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PubMed1984

[Deferoxamine in hemosiderosis. Fecal iron excretion during continuous subcutaneous infusion].

Faecal iron excretion during continuous subcutaneous deferoxamine infusion was measured by flameless atom-absorption spectral photometry in 21 patients with homozygous ss-thalassaemia and one patient with Blackfan-Diamond anaemia. Taking into account faecal and urinary iron excretion, more iron was eliminated than was taken up during transfusion and by intestinal absorption. Urine contained 37.9%, faeces 62.1% of the amount of iron eliminated.

Adolescent↗
PubMed1998

Clinical, gross morphological and neurochemical follow-up in normal, hydrocephalic and hydrocephalic-shunted rats.

The optimal time for treating congenital hydrocephalus is still unknown. The following study tries to answer this question by shunting hydrocephalic rats around the 10th day of life when the brain development corresponds to that of humans at birth. The animals were allocated to 4 groups: normal, hydrocephalic, normal sham-operated and hydrocephalic-shunted rats (groups 1-4). Body weight and neurological development was tested every 2nd-3rd day. X-rays of the skull were performed in groups 2, 3 and 4 at 10 days and in all at 20 days. In groups 2 and 4 0.1 ml lohexolum was instilled into the lateral ventricle at 10 days and in all at 20 days. X-rays were used to calculate the volume of the neurocranium and to identify ventriculomegaly and its grade (1-3). The x-rays and shunting were performed under general anesthesia, the latter using Kausch's technique. At 20 days of age the animals were sacrificed, the homogenized brain tissue was used to measure Protein, DNA, GAD and CNP by photometry, fluorometry and enzymatically. Time-course of neurological development in group 1: The locomotor patterns were attained gradually up to the ability to hold on to a bar. In group 2 there were larger variations and holding on to a bar was possible only in 6/13. In addition, a distinct tremor was observed in 34%. In group 4 holding on to a bar was possible in all, tremor was observed in 20% of examinations and diminishing following surgery. The volume of the neurocranium in groups 2 and 4 was significantly larger than in groups 3 at 10 days; and in group 2 significantly larger than in groups 1, 3 and 4 at 20 days. In group 4 the ventriculomegaly was distinctly reduced in 7 and remained the same in 1. Protein was significantly lower in group 4. DNA was the same in all groups. GAD was the lowest and the CNP significantly low in group 2. The study shows that an effective shunt may reverse the progressive clinical, radiological and neurochemical changes of hydrocephalus, and that surgery must not be performed necessarily earlier (e.g. prenatally in humans).

Age Factors↗
PubMed2001

In vitro complement activation favoring soluble C5b-9 complex formation alters myocellular sodium homeostasis.

BACKGROUND: Deranged Na(+) homeostasis in skeletal muscle is closely associated with excessive complement activation that is encountered during sepsis. Recent evidence suggests that soluble C5b-9 complexes (SC5b-9), which are readily detected in plasma during sepsis and have long been considered irrelevant nonmembrane binding end products of complement activation, may have numerous biologic effects. The purpose of this study, therefore, was to determine the effects of SC5b-9 on myocellular ion homeostasis and its mechanism(s) of action. METHODS: Hindlimb fast-twitch extensor digitorum longus (EDL) was freshly isolated from rats weighing 50 to 70 g and then incubated at 30 degrees C for 60 minutes in normal Krebs-Henseleit buffer (KHB, pH 7.4) containing 10% zymosan-activated rat serum (10 mg/mL at 37 degrees C for 60 minutes) as a source of SC5b-9. Zymosan particles were removed by centrifugation after activation to exclude any noncomplement direct effects. Heat-inactivated rat serum (56 degrees C for 30 minutes) was used as control. EDL muscle was also incubated with pertussis toxin (1 microg/mL), in Ca(2+)-free KHB, with thapsigargin (0.3 or 3 micromol/L), or with ouabain (0.01, 0.1 or 1 micromol/L) before and/or during incubation with 10% zymosan-activated or heat-inactivated rat serum. Intracellular Na(+) and K(+) contents ([Na(+)](i) or [K(+)](i)) of EDL muscle were determined by using flame photometry after washing in ice-cold Na(+)-free Tris-sucrose buffer. SC5b-9 in zymosan-activated human serum was determined by SC5b-9 enzyme-linked immunoassay. RESULTS: SC5b-9 in zymosan-activated human serum significantly increased by 400% as compared with nonactivated, normal human serum. Zymosan-activated rat serum markedly increased [Na+]i without affecting [K(+)](i) in fast-twitch EDL muscle, which was completely inhibited by pertussis toxin, removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) with thapsigargin. The addition of ouabain (at micromolar concentrations) increased myocellular [Na(+)](i) and decreased myocellular [K(+)](i) in both the zymosan-activated and the heat-inactivated rat serum groups. The effects of ouabain on myocellular [Na(+)](i) and [K(+)](i) were equivalent in these 2 groups. Zymosan-activated and heat-inactivated rat serum had similar effects on myocellular [K(+)](i) in the presence or absence of pertussis toxin, removal of extracellular Ca(2+) or depletion of intracellular Ca(2+). CONCLUSIONS: Zymosan-activated rat serum (presumed SC5b-9 enriched) selectively alters Na(+) homeostasis in isolated fast-twitch skeletal muscle. The mechanisms for such effects may be linked to G-proteins, Ca(2+) flux and Na(+),K(+)-adenosine triphosphatase pump binding site blockade.

Animals↗
PubMed2001

Human vascular smooth muscle cells of diabetic origin exhibit increased proliferation, adhesion, and migration.

PURPOSE: Patients with diabetes mellitus (DM) experience progressive macrovascular atherosclerosis and intimal hyperplastic restenosis with increased frequency as compared with nondiabetic patients. These observations suggest that vascular smooth muscle cells (VSMCs) behave in a phenotypically different and more aggressive manner in diabetic patients. In this study, we compared the in vitro rates of proliferation, adhesion, and migration of human VSMCs obtained from diabetic and nondiabetic patients. METHODS: Human VSMC cultures were isolated from 23 diabetic patients (9 artery, 14 vein) and 15 nondiabetic patients (9 artery, 6 vein) with extensive lower extremity atherosclerosis. All patients were between 61 and 78 years of age (average: 68.4 years [diabetic]; 67.3 years [nondiabetic]). All diabetic patients had type 2 DM. Vascular specimens were obtained at the time of amputation from infragenicular arteries and during arterial revascularization from saphenous veins. Cells from passages 2 and 3 were assayed for their proliferative capacity with total DNA fluorescence photometry and for adhesion and migration with a modified Boyden chamber. RESULTS: The average duration of diabetes was 11.6 +/- 4.1 years. The average number of diabetic complications (retinopathy, neuropathy, nephropathy, coronary artery disease) was 2.8 +/- 0.7 per patient. Diabetic VSMCs exhibited abnormal morphology in cell culture with loss of the normal hill and valley configuration. Proliferation was significantly increased in VSMCs of diabetic origin (156 +/- 57 absorption units) as compared with those of nondiabetic origin (116 +/- 42 absorption units) (P <.001). Diabetic VSMCs demonstrated significantly greater adhesion (63.6 +/- 24 per high-power field vs 37.9 +/- 13 per high-power field; P =.002) and migration (397 +/- 151 per low-power field vs 121 +/- 99 per low-power field; P =.001) rates. CONCLUSIONS: Diabetic VSMCs exhibit significantly increased rates of proliferation, adhesion, and migration as well as abnormal cell culture morphology suggestive of abnormal contact inhibition. These observations of human VSMCs in culture are consistent with the increased rate of infragenicular atherosclerosis and the increased rates of restenosis observed clinically in diabetic patients. The atherosclerosis- and intimal hyperplasia-promoting behavior exhibited appears to be intrinsic to the DM-VSMC phenotype and must be considered when designing methods to limit atherosclerosis and intimal hyperplasia in diabetic patients.

Aged↗
PubMed2005

The cannabinoid agonist WIN55,212-2 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins.

Central nervous system responses to cannabis are primarily mediated by CB(1) receptors, which couple preferentially to G(i/o) G proteins. Here, we used calcium photometry to monitor the effect of CB(1) activation on intracellular calcium concentration. Perfusion with 5 microM CB(1) aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB(1) and in cultured hippocampal neurons. The increase was blocked by coincubation with the CB(1) antagonist, SR141716A, and was absent in nontransfected human embryonic kidney 293 cells. The calcium rise was WIN-specific, being essentially absent in cells treated with other classes of cannabinoid agonists, including Delta(9)-tetrahydrocannabinol, HU-210, CP55,940, 2-arachidonoylglycerol, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was independent of G(i/o), because it was present in pertussis toxin-treated cells. Indeed, pertussis toxin pretreatment enhanced the potency and efficacy of WIN to increase intracellular calcium. The calcium increases appeared to be mediated by G(q) G proteins and phospholipase C, because they were markedly attenuated in cells expressing dominant-negative G(q) or treated with the phospholipase C inhibitors U73122 and ET-18-OCH(3) and were accompanied by an increase in inositol phosphates. The calcium increase was blocked by the sarco/endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin, the inositol trisphosphate receptor inhibitor xestospongin D, and the ryanodine receptor inhibitors dantrolene and 1,1'-diheptyl-4,4'-bipyridinium dibromide, but not by removal of extracellular calcium, showing that WIN releases calcium from intracellular stores. In summary, these results suggest that WIN stabilizes CB(1) receptors in a conformation that enables G(q) signaling, thus shifting the G protein specificity of the receptor.

Analgesics↗
PubMed2001

Electroretinogram analysis of relative spectral sensitivity in genetically identified dichromatic macaques.

The retinas of macaque monkeys usually contain three types of photopigment, providing them with trichromatic color vision homologous to that of humans. However, we recently used molecular genetic analysis to identify several macaques with a dichromatic genotype. The affected X chromosome of these animals contains a hybrid gene of long-wavelength-sensitive (L) and middle-wavelength-sensitive (M) photopigments instead of separate genes encoding L and M photopigments. The product of the hybrid gene exhibits a spectral sensitivity close to that of M photopigment; consequently, male monkeys carrying the hybrid gene are genetic protanopes, effectively lacking L photopigment. In the present study, we assessed retinal expression of L photopigment in monkeys carrying the hybrid gene. The relative sensitivities to middle-wavelength (green) and long-wavelength (red) light were measured by electroretinogram flicker photometry. We found the sensitivity to red light to be extremely low in protanopic male monkeys compared with monkeys with the normal genotype. In female heterozygotes, sensitivity to red light was intermediate between the genetic protanopes and normal monkeys. Decreased sensitivity to long wavelengths was thus consistent with genetic loss of L photopigment.

Animals↗
PubMed1987

On-line visualization of dendritic release of acetylcholinesterase from mammalian substantia nigra neurons.

This study presents, to our knowledge, the first on-line measurement of acetylcholinesterase (AcChoEase) release from brain tissue. It is now well established that a soluble form of the enzyme is released from central nervous system neurons, and it has been proposed on indirect grounds that such release may occur not only from presynaptic terminals but also from the dendrites of dopamine-containing nigrostriatal neurons. We have used a chemiluminescent reaction to examine the real-time release of AcChoEase from the substantia nigra in vitro in brainstem slices. The light emission was captured by two fiber optic systems, one in direct contact with the brain slice from below and the second 4-mm above the slice, allowing simultaneous imaging of the emitted light and quantitative photometry. It was determined that the light signals are not due to the spontaneous hydrolysis of acetylcholine or the presence of free choline, but are caused by the enzymatic action of AcChoEase. Using this technique, it can be directly shown that AcChoEase is spontaneously released from the soma or dendrites of nigral neurons. The release of the enzyme, which is stored in the subcisternal dendritic compartment, is resistant to blockade of voltage-dependent sodium conductances, is calcium dependent, and can be increased by addition of potassium to the bathing solution. The procedure we describe here will make it possible to study the release of endogenous AcChoEase on a time-scale close to that over which it functions.

Acetylcholinesterase↗
PubMed1993

Peptide analogues alter the progression of premalignant lesions, as measured by Photofrin fluorescence.

Somatostatin analogue RC-160 and bombesin/gastrin-releasing peptide antagonist RC-3095 were infused at 2 micrograms per day via miniosmotic pumps implanted s.c. in hamsters with premalignant disease to examine the effect of these peptides on cancer promotion and progression. These analogues have been shown to inhibit growth of certain tumors, especially those that overexpress tyrosine kinase activity. Progression of premalignant lesions initiated by applying 0.5% 9,10-dimethyl-1,2-benzanthracene (DMBA) to the hamster buccal cheek pouch was measured by Photofrin-induced fluorescence 24 hr after injecting the porphyrin (1.0 mg/kg) by using in vivo fluorescence photometry. This method of monitoring progression was reaffirmed by the observations that fluorescence increased significantly as compared with controls in lesions receiving 4 additional weeks of continuous promotion by DMBA application (P < 0.01 in two independent trials) and in lesions receiving transient promotion by laser incision (P < 0.01 and < 0.05 at the same time in the two trials). Twelve weeks after treatment, fluorescence had decreased significantly among animals treated for 2 weeks with RC-3095 (control, 0.53 +/- 0.03 V vs. RC-3095, 0.28 +/- 0.03 V; P < 0.0005) or with RC-160 (control, 0.85 +/- 0.03 V vs. RC-160, 0.24 +/- 0.03 V; P < 0.0001). These data were obtained 20 weeks after DMBA initiation. Thus, treatment with RC-160 and RC-3095 decreased the progression, measured by fluorescence, compared with control animals. In addition, there was also an absolute continuous decrease in fluorescence for the 22 weeks after the cessation of RC-160 treatment. That the changes in tumor progression produced by RC-160 extended beyond the treatment period supports the hypothesis that the changes were irreversible. Histopathological analysis revealed normal tissue and/or mild-moderate dysplasia in hamster buccal mucosa treated with the RC-160 (an improvement compared to pretreatment), whereas 40% of the animals receiving no treatment after DMBA initiation developed invasive squamous cell carcinomas after 20 weeks. These results show that the antagonists of bombesin/gastrin-releasing peptide can delay the development of malignancies and the agonists of somatostatin can potentially reverse this development.

Animals↗
PubMed1998

Membrane voltage initiates Ca2+ waves and potentiates Ca2+ increases with abscisic acid in stomatal guard cells.

In higher plants changes and oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) are central to hormonal physiology, including that of abscisic acid (ABA), which signals conditions of water stress and alters ion channel activities in guard cells of higher-plant leaves. Such changes in [Ca2+]i are thought to encode for cellular responses to different stimuli, but their origins and functions are poorly understood. Because transients and oscillations in membrane voltage also occur in guard cells and are elicited by hormones, including ABA, we suspected a coupling of [Ca2+]i to voltage and its interaction with ABA. We recorded [Ca2+]i by Fura2 fluorescence ratio imaging and photometry while bringing membrane voltage under experimental control with a two-electrode voltage clamp in intact Vicia guard cells. Free-running oscillations between voltages near -50 mV and -200 mV were associated with oscillations in [Ca2+]i, and, under voltage clamp, equivalent membrane hyperpolarizations caused [Ca2+]i to increase, often in excess of 1 microM, from resting values near 100 nM. Image analysis showed that the voltage stimulus evoked a wave of high [Ca2+]i that spread centripetally from the peripheral cytoplasm within 5-10 s and relaxed over 40-60 s thereafter. The [Ca2+]i increases showed a voltage threshold near -120 mV and were sensitive to external Ca2+ concentration. Substituting Mn2+ for Ca2+ to quench Fura2 fluorescence showed that membrane hyperpolarization triggered a divalent influx. ABA affected the voltage threshold for the [Ca2+]i rise, its amplitude, and its duration. In turn, membrane voltage determined the ability of ABA to raise [Ca2+]i. These results demonstrate a capacity for voltage to evoke [Ca2+]i increases, they point to a dual interaction with ABA in triggering and propagating [Ca2+]i increases, and they implicate a role for voltage in "conditioning" [Ca2+]i signals that regulate ion channels for stomatal function.

Journal Article↗
PubMed1999

A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease.

Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals.

Amino Acid Sequence↗
PubMed1999

Afterglows from the largest explosions in the universe.

The distinction of "largest explosions in the universe" has been bestowed on cosmic gamma-ray bursts. Their afterglows are brighter than supernovae and therefore are called hypernovae. Photometry and spectroscopy of these afterglows have provided major breakthroughs in our understanding of this mysterious phenomenon.

Journal Article↗
PubMed1995

Regulation of cytoplasmic pH in osteoclasts. Contribution of proton pumps and a proton-selective conductance.

Osteoclasts resorb bone by secreting protons into an extracellular resorption zone through vacuolar-type proton pumps located in the ruffled border. The present study was undertaken to evaluate whether proton pumps also contribute to intracellular pH (pHi) regulation. Fluorescence imaging and photometry, and electrophysiological methods were used to characterize the mechanisms of pH regulation in isolated rabbit osteoclasts. The fluorescence of single osteoclasts cultured on glass coverslips and loaded with a pH-sensitive indicator was measured in nominally HCO(3-)-free solutions. When suspended in Na(+)-rich medium, the cells recovered from an acute acid load primarily by means of an amiloride-sensitive Na+/H+ antiporter. However, rapid recovery was also observed in Na(+)-free medium when K+ was used as the substitute. Bafilomycin-sensitive, vacuolar-type pumps were found to contribute marginally to pH regulation and no evidence was found for K+/H+ exchange. In contrast, pHi recovery in high K+ medium was largely attributed to a Zn(2+)-sensitive proton conductive pathway. The properties of this conductance were analyzed by patch-clamping osteoclasts in the whole-cell configuration. Depolarizing pulses induced a slowly developing outward current and a concomitant cytosolic alkalinization. Determination of the reversal potential during ion substitution experiments indicated that the current was due to H+ (equivalent) translocation across the membrane. The H+ current was greatly stimulated by reducing pHi, consistent with a homeostatic role of the conductive pathway during intracellular acidosis. These results suggest that vacuolar-type proton pumps contribute minimally to the recovery of cytoplasmic pH from intracellular acid loads. Instead, the data indicate the presence of a pH- and membrane potential-sensitive H+ conductance in the plasma membrane of osteoclasts. This conductance may contribute to translocation of charges and acid equivalents during bone resorption and/or generation of reactive oxygen intermediates by osteoclasts.

Animals↗
PubMed1997

Correlating Ca2+ responses and secretion in individual RBL-2H3 mucosal mast cells.

The role of Ca2+ in stimulus-response coupling in nonexcitable cells is still not well understood. The Ca2+ responses of individual cells are extremely diverse, often displaying marked oscillations, and almost nothing is known about the specific features of these Ca2+ signals that are important for the functional response of a cell. Using the RBL-2H3 mucosal mast cell as a model, we have studied the temporal relationship between changes in intracellular Ca2+ and serotonin secretion at the single-cell level using simultaneous indo-1 photometry and constant potential amperometry. Secretion in response to antigen never occurs until intracellular Ca2+ is elevated, nor is it seen during the first few oscillations in Ca2+. Exocytotic events tend to be clustered around the peaks of oscillations, but excellent secretion is also seen in cells with sustained elevations in Ca2+. Ca2+ release from stores in the absence of influx fails to elicit secretion. If refilling and continued release of Ca2+ from stores is prevented with thapsigargin, Ca2+ influx can still trigger secretion, suggesting that store-associated microdomains of Ca2+ are not required for exocytosis. Our findings demonstrate the importance of an amplitude-encoded Ca2+ signal and Ca2+ influx for stimulus-secretion coupling in these nonexcitable cells.

Animals↗
PubMed2000

CaV2.2 and CaV2.3 (N- and R-type) Ca2+ channels in depolarization-evoked entry of Ca2+ into mouse sperm.

As sperm prepare for fertilization, surface Ca(2+) channels must open to initiate required, Ca(2+)-mediated events. However, the molecular identity and functional properties of sperm Ca(2+) channels remain uncertain. Here, we use rapid local perfusion and single-cell photometry to examine the kinetics of calcium responses of mouse sperm to depolarizing stimuli. The linear rise of intracellular [Ca(2+)] evoked by approximately 10-s applications of an alkaline high [K(+)] medium directly reports activity of voltage-gated Ca(2+) channels. Little response occurs if external Ca(2+) is removed or if external or internal pH is elevated without depolarization. Responses are inhibited 30-40% by 30-100 micrometer Ni(2+) and more completely by 100-300 micrometer Cd(2+). They resist the dihydropyridines nitrendipine and PN200-110, but 1-10 micrometer mibefradil inhibits reversibly. They also resist the venom toxins calciseptine, omega-conotoxin MVIIC, and kurtoxin, but omega-conotoxin GVIA (5 micrometer) inhibits approximately 50%. GVIA also partially blocks transient, low voltage activated Ca(2+) currents of patch-clamped spermatids. Differential sensitivity of sperm responses to Ni(2+) and Cd(2+) and partial blockade by GVIA indicate that depolarization opens at least two types of voltage-gated Ca(2+) channels in epididymal sperm examined prior to capacitation. Involvement of a previously undetected Ca(V)2.2 (N-type) channel, suggested by the action of GVIA, is substantiated by immunodetection of Ca(2+) channel alpha(1B) subunits in sperm and sperm extracts. Resistance to dihydropyridines, calciseptine, MVIIC, and kurtoxin indicates that Ca(V)1, Ca(V)2.1, and Ca(V)3 (L-, P/Q-, and T-type) channels contribute little to this evoked response. Partial sensitivity to 1 micrometer mibefradil and an enhanced sensitivity of the GVIA-resistant component of response to Ni(2+) suggest participation of a Ca(V)2.3 (R-type) channel specified by previously found alpha(1E) subunits. Our examination of depolarization-evoked Ca(2+) entry indicates that mature sperm possess a larger palette of voltage-gated Ca(2+) channels than previously thought. Such diversity may permit specific responses to multiple cues encountered on the path to fertilization.

Animals↗
PubMed2001

Functional characterization of mutants in the predicted pore region of the rabbit cardiac muscle Ca(2+) release channel (ryanodine receptor isoform 2).

A highly conserved amino acid sequence, GVRAGGGIGD(4831), which may form part of the Ca(2+) release channel pore in RyR2, was subjected to Ala scanning or Ala to Val mutagenesis; function was then measured by expression in HEK-293 cells, followed by Ca(2+) photometry, high affinity [(3)H]ryanodine binding, and single-channel recording. All mutants except I4829A and I4829T (corresponding to the I4897T central core disease mutant in RyR1) displayed caffeine-induced Ca(2+) release in HEK-293 cells; only mutants G4826A, I4829V, and G4830A retained high affinity [(3)H]ryanodine binding; and single-channel function was found for all mutants tested, except for G4822A and A4825V. EC(50) values for caffeine-induced Ca(2+) release were increased for G4822A, R4824A, G4826A, G4828A, and D4831A; decreased for V4823A; and unchanged for A4825V, G4827A, I4829V, and G4830A. Ryanodine (10 microm), which did not stimulate Ca(2+) release in wild type (wt), did so in Ala mutants in amino acids 4823-4827. It inhibited the caffeine response in wt and most mutants, but enhanced the amplitude of caffeine-induced Ca(2+) release in mutant G4828A. It also restored caffeine-induced Ca(2+) release in mutants I4829A and I4829T. In single-channel recordings, mutants I4829V and G4830A retained normal conductance, whereas all others had decreased unitary channel conductances ranging from 27 to 540 picosiemens. Single-channel modulation was retained in G4826A, I4829V, and G4830A, but was lost in other mutants. In contrast to wt and G4826A, I4829V, and G4830A, in which divalent metals were preferentially conducted, mutants with loss of modulation had no selectivity of divalent cations over a monovalent cation. Analysis of Gly(4822) to Asp(4831) mutants in RyR2 supports the view that this highly conserved sequence constitutes part of the ion-conducting pore of the Ca(2+) release channel and plays a key role in ryanodine and caffeine binding and activation.

Amino Acid Sequence↗
PubMed2001

Biosynthesis of riboflavin. Single turnover kinetic analysis of GTP cyclohydrolase II.

GTP cyclohydrolase II catalyzes the conversion of GTP into a mixture of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (Compound 2), formate, and pyrophosphate. Moreover, GMP was recently shown to be formed as a minor product. The major product (Compound 2) serves as the first committed intermediate in the biosynthesis of the vitamin, riboflavin. Numerous pathogenic microorganisms are absolutely dependent on endogenous synthesis of riboflavin. The enzymes of this pathway are therefore potential drug targets, and mechanistic studies appear relevant for development of bactericidal inhibitors. Pre-steady state quenched flow analysis of GTP cyclohydrolase II shows the rate-determining step to be located at the beginning of the reaction sequence catalyzed by the enzyme. Thus, GTP is consumed at a rate constant of 0.064 s(-1), and the reaction product, Compound 2, is formed at an apparent rate constant of 0.062 s(-1). Stopped flow experiments monitored by multiwavelength photometry are well in line with these data. 2-Amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate can serve as substrate for GTP cyclohydrolase II but does not fulfill the criteria for a kinetically competent intermediate. A hypothetical reaction mechanism involves the slow formation of a phosphoguanosyl derivative of the enzyme under release of pyrophosphate. The covalently bound phosphoguanosyl moiety is proposed to undergo rapid hydrolytic release of formate from the imidazole ring and/or hydrolytic cleavage of the phosphodiester bond.

Chromatography, High Pressure Liquid↗
PubMed2003

Temperature-dependent presteady state kinetics of lumazine synthase from the hyperthermophilic eubacterium Aquifex aeolicus.

6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Presteady state kinetic experiments using the enzyme from the hyperthermophilic bacterium Aquifex aeolicus were monitored by multiwavelength photometry. An early optical transient absorbing around 330 nm is interpreted as a Schiff base intermediate obtained by reaction of the position 5 amino group of the heterocyclic substrate with the carbonyl group of 3,4-dihydroxy-2-butanone 4-phosphate. A second transient with an absorption maximum at 445 nm represents an intermediate resulting from the elimination of orthophosphate from the Schiff base. The rate-determining step is the subsequent formation of the 7-exomethylene type anion of 6,7-dimethyl-8-ribityllumazine. The rate constants for the three partial reactions identified by the stopped flow experiments show linear Arrhenius relations in the temperature range of 15-70 degrees C.

Eubacterium↗
PubMed2004

Oligomeric state of the Escherichia coli metal transporter YiiP.

YiiP is a 32.9-kDa metal transporter found in the plasma membrane of Escherichia coli (Chao, Y., and Fu, D. (2004) J. Biol. Chem. 279, 17173-17180). Here we report the determination of the YiiP oligomeric state in detergent-lipid micelles and in membranes. Molecular masses of YiiP solubilized with dodecyl-, undecyl-, decyl-, or nonyl-beta-d-maltoside were measured directly using size-exclusion chromatography coupled with laser light-scattering photometry, yielding a mass distribution of YiiP homo-oligomers within a narrow range (68.0-68.8 kDa) that equals the predicted mass of a YiiP dimer within experimental error. The detergent-lipid masses associated with YiiP in the mixed micelles were found to increase from 135.5 to 232.6 kDa, with an apparent correlation with the alkyl chain length of the maltoside detergents. Cross-linking the detergent-solubilized YiiP with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) resulted in a dimeric cross-linked product in an EDC concentration-dependent manner. The oligomeric state of the purified YiiP in reconstituted membranes was determined by electron microscopic analysis of two-dimensional YiiP crystals in negative stain. A projection structure calculated from measurable optical diffractions to 25 A revealed a pseudo-2-fold symmetry within a molecular boundary of approximately 75 x 40 A, indicative of the presence of YiiP dimers in membranes. These data provide direct structural evidence for a dimeric association of YiiP both in detergent-lipid micelles and in the reconstituted lipid bilayer. The functional relevance of the dimeric association in YiiP is discussed.

Calibration↗