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PubMed2003

Trans allele methylation and paramutation-like effects in mice.

In mammals, imprinted genes have parent-of-origin-specific patterns of DNA methylation that cause allele-specific expression. At Rasgrf1 (encoding RAS protein-specific guanine nucleotide-releasing factor 1), a repeated DNA element is needed to establish methylation and expression of the active paternal allele. At Igf2r (encoding insulin-like growth factor 2 receptor), a sequence called region 2 is needed for methylation of the active maternal allele. Here we show that replacing the Rasgrf1 repeats on the paternal allele with region 2 allows both methylation and expression of the paternal copy of Rasgrf1, indicating that sequences that control methylation can function ectopically. Paternal transmission of the mutated allele also induced methylation and expression in trans of the normally unmethylated and silent wild-type maternal allele. Once activated, the wild-type maternal Rasgrf1 allele maintained its activated state in the next generation independently of the paternal allele. These results recapitulate in mice several features in common with paramutation described in plants.

Alleles↗

PubMed2006

The origin and evolution of stereotyped patterns of macrochaetes on the nota of cyclorraphous Diptera.

A long-standing problem in evolutionary biology is how genetic variation arises within populations and evolves to make species anatomically different. Many of the morphological differences in body plans between animal groups are thought to result from changes in gene expression during development. The rules governing the structure and evolution of cis-regulatory gene sequences are unknown, however, and the evolution of traits between closely related species remains relatively unexplored at a molecular level. To study the evolution of gene regulation, it is necessary to find a tractable trait that varies between species and for which the genetic regulation is well known in at least one of the species. The stereotyped, two-dimensional pattern of bristles on the thorax of Drosophila has been intensively investigated and is due to a precise spatial expression of proneural genes. Other species of flies have different bristle patterns and so comparisons between them provide a good paradigm for the study of changes in gene regulation. Here, we review the current state of understanding of these changes.

Animals↗

PubMed1998

cDNA sequence and heterologous expression of monomeric spinach pullulanase: multiple isomeric forms arise from the same polypeptide.

The spinach pullulanase gene was cloned and sequenced using peptide sequences of the purified enzyme as a starting point and employing PCR techniques and cDNA library screening. Its open reading frame codes for a protein of 964 amino acids which represents a precursor of the pullulanase. The N-terminal transit peptide consists of 65 amino acids, and the mature protein, comprising 899 amino acids, has a calculated molecular mass of 99kDa. Pullulanase is a member of the alpha-amylase family. In addition to a characteristic catalytic (beta/alpha)8-barrel domain, it contains a domain, F, that is specific for branching and debranching enzymes. Pullulanase cDNA was expressed in Escherichia coli, and the purified protein was compared with the enzyme from spinach leaves. Identity of the two proteins was confirmed in terms of catalytic properties, N-terminal amino acid sequences and molecular masses. The pullulanase produced by E. coli showed the same microheterogeneity as the spinach leaf enzyme: it could be resolved into two substrate-induced forms by electrophoresis in amylopectin-containing polyacrylamide gels, and, in the absence of substrate, into several free forms (charge isomers) by isoelectric focusing or chromatofocusing. Rechromatofocusing of single free forms resulted in the originally observed pattern of molecular forms. However, heterogeneity of the protein disappeared on isoelectric focusing under completely denaturing conditions when only one protein band was observed. Post-translational modifications such as glycosylation and phosphorylation could be excluded as potential explanations for the protein heterogeneity. Therefore the microheterogeneity of spinach leaf pullulanase results from neither genetic variation nor post-translational modifications, but is a property of the single unmodified gene product. The different interconvertible forms of the pullulanase represent protein populations of different tertiary structure of the same polypeptide.

Amino Acid Sequence↗

PubMed2003

The origin and course of the greater, lesser and least thoracic splanchnic nerves in New Zealand rabbits.

In this study, the origin and course of the greater, lesser and least splanchnic nerves was investigated in 12 adult New Zealand rabbits with regard to sex and the side of the body. There were no significant differences between the female and male rabbits. The greater and lesser splanchnic nerves were present in all the cadavers examined. However, the least splanchnic nerve was found on the right side in 50% of the cadavers (nos. 2, 4, 6, 7, 11 and 12), and on the left side in 75% (1, 2, 4, 6, 7, 8, 9, 11 and 12). With regard to the origin and pattern of the greater, lesser and least splanchnic nerves, there were significant differences between the right and left sides of the body even in the same rabbit. No significant difference was observed between sexes.

Animals↗

PubMed2001

Spontaneous, electrically, and cesium chloride induced arrhythmia and afterdepolarizations in the rapidly paced dog heart.

Despite frequent arrhythmia and sudden death in heart failure, attempts to study arrhythmia mechanisms in patients are difficult. The dog heart, paced for several weeks at a fast rate to induce heart failure is prone to arrhythmia. The aim of this study was to determine the activation patterns of spontaneous and electrically induced arrhythmia and the susceptibility of the failing dog heart to arrhythmia and early afterdepolarization (EAD) induced triggered activity elicited by exogenous administration of cesium chloride (CsCl). The hearts of 56 mongrel dogs were paced at 240 beats/min for 3-5 weeks (heart failure group). Twenty-one similarly operated, but not paced dogs served as the control group. At baseline, all dogs were healthy as assessed electrophysiologically and hemodynamically. Spontaneous (bradycardia, tachycardia, and arrhythmic deaths) and electrically induced arrhythmia was frequent in dogs with heart failure. Also, the minimal dose of CsCl that produced ventricular tachycardia was significantly lower in the heart failure than the control dogs (1.02 +/- 0.02 vs 1.21 +/- 0.07 mMol/kg, P < 0.05). Epicardial mapping during spontaneous and electrically induced arrhythmia in the heart failure dogs showed initiation patterns with focal origin, often from multiple sites. This pattern was consistent with the patterns observed with CsCl induced ventricular tachycardia. In in vitro microelectrode studies, CsCl superfusion (2.5-5 mMol/L) induced triggered activity due to EADs within 30 minutes, in seven of the eight Purkinje fibers from four heart failure dogs. EADs were also found in ventricular myocytes of papillary muscle from two heart failure dogs. In contrast, 5 mMol/L CsCl induced EADs in only one of eight Purkinje fibers from the hearts of four control dogs and no papillary myocytes even with continuous superfusion for up to 60 minutes (P < 0.01). These results demonstrate that pacing induced heart failure in the dog has an increased tendency to develop ventricular tachycardia and triggered activity unmasked by CsCl.

Animals↗

PubMed1994

Human pharmacokinetics of glycosaminoglycans using deuterium-labeled and unlabeled substances: evidence for oral absorption.

In the present study, the pharmacokinetics of extractive GAGs used as therapeutic agents have been studied after intravenous and oral administration on volunteers. The use of native or deuterium-labeled compounds, followed by HPLC/MS detection, allowed the quantitation of exogenous heparin and DS as major disaccharide fragments, obtained either by enzymatic or chemical depolymerization. In particular the high level of labeling reached in DS allowed its differentiation from structurally related endogenous species. The estimated plasmatic bioavailability was about 18% for DS. Notwithstanding the impossibility of evaluating the same parameters for heparin species, due to the interferences of endogenous GAGs, the results obtained provided clear evidence of oral availability of heparin and DS through detection and quantitation of structures specifically related to these GAGs. Due to the selectivity of the lyases used, the enzymatic degradation specifically allowed the detection of both DS and heparin species still retaining the original sulfation pattern. Additionally, the chemical degradation could detect the main metabolites of the drugs, consisting of partially to totally desulfated GAGs showing a more or less marked reduction in their molecular weight.

Administration, Oral↗

PubMed2003

Identification of severe acute respiratory syndrome in Canada.

BACKGROUND: Severe acute respiratory syndrome (SARS) is a condition of unknown cause that has recently been recognized in patients in Asia, North America, and Europe. This report summarizes the initial epidemiologic findings, clinical description, and diagnostic findings that followed the identification of SARS in Canada. METHODS: SARS was first identified in Canada in early March 2003. We collected epidemiologic, clinical, and diagnostic data from each of the first 10 cases prospectively as they were identified. Specimens from all cases were sent to local, provincial, national, and international laboratories for studies to identify an etiologic agent. RESULTS: The patients ranged from 24 to 78 years old; 60 percent were men. Transmission occurred only after close contact. The most common presenting symptoms were fever (in 100 percent of cases) and malaise (in 70 percent), followed by nonproductive cough (in 100 percent) and dyspnea (in 80 percent) associated with infiltrates on chest radiography (in 100 percent). Lymphopenia (in 89 percent of those for whom data were available), elevated lactate dehydrogenase levels (in 80 percent), elevated aspartate aminotransferase levels (in 78 percent), and elevated creatinine kinase levels (in 56 percent) were common. Empirical therapy most commonly included antibiotics, oseltamivir, and intravenous ribavirin. Mechanical ventilation was required in five patients. Three patients died, and five have had clinical improvement. The results of laboratory investigations were negative or not clinically significant except for the amplification of human metapneumovirus from respiratory specimens from five of nine patients and the isolation and amplification of a novel coronavirus from five of nine patients. In four cases both pathogens were isolated. CONCLUSIONS: SARS is a condition associated with substantial morbidity and mortality. It appears to be of viral origin, with patterns suggesting droplet or contact transmission. The role of human metapneumovirus, a novel coronavirus, or both requires further investigation.

Adult↗

PubMed1979

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.

A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.

Animals↗

PubMed2004

Duodenal ulcers preceding cervical tuberculous lymphadenitis.

Lymphadenitis is the most common presentation of extra pulmonary tuberculosis, whereas gastrointestinal localization, particularly duodenal involvement, is rare. We report a case of extra pulmonary tuberculosis with association between cervical lymphadenitis and duodenitis with multiple ulcers, not responsive to treatment with protonic pump inhibitors, in a human immunodeficiency virus-seronegative adult woman of Eritrean origin. Clinical patterns of duodenal TB, diagnostic difficulties and aetiopathogenesis are discussed according to the literature. In this case report it is suggested that tuberculous infection must be considered when duodenal ulcers fail to respond to proton pump inhibitors, especially when the patient comes from an endemic area.

Duodenal Ulcer↗

PubMed1999

Expression of VEGF splice variants 144/145 and 205/206 in adult male tissues.

Currently, at least five different mRNA species encoding vascular endothelial growth factor-A (VEGF-A) have been characterized. These variants result from alternative splicing of the VEGF-A transcript and encode human isoforms of VEGF protein of 121, 145, 165, 189, and 206 amino acids. In the rat, a similar profile of VEGF-A splice variants has been described, each encoding one fewer amino acid than the human species. Studies of mammalian tissues have shown that these mRNA isoforms vary in abundance. Whereas VEGF 188/189, 164/165, and 120/121 (rat/human, respectively) are the predominant forms expressed in most tissues and cells examined, VEGF 144/145 and 205/206 are rare variants. Previously, VEGF 144/145 had been detected only in placental and uterine tissues and endometrial carcinoma cell lines, whereas VEGF 205/206 was detected only in fetal liver and placenta. Using an RT-PCR technique, cDNA cloning, and sequencing, we have detected and confirmed the presence of mRNA encoding VEGF 144/145 and 205/206 in both adult rat lung and penis. Therefore, these low-abundance VEGF splice variants have a more diverse expression pattern than originally predicted.

Adult↗

PubMed1979

Immunofluorescent and histochemical localization of AMP deaminase in skeletal muscle.

Fluorescent antibody staining experiments with both isolated myofibrils and muscle fibers grown in culture show that AMP deaminase is bound to the myofibril in the A band. The strongest staining occurs at each end of the A band. The approximate width of the fluorescent stripes and their relation to the A band remains constant as a function of sarcomere length. Removal of enzyme from the myofibrils leads to loss of staining, and readdition of purified enzyme restores the original staining pattern. A histoenzymatic method for the detection of AMP deaminase activity in cultured fibers gives comparable localization. The results are consistent with the previous observation (Ashby, B. and C. Frieden. 1977.J. Biol. Chem. 252:1869--1872) that AMP deaminase forms a tight complex in solution with subfragment-2 (S-2) of myosin or with heavy meromyosin (HMM).

AMP Deaminase↗

PubMed2004

Convergence, divergence, and homogenization in the ecological structure of emydid turtle communities: the effects of phylogeny and dispersal.

Studies that have explored the origins of patterns of community structure from a phylogenetic perspective have generally found either convergence (similarity) in community structure between regions through adaptive evolution or lack of convergence (dissimilarity) due to phylogenetic conservatism in the divergent ecological characteristics of lineages inhabiting different regions. We used a phylogenetic approach to document a third pattern in the structure of emydid turtle communities. Emydid communities in southeastern North America tend to have a higher proportion of aquatic species than those in the northeast. This pattern reflects phylogenetic conservatism in the ecology and biogeography of two basal emydid clades, limiting convergence in community structure between these regions. However, differences in community structure between northeastern and southeastern North America have also been homogenized considerably by the dispersal of species with phylogenetically conserved ecological characteristics between regions. This pattern of ecologically conservative dispersal may be important in many continental and oceanic systems.

Animals↗

PubMed1982

A simple and efficient method for the separation and detection of small DNA fragments by electrophoresis in formamide containing agarose gels and Southern blotting to DBM-paper.

We show in this report that DNA fragments smaller than 300 bp are separated with high resolution by electrophoresis in concentrated (up to 7%) agarose gels containing 50% formamide. The separated DNA fragments can subsequently be quantitatively transferred to DBM-paper [Alwine, J.C. et al., Proc. Natl. Acad. Sci. USA (1977) 74, 5350-54] using the Southern technique [Southern, E.M., J. Mol. Biol. (1975) 98, 503-517], while preserving the sharpness of the original gel pattern. Since thin (0.2-0.4 mm) and thick (up to 5 mm) agarose slab gels can be easily handled in vertical or horizontal apparatus, this method should prove to be a very useful extention of the Southern technique, applicable to a variety of analytical and preparative purposes.

Base Composition↗

PubMed1980

The detection of DNA-binding proteins by protein blotting.

A method, called "protein blotting," for the detection of DNA-binding proteins is described. Proteins are separated on an SDA-polyacrylamide gel. The gel is sandwiched between 2 nitrocellulose filters and the proteins allowed to diffuse out of the gel and onto the filters. The proteins are tightly bound to each filter, producing a replica of the original gel pattern. The replica is used to detect DNA-binding proteins, RNA-binding proteins or histone-binding proteins by incubation of the filter with [32P]DNA, [125I]RNA, or [125I] histone. Evidence is also presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator. Strategies for the detection of specific protein-DNA interactions are discussed.

Carrier Proteins↗

PubMed1994

Structure and iron transport activity of vibrioferrin, a new siderophore of Vibrio parahaemolyticus.

The structure of vibrioferrin, a siderophore from Vibrio parahaemolyticus, was elucidated based on a combination of partial hydrolysis and spectroscopic techniques. HPLC of purified vibrioferrin showed two peaks with an area ratio of approximately 2:1. However, upon reinjection of each of those isolated compounds, the original chromatographic pattern was obtained, indicating an equilibrium between two compounds in aqueous solution. Consistent with this finding, most of the NMR signals of vibrioferrin were duplicated. The structure was determined as 1-(2-[2-(5-carboxy-5-hydroxy-2-oxo-1-pyrrolidinyl)propionamide]ethyl) citrate, which exists in two epimeric forms resulting from cyclization between an amidic nitrogen of the alanine residue and a keto group of the 2-ketoglutaric acid residue. Transport experiments with 55Fe-labeled vibrioferrin demonstrated the function of vibrioferrin as a siderophore in V. parahaemolyticus. Kinetic studies with mid-log phase cells revealed that the iron uptake system was receptor-mediated, with Km and Vmax values of 67 nM and 54 pmol Fe/mg cell protein/min, respectively. Moreover, iron uptake mediated by vibrioferrin was blocked both by uncouplers and by ATPase inhibitors.

Biological Transport↗

PubMed2006

The anatomic landmarks of ethmoidal arteries for the surgical approaches.

Knowledge of variations in the possible patterns of origins, courses, and distributions of the ethmoidal arteries are necessary for the diagnosis and important for the treatment of orbital disorders. Ethmoidal arteries are damaged in endonasal surgical interventions and in operations performed on the inner wall of the orbita.A description of the anatomic landmarks of the ethmoidal arteries and ethmoidal canals is presented, based on data from microdissection in 19 adult cadavers studied after injection of red-dyed latex into the arterial bed. In all subjects, each of ethmoidal arteries originated from ophthalmic artery. The anterior ethmoidal artery was observed in all specimens except for one case. The diameter of the artery thicker than the posterior ethmoidal artery was 0.92 +/- 0.2 mm on the right and 0.88 +/- 0.15 mm on the left. The branching of the anterior ethmoidal artery from the ophthalmic artery was determined in four different types. The diameter of the posterior ethmoidal artery was measured as 0.66 +/- 0.21 mm on the right and 0.63 +/- 0.19 mm on the left. The anterior ethmoidal canal was located between the second and third lamella in 29 of 38 cases. The mean distance between the limen nasi and anterior ethmoidal canal was 48.1 +/- 3.2 mm.The article confirms the well-known variability of the ethmoidal arteries and their topographic relation to the ethmoidal canals. Advances in surgical techniques, instrumentation, and regional arterial anatomy have resulted in functional operations of endoscopic sinus and orbital surgery with fewer complications.

Adult↗

PubMed1996

An anatomic study of neuropeptide immunoreactivities in the lumbar dura mater after lumbar sympathectomy.

STUDY DESIGN: The distribution and density of nerve innervation in the lumbar dura mater after lumbar sympathectomy were assessed in wistar rats. OBJECTIVE: To provide basic information on the interaction between sympathetic and sensory nerves in patients with low back pain. SUMMARY OF BACKGROUND DATA: Many studies have indicated that the sinuvertebral nerve has an important role in innervating the tissues around the vertebrae. However, the origin, innervating pattern, and connections between the nerves are still controversial. It is well known that pain is often accompanied with sympathetic symptoms and exaggerated by sympathetic stimuli. Occasionally, anesthetic block at the L2 or L3 sympathetic ganglion relieves low back pain or symptoms associated with low back pain. The authors assessed the changes of the density and distribution of nerve innervation of the lumbar dura mater after lumbar sympathectomy. METHODS: Normal adult rats were sympathectomized at L2-L3. The threshold for thermal noxious pain by hot-plate analgesia test and changes in neuropeptides in the lumbar dura mater and dorsal root ganglia using light microscopic immunohistochemistry were assessed and compared with control rats. RESULTS: In the hot-plate analgesia test, sympathectomized rats increased their hot-plate latency time compared with that of sham-operated rats. Density of calcitonin gene-related peptide immunoreactive fibers in sympathectomy side of the lumbar dura mater decreased to 45.5% compared with the contralateral side. The number and size of calcitonin gene-related peptide immunoreactive cells in dorsal root ganglia showed no difference between sympathectomized and contralateral side. CONCLUSION: Sympathectomy increased the pain threshold and made the sympathectomized rats hypesthetic. A large numbers of sensory fibers innervated the lumbar dura mater via L2-L3 sympathetic nerve in rats. Sympathectomy reduced the number of these nerve fibers in the lumbar dura mater. Sympathetic nerves may play an important role for low back pain involving the lumbar dura mater.

Animals↗

PubMed2005

Importance of the anatomic features of the lacrimal artery for orbital approaches.

Knowledge of variations in the possible patterns of origin, course, and distribution of the lacrimal artery are necessary for the diagnosis and important for the treatment of orbital disorders. The vascularization of 38 lacrimal glands was studied by orbital dissection subsequent to injection of the arterial bed with red-dyed latex. The origin, calibration, and branches of the lacrimal artery and its topographic relations were investigated. In all subjects, arteria lacrimalis originated from ophthalmic artery. On the right, the lacrimal artery sprang from the angle of the ophthalmic artery in 63.15% of the cases, from the curve of the ophthalmic artery in 26.31%, and from the first part the ophthalmic artery in 5.26%. The outer diameter of the lacrimal artery was measured as 1.02 +/- 0.17 mm on the right and 1.03 +/- 0.16 mm on the left. In 68.42 of the cases on the right and in 52.63 of the cases on the left, the lacrimal artery was present, and the lacrimal nerve was seen in a superolateral position with respect to the origin of the artery. Variability of the glandular branch in its course toward lacrimal gland was observed. Recurrent meningeal branch was seen in six cases on the right and in five on the left. On the right, of the six cases, two passed through meningoorbital foramen, and four passed through superior orbital fissure and entered middle cranial fossa. On the left, of the five cases, two passed through meningoorbital foramen, and three passed through superior orbital fissure and entered middle cranial fossa. In this case, the lacrimal gland is the site of an intraorbital anastomosis between internal and external carotid systems. This article confirms the well-known variability of the lacrimal arterial branches and their relation to the lacrimal gland. These variations have been discussed and described with respect to the embryonic development. A better understanding of the vascular anatomy of the lacrimal gland should allow modification of surgical techniques to reduce bleeding during biopsy or excision of the lacrimal gland.

Adult↗