Single Ligation Research Articles

Molecular cloning of rabbit matrix metalloproteinase-2 and its broad expression at several tissues

We have cloned cDNA encoding rabbit matrix metalloproteinase-2 (MMP-2, 72 kDa type IV collagenase) by a combination of conventional library screening, the ‘single strand ligation to single-stranded cDNA (SLIC)’ method and ‘long and accurate PCR (LA-PCR)’. Deduced amino acid sequence was highly conserved through mammalian species. Northern blot analysis revealed that rabbit MMP-2 had 2 species of mRNA, 2.8 kbp and 3.5 kbp, and were expressed constitutively in all the tissues tested. This was totally different from mRNA expression of rabbit MMP-1, -3 and -9.

Early Gene Response to Hepatic Ischemia/Reperfusion

The purpose of this study was to define the differences in heat shock protein (hsp)70, albumin, α1-acid glycoprotein (AGP), and CCAAT enhancer binding proteins (C/EBP) α and β mRNA between hepatic ischemia and reperfusion, and to begin to explore C/EBP protein production. These genes have been found important in the hepatic response to lipopolysaccharide and inflammation. In two experiments, Sprague-Dawley rats underwent temporary occlusion of the median and left hepatic lobe vasculature. The first experiment included a single sham-operated group and ligation of the right hepatic lobes during reperfusion. It compared 30 and 60 min ischemia to 2 h reperfusion. The second experiment included a sham-operated group for every time point, and the right hepatic lobes were not ligated during reperfusion; a 30-min ischemia group was compared to 2-, 5-, and 24-h reperfusion groups. Total RNA from the ischemic lobes was analyzed by Northern hybridization for hsp70, albumin, AGP, and C/EBPα and β. C/EBPα and β proteins were compared by Western blotting. Differences in experimental design played an important role in interpretation of results. hsp70 mRNA began to increase during ischemia. Albumin mRNA remained constant during ischemia and reperfusion. The ischemic hepatocyte nucleus is not quiescent and retains the ability to upregulate certain genes, e.g., hsp70. Changes in mRNA in response to hepatic ischemia/reperfusion occur rapidly. Hepatic ischemia/reperfusion does not recapitulate the classic acute phase response; albumin is not downregulated during reperfusion.

Single-Site Polymerase Chain Reaction through Single Oligonucleotide Ligation

Single-Site Polymerase Chain Reaction through Single Oligonucleotide Ligation

The effect of radicular arteries ligation on the spinal cord blood supply in cats.

In order to study the contribution of the radicular arteries to the blood supply of the spinal cord, we performed experimental occlusion of these arteries in cats. In 43 cats, under thiopental anaesthesia, unilateral, bilateral, single or multiple ligations of dorsal radicular arteries were carried out. The animals were killed at the 1st, 2nd and 7th postoperative day. The spinal cords were removed and preserved in formalin solution for 1 month and then examined, using light microscopy. We conclude that bilateral or multiple ligations are capable of producing ischaemic lesions in the spinal cord, more profound by the seventh postoperative day, while single ligations do not produce such lesions, unless a medullary artery is involved. The ischaemic lesions which were observed were associated with clinical neurological deficits.

Investigation of the influence of cytosine methylation on DNA flexibility.

To test the influence of pyrimidine methyl groups on DNA flexibility and helix repeat, two sets of 14 mixed sequence DNA molecules, spanning a range of lengths from 158 to 180 base pairs, were cyclized with T4 DNA ligase. The two sets differed only in that the Cyt-5 positions of all cytosines (80-90 cytosine residues per molecule) were fully methylated in the members of one set. Determination of the molar cyclization factors, persistence lengths, helix repeats, and torsional elastic constants revealed no significant differences between the two sets. These results imply that, at least for mixed sequence DNA, the biological consequences of cytosine methylation are likely to derive from either local structural distortions in the helix, which do not propagate as altered twist, or from direct protein-methyl cytosine interactions.

Passive immunization against tumor necrosis factor and interleukin-1 fails to reduce lung neutrophil sequestration in chronic sepsis.

The proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) are produced within the lung during sepsis, and may induce neutrophil sequestration resulting in neutrophil-mediated lung injury. We hypothesized that, if there is a cause and effect between TNF alpha or IL-1 production and lung neutrophil sequestration during chronic sepsis, TNF alpha mRNA and IL-1 mRNA levels in the lung after cecal ligation and puncture should correlate with the number of sequestered neutrophils as measured by the myeloperoxidase (MPO) content of the lung. To test this hypothesis, Swiss Webster mice were subjected to varying degrees of infectious challenge by single and double-puncture cecal ligation and puncture, or simultaneous antibiotic treatment, and their lungs and blood were harvested at 24 h. Lung TNF alpha and IL-1 beta mRNAs were measured by the reverse-transcription differential polymerase chain reaction, and MPO was measured by colorimetric assay. TNF alpha serum levels showed no correlation with the MPO content of the lung, whereas IL-1 levels were undetectable. Lung TNF alpha mRNA correlated weakly, and IL-1 beta mRNA exhibited a strong correlation with lung MPO (r = .9, p < .01), but administration of anti-TNF alpha- or anti-IL-1-neutralizing antibodies did not prevent a rise in lung MPO. IL-1 beta mRNA in bronchoalveolar macrophages correlated well with whole lung tissue IL-1 beta mRNA levels (r = .91, p < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Use of transcolonic portal scintigraphy to monitor blood flow and progressive postoperative attenuation of partially ligated single extrahepatic portosystemic shunts in dogs

Summary Transcolonic portal scintigraphy was used to evaluate immediate and long-term changes in shunt blood flow after partial ligation of single extrahepatic portosystemic shunts in 8 dogs. Scintigraphy was performed before surgery, within the first 7 days after surgery, and 3 to 9 months after surgery. Shunt fraction values for this group of dogs before surgery ranged from 67 to 87% (normal reference range, ≤ 15%). On the basis of postoperative scintigraphy, 4 dogs had immediate and lasting occlusion of shunt blood flow (shunt fraction, ≤ 15%). One dog had a shunt fraction value within the reference range immediately after surgery, had evidence of recurrent shunt blood flow (shunt fraction, 23%) at 3 months after surgery, and again had a shunt fraction value within the reference range 9 months after surgery. Persistent, though reduced, shunt blood flow (shunt fractions, 23 and 2 7%) was evident immediately after surgery in 2 dogs. One of these dogs had a shunt fraction within the reference range 3 months after surgery, whereas shunt blood flow in the other dog had increased to 41%. One dog had no appreciable change in shunt blood flow immediately after surgery (shunt fraction, 70%), but the shunt fraction value had decreased to 41% at the time of the final scintigraphic examination. As assessed by transcolonic portal scintigraphy, partial single extrahepatic portosystemic ligation caused a significant decrease in mean shunt blood flow in this group of 8 dogs and resulted in eventual shunt occlusion in a majority of the dogs (6 of 8 dogs). Partial ligation with progressive attenuation of shunt blood flow may obviate the need to perform additional surgery.

Bridging the gap. Joining of nonhomologous ends by DNA polymerases.

DNA double strand breaks with noncomplementary ends can be joined by mechanisms of nonhomologous recombination. In some systems a DNA end with a 3'-protruding single strand (PSS), which does not have a recessed 3'-hydroxyl that can allow for fill-in DNA synthesis, is joined to a blunt end with preservation of the 3'-PSS. It has been proposed that this process occurs via single strand ligation or is facilitated by an alignment protein. We were interested in testing the hypothesis that a DNA polymerase could function as this putative alignment protein. To characterize polymerase activities in this type of reaction, we incubated short double-stranded oligonucleotides that had an excess of one of the strands with an exonuclease-free Klenow fragment of Escherichia coli polymerase I, Taq DNA polymerase from Thermus aquaticus, or an exonuclease-free Stoffel fragment of Taq DNA polymerase. Products were analyzed by using biotinylated oligonucleotides separated by denaturing polyacrylamide gel electrophoresis. To further assess the effect of DNA polymerases on the joining of 3'-PSS ends to blunt ends, we incubated linear plasmid DNA with the polymerases and subjected the DNA to Southern blot and sequence analysis. We determined that these DNA polymerases can use a 3'-PSS end as a template after priming off the 3'-hydroxyl of a blunt end. This implies that the joining of noncomplementary ends in eukaryotic cells could proceed by a similar mechanism.

Spatial Distribution of Nerve Injury after Occlusion of Individual Major Vessels in Rat Sciatic Nerves

In an attempt to better understand the spatial distribution of ischemic injury secondary to occlusion of major arteries, we measured nerve blood flow (NBF) and studied morphologic changes at various levels distal to the ligature site. Arterial ligation of the femoral, internal iliac, or superior gluteal artery was preceded and followed by measurement of NBF using laser Doppler flowmetry which helped identify "watershed areas" and guided the sampling process as nerves were examined pathologically and areas of injury were identified. Femoral artery ligation produced the most severe ischemia, focally reducing NBF by 80% in the tibial nerve at a level just below the knee. Within these ischemic nerve segments there were degenerative changes of nerve fibers seen mainly in the subperineurial region. Ligation of the internal iliac artery caused an approximately 60% reduction in NBF at the upper and mid-thigh levels of the sciatic nerve which resulted only in endoneurial edema in tissue taken at this level. Following superior gluteal artery ligation. NBF was reduced by only 20% at the pelvic level of the sciatic nerve and there was neither endoneurial edema nor fiber abnormalities. This study demonstrates the watershed pattern of ischemic injury associated with single vessel ligation by correlating neuropathologic change with quantitative measures of local nerve blood flow. The data further support the concept that mild levels of ischemia cause endoneurial edema, while moderate levels of ischemia produce demyelination and severe ischemia produces Wallerian degeneration.

Secretion of ?-lactamase from K. lactis using pEPS1, a convenient episomal vector designed for the secretion of foreign proteins

A convenient shuttle vector that enables high level secretion of proteins from Kluyveromyces lactis has been developed. The vector, pEPS1, contains a unique cloning site that allows the construction, in a single ligation step, of episomal plasmids capable of directing secretion of foreign gene products from K. lactis. As an example we demonstrate the production of β-lactamase and determine optimal conditions for its secretion into the culture media.

Efficient parvovirus B19 DNA purification and molecular cloning

A simple and efficient method of purification and molecular cloning of Parvovirus B19 DNA directly from small quantities of viremic sera was developed. Purified virions were lysed in annealing conditions, then viral DNA purification in double strand (ds) DNA form was achieved using an affinity DNA binding matrix. Affinity purification yielded a consistently high recovery of viral DNA. Using affinity purified ds viral DNA, we efficiently and stably cloned the complete coding internal unique sequence of B19 DNA. In our cloning strategy AatII and BamHI restriction endonuclease sites were exploited. This permitted cleavage of the 5.0 kbp AatII fragment in two AatII- BamHI fragments which could be efficiently cloned in a directional way in pUCI8 plasmid vector. The availability of the two cloned AatII- BamHI fragments thus allowed the construction of a full length clone in a single ligation reaction.

Cloning of two different 5′ untranslated exons of bovine acidic fibroblast growth factor by the single strand ligation to single-stranded cDNA methodology

In an attempt to characterize the 5′ UTR of the aFGF mRNAs we used the new anchored PCR methodology, single strand ligation to ss-cDNAs (SLIC). In bovine brain and retina, two kinds of aFGF cDNA clones were isolated. They contained two alternative exons located 34 by upstream to the translation initiation codon ATG. Taking into account the number of clones specific for each exon, the two mRNAs are expressed with the same ratio in both tissues. One of these bovine 5′ UTR exons (136 bp) showed 81% identity to a human 5′ UTR exon, the second one (323 bp) was 70% identical to the second human 5′ UTR exon with a central region of 90 nucleotides showing 41% identity. The conservation of the splicing positions for these 5′ UTR alternate exons in both bovine and human species, suggests that the overall structure of the aFGF gene is conserved in mammals. Furthermore, the conservation of the nucleotide sequences and of the localization of these 5′ UTR exons suggests that these non-coding regions may be involved in the control of aFGF gene expression.

Assembly and expression of a synthetic gene encoding the bovine glycoprotein hormone α-subunit

The glycoprotein hormones are a family of αβ heterodimeric proteins which are responsible for gonadal and thyroid function. In previous studies we employed chimeric glycoprotein hormone β-subunits to identify amino acid residues critical for binding to receptors and antibodies. To facilitate similar studies of the α-subunit of these hormones, we assembled a 406 bp synthetic gene which encodes the human α-subunit leader sequence and the secreted portion of the bovine α-subunit. It contains unique restriction sites that can be used for cassette mutagenesis or for making human/bovine α-subunit chimeras. The gene was assembled from eight long oligodeoxynucleotides in a single ligation and its structure verified by DNA sequencing. Co-transfection of COS-7 cells with the synthetic gene and the cDNA for human chorionic gonadotropin (hCG) β-subunit resulted in the secretion of a functional αβ heterodimer which bound to luteinizing hormone receptors. The protein was recognized by several monoclonal antibodies including B109, an antibody to a conformational epitope which binds hCG but not the free bovine α-, human α-, or hCG β-subunits. This suggests that the binding site for B109 may be formed by residues located primarily within the hCG β-subunit and that formation of this epitope requires a change in conformation of the β-subunit when it combines with the α-subunit.

Tratamento cirúrgico da compressão do esôfago por anomalia do arco aórtico: relato de caso

An adult patient presenting a congenital vascular anomaly of the aortic arch and ring formation, surrounding trachea and esophagus, by compression of the latest was complaining of progressive dysphagia. A surgical approach was performed by right lateral thoracotomy with ressection of the Kommerell diverticulum, section of the arterious ligament and single ligation of the subclavian artery which was followed by total regression of the symptoms. No subclavian steal syndrome or ischemia was observed on the left upper limb. The authors discuss the manner of approach and surgical treatment of this rare anomaly.

A Comparative Study of Different Treatments of Hemorrhoids

Hemorrhoid is a common anal problem in our country that is currently treated by many different methods. Three modalities were assessed in a randomized prospective trial in which 848 patients with a second-degree hemorrhoid were treated by rubber band ligation (470 patients), injection sclerotherapy (280 patients), or cryosurgery (98 patients). The effectiveness of each method was assessed clinically at 3, 6, 9, and 12 months after initial treatment, and determined by improvement in presenting symptoms. All patients complained of rectal bleeding and prolapse of the hemorrhoid on defecation, but only 69% had anal pain and 14% suffered from pruritus ani. Rubber band ligation was found to be superior to the other methods, and displayed statistical significance when compared to injection sclerotherapy and cryosurgery.

Heparin Prevents Stasis-Induced Thrombosis through Protection of the Venous Endothelial Cells: An Electron Microscopic Study in Rabbits

This study was performed to see whether or not protection of the endothelial cells contributes to the antithrombotic effects of heparin. New Zealand albino rabbits were subjected to jugular vein stasis by single caudal ligation for 2 h. Three treatments were given: saline (control group), heparin (0.2 mg/kg) 5 or 45 min before ligature of the vein. Groups of 6-8 animals were killed at 0, 5, 15, 30 and 120 min. The following parameters were determined: (1) involution and damage of the endothelial cells by scanning and transmission electron microscopy; (2) incidence and weight of thrombi in the lumens of the veins after 2 h stasis, and (3) effects of heparin on APTT and anti-Xa activity. In the control group, stasis caused a considerable involution of the endothelial cells in the first 30 min, followed by fibrin deposition and thrombus generation. Heparin strongly reduced the damage to the endothelial cells, with very evident protection of the cell membranes, and prevented thrombus generation: there were significant decreases in both incidence and weight of thrombi. These effects of heparin were evident both shortly after (maximal anticoagulant effect) and long after (no anticoagulant effect) pretreatment. We think that, under the experimental conditions we used, heparin prevented venous thrombosis at least partially by protection of the endothelial cells, through unknown mechanisms.

Application of the method of phage T4 DNA ligase-catalyzed ring-closure to the study of DNA structure: II. NaCl-dependence of DNA flexibility and helical repeat

In this work, we demonstrate that it is possible to determine the molar cyclization factor j M from single ligation reactions in which both circular and linear dimer DNA species are formed concurrently from linear monomers. This approach represents a significant improvement over previous methods, in which j M is evaluated from the ratio of the rate constants for two separate processes; namely (1) the cyclization of linear DNA and (2) the association of two linear molecules to form linear dimers. Determination of j M for a 366 base-pair molecule yields 5.8 × 10 −8 m, in close agreement with the value of 5.6 × 10 −8 m determined by Shore et al. for the same molecule. Using the current approach for the determination of j M, we have investigated the dependence on NaCl concentration (0 to 162 m m-NaCl, 1 m m-MgCl 2) of both the lateral and torsional flexibilities of DNA. The principal observation is that both quantities are essentially constant over the above range of NaCl concentrations, with the persistence length P ≈ 450 (±15) A ̊ , and the torsional elastic constant C ≈ 2.0 (±0.2) × 10 −19 erg cm. These observations are in accord with the previous theoretical prediction that P becomes essentially independent of NaCl concentration above 10 to 20 m m. We have examined the dependence of the helical repeat of DNA on NaCl concentration over the above range, and have found the value of 10.44 base-pairs per turn to be essentially constant over that range. This last result suggests that earlier studies have overestimated the dependence of DNA helical twist on salt concentration.

Rational Methods of DNA Synthesis, Exemplified for the Preparation of Parts of the Human Proopiomelanocortin Gene

Abstract Complete genes can now be prepared in single-batch procedures a) as one long oligonucleotide strand, b) as a number of simultaneously synthesized fragments. For further increase of overall efficiency DNA fragments terminated by a 3′-ribonucleoside can be joined by solid-phase single strand ligation using RNA ligase. This paper describes the application of these techniques to the preparation of parts of the human proopiomelanocortin gene.

Chemical synthesis and expression in yeast of a gene encoding connective tissue activating peptide-III. A novel approach for the facile assembly of a gene encoding a human platelet-derived mitogen.

A synthetic gene encoding the platelet-derived factor, connective tissue activating peptide-III (CTAP-III) (Castor, C.W., Miller, J.W., and Walz, D.A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 765-769), has been expressed and secreted from Saccharomyces cerevisiae by using a yeast expression vector and an alpha-factor leader segment. Mitogenic activity reported for naturally derived CTAP-III has been demonstrated here for recombinant CTAP-III. Active CTAP-III has been purified to apparent homogeneity. Structural studies have confirmed its identity. A general approach for the facile synthesis of genes is presented which has permitted the assembly of the entire structural gene and flanking regions (280 base pairs) from 20 oligomers in a single annealing and ligation reaction pool.

Varicose veins as a source of adult human endothelial cells

Endothelial cells can be harvested from segments of adult human saphenous vein in a varicose condition removed from patients having single or bilateral vein ligation and stripping. The cells are harvested by scraping with a scalpel, seeded on to gelatin coated or Primaria flasks and are passaged by removal with a rubber policeman. The cells cultured in this manner are maintained in a growth medium that is not supplemented with growth factors. The cells grow with a cobblestone monolayer morphology, possess angiotensin converting enzyme activity and react with antibodies to Factor VIII antigen. The cells fluoresce brightly after reaction with monoclonal antibodies specific for human endothelial cells. Thus, stripped varicose vein segments provide a readily available source of endothelial cells.