Biomedical subjects

J M Fernandez

Publications and source records attributed to J M Fernandez.

At least 55 records · Page 3Linked to original sources

PubMed2000

Stretching single molecules into novel conformations using the atomic force microscope.

A dense network of interconnected proteins and carbohydrates forms the complex mechanical scaffold of living tissues. The recently developed technique of single molecule force spectroscopy using the atomic force microscope (AFM) has enabled a detailed analysis of the force-induced conformations of these molecules and the determinants of their mechanical stability. These studies provide some of the basic knowledge required to understand the mechanical interactions that define all biological organisms.

Animals↗

PubMed2000

Point mutations alter the mechanical stability of immunoglobulin modules.

Immunoglobulin-like modules are common components of proteins that play mechanical roles in cells such as muscle elasticity and cell adhesion. Mutations in these proteins may affect their mechanical stability and thus may compromise their function. Using single molecule atomic force microscopy (AFM) and protein engineering, we demonstrate that point mutations in two beta-strands of an immunoglobulin module in human cardiac titin alter the mechanical stability of the protein, resulting in mechanical phenotypes. Our results demonstrate a previously unrecognized class of phenotypes that may be common in cell adhesion and muscle proteins.

Amino Acid Substitution↗

PubMed2000

Effects of dietary inclusion of chromium propionate and calcium propionate on glucose disposal and gastrointestinal development in dairy calves.

In experiment 1, 21 male Holstein calves (43.9 kg) were fed only milk replacer at 1.4% of their body weight as dry matter for 6 wk. Dietary treatments included a commercial milk replacer (22% protein, 15% fat) containing (dry basis) either 6.4% Ca propionate or 6.4% dextrose (control) and either 0 or 0.5 mg/kg of supplemental Cr as Cr propionate. Neither Cr nor Ca propionate affected body weight gain; however, Ca propionate tended to increase the growth of the entire foregut measured after slaughter at 6 wk of age. A Minimal Model glucose tolerance test indicated that insulin sensitivity was not affected by treatment. However, calves fed Cr had higher glucose disappearance indexes than controls when propionate was not fed (0.013 vs. 0.019 units) but similar clearance when propionate was included (0.018 vs. 0.018 units, Cr x P interaction). The area under the glucose response curves after propionate-loading tests was much greater for calves fed the Cr versus control replacer when propionate was not present; however, when propionate was included, the response was less dramatic. In experiment 2, 25 Holstein calves were used to study performance and metabolic responses when milk replacer, and then postweaning starter, were supplemented with 0.5 mg/kg of Cr as Cr propionate. The metabolic responses of these calves were not affected by treatment. Overall, combined data suggested that supplemental Cr may improve glucose effectiveness; however, these responses seemed to be attenuated by supplemental propionate.

Animal Feed↗

PubMed1999

Mechanical unfolding intermediates in titin modules.

The modular protein titin, which is responsible for the passive elasticity of muscle, is subjected to stretching forces. Previous work on the experimental elongation of single titin molecules has suggested that force causes consecutive unfolding of each domain in an all-or-none fashion. To avoid problems associated with the heterogeneity of the modular, naturally occurring titin, we engineered single proteins to have multiple copies of single immunoglobulin domains of human cardiac titin. Here we report the elongation of these molecules using the atomic force microscope. We find an abrupt extension of each domain by approximately 7 A before the first unfolding event. This fast initial extension before a full unfolding event produces a reversible 'unfolding intermediate' Steered molecular dynamics simulations show that the rupture of a pair of hydrogen bonds near the amino terminus of the protein domain causes an extension of about 6 A, which is in good agreement with our observations. Disruption of these hydrogen bonds by site-directed mutagenesis eliminates the unfolding intermediate. The unfolding intermediate extends titin domains by approximately 15% of their slack length, and is therefore likely to be an important previously unrecognized component of titin elasticity.

Biomechanical Phenomena↗

PubMed1999

The micro-mechanics of single molecules studied with atomic force microscopy.

The atomic force microscope (AFM) in its force-measuring mode is capable of effecting displacements on an angstrom scale (10 A = 1 nm) and measuring forces of a few piconewtons. Recent experiments have applied AFM techniques to study the mechanical properties of single biological polymers. These properties contribute to the function of many proteins exposed to mechanical strain, including components of the extracellular matrix (ECM). The force-bearing proteins of the ECM typically contain multiple tandem repeats of independently folded domains, a common feature of proteins with structural and mechanical roles. Polysaccharide moieties of adhesion glycoproteins such as the selectins are also subject to strain. Force-induced extension of both types of molecules with the AFM results in conformational changes that could contribute to their mechanical function. The force-extension curve for amylose exhibits a transition in elasticity caused by the conversion of its glucopyranose rings from the chair to the boat conformation. Extension of multi-domain proteins causes sequential unraveling of domains, resulting in a force-extension curve displaying a saw tooth pattern of peaks. The engineering of multimeric proteins consisting of repeats of identical domains has allowed detailed analysis of the mechanical properties of single protein domains. Repetitive extension and relaxation has enabled direct measurement of rates of domain unfolding and refolding. The combination of site-directed mutagenesis with AFM can be used to elucidate the amino acid sequences that determine mechanical stability. The AFM thus offers a novel way to explore the mechanical functions of proteins and will be a useful tool for studying the micro-mechanics of exocytosis.

Animals↗

PubMed1999

Atomic force microscopy captures length phenotypes in single proteins.

We use single-protein atomic force microscopy techniques to detect length phenotypes in an Ig module. To gain amino acid resolution, we amplify the mechanical features of a single module by engineering polyproteins composed of up to 12 identical repeats. We show that on mechanical unfolding, mutant polyproteins containing five extra glycine residues added to the folded core of the module extend 20 A per module farther than the wild-type polyproteins. By contrast, similar insertions near the N or C termini have no effect. Hence, our atomic force microscopy measurements readily discriminate the location of the insert and measure its size with a resolution similar to that of NMR and x-ray crystallography.

Connectin↗

PubMed1999

Pulsed laser imaging of Ca(2+) influx in a neuroendocrine terminal.

The surge of Ca(2+) that triggers vesicle fusion is shaped by the distribution of Ca(2+) channels and the physical relationship between those channels and the exocytotic apparatus. Although channels and the release apparatus are thought to be tightly associated at fast synapses, the arrangement at neuroendocrine cells is less clear. The distribution of Ca(2+) influx near release sites is difficult to determine because of spatial and temporal limitations on Ca(2+) imaging techniques. We now present spatially resolved images of Ca(2+) influx into rat neuroendocrine terminals on a millisecond time scale. Images of voltage-dependent Ca(2+) influx into neurohypophysial terminals were captured after excitation of Ca(2+)-sensitive dyes with pulses of laser light lasting a fraction of a microsecond. Submembranous Ca(2+) increases were detected during the first millisecond of an evoked Ca(2+) tail current. Steep gradients of Ca(2+) were evident, with concentrations near the membrane reaching above 1 microM during a 30 msec depolarization. Ca(2+) influx appeared evenly distributed, even when diffusion was restricted with an exogenous Ca(2+) chelator. During longer depolarizations, mean and peak Ca(2+) concentrations reached an asymptote in parallel, suggesting that Ca(2+) binding proteins near the membrane rapidly buffer Ca(2+) and do not become saturated during prolonged influx. These data support the hypothesis that exocytosis is activated in these terminals by the summation of influx through multiple, randomly spaced Ca(2+) channels.

Animals↗

PubMed1999

Atomic levers control pyranose ring conformations.

Atomic force microscope manipulations of single polysaccharide molecules have recently expanded conformational chemistry to include force-driven transitions between the chair and boat conformers of the pyranose ring structure. We now expand these observations to include chair inversion, a common phenomenon in the conformational chemistry of six-membered ring molecules. We demonstrate that by stretching single pectin molecules (1 --> 4-linked alpha-D-galactouronic acid polymer), we could change the pyranose ring conformation from a chair to a boat and then to an inverted chair in a clearly resolved two-step conversion: 4C1 right arrow over left arrow boat right arrow over left arrow 1C4. The two-step extension of the distance between the glycosidic oxygen atoms O1 and O4 determined by atomic force microscope manipulations is corroborated by ab initio calculations of the increase in length of the residue vector O1O4 on chair inversion. We postulate that this conformational change results from the torque generated by the glycosidic bonds when a force is applied to the pectin molecule. Hence, the glycosidic bonds act as mechanical levers, driving the conformational transitions of the pyranose ring. When the glycosidic bonds are equatorial (e), the torque is zero, causing no conformational change. However, when the glycosidic bond is axial (a), torque is generated, causing a rotation around C---C bonds and a conformational change. This hypothesis readily predicts the number of transitions observed in pyranose monomers with 1a-4a linkages (two), 1a-4e (one), and 1e-4e (none). Our results demonstrate single-molecule mechanochemistry with the capability of resolving complex conformational transitions.

Carbohydrate Conformation↗

PubMed1999

Mechanical and chemical unfolding of a single protein: a comparison.

Is the mechanical unraveling of protein domains by atomic force microscopy (AFM) just a technological feat or a true measurement of their unfolding? By engineering a protein made of tandem repeats of identical Ig modules, we were able to get explicit AFM data on the unfolding rate of a single protein domain that can be accurately extrapolated to zero force. We compare this with chemical unfolding rates for untethered modules extrapolated to 0 M denaturant. The unfolding rates obtained by the two methods are the same. Furthermore, the transition state for unfolding appears at the same position on the folding pathway when assessed by either method. These results indicate that mechanical unfolding of a single protein by AFM does indeed reflect the same event that is observed in traditional unfolding experiments. The way is now open for the extensive use of AFM to measure folding reactions at the single-molecule level. Single-molecule AFM recordings have the added advantage that they define the reaction coordinate and expose rare unfolding events that cannot be observed in the absence of chemical denaturants.

Calmodulin-Binding Proteins↗

PubMed1999

The study of protein mechanics with the atomic force microscope.

The unfolding and folding of single protein molecules can be studied with an atomic force microscope (AFM). Many proteins with mechanical functions contain multiple, individually folded domains with similar structures. Protein engineering techniques have enabled the construction and expression of recombinant proteins that contain multiple copies of identical domains. Thus, the AFM in combination with protein engineering has enabled the kinetic analysis of the force-induced unfolding and refolding of individual domains as well as the study of the determinants of mechanical stability.

Entropy↗

PubMed1999

Single protein misfolding events captured by atomic force microscopy.

Using single protein atomic force microscopy (AFM) techniques we demonstrate that after repeated mechanical extension/relaxation cycles, tandem modular proteins can misfold into a structure formed by two neighboring modules. The misfolding is fully reversible and alters the mechanical topology of the modules while it is about as stable as the original fold. Our results show that modular proteins can assume a novel misfolded state and demonstrate that AFM is able to capture, in real time, rare misfolding events at the level of a single protein.

Connectin↗

PubMed1999

The effect of bovine somatotropin treatment on production of lactating angora does with kids.

Fourteen Angora does (35+/-2 kg), each with a single kid and in the first month of lactation, were used to determine ongoing (Period 1) and residual (Period 2) effects of chronic bovine somatotropin (bST) treatment. Specifically, we sought to determine whether chronic bST treatment was capable of improving milk yield, and thus kid growth, and mohair production of nursing does. The experiment consisted of a 2-wk pretreatment period, 5 wk of weekly subcutaneous treatment of slow-release bST (n = 7; Period 1), and a 4-wk posttreatment period (Period 2). The weekly dose of bST was calculated to release 100 microg/(kg BW.d(-1)). To estimate milk production, kids were separated from the does daily for 5 h, and their BW was recorded before and after suckling. The difference in BW was taken as milk production for 5 h. Fiber growth was measured by shearing does at the start of the experiment and at the end of Periods 1 and 2. Dry matter intake and BW of does were not affected by bST (P>.05). Average daily gain of kids that were suckling bST-treated does was higher (P<.05) than for kids of untreated does during Period 1 (184 vs. 139 g/d) but not during Period 2 (140 vs. 136 g/d; P>.10). Treatment with bST did not affect (P>.10) milk composition or clean fleece production in either period. Injection of bST did not affect (P>.10) plasma concentrations of glucose (mean = 49.5 mg/dL), urea N (mean = 19 mg/dL), total protein (mean = 72.5 g/d), or NEFA (mean = 122 microEq/L). During the period of bST treatment, plasma concentrations of somatotropin and IGF-I were increased (P<.05), concentrations of thyroxine and cortisol were decreased (P<.10), and plasma insulin levels were unchanged (P>.10) by bST. In conclusion, treatment of Angora dams with bST did not change DMI or mohair growth, but it improved growth of their kids.

Animals↗

PubMed1999

Dietary protein and chromium tripicolinate in Suffolk wether lambs: effects on production characteristics, metabolic and hormonal responses, and immune status.

Thirty-two Suffolk wether lambs were fed for 84 d in a 2 x 2 factorial experiment using two levels of dietary protein (9.0 to 12.1% CP, low protein, LP; or 12.8 to 14.4% CP, high protein, HP) and supplemental Cr (none, C; or 400 ppb Cr as chromium tripicolinate, Cr). At 14- to 21-d intervals, lambs were weighed, and jugular blood samples were collected. Mean ADG and carcass weight (P > .10) did not differ. In lambs fed HP, Cr reduced liver weight and increased kidney weight (P < .01). Lambs fed HP had elevated plasma urea N (PUN; P < .01) and albumin (P < .04). During an i.v. epinephrine challenge on d 43, plasma cortisol declined in lambs fed Cr (Cr x time, P < .03) and in lambs fed LP (CP x time, P < .001). An i.v. glucose tolerance test conducted 3 h later showed that supplemental Cr decreased glucose clearance rate in lambs fed HP (CP x Cr, P < .10) but not in lambs fed LP. On d 62, PUN was increased in lambs fed HP (P < .001) between 0 and 3 h postprandial, and there was a Cr x CP interaction (P < .04). Postprandial plasma NEFA declined with Cr vs C (Cr x time, P < .07) and with HP vs LP (CP x time, P < .10). By d 66, lambs fed Cr had an elevated (P < .03) blood platelet and fibrinogen content. Chromium increased erythrocyte count in lambs fed HP (Cr x CP, P < .08), and isolated peripheral lymphocytes had greater blastogenic response to 4 microg/mL of phytohemagglutinin (Cr x CP, P < .001). The lymphocyte response to pokeweed mitogen (.2 microg/mL) was reduced in lambs fed Cr (P < .10). In the present experiment, Cr supplementation had minimal and inconsistent effects on production and metabolic criteria of lambs.

Animals↗

PubMed1999

Effects of dietary fiber on intake, milk yield, and digestion by lactating dairy cows during cool or hot, humid weather.

Lactating cows were offered diets with increasing neutral detergent fiber concentrations to determine the effects on intake, milk yield and composition, blood hormones, and nutrient digestion during cool or hot weather conditions. Tifton 85 bermudagrass hay was substituted for corn silage so that the forage portion of diets were: 1) 40% corn silage (control), 2) 32.4% corn silage, 7.6% bermudagrass, 3) 24.8% corn silage, 15.2% bermudagrass, or 4) 17.2% corn silage, 22.8% bermudagrass (dry basis). Dietary neutral detergent fiber concentrations (% dry matter) were 30.2, 33.8, 37.7, and 42.0, respectively. Intake of dry matter declined with increasing dietary neutral detergent fiber during cool and hot periods, but intake adjusted for cool weather treatment differences did not change further during hot weather. Milk yield declined linearly with increasing neutral detergent fiber during cool weather and changed quadratically during hot weather. Milk temperature declined with increasing dietary neutral detergent fiber for the p.m. milking during the cool period and declined with increasing dietary neutral detergent fiber for the a.m. and p.m. milkings during the hot period. Digestibility of neutral detergent fiber improved and ruminal turnover of particulate digesta was increased with greater dietary neutral detergent fiber content. No dietary fiber level by hot weather interaction was observed, suggesting that total energy intake may have and a greater effect on milk yield than dietary fiber content during hot, humid weather.

Animals↗

PubMed1999

Crude protein and rumen undergradable protein effects on reproduction and lactation performance of Holstein cows.

We conducted a study to determine the effects of excess dietary crude protein (CP) and rumen undegradable protein (RUP) on reproduction and lactation performance of Holstein cows. During each of three yearly replicates, cows were blocked by previous mature equivalent milk production and randomly assigned at calving (n = 47; partum group) or at 42 +/- 21 d postpartum (n = 134; postpartum group) to the following dietary treatments: 1) ryegrass pasture supplemented with a corn and soybean meal grain mix (high CP, moderate RUP); 2) ryegrass pasture mornings and corn silage evenings, supplemented with grain as in diet 1 (moderate CP, moderate RUP control diet), and 3) ryegrass pasture mornings and corn silage evenings, supplemented with a grain mix containing corn, soybean meal, corn gluten meal, and blood meal (moderate CP, high RUP). Dietary CP and RUP concentrations were approximately 23.1, 5.8; 17.7, 5.0; and 17.2, 6.8% of dry matter for diets 1 to 3, respectively. Plasma urea N concentrations were highest in cows fed diet 1 (25.0 mg/dl), intermediate in cows on diet 2 (20.1 mg/dl), and lowest in cows on diet 3 (18.5 mg/dl). Cows fed excess dietary protein (diet 1) exhibited lower first breeding pregnancy rates (24.1 vs. 41.0%) and lower overall pregnancy rates (53.4 vs. 75.4%) than did cows fed diet 2, increasing time nonpregnant by an average of 15.1 d per cow. Reproductive performance was similar between cows fed diets 2 and 3. Mean fat-corrected milk (FCM) yield was not affected by protein concentration (diet 1 vs. 2); however, partum group cows that received supplemental RUP (diet 3) produced more 3.5% FCM than controls in early lactation. Feeding grain diets that contained excess dietary protein impaired the reproductive performance of dairy cows grazing ryegrass.

Ammonia↗

PubMed1998

Polysaccharide elasticity governed by chair-boat transitions of the glucopyranose ring.

Many common, biologically important polysaccharides contain pyranose rings made of five carbon atoms and one oxygen atom. They occur in a variety of cellular structures, where they are often subjected to considerable tensile stress. The polysaccharides are thought to respond to this stress by elastic deformation, but the underlying molecular rearrangements allowing such a response remain poorly understood. It is typically assumed, however, that the pyranose ring structure is inelastic and locked into a chair-like conformation. Here we describe single-molecule force measurements on individual polysaccharides that identify the pyranose rings as the structural unit controlling the molecule's elasticity. In particular, we find that the enthalpic component of the polymer elasticity of amylose, dextran and pullulan is eliminated once their pyranose rings are cleaved. We interpret these observations as indicating that the elasticity of the three polysaccharides results from a force-induced elongation of the ring structure and a final transition from a chair-like to a boat-like conformation. We expect that the force-induced deformation of pyranose rings reported here plays an important role in accommodating mechanical stresses and modulating ligand binding in biological systems.

Amylose↗

PubMed1998

The molecular elasticity of the extracellular matrix protein tenascin.

Extracellular matrix proteins are thought to provide a rigid mechanical anchor that supports and guides migrating and rolling cells. Here we examine the mechanical properties of the extracellular matrix protein tenascin by using atomic-force-microscopy techniques. Our results indicate that tenascin is an elastic protein. Single molecules of tenascin could be stretched to several times their resting length. Force-extension curves showed a saw-tooth pattern, with peaks of force at 137pN. These peaks were approximately 25 nm apart. Similar results have been obtained by study of titin. We also found similar results by studying recombinant tenascin fragments encompassing the 15 fibronectin type III domains of tenascin. This indicates that the extensibility of tenascin may be due to the stretch-induced unfolding of its fibronectin type III domains. Refolding of tenascin after stretching, observed when the force was reduced to near zero, showed a double-exponential recovery with time constants of 42 domains refolded per second and 0.5 domains per second. The former speed of refolding is more than twice as fast as any previously reported speed of refolding of a fibronectin type III domain. We suggest that the extensibility of the modular fibronectin type III region may be important in allowing tenascin-ligand bonds to persist over long extensions. These properties of fibronectin type III modules may be of widespread use in extracellular proteins containing such domain.

Alternative Splicing↗

PubMed1998

Influence of intravenous L-carnitine administration in sheep preceding an oral urea drench.

Two experiments were conducted to investigate the effect of i.v. administration of L-carnitine on selected metabolites in sheep and to determine the feasibility of using L-carnitine to ameliorate the deleterious effects of hyperammonemia in sheep. In Exp. 1, i.v. L-carnitine solutions were administered at three levels in a replicated Latin square: 0 (CONT), 6.36 (CAR 1), and 12.72 (CAR 2) mmol L-carnitine/kg x (75) BW using Suffolk ewes (n = 6; average BW 75+/-3 kg). Plasma L-carnitine concentration was increased (P<.05) by treatment (51.9 vs 102.3, and 96.4 micromol/L in CONT, CAR 1, and CAR 2, respectively). Plasma glucose concentration was elevated (P<.05) in CAR 2 and CAR 1. Plasma NEFA concentration was highest (P<.05) in CAR 2. Area under the response curve for glucose was greater (P<.02) in CAR 2. In Exp. 2, Suffolk ewes (n = 16; average BW 48+/-2 kg) were used in a randomized complete block design with a 2x2 factorial treatment arrangement to determine the effects of i.v. L-carnitine administration during an oral urea load test (OULT). L-Carnitine (0 and 6.36 mmol/kg x (75) BW) was administered i.v. at 30 min, and an oral urea drench (50% wt/vol; 0 and 300 mg/kg BW) was administered at 60 min. Plasma L-carnitine was increased (P<.0001) by i.v. L-carnitine. Plasma ammonia N was highest (P<.0001) in the UREA treatment compared with the CONT, CARN, and CARN + UREA treatments (148 vs 95, 101, and 108 micromol/L, respectively). Intravenous L-carnitine administration influenced plasma glucose and NEFA concentrations in sheep and, when administered 30 min preceding an OULT, prevented the development of subclinical hyperammonemia in sheep.

Administration, Oral↗