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PubMed1996

Structural features and light-dependent changes in the cytoplasmic interhelical E-F loop region of rhodopsin: a site-directed spin-labeling study.

Thirty consecutive single cysteine substitution mutants in the amino acids Q225-I256 of bovine rhodopsin have been prepared and modified with a sulfhydryl specific nitroxide reagent. This sequence includes the E-F interhelical loop, a transducin interaction site. The accessibilities of the attached nitroxides to collisions with hydrophilic and hydrophobic paramagnetic probes in solution were determined, and the electron paramagnetic resonance spectra analyzed in terms of side chain mobility, both in the dark and after photoactivation. Accessibility cata shows that the rhodopsin polypeptide chain crosses an aqueous/ hydrophobic boundary in the range V227-K231 and again in the range V250-V254. In the hydrophobic segments, both the accessibility and mobility data are consistent with helical structures. In the regions of the sequence located within the aqueous phase, periodic variation in both accessibility and mobility of the spin-labeled side chains indicates that the E-F interhelical loop is largely alpha-helical, being formed by regular extensions of the E and F helices by about 1.5 and 3 turns, respectively. Judging from nitroxide mobilities, the putative extension of helix E in the aqueous phase is more dynamic than that of helix F. Changes in the electron paramagnetic resonance characteristics of the spin-labeled rhodopsin upon photoactivation indicate that chromophore isomerization results in patterns of structural changes that can be interpreted in terms of movements of helices that extend into the aqueous loop regions.

Amino Acid Sequence↗

PubMed1990

Biochemical variations in Gigantocotyle explanatum and Gastrothylax crumenifer with respect to their seasonal reproduction.

Biochemical components, glycogen, protein, nucleic acids and lipid fractions were analysed every month from January to December 1986, in the liver and rumen amphistomes Gigantocotyle explanatum and Gastrothylax crumenifer, respectively. The results reveal a considerable seasonal variation. In the rumen amphistome the components reach their maximum level only once a year, whereas in the liver amphistome, more than one peak is observed in a year. In G. crumenifer the content of glycogen and nucleic acids increases before the onset of egg production while protein and lipids reach their maximum level during the egg production phase. The variations in biochemical components were associated with the reproductive cycles and gonad recrudescence of these parasites.

Animals↗

PubMed1996

Multiplex, fluorescent, solid-phase minisequencing for efficient screening of DNA sequence variation.

We developed a multiplex, solid-phase minisequencing method to detect multiple single-nucleotide polymorphisms in an undivided sample. The amplified DNA templates are first captured on a manifold. Then, with multiple minisequencing primers of various sizes, single-nucleotide extension reactions are carried out simultaneously with fluorescently labeled dideoxynucleotides. The size of the extended product, determined by using a DNA sequencing instrument, defines the site of the polymorphisms, and the incorporated nucleotide gives the identity of the nucleotide at each site. HLA-DQA1 typing was used as a model system to evaluate the method. The DR2 subgroup of the HLA-DRB1 gene was typed along with the DQA1 gene to demonstrate the feasibility of the method in analyzing multiple genes at multiple sites simultaneously. The method is generally applicable for screening any single-nucleotide polymorphisms or point mutations, and its manifold format allows practical handling of large numbers of samples.

Alleles↗

PubMed1988

[Comparative study of the effects of metoclopramide and metopimazine on the duodenojejunal motility during the interdigestive period: a manometric study in healthy subjects].

The objective of this paper was to compare the effects of metoclopramide (MTC) and of metopimazine (MTP) on intestinal motility in normal subjects in the period between digestion by injecting these two agents at a well-defined time of the migrating motor complex (MMC). Duodenojejunal motility was recorded by manometry (four microperfused catheters; study segment: 30 cm) during the period between phases of digestion (fasting greater than 12 hrs), for 4.6 hrs on average (3, 4, 6 hrs). 14 normal volunteer subjects (6 males, and 8 females 19 to 49 years of age) were randomly assigned to 2 study groups and were given MTC (10 mg) or MTP (10 mg) in slow IV injection (5 mins.) at a controlled rate, performed 25 mins after onset of a phase 3 (P3) in the study segment. As a reference, results obtained in a control group of seven subjects recorded under the same conditions are reported, in addition. Changes in MMC were evaluated in each group by the mean number of P3 hourly and the percent of subjects presenting a P3 within the two hours following injection of the test drug. Variations in phase 2 type motility (P2) were measured using motility indices (MI): the sum of the amplitudes, number of waves, per 5 minute interval. The number of hourly P3 was 0.40 for MTC vs 0.28 for MTP (control 0.47); 90 minutes after an injection, the first P3 appeared in 71% of patients in the MTC groups vs. 14% in the MTP group (control 57%). P3 recorded following injection were not different from the preceding in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1998

Variations in erythrocyte antioxidant glutathione peroxidase activity during the menstrual cycle.

OBJECTIVE: Antioxidant enzymes are an important part of the defence mechanisms against free radical damage. Little is known, however, about the relationship between sex steroid hormones and cellular antioxidant systems. We have investigated the effect of physiological sex steroid changes on the erythrocyte antioxidant glutathione peroxidase (GSH-Px) activity during the menstrual cycle in a population of healthy normomenorrhoic women. DESIGN: Prospective, controlled. PATIENTS: Ten normally cycling women (age range: 19-28 years; mean: 23.3 years) were recruited for alternate-day blood sampling from the first day of one menstrual bleed until the first day of the subsequent menstrual phase. MEASUREMENTS: Plasma was analysed for LH, FSH, oestradiol (E2) and progesterone (P4) concentrations. Erythrocyte GSH-Px activity was evaluated on the same days in all subjects. Pyruvate-kinase (PK) activity, as an index of red blood cell population age, was also determined. RESULTS: All the women examined had a normal ovulatory cycle, as indicated by the hormone plasma pattern. Cycle length was standardized on the basis of the preovulatory E2 peak. Significant cycle phase-related changes in GSH-Px (P < 0.03) were observed, with higher GSH-Px activity levels being found from the later follicular to early luteal phase as compared to early follicular phase (chi 2 = 8.53, P < 0.001 and chi 2 = 5.54, P < 0.002, respectively). A significant positive correlation was observed between mean E2 and GSH-Px cycle-related changes (r = 0.78, P = 0.001). Conversely, no significant cycle phase-dependent variations were detected in PK. CONCLUSIONS: The findings support the hypothesis that physiological ovarian E2 production during the menstrual cycle may play an important role in regulating erythrocyte glutathione peroxidase activity.

Adult↗

PubMed1994

Effect of fasting and thyroidectomy on cysteine proteinase activities in liver and muscle.

Prolonged starvation mimics chronic negative nitrogen balance observed in many physiopathological situations. During starvation, an initial decrease in protein utilization (phase I) is followed by a long period of protein sparing (phase II) that ends with a marked rise in nitrogen excretion (phase III). Variations in protein metabolism during starvation are determined by changes in protein synthesis and degradation rates (Cherel, Y., Attaix, D. Rosolowska-Huszcz, D., Belkhou, R., Robin, J.P., Arnal, M. and Le Maho, Y. (1991) Clin. Sci. 81, 611-619), but little information is available on expression of proteolytic systems. In this study, cathepsin B, H and L activities were compared in hindlimb muscles and liver at various phases of starvation in thyroidectomized and sham-operated rats. In muscle, cathepsin activities fell from the fed state to phase II, which suggests that cathepsins may play a role in the curtailment of muscle proteolysis during protein sparing phase. This decrease of muscle cathepsin activities was reproduced by thyroidectomy alone. In contrast, liver cathepsin B and H activities fell during starvation, but were not affected by thyroidectomy alone. Liver cathepsin L decreased only during starvation in thyroidectomized animals. These observations emphasize that different mechanisms modulate cathepsin expression in skeletal muscle and liver.

Animals↗

PubMed2000

Neuropathological heterogeneity in Alzheimer's disease: a study of 80 cases using principal components analysis.

Three hypotheses have been proposed to explain neuropathological heterogeneity in Alzheimer's disease (AD): the presence of distinct subtypes ('subtype hypothesis'), variation in the stage of the disease ('phase hypothesis') and variation in the origin and progression of the disease ('compensation hypothesis'). To test these hypotheses, variation in the distribution and severity of senile plaques (SP) and neurofibrillary tangles (NFT) was studied in 80 cases of AD using principal components analysis (PCA). Principal components analysis using the cases as variables (Q-type analysis) suggested that individual differences between patients were continuously distributed rather than the cases being clustered into distinct subtypes. In addition, PCA using the abundances of SP and NFT as variables (R-type analysis) suggested that variations in the presence and abundance of lesions in the frontal and occipital lobes, the cingulate gyrus and the posterior parahippocampal gyrus were the most important sources of heterogeneity consistent with the presence of different stages of the disease. In addition, in a subgroup of patients, individual differences were related to apolipoprotein E (ApoE) genotype, the presence and severity of SP in the frontal and occipital cortex being significantly increased in patients expressing apolipoprotein (Apo)E allele epsilon4. It was concluded that some of the neuropathological heterogeneity in our AD cases may be consistent with the 'phase hypothesis'. A major factor determining this variation in late-onset cases was ApoE genotype with accelerated rates of spread of the pathology in patients expressing allele epsilon4.

Age of Onset↗

PubMed2003

Ca/P ratio effects on the degradation of hydroxyapatite in vitro.

Phase purity is a well-recognized but not well-understood variable affecting the biological integration of hydroxyapatite (HA)-based biomaterials. Minor amounts of specific, relevant impurities--calcium oxide (CaO) and tricalcium phosphate (TCP)--may often be present either as deliberate additions or as a result of decomposition during sintering. We investigated the influence of these two impurities in terms of their effects on surface morphology, weight loss/gain, and microstructural-level degradation. Phase purity variations were deliberately introduced into an otherwise-standardized HA matrix--the parent HA grain size and bulk density were relatively constant--produced using identical fabrication conditions. Stability varied markedly during exposure to mildly acidic, neutral, and pH 7.4 phosphate-buffered saline. Equivalent molar variations in the Ca/P ratio (1.62 vs 1.72) on either side of the stoichiometric ratio produce relatively small volumetric amounts of CaO (1.6 vol%) versus TCP (27 vol%) in HA. However, the relatively small amounts of CaO render the bulk more susceptible to degradation and more likely to have negative effects on a biological milieu. Interestingly, the presence of CaO is also a potent nucleating agent for the precipitation of new surface phases and detectable weight gain. The TCP-containing ceramic, in contrast, paradoxically exhibited slightly greater resistance to degradation than HA.

Biocompatible Materials↗

PubMed1977

Variations in the adenylate energy charge during phased growth (cell cycle) of Candida utilis under energy excess and energy-limiting growth conditions.

The variations in the levels of adenine nucleotides during the phased growth (cell cycle) of the yeast Candida utilis growing under nitrogen, sulfate, or iron limitation with glycerol as carbon source have been determined. Synchronous cultures were obtained by the continuous phasing technique, and the results were compared with those of chemostat cultures growing at similar growth rates and under the same types of nutrient limitation. Whereas the chemostat experiments indicated only the average energy status of cultures growing at random, results from phased cultures showed that the adenylate energy charge, defined as (ATP + (1/2)ADP)/(ATP + ADP + AMP) (where ATP, ADP, and AMP signify adenosine 5'-triphosphate, -diphosphate, and -monophosphate, respectively), varied during the phased growth of the yeast. These variations were related to the stage of development of the cells and to the type of nutrient limitation. In every case the energy charge dropped to a low value during the first half of the phasing cycle (cell cycle). Whereas the energy charge was maintained at relatively high levels (ranging from 0.78 to 0.94), for sulfate- or nitrogen-limited cultures, it was very low when iron was the growth-limiting nutrient (0.44 to 0.78). In spite of the low energy charge, the yeast continued to grow under iron limitation. The main component of the adenylate pool of the iron-limited culture was ADP and not ATP as observed with other types of nutrient limitation. It is concluded that under iron limitation the growth of the organism is limited by energy and that under energy-limited growth the energy charge of a growing organism is maintained at low levels. The reason for maintaining a low energy charge in an energy-limited culture is discussed.

Adenine Nucleotides↗

PubMed2006

Role of molecular weight and phase sequence in the temperature variation of film tension above the bulk isotropic transition in freestanding liquid-crystal films.

Building upon our previous report [Veum, Phys. Rev. E 17, 020701(R) (2005)] involving two compounds, we have performed a systematic study of the temperature variation of film tension above the bulk isotropic transition in freestanding films to include a total of six smectic liquid-crystal compounds. Consistent with the previous results, the tension increases sharply with temperature above the transition, the tension-temperature slope is proportional to the film's thickness, and the data can be interpreted in the context of theoretical models for layer thinning. Our data suggest that both molecular weight and bulk phase appearing below its isotropic phase play important roles in the slope values.

Journal Article↗

PubMed1996

Analysis of blood pressure rhythms for drug efficacy evaluation.

This study analyzes some methods of evaluating the effects of antihypertensive drugs on blood pressure circadian rhythm. We reviewed four different approaches: hourly averages, trough-to-peak ratio, cosinor method, and Fourier series applied to the same data to prove the time course of the effects of isradipine administered once daily. A total of 141 patients of both sexes (mean age 53 years, range 30-76) with mild to moderate essential hypertension were enrolled in this multicenter trial after a 2-week placebo run-in. Treatment with isradipine SRO 5 mg/day administered between 8 and 9 AM was started. Each patient underwent ambulatory BP monitoring at the time of entry and after 6 weeks of treatment. Calculation of hourly averages showed decreases after 4 AM, and from about 8-9 AM, when the drug was administered, and the decreases practically did not vary until about 10 PM. Subsequently, the decreases became smaller and indicated reduced drug activity. However, this hypothesis no longer held after 4 AM. The trough-to-peak ratio was calculated including hourly averages after the dose divided by the lowest hourly average. Both systolic and diastolic blood pressure showed constant reduction from 3 PM (time of peak) to 11 PM. However, after 11 PM, higher trough-to-peak ratios paradoxically occurred due to a major reduction obtained with placebo, and the negative percentages just before the next dose cannot be attributed to treatment. Applying the cosinor method, maximal values were greatly underestimated, nocturnal values were overestimated, and the absolute maximum occurred in proximity to the minimum relating to postprandial dip. The generalized cosinor model, known as Fourier partial series, was always curtailed to the third harmonic. Fourier analysis was able to describe the daily trend of BP both before and after isradipine administration. We used statistical tests to determine if the differences described by the models were significant. The tests indicated significant mean level reductions after therapy and no appreciable amplitude and phase-related variations. The nocturnal and periawakening intervals, in which BP changed linearly, oscillated between 3 and 4 hours. Within these intervals BP rose by 13-16 mm Hg or fell by 14-19 mm Hg. The intervals, ranges, and speed differences were tested and were never significant. The medication tested was effective only at the mean level, the variables used to characterize the time course of its effect remaining unchanged. There was no significant phase shift of the curves, and BP rhythm was preserved.

Adult↗

PubMed2004

Cell cycle-related variation in subcellular localization of eIF3e/INT6 in human fibroblasts.

The Int-6 gene is a site of mouse mammary tumour virus (MMTV) integration in murine tumours and INT6 protein has been identified independently as a subunit (eIF3e) of the eukaryotic translation initiation factor eIF3. In addition, the protein can interact with two other multi-subunit complexes: the COP9 signalosome (CSN) and the proteasome. The role of INT6 in tumourigenesis is nonetheless currently unclear. Here, using immunofluorescence microscopy, we show that eIF3e/INT6 is localized in part to the nucleus, while other eIF3 components are cytoplasmic. Primary human fibroblasts, but not their transformed counterparts, showed reduced nuclear INT6 staining in some cells, and this reduction was maximal in early S phase. This variation in eIF3e/INT6 may indicate regulated shuttling between cellular compartments and would be consistent with the presence of a nuclear export signal as well as a nuclear localization signal in the protein sequence. Loss of regulation of eIF3e/INT6 redistribution may therefore be a significant feature of malignancy in human cells.

Active Transport, Cell Nucleus↗

PubMed1983

Secreted collagen ratios in normal human and osteogenesis imperfecta skin fibroblasts.

We examined the effects of several variables on the ratio of type I:type III collagen secreted by human caucasian skin fibroblasts in normal and osteogenesis imperfecta (OI) phenotypes. Isotopically labelled collagen extracted from fibroblast medium was analyzed by DEAE-cellulose chromatography and identified by appropriate methods. Type I procollagen was the major form of collagen secreted into the medium by normal cells cultured from one mid-term fetus, infants (n = 3), children (n = 3), adolescents (n = 2), and adults (n = 3). Interstrain differences in collagen production under standardized conditions were significantly greater than intrastrain variation (anova, p = 0.0051). There was no significant alteration in the type I:type III collagen ratio due to variation in: phase of cell growth, doublings (between 13th and 22nd), rate of isotope incorporation, labelling time (24-72 hrs) in the presence of ascorbic acid (50 micrograms/ml), age of donor (with the possible exception of adolescence), and site of biopsy (genital and non-genital sites). Variable conversion of type I procollagen to collagen did not perturb the type I:type III collagen ratio. Cell strains from OI patients (Sillence classification): type I (one strain); type II, III and IV (3 strains each) had greater interstrain than intrastrain variation in the collagen ratio (p = 0.0149). Interstrain differences were greater in OI cell strains relative to normal cell strains (p less than 0.01). In the aggregate, OI cells had significantly lower type I collagen production relative to type III (I/III ratio = 1.18) when compared with normal cells (I/III ratio = 2.90; t test, p less than 0.0001). These findings imply abnormal synthesis, secretion or stability of type I procollagen and greater phenotypic heterogeneity in OI skin fibroblasts relative to normal cells.

Adolescent↗

PubMed1978

Postural and kinetic coordination following cortical stimuli which induce flexion movements in the cat's limbs.

The relationship between movement and the postural adjustment associated with it has been studied following flexion movements of a limb induced by cortical stimulation in chronically implanted cats. Variations in the vertical force exerted by each limb, as well as the displacement of the centre of gravity (projected onto the plane of stance), were investigated. It was established that: The pattern of postural adjustment observed in these conditions is diagonal; that is to say, flexion of one limb is accompanied by diminution in the vertical force exerted by the diagonally opposite limb and an increase in the force exerted by the other two. The displacement of the projected centre of gravity is sterotyped during the first phase of variation in forces. Its extent may be considered as an index of the efficiency of postural adjustment. The study of the latencies of the changes in force indicates, at least in the case of hindlimb movements, that postural adjustment is an active phenomenon and does not arise passively from the interplay of purely mechanical forces. In the same conditions, an analysis of the time course and latency of recorded changes in force indicates that postural adjustment is not triggered reflexly by the disequilibrium brought about by movement but is a result of a central nervous command.

Animals↗

PubMed2004

An ultrastructural study of cellular response to variation in porosity in phase-pure hydroxyapatite.

Hydroxyapatite has been shown to be biocompatible and bioactive. Incorporation of porosity has been shown to enhance osteointegration; however, difficulty in controlling the extent and type of porosity has limited investigation into determining the role of both macro- and microporosity. The current investigation reports on the synthesis of four types of phase-pure hydroxyapatite with varying levels of porosity (HA1-HA4), and with defined levels of macro- and microporosities. Transmission electron microscopy was used to evaluate qualitatively the effect of these two parameters on cell-material interactions following a 30-day incubation period. Biological mineralization was observed within vesicles and the needle-like minerals were confirmed as hydroxyapatite using X-ray microanalysis. This demonstrated the suitability of primary human osteoblast-like cells as a tool to assess the extent of mineralization. Furthermore, internalization of hydroxyapatite particles was observed. Our findings show that the variation in macro- and microporosity does not affect the extent of cell-material interaction, with collagen synthesis evident in all samples.

Cell Communication↗

PubMed2005

Interfacial stabilization of organic-aqueous two-phase microflows for a miniaturized DNA extraction module.

Organic-aqueous liquid (phenol) extraction is one of many standard techniques to efficiently purify DNA directly from cells. The cell components naturally distribute themselves into the two fluid phases in order to minimize interaction energies of the biological components with the surrounding solvents. The membrane components and protein partition to the interface between the organic and aqueous phases while the DNA stays in the aqueous phase. The aqueous phase is then removed with a purified DNA sample. This work studies the first steps towards miniaturizing this liquid extraction technique in a microfluidic device. The first step is to understand how the two liquid phases behave in microchannels. Due to the interfacial tension between the two liquid phases, novel approaches must be examined in order to obtain interfacial stability under flow conditions. The stability of the organic-aqueous interface is improved by reducing the interfacial tension between the two phases by incorporating a surfactant into the aqueous phase. The variation of the interfacial tension as a function of surfactant concentration is also quantified in this work. This has led to the ability to create stable stratified microflows in both a dual inlet and three inlet microfluidic systems. Also, the first step in understanding biological interactions at the organic-aqueous interface is investigated using a fluorescently labeled bovine serum albumin protein.

Cell Membrane↗

PubMed1980

Antagonism by chlorisondamine and propranolol, but not by atenolol, of the circadian phase-dependent phentolamine-induced changes in the cardiac noradrenaline turnover in the rat.

The effects of phentolamine alone or in combination with propranolol, atenolol and chlorisondamine were studied on the concentration and turnover of noradrenaline in the heart of light-dark (L:D = 12:12 h) synchronized rats. In order to detect possible circadian phase-dependent variations in the drug effects, the same experiments were performed in the light-period and dark-period, respectively. The parameters of the turnover were calculated from the exponential decline of i.v. injected 3H-(-)-noradrenaline. Phentolamine significantly decreased the noradrenaline concentration during L, but not during D. Reduction in 3H-noradrenaline accumulation by phentolamine was 42.3% during L and 22.2% during D. Phentolamine increased the turnover rate of cardiac noradrenaline more than 3-fold in either photoperiod. Chlorisondamine reversed all the effects of phentolamine studied. Propranolol, but not atenolol, antagonized the effects of phentolamine in a dose-dependent and stereospecific way, being more effective when applied during D. Thus, the chronopharmacological studies in unrestrained rats show a circadian phase-dependency of the effects of adrenoceptor blocking drugs. It is concluded that a central site of action is responsible for the antagonism by propranolol of the phentolamine-induced increase in the turnover of the cardiac noradrenaline in vivo.

Animals↗

PubMed1998

Glucocorticoids induce G1 as well as S-phase lengthening in normal human stimulated lymphocytes: differential effects on cell cycle regulatory proteins.

In order to analyze dexamethasone effects on peripheral blood lymphocyte proliferation, we defined various experimental conditions: dexamethasone introduced (i) at the time of phytohemagglutinin stimulation, (ii) 48 h after the beginning of phytohemagglutinin stimulation, and (iii) on unstimulated lymphocytes. In stimulated lymphocytes, we observed an early G1 accumulation (P < 0.005), a delayed increase in the duration of S-phase (P < 0.03), and a consequent increase in cell-cycle duration. The expression of several cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) was modified. Cyclin D3, CDK4, and CDK6 involved in G1-phase control were significantly decreased under dexamethasone treatment whatever the level of stimulation of lymphocytes (stimulated or unstimulated PBL). Cyclin E and CDK2, acting in G1/ S-phase transition and S-phase regulation, decreased in stimulated lymphocytes before any modification of S-phase (P < 0.002). The expression of CKIs, mainly of p27Kip1, appeared to vary with the degree of cell stimulation: a decrease was observed on treated unstimulated lymphocytes, while p27Kip1 increased in dexamethasone-treated cells during stimulation. Our results indicate sequential modifications of the cell-cycle regulation by dexamethasone starting with an action on G1 followed by S-phase control modifications. The protein analysis pinpoints the major complexes concerned: CDK4 and CDK6/cyclin D are mainly involved in G1-phase modifications, while CDK2 and its partner, cyclin E, might be specifically involved in the lengthening of S-phase. The variations observed for p27Kip1 might amplify the functional effects of dexamethasone on kinasic complexes.

Apoptosis↗