito from inactivation was significantly prolonged: it was 531+/-80 ms (n=10) in LTM and 27+/-6 ms (n=9) in C (P<0.05) at -65 mV. CONCLUSIONS: Ito changes are associated with and can provide at least a partial explanation for action-potential and T-wave changes occurring with LTM.

Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes.

In the experiments here, the developmental expression of the functional Ca(2+)-independent, depolarization-activated K+ channel currents, Ito and IK, and of the voltage-gated K+ channel (Kv) alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 in rat ventricular myocytes were examined quantitatively. Using the whole-cell patch clamp recording method, the properties and the densities of Ito and IK in ventricular myocytes isolated from postnatal day 5 (P5), 10 (P10), 15 (P15), 20 (P20), 25 (P25), 30 (P30), and adult (8-12 wk) rats were characterized and compared. These experiments revealed that mean Ito densities increase fourfold between birth and P30, whereas IK densities vary only slightly. Neither the time- nor the voltage-dependent properties of the currents vary measurably, suggesting that the subunits underlying functional Ito and IK channels are the same throughout postnatal development. In parallel experiments, the developmental expression of each of the voltage-gated K+ channel alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, was examined quantitatively at the mRNA and protein levels using subunit-specific probes. RNase protection assays revealed that Kv1.4 message levels are high at birth, increase between P0 and P10, and subsequently decrease to very low levels in adult rat ventricles. The decrease in message is accompanied by a marked reduction in Kv1.4 protein, consistent with our previous suggestion that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes. In contrast to Kv1.4, the mRNA levels of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 increase (three- to five-fold) between birth and adult. Western analyses, however, revealed that the expression patterns of these subunits proteins vary in distinct ways: Kv1.2 and Kv4.2, for example, increase between P5 and adult, whereas Kv1.5 remains constant and Kv2.1 decreases. Throughout development, therefore, there is a mismatch between the numbers of Kv alpha subunits expressed and the functional voltage-gated K+ channel currents distinguished electrophysiologically in rat ventricular myocytes. Alternative experimental approaches will be required to define directly the Kv alpha subunits that underlie functional voltage-gated K+ channels in these (and other) cells. In addition, the finding that Kv alpha subunit protein expression levels do not necessarily mirror mRNA levels suggests that caution should be exercised in attempting functional interpretations of observed changes in mRNA levels alone.

Effects of pentobarbitone on acetylcholine-activated channels in mammalian muscle.

Acetylcholine-activated single channel currents were recorded from the extrajunctional region of chronically denervated skeletal muscle of the rat by the patch clamp technique. In control experiments, the cumulative open-time, closed-time and burst length distributions could be well described by the sum of two exponentials. Pentobarbitone decreased the mean open time and increased the time constant of the fast component of the closed time distribution. These effects increased with drug concentration. The mean burst length was relatively independent of pentobarbitone concentration over the range of concentrations used (10-500 microM). These observations are inconsistent with a simple sequential blocking model and it is suggested that pentobarbitone has an allosteric site of action on receptor-channel complexes that makes the open state less stable.

Correlation between CD4+ lymphocyte counts, concurrent antigen skin test and tuberculin skin test reactivity in human immunodeficiency virus type 1-infected and -uninfected children with tuberculosis.

BACKGROUND: HIV-infected children are at high risk of developing tuberculosis after infection by Mycobacterium tuberculosis. Emphasis is placed on tuberculin skin testing (TST) for diagnosing tuberculosis in children; however, its value in HIV-infected children is controversial. OBJECTIVES: To determine whether concurrent antigen testing and/or CD4+ lymphocyte counts help in the interpretation of the TST in children with tuberculosis. METHODS: Children eligible for the study were diagnosed as having tuberculosis on clinical criteria. CD4+ lymphocyte counts and delayed-type hypersensitivity (DTH) test, using the CMI Multitest were performed when tuberculosis was diagnosed. RESULTS: One hundred thirty children were enrolled. Tuberculin reactivity was lower in HIV-infected children at all cutoff levels than in HIV-uninfected children (P < 0.0001). The positive predictive value of normal CD4+ lymphocyte counts in predicting tuberculin reactions of > or =5 mm (in HIV-1-infected) and > or =10 mm (in HIV-uninfected patients) were 50 and 80.3%, respectively (P < 0.0001). An intact DTH reaction to the CMI Multitest in predicting reactions of > or =5 mm and > or =10 mm to tuberculin in HIV-infected and -uninfected children were 55 and 76%, respectively (P < 0.001). Kwashiorkor was responsible for 53.3% of false-negative TST in HIV-uninfected children with normal CD4+ lymphocyte counts. CONCLUSION: TST is of limited value as an adjunct in diagnosing tuberculosis in HIV-infected children. CD4+ lymphocyte counts and concurrent DTH testing are not useful for predicting tuberculin reactivity in HIV-infected patients with tuberculosis.

Use of potassium sorbate for treatment of fungal infections.

One hundred and twenty-two cases of vaginal fungal infections treated with potassium sorbate are presented. A new method of follow-up home application by means of vaginal tampons is tried. Relief of symptoms is prompt, and yeast organism disappear; the safety and superior efficacy of a strengthened (3%) solution is established. Treatment of fungal infections in males is also discussed.

The distribution of mRNA encoded by a new member of the neuronal nicotinic acetylcholine receptor gene family (alpha 5) in the rat central nervous system.

The cellular localization of transcripts for a new putative agonist-binding subunit of the neuronal nicotinic acetylcholine receptor (nAChR), alpha 5, was examined using in situ hybridization in the rat central nervous system (CNS), alpha 5 subunit mRNA was localized to a small number of regions when compared with two of the other known agonist-binding subunits, alpha 3 and alpha 4, alpha 5 mRNA is expressed at relatively high levels in neurons of the subiculum (pyramidal layer), presubiculum and parasubiculum (layers IV and VI), which are components of the hippocampal formation, in the substantia nigra pars compacta and ventral tegmental area, in the interpeduncular nucleus, and in the dorsal motor nucleus of the vagus nerve. Moderate hybridization signals were detected in neurons of the isocortex (layer VIb), anterior olfactory nucleus, trigeminal ganglion, superior olivary complex, nucleus of the solitary tract, and area postrema. No hybridization above background levels was seen in the amygdala, septum, thalamus, hypothalamus, or cerebellum. These results suggest that the alpha 5 subunit differs from other known agonist-binding subunits in its distribution.

Effects of NGF overexpression on anatomical and physiological properties of sympathetic postganglionic neurons.

To examine the effects of increased target derived nerve growth factor (NGF) on the sympathetic nervous system, the superior cervical ganglion was characterized in transgenic mice overexpressing NGF in keratinized epithelium (e.g. skin, tongue and oral cavity). In these mice NGF overexpression was achieved via expression of an NGF transgene driven by the K14 keratin promoter. This promoter is expressed at approximately embryonic day 11 and thereafter expressed constitutively in the adult. This expression results in supranormal levels of NGF in targets of sympathetic postganglionic neurons prior to the period of programmed cell death. Examination of postnatal day 6 (PN6) and adult transgenic mice shows ca. 2.5-fold increase in neuron number in the superior cervical ganglion (SCG). Analysis of SCG neuronal size revealed a dramatic hypertrophy in the transgenic mice that is present by PN6 and is maintained in the adult. Intracellular physiological measurements of control superior cervical ganglia identified two distinct types of neurons identified on the basis of their response to depolarizing current; 'phasic' neurons fire a single action potential while 'tonic' neurons fire continuously. In adult transgenic mice the phasic neurons were 102% larger than control phasic neurons while the tonic neurons only increased 44% relative to controls. The hypertrophy of sympathetic ganglia in the transgenic mice was correlated with an increased innervation of skin and dorsal root ganglia, structures that either express the transgene or concentrate NGF produced by the skin.

Expression of the trk gene family of neurotrophin receptors in prevertebral sympathetic ganglia.

When the phenotype of neurons in pre- and paravertebral sympathetic ganglia are compared, there are marked differences in NGF dependence, neuropeptide content, connectivity and electrophysiological properties. The trophic interactions that induce these differences are currently poorly understood. One explanation is that prevertebral neurons receive a second neurotrophic signal, other than NGF, from their target of innervation. If this is the case, neurons in the prevertebral ganglia should express another neurotrophin receptor, in addition to the NGF receptor (trkA). To test this prediction, the level of expression of three neurotrophin receptors, trkA, trkB and trkC, were examined in one paravertebral sympathetic ganglia, the SCG, and two prevertebral ganglia, the celiac and superior mesenteric ganglia. It was found that mRNA encoding the full-length form of the trkB receptor was barely expressed in the SCG. Significantly higher levels of full-length trkB mRNA expression were found in the prevertebral ganglia. Ligands of the trkB receptor may, therefore, contribute to the differentiation and/or survival of some prevertebral sympathetic neurons.

Galanin-like peptide (GALP) neurone-specific phosphoinositide 3-kinase signalling regulates GALP mRNA levels in the hypothalamus of males and luteinising hormone levels in both sexes.

Galanin-like peptide (GALP) neurones participate in the metabolic control of reproduction and are targets of insulin and leptin regulation. Phosphoinositide 3-kinase (PI3K) is common to the signalling pathways utilised by both insulin and leptin. Therefore, we investigated whether PI3K signalling in neurones expressing GALP plays a role in the transcriptional regulation of the GALP gene and in the metabolic control of luteinising hormone (LH) release. Accordingly, we deleted PI3K catalytic subunits p110α and p110ß via conditional gene targeting (cKO) in mice (GALP-p110α/ß cKO). To monitor PI3K signalling in GALP neurones, these animals were also crossed with Cre-dependent FoxO1GFP reporter mice. Compared to insulin-infused control animals, the PI3K-Akt-dependent FoxO1GFP nuclear exclusion in GALP neurones was abolished in GALP-p110α/ß cKO mice. We next used food deprivation to investigate whether the GALP-neurone specific ablation of PI3K activity affected the susceptibility of the gonadotrophic axis to negative energy balance. Treatment did not affect LH levels in either sex. However, a significant genotype effect on LH levels was observed in females. By contrast, no genotype effect on LH levels was observed in males. A sex-specific genotype effect on hypothalamic GALP mRNA was observed, with fed and fasted GALP-p110α/ß cKO males having lower GALP mRNA expression compared to wild-type fed males. Finally, the effects of gonadectomy and steroid hormone replacement on GALP mRNA levels were investigated. Compared to vehicle-treated mice, steroid hormone replacement reduced mediobasal hypothalamus GALP expression in wild-type and GALP-p110α/ß cKO animals. In addition, within the castrated and vehicle-treated group and compared to wild-type mice, LH levels were lower in GALP-p110α/ß cKO males. Double immunofluorescence using GALP-Cre/R26-YFP mice showed androgen and oestrogen receptor co-localisation within GALP neurones. Our data demonstrate that GALP neurones are direct targets of steroid hormones and that PI3K signalling regulates hypothalamic GALP mRNA expression and LH levels in a sex-specific fashion.

Molecular identification of prey in the stomach contents of Harp Seals (Pagophilus groenlandicus) using species-specific oligonucleotides.

All methods of diet analysis in marine mammals, including hard part analysis (HPA), have biases affecting the accuracy of prey-species identification and frequency in the estimated diet due to differential consumption, digestion and retention. Using PCR amplification of specific prey DNA with species-specific primers, we developed a DNA-based method that complements HPA and provides an alternative means to detect prey from stomach contents of Harp Seals (Pagophilus groenlandicus). The target size that could be reliably amplified was determined using a digestion time-series of Atlantic Cod (Gadus morhua) tissue in simulated seal stomachs. Various target lengths were trialed using general teleost primers; amplicons of approximately 800 bp or less were consistently obtained. Prey species-specific PCR primers for Atlantic Cod, Arctic Cod (Boreogadus saida) and Capelin (Mallotus villosus) were designed and tested with DNA from the stomach contents of 31 Harp Seals. Amplicons were obtained for all three species-specific primer sets. Amplification results compared with HPA revealed: (i) Atlantic Cod hard parts were found in five stomachs where no Atlantic Cod DNA amplified, suggesting that Atlantic Cod may be over-represented in the estimated diet, (ii) amplification of Arctic Cod DNA occurred for 17 stomachs, including all 12 stomachs with, and five stomachs without, Arctic Cod hard parts, and (iii) Capelin DNA amplified for four of five stomachs with Capelin hard parts and for one stomach without Capelin hard parts. We conclude that PCR amplification of specific prey DNA provides a viable means to complement Harp Seal diet analysis by HPA, but suggest that valuable information for quantitative diet analysis rests in a quantitative PCR approach.

Paediatric emergency department anaphylaxis: different patterns from adults.

BACKGROUND AND AIMS: Data on acute paediatric anaphylaxis presentations to the emergency department (ED) are limited. All allergic presentations to one Australian paediatric ED were studied to determine epidemiological, clinical, and outcome data. METHODS: Retrospective, case based study of patients under 16 years attending one metropolitan, paediatric teaching hospital ED in Australia over three years. The medical records of patients presenting with generalised allergic reactions and anaphylaxis satisfying relevant ICD-9-CM diagnostic codes were studied. The incidence, age, sex ratio, co-morbidities, likely aetiology, clinical features, management, and disposal were determined. RESULTS: A total of 526 children with generalised allergic reactions, and 57 with anaphylaxis were included in the study. This represented incidences of 9.3:1000 ED presentations for generalised allergic reactions and 1:1000 for anaphylaxis. There were no fatalities. In anaphylaxis cases, a cause was recognised in 68.4%. Cutaneous features were present in 82.5%. A past history of asthma was reported in 36.8%. Adrenaline was used in 39.3% of severe anaphylaxis cases. The ED alone definitively cared for 97.8% of all patients. Follow up was inadequate in cases of anaphylaxis. CONCLUSIONS: This is the first reported incidence figure for paediatric anaphylaxis ED presentations in Australia, and is less than that reported in adults in the same local population. However, the incidence of generalised allergic reactions of 9.3:1000 was greater than in the adults. Virtually all paediatric allergic cases may be managed in the ED alone, provided that the importance of specialist follow up, particularly for severe anaphylaxis, is recognised.