Filtration of dermal fibroblast-conditioned culture media is required for the reliable quantitation of cleaved carboxy-terminal peptide of collagen type I (CICP) by ELISA

Cleavage of the collagen type I carboxy-terminal peptide (CICP) from the procollagen molecule is an essential step in collagen biosynthesis. The commercial CICP ELISA (Quidel Corporation, USA), developed for quantifying CICP in serum in clinical monitoring, is often also applied to cellular studies as a measure of collagen synthesis. However, unlike in serum samples, which contain only cleaved CICP, cell-conditioned culture media also contains “uncleaved CICP”, namely procollagen, and there is no specific guidance on how to interpret the ELISA data obtained with such samples. Here we attempted to reliably quantify cleaved CICP in human dermal fibroblast-conditioned cell culture media using the CICP ELISA. CICP concentration was determined in the parent and filtered samples of culture media of dermal fibroblasts (CCD-25SK). Gel-separated samples were also subjected to protein staining or analyzed by Western blot using the anti-CICP antibodies supplied in the ELISA kit. The derived concentrations of CICP in the filtered aliquots and the parent unfiltered samples increased over time. The increase in CICP in the unfiltered samples was not proportional to the increase seen in the filtered aliquot. CICP ELISA antibodies recognized both the cleaved CICP trimer and procollagen molecule. The data presented show that (a) the commercial CICP ELISA recognizes both procollagen and cleaved CICP in cell-conditioned culture media and thus attention should be paid in interpreting data from cell culture studies using this ELISA and (b) the filtration method described herein can be used to exclusively and reliably monitor cleaved CICP.     

Hypoxia/reoxygenation induces apoptosis through biphasic induction of protein synthesis in cultured rat brain neurons

To investigate biochemical events accounting for the outcome of central neurons following hypoxia/reoxygenation, cultured neurons from fetal rat forebrain were exposed to hypoxia (95% N₂/5% CO₂) for 6 h, and then reoxygenated for up to 96 h. Time-dependent changes in macromolecular biosynthesis were analysed by incorporation of [³H]uridine and [³H]leucine and were coupled to cell viability and lactate dehydrogenase leakage. Morphological features of necrosis and apoptosis were scored following nuclear incorporation of the fluorescent dye 4,6-diamidino-2-phenylindole. Hypoxia led to a 36% reduction of cell viability at the end of the reoxygenation period, while 23% of the neurons exhibited apoptosis. A biphasic increase in the rates of protein synthesis was measured 1 h after the onset of hypoxia (77% above controls) and by 48-h postreoxygenation (72%). The presence of cycloheximide during hypoxia inhibited both peaks of synthesis and prevented the development of apoptosis. Protein electrophoresis outlined specific alterations in constitutive proteins, and immunohistochemistry revealed an overexpression of the pro-apoptotic gene products Bax and ICE. Therefore, hypoxia followed by reoxygenation would trigger sequential changes in synthesis of specific proteins, leading to delayed and mainly apoptotic neuronal death.     

Brain volumes in late life: gender,hormone treatment,and estrogen receptor variants

Structural imaging studies suggest gender differences in brain volumes; however, whether hormone treatment (HT) can protect against age-related structural changes remains unknown, and no prior neuroimaging study has investigated potential interactions between HT and estrogen receptor (ESR) polymorphisms. Magnetic resonance imaging was used to measure gray and white matter, hippocampal volume, corpus callosum, cerebrospinal fluid (CSF), total intracranial volume (ICV) and white matter lesions (WML) in 582 non-demented older adults. In multivariable analysis, when compared to women who had never used HT, men and women currently on treatment, but not past users, had significantly smaller ratios of gray matter to ICV and increased atrophy (CSF/ICV ratio). Hippocampal and white matter volume as well as the corpus callosum area were not significantly different across groups. ESR2 variants were not significantly associated with brain measures, but women with the ESR1 rs2234693 C allele had significantly smaller WML. Furthermore this association was modified by HT use. Our results do not support a beneficial effect of HT on brain volumes in older women, but suggest the potential involvement of ESR1 in WML.     

Exposure to phthalates and phenols during pregnancy and offspring size at birth

Background: Data concerning the effects of prenatal exposures to phthalates and phenols on fetal growth are limited in humans. Previous findings suggest possible effects of some phenols on male birth weight.Objective: Our aim was to assess the relationships between prenatal exposures to phthalates and phenols and fetal growth among male newborns.Methods: We conducted a case–control study on male malformations of the genitalia nested in two French mother–child cohorts with recruitment between 2002 and 2006. We measured, in maternal urinary samples collected between 6 and 30 gestational weeks, the concentrations (micrograms per liter) of 9 phenol (n = 191 pregnant women) and 11 phthalate metabolites (n = 287). Weight, length, and head circumference at birth were collected from maternity records. Statistical analyses were corrected for the oversampling of malformation cases.Results: Adjusted birth weight decreased by 77 g [95% confidence interval (CI): –129, –25] and by 49 g (95% CI: –86, –13) in association with a 1-unit increase in ln-transformed 2,4-dichlorophenol (DCP) and 2,5-DCP urinary concentrations, respectively. Benzophenone-3 (BP3) ln-transformed concentrations were positively associated with weight (26 g; 95% CI: –2, 54) and head circumference at birth (0.1 cm; 95% CI: 0.0, 0.2). Head circumference increased by 0.3 cm (95% CI: 0.0, 0.7) in association with a 1-unit increase in ln-transformed BPA concentration. For phthalate metabolites there was no evidence of monotonic associations with birth weight.Conclusions: Consistent with findings of a previous study, we observed evidence of an inverse association of 2,5-DCP and a positive association of BP3 with male birth weight.     

Environmental and urinary markers of prenatal exposure to drinking water disinfection by-products, fetal growth, and duration of gestation in the PELAGIE birth cohort (Brittany, France, 2002-2006)

Although prenatal exposure to water disinfection by-products does not appear to affect the duration of gestation, its impact on fetal growth remains an open question. The authors studied the associations between prenatal exposure to disinfection by-products and fetal growth restriction (FGR) and preterm birth in the PELAGIE cohort, a French birth cohort comprising 3,421 pregnant women recruited between 2002 and 2006. Exposure was assessed by estimating levels of trihalomethanes (THMs) in tap water during pregnancy and maternal water use and by measuring maternal urinary levels of trichloroacetic acid (TCAA) during early pregnancy in a nested case-control design that compared 174 FGR cases, 114 preterm births, and 399 controls. Higher uptake of THMs (especially brominated THMs) was associated with a higher risk of FGR. Women with TCAA detected in their urine (>0.01 mg/L) had a higher risk of FGR than those with TCAA levels below the detection limit (adjusted odds ratio = 1.8, 95% confidence interval: 0.9, 3.7) and had an odds ratio for preterm birth below 1 (adjusted odds ratio = 0.8, 95% confidence interval: 0.3, 2.6). Results from this prospective study, the first to use a biomarker of disinfection by-product exposure, suggest that prenatal exposure affects fetal growth, but the causal agent or agents remain to be identified.     

Phosphorylation of the Cav3.2 T-type calcium channel directly regulates its gating properties

Phosphorylation is a major mechanism regulating the activity of ion channels that remains poorly understood with respect to T-type calcium channels (Cav3). These channels are low voltage-activated calcium channels that play a key role in cellular excitability and various physiological functions. Their dysfunction has been linked to several neurological disorders, including absence epilepsy and neuropathic pain. Recent studies have revealed that T-type channels are modulated by a variety of serine/threonine protein kinase pathways, which indicates the need for a systematic analysis of T-type channel phosphorylation. Here, we immunopurified Cav3.2 channels from rat brain, and we used high-resolution MS to construct the first, to our knowledge, in vivo phosphorylation map of a voltage-gated calcium channel in a mammalian brain. We identified as many as 34 phosphorylation sites, and we show that the vast majority of these sites are also phosphorylated on the human Cav3.2 expressed in HEK293T cells. In patch-clamp studies, treatment of the channel with alkaline phosphatase as well as analysis of dephosphomimetic mutants revealed that phosphorylation regulates important functional properties of Cav3.2 channels, including voltage-dependent activation and inactivation and kinetics. We also identified that the phosphorylation of a locus situated in the loop I-II S442/S445/T446 is crucial for this regulation. Our data show that Cav3.2 channels are highly phosphorylated in the mammalian brain and establish phosphorylation as an important mechanism involved in the dynamic regulation of Cav3.2 channel gating properties.Voltage-gated calcium channels (L-, N-, P/Q-, R-, and T-types) mediate calcium entry in many different cell types in response to membrane depolarization and action potentials. Calcium influx through these channels serves as an important second messenger of electrical signaling, initiating a variety of cellular events and physiological functions (1, 2). Among the family of voltage-gated calcium channels, T-type calcium channels (Cav3 family) have unique electrophysiological properties, because they display low voltage-activated calcium currents with rapid activation/inactivation kinetics. In neurons, small changes in the membrane potential near the resting potential can activate T-type channels, favoring further membrane depolarization and repetitive firing of action potentials (3–5). These unique gating properties of T-type channels make them important in many different processes, including neuronal spontaneous firing and pacemaker activities, rebound burst firing, sleep rhythms, sensory processing, and neuronal differentiation, as well as in pathological conditions, such as epilepsy and neuropathic pain (6).To properly assure this plurality of physiological functions, a tight control of T-type calcium channels is necessary. An important regulatory mechanism is phosphorylation, the fastest and most frequent posttranslational modification for a protein. Ion channels, especially voltage-gated channels, are critically regulated by phosphorylation. Voltage-dependent sodium and potassium channels have been shown to be the target of multiple phosphorylation events, regulating different channel functions and being involved in pathological states, like epilepsy (7–11). Also, several studies have shown the crucial role of the L-type/Cav1 phosphorylation in important physiological functions, like the fight or flight response (12–15).Regarding T-type channels, phosphorylation remains poorly understood. There are three Cav3 pore-forming proteins (Cav3.1, Cav3.2, and Cav3.3 subunits) all displaying typical properties of T-type channels when expressed in heterologous cell systems (3, 16). Among them, the Cav3.2 channel seems to be particularly sensitive to various types of regulation, including phosphorylation. To date, several serine/threonine kinases, like PKA, PKC, or CamKII, have been shown to regulate Cav3.2 activity (reviewed in refs. 17–20); however, in most cases, this regulation is tissue-dependent, and little is still known about its molecular basis. Cav3.2 channels bear more than 100 multiple intracellular serine and threonine residues that are predicted to be phosphorylated by common prediction algorithms, like NetPhos2.0 (21). However, which of these residues are actually phosphorylated and what functional impact this phosphorylation will have remain to be determined.In this study, we investigate the phosphorylation pattern of the Cav3.2 isoform of the T-type channels and its role in Cav3.2 channel properties. Using an MS approach, we have established the first, to our knowledge, phosphorylation map of the Cav3.2 channel in a mammalian brain and a human cell line. Then, by using alkaline phosphatase (AP) and dephosphomimetic mutants in patch-clamp experiments, we reveal the importance of phosphorylation in modulating Cav3.2 gating properties. We have also identified a phosphorylation hot spot situated in the loop connecting domains I and II of the channel that plays a crucial role in this regulation. Altogether, this study provides important insights regarding how phosphorylation regulates Cav3.2 channels.     

Comprehensive psychological intervention to improve outcome in functional gastrointestinal disorder: a cohort study

INTRODUCTION

Patients with functional gastrointestinal disorders (FGIDs) have a decreased quality of life (QoL). Psychological illnesses are strongly associated with FGIDs. This study examined the effect of a comprehensive psychological intervention programme designed for refractory FGID patients.

METHODS

Refractory FGID patients at a tertiary gastroenterology unit were encouraged to participate in a psychological intervention programme, which included screening for anxiety and depression in patients, educating patients and physicians on FGIDs, and providing early access to psychiatric consultation for patients with significant psychological illnesses. The duration of follow-up was six months. Outcomes were measured using the Irritable Bowel Syndrome-QoL (IBS-QoL) instrument and the EuroQol five dimensions (EQ-5D) questionnaire.

RESULTS

A total of 1,189 patients (68% female, 80% Chinese, mean age 48.6 years) participated in the programme. Among these participants, 51% had a significant psychological disorder (Hospital Anxiety and Depression Scale [HADS] anxiety or depression score > 7). These participants had a significantly poorer QoL (IBS-QoL and EQ-5D, both p < 0.0001), and were more likely to be single or English-speaking, as compared to the participants without psychological disorders. Participants who completed ≥ 3 months of follow-up (n = 906) showed significant and durable improvement. High baseline HADS anxiety score predicted improvement (p < 0.001), with participant IBS-QoL and EQ-5D scores decreasing over time.

CONCLUSION

The intervention programme was associated with a clinically meaningful improvement in the QoL of patients with refractory FGIDs. High baseline anxiety was predictive of improvement.