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Biomedical subjects

D Manor

Publications and source records attributed to D Manor.

At least 37 records · Page 2Linked to original sources
PubMed1997

Improved method for diagnosis of polycythemia vera based on flow cytometric analysis of autonomous growth of erythroid precursors in liquid culture.

"Autonomous" development of erythroid colonies in erythropoietin (EPO)-free semi-solid culture has been used as an in vitro assay for diagnosis of polycythemia vera (PV). These colonies, however, are small and poorly hemoglobinized, rendering the assay in many cases unreliable. We report here on the use of a novel assay; it combines a modified culture procedure that maximizes the growth of EPO-independent erythroid cells, and immunofluorescence flow cytometry for their detection and quantitation. Peripheral blood mononuclear cells are cultured for 2-5 days in the presence of a combination of growth factors. During this phase, early erythroid committed progenitors, burst forming units (BFUe), proliferate and differentiate into colony forming units (CFUe)-like progenitors. In the second phase, the latter cells, in the presence of stem cell factor, hemin, and iron-saturated transferrin, continue to proliferate and mature into hemoglobin (Hb)-containing orthochromatic normoblasts. Neither phases contained EPO. The culture produced large, pure, and synchronized erythroid cell populations. The cells were then dually labeled with fluorescent probes, nuclear DNA with thiazole orange and intracellular hemoglobin (Hb) with phycoerythrin-conjugated monoclonal antibodies against human Hb. Cells positive for both labels were assigned as Hb-containing nucleated precursors. The presence of such cells in EPO-free cultures indicated "autonomous growth." None of the EPO-free cultures derived from normal donors or patients with secondary polycythemia contained such cells. Cultures derived from PV patients contained from 5 to 92% "autonomously grown" cells. These culture and analysis methods should minimize false negative results with PV patients and provide objective and quantitative data.

Cells, Cultured↗
PubMed1997

The presence of a sponsoring embryo in a batch of poor quality thawed embryos significantly increases pregnancy and implantation rate.

OBJECTIVE: To evaluate quantitatively the effect of one good-quality (sponsoring) embryo in a batch of low-quality thawed embryos on the implantation and pregnancy rates (PR). DESIGN: Retrospective analysis of data. SETTING: Tertiary care center IVF clinic affiliated with a university medical school. PATIENT(S): Between March 1988 and April 1995, 392 IVF patients underwent a total of 440 thawing and ET cycles of 1,436 multicellular embryos. MAIN OUTCOME MEASURE(S): Implantation, clinical pregnancy, and multiple pregnancy rates. RESULT(S): In the absence of sponsoring embryos in the thawed batch of embryos, a PR of 9.8% with an implantation rate of 3.1% was achieved. In the presence of a single sponsoring embryo, the PR nearly doubled (18.2%), with a significantly higher implantation rate of 7.0%. Only singleton pregnancies were achieved in the absence of sponsoring embryos compared with 21.7% multiple pregnancies in the single sponsoring embryo group. CONCLUSION(S): The presence of a sponsoring embryo in a batch of poor quality thawed embryos is an important factor that significantly increased pregnancy and implantation rates. The optimal strategy for planning batches of multicellular frozen embryos is to include at least one sponsoring embryo in each batch when possible. We speculate that the sponsoring embryo may favorably influence the chances of low-quality embryos to undergo successful implantation.

Adult↗
PubMed1997

Assisted implantation: direct intraendometrial embryo transfer.

We report herein a technique for direct intraendometrial transfer (DIET) of human embryos. In this study we evaluated whether 2-day embryos injected into the endometrial stroma can develop normally into viable pregnancies. After in vitro fertilization the embryos were injected into the endometrium of 14 women under direct visualization using a CO2-pulsed flexible hysteroscope. A total of 44 embryos were transferred, resulting in 2 clinical (14.3% per cycle) and 2 chemical pregnancies. In 1 patient, amniocentesis revealed monosomy X and the pregnancy was terminated at 18 weeks. The 2nd patient had an uneventful pregnancy and delivered a healthy baby. The results from the present study, demonstrating a low implantation rate after DIET of 2-day embryos, suggest that the endometrial stroma does not provide an optimal environment for early embryonic development. The acidifying effect of CO2 used for insufflation may also explain the low pregnancy rate after DIET. We conclude that it is possible to achieve pregnancy by DIET in humans, but presently this procedure can be considered only in cases where the implantation site needs to be precisely determined.

Adult↗
PubMed1996

Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using extrinsic reporter group fluorescence.

The overall goal of these studies was to examine the applicability of extrinsic reporter group fluorescence in monitoring the GTP-binding/GTPase cycle of a Ras-like GTP-binding protein. Toward this end, we have labeled the GTP-binding protein Cdc42Hs with the environmentally sensitive fluorophore succinimidyl 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoate (sNBD) at a single reactive lysine residue. We find that the sNBD-labeled Cdc42Hs undergoes a fluorescence enhancement at 545 nm when Cdc42Hs exchanges bound GDP for GTP. This enhancement is then fully reversed upon GTP hydrolysis. The specific GTPase-activating protein for Cdc42Hs, the Cdc42Hs-GAP, strongly stimulates the rate of reversal of the fluorescence enhancement at 545 nm, consistent with its ability to fully catalyze the GTPase reaction of Cdc42Hs. Conversely, the specific guanine nucleotide exchange factor (GEF), Cdc24, strongly stimulates the fluorescence enhancement that accompanies GTP binding, consistent with its ability to stimulate the GDP-GTP exchange reaction on Cdc42Hs. Resonance energy transfer measurements yielded a distance of approximately 32 A for the sNBD moiety and the guanine nucleotide binding site occupied with either N-methylanthraniloyl- (Mant) dGDP or MantdGTP. Taken together, these results identify a conformationally sensitive reporter site on the Cdc42Hs molecule that is located some distance away from the guanine nucleotide binding site but nonetheless provides a highly sensitive monitor for GTP-binding, GTPase activity, and the interactions of key regulatory proteins.

Animals↗
PubMed1996

The rate of turnover of cortical GABA from [1-13C]glucose is reduced in rats treated with the GABA-transaminase inhibitor vigabatrin (gamma-vinyl GABA).

Brain GABA levels rise and plateau following prolonged administration of the irreversible GABA-transaminase inhibitor vigabatrin (gamma-vinylGABA). Recently it has been shown that increased GABA levels reduces GAD67 protein, one of two major isoforms of glutamic acid decarboxylase (GAD). The effects of GABA elevation on GABA synthesis were assessed in vivo using 1H and 13C-edited NMR spectroscopy. Rates of turnover of cortical glutamate and GABA from intravenously administered [1-13C]glucose were measured in alpha-chloralose anesthetized rats 24 hours after receiving vigabatrin (500 mg/kg, i.p.) and in non-treated controls. GABA concentration was increased 2-fold at 24 hours (from 1.3 +/- 0.4 to 2.7 +/- 0.9 mumol/g) and GABA-T activity was inhibited by 60%. Tricarboxylic acid cycle flux was not affected by vigabatrin treatment compared to non-treated rats (0.47 +/- 0.19 versus 0.52 +/- 0.18 mumol/g, respectively). GABA-C2 fractional enrichment (FE) measured in acid extracts rose more slowly in vigabatrin-treated compared to non-treated rats, reaching > 90% of the glutamate FE after 3 hours. In contrast, GABA FE > or = glutamate FE in non-treated rats. A metabolic model consisting of a single glutamate pool failed to account for the rapid labeling of GABA from glutamate. Metabolic modelling analysis based on two (non-communicating) glutamate pools revealed a approximately 70% decrease in the rate of GABA synthesis following vigabatrin-treatment, from 0.14 (non-treated) to 0.04 mumol/g/min (vigabatrin-treated). These findings, in conjunction with the previously reported differential effects of elevated GABA on the GAD isoforms, suggests that GAD67 may account for a major fraction of cortical GABA synthesis in the alpha-chloralose anesthetized rat brain in vivo.

4-Aminobutyrate Transaminase↗
PubMed1996

The incidence and possible relevance of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fathers.

Microdeletions linked to deletion intervals 5 and 6 of the Y chromosome have been associated with male factor infertility. Members from at least two gene families lie in the region containing azoospermia factor (AZF), namely YRRM and DAZ. With the advent of intracytoplasmic sperm injection (ICSI), it is possible for men with severe male factor infertility to produce a child. The genetic consequences of such a procedure have been questioned. This report describes the first study of a population (32 couples) of infertile fathers and their sons born after ICSI. The objectives were firstly to determine the incidence and map location of Y chromosome microdeletions and to compare the frequencies with other population studies involving severe male factor infertility, and secondly to formulate a working hypothesis concerning developmental aetiology of Y chromosome microdeletions. The incidence of microdeletions in the ICSI population was shown to be 9.4% (within the range 9-18% reported for populations of severe male factor infertility patients). Microdeletions in two out of three affected father/son pairs mapped in the region between AZFb and AZFc and the third involved a large microdeletion in AZFb and AZFc. Of three affected father/son pairs, microdeletions were detected in the blood of one infertile propositus father and three babies. Assuming that the gonomes of the ICSI-derived babies are direct reflections of those of their fathers germ lines, it is possible that two of three infertile fathers were mosaic for intact Y and microdeleted Y chromosomes. In such cases, the developmental aetiology of the microdeletion may be due to a de-novo microdeletion arising as a post-zygotic mitotic error in the infertile propositus father, thus producing a mosaic individual who may or may not transmit the deletion to his ICSI-derived sons depending on the extent of primordial germ cell mosaicism. In one of three affected fathers, the microdeletion detected in his blood was also detected in his ICSI-derived son. In this case the de-novo event giving rise to the microdeletion may have occurred due to a post- (or pre-) meiotic error in the germ line of this father's normally fertile father (i.e. the ICSI-derived baby's grandfather).

Adult↗
PubMed1996

Undocumented embryos: do not trash them, FISH them.

Pronuclei formation is routinely assessed 16-20 h after oocyte insemination in in-vitro fertilization (IVF). Occasionally, the pronuclei disappear before this time, rendering them as 'undocumented'. Since the number of pronuclei detected is used to distinguish normal from abnormal embryos in the context of ploidy, the diploidy of undocumented embryos is questionable, and therefore they are routinely discarded. The introduction of fluorescent in-situ hybridization (FISH) technology allows the assessment of ploidy status in undocumented embryos that continue to cleave to form blostomeres. In this study, we used FISH to analyse the chromosomal status of 23 undocumented embryos obtained from 10 patients. Biopsied blastomeres were fixed and probed for five chromosomes (X, Y, 13, 18, 21). Diploidy was confirmed in 13 (57%) embryos while the remaining 10 embryos displayed various chromosomal anomalies. Six of the diploid embryos were transferred subsequently to the patients. One ongoing pregnancy was achieved following transfer of an undocumented, analysed embryo, which was already cleaved when assessed 20 h after insemination. We suggest that accelerated dismantling of the pronuclear membrane and subsequent cleavage do not necessarily indicate abnormal chromosomal content and may result in normal pregnancy. In a patient with a small number of embryos, FISH may be used to ascertain diploidy of undocumented embryos, thereby increasing the number of available embryos for transfer.

Blastomeres↗
PubMed1996

Comparison of gonadotrophin-releasing hormone analogues and human chorionic gonadotrophin for the induction of ovulation and prevention of ovarian hyperstimulation syndrome: a case-control study.

Gonadotrophin-releasing hormone analogue (GnRHa) has been suggested as an alternative to human chorionic gonadotrophin (HCG) for triggering ovulation, while preventing ovarian hyperstimulation syndrome (OHSS). Since a prospective, controlled study would be unethical at this point, we used a retrospective, case-self control approach to compare GnRHa with HCG in that context. A group of 16 in-vitro fertilization (IVF) patients who had severe OHSS in previous cycles, in which HCG was given to trigger ovulation, were studied in subsequent cycles in which GnRHa was used. Each GnRHa cycle (case) was compared to a previous HCG cycle that resulted in OHSS (self control). None of these subsequent cycles resulted in severe OHSS. The use of GnRHa did not affect the number of oocytes retrieved or their quality. Serum oestradiol concentrations on the day of ovulation triggering were significantly (P < 0.01) higher in the GnRHa cycles compared to HCG cycles. Exogenous progesterone and oestradiol were effective in maintaining relatively constant serum oestradiol and progesterone serum concentrations during the luteal phase. Pregnancy rate per cycle was similar in the two groups. In conclusion, the use of GnRHa to induce ovulation in IVF patients, who are at high risk for developing OHSS, effectively eliminates this risk without affecting other parameters of the stimulation cycle.

Case-Control Studies↗
PubMed1995

Modulation of the arterial coronary blood flow by asynchronous activation with ventricular pacing.

UNLABELLED: This study aims to test the assumptions that: (1) coronary arterial flow is attenuated in an early activated region by ventricular pacing; (2) asynchronous mechanical activation caused by ventricular pacing under controlled perfusion pressure and intact coronary tone is associated with reduced coronary flow compared to atrial pacing; and (3) abolishment of vascular tone under controlled perfusion pressure diminishes the expected difference in blood flow between atrial and ventricular pacing. Blood flow velocity (BFV) in the left anterior descending (LAD) and the left circumflex arteries (CFX) and a wall thickening index were measured in 14 open-chest dogs under normal conditions, and constant perfusion pressure. Four pacing sites were used: right atrium (RAp), mid-right ventricle (RVp), mid-left ventricle (LVp), and left ventricular apex (Apexp). Pacing modes were either sequential ventriculoatrial (VA) (protocol A, n = 7), or sequential atrioventricular (AV) (protocol B, n = 7), with a shorter AV difference (30 msec) than normal. RESULTS: BFV was decreased in the LAD during RVp and Apexp pacing by 9.7%-12.9% versus RAp and by 11.6%-14.6% versus LVp (P < 0.05). No BFV variations were observed in the CFX. Flow velocity conductance (FVC = mean blood flow velocity divided by the mean aortic pressure) was higher by 16%-28% in the CFX for the three ventricular pacing sites versus the atrial pacing, and higher by 14.1% +/- 6.1% only in LVp versus RAp pacing in the LAD (P < 0.05). Wall thickening index reduced during ventricular pacing in all three ventricular sites by 50%-64% (P < 0.05) compared to atrial pacing. Under constant perfusion pressure, LAD blood flow decreased with ventricular pacing as compared to right atrial pacing; this was particularly pronounced during the diastolic phase (16.6%-45.5%, P < 0.02). Normalized oscillatory flow amplitude (OFAn) was reduced in RVp pacing compared to RAp and LVp pacing (16.2 +/- 3.5 and 21.7% +/- 4.1%, respectively, P < 0.03). The variations in blood flow and OFAn disappeared with adenosine-mediated maximum vasodilatation. SUMMARY: (1) Mean and phasic flows are reduced in the early activated LAD region by ventricular pacing (RVp, Apexp). (2) Under controlled perfusion pressure and intact vascular tone, ventricular pacing compromises blood flow compared with atrial pacing. (3) This effect disappears when vascular tone is eliminated by intracoronary injection of adenosine, suggesting that the coronary autoregulation is responsible for some of the effects.

Animals↗
PubMed1995

Left ventricular mechanics in arrested dog heart: effects of ventricular interaction and vascular volumes.

The present study was designed to determine the effects of right heart pressure on the compliance of the left ventricle (LV). The studies were conducted on isolated, blood-perfused, potassium-arrested dog hearts with vasomotor tone either present (n = 5) or absent (n = 8). A balloon was used to control LV volume, whereas right heart (RHP) or coronary sinus (CSP) pressures were controlled via a column placed in the right heart or coronary sinus, respectively. Control of CSP independently of RHP allowed us to assess the relative contribution of coronary venous pressure to changes in LV compliance under conditions of elevated RHP. LV volume and compliance at a LV pressure of 15 mmHg (V15 and C15, respectively) were calculated to quantify the shift and slope changes of the LV pressure-volume (P-V) relationships. V15 and C15 decreased with vasomotor tone present from 52.8 +/- 1.4 ml and 1.7 +/- 0.1 ml/mmHg at control, to 43.3 +/- 2.1 ml and 1.4 +/- 0.1 ml/mmHg (P < 0.05) with RHP = 0 and 20 mmHg, respectively. Similar effects were obtained with vasodilation, but C15 was significantly lower relative to autoregulation but C15 was significantly lower relative to autoregulation (1.0 +/- 0.1 at control RHP, P < 0.05). Elevation of CSP with vasomotor tone resulted in an upward shift in the LV P-V relationship: V15 decreased from 53.4 +/- 2.1 at CSP = 0 mmHg to 50.9 +/- 1.6 ml at CSP = 20 mmHg (P < 0.05). After vasodilation there was no detectable shift in the LV P-V relationship with elevation of CSP.

Animals↗
PubMed1995

Modulation of coronary flow by left ventricular volume in the presence and absence of vasomotor tone.

This study was undertaken to determine the relationship between left ventricular (LV) volume and coronary flow in the presence and absence of coronary vasomotor tone in arrested dog hearts. We utilized an isolated, blood-perfused, potassium-arrested dog heart preparation with vascular vasomotor tone present (n = 5) or after maximal vasodilation with adenosine (n = 7). LV volume was controlled with a balloon while left and right coronary flows were recorded. Left and right coronary flows were plotted as a function of LV volume, and the degree of interdependency was quantitatively assessed by the slope of the linear regression and the correlation coefficient (r) between coronary flow and LV volume. With vasomotor tone present, both left (slope = 0.01 +/- 0.06 min-1) and right (slope = -0.01 +/- 0.01 min-1) coronary arterial flows were maintained relatively constant over a wide range of LV volumes. After maximal vasodilation, left coronary flow decreased linearly with LV volume loading (slope = -2.51 +/- 0.47 min-1, r2 = 0.96 +/- 0.02), whereas right coronary flow, similar to the response with tone present, did not change relative to control in most cases. We conclude that changes in coronary vasomotor tone may take place with LV volume loading to compensate for the mechanical vascular resistance changes secondary to myocardial stretch.

Animals↗
PubMed1995

Successful treatment of pure red cell aplasia in systemic lupus erythematosus with erythropoietin.

We describe a 32-year-old woman who developed severe anemia due to pure red cell aplasia in the course of systemic lupus erythematosus (SLE). After failure of therapy with high doses of glucocorticoids and immunoglobulins, and despite high levels of endogenous erythropoietin, she was treated with human recombinant erythropoietin with dramatic and sustained improvement. Based on this case and on the literature review of erythropoietin therapy in pure red cell aplasia, we suggest that erythropoietin should be used in SLE associated pure red cell aplasia before cytotoxic therapy.

Adult↗
PubMed1994

Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy.

We have developed several high-resolution assays for the nucleotide state of a rho-subfamily low molecular weight GTP-binding protein, Cdc42Hs. The first involves the use of the fluorescent N-methylanthraniloyl derivative of GDP (mant-GDP). As has been shown for the ras protein, mant-dGDP fluorescence is significantly enhanced (approximately 20%) upon binding to Cdc42Hs. It was further found that the binding of mant-nucleotides results in an efficient energy transfer between the single tryptophan residue of Cdc42Hs and the mant moiety. The exchange of mant-dGDP for GDP bound to Cdc42Hs, as read-out either by the enhancement of the mant fluorescence or by energy transfer, is inhibited by physiological (mM) Mg2+ concentrations and correlates exactly to the rate of [3H]GDP exchange observed in filter-binding assays. Moreover, changes in the fluorescence of mant-dGDP are also sensitive to nucleotide dissociation induced by the dbl-oncogene product, a known nucleotide exchange factor for Cdc42Hs. A second fluorescence read-out for the nucleotide-bound state of Cdc42Hs involves the measurements of intrinsic fluorescence of a single tryptophan residue (W97) which is highly sensitive to whether GDP or GTP is bound in the nucleotide pocket. The hydrolysis of GTP to GDP by Cdc42Hs results in an approximately 30% enhancement of the protein fluorescence. The rate of this fluorescence change corresponds well to the rate of conversion of [gamma-32P]GTP to GDP plus [32P]Pi as measured by filter-binding assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed1994

Interactions among calcium compartments in C6 rat glioma cells: involvement of potassium channels.

1. Variations in intracellular free Ca2+ concentration ([Ca2+]i) induced by alteration of the extracellular concentrations of Ca2+ ([Ca2+]o) and K+ ([K+]o) were imaged in single fluo-3-loaded C6 glioma cells. In addition, the effect of membrane potential on [Ca2+]i was investigated in fura-2-loaded, voltage-clamped cells. 2. Step alterations of [Ca2+]o from 0 to 10 nM were followed by proportional variations in [Ca2+]i, with a maximum 7-fold increase and an apparent half-maximum at [Ca2+]o of 1.5 mM. 3. The time to half-maximum change (t1/2) of [Ca2+]o-associated [Ca2+]i variations ranged between 10 and 50 s, and was inversely related to the amplitude of [Ca2+]o steps. 4. Transient, serotonin-induced [Ca2+]i elevations, used as a measure of Ca2+ availability in inositol 1,4,5-trisphosphate-sensitive stores, were diminished within 10 min in 0 mM [Ca2+]o, but were unaffected by [Ca2+]o changes in the 1-5 mM range. 5. Restoration of normal [Ca2+]i following its elevation by serotonin was delayed by removal of external Na+ or Cl- and was enhanced by warming the medium to 37 degrees C. These conditions did not affect [Ca2+]o-associated [Ca2+]i variations. 6. [Ca2+]o-associated [Ca2+]i variations were depressed by La3+ and Ba2+, while blockers of voltage-activated Ca2+ channels were ineffective. 7. Elevated [K+]o depressed the basal level of [Ca2+]i, and in high concentrations (70-140 mM) also diminished the response to serotonin. 8. Depolarizing the membrane potential of voltage-clamped cells reversibly reduced [Ca2+]i. These membrane-potential associated [Ca2+]i variations were blocked by La3+, Ba2+ and TEA, all of which also depolarized membrane resting potential. 9. Apamin (at 1-10 microM), a blocker of [Ca2+]i-activated K+ channel, totally and reversibly prevented [Ca2+]o-associated [Ca2+]i variations. 10. These studies indicate that C6 cells are responsive to variations in [Ca2+]o, and that a K+ channel is a possible path through which Ca2+ penetrates into the cell.

Animals↗
PubMed1994

Discrete activation of transduction pathways associated with acetylcholine m1 receptor by several muscarinic ligands.

Activation of transfected muscarinic m1 acetylcholine receptors (m1AChR) has been linked to several signal transduction pathways which include phosphoinositide hydrolysis, arachidonic acid release and cAMP accumulation. In Chinese hamster ovary cells stably transfected with the rat m1AChR gene, carbachol elicited all three responses with EC50 values of 2.6, 3.8 and 76 microM, respectively. However, pilocarpine and the selective muscarinic agonist AF102B activated phosphoinositide hydrolysis (by 94 and 27% vs. carbachol, respectively), while antagonizing carbachol-mediated cAMP accumulation. Carbachol also activated (by 4-fold) adenylyl cyclase in membranes prepared from these cells, indicating independence of this signal from intracellular mediators. Moreover, carbachol and AF102B similarly elevated cytosolic Ca2+ in intact m1AChR-transfected cells. The ligand-selective cAMP accumulation, its independence from Ca2+ and the carbachol-activated adenylyl cyclase in membranes suggest that it represents an independent m1AChR-mediated signal, unrelated to phosphoinositide hydrolysis. Selective muscarinic ligands such as AF102B may independently activate distinct signalling pathways, which may be important for designing cholinergic replacement therapy for treating Alzheimer's disease.

Adenylyl Cyclases↗
PubMed1994

Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides.

We have utilized Raman difference spectroscopy to investigate hydrogen bonding interactions of the guanine moiety in guanine nucleotides with the binding site of two G proteins, EF-Tu (elongation factor Tu from Escherichia coli) and the c-Harvey ras protein, p21 (the gene product of the human c-H-ras proto-oncogene). Raman spectra of proteins complexed with GDP (guanosine 5' diphosphate), IDP (inosine 5' diphosphate), 6-thio-GDP, and 6-18O-GDP were measured, and the various difference spectra were determined. These were compared to the difference spectra obtained in solution, revealing vibrational features of the nucleotide that are altered upon binding. Specifically, we observed significant frequency shifts in the vibrational modes associated with the 6-keto and 2-amino positions of the guanine group of GDP and IDP that result from hydrogen bonding interactions between these groups and the two proteins. These shifts are interpreted as being proportional to the local energy of interaction (delta H) between the two groups and protein residues at the nucleotide binding site. Consistent with the tight binding between the nucleotides and the two proteins, the shifts indicate that the enthalpic interactions are stronger between these two polar groups and protein than with water. In general, the spectral shifts provide a rationale for the stronger binding of GDP and IDP with p21 compared to EF-Tu. Despite the structural similarity of the binding sites of EF-Tu and p21, the strengths of the observed hydrogen bonds at the 6-keto and 2-amino positions vary substantially, by up to a factor of 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1994

Modulation of small conductance calcium-activated potassium channels in C6 glioma cells.

Using the patch clamp technique, we have characterized a small conductance, calcium-activated potassium (SK) channel in the C6 glioma cell line. Elevation of cytosolic Ca2+ concentration ([Ca2+]i) by applications of serotonin or ionomycin induced bursts of channel opening recorded in the cell-attached configuration. These channels underlie the serotonin-induced, [Ca2+]i-activated whole-cell K+ conductance described previously. [Ca2+]i directly activated SK channels in inside-out patches with a biphasic concentration dependence. Submicromolar [Ca2+]i induced bursts of channel openings with a unitary conductance of about 25 pS, similar to that of the serotonin-induced channels. Supramicromolar [Ca2+]i caused prolonged openings with a unitary conductance of about 35 pS, resulting in a pronounced increase of the average current in patches exposed to [Ca2+]i above 100 microM. The two modes of opening reflect the activity of the same SK channel. The channel conductance depended on external K+ concentration with KD of 5 mM. The channel was slightly permeable to cations other than K+, with a permeability ratio for K+:Ca2+:Na+ of 1:0.040:0.030, respectively. ATP was required to maintain channel activity in outside-out patches but was not essential in inside-out patches. The modulations of SK channels in C6 cells by components in their microenvironment may be related to the role of glial cells in controlling the extracellular milieu in the CNS.

Adenosine Triphosphate↗