Is the activity of the fusion pore scaffold regulated by a coincidence detector?
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Amino Acid Sequence↗
Biomedical subjects
J M Fernandez
Publications and source records attributed to J M Fernandez.
Localization of the site of Ca2+ release at the level of a single sarcomere in skeletal muscle fibres.
The development of mechanical force in skeletal muscle fibres is brought about by rapid increases in the intracellular calcium concentration (Ca2+ transients) which can be detected by optical methods. Local stimulation experiments and ultrastructural evidence suggest that, at a microscopic level, these Ca2+ transients are generated by the release of Ca2+ ions from the terminal cisternae of the sarcoplasmic reticulum in response to the depolarization of the transverse tubules (t-tubules). Nevertheless, to date, there is no functional information on the exact location at which Ca2+ release takes place. The present experiments were designed to obtain direct evidence about dynamic changes in localization and microscopic distribution of Ca2+ in a single sarcomere using two independent novel methodologies: confocal spot detection of Ca2+ transients and Ca2+ imaging with pulsed laser excitation.
Action Potentials↗
RT-PCR cloning of Rab3 isoforms expressed in peritoneal mast cells.
Rab proteins are ras-like low molecular mass GTP-binding proteins, which are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. We have previously shown that synthetic peptides, corresponding to the effector domain of Rab3 proteins, stimulate a complete exocytotic response in mast cells. We have used a PCR-cloning strategy to investigate the presence of mRNA encoding Rab3 in mast cells. RNA based PCR was then performed on mast cell RNA using degenerate oligonucleotide primers based on two conserved sequences among Rab3 proteins. However, no PCR products were obtained, even for proteins known to be expressed in high copy numbers in mast cells (beta-actin and Fc receptor). We have found that the problem resides in the presence of mast cell secretory granule derived heparin, that copurifies with the RNA; heparin has been shown to inhibit the activity of reverse transcriptase and Taq polymerase in PCR. After treating the RNA (obtained from about 500 mast cells) with heparinase, several PCR products of varying size were obtained using primers specific for Rab3 proteins. These products were cloned and sequenced. We have found clones containing sequences that had a 100% homology at the deduced amino acid level to a portion of Rab3B and Rab3D (amino acids 16 to 83).
Amino Acid Sequence↗
Primary structure of a new HLA-A2 subtype: HLA-A*0213.
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Amino Acid Sequence↗
The exocytotic fusion pore and neurotransmitter release.
Membrane fusion is ubiquitous in biological systems, occurring in the simplest of unicellular eukaryotes as well as higher eukaryotes. As soon as the first primitive eukaryotic cell utilized a lipid bilayer as an outer membrane, membrane fusion (and fission) became necessary for the traffic of material from the outside to the inside, the inside to the outside, and between different intracellular membrane-bounded compartments. The earliest cells would have made use of the intrinsic ability of lipid bilayers to fuse under certain conditions. Although this fusogenic property of bilayers has been known for some time, it is has become clear only relatively recently that two phospholipid bilayers will fuse spontaneously, owing to a hydrophobic force, when the bilayers are brought close together under conditions of membrane tension or high curvature (Helm and Israelachvili, 1993). The primeval cell would have used proteins to develop the appropriate architecture in which such fusion would occur in a regulated manner. During the course of evolution, ever more sophisticated ways of regulating this basic process would evolve, but the underlying fusion mechanism would remain unchanged. We have proposed that a macromolecular scaffold of proteins is responsible for bringing the plasma membrane close to the secretory granule membranes and creating the architecture that enables the hydrophobic force to cause fusion (Figure 1; Nanavati et al., 1992; Monck and Fernandez, 1992; Oberhauser and Fernandez, 1993). Evidence is now accumulating that there are several highly conserved families of proteins associated with vesicle fusion events, from yeast to mammalian cells, and with intracellular traffic, as well as with regulated exocytosis and synaptic transmission (Bennett and Scheller, 1993; Sollner et al., 1993; Südhof et al., 1993). The molecular structures (or scaffolds) that regulate membrane fusion are likely to contain related proteins and share certain fundamental properties.
Calcium-Binding Proteins↗
The fusion pore interface: a new biological frontier.
The development of micro-voltammetry to detect the release of secretory products from single cells has yielded surprising information, which suggests that the release of secretory products is regulated after the fusion of secretory vesicles with the plasma membrane. This technique has also been used to demonstrate that the release of secretory products can occur during transient fusion events, which leads one to question the current models for membrane recycling. In the past year, strong evidence has emerged in support of a role for rab3 and G alpha i3 proteins in regulating a putative scaffold of proteins that cause bilayer fusion during exocytosis. These findings parallel the biochemical identification of several new cytosolic, secretory vesicle and plasma membrane proteins that may also play a role in regulating fusion.
Animals↗
Pulsed laser imaging of rapid Ca2+ gradients in excitable cells.
Excitable cells are thought to respond to action potentials by forming short lived and highly localized Ca2+ gradients near sites of Ca2+ entry or near the site of Ca2+ release by intracellular stores. However, conventional imaging techniques lack the spatial and temporal resolution to capture these gradients. Here we demonstrate the use of pulsed-laser microscopy to measure Ca2+ gradients with submicron spatial resolution and millisecond time resolution in two preparations where the Ca2+ signal is thought to be fast and highly localized: adrenal chromaffin cells, where the entry of Ca2+ through voltage dependent Ca2+ channels triggers exocytotic fusion; and skeletal muscle fibers, where intracellular Ca2+ release from the sarcoplasmic reticulum initiates contraction. In chromaffin cells, Ca2+ gradients developed over 10-100 ms and were initially restricted to discrete submembrane domains, or hot spots, before developing into complete rings of elevated Ca2+ concentration. In frog skeletal muscle large, short-lived (approximately 6 ms) Ca2+ gradients were observed within individual sarcomeres following induction of action potentials. The pulsed laser imaging approach permits, for the first time, the capture and critical examination of rapid Ca2+ signaling events such as those underlying excitation-secretion and excitation-contraction coupling.
Action Potentials↗
Blood metabolite and regulatory hormone concentrations and response to metabolic challenges during the infusion of mimosine and 2,3-dihydroxypyridine in alpine goats.
Sixteen Alpine wethers (average BW 35 +/- 2 kg) were used to evaluate the effect of continuous 48-h intravenous infusions of saline (CON), mimosine (MIM; 200 mg.kg.75.d-1), 2-hydroxy-3(1H)-pyridine (2,3-DHP; 200 mg.kg.75.d-1, or MIM+2,3-DHP (100 mg of MIM plus 100 mg of 2,3-DHP-kg.75.d-1) on hepatic function and selected blood metabolite and circulating hormone concentrations. Neither MIM nor 2,3-DHP affected plasma ammonia N, glucose, cortisol, or insulin concentrations over time (P > .10). Jugular plasma total protein concentration was greater in the MIM group (P < .07). Plasma triiodothyronine (P < .01) and thyroxine (P < .08) concentrations were higher in the goats receiving the MIM, 2,3-DHP, and MIM+2,3-DHP infusions than in the goats receiving the CON infusion. Plasma urea N concentration was decreased by MIM (P < .10) and MIM+2,3-DHP (P < .03) compared with the CON infusion. A Propionate Load Test was conducted at 24 to 28 h into the infusion to assess the toxins' effects on the liver's ability to increase circulating glucose concentrations in the presence of elevated propionate levels. The results indicated that neither 2,3-DHP nor MIM reduced the liver's ability to respond to a bolus dose of propionate (P > .10). Following a Urea Load Test, circulating ammonia N and glucose concentrations in the MIM, 2,3-DHP, and MIM+2,3-DHP treatments had lower peak values than that in the CON treatment (P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)
Ammonia↗
Changes in concentrations of hormones, metabolites, and amino acids in plasma of adult horses relative to overnight feed deprivation followed by a pellet-hay meal fed at noon.
Experiment 1 was conducted to characterize the concentrations of prolactin, growth hormone (GH), cortisol, insulin, glucagon, glucose, nonesterified fatty acids (NEFA), urea N, and 10 indispensable amino acids in the plasma of mares (n = 8) and stallions (n = 8) during the last 4 h of a 19-h period of feed deprivation and for 8 h after a noon meal. Experiment 2 was similar to Exp. 1 except that only stallions (n = 8) were used, and they were either fed (n = 4) or not fed (n = 4) at noon in a 2 x 2 Latin square design conducted over two sampling days 7 d apart. In Exp. 1, increases (P < .01) after feeding were observed for plasma concentrations of prolactin, cortisol, insulin, glucagon, glucose, urea N, and all amino acids except methionine; NEFA concentrations decreased (P < .01) after feeding. Episodic increases in GH concentrations were observed for most horses but were not associated with either feeding or gender (P > .1). Plasma urea N concentrations were higher (P < .025) overall in stallions than in mares, and the rise in prolactin concentrations after feeding was greater (P < .01) in stallions than in mares. In Exp. 2, meal-associated increases (P < .01) were observed for plasma concentrations of prolactin, insulin, glucagon, and glucose; NEFA concentrations decreased (P < .01). Except for cortisol, no hormone or metabolite varied with time across days when the stallions were not fed (P > .1), indicating that there was no inherent diurnal or feeding schedule-associated fluctuations in their concentrations. Cortisol concentrations varied (P < .02) over time but did not differ (P > .1) between fed and nonfed stallions. Again, GH concentrations were episodic but did not differ (P > .1) between fed and nonfed stallions. The lack of feeding effects on GH secretion in horses is similar to the response in pigs but differs from that in ruminants, in which GH concentrations generally decline after feeding.
Amino Acids↗
Influence of chromium picolinate on glucose usage and metabolic criteria in growing Holstein calves.
Holstein calves, 10 steers (98 +/- 16 kg; Exp. 1) and 14 heifers (122 +/- 7 kg; Exp. 2), were fed either corn-cottonseed hull basal diets (CON; 15.1 and 13.2% CP, respectively) or CON plus 370 micrograms/kg of Cr as Cr-tripicolinate (CrP). After either 58 (Exp. 1) or 56 (Exp. 2) days of ADG and DMI measurement, calves were individually fed 1.4% of their BW every 12 h through the end of each experiment. Nitrogen balance, i.v. glucose tolerance tests (IVGTT), i.v. insulin challenge tests (IVICT), baseline growth hormone (GH) measurements, and GH-releasing hormone challenges were then performed on calves in split groups on d 70 through 87 in Exp. 1. In Exp. 2, all challenges began on d 78 and N balance was not determined. Inclusion of CrP did not affect (P > .10) ADG, DMI, or ADG/DMI of pooled data from Exp. 1 and 2 or N balance criteria in Exp. 1. Plasma glucose, insulin, and NEFA concentrations were not affected (P > .10) by CrP. Total plasma cholesterol was lower (P < .05) for CrP- than for CON-fed calves at wk 4 in Exp. 1 and wk 6 in Exp. 2. During IVGTT, calves fed CrP vs CON had higher (P < .05) clearance rates for glucose in Exp. 1 (2.68 vs 1.88%/min) and in Exp. 2 (2.64 vs 2.08%/min). During IVICT, calves fed CrP vs CON had higher clearance rates for glucose in Exp. 1 (2.98 vs 2.34%/min; P = .12) and in Exp. 2 (2.89 vs 2.00%/min; P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)
Animal Feed↗
Plasma concentrations of prolactin, glucose, insulin, urea nitrogen, and total amino acids in stallions after ingestion of feed or gastric administration of feed components.
Concentrations of prolactin, glucose, insulin, urea N, and total amino acids in plasma of stallions after ingestion of pelleted feed were compared to those after direct gastric administration of water, NaCl, egg albumin, or corn starch (Exp. 1) or water, egg albumin, hydrolyzed casein (Amicase), or a mixture of indispensable amino acids (Exp. 2). Stallions were fed once daily (75% pellet and 25% hay) at 1500 for 30 d. On d 22, 24, 26, 28, and 30, blood samples were collected every 30 min from 1 h before through 4 h after treatment, which occurred at 1100. In Exp. 1, there was a positive secretory response for prolactin (P = .013) only after the meal. Positive glucose and insulin responses were observed after the meal (P < .055) and after gastric administration of corn starch (P < .001). Total amino acids increased (P = .008) only after the meal. In Exp. 2, a positive prolactin response (P < .001) occurred after the meal and a negative response (P = .023) after administration of water; administration of Amicase increased (P = .061) prolactin concentrations after a 2.5-h delay. Positive responses were observed for glucose, insulin, and total amino acids after the meal (P < .001) and after administration of Amicase or the amino acid mixture (P < .026). Positive urea N responses were observed after administration of Amicase and the amino acid mixture (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acids↗
The secretory granule matrix: a fast-acting smart polymer.
The secretory granule matrix is a miniature biopolymer that consists of a charged polymer network that traps peptides and transmitters when it condenses and releases them on exocytotic decondensation. Models of exocytotic fusion have treated this matrix as a short circuit and have neglected its electrical contributions. This matrix responded to negative voltages by swelling, which was accompanied by a large increase in conductance, and to positive voltages by condensing. Thus, the matrix resembled a diode. The swollen matrix exerted large pressures on the order of 12 bar. The responses took place within milliseconds of the application of the electric field. These findings suggest that matrix decondensation, and therefore product release, is controlled by potential gradients.
Animals↗
When is the optimum time to harvest peripheral blood stem cells in children following standard dose chemotherapy?
The optimal time for peripheral stem cell harvest may be predicted by recovery of blood count following priming doses of chemotherapy. Bone marrow growth factors have also been used to enhance yield. Both these manoeuvres may necessitate alterations to standard treatment programmes. In order to determine whether adequate peripheral stem cell yields could be obtained without altering chemotherapy regimens, 18 children with either leukaemia or solid tumours were studied. Circulating bone marrow progenitor cells were estimated by CFU-GM cultures in soft agar. There were 8 males and 10 females, median age 5.6 years (range 2-13 years). Six out of 13 studies in leukaemia patients were done when the bone marrow contained significant leukaemic blasts, and in 2 of 9 studies in patients with solid tumours, there was active infiltration of the bone marrow. In 15 of 23 studies, a rise in CFU-GM levels to greater than 1,000 x 10(3)/l were observed following postchemotherapy aplasia. Maximum CFU-GM levels were maintained for only 1-2 days and could be predicted by the achievement of a total white cell count in the region of 2 x 10(9)/l and platelet count around 100 x 10(9)/l. Neutrophil recovery did not predict the rise in CFU-GM levels. A rapid rise in monocyte levels also preceded maximum CFU-GM levels. Predictably, the CFU-GM counts were lower in patients who were heavily pretreated, but in several children, the counts were sufficient to predict an adequate harvest to be used in conjunction with bone marrow autotransplantation. It is concluded that in children with active bone marrow recovery, chemotherapy priming or the use of bone marrow growth factors may not be necessary to achieve useful stem cell harvests.
Adolescent↗
GTP gamma S stimulates exocytosis in patch-clamped rat melanotrophs.
We investigated the molecular mechanisms regulating exocytosis in patch-clamped melanotrophs by measuring the membrane capacitance. Ca(2+)-dependent exocytosis could be induced by membrane depolarization or by including solutions containing 2 microM free Ca2+ in the patch pipette. Ca(2+)-dependent exocytosis was inhibited by GDP beta S, suggesting involvement of a GTP-binding protein. The hydrolysis-resistant GTP analogs, GTP gamma S and GppNHp, were able to stimulate exocytosis at low free Ca2+ concentrations. The stimulatory response to GTP gamma S was abolished by both GDP beta S and GTP. The latter suggests that a sustained activation of a GTP-binding protein is necessary for exocytosis. This behavior is similar to the stimulation of exocytosis by guanine nucleotides in mast cells and other nonexcitable cells and suggests a common regulatory mechanism.
Animals↗
Patch clamp studies of single intact secretory granules.
The membrane of secretory granules is involved in the molecular events that cause exocytotic fusion. Several of the proteins that have been purified from the membrane of secretory granules form ion channels when they are reconstituted in lipid bilayers and, therefore, have been thought to form part of the molecular structure of the exocytotic fusion pore. We have used the patch clamp technique to study ion conductances in single isolated secretory granules from beige mouse mast cells. We found that the membrane of the intact granule had a conductance of < 50 pS. No abrupt changes in current corresponding to the opening and closing of ion channels were observed, even under conditions where exocytotic fusion occurred. However, mechanical tension or a large voltage pulse caused the breakdown of the granule membrane resulting in the abrupt opening of a pore with an ion conductance of about 1 nS that fluctuated rapidly and could expand to an immeasurably large conductance or close completely. Surprisingly, the behavior of these pores resembled the pattern of conductance changes of exocytotic fusion pores observed in degranulating beige mast cells. This similarity supports the view that the earliest fusion pore is formed upon the breakdown of a bilayer such as that formed during hemifusion.
Animals↗
The expression of GAP-43 mRNA in developing embryonic striatal tissue grafts.
We have studied by quantitative in situ hybridization histochemistry the mRNA expression of the growth associated phosphoprotein (GAP-43) in grafts of foetal striatal neurones implanted into the ibotenic acid lesioned adult rat neostriatum. Animals were studied at 7, 15, 30 and 90 days after implantation. Using a 35S-labelled specific oligonucleotide probe we observed the highest levels of GAP-43 mRNA in 15-day-old grafts; levels were significantly reduced at 30 days and in 3-month-old grafts GAP-43 mRNA expression was not significantly different from the very low levels in the normal rat striatum. The data indicate that GAP-43 mRNA is highly expressed in developing but not mature neurones and suggest that embryonic striatal grafts may develop a relatively adult phenotype within the host brain by about 4 weeks; by which time most of the synaptic connections are established.
Animals↗
The localization of cholecystokinin mRNA in embryonic striatal tissue grafts: further evidence for the presence of non-striatal cells.
We have studied by in situ hybridization histochemistry the mRNA expression of preprocholecystokinin (CCK) in grafts of foetal striatal tissue neurones implanted into the ibotenic acid lesioned adult rat neostriatum. Animals were studied at 30 and 90 days after implantation. Using a 35S-labelled 'antisense' oligonucleotide (45-mer) probe complementary to the rat CCK cDNA we observed a dense hybridization signal for CCK mRNA in the striatal eminence in developing embryos and in all the striatal grafts examined; no CCK mRNA expressing cells were found in the normal neonatal or adult rat neostriatum. However, CCK mRNA was heavily expressed in other areas of the host brain including the somatosensory cortex, the claustrum, primary olfactory cortex and endopiriform cortex. The germinal cell layer of the striatal eminence is the source of origin for these non-striatal cell groups as well as for cells destined to form the neostriatum. The present results suggest that CCK mRNA expression provides an unambiguous marker for many of the non-striatal cells in 'striatal' grafts.
Animals↗
Effect of source and amount of protein on milk production in dairy cows.
Forty multiparous Alpine does (mean BW of 61.5 kg) were utilized in a 13-wk trial to investigate the effects of a TMR differing in CP amount (13 or 17%) and source (solvent-extracted soybean meal or heat-treated soybean meal with or without urea) on lactational performance. Protein supplements contributed 30% of the N in 13% CP diets and 50% of the N in 17% CP diets. All diets were isoenergetic (2.5 Mcal of metabolizable energy/kg of DM) and were fed for ad libitum intake for the entire trial. Mean DMI (2.88 kg/d), milk production (2.65 kg/d), milk fat (4.05%), milk protein (2.68%), milk lactose (4.54%), and milk SNF (7.81%) did not differ among dietary treatments. Plasma urea N was greater (23.2 vs. 10.9 mg/dl) in does receiving the 17% CP diets; however, blood hematocrit (27.4%), beta-hydroxybutyrate (843 microM), plasma glucose (68.8 mg/dl), NEFA (600 mu eq/dl), and plasma total protein (74.% g/L) were not significantly affected by treatment. The apparent absence of a dietary effect on lactational performance may be due to the high DMI of the does (4.7% when expressed as DMI per kilogram of BW) and high CP intake providing a surfeit of protein relative to requirements.
3-Hydroxybutyric Acid↗