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Biomedical subjects

J M Fernandez

Publications and source records attributed to J M Fernandez.

At least 19 recordsLinked to original sources
PubMed1992

Exocytotic fusion is activated by Rab3a peptides.

Studies of intracellular traffic in yeast and mammalian systems have implicated members of the Rab family of small GTP-binding proteins as regulators of membrane fusion. We have used the patch clamp technique to measure exocytotic fusion events directly and investigate the role of GTP-binding proteins in regulating exocytosis in mast cells. Intracellular perfusion of mast cells with GTP-gamma S is sufficient to trigger complete exocytotic degranulation in the absence of other intracellular messengers. Here we show that GTP is a potent inhibitor of GTP-gamma S-induced degranulation, indicating that sustained activation of a GTP-binding protein is sufficient for membrane fusion. We have found that synthetic oligopeptides, corresponding to part of the effector domain of Rab3a, stimulate complete exocytotic degranulation, similar to that induced by GTP-gamma S. The response is selective for Rab3a sequence and is strictly dependent on Mg2+ and ATP. This suggests that sustained activation of a Rab3 protein causes exocytotic fusion. The peptide response can be accelerated by GDP-beta S, suggesting that Rab3a peptides compete with endogenous Rab3 proteins for a binding site on a target effector protein, which causes fusion on activation.

Amino Acid Sequence↗
PubMed1992

Acute nonlymphoblastic leukemia in children treated for acute lymphoblastic leukemia with an intensive regimen including teniposide.

Some cases of conversion from acute lymphoblastic leukemia (ALL) to acute nonlymphoblastic leukemia (ANLL) at relapse have been reported recently. We report three cases initially diagnosed as having ALL and showing morphological, cytochemical, and immunophenotypic features of ANLL at relapse (lineage switch). Conversion was observed among 14 patients who developed bone marrow relapse while undergoing intensive treatment with our ALL protocol, which includes teniposide, and that had been administered to 62 patients. The three cases converted at first relapse, with a mean time of 20 months (13-29 months). Clinical and immunologic characteristics of T-cell leukemia were present in one patient. Changes documented in cytogenetic studies are discussed. The underlying mechanisms for the lineage switch remain unclear as does its relation with mixed lineage leukemias, but we believe that drugs employed in our therapy protocol could have had an influence on this conversion.

Child, Preschool↗
PubMed1992

The exocytotic fusion pore modeled as a lipidic pore.

Freeze-fracture electron micrographs from degranulating cells show that the lumen of the secretory granule is connected to the extracellular compartment via large (20 to 150 nm diameter) aqueous pores. These exocytotic fusion pores appear to be made up of a highly curved bilayer that spans the plasma and granule membranes. Conductance measurements, using the patch-clamp technique, have been used to study the fusion pore from the instant it conducts ions. These measurements reveal the presence of early fusion pores that are much smaller than those observed in electron micrographs. Early fusion pores open abruptly, fluctuate, and then either expand irreversibly or close. The molecular structure of these early fusion pores is unknown. In the simplest extremes, these early fusion pores could be either ion channel like protein pores or lipidic pores. Here, we explored the latter possibility, namely that of the early exocytotic fusion pore modeled as a lipid-lined pore whose free energy was composed of curvature elastic energy and work done by tension. Like early exocytotic fusion pores, we found that these lipidic pores could open abruptly, fluctuate, and expand irreversibly. Closure of these lipidic pores could be caused by slight changes in lipid composition. Conductance distributions for stable lipidic pores matched those of exocytotic fusion pores. These findings demonstrate that lipidic pores can exhibit the properties of exocytotic fusion pores, thus providing an alternate framework with which to understand and interpret exocytotic fusion pore data.

Animals↗
PubMed1992

Events leading to the opening and closing of the exocytotic fusion pore have markedly different temperature dependencies. Kinetic analysis of single fusion events in patch-clamped mouse mast cells.

The earliest event in exocytosis is the formation of a fusion pore, an aqueous channel that connects the lumen of a secretory granule with the extracellular space. We can observe the formation of individual fusion pores and their subsequent dilation or closure by measuring the changes in the admittance of patch-clamped mast cells during GTP gamma S-stimulated exocytotic fusion. To investigate the molecular structure of the fusion pore, we have studied the temperature dependency of the rate constants for fusion pore formation and closure. An Arrhenius plot of the rate of fusion pore formation shows a simple linear relationship with an apparent activation energy of 23 kcal/mol. In contrast, the Arrhenius plot of the rate of closure of the fusion pore is discontinuous, with the break at approximately 13 degrees C. Above the break point, the rate of closure has a weak temperature dependence (7 kcal/mol), whereas below 13 degrees C the rate of closure is temperature independent. This type of temperature dependency is characteristic of events that depend on diffusion in a lipid phase that undergoes a fluid-solid phase transition. We propose that the formation of the fusion pore is regulated by the conformational change of a molecular structure with a high activation energy, whereas the closure of the fusion pore is regulated by lipids that become phase separated at 13 degrees C.

Animals↗
PubMed1992

Thin-section ratiometric Ca2+ images obtained by optical sectioning of fura-2 loaded mast cells.

The availability of the ratiometric Ca2+ indicator dyes, fura-2, and indo-1, and advances in digital imaging and computer technology have made it possible to detect Ca2+ changes in single cells with high temporal and spatial resolution. However, the optical properties of the conventional epifluorescence microscope do not produce a perfect image of the specimen. Instead, the observed image is a spatial low pass filtered version of the object and is contaminated with out of focus information. As a result, the image has reduced contrast and an increased depth of field. This problem is especially important for measurements of localized Ca2+ concentrations. One solution to this problem is to use a scanning confocal microscope which only detects in focus information, but this approach has several disadvantages for low light fluorescence measurements in living cells. An alternative approach is to use digital image processing and a deblurring algorithm to remove the out of focus information by using a knowledge of the point spread function of the microscope. All of these algorithms require a stack of two-dimensional images taken at different focal planes, although the "nearest neighbor deblurring" algorithm only requires one image above and below the image plane. We have used a modification of this scheme to construct a simple inverse filter, which extracts optical sections comparable to those of the nearest neighbors scheme, but without the need for adjacent image sections. We have used this "no neighbors" processing scheme to deblur images of fura-2-loaded mast cells from beige mice and generate high resolution ratiometric Ca2+ images of thin sections through the cell. The shallow depth of field of these images is demonstrated by taking pairs of images at different focal planes, 0.5-microns apart. The secretory granules, which exclude the fura-2, appear in focus in all sections and distinct changes in their size and shape can be seen in adjacent sections. In addition, we show, with the aid of model objects, how the combination of inverse filtering and ratiometric imaging corrects for some of the inherent limitations of using an inverse filter and can be used for quantitative measurements of localized Ca2+ gradients. With this technique, we can observe Ca2+ transients in narrow regions of cytosol between the secretory granules and plasma membrane that can be less than 0.5-microns wide. Moreover, these Ca2+ increases can be seen to coincide with the swelling of the secretory granules that follows exocytotic fusion.

Algorithms↗
PubMed1992

Effect of intraperitoneal administration of lysine and methionine on mohair yield and quality in Angora goats.

Eight mature Angora wethers (average BW 47.2 kg) were used in a 4 x 4 replicated Latin square design to evaluate responses to intraperitoneal (IP) administration of amino acids. The IP treatments consisted of saline (Control), methionine 1 g/d (Met), lysine 2 g/d (Lys), and methionine + lysine (Met + Lys). The amino acids dissolved in Control were infused continuously for the first 14 d of each 28-d period using peristaltic infusion pumps. Average d-28 grease and clean mohair yields (grams/100 square centimeters), and fiber diameter (micrometers) and length (centimeters) measurements during Control administration were 8.8, 7.6, 40, and 2.3, respectively. Mean clean mohair yield and fiber diameter increased by 5.3% (P less than .039) and 2.5% (P less than .067), respectively, with Met administration but were decreased by 9.2% (P less than .033) and 3.8% (P less than .001), respectively, by Lys administration; however, mean fiber length was increased (P less than .014) 21.7% by Lys infusion. The goats did not exhibit increased grease (P greater than .939) and clean (P greater than .477) mohair yields and fiber diameter (P greater than .619) when treated with Met+Lys. A N balance trial was conducted during d 10 through 14 of each period. Total retained N (grams per day) during Control administration was 18.4 and 24% greater than Met (P greater than .281) and Lys (P less than .061), respectively. When expressed as a proportion of N intake, retained N was lowest (P less than .127) in the Lys infusion group. Jugular blood ammonia N and plasma glucose and total protein concentrations were not affected (P greater than .10) by treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Dietary protein level and ruminal degradability for mohair production in Angora goats.

Twenty-eight Angora goat doelings (average BW 22.1 kg) were used in a 150-d study to examine the effects of dietary CP level and degradability on mohair fiber production. A 2 x 2 factorial arrangement was instituted using conventional, solvent-extracted soybean meal (high degradability) or expelled, heat-treated soybean meal (low degradability) incorporated into low- (12%) or high- (19%) CP diets. Grease and clean mohair weights were greater (P less than .05) in goats fed the diets containing 19% CP. Mohair fiber diameter was not affected (P greater than .10) by dietary CP level. Clean mohair weight tended (P less than .08) to be higher in the goats fed diets containing expelled, heat-treated soybean meal. Body weight gains were not affected (P greater than .10) by CP level or degradability, whereas DMI increased (P less than .01) with increasing CP level. Ruminal fluid pH and total VFA concentrations were not affected (P greater than .10) by diet. Ruminal ammonia N concentration increased (P less than .05) as CP level in the diet increased, and postprandial changes in concentrations were less noticeable in the group fed expelled, heat-treated soybean meal. Plasma urea N (P less than .001) and total protein (P less than .01) concentration increased as dietary CP level increased. Plasma glucose was elevated (P less than .001) 2 h after feeding in the goats fed conventional, solvent-extracted soybean meal, whereas glucagon concentrations were greater at 0 and 4 h in the group fed expelled, heat-treated soybean meal.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗
PubMed1992

Dietary fiber and milk yield, mastication, digestion, and the rate of passage in goats fed alfalfa hay.

Two experiments were conducted to study the effect of intake of fiber on productive performance of high producing dairy goats during early to midlactation. Four dietary treatments were isonitrogenous and consisted of combinations of chopped alfalfa hay and concentrate, yielding 14, 18, 22, and 26% ADF. In Experiment 1, 40 multiparous Alpine does were used in a completely randomized block design. Milk fat content and total chewing time increased, and milk yield tended to decrease, as dietary ADF intake increased. Chewing efficiency [min/(g x kg BW.75)] for DM decreased, whereas that for ADF increased as ADF intake increased. Prediction equations were the following: milk fat yield, g/d = 115.78 - .128 x ADF intake, g/d + .00021 X (ADF intake)2 (r = .55); total chewing time, min/d = 345.33 + .32 x ADF intake, g/d (r = .60). In Experiment 2, 20 does were used in a completely randomized design. Apparent digestibilities of DM and energy decreased as dietary ADF intake increased. Rumen turnover rate and transit time of liquid were affected by ADF intake. Transit time of hay decreased as ADF intake increased. Intake of ADF affected pH and ammonia, acetate, and butyrate concentrations in the rumen. Acetate to propionate ration increased with ADF intake. No apparent trends were observed in whole blood beta-hydroxybutyrate or in plasma NEFA concentrations related to ADF intake. It appeared that DMI and milk fat yield leveled at 22% ADF or 43% NDF. For lactating dairy goats producing more than 3.5 kg/d of milk, calculated fat output reached a plateau when they consumed 587 g/d of ADF and spent 512 min/d chewing.

3-Hydroxybutyric Acid↗
PubMed1992

Influence of dietary protein on performance of dairy goats during pregnancy.

An experiment was conducted utilizing 44 Alpine does (48 kg of BW) to examine the effects of dietary protein intake on nutritional status and performance of does during the latter half of pregnancy and parturition. All dry does were bred, confirmed pregnant, and fed a diet containing 9% CP and 2.04 Mcal of metabolizable energy/kg of DM through wk 11 of gestation. On wk 12, does were blocked by BW and assigned to one of three dietary treatments: 8.8% (low) CP, 11.0% (medium) CP, and 14.3% (high) CP. All diets were isocaloric (2.04 Mcal of metabolizable energy/kg of DM) and fed for ad libitum intake from wk 12 of gestation until parturition. Average CP intakes for low, medium, and high protein were 164, 220, and 303 g/d, respectively. Body weight gains increased with increasing CP intake. Kidding rates were 79, 100, and 93% for the low, medium, and high protein groups. Gestation length, litter size, and litter weight did not differ among treatment groups. Urea N in jugular blood plasma increased with increasing CP intake. Plasma glucose and whole blood beta-hydroxybutyrate tended to increase with increasing CP intake. Plasma glucose decreased 7, 6, and 3%, and NEFA increased 97, 400, and 187% between wk 13 and 21 of gestation for the low, medium, and high protein diets, respectively. The data suggest that the low protein diet may be inadequate to meet the does' protein requirement during late gestation, but there was no apparent advantage in feeding the high rather than medium protein diet. Intake of CP for the medium protein diet, 9.8% g/kg of BW.75, was 40% higher than that recommended by the NRC for pregnant goats.

Animal Nutritional Physiological Phenomena↗
PubMed1992

Production performance and physiological responses of Angora goat kids fed acidified milk replacer.

Angora kids were blocked by birth weight and sex and assigned randomly to goat milk or acidified milk replacer. Daily milk intake, weekly BW, and heart girth measurements, and blood parameters (packed cell volume, total protein, glucose, and NEFA) were monitored at 3 d (initial) and at 4, 6, 8, and 9 wk of age. Both groups were fed their respective milks for ad libitum intake for 6 wk and then reduced to 75, 50, 25, and 0% of wk-6 intake during wk 7, 8, 9, and 10, respectively. Solid feed (20% CP and 3.1. Mcal of metabolizable energy/kg of DM) was provided for ad libitum intake starting on wk 3. Pretreatment BW (average 2.4 kg) and blood parameters were similar for milk and replacer groups. Packed cell volume (21.8 and 34.2%), total protein (50.3 and 46.6 g/L), and NEFA (.52 and .69 meq/L) for goat milk and acidified milk replacer groups, respectively, were affected by dietary treatment. Final BW (average 10.5 kg) and mean plasma glucose concentration (84 to 88 mg/dl) were similar between treatments; however, kids fed goat milk produced more mohair (13.8%) than those fed acidified milk replacer. Despite physiological differences, acidified milk replacer can be used successfully to raise Angora kids.

Animal Feed↗
PubMed1992

Developmental stages of the vomeronasal organ in the rat: a light and electron microscopic study.

This paper describes the development of the rat vomeronasal organ from the stage of anlage until adulthood. Groups of four rats were sacrificed daily from prenatal day 13 (E13) until birth; at days 2, 4, 7, 10, 14 and 16 after birth; weekly from day P21 to P42 plus an additional group of adults. The vomeronasal organs were processed for light microscopy, including alcian blue-PAS and NADH-diaphorase reactions, and also for electron microscopy. For summarizing our results we propose the following developmental stages: 1. Anlage (E13). 2. Early morphogenesis (E14-16). 3. Late morphogenesis (E17 to birth). 4. Initiation of secretory activity (First postnatal week). 5. Cytoarchitectural maturity (2nd postnatal week). 6. Complete maturity (From 3rd postnatal week onwards). Our results on the maturation of the histological structure and the histochemical reactions, indicate that there may be some functional activity at birth but the development of the organ still continues during the first three postnatal weeks to acquire its full functional capability.

Animals↗
PubMed1991

Is swelling of the secretory granule matrix the force that dilates the exocytotic fusion pore?

The swelling of the secretory granule matrix which follows fusion has been proposed as the driving force for the rapid expansion of the fusion pore necessary for exocytosis. To test this hypothesis, we have combined simultaneous measurements of secretory granule swelling using videomicroscopy with patch clamp measurements of the time course of the exocytotic fusion pore in mast cells from the beige mouse. We show that isotonic acidic histamine solutions are able to inhibit swelling of the secretory granule matrix both in purified secretory granules lysed by electroporation and in intact cells stimulated to exocytose by guanine nucleotides. In contrast to the inhibitory effects on granule swelling, the rate of expansion of the exocytotic fusion pore is unaffected. Therefore, as the rate of granule swelling was more than 20 times slower under these conditions, swelling of the secretory granule matrix due to water entry through the fusion pore cannot be the force responsible for the characteristic rapid expansion of the exocytotic fusion pore. We suggest that tension in the secretory granule membrane, which has recently been demonstrated in fused secretory granules, might be the force that drives the irreversible expansion of the fusion pore.

Animals↗
PubMed1991

Reversible condensation of mast cell secretory products in vitro.

We have investigated the mechanisms responsible for the condensation and decondensation of secretory products that occur in mast cell secretion. We show here that the hydrated matrix of an exocytosed secretory granule can be recondensed to its original volume by exposure to acidic solutions containing histamine at concentrations that mimic those found in vivo. Recondensation by acidic histamine began in the range of 1-10 mM with a dose response curve that was accurately predicted by a Hill type equation with four highly cooperative binding sites and a half maximum concentration of [Hi++] = 3.9 mM. Recondensation by histamine showed a sigmoidal dependency on pH (critical range pH 5.5-6.5) and was fully reversible. These experiments suggest that histamine, possibly by binding to anionic sites in the protein-heparin complex of the granule matrix, triggers a change in the polymeric structures of the granule matrix from an extended coil to a collapsed globular state. This may be a useful model for understanding the condensation of secretory products into dense core granules and their subsequent decondensation upon exocytosis.

Animals↗
PubMed1990

Tension in secretory granule membranes causes extensive membrane transfer through the exocytotic fusion pore.

For fusion to occur the repulsive forces between two interacting phospholipid bilayers must be reduced. In model systems, this can be achieved by increasing the surface tension of at least one of the membranes. However, there has so far been no evidence that the secretory granule membrane is under tension. We have been studying exocytosis by using the patch-clamp technique to measure the surface area of the plasma membrane of degranulating mast cells. When a secretory granule fuses with the plasma membrane there is a step increase in the cell surface area. Some fusion events are reversible, in which case we have found that the backstep is larger than the initial step, indicating that there is a net decrease in the area of the plasma membrane. The decrease has the following properties: (i) the magnitude is strongly dependent on the lifetime of the fusion event and can be extensive, representing as much as 40% of the initial granule surface area; (ii) the rate of decrease is independent of granule size; and (iii) the decrease is not dependent on swelling of the secretory granule matrix. We conclude that the granule membrane is under tension and that this tension causes a net transfer of membrane from the plasma membrane to the secretory granule, while they are connected by the fusion pore. The high membrane tension in the secretory granule may be the critical stress necessary for bringing about exocytotic fusion.

Animals↗
PubMed1990

Patch-clamp measurements reveal multimodal distribution of granule sizes in rat mast cells.

Using patch-clamp techniques, we have followed the attributes of the secretory granules of peritoneal mast cells obtained from rats of different ages. The granule attributes were determined by following the step increases in the cell surface membrane area caused by the exocytosis of the granules in GTP gamma S stimulated mast cells. Our data show that the amount of granule membrane available for exocytosis depends exponentially on the weight (age) of the donor rat, reaching a maximum at approximately 300 g. The data are consistent with an exponential growth in the number of granules contained by mast cells of maturing animals. Histograms of the sizes of the step increases in surface area caused by exocytosis of the granules showed at least four equally spaced peaks of similar variance where the position of the first peak and the spacing between peaks averaged 1.3 +/- 0.4 micron2. In all cells recorded, no more than seven peaks could be found, the higher order peaks having a lower probability of occurrence. The distribution of granule sizes did not change measurably between young and adult animals. This study suggests that at least two separate steps may determine the size of a secretory granule: granule to granule fusion that may account for the subunit composition of granule sizes and traffic of microvesicles through the maturing granules that may account for the variance observed in the granule sizes. This study also demonstrates a novel way to study granulo-genesis in living cells.

Aging↗
PubMed1990

Compound versus multigranular exocytosis in peritoneal mast cells.

We have used the whole-cell patch-pipette technique to measure the step increases in the cell membrane capacitance (equivalent to the membrane area) caused by the fusion of secretory granules in degranulating murine mast cells. We have observed that up to 30% of the total membrane expansion caused by degranulation results from large fusion events that cannot be explained by the fusion of single secretory granules. These large events are observed mainly in the initial phase of a degranulation. We have developed a simple mathematical model for a mast cell to test whether these large events are caused by a stimulus-induced, granule-to-granule fusion that occurs before their exocytosis (multigranular exocytosis). Our results suggest that the large fusion events are caused by the exocytosis of granule aggregates that existed before stimulation and that are located at the cell's periphery. We propose a novel mechanism by which granule aggregates can be formed at the periphery of the cell. This mechanism relies on the ability of a transiently fused granule ("flicker") to fuse with more internally located granules in a sequential manner. This pattern may result in the formation of larger peripheral granules that later on can fuse with the membrane. The formation of peripheral granule aggregates may potentiate a subsequent secretory response.

Animals↗
PubMed1990

Subclinical ammonia toxicity in steers: effects on hepatic and portal-drained visceral flux of metabolites and regulatory hormones.

Four calves (avg wt 161 kg) were surgically fitted with indwelling catheters in the femoral artery and femoral, portal, hepatic and mesenteric veins to study the effects of subclinical ammonia toxicity on portal-drained viscera (PDV) and hepatic (HEP) net flux of key metabolites and pancreatic hormones. Hyperammonemia was induced via administration of ammonium chloride (NH4Cl; 12 mumol.kg BW-1.min-1) via the femoral vein catheter for 240 min; infusions were preceded (PRE) and followed (POST) by 60- and 180-min control periods, respectively. Blood samples were obtained from the arterial catheters, and portal and hepatic vein catheters. Net flux rates were calculated by multiplying venoarterial differences by blood flow. Arterial plasma ammonia N peaked (P less than .01) at 327 micrograms/dl; hepatic ammonia extraction increased (P less than .01) from 10 to 23% during NH4Cl infusion. Arterial plasma glucose concentrations increased (P less than .05) during NH4Cl infusion (90.5 vs 82.6 mg/dl) concomitant with trends toward a reduction in net HEP glucose output. Portal-drained visceral release of insulin did not increase (P greater than .10) during NH4Cl infusion despite the steady rise in circulating glucose concentration; however, cessation of NH4Cl infusion resulted in a 109% increase (P less than .05) in PDV insulin release at +60 min POST. Plasma L-lactate, nonesterified fatty acids, urea N and glucagon concentrations and net fluxes were variable throughout the experiment. Results tend to indicate that hyperammonemia reduced hepatic glucose output and glucose-mediated pancreatic insulin release.

Ammonia↗