Peroxidasin is required for full viability in development and for maintenance of tissue mechanics in adults.

Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membranes that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved throughout the animal kingdom, indicating they are important, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a new CRISPR-generated catalytic null, and found that homozygotes were mostly lethal with 13 % viable escapers. Mouse mutants also show semi-lethality, with Mendelian analysis demonstrating ∼50 % lethality and ∼50 % escapers. Despite the strong mutations, the homozygous fly and mouse escapers had low but detectable levels of Col4 crosslinking, indicating the existence of inefficient alternative crosslinking mechanisms, probably responsible for the viable escapers. Fly mutant phenotypes are consistent with decreased basement membrane stiffness. Interestingly, we found that even after basement membranes are assembled and crosslinked in wild-type animals, continuing Peroxidasin activity is required in adults to maintain tissue stiffness over time. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.

Analysis of Drosophila and mouse mutants reveals that Peroxidasin is required for tissue mechanics and full viability.

Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membrane that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved, indicating they are essential, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a newly generated catalytic null, and found that homozygotes were mostly lethal with 13% viable escapers. A Mendelian analysis of mouse mutants shows a similar pattern, with homozygotes displaying ~50% lethality and ~50% escapers. Despite the strong mutations, the homozygous escapers had low but detectable levels of Col4 crosslinking, indicating that inefficient alternative mechanisms exist and that are probably responsible for the viable escapers. Further, fly mutants have phenotypes consistent with a decrease in stiffness. Interestingly, we found that even after adult basement membranes are assembled and crosslinked, Peroxidasin is still required to maintain stiffness. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.

Interactions between breathing rate and low-frequency fluctuations in blood pressure and cardiac intervals.

Evidence derived from spontaneous measures of cardiovagal baroreflex sensitivity (BRS) suggests that slow breathing at 6 breaths/min augments BRS. However, increases in BRS associated with slow breathing may simply reflect the frequency-dependent nature of the baroreflex rather than the modulation of baroreflex function by changes in breathing rate per se. To test this hypothesis we employed a crossover study design (n = 14) wherein breathing rate and systolic arterial blood pressure (SAP) oscillation induced via the application of oscillating lower body negative pressure (OLBNP) were independently varied at fixed frequencies. Breathing rate was controlled at 6 or 10 breaths/min with the aid of a metronome, and SAP oscillations were driven at 0.06 Hz and 0.1 Hz using OLBNP. The magnitudes of SAP and R-R interval (cardiac period) oscillations were quantified using power spectral analysis, and the transfer function gain between SAP and R-R interval was used to estimate BRS. Linear mixed-effects models were used to examine the main effects and interactions between breathing rate and OLBNP frequency. There was no statistical interaction between breathing and OLBNP frequency (P = 0.59), indicating that the effect of breathing rate on BRS did not differ according to OLBNP frequency (and vice versa). Additionally, there was no main effect for breathing rate (P = 0.28). However, we observed a significant main effect for OLBNP frequency (P = 0.01) consistent with the frequency-dependent nature of baroreflex. These findings suggest that increases in spectral indices of BRS reflect the frequency dependence of the baroreflex and are not due to slow breathing per se.

The repeated sit-to-stand maneuver is a superior method for cardiac baroreflex assessment: a comparison with the modified Oxford method and Valsalva maneuver.

Baroreflex assessment has diagnostic and prognostic utility in the clinical and research environments, and there is a need for a reliable, simple, noninvasive method of assessment. The repeated sit-to-stand method induces oscillatory changes in blood pressure (BP) at a desired frequency and is suitable for assessing dynamic baroreflex sensitivity (BRS). However, little is known about the reliability of this method and its ability to discern fundamental properties of the baroreflex. In this study we sought to: 1) evaluate the reliability of the sit-to-stand method for assessing BRS and compare its performance against two established methods (Oxford method and Valsalva maneuver), and 2) examine whether the frequency of the sit-to-stand method influences hysteresis. Sixteen healthy participants underwent three trials of each method. For the sit-to-stand method, which was performed at 0.1 and 0.05 Hz, BRS was quantified as an integrated response (BRSINT) and in response to falling and rising BP (BRSDOWN and BRSUP, respectively). Test retest reliability was assessed using the intraclass correlation coefficient (ICC). Irrespective of frequency, the ICC for BRSINT during the sit-to-stand method was ≥0.88. The ICC for a rising BP evoked by phenylephrine (PEGAIN) in the Oxford method was 0.78 and ≤0.5 for the remaining measures. During the sit-to-stand method, hysteresis was apparent in all participants at 0.1 Hz but was absent at 0.05 Hz. These findings indicate the sit-to-stand method is a statistically reliable BRS assessment tool and suitable for the examination of baroreflex hysteresis. Using this approach we showed that baroreflex hysteresis is a frequency-dependent phenomenon.

Assessment of cerebral autoregulation: the quandary of quantification.

We assessed the convergent validity of commonly applied metrics of cerebral autoregulation (CA) to determine the extent to which the metrics can be used interchangeably. To examine between-subject relationships among low-frequency (LF; 0.07-0.2 Hz) and very-low-frequency (VLF; 0.02-0.07 Hz) transfer function coherence, phase, gain, and normalized gain, we performed retrospective transfer function analysis on spontaneous blood pressure and middle cerebral artery blood velocity recordings from 105 individuals. We characterized the relationships (n = 29) among spontaneous transfer function metrics and the rate of regulation index and autoregulatory index derived from bilateral thigh-cuff deflation tests. In addition, we analyzed data from subjects (n = 29) who underwent a repeated squat-to-stand protocol to determine the relationships between transfer function metrics during forced blood pressure fluctuations. Finally, data from subjects (n = 16) who underwent step changes in end-tidal P(CO2) (P(ET)(CO2) were analyzed to determine whether transfer function metrics could reliably track the modulation of CA within individuals. CA metrics were generally unrelated or showed only weak to moderate correlations. Changes in P(ET)(CO2) were positively related to coherence [LF: ß = 0.0065 arbitrary units (AU)/mmHg and VLF: ß = 0.011 AU/mmHg, both P < 0.01] and inversely related to phase (LF: ß = -0.026 rad/mmHg and VLF: ß = -0.018 rad/mmHg, both P < 0.01) and normalized gain (LF: ß = -0.042%/mmHg(2) and VLF: ß = -0.013%/mmHg(2), both P < 0.01). However, Pet(CO(2)) was positively associated with gain (LF: ß = 0.0070 cm·s(-1)·mmHg(-2), P < 0.05; and VLF: ß = 0.014 cm·s(-1)·mmHg(-2), P < 0.01). Thus, during changes in P(ET)(CO2), LF phase was inversely related to LF gain (ß = -0.29 cm·s(-1)·mmHg(-1)·rad(-1), P < 0.01) but positively related to LF normalized gain (ß = 1.3% mmHg(-1)/rad, P < 0.01). These findings collectively suggest that only select CA metrics can be used interchangeably and that interpretation of these measures should be done cautiously.

Sympathetic regulation of the human cerebrovascular response to carbon dioxide.

Although the cerebrovasculature is known to be exquisitely sensitive to CO(2), there is no consensus on whether the sympathetic nervous system plays a role in regulating cerebrovascular responses to changes in arterial CO(2). To address this question, we investigated human cerebrovascular CO(2) reactivity in healthy participants randomly assigned to the α(1)-adrenoreceptor blockade group (9 participants; oral prazosin, 0.05 mg/kg) or the placebo control (9 participants) group. We recorded mean arterial blood pressure (MAP), heart rate (HR), mean middle cerebral artery flow velocity (MCA(V mean)), and partial pressure of end-tidal CO(2) (Pet(CO(2))) during 5% CO(2) inhalation and voluntary hyperventilation. CO(2) reactivity was quantified as the slope of the linear relationship between breath-to-breath Pet(CO(2)) and the average MCAv(mean) within successive breathes after accounting for MAP as a covariate. Prazosin did not alter resting HR, Pet(CO(2)), MAP, or MCA(V mean). The reduction in hypocapnic CO(2) reactivity following prazosin (-0.48 ± 0.093 cm·s(-1) · mmHg(-1)) was greater compared with placebo (-0.19 ± 0.087 cm · s(-1) · mmHg(-1); P < 0.05 for interaction). In contrast, the change in hypercapnic CO(2) reactivity following prazosin (-0.23 cm · s(-1) · mmHg(-1)) was similar to placebo (-0.31 cm · s(-1) · mmHg(-1); P = 0.50 for interaction). These data indicate that the sympathetic nervous system contributes to CO(2) reactivity via α(1)-adrenoreceptors; blocking this pathway with prazosin reduces CO(2) reactivity to hypocapnia but not hypercapnia.

Self-injurious behaviour in intellectual disability syndromes: evidence for aberrant pain signalling as a contributing factor.

BACKGROUND: In most individuals, injury results in activation of peripheral nociceptors (pain-sensing neurons of the peripheral nervous system) and amplification of central nervous system (CNS) pain pathways that serve as a disincentive to continue harmful behaviour; however, this may not be the case in some developmental disorders that cause intellectual disability (ID). Moreover, individuals affected by ID disorders may initiate self-injurious behaviour to address irritating or painful sensations. In normal individuals, a negative feedback loop decreases sensation of pain, which involves descending inhibitory neurons in the CNS that attenuate spinal nociceptive processing. If spinal nociceptive signalling is impaired in these developmental disorders, an exaggerated painful stimulus may be required in order to engage descending anti-nociceptive signals. METHODS: Using electronic databases, we conducted a review of publications regarding the incidence of chronic pain or altered pain sensation in ID patients or corresponding preclinical models. RESULTS: There is a body of evidence indicating that individuals with fragile X mental retardation and/or Rett syndrome have altered pain sensation. These findings in humans are supported by mechanistic studies using genetically modified mice harbouring mutations consistent with the human disease. Thus, once self-injurious behaviour is initiated, the signal to stop may be missing. Several developmental disorders that cause ID are associated with increased incidence of gastroesophageal reflux disease (GERD), which can cause severe visceral pain. Individuals affected by these disorders who also have GERD may self-injure as a mechanism to engage descending inhibitory circuits to quell visceral pain. In keeping with this hypothesis, pharmacological treatment of GERD has been shown to be effective for reducing self-injurious behaviour in some patients. Hence, multiple lines of evidence suggest aberrant nociceptive processing in developmental disorders that cause ID. CONCLUSIONS: There is evidence that pain pathways and pain amplification mechanisms are altered in several preclinical models of developmental disorders that cause ID. We present hypotheses regarding how impaired pain pathways or chronic pain might contribute to self-injurious behaviour. Studies evaluating the relationship between pain and self-injurious behaviour will provide better understanding of the mechanisms underlying self-injurious behaviour in the ID population and may lead to more effective treatments.

Genome-wide scan of resistin mRNA expression in omental adipose tissue of baboons.

INTRODUCTION: The hormone resistin was recently discovered in adipose tissue of mice. Functional tests suggest a role for resistin in the regulation of insulin sensitivity. However, human studies have reported controversial results on the metabolic function of this hormone. METHODS: A 1 g omental adipose tissue biopsy was obtained from 404 adult baboons. Resistin mRNA expression was assayed by real-time, quantitative RT-PCR, and univariate and bivariate quantitative genetic analyses were performed, via the variance decomposition approach. A genome scan analysis was conducted using resistin mRNA abundance in omental adipose tissue as a quantitative phenotype. RESULTS: A significant heritability of h2 = 0.23 (P = 0.003) was found for resistin mRNA abundance in omental adipose tissue. A genome scan detected a quantitative trait locus for resistin expression with an LOD score of 3.8, in the region between markers D19S431 and D19S714, corresponding to human chromosome 19 p13. This chromosomal region contains genes related to insulin resistance phenotypes, such as resistin, insulin receptor, angiopoietin-like 4 protein and LDL receptor. CONCLUSIONS: Individual variation in resistin mRNA expression has a significant genetic component, and a gene or genes on chromosome 19 p13 may regulate resistin mRNA levels in baboon omental adipose tissue.

P2 receptors modulate respiratory rhythm but do not contribute to central CO2 sensitivity in vitro.

Multiple brainstem sites are proposed to contribute to central respiratory chemosensitivity, however, the underlying molecular mechanisms remain unknown. P2X2 subunit-containing ATP receptors, which mediate pH-sensitive currents, appear to contribute to central chemosensitivity in vivo [J. Physiol. 523 (2000) 441]. However, recent data from P2X2 knockout mice [J. Neurosci. 23 (2003) 11315] indicate that they are not essential. To further explore the role of P2 receptors in central chemosensitivity, we examined the effects of P2 receptor agonists/antagonists on respiratory-related activity and CO2-sensitivity of rhythmically-active in vitro preparations from neonatal rat. Our main findings: (i) that putative chemosensitive regions of the ventrolateral medulla are immunoreactive for the P2X2 subunit; (ii) that ATP potentiates respiratory frequency in a dose-dependent, and PPADS-sensitive (P2 receptor antagonist), manner; and (iii) that the increase in burst frequency produced by increasing CO2 is unaffected by PPADS, indicate that ATP is a potent modulator of respiratory activity, but that P2 receptors do not contribute to central chemosensitivity in vitro.

Genetics of leptin expression in baboons.

OBJECTIVE: Leptin gene expression is higher in females than in males, and is regulated by many factors including energy intake and insulin, but little is known about the inheritance of leptin gene expression. We have investigated leptin (LEP) gene express-ion, to determine whether it is heritable, and whether the difference in LEP expression between males and females has a genetic component. STUDY POPULATION: A total of 319 baboons (Papio hamadryas) (220 females, 99 males) from a captive, pedigreed colony. MEASUREMENTS AND METHODS: We cloned a baboon LEP cDNA, and quantified LEP mRNA expression in baboon omental adipose tissue using a ribonuclease protection assay. In addition, we assayed circulating leptin levels, adipocyte cell volume, and weight. We used maximum likelihood-based variance decomposition methods to determine the genetic architecture of LEP levels, including testing for genotype-by-sex interaction. RESULTS: Omental LEP mRNA expression was significantly and positively correlated with weight and adipocyte cell volume in baboons. Both mRNA and plasma levels of leptin were higher in females than in males, and both measures were heritable. The results of our genetic analysis show that there was a genotype-by-sex interaction in the levels of plasma leptin, but not in omental LEP mRNA. CONCLUSIONS: As in humans, baboon leptin mRNA and protein levels are expressed at a higher level in females than in males. We detected evidence that the plasma levels were affected by genes that are differentially expressed in males and females, while the omental mRNA levels were not. This finding suggests that the genes that differentially regulate plasma leptin levels between males and females may exert their effects on post-transcriptional processes.

Catalytic inhibition of human DNA topoisomerase IIalpha by hypericin, a naphthodianthrone from St. John's wort (Hypericum perforatum).

St. John's wort (Hypericum perforatum) is the most widely used herbal medicine for the treatment of depression. However, concerns have arisen about the potential of its interaction with other drugs due to the induction of cytochrome P450 isozymes 1A2 and 3A4 by the components hypericin and hyperforin, respectively. Structurally similar natural products are often employed as antitumor agents due to their action as inhibitors of DNA topoisomerases, nuclear enzymes that modify DNA during cellular proliferation. Preliminary findings that hypericin inhibited the DNA relaxation activity of topoisomerase IIalpha (topo II; EC 5.99.1.3) led us to investigate the mechanism of enzyme inhibition. Rather than stabilizing the enzyme in covalent complexes with DNA (cleavage complexes), hypericin inhibited the enzyme prior to DNA cleavage. In vitro assays indicate that hypericin is a potent antagonist of cleavage complex stabilization by the chemotherapeutics etoposide and amsacrine. This antagonism appears to be due to the ability of hypericin to intercalate or distort DNA structure, thereby precluding topo II binding and/or DNA cleavage. Supporting its non-DNA damaging, catalytic inhibition of topo II, hypericin was shown to be equitoxic to both wild-type and amsacrine-resistant HL-60 leukemia cell lines. Moreover, hypericin was incapable of stimulating DNA damage-responsive gene promoters that are activated by etoposide. As with the in vitro topo II assay, antagonism of DNA damage stimulated by 30 microM etoposide was evident in leukemia cells pretreated with 5 microM hypericin. Since many cancer patients experience clinical depression and concomitantly self-medicate with herbal remedies, extracts of St. John's wort should be investigated further for their potential to antagonize topo II-directed chemotherapy regimens.

Development of the ventilatory response to hypoxia in Swiss CD-1 mice.

We examined developmental changes in breathing pattern and the ventilatory response to hypoxia (7.4% O(2)) in unanesthetized Swiss CD-1 mice ranging in age from postnatal day 0 to 42 (P(0)-P(42)) using head-out plethysmography. The breathing pattern of P(0) mice was unstable. Apneas were frequent at P(0) (occupying 29 +/- 6% of total time) but rare by P(3) (5 +/- 2% of total time). Tidal volume increased in proportion to body mass ( approximately 10-13 ml/kg), but increases in respiratory frequency (f) (55 +/- 7, 130 +/- 13, and 207 +/- 20 cycles/min for P(0), P(3), and P(42), respectively) were responsible for developmental increases in minute ventilation (690 +/- 90, 1,530 +/- 250, and 2,170 +/- 430 ml. min(-1). kg(-1) for P(0), P(3), and P(42), respectively). Between P(0) and P(3), increases in f were mediated by reductions in apnea and inspiratory and expiratory times; beyond P(3), increases were due to reductions in expiratory time. Mice of all ages showed a biphasic hypoxic ventilatory response, which differed in two respects from the response typical of most mammals. First, the initial hyperpnea, which was greatest in mature animals, decreased developmentally from a maximum, relative to control, of 2.58 +/- 0.29 in P(0) mice to 1. 32 +/- 0.09 in P(42) mice. Second, whereas ventilation typically falls to or below control in most neonatal mammals, ventilation remained elevated relative to control throughout the hypoxic exposure in P(0) (1.73 +/- 0.31), P(3) (1.64 +/- 0.29), and P(9) (1. 34 +/- 0.17) mice but not in P(19) or P(42) mice.

Synaptic control of motoneuron excitability in rodents: from months to milliseconds.

1. Motoneurons (MN) shape motor patterns by transforming inputs into action potential output. This transformation, excitability, is determined by an interaction between synaptic inputs and intrinsic membrane properties. Excitability is not static, but changes over multiple time scales. The purpose of the present paper is to review our recent data on synaptic factors important in the dynamic control of MN excitability over time scales ranging from weeks to milliseconds. 2. Developmental changes in modulation of MN excitability are well established. Noradrenergic potentiation of hypoglossal (XII) MN inspiratory activity in rhythmically active medullary slice preparations from rodents increases during the first two postnatal weeks. This is due to increasing alpha 1- and beta-adrenoceptor excitatory mechanisms and to a decreasing inhibitory mechanism mediated by alpha 2-adrenoceptors. Over a similar period, ATP potentiation of XII inspiratory activity does not change. 3. Motoneuron excitability may also change on a faster time scale, such as between different behaviours or different phases of a behaviour. Examination of this has been confounded by the fact that excitatory synaptic drives underlying behaviour can obscure smaller concurrent changes in excitability. Using the rhythmically active neonatal rat brain-stem-spinal cord preparation, we blocked excitatory inspiratory drive to phrenic MN (PMN) to reveal a reduction in PMN excitability specific to the inspiratory phase that: (i) arises from an inhibitory GABAergic input; (ii) is not mediated by recurrent pathways; and (iii) is proportional to and synchronous with the excitatory inspiratory input. We propose that the proportionality of the concurrent inhibitory and excitatory drives provides a means for phase-specific modulation of PMN gain. 4. Modulation across such diverse time scales emphasizes the active role that synaptic factors play in controlling MN excitability and shaping behaviour.