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PubMed1989

Alpha 1 chain of chick type VI collagen. The complete cDNA sequence reveals a hybrid molecule made of one short collagen and three von Willebrand factor type A-like domains.

A cDNA library constructed from chick aorta poly(A+) RNA in the expression vector pEX1 was screened with rabbit polyclonal antisera. Additional clones were obtained by DNA-DNA hybridization with subclones from the most 5'- and 3'-ends. The overlapping clones span 4.6 kilobases and code for the entire alpha 1 (VI) chain. The nucleotide sequence reveals a 3057-base pair open reading frame that codes for 1019 amino acids. Analysis of the deduced amino acid sequence predicts that alpha 1 (VI) has one collagenous domain (COL) of 336 residues flanked by three repeated domains of about 200 residues each, one at the amino (A'3) and two at the carboxyl ends (A'2 and A'1), respectively, that are similar to the type A repeats of von Willebrand Factor. The COL domain presents two short interruptions near the carboxyl end of the triple helix and three of the six potential N-asparaginyl-linked carbohydrate attachment sites (Asn-Xaa-Ser/Thr). Furthermore, it contains 1 cysteine at position 89 that could participate in the formation of dimers and 3 Arg-Gly-Asp sequences that might be potential sites for cell adhesion. The COL domain shows an extended region, starting from position 40, within the triple helix, made of 14 Gly-Xaa-Yaa triplets that lack proline in the Y position, suggesting that it might be more flexible than the rest of the domain. At the junction of the COL with the N- and C-terminal domains, there are several cysteines that could confer the well known resistance of type VI collagen to pepsin and collagenase digestion under nonreducing conditions. The present sequence data allow a structural model for type VI collagen assembly to be proposed that is consistent with the structure implied from previous electron microscopic observation by Furthmayr et al. (Furthmayr, H., Wiedemann, H., Timpl, R., Odermatt, R., and Engel, J. (1983) Biochem. J. 221, 303-311).

Amino Acid Sequence↗
PubMed1996

Genetically unstable CXG repeats are structurally dynamic and have a high propensity for folding. An NMR and UV spectroscopic study.

Recent molecular genetics studies have revealed a correlation between spontaneous, progressive expansion of several DNA trinucleotide repeats and certain hereditary neurodegenerative diseases. Triplet repeat (TR) sequences may be present in structured forms that can mediate the processes interrupting normal cellular replication, transcription, or repair activities, eventually leading to gene mutation. Using high resolution NMR spectroscopy and other biophysical methods, we probed the solution structures and properties of single-stranded TR sequences. These studies have led to the discovery of a new duplex motif (e-motif), present in CCG repeats, and to the elucidation of the structure of the (CTG)3 duplex. In this paper we provide a global picture of the solution behavior of the human disease-related CXG (X = A, C, G, or T) and the comparison GXC (X = A, or T) TR sequences. All six triplet repeats form antiparallel duplexes. The mismatched bases in CAG and CGG repeat duplexes are rather flexible and they do not appear to form stable, paired conformations. By comparison, GAC repeat duplexes and their mismatched A residues are well-structured. Most interestingly, the structures of the disease-related CXG repeats exhibit a propensity for folding at chain lengths as short as 12 residues. Furthermore, the energy barrier for the formation of homo-duplexes from the corresponding complementary hetero-duplexes are much lower for the CXG TR sequences than for the GAC or GTC TR sequences. These results provide insights into the conformation and physiochemical properties of TR sequences. Thus, a basis is provided for further studies of the behavior of long TR sequences in an effort to elucidate the molecular mechanisms of in vivo expansion and function of TR sequences.

Base Composition↗
PubMed1995

Human ribosomal RNA gene cluster: identification of the proximal end containing a novel tandem repeat sequence.

Human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters on the short arms of five pairs of acrocentric chromosomes. We have demonstrated that a majority of the rDNA clusters are detected as 3-Mb DNA fragments when released from human genomic DNA by EcoRV digestion. This indicated the absence of the EcoRV restriction site within the rDNA clusters. We then screened for rDNA-positive cosmid clones using a chromosome 22-specific cosmid library that was constructed from MboI partial digests of the flow-sorted chromosomes. Three hundred twenty rDNA-positive clones negative for the previously reported distal flanking sequence (pACR1) were chosen and subjected to EcoRV digestion. Seven clones susceptible to EcoRV were further characterized as candidate clones that might have been derived from the junctions of the 3-Mb rDNA cluster. We identified one clone containing part of the rDNA unit sequence and a novel flanking sequence. Detailed analysis of this unique clone revealed that the coding region of the last rRNA gene located at the proximal end of the cluster is interrupted with a novel sequence of approximately 147 bp that is tandemly repeated and is connected with an intervening 68-bp unique sequence. This junction sequence was readily amplified from chromosomes 21 and 15 as well as 22 using the polymerase chain reaction. Fluorescence in situ hybridization further indicated that the approximately 147-bp sequence repeat is commonly distributed among all the acrocentric short arms.

Animals↗
PubMed1999

Interleukin-6 increases sensitivity to the locomotor-stimulating effects of amphetamine in rats.

Interleukin (IL)-6 mediates brain-immune interactions, influences the survival of postnatal mesencephalic and basal forebrain cells, influences mesocorticolimbic dopamine and serotonin neurotransmission, and is linked with various central nervous system disorders. In the present study, single injections of IL-6 (1 or 2 microg/Long-Evans rat, i.p.) induced modest elevations of locomotor activity. The locomotor increases were not augmented by repeated intermittent injections of IL-6 (five daily injections; 1 microg/rat), however. Nonetheless, repeated IL-6 treatment increased sensitivity to the locomotor-stimulating effects of 1.0 and 0.5 mg/kg amphetamine, when tested 5, 7, or 14 days following interruption of the cytokine treatment. The ability of acute IL-6 injections to alter locomotor activity and the ability of repeated IL-6 injections to produce long-lasting sensitization to the locomotor-stimulating effects of amphetamine suggest an interaction of this cytokine with the mesolimbic dopamine system, a system implicated in aspects of schizophrenia, addiction, and movement disorders. The fact that IL-6 caused a lasting change in responsiveness to amphetamine implies a mechanism by which immunogenic stimuli can alter brain circuitry, changing its sensitivity to seemingly unrelated subsequent stimuli or events.

Amphetamine↗
PubMed1993

Haemophilia B due to a de novo insertion of a human-specific Alu subfamily member within the coding region of the factor IX gene.

A de novo insertion of an Alu repeated DNA element was found within exon V of the factor IX gene in a patient with severe haemophilia B. The element interrupts the reading frame of the mature factor IX at glutamic acid 96 resulting in a stop codon within the inserted sequence. The Alu repeat is 322 bp long, and the 5' region is shortened by 38 bp. The insertion created a target site duplication of 15 bp consistent with retroposition, and contains a pure polyadenine tract of at least 78 resides at the 3' end. The nucleotide sequence agrees with a consensus for an Alu subfamily which is evolutionarily the most recently inserted, suggesting that it is an exact copy of a putative source gene. These observations indicate that retroposition of Alu elements is a continual process and a mechanism for generating human genetic defects.

Base Sequence↗
PubMed1998

Analysis of striated fiber formation by recombinant SF-assemblin in vitro.

The basal bodies of green flagellates are often connected to striated microtubule-associated fibers (SMAFs), which are highly ordered bundles of 2 nm filaments. SF-assemblin (33 kDa) is the principal structural subunit of the SMAFs and consists of a non-helical head domain of approximately 32 residues and an alpha-helical rod domain that shows a pronounced coiled-coil forming ability. To investigate the functional role of the head domain we expressed N-terminally truncated molecules using a cDNA coding for SF-assemblin from Chlamydomonas reinhardtii. Recombinant wild-type SF-assemblin or molecules with an N-terminal deletion of ten residues formed striated fibers with an axial repeat of 28 nm. N-terminal truncations of 19 and 29 residues yielded assembly-incompetent molecules, revealing that the head domain is necessary for the constitution of striated fibers. Further, molecules with an internal deletion of 24 residues or with duplicated segments corresponding to insertions of 29 and 58 residues were constructed. The resulting fibers had altered cross-striation patterns and axial repeats. The observed shifts in the axial repeat corresponded well to the number of inserted or deleted residues, indicating a linear coherence between molecule length and axial repeat. The heptad pattern of the rod domain of SF-assemblin is regularly interrupted by skip residues. The removal of one or two skip residues had no significant effect on the ultrastructure of the striated fibers. Substitution of skip no. 2 with alanine resulted in a modified, asymmetric cross-striation pattern, indicating a polar architecture of the striated fibers. In summary, various mutations of SF-assemblin effected the solubility of the molecules, and the axial repeat, cross-striation pattern, or overall appearance of the fibers. Thus, analysis of SF-assemblin may represent a valuable system to study the interactions involved in the polymerization of fibrous coiled-coil proteins. A model of the SMAFs based on staggered protofilaments consisting of overlapping 36 nm subunits is presented.

Amino Acid Sequence↗
PubMed1998

A methylated Neurospora 5S rRNA pseudogene contains a transposable element inactivated by repeat-induced point mutation.

In an analysis of 22 of the roughly 100 dispersed 5S rRNA genes in Neurospora crassa, a methylated 5S rRNA pseudogene, Psi63, was identified. We characterized the Psi63 region to better understand the control and function of DNA methylation. The 120-bp 5S rRNA-like region of Psi63 is interrupted by a 1.9-kb insertion that has characteristics of sequences that have been modified by repeat-induced point mutation (RIP). We found sequences related to this insertion in wild-type strains of N. crassa and other Neurospora species. Most showed evidence of RIP; but one, isolated from the N. crassa host of Psi63, showed no evidence of RIP. A deletion from near the center of this sequence apparently rendered it incapable of participating in RIP with the related full-length copies. The Psi63 insertion and the related sequences have features of transposons and are related to the Fot1 class of fungal transposable elements. Apparently Psi63 was generated by insertion of a previously unrecognized Neurospora transposable element into a 5S rRNA gene, followed by RIP. We name the resulting inactivated Neurospora transposon PuntRIP1 and the related sequence showing no evidence of RIP, but harboring a deletion that presumably rendered it defective for transposition, dPunt.

Amino Acid Sequence↗
PubMed2003

Chronotherapy of high-dose active vitamin D3 in haemodialysis patients with secondary hyperparathyroidsm: a repeated dosing study.

AIMS: Renal osteodystrophy is the major complication in patients with end-stage renal failure. Oral or intravenous vitamin D3 (D3) is given to these patients, but severe hypercalcaemia sometimes interrupts this therapy. This study was undertaken to determine whether the effectiveness and safety of D3 also depend on its dosing time during a repeated treatment. METHODS: A higher dose (3 micro g) was given orally to 13 haemodialysis patients at 08.00 h or 20.00 h for 12 months by a randomized, cross-over design. RESULTS: Three patients were withdrawn due to severe hypercalcaemia after switching from 08.00 h to 20.00 h dosings. The elevation in serum calcium concentration was significantly (P < 0.001) greater during the 08.00 h dosing in the remaining ten patients. Mean serum Ca concentration after the trial was 10.92 (95% confidence interval (CI) 10.79, 11.06) and 9.55 mg dl-1 (95% CI 9.30, 9.71) by 08.00 h and 20.00 h dosing, respectively. On the other hand, the suppression of the elevated serum parathyroid hormone (PTH) and subsequent increment in bone density were significantly greater during the 08.00 h dosing. Mean PTH concentration after the trial was 414 (95% CI 360, 475) and 220 pg ml-1 (95% CI 202, 249) by 08.00 h and 20.00 h dosing, respectively (P = 0.02). Mean increment of bone density after the trial was 22 (95% CI 8, 32) and 57 g cm-3 (95% CI 43, 83) by 08.00 h and 20.00 h dosing, respectively (P = 0.04). CONCLUSION: These results indicate that a higher dose of oral D3 is more effective and safe after dosing at evening in patients with renal osteodystrophy.

Administration, Oral↗
PubMed2006

Efficient in toto targeted recombination in mouse liver by meganuclease-induced double-strand break.

BACKGROUND: Sequence-specific endonucleases with large recognition sites can cleave DNA in living cells, and, as a consequence, stimulate homologous recombination (HR) up to 10 000-fold. The recent development of artificial meganucleases with chosen specificities has provided the potential to target any chromosomal locus. Thus, they may represent a universal genome engineering tool and seem to be very promising for acute gene therapy. However, in toto applications depend on the ability to target somatic tissues as well as the proficiency of somatic cells to perform double-strand break (DSB)-induced HR. METHODS: In order to investigate DSB-induced HR in toto, we have designed transgenic mouse lines carrying a LagoZ gene interrupted by one I-SceI cleavage site surrounded by two direct repeats. The LagoZ gene can be rescued upon cleavage by I-SceI and HR between the two repeats in a process called single-strand annealing. beta-Galactosidase activity is monitored in liver after tail vein injection of adenovirus expressing the meganuclease I-SceI. RESULTS: In toto staining revealed a strong dotted pattern in all animals injected with adenovirus expressing I-SceI. In contrast, no staining could be detected in the control. beta-Galactosidase activity in liver extract, tissue section staining, and PCR analysis confirmed the presence of the recombined LagoZ gene. CONCLUSIONS: We demonstrate for the first time that meganucleases can be successfully delivered in animal and induce targeted genomic recombination in mice liver in toto. These results are an essential step towards the use of designed meganucleases and show the high potential of this technology in the field of gene therapy.

Animals↗
PubMed1981

Orienting behavior by rats with visual cortical and subcortical lesions.

The effect of visual cortical and subcortical lesions on orienting behavior was assessed bu examining the rats' ability to interrupt an ongoing response and perform appropriate head and postural adjustments to repeatedly presented auditory or apparently moving visual stimuli. Large lesions of the entire superior colliculus (SC) or the deep layers of the SC did not result in visual agnosia or the inability to perform the motor responses involved in orienting. Rather, the orienting response simply ws not emitted to visual stimuli that the intact rat treated as less salient, but was to those it treated as more salient. Lesions of either the superficial layers of the SC or visual cortex also did not completely prevent orienting to very salient, apparently moving visual stimuli, but did produce changes in the number of responses made to such stimuli and in the occurrence of other components of orienting behavior. It was suggested that the SC and visual cortex play a modulatory role in orienting behavior and that stimulus characteristics must be considered in the development of neuronal models of orienting behavior.

Animals↗
PubMed2005

Comparative sequence analysis of the phytochrome C gene and its upstream region in allohexaploid wheat reveals new data on the evolution of its three constituent genomes.

Bread wheat is an allohexaploid with genome composition AABBDD. Phytochrome C is a gene involved in photomorphogenesis that has been used extensively for phylogenetic analyses. In wheat, the PhyC genes are single copy in each of the three homoeologous genomes and map to orthologous positions on the long arms of the group 5 chromosomes. Comparative sequence analysis of the three homoeologous copies of the wheat PhyC gene and of some 5 kb of upstream region has demonstrated a high level of conservation of PhyC, but frequent interruption of the upstream regions by the insertion of retroelements and other repeats. One of the repeats in the region under investigation appeared to have inserted before the divergence of the diploid wheat genomes, but was degraded to the extent that similarity between the A and D copies could only be observed at the amino acid level. Evidence was found for the differential presence of a foldback element and a miniature inverted-repeat transposable element (MITE) 5' to PhyC in different wheat cultivars. The latter may represent the first example of an active MITE family in the wheat genome. Several conserved non-coding sequences were also identified that may represent functional regulatory elements. The level of sequence divergence (Ks) between the three wheat PhyC homoeologs suggests that the divergence of the diploid wheat ancestors occurred some 6.9 Mya, which is considerably earlier than the previously estimated 2.5-4.5 Mya. Ka/Ks ratios were <0.15 indicating that all three homoeologs are under purifying selection and presumably represent functional PhyC genes. RT-PCR confirmed expression of the A, B and D copies. The discrepancy in evolutionary age of the wheat genomes estimated using sequences from different parts of the genome may reflect a mosaic origin of some of the Triticeae genomes.

5' Flanking Region↗
PubMed1992

Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus.

The Saccharomyces cerevisiae mitochondrial endonuclease I-SceI creates a double-strand break as the initiating step in the gene conversional transfer of the omega+ intron to omega- DNA. We have expressed a galactose-inducible synthetic I-SceI gene in the nucleus of yeast that also carries the I-SceI recognition site on a plasmid substrate. We find that the galactose-induced I-SceI protein can be active in the nucleus and efficiently catalyze recombination. With a target plasmid containing direct repeats of the Escherichia coli lacZ gene, one copy of which is interrupted by a 24-bp cutting site, galactose induction produces both deletions and gene conversions. Both the kinetics and the proportion of deletions and gene conversions are very similar to analogous events initiated by a galactose-inducible HO endonuclease gene. We also find that, in a rad52 mutant strain, the repair of double-strand breaks initiated by I-SceI and by HO are similarly affected: the formation of deletions is reduced, but not eliminated. Altogether, these results suggest either that the two endonucleases act in the same way after double-strand break formation or that the two endonucleases are not involved in subsequent steps.

Base Sequence↗
PubMed1991

Macronuclear and micronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1 alpha in Stylonychia lemnae.

The micronuclear and macronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1 alpha in the hypotrich ciliate Stylonychia lemnae were compared. The two sequences are generally colinear. The coding sequence of the micronuclear gene is, however, interrupted by a 64 bp insert flanked by a 2 bp direct repeat in a gene region which is moderately conserved among EF 1 alpha genes of different organisms. The insertion site is distinct from known intron positions in eukaryotic EF 1 alpha genes. The insert sequence shows inverted repeats at its ends and thus exhibits typical features of an internal eliminated sequence (IES). Comparison with other such sequences in the related organism Oyxtricha nova shows that the IES falls into a new group of such elements. The macronuclear gene exhibits a strikingly limited codon usage, which cannot be simply explained by the overall base composition of the DNA but probably also relates to the very high copy number of the macronuclear gene and the putative high amount of the gene product.

Amino Acid Sequence↗
PubMed1987

External ATP mimics carbachol in initiating calcium mobilization from pancreatic beta-cells conditioned by previous exposure to glucose.

1 Exposure to ATP (2-200 microM) resulted in a prominent peak of 45Ca efflux, when beta-cell-rich pancreatic islets from ob/ob-mice were perifused with a Ca2+-deficient medium. ADP and the stable alpha/beta-methylene analogues of ATP and ADP also had stimulatory effects. 2 The nucleotide initiation of 45Ca efflux mimicked that obtained with carbachol both in requiring previous exposure to glucose and in being more pronounced after replacing extracellular Na+ by K+. 3 It was possible to induce repeated peaks of stimulated 45Ca efflux, when the exposure to ATP was interrupted with intervals of perifusion with glucose-containing media. 4 The observations are consistent with the existence of P2-purinoceptors in islets, suggesting that these receptors mediate a similar mobilization of calcium as noted when activating polyphosphoinositide breakdown with carbachol. In view of the high contents of ATP and ADP in the beta-cell secretory granules, activation of P2-purinoceptors should be considered as a possible mechanism for amplification of the initial insulin secretory response.

Adenine Nucleotides↗
PubMed1989

Local complement activation, thromboxane-mediated vasoconstriction, and vascular leakage in isolated lungs. Role of the terminal complement sequence.

In situ complement activation was induced in isolated, ventilated, and blood-free perfused rabbit lungs. Six to 11% human serum (vol/vol) was repeatedly admixed to the recirculating Krebs Henseleit buffer during 10-min periods, interrupted by rinsing phases with serum-free medium. This caused dose-dependent, reversible, and reproducible pulmonary artery pressor responses with pressure peaks or plateaus of 8 to 22 mm Hg. Pressor responses were paralleled by a marked release of thromboxane (TxA2) and prostaglandin I2 into the medium. Heat-inactivated serum, purified fluid-phase SC5b-9, and serum that had been activated with inulin in the absence or presence of a carboxypeptidase inhibitor all failed to elicit pressor responses. Both pressor response and prostanoid release were strictly dependent on complement factor C8, and evidence for C8 binding was obtained with the use of 125I-C8. Inhibitor studies collectively indicated that TxA2 was the predominant vasoconstrictive agent. Although repetitive short-term application of human serum induced no major lung weight gain, prolonged exposure to serum (40 to 80 min) without intermittent rinsing phases caused a delayed increase in the capillary filtration coefficient to maximally 3- to 4-fold values, with marked lung edema formation. We conclude that in situ activation of complement in the rabbit lung vasculature causes immediate intrapulmonary prostanoid generation and thromboxane-mediated vasoconstriction, independent of circulating leukocytes, but dependent on the formation of terminal membrane-complement complexes. Continuous complement activation results in a delayed increase in lung vascular permeability.

Animals↗
PubMed1992

Is cerebral arteritis the cause of the Landau-Kleffner syndrome? Four cases in childhood with angiographic study.

Four children with Landau-Kleffner syndrome were studied over a six year period. They presented with acquired aphasia, epilepsy, and focal or generalized EEG discharges which were exacerbated during sleep. In addition, cerebral angiography demonstrated isolated arteritis of some branches of the carotid arteries in all cases. Computed tomographic and magnetic resonance images were normal. Nicardipine in a dose of 1 to 2 mg/kg/day, added to conventional anticonvulsant drugs provided effective supplementary control of seizures, of paroxysmal EEG discharges, and of language and behavioural disturbances, even several years after the onset of the disorder and in patients whose response to other medications, including steroids, had been poor. Interruption of nicardipine administration was followed by relapse of the language disorder. Repeat angiography was performed in all four patients and showed recanalization of obstructed vessels in two cases. Focal cerebral vasculitis may be the pathogenesis of the Landau-Kleffner syndrome and calcium channel blockers such as nicardipine may be effective and specific therapy.

Adolescent↗
PubMed1990

[Aberrometry in the diagnosis of eye diseases].

Four methods (by means of Smirnova-Korniushina's aberrometer; with the help of aberroscope--Cherning's lattice; by presenting a figure of a cross; and by automatic refractometry through different portions of the pupil) were used for studying aberration of the optic system of the eye in 490 persons. Among them, 37 were healthy persons, 27--patients with ametropia of moderate degree, 24--with high myopia, 9--with astigmatism above 3.0 D, 16--with monocular diplopia, 327--with keratoconus, 7--with scars of the cornea, 21--with initial cataract, 47--after keratotomy. In norm and in ametropia to 6.0 D, a histogram of refraction distribution by the pupil has a small disperse and a pronounced peak, the Cherning's lattice is straight. In high myopia, the disperse of refraction increases, the peak is slightly pronounced, the lattice is curved along the margins. In astigmatism, the disperse of refraction is great, but there can be peaks. The whole lattice is curved. In monocular diplopia, two more peaks can be seen on the histogram. The lattice is greatly curved. In keratoconus, aberrometry without correction is possible only at the initial stage and shows a rather mixed picture. The histogram of refraction represents a low irregular row. In aberroscopy, the bands are greatly curved, frequently overstep the bounds of the frame that itself looses a form of a circle, becomes oval or polygonal. Contact correction improves the aberration picture considerably. In scars of the cornea, the lattice is distorted and infrequently repeats their forms. In initial cataract, the course of the bands becomes interrupted and rounded spots appear between them.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1985

Organization of a type I keratin gene. Evidence for evolution of intermediate filaments from a common ancestral gene.

The genomic structure of the mouse 59-kDa keratin gene, a Type I intermediate filament (IF) gene is presented. A comparison of the organization of this gene with that of the human 67-kDa keratin, a Type II IF gene, and hamster vimentin, a Type III IF gene, suggests a common evolutionary origin for Type I, II, and III IF genes. Most introns in these three types of IF genes occur at similar positions within the region encoding sequences predicted to form coiled-coils, but do not delineate structural subdomains. Interestingly though, most of the introns interrupt at or near the beginning of the characteristic 7-residue (heptad) repeat of sequences which form the coiled-coil. These data suggest that the three types of IF genes arose from a common ancestor which may have been assembled from smaller units containing multiple heptad repeats. Subsequent duplication events may then have formed the three known alpha-helical types and each of their various members.

Animals↗