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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
PubMed2001

Detection of the 5'-cap structure of messenger RNAs with the use of the cap-jumping approach.

An effective procedure for specific determination of the cap structure at the 5'-terminus of mRNA and for isolation of the corresponding full-length cDNA has been developed. The procedure involves covalent attachment of an oligonucleotide template extender to the 5'-cap structure of mRNA followed by RT-PCR using M-MLV SuperScript II reverse transcriptase. In the course of reverse transcription, the enzyme 'jumps over' the cap structure and includes the sequence complementary to the oligonucleotide template extender into the 3'-end of the first cDNA strand. The cap-jumping method was successfully tested using some mammalian cellular mRNAs, genomic RNAs of tobacco mosaic virus (TMV) U1 and the recently isolated crucifer-infecting tobamovirus. Moreover, cDNA products corresponding to the genomic tobamovirus RNA were obtained from total RNA extracted from tobacco plants infected by crucifer-infecting tobamovirus or tobacco mosaic virus. Using the cap-jumping method, we have shown for the first time that genomic crucifer-infecting tobamovirus (crTMV) RNA contains a 5'-cap structure. This improved method can be recommended for the construction of full-length and 5'-end enriched cDNA libraries, identification of capped RNAs and determination of their 5'-terminal sequences.

5' Untranslated Regions↗
PubMed2001

Single molecules.

Explore the source record for details and available documents.

Journal Article↗
PubMed2001

Multiple conformations of PEVK proteins detected by single-molecule techniques.

An important component of muscle elasticity is the PEVK region of titin, so named because of the preponderance of these amino acids. However, the PEVK region, similar to other elastomeric proteins, is thought to form a random coil and therefore its structure cannot be determined by standard techniques. Here we combine single-molecule electron microscopy and atomic force microscopy to examine the conformations of the human cardiac titin PEVK region. In contrast to a simple random coil, we have found that cardiac PEVK shows a wide range of elastic conformations with end-to-end distances ranging from 9 to 24 nm and persistence lengths from 0.4 to 2.5 nm. Individual PEVK molecules retained their distinctive elastic conformations through many stretch-relaxation cycles, consistent with the view that these PEVK conformers cannot be interconverted by force. The multiple elastic conformations of cardiac PEVK may result from varying degrees of proline isomerization. The single-molecule techniques demonstrated here may help elucidate the conformation of other proteins that lack a well-defined structure.

Connectin↗
PubMed2001

Autosomal dominant limb-girdle muscular dystrophy: a large kindred with evidence for anticipation.

BACKGROUND: Fourteen genetically distinct forms of limb-girdle muscular dystrophy (LGMD) have been identified, including five types of autosomal dominant LGMD (AD-LGMD). OBJECTIVE: To describe clinical, histologic, and genetic features of a large Spanish kindred with LGMD and apparent autosomal dominant inheritance spanning five generations. METHOD: The authors examined 61 members of the family; muscle biopsies were performed on five patients. Linkage analysis assessed chromosomal loci associated with other forms of AD-LGMD. RESULTS: A total of 32 individuals had weakness of the pelvic and shoulder girdles. Severity appeared to worsen in successive generations. Muscle biopsy findings were nonspecific and compatible with MD. Linkage analysis to chromosomes 5q31, 1q11-q21, 3p25, 6q23, and 7q demonstrated that this disease is not allelic to LGMD forms 1A, 1B, 1C, 1D, and 1E. CONCLUSIONS: This family has a genetically distinct form of AD-LGMD. The authors are currently performing a genome-wide scan to identify the disease locus.

Adolescent↗
PubMed2001

Stepwise unfolding of titin under force-clamp atomic force microscopy.

Here we demonstrate the implementation of a single-molecule force clamp adapted for use with an atomic force microscope. We show that under force-clamp conditions, an engineered titin protein elongates in steps because of the unfolding of its modules and that the waiting times to unfold are exponentially distributed. Force-clamp measurements directly measure the force dependence of the unfolding probability and readily captures the different mechanical stability of the I27 and I28 modules of human cardiac titin. Force-clamp spectroscopy promises to be a direct way to probe the mechanical stability of elastic proteins such as those found in muscle, the extracellular matrix, and cell adhesion.

Animals↗
PubMed2001

Unfolding of titin domains explains the viscoelastic behavior of skeletal myofibrils.

The elastic section of the giant muscle protein titin contains many immunoglobulin-like domains, which have been shown by single-molecule mechanical studies to unfold and refold upon stretch-release. Here we asked whether the mechanical properties of Ig domains and/or other titin regions could be responsible for the viscoelasticity of nonactivated skeletal-muscle sarcomeres, particularly for stress relaxation and force hysteresis. We show that isolated psoas myofibrils respond to a stretch-hold protocol with a characteristic force decay that becomes more pronounced following stretch to above 2.6-microm sarcomere length. The force decay was readily reproducible by a Monte Carlo simulation taking into account both the kinetics of Ig-domain unfolding and the worm-like-chain model of entropic elasticity used to describe titin's elastic behavior. The modeling indicated that the force decay is explainable by the unfolding of only a very small number of Ig domains per titin molecule. The simulation also predicted that a unique sequence in titin, the PEVK domain, may undergo minor structural changes during sarcomere extension. Myofibrils subjected to 1-Hz cycles of stretch-release exhibited distinct hysteresis that persisted during repetitive measurements. Quick stretch-release protocols, in which variable pauses were introduced after the release, revealed a two-exponential time course of hysteresis recovery. The rate constants of recovery compared well with the refolding rates of Ig-like or fibronectin-like domains measured by single-protein mechanical analysis. These findings suggest that in the sarcomere, titin's Ig-domain regions may act as entropic springs capable of adjusting their contour length in response to a stretch.

Amino Acid Sequence↗
PubMed2001

Fingerprinting polysaccharides with single-molecule atomic force microscopy.

We report the use of an atomic force microscopy (AFM)-based force spectroscopy technique to identify, at the single-molecule level, the components of mixtures of polysaccharides. Previously, we showed that the elasticity of certain types of polysaccharides is governed by force-induced conformational transitions of the pyranose ring. These transitions produce atomic fingerprints in the force-extension spectrum that are characteristic of the ground-energy conformation of the pyranose ring and the type of glycosidic linkages. Using this approach we find that commercially available agarose and lambda-carrageenan contain molecules that, when stretched in an atomic force microscope, produce a force spectrum characteristic of alpha-(1-->4) d-glucans. We have identified these molecules as amylopectin or floridean starch, a storage polysaccharide in algae. Our methodology can identify individual polysaccharide molecules in solution, which is not possible by any other spectroscopic technique, and therefore is an important addition to the arsenal of analytical techniques used in carbohydrate research.

Amylopectin↗
PubMed2001

Effect of perezone, aminoperezone and their corresponding isomers isoperezone and isoaminoperezone upon in vitro platelet aggregation.

The effect on platelet aggregation of perezone, isoperezone, aminoperezone and isoaminoperezone has been determined in human platelets, using adenosinediphosphate (ADP), epinephrine and collagen as inducers. Perezone inhibited ADP- epinephrine- and collagen-induced platelet aggregation; isoperezone, aminoperezone and isoaminoperezone did not. The difference in biological responses could be the result of their structures. While isoperezone, aminoperezone and isoaminoperezone have carbonyl groups in the vicinity of other functional groups, this is not the case for perezone, in which one of the carbonyl groups has an adjacent free position.

Adenosine Diphosphate↗
PubMed2001

Skin dose and dose-area product values for interventional cardiology procedures.

Coronary angiography and percutaneous transluminal coronary angioplasty procedures performed in four different facilities were monitored in the present study by measuring maximum skin dose, dose-area product and other operational parameters. Radiographic slow film, thermoluminescent dosemeters and transmission ion chambers were used to measure dose related quantities. Values of 107-711 mGy for maximum skin dose and 27.3-370.6 Gy cm2 for dose-area product were found, together with cumulative skin dose estimates of 110-3706 mGy. A discussion of the relationship of measured dose-area product and skin dose values is made using a field concentration factor defined as a way to interpret the findings. No general correlation was observed between dose-area product and maximum skin dose. Cumulative skin dose estimates throughout a procedure should be discarded as a realistic method for assessing deterministic risk in cardiology procedures. Slow film in addition to thermoluminescent dosemeters for measurement of maximum skin dose is a good alternative, especially for complex interventional procedures. For repeated procedures, combining film and dose-area product monitoring favours optimization of radiation protection for the patient.

Angioplasty, Balloon, Coronary↗
PubMed2001

Effect of chromium picolinate and chromium propionate on glucose and insulin kinetics of growing barrows and on growth and carcass traits of growing-finishing barrows.

Two experiments were conducted to determine the effects of dietary Cr tripicolinate (CrPic) or Cr propionate (CrProp) on growth, carcass traits, plasma metabolites, glucose tolerance, and insulin sensitivity in pigs. In Exp. 1, 36 barrows (12 per treatment; initial and final BW were 20 and 38 kg) were allotted to the following treatments: 1) corn-soybean meal basal diet (control), 2) as 1 + 200 ppb Cr as CrPic, or 3) as 1 + 200 ppb Cr as CrProp. Growth performance data were collected for 28 d, and then 23 pigs (seven, eight, and eight pigs for treatments 1, 2, and 3, respectively) were fitted with jugular catheters and a glucose tolerance test (500 mg glucose/kg BW) and an insulin challenge test (0.1 IU of porcine insulin/kg BW) were conducted. Both CrPic and CrProp decreased (P < 0.05) ADG and ADFI but did not affect gain:feed (P > 0.10). Fasting plasma glucose, total cholesterol, urea N, insulin, and high-density lipoprotein cholesterol:total cholesterol concentrations were not affected (P > 0.10) by either Cr source. Pigs fed CrPic had lower (P < 0.02) fasting plasma NEFA concentrations than control pigs, but plasma NEFA concentrations of pigs fed CrProp were not affected (P > 0.10). During the glucose tolerance test, glucose and insulin kinetics were not affected by treatment (P > 0.10). During the insulin challenge test, glucose clearance was increased (P < 0.01) in pigs fed CrProp but not affected (P > 0.10) in pigs fed CrPic. Glucose half-life was decreased (P < 0.03) in pigs fed CrPic or CrProp, but insulin kinetics were not affected (P > 0.10). In Exp. 2, 48 barrows (four replicates of four pigs per replicate; initial and final BW were 23 and 115 kg) were allotted to the same dietary treatments in a growing-finishing study. Average daily gain, ADFI, and gain:feed were not affected (P > 0.10) by treatments. Carcass length tended (P = 0.10) to be greater in pigs fed CrPic than in pigs fed CrProp, but other carcass measurements were not affected (P > 0.10). Glucose kinetics from the insulin challenge test indicate that both CrPic and CrProp increase insulin sensitivity and that both Cr sources are bioavailable.

Animals↗
PubMed2001

Supplemental dietary protein for grazing dairy cows: effect on pasture intake and lactation performance.

One hundred twenty-four cows (92 multiparous and 32 primiparous) were used to evaluate the effect of grain supplements containing high crude protein [(22.8% CP, 5.3% rumen undegradable protein (RUP), dry matter basis], moderate CP (16.6% CP, 6.1% RUP), and moderate CP with supplemental RUP (16.2% CP, 10.8% RUP) on lactation performance of Holstein cows rotationally grazing annual ryegrass-oat pastures. Supplemental protein was provided by solvent extracted soybean meal in the high CP and moderate CP supplements and as a corn gluten meal-blood meal mixture (2.8:1) in the moderate CP, high RUP supplement. Cows were blocked according to previous mature milk equivalent production and calving date (partum group; 0 d in milk or postpartum group; 21 to 65 d in milk) and randomly assigned to dietary treatments. Grain was individually fed, at approximately a 1:3 grain to milk ratio, before a.m. and p.m milkings. The study was replicated during two grazing seasons that averaged 199 d. Cows had ad libitum access to bermudagrass hay while on pasture (dry matter intake = 1.3 kg/d). Protein supplementation had no effect on study long pasture dry matter (12.7 +/- 1.0 kg/d) or total dry matter (23.9 +/- 1.2 kg/d) consumption. Protein concentration did not affect actual milk yield of either calving group (high CP vs. moderate CP); however, postpartum group cows receiving high CP grain supplements maintained greater milk fat concentrations (3.34 vs. 3.11%), which led to higher fat-corrected milk (FCM) yields than control cows receiving moderate CP grain diets (30.3 vs. 28.9 kg/d). Crude protein concentration in milk of high CP-supplemented, postpartum group cows was also higher than moderate CP cows (3.42 vs. 3.27%). Additional RUP did not increase FCM yield above that generated by moderate CP grain diets for partum (34.3 vs. 32.9 kg/d) or postpartum-group cows (28.9 vs. 28.2 kg/d). Increasing CP concentration of grain supplement did not affect milk yield of Holstein cows grazing immature winter annual pastures. Supplementing additional RUP was without benefit, indicating that in this study energy deprivation may have been the major nutritional constraint for high-producing dairy cows grazing lush pastures.

Animal Feed↗
PubMed2001

Supplemental dietary protein for grazing dairy cows: reproduction, condition loss, plasma metabolites, and insulin.

An experiment was conducted over a 2-yr period to investigate the influence of grain crude protein (CP) and rumen undegradable protein (RUP) concentration on reproduction and energy status of dairy cows grazing annual ryegrass (Lolium multiflorum) and oats (Avena sativa). Holstein cows (n = 122) were blocked by calving group [partum (0 d postpartum) vs. postpartum (41 +/- 19 d postpartum at study initiation)] and assigned to grain supplements containing high CP [22.8% of dry matter (DM)], moderate CP (16.6%), or moderate CP (16.2%)] supplemented with RUP from blood meal and corn gluten meal. Postpartum condition loss was greater and first-service pregnancy rate was lower for partum-group cows receiving high CP grain supplements compared with control cows receiving moderate CP supplements. The RUP supplements reduced grain consumption, increased days to first estrus, and reduced first-service pregnancy rate of partum-group cows. The reproduction of postpartum group cows was unaffected by protein supplements. Plasma urea nitrogen was higher for cows fed high CP diets, but plasma ammonia nitrogen, glycated hemoglobin, nonesterified fatty acids, beta-hydoxybutyrate, glucose, and insulin concentrations were similar to cows fed moderate CP. Excess postpartum condition loss, coupled with inconsistent protein supplement effects on days to first service and first-service pregnancy rate, suggest that energy deprivation may have contributed to the low fertility experienced by grazing cows in this study.

Ammonia↗
PubMed2000

Intracellular Ca(2+) channel immunoreactivity in neuroendocrine axon terminals.

The concentration of neuroendocrine terminals in the neurohypophysis facilitates the identification and localization of Ca(2+) channel subtypes near neuroendocrine release sites. Immunoblots of rat neurohypophysial tissue identified the alpha(1)1.3, alpha(1)2.1, alpha(1)2.2, and alpha(1)2.3 Ca(2+) channel subunits. Immunofluorescence staining of axon terminal plasma membranes was weak, suggesting that Ca(2+) channels are dispersed. This contrasts with the highly punctate alpha(1)2.2 immunoreactivity in bovine chromaffin cells; the neurohypophysial terminals may therefore lack the specialized release zones found in those cells. Immunofluorescence and immunogold labeling identify dense core granule-like structures in the terminal cytoplasm containing multiple Ca(2+) channel types. Ca(2+) channels in internal membranes may play an important role in channel targeting and distribution in neuroendocrine cells.

Animals↗
PubMed2000

Atomic force microscopy reveals the mechanical design of a modular protein.

Tandem modular proteins underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. The fundamental unit of elastic proteins is their individually folded modules. Here, we use protein engineering to construct multimodular proteins composed of Ig modules of different mechanical strength. We examine the mechanical properties of the resulting tandem modular proteins by using single protein atomic force microscopy. We show that by combining modules of known mechanical strength, we can generate proteins with novel elastic properties. Our experiments reveal the simple mechanical design of modular proteins and open the way for the engineering of elastic proteins with defined mechanical properties, which can be used in tissue and fiber engineering.

Biomechanical Phenomena↗
PubMed2000

Atomic force microscopy captures quantized plastic deformation in gold nanowires.

Scanning probe microscopy has become a powerful tool to detect structural changes in small clusters of atoms. Herein, we use an atomic force microscope to measure the length of gold nanowire structures during extension and compression cycles. We have found that nanowires elongate under force in quantized steps of up to three integer multiples of 1.76 A and that they shorten spontaneously in steps of 1.52 A. Our results can be explained by the sliding of crystal planes within the gold nanowires creating stacking faults that change the local structure from face-centered cubic to hexagonal close packed. Our data also show that there can be up to three simultaneous slip events, in good agreement with the tetrahedral arrangement of slip planes in a gold crystal. These experiments provide direct evidence for the mechanism underlying the plastic deformation of a nanowire. A similar approach can be used to examine the atomic events underlying the plastic failure of other metals and their alloys.

Journal Article↗
PubMed2000

Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering.

Mechanical unfolding and refolding may regulate the molecular elasticity of modular proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein engineering techniques allow the construction of homomeric polyproteins for the precise analysis of the mechanical unfolding of single domains. alpha-Helical domains are mechanically compliant, whereas beta-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp).

Elasticity↗