Quantifying acyl-chain diversity in isobaric compound lipids containing monomethyl branched-chain fatty acids.

Compound lipids comprise a diverse group of metabolites present in living systems, and metabolic- and environmentally-driven structural distinctions across this family is increasingly linked to biological function. However, methods for deconvoluting these often isobaric lipid species are lacking or require specialized instrumentation. Notably, acyl-chain diversity within cells may be influenced by nutritional states, metabolic dysregulation, or genetic alterations. Therefore, a reliable, validated method of quantifying structurally similar even-, odd-, and branched-chain acyl groups within intact compound lipids will be invaluable for gaining molecular insights into their biological functions. Here we demonstrate the chromatographic resolution of isobaric lipids containing distinct combinations of straight-chain and branched-chain acyl groups via ultra-high-pressure liquid chromatography (UHPLC)-mass spectrometry (MS) using a C30 liquid chromatography column. Using metabolically-engineered adipocytes lacking branched-keto acid dehydrogenase A (Bckdha), we validate this approach through a combination of fatty acid supplementation and metabolic tracing using monomethyl branched-chain fatty acids and valine. We observe resolution of numerous isobaric triacylglycerols and other compound lipids, demonstrating the resolving utility of this method. This approach strengthens our ability to quantify and characterize the inherent diversity of acyl chains across the lipidome.

Oculomotor control in children with fetal alcohol spectrum disorders assessed using a mobile eye-tracking laboratory.

Prenatal exposure to alcohol can result in a spectrum of adverse developmental outcomes, collectively termed fetal alcohol spectrum disorders (FASDs). This study evaluated deficits in sensory, motor and cognitive processing in children with FASD that can be identified using eye movement testing. Our study group was composed of 89 children aged 8-15 years with a diagnosis within the FASD spectrum [i.e. fetal alcohol syndrome (FAS), partial fetal alcohol syndrome (pFAS), and alcohol-related neurodevelopmental disorder (ARND)], and 92 controls. Subjects looked either towards (prosaccade) or away from (antisaccade) a peripheral target that appeared on a computer monitor, and eye movements were recorded with a mobile, video-based eye tracker. We hypothesized that: (i) differences in the magnitude of deficits in eye movement control exist across the three diagnostic subgroups; and (ii) children with FASD display a developmental delay in oculomotor control. Children with FASD had increased saccadic reaction times (SRTs), increased intra-subject variability in SRTs, and increased direction errors in both the prosaccade and antisaccade tasks. Although development was associated with improvements across tasks, children with FASD failed to achieve age-matched control levels of performance at any of the ages tested. Moreover, children with ARND had faster SRTs and made fewer direction errors in the antisaccade task than children with pFAS or FAS, although all subgroups were different from controls. Our results demonstrate that eye tracking can be used as an objective measure of brain injury in FASD, revealing behavioral deficits in all three diagnostic subgroups independent of facial dysmorphology.

Gap junction connexon configuration in rapidly frozen myocardium and isolated intercalated disks.

By using two ultrarapid freezing techniques, we have captured the structure of rat and rabbit cardiac gap junctions in a condition closer to that existing in vivo than to that previously achieved. Our results, which include those from fully functional hearts frozen in situ in the living animal, show that the junctions characteristically consist of multiple small hexagonal arrays of connexons. In tissue frozen 10 min after animal death, however, unordered arrays are common. Examination of junction structure at intervals up to 40 min after death reveals a variety of configurations including dispersed and close-packed unordered arrays, and hexagonal arrays. By use of an isolated intercalated disk preparation, we show that the configuration of cardiac gap junctions in vitro cannot be altered by factors normally considered to induce functional uncoupling. These experiments demonstrate that, contrary to the conclusions of some earlier studies (Baldwin, K. M., 1979, J. Cell Biol., 82:66-75; Peracchia, C., and L. L. Peracchia, 1980, J. Cell Biol., 87:708-718), the arrangement of gap junction connexons, in cardiac tissue at least, cannot be used as a reliable guide to the functional state of the junctions.

Selective disruption of gap junctional communication interferes with a patterning process in hydra.

The cells that make up the body column of hydra are extensively joined by gap junctions, capable of mediating the rapid exchange of small hydrophilic molecules between the cytoplasms of neighboring cells. Both the rate of transfer of small molecules through the gap junctions and the rate of return of gap junction coupling after grafting experiments are sufficiently rapid to mediate events in the patterning of hydra tissue. Antibodies to the major rat liver gap junction protein (27,000 daltons) recognize a gap junction antigen in hydra and are effective in eliminating junctional communication between hydra cells. The antibodies perturb the head inhibition gradient in grafting operations, suggesting that cell-cell communication via gap junctions is important in this defined tissue patterning process.

Executive function deficits in children with fetal alcohol spectrum disorders (FASD) measured using the Cambridge Neuropsychological Tests Automated Battery (CANTAB).

BACKGROUND: Chronic prenatal alcohol exposure causes a spectrum of deleterious effects in offspring, collectively termed fetal alcohol spectrum disorders (FASD), and deficits in executive function are prevalent in FASD. The goal of this research was to test the hypothesis that children with FASD exhibit performance deficits in tasks that assess attention, planning and spatial working memory. METHODS: Subjects (8-15 years male and female children) with a diagnosis of fetal alcohol syndrome (FAS), partial FAS (pFAS), or alcohol-related neurodevelopmental disorder (ARND), and age- and sex-matched controls, completed four tasks selected from the Cambridge Neuropsychological Tests Automated Battery (CANTAB). RESULTS: Compared with age-matched control children (n = 92), subjects with FASD (n = 89) exhibited longer reaction and decision times (effect size range; Cohen's d = .51 to .73), suggesting deficits in attention. Children with FASD demonstrated deficits in planning and spatial working memory that became more pronounced when task difficulty increased. The largest effect size in this study population (Cohen's d = 1.1) occurred in the spatial working memory task. Only one outcome measure revealed differences across the diagnostic subgroups, although all groups were different from control. CONCLUSION: This study demonstrates that deficits in multiple executive function domains, including set shifting, planning and strategy use, attention and spatial working memory, can be assessed in children with FASD using an easy to administer, brief battery of computer-based neuropsychological tasks. The tasks appear to be equally sensitive for brain injury resulting from prenatal exposure to alcohol, regardless of the presence of facial dysmorphology.

Antisense down regulation of connexin31.1 reduces apoptosis and increases thickness of human and animal corneal epithelia.

The roles of the gap junction protein connexin31.1 (Cx31.1) are poorly understood, especially as the protein appears to form non-functional channels. Cx31.1 specific antisense oligodeoxynucleotides (ODNs) were designed to evaluate its roles in a corneal epithelium model. Expression of Cx31.1 in corneal epithelium extends from the suprabasal layers of polyhedral wing cells through to the flat squamous cells of superficial layers which are shed into the tear film. Deoxyribozymes (Dzs) were tested for cleavage efficacy using in vitro transcribed Cx31.1 mRNA. Cleavage results showed a putative tertiary structure for Cx31.1 mRNA with one region appearing to have a higher potential for antisense targeting. Application of antisense ODNs designed to this region caused Cx31.1 knockdown in rat and human corneal organotypic culture models, leading to a reduction in apoptosis and a thickening of the corneal epithelium (p=0.0045). Cx31.1 appears to play a role in triggering cell death; knocking it down may provide a novel approach for tissue repair and engineering.

Effects of chronic prenatal ethanol exposure on mitochondrial glutathione and 8-iso-prostaglandin F2alpha concentrations in the hippocampus of the perinatal guinea pig.

It is hypothesised that oxidative stress is a key mechanism of ethanol neurobehavioural teratogenicity, resulting in altered endogenous antioxidant status and increased membrane lipid peroxidation in the hippocampus of chronic prenatal ethanol exposure (CPEE) offspring. To test this hypothesis, timed pregnant guinea-pigs (term, approximately gestational day (GD) 68) received chronic daily oral administration of (i) 4 g ethanol kg(-1) maternal bodyweight, (ii) isocaloric sucrose with pair feeding, or (iii) water. At GD 65 (term fetus) and postnatal day (PD) 0 (neonate), individual offspring were killed, the brain was excised and the hippocampi were dissected. Glutathione (GSH) concentration was measured in the cytosolic and mitochondrial fractions of hippocampal homogenate. The occurrence of lipid peroxidation was determined by measuring the concentration of 8-iso-prostaglandin F2+/- (8-iso-PGF2+/-). There was CPEE-induced decreased brain weight and hippocampal weight at GD 65 and PD 0, decreased mitochondrial GSH concentration in the hippocampus at PD 0, with no change in mitochondrial GSH concentration at GD 65 or cytosolic GSH concentration at GD 65 or PD 0, and no change in mitochondrial or whole-homogenate 8-iso-PGF2+/- concentration in the hippocampus at GD 65 or PD 0. The data demonstrate that CPEE produces selective mitochondrial dysfunction in the hippocampus of the neonatal guinea-pig, involving GSH depletion.

Chronic prenatal ethanol exposure increases apoptosis in the hippocampus of the term fetal guinea pig.

It is hypothesized that chronic prenatal ethanol exposure (CPEE), via maternal ethanol administration, increases mitochondrial-directed apoptosis in the hippocampus of the term fetus that precedes loss of hippocampal CA1 pyramidal cells. To test this hypothesis, timed pregnant guinea pigs received chronic oral administration of: 4 g ethanol/kg maternal body weight/day, isocaloric-sucrose/pair-feeding or water throughout gestation. At gestational day 65 (term fetus) and postnatal day 0 (neonate), individual offspring were euthanized, and the brain was excised and dissected. CPEE, compared with the isocaloric-sucrose/pair-fed and water control groups, decreased the brain weight of the term fetus and neonate. CPEE did not alter the density of CA1 pyramidal cells in the hippocampus of the term fetus and neonate. In the term fetus, CPEE increased cytochrome c content in the cytosolic fraction of the hippocampus, altered the mitochondrial localization of cytochrome c in cells of the dorsal hippocampus, and increased the percentage of cells in the dorsal hippocampus containing activated caspase-3 and cleaved poly(ADP-ribose) polymerase. The data indicate that CPEE increases neuroapoptosis in the hippocampus of term fetus, which appears to occur via an intrinsic, mitochondrial-directed mechanism initiated by leakage of pro-apoptotic cytochrome c from mitochondria into the cytoplasm.

Cognitive function, quality of life and mental health in survivors of our-of-hospital cardiac arrest: a review.

There is growing interest in the long-term outcomes of patients surviving out-of-hospital cardiac arrest (OHCA). This paper aims to summarise the available literature on the long-term cognitive, health-related quality of life (QoL) and mental health outcomes of survivors of OHCA. Between 30% and 50% of survivors of OHCA experience cognitive deficits for up to several years post-discharge. Deficits of attention, declarative memory, executive function, visuospatial abilities and verbal fluency are commonly reported. Survivors of OHCA appear to report high rates of mental illness, with up to 61% experiencing anxiety, 45% experiencing depression and 27% experiencing post-traumatic stress. Fatigue appears to be a commonly reported long-term outcome for survivors of OHCA. Investigations of long-term QoL for these patients have produced mixed findings. Carers of survivors of OHCA report high rates of depression, anxiety and post-traumatic stress, with insufficient social and financial support. The heterogeneous range of instruments used to assess cognitive function and QoL prevent any clear conclusions being drawn from the available literature. The potential biases inherent in this patient population and the interaction between QoL, cognitive performance and mental health warrant further investigation, as does the role of post-discharge support services in improving long-term patient outcomes.

Battle of the hemichannels--Connexins and Pannexins in ischemic brain injury.

Perinatal ischemic brain injury can occur as a result of a global ischemic insult or focal ischemic stroke in the preterm or full-term neonate. One of the most striking features of HI injury is that, after initial recovery of cellular oxidative metabolism, there is a delayed, 'secondary' mitochondrial failure that spreads over time from the most severely damaged areas outwards, into previously undamaged regions. This secondary failure is accompanied by transient seizure activity and cytotoxic edema. The specific mechanisms of this spread are poorly understood, but it is at least partly associated with spreading waves of depression that can trigger cell death in neighboring uninjured tissues. Both Connexin and Pannexin hemichannels may mediate release of paracrine molecules that in turn propagate cell death messages by releasing intracellular mediators, such as ATP, NAD(+), or glutamate or by abnormally prolonged opening to allow cell edema. This review will discuss the controversy around the relative contribution of both Connexin and Pannexin hemichannels and mechanisms by which they may contribute to the spread of ischemic brain injury.

Validation of immunohistochemical quantification in confocal scanning laser microscopy: a comparative assessment of gap junction size with confocal and ultrastructural techniques.

Confocal scanning laser microscopy (CSLM) is increasingly being used to image antibody-labeled structures visualized with a fluorescent secondary antibody. Such digital images are routinely stored on computer and are well suited to quantitative analysis. Although theoretical aspects of CSLM imaging and resolution are well defined, information is lacking on the relationship observed between measurements of fluorescent antibody-labeled structures and the size of the same structures as determined by electron microscopy (EM). In the present study we examined this relationship for the cardiac gap junction. Data on the size of immunofluorescent-labeled gap junctions were acquired by two methods of analysis from CSLM images and compared statistically with measurements of gap junction size obtained by freeze-fracture EM. The freeze-fracture data were compared before and after exclusion of small junctions, corresponding to those that theoretically would not have been detected in CSLM analysis. The data obtained by the different methods were similar but not identical, reflecting the advantages and limitations of each technique. However, the comparison did indicate that with appropriate sample preparation and orientation, accurate and rapid analysis can be achieved by CSLM, particularly when digital semi-automated techniques are employed.

Functional decline in Alzheimer's disease: a longitudinal study.

OBJECTIVE: To assess the reliability of activities of daily living (ADL) measures and determine the rate and pattern of longitudinal ADL change in patients with Alzheimer's disease (AD). DESIGN: Longitudinal study with evaluations every 6 months. SETTING: Alzheimer's Disease Research Center Clinics at Mount Sinai Medical Center, New York, and the Veterans Affairs Medical Center, Bronx, New York. PATIENTS: 104 patients meeting NINCDS-ADRDA criteria for probable AD were followed an average of 31 months. MEASUREMENTS: Physical Self-Maintenance Scale (PSMS) and Instrumental Activities of Daily Living Scale (IADLS) of Lawton and Brody and the Blessed Test of Information, Memory, and Concentration. RESULTS: For men there was inconsistency over time in the decision about whether or not some IADLS items such as "Laundry" were appropriate for a given patient. Test-retest and inter-rater reliabilities were high for all PSMS items and for IADLS items that were judged to be appropriate. IADLS scores changed an average of 2.06 (+/- 3.27) points annually, and change scores were smaller for patients with severe dementia. PSMS scores changed an average of 2.44 (+/- 3.87) points annually with virtually all change occurring in severely demented patients. There was a marked ceiling effect for the IADLS and a curvilinear relationship of PSMS annual change with PSMS baseline. CONCLUSIONS: Both PSMS and IADLS measures can be reliable in AD patients, but better IADLS items for males need to be developed. IADLS scores are sensitive to change in mild and moderately demented AD patients, while PSMS scores are sensitive to change in more severely demented patients.

Noncognitive disturbances in Alzheimer's disease: frequency, longitudinal course, and relationship to cognitive symptoms.

OBJECTIVE: To investigate the frequency and longitudinal course of symptoms of depression, agitation, and psychosis in a longitudinally studied sample of patients with Alzheimer's disease (AD). DESIGN: Longitudinal study of AD patients with follow-up assessments at 6-month intervals for an average of more than 3 years. SETTING: Alzheimer's Disease Research Center of the Mount Sinai Medical Center and the Bronx VA Medical Center, New York. PARTICIPANTS: A total of 153 AD patients. MEASUREMENTS: Blessed Test of Information, Memory and Concentration (BIMC) and the Alzheimer's Disease Assessment Scale cognitive (ADAS-Cog) and noncognitive (ADAS-NC) subscales. RESULTS: At entry into the study, more than 90% of patients had a behavioral disturbance that was rated as mild or worse on one of the 10 ADAS noncognitive items; and 40% had at least one rating that was moderate or severe. Correlational analyses indicated that, with the exception of the two mood-related items, noncognitive symptoms on the ADAS were not highly correlated with one another. Only one of the noncognitive items, concentration, was strongly correlated with the severity of cognitive impairment. On average, patients showed progressively worse cognitive functioning over time as measured both by the ADAS-Cog and the BIMC. The mean severity of noncognitive symptoms did not change during the course of a 5-year follow up. The severity of behavioral disturbance at any one evaluation was negatively correlated with change in behavior during the next 6 months and was not correlated with cognitive decline. CONCLUSION: Mild behavioral disturbances are common, whereas moderate to severe behavioral symptoms are less frequent in this population of AD patients. Disturbances in mood and manifestations of agitation and psychotic symptoms are not closely related to one another and show little progressive worsening over time. Rather, they tend to be episodic such that increasing severity at one time is usually followed by improvement later. Concentration problems are a manifestation of cognitive dysfunction rather than behavioral disturbance in AD. Implications of these results for treatment of noncognitive disturbances in AD are discussed.

Do physical and sexual abuse differentially affect chronic pain states in women?

To evaluate the role of physical and/or sexual abuse on chronic pain symptoms and health care utilization in women, 104 consecutive female patients presenting to a multidisciplinary pain center for management of chronic pain were surveyed. Outcomes included a measure of sexual or physical abuse history (Drossman Sexual-Physical Abuse Survey), and measures of anxiety, health care utilization, substance abuse, and somatic symptoms. Forty-eight percent of the sample reported a history of physical abuse (PA) or sexual abuse (SA). Forty percent of the abused patients reported both PA and SA and the remainder reported SA (37%) or PA (23%) alone. The women who reported abuse had increased pain, physical symptoms, anxiety symptoms, and mental health care utilization compared to nonabused women. The women who reported abuse were also more likely to smoke and abuse street drugs. Women who reported both PA and SA were more likely to report head pain when compared to those who reported only PA or SA. Given the impact of abuse, particularly SA, on the presentation of chronic pain, queries regarding abuse should become a routine component of the patient interview. Abused patients should be referred to mental health care practitioners as a component of successful pain management if unresolved issues persist.

The role of childhood and adulthood abuse among women presenting for chronic pain management.

OBJECTIVE: This study investigated the association between repeated childhood and adulthood abuse and somatic symptom reporting, mental health care use, and substance use among women with chronic pain. DESIGN: A survey of a consecutive sample. PATIENTS: Ninety consecutive women patients presenting for chronic pain management at a multidisciplinary pain management center. OUTCOME MEASURES: The authors assessed the presence or absence of physical or sexual abuse (using the Drossman Physical-Sexual Abuse Survey), period of abuse, demographics, mental health care use, drug or alcohol use and substance abuse, and the presence or absence of physical, pain, and anxiety (somatic) symptoms. RESULTS: The response rate among patients surveyed was 64%. Of the 43 respondents (48%) who reported abuse, 17 (40%) cited childhood abuse, 12 (28%) cited adulthood abuse, and 14 (33%) cited repeated abuse. Women describing long-term abuse reported a significantly greater number of physical, pain, and anxiety symptoms and were more likely to report a history of substance abuse than women reporting abuse during childhood or adulthood alone. CONCLUSIONS: These data indicate a significant association between health status and reported abuse among women presenting to a multidisciplinary pain center for pain management. This finding is consistent with those of previous investigators, and emphasizes the importance of routine evaluation of the presence of long-term abuse as a possible predictor of the onset of chronic pain states.

Neoplasia in the rat induced by N-hydroxyphenacetin, a metabolite of phenacetin.

N-hydroxyphenacetin, a phenacetin metabolite, was fed to rats as a 0.05-0.5% dietary supplement. After 9 months, tumours of the liver were found in 36 of 64 animals. One animal also developed a renal tumour. No tumours were found in control animals. The findings implicate phenacetin as a carcinogen and suggest that N-hydroxyphenacetin may be the metabolite responsible.

Immunolabelling patterns of gap junction connexins in the developing and mature rat heart.

The distribution of gap junctions in prenatal, postnatal, and adult rat hearts was studied by laser scanning confocal microscopy, using antiserum raised to a peptide (HJ) matching part of the sequence of connexin43 (a cardiac gap junction protein). Using digital reconstruction of optically-sectioned tissue volumes, a highly sensitive detection of immunolabelled gap junctions was achieved. The distribution of positive anti-HJ immunolabelling was regionalised in the prenatal heart from its first detection at 10 days post-coitus. High levels of immunopositive staining occurred in the trabeculae of the embryonic ventricles. Other zones of the early myocardium including early central conduction tissues had no detectable signal. The prenatal outflow tract, interventricular septum and a narrow zone of myocardium subjacent to the epicardial free wall also had low levels of immunopositive signal. During postnatal growth and in the adult rat heart, a marked distinction emerged between the central conducting tissues of the atria and ventricles. Whilst small immunostained gap junctions became detectable within the atrioventricular node on the atrial side of the junction, between the interatrial and interventricular septa, no immunolabelling was found within the ventricular branching bundle. This difference between the atrioventricular node and branching bundle is consistent with potential functional distinctions between these two structures, and is not consistent with the recent proposal that the His bundle and its branches act as an extended atrioventricular node in smaller mammals such as the rat. Ventricular Purkinje fibres, distal to the branching bundle, showed high levels of anti-HJ immunostaining. Organisation of gap junctions into intercalated disks within the ventricle proceeded late into intercalated disks within the ventricle proceeded late into the adolescent stages of heart growth. The distribution of a second connexin protein, MP70, not previously characterised in the heart, was studied using monoclonal antibodies. MP70 was transiently immunolabelled in the heart during the postnatal period, but only within valves. Previously, this protein has been reported only in the eye lens. MP70-containing gap junctions may represent a specialisation in avascular tissues, since blood vessels are not present in either the eye lens or the cusps of heart valves.

Resolution of refractory symptoms of secondary erythermalgia with intermittent epidural bupivacaine.

BACKGROUND AND OBJECTIVES: Erythermalgia is a rare disorder characterized by erythema, edema, elevated skin temperature, and burning pain of the hands and/or feet. The etiology of primary erythermalgia is idiopathic while secondary erythermalgia is associated with autoimmune and rheumatologic factors. Symptoms are typically refractory to medical management. We report the use of epidural bupivacaine as a treatment option for secondary erythermalgia. CASE REPORT: A 28-year-old woman presented with secondary erythromelalgia that was characterized by severe pain, swelling, and erythema. Medical management failed and a lumbar epidural dosed with bupivacaine was used. This resulted in complete resolution of the patient's symptoms. CONCLUSION: Intermittent epidural bupivacaine may provide treatment of the refractory symptoms of secondary erythermalgia.

Calciphylaxis treated with neurolytic lumbar sympathetic block: case report and review of the literature.

BACKGROUND AND OBJECTIVE: Calciphylaxis is a painful complication of end-stage renal disease and secondary hyperparathyroidism. Calcification most commonly affects skin and soft tissue of the lower extremities resulting in excruciatingly painful skin ulcers. Treatment involves correction of hypercalcemia and hyperphosphatemia, parathyroidectomy, and supportive measures. METHODS: The literature and the merits of neurolytic lumbar sympathetic blockade (LSB) for the treatment of pain associated with calciphylaxis are reviewed. CONCLUSIONS: The neurolytic LSB provided pain relief and is a treatment modality to be considered in managing the pain associated with calciphylaxis.

A variation of the smooth septate junction in sea spiders (pycnogonida).

A new type of septate junction considered to be a variation of the arthropod smooth septate junction is described in pycnogonid (sea spider) endothermal tissue based on the use of conventional thin-section, lanthanum tracer and freeze-fracture techniques. This new type of septate junction is apparently unique to the Pycnogonida but closely resembles septate junctions previously described in the Merostomata and Collembola. This in conjunction with previous work suggests that the septa of smooth septate junctions may not be as 'smooth' as generally thought and probably have a complex substructure.