Alpha-oestrogen and progestin receptor expression in the hypothalamus and preoptic area dopaminergic neurones during oestrous in cycling rats

A secretory surge of prolactin occurs on the afternoon of oestrous in cycling rats. Although prolactin is regulated by ovarian steroids, plasma oestradiol and progesterone levels do not vary during oestrous. Because prolactin release is tonically inhibited by hypothalamic dopamine and modulated by dopamine transmission in the preoptic area (POA), the present study aimed to evaluate whether oestrogen receptor (ER)-alpha and progestin receptor (PR) expression in the dopaminergic neurones of arcuate (ARC), periventricular, anteroventral periventricular (AVPe) and ventromedial preoptic (VMPO) nuclei changes during the day of oestrous. Cycling rats were perfused every 2 h from 10-20 h on oestrous. Brain sections were double-labelled to ER alpha or PR and tyrosine hydroxylase (TH). The number of TH-immunoreactive (ir) neurones did not vary significantly in any area evaluated. ER alpha expression in TH-ir neurones increased at 14 and 16 h in the rostral-ARC and dorsomedial-ARC, 14 h in the caudal-ARC and 16 h in the VMPO, whereas it was unaltered in the ventrolateral-ARC, periventricular and AVPe. PR expression in TH-ir neurones of the periventricular and rostral, dorsomedial, ventrolateral and caudal-ARC decreased transitorily during the afternoon, showing the lowest levels between 14 and 16 h; but it did not vary in the AVPe and VMPO. Plasma oestradiol and progesterone concentrations were low and unaltered during oestrous, indicating that the changes in receptors expression were probably not due to variation in ligand levels. Thus, our data suggest that variations in ER alpha and PR expression may promote changes in the activity of medial basal hypothalamus and POA dopaminergic neurones, even under unaltered secretion of ovarian steroids, which could facilitate the occurrence and modulate the magnitude of the prolactin surge on oestrous.

Binding and functional studies with the growth hormone receptor antagonist, B2036-PEG (pegvisomant), reveal effects of pegylation and evidence that it binds to a receptor dimers

GH actions are dependent on receptor dimerization. The GH receptor antagonist, B2036-PEG, has been developed for treating acromegaly. B2036 has mutations in site 1 to enhance receptor binding and in site 2 to block receptor dimerization. Pegylation (B2036-PEG) increases half-life and lowers immunogenicity, but high concentrations are required to control insulin-like growth factor-I levels. We examined antagonist structure and function and the impact of pegylation on biological efficacy. Unpegylated B2036 had a 4.5-fold greater affinity for GH binding protein (GHBP) than GH but similar affinity for membrane receptor. Pegylation substantially reduced membrane binding affinity and receptor antagonism, as assessed by a transcription assay, by 39- and 20-fold, respectively. GHBP reduced antagonist activity of unpegylated B2036 but did not effect antagonism by B2036-PEG. B2036 down-regulated receptors, and membrane binding sites doubled in the presence of dimerization-blocking antibodies, suggesting that B2036 binds to a receptor dimer. It is concluded that the high concentration requirement of B2036-PEG for clinical efficacy relates to pegylation, which decreases binding to membrane receptor but has the advantages of reduced clearance, immunogenicity, and interactions with GHBP. Our studies suggest that B2036 binds to a receptor dimer and induces internalization but not signaling.

Crystallization and preliminary diffraction studies of native and selenomethionine CcmG (CycY, DsbE)

t Disulfide-bond (Dsb) proteins are a family of redox proteins containing a Cys-X-X-Cys motif. They are essential for disulfide-bond exchange in the bacterial periplasm and are necessary for the correct folding and function of many secreted proteins. CcmG (DsbE) is a reducing Dsb protein required for cytochrome c maturation. Crystals of Bradyrhizobium japonicum CcmG have been obtained that diffract X-rays to 1.14 Angstrom resolution. The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit-cell parameters a = 35.1, b = 48.2, c = 90.2 Angstrom. Selenomethionine CcmG was expressed without using a methionine auxotroph or methionine-pathway inhibition and was purified without reducing agents.

Development of response-selective agonists of human C5a anaphylatoxin: Conformational, biological, and therapeutic considerations

Numerous studies on the relationship between the structure and function of peptide agonists derived from the biologically active, C-terminal region of human C5a anaphylatoxin have been reported over the past decade. These studies have been performed with the objective of parlaying this structure-function information into the design of peptide/peptidomimetic modulators of C5a receptor (C5aR)-mediated function. In this review, we describe a rational approach for the development of conformationally biased, decapeptide agonists of C5a and described how these stabilized and specific conformational features relate to the expression of specific C5a-like activities in vitro and in vivo. The therapeutic potential of such response-selective C5a agonists is discussed and underscored by the results of one such response-selective C5a agonist that was used in vivo as an effective molecular adjuvant capable of generating antigen-specific humoral and cellular immune responses. Finally, we describe the synthesis of a new generation of highly response-selective, conformationally biased C5a agonist and discuss the in vitro and in vivo biologic results that so indicate this biologic selectivity.

A cross-sectional study of the association between Heberden's nodes, radiographic osteoarthritis of the hands, grip strength, disability and pain

Objective: To describe the associations between hand osteoarthritis (OA), pain and disability in males and females and to further validate the Australian/Canadian CA hand index (AUSCAN LK3.0). Design: Cross-sectional study of 522 subjects from 101 Tasmanian families (males N=174, females N=348). Hand OA was assessed by two observers using the Altman atlas for joint space narrowing and osteophytes at distal interphalangeal and first carpometacarpal joints as well as a score for Heberden's nodes based on hand photography. Hand pain and function were assessed by the AUSCAN LK3.0 and grip strength by dynamometry in both hands on two occasions. Results: The prevalence of hand CA was high in this sample at 44-71% (depending on site). Pain and dysfunction increased with age while grip strength decreased (all P <0.001). All three measures were markedly worse in women, even after taking the severity of arthritis into account. Hand CA explained 5.7-10% of the variation in function, grip strength and pain scores, even after adjustment for age and sex. Further adjustment suggested that the osteoarthritic associations with function and grip strength were largely mediated by pain. Severity of disease was more strongly associated with these scores than presence or absence. Lastly, the AUSCAN LK3.0 showed a comparable association to grip strength with structural damage providing further evidence of index validity. Conclusions: Hand CA at these two sites makes substantial contributions to hand function, strength and pain. The associations with function and strength measures appear mediated by pain. Gender differences in all three measures persist after adjustment for variation in age and CA severity indicating that factors apart from radiographic disease are responsible. (C) 2001 OsteoArthritis Research Society International.

Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

We have investigated the expression and function of the isoforms of laminin bearing the alpha(5) chain, i.e. laminin-10/11 in neonatal and adult human skin. By immunostaining human skin derived from a variety of anatomic sites, we found that the laminin-alpha(5) chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well-studied laminin-5 isoform did not change significantly with age, laminin-10/11 (a5 chain) appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-10/11 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. Importantly, in vitro cell adhesion assays demonstrated that laminin-10/11 is a potent adhesive substrate for both neonatal and adult keratinocytes and that this adhesion is mediated by the alpha(3)beta(1), and alpha(6)beta(4) integrins. Adhesion assays performed with fractionated basal keratinocytes showed that stem cells, transit amplifying cells and early differentiating cells all adhere to purified laminin-10/11 via these receptors. Further, laminin-10/11 provided a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type-18 transformed tumorigenic keratinocyte cell line in vitro. Finally, laminin-10/11 was shown to stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin-10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.

Myocardial abnormalities in hypertensive patients with normal and abnormal left ventricular filling: a study of ultrasound tissue characterization and strain

Abnormal left ventricular (LV) filling is common, but not universal, in hypertensive LV hypertrophy (LVH). We sought to elucidate the relative contributions of myocardial structural changes, loading and hypertrophy to LV dysfunction in 113 patients: 85 with hypertensive LVH and 28 controls without LVH and with normal filling. Patients with normal dobutamine stress echocardiography and no history of coronary artery disease were selected, in order to exclude a contribution from ischaemia or scar. Abnormal LV filling was identified in 65 LVH patients, based on Doppler measurement of transmitral filling and annular velocities. All patients underwent grey-scale and colour tissue Doppler imaging from three apical views, which were stored and analysed off line. Integrated backscatter (113) and strain rate imaging were used to detect changes in structure and function; average cyclic variation of 113, strain rate and peak systolic strain were calculated by averaging each segment. Calibrated 113 intensity, corrected for pericardial 113 intensity, was measured in the septum and posterior wall from the parasternal long-axis view. Patients with LVH differed significantly from controls with respect to all backscatter and strain parameters, irrespective of the presence or absence of abnormal LV filling. LVH patients with and without abnormal LV filling differed with regard to age, LV mass and incidence of diabetes mellitus, but also showed significant differences in cyclic variation (P < 0.01), calibrated 113 in the posterior wall (P < 0.05) and strain rate (P < 0.01), although blood pressure, heart rate and LV systolic function were similar. Multivariate logistic regression analysis demonstrated that age, LV mass index and calibrated IB in the posterior wall were independent determinants of abnormal LV filling in patients with LVH. Thus structural and functional abnormalities can be detected in hypertensive patients with LVH with and without abnormal LV filling. In addition to age and LVH, structural (not functional) abnormalities are likely to contribute to abnormal LV filling, and may be an early sign of LV damage. 113 is useful for the detection of myocardial abnormalities in patients with hypertensive LVH.

Characterisation of chronic lateral epicondylalgia using the McGill pain questionnaire, visual analog scales, and quantitative sensory tests

Aims: To characterise chronic lateral epicondylalgia using the McGill Pain Questionnaire, Visual Analog Scales for pain and function, and Quantitative Sensory Tests; and to examine the relationship between these tests in a population with chronic lateral epicondylalgia. Method: Fifty-six patients (29 female, 27 male) diagnosed with unilateral lateral epicondylalgia of 18.7 months (mean) duration (range 1-300), with a mean age of 50.7 years (range 27-73) participated in this study. Each participant underwent assessment with the McGill Pain Questionnaire (MPQ), Visual Analog Scales (VAS) for pain and function. and Quantitative Sensory Tests (QST) including thermal and pressure pain thresholds, pain free grip strength, and neuromeningeal tissue testing via the upper limb tension test 2b (ULTT 2b). Results: Moderate correlation (r = .338-.514, p = .000-.013) was found between all indices of the MPQ and VAS for pain experienced in the previous 24 hours and week. Thermal pain threshold was found to be significantly higher in males. A significant poor to moderate correlation was found between the Pain Rating Index (PRI) in the sensory category of the MPQ and ULTT2b scores (r = .353, p = .038). There was no other significant correlation between MPQ and QST data. Pain free grip strength was poorly yet significantly correlated with duration of pathology (r = 318, p = .038). Conclusion: The findings of this study are in agreement with others (Melzack and Katz, 1994) regarding the multidimensional nature of pain, in a condition conventionally conceived as a musculoskeletal pain state. The findings also suggest that utilisation of only one pain measurement tool is unlikely to provide a thorough clinical picture of pain experienced with chronic lateral epicondylalgia.

Occlusable corneas in toadfishes: light transmission, movement and ultrastruture of pigment during light- and dark-adaptation

The toadfishes Tetractenos hamiltoni and Torquigener pleurogramma (Tetraodontidae) possess occlusable yellow corneas. We examine the light transmission and location of the yellow/orange pigment throughout the cornea, the temporal properties of pigment migration and the ultrastructure of the pigmented processes during light- and dark-adaptation. Each species was dark-adapted during the day and light-adapted during the night and then exposed to either sun illumination or darkness for different lengths of time (0-70 min). Movement of corneal pigment could be induced in both species regardless of time of day or night. The pigment was able to migrate in a dorsal or ventral direction and changed from minimal to maximal pigmentation within 60 min. Three types of transmission curves were found with varying degrees of transmission in the 400-500 nm waveband, indicating that the pigment distribution is not uniform across the cornea; some areas of the cornea transmit near UV light, while others absorb blue light. The gradual change of the transmission characteristics in different areas of the cornea indicates the presence of different concentrations of a single type of pigment. Ultrastructural examination of the corneas showed that the layer containing the pigment is situated within the scleral cornea either surrounding (T. pleurogramma) or abutting (T. hamiltoni) an iridescent layer. Long sheet-like processes or chromatophores extending centrally from dorsal and ventral reservoirs are filled with pigment during the light-adapted state but empty in the dark-adapted state.

Immunological changes after cancer treatment and participation in an exercise program

Purpose: The purpose of this investigation was to evaluate the impact of undertaking peripheral blood stem cell transplantation (PBST) on T-cell number and function, and to determine the role of a mixed type, moderate intensity exercise program in facilitating the recovery of T-cell number and function. Methods: Immunological measures of white blood cell, lymphocyte, CD3(+), CD4(+), and CD8(+) counts, and CD3(+) cell function were assessed pretransplant (PI), immediately posttransplant (PII), and 1 month (II), 2 months (12) and 3 months (PIII) posttransplant. After PII, 12 patients were divided equally into a control group (CG) or exercise intervention group (EG). Results: Lower total T-cell, helper T-cell, and suppressor T-cell counts (P < 0.01), as well as lower T-cell function (P < 0.01), when compared with normative data, were found at PI. More specifically, 88% of the group had CD3(+), CD4(+), and CD8(+) counts that were more than 40%, 20%, and 50% below normal at PI, respectively. Undertaking a PBST caused further adverse changes to the total leukocyte, lymphocyte, CD3(+), CD4(+) and CD8(+) count. and the helper/suppressor ratio. Although CD8(+) counts had returned to normal by PIII, CD3(+), CD4(+), and the CD4(+)/CD8(+) ratio remained significantly lower than normative data (P < 0.01), with 66%, 100%, and 100% of the subject group reporting counts and ratios, respectively, below the normal range. Conclusion: The PBST patients were immunocompromised before undertaking the transplant, and the transplant procedure imposed further adverse changes to the leukocyte and lymphocyte counts. The leukocyte and CD8(+) counts returned to normal within 3 months posttransplant; however, the other immunological parameters assessed demonstrated a delayed recovery. Although participation in the exercise program did not facilitate a faster immune cell recovery, neither did the exercise program hinder or delay recovery.

Asymmetric contribution of α and β subunits to the activation of αβ heteromeric glycine receptors

This study investigated the role of beta subunits in the activation of alphabeta heteromeric glycine receptor (GlyR) chloride channels recombinantly expressed in HEK293 cells. The approach involved incorporating mutations into corresponding positions in alpha and beta subunits and comparing their effects on receptor function. Although cysteine-substitution mutations to residues in the N-terminal half of the alpha subunit M2-M3 loop dramatically impaired the gating efficacy, the same mutations exerted little effect when incorporated into corresponding positions of the beta subunit. Furthermore, although the alpha subunit M2-M3 loop cysteines were modified by a cysteine-specific reagent, the corresponding beta subunit cysteines showed no evidence of reactivity. These observations suggest structural or functional differences between alpha and beta subunit M2-M3 loops. In addition, a threonine-->leucine mutation at the 9' position in the beta subunit M2 pore-lining domain dramatically increased the glycine sensitivity. By analogy with the effects of the same mutation in other ligand-gated ion channels, it was concluded that the mutation affected the GlyR activation mechanism. This supports the idea that the GlyR beta subunit is involved in receptor gating. In conclusion, this study demonstrates that beta subunits contribute to the activation of the GlyR, but that their involvement in this process is significantly different to that of the alpha subunit.

Accessing Planktothrix species diversity and associated toxins using quantitative real-time PCR in natural waters

Tese de Doutoramento em Biologia apresentada à Faculdade de Ciências da Universidade do Porto, 2015.

Microbiological quality of drinking water of urban and rural communities, Brazil

OBJECTIVE: To evaluate the microbiological quality of treated and untreated water samples came from urban and rural communities and to examine the relationship between coliforms occurrence and average water temperature, and a comparison of the rainfall levels. METHODS: A sample of 3,073 untreated and treated (chlorinated) water from taps (1,594), reservoir used to store treated water (1,033), spring water (96) and private well (350) collected for routine testing between 1996 and 1999 was analyzed by the multiple dilution tube methods used to detect the most probable number of total and fecal coliforms. These samples were obtained in the region of Maringá, state of Paraná, Brazil. RESULTS: The highest numbers water samples contaminated by TC (83%) and FC (48%) were found in the untreated water. TC and FC in samples taken from reservoirs used to store treated water was higher than that from taps midway along distribution lines. Among the treated water samples examined, coliform bacteria were found in 171 of the 1,033 sampling reservoirs. CONCLUSIONS: Insufficient treatment or regrowth is suggested by the observation that more than 17% of these treated potable water contained coliform. TC and FC positive samples appear to be similar and seasonally influenced in treated water. Two different periods must be considered for the occurrence of both TC and FC positive samples: (i) a warm-weather period (September-March) with high percentage of contaminated samples; and (ii) cold-weather period (April-August) were they are lower. Both TC and TF positive samples declined with the decreased of water temperature.

Analysis of polycyclic aromatic hydrocarbons in fish: optimisation and validation of microwave-assisted extraction

An accurate and sensitive method for determination of 18 polycyclic aromatic hydrocarbons (PAHs) (16 PAHs considered by USEPA as priority pollutants, dibenzo[a,l]pyrene and benzo[j]fluoranthene) in fish samples was validated. Analysis was performed by microwave-assisted extraction and liquid chromatography with photodiode array and fluorescence detection. Response surface methodology was used to find the optimal extraction parameters. Validation of the overall methodology was performed by spiking assays at four levels and using SRM 2977. Quantification limits ranging from 0.15–27.16 ng/g wet weight were obtained. The established method was applied in edible tissues of three commonly consumed and commercially valuable fish species (sardine, chub mackerel and horse mackerel) originated from Atlantic Ocean. Variable levels of naphthalene (1.03–2.95 ng/g wet weight), fluorene (0.34–1.09 ng/g wet weight) and phenanthrene (0.34–3.54 ng/g wet weight) were detected in the analysed samples. None of the samples contained detectable amounts of benzo[a]pyrene, the marker used for evaluating the occurrence and carcinogenic effects of PAHs in food.

Happiness, hope, and affection as predictors of quality of life and functionality of individuals with heart failure at three-month follow-up

The scientific evidence supporting the management of the chronically ill in a positive psychological perspective in opposition to traditional pathological approach is scarce. This study examines issues associated with recovery of health status in heart failure, in particular hope, affection, and happiness. We use a longitudinal study of 128 symptomatic patients who after medical intervention reported improved quality of life and function at 3-month follow-up. We evaluated the contribution of happiness, hope and affection, individually and as a whole, in the quality of life and functionality of individuals with heart failure. Happiness (Subjective Happiness Scale), Hope (HOPE Scale), and affection (PANAS (positive and negative affect schedule)) were determined before medical intervention. Individually, we found that happiness is correlated with the quality of life and functionality, hope to self-efficacy dimension of the quality of life scale, positive affect to functionality and negative affect with symptoms dimension, quality of life dimension, and overall sum of the quality of life scale. Overall, we found that happiness has a unique contribution to the quality of life, except in self-efficacy dimension where hope takes this contribution and positive affect has a unique contribution to the functionality in this short-term follow-up. The results highlight the importance of positive variables to health outcomes for people with heart failure and should be considered in intervention programs for this syndrome.