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PubMed1983

The influence of systemic arterial pressure and intracranial pressure on the development of cerebral vasogenic edema.

The influence of intracranial pressure (ICP), systemic arterial pressure (SAP), and cerebral perfusion pressure (CPP) upon the development of vasogenic cerebral edema is largely unknown. To study their relationship, the authors have produced an osmotic disruption of the blood-brain barrier unilaterally in rabbits by injecting 1 cc/kg of 2M NaCl into the left internal carotid artery. The amount of vasogenic edema produced was assessed by quantitation of the extravasation of Evans blue dye into the area of maximum blood-brain barrier breakdown by means of optical densitometry following formamide extraction. The ICP was measured using a cisterna magna catheter into which mock cerebrospinal fluid could be infused at a predetermined pressure. The SAP was controlled by exsanguination from a femoral artery catheter. In 18 animals in which blood pressure was not controlled, no significant relationship between the ICP and the degree of Evans blue dye extravasation was noted. In these animals, however, a direct relationship between CPP (defined as mean arterial pressure minus mean ICP) and extravasation of Evans blue dye was found (correlation coefficient 0.630; p less than 0.001). When ICP was held constant at 0 to 5 mm Hg in another group of 16 animals and different levels of blood pressure were produced by exsanguination, a significant direct relationship between extravasation of Evans blue dye and the SAP was found (correlation coefficient 0.786; p less than 0.001). In a third group of 20 animals, the blood pressure was held constant at 90 to 100 mm Hg and the ICP was varied between 0 and 75 mm Hg. There was a highly significant result indicating increasing Evans blue dye extravasation with lower levels of ICP (p less than 0.001). Cerebral blood flow determinations by the hydrogen clearance method indicated loss of autoregulation in all animals in the areas of brain injured by intracarotid hypertonic saline. These results indicate that high SAP and low ICP (that is, a large CPP) promote Evans blue dye extravasation in this model of blood-brain barrier disruption. This finding has implications for the management of patients with vasogenic edema.

Animals↗
PubMed2004

Pharmacokinetics of controlled-release polymers in the subarachnoid space after subarachnoid hemorrhage in rabbits.

OBJECT: Implantation of controlled-release polymers into the subarachnoid space to deliver drugs for treatment of vasospasm after subarachnoid hemorrhage (SAH) is currently of interest. Among the issues regarding local delivery of drugs in the subarachnoid space, however, are the extent of diffusion and the rate of release of the loaded agents. In this study Evans blue dye (EBD) was loaded into controlled-release polymers and its pharmacokinetic properties were determined in vitro and in vivo by using a rabbit model of SAH. METHODS: Ethylene-vinyl acetate copolymer (EVAc) was loaded 40% (w:w) with EBD and its pharmacokinetics were spectrophotometrically determined in vitro by examining three EBD-EVAc polymers. Additional polymers were implanted either into the frontal lobe or into the cisterna magna of 16 New Zealand White rabbits. Subarachnoid hemorrhage was induced in eight of the animals by an injection of 1.5 ml of arterial blood into the cisterna magna. The animals were killed 3 or 14 days postoperatively, their brains and spinal cords were harvested, and samples of each were placed in formamide for dye extraction and quantification. Specimens were examined macroscopically and the concentrations of EBD were determined with the aid of a spectrophotometer. The EBD-EVAc polymers continuously released EBD over a 133-day period. The controlled release of the dye into the subarachnoid space in either location resulted in staining of the entire central nervous system (CNS) in rabbits when the polymers were placed either on the frontal lobe or in the cisterna magna. The EBD diffusion covered a distance of at least 40 cm. The presence of blood in the subarachnoid space did not interfere with the diffusion. CONCLUSIONS: In this study the authors define the rate and extent of diffusion of EBD from controlled-release polymers placed in the subarachnoid space under conditions of SAH. Evans blue dye diffused through the entire rabbit CNS, covering a distance greater than that of the longest dimension of the hemicircumference of the subarachnoid space around the human brain. The pharmacokinetic properties of EBD-EVAc polymers are comparable to those of antivasospasm agents that are successfully used in animal models of SAH.

Animals↗
PubMed1995

An alternative synthesis of the NMDA antagonist CGS 19755 via free radical carbamoylation of ethyl isonicotinate.

The NMDA antagonist CGS 19755 (cis-4-phosphonomethyl-2-piperidinecarboxylic acid) has been prepared by applying Minisci reaction conditions [formamide, hydrogen peroxide, iron(II) sulfate] to ethyl isonicotinate, reduction of the ester with sodium borohydride, alcoholysis of the 2-carboxamide, formation of 4-(diethylphosphonomethyl)-2-pyridinecarboxylate, hydrogenation of the pyridine nucleus, and acid hydrolysis. The overall, unoptimized yield was around 11%. The procedure employs cheap starting materials, is practical and avoids the use of toxic and hazardous cyanotrimethylsilane which is used in the published procedure.

Free Radicals↗
PubMed2006

Structural basis for the interaction of CCR5 with a small molecule, functionally selective CCR5 agonist.

The chemokine receptor CCR5 is an attractive target for HIV-1 drug development, as individuals whose cells lack surface CCR5 expression are highly resistant to HIV-1 infection. CCR5 ligands, such as CCL5/RANTES, effectively inhibit HIV-1 infection by competing for binding opportunities to the CCR5 and inducing its internalization. However, the inherent proinflammatory activity of the chemotactic response of CCR5 ligands has limited their clinical use. In this study, we found that a novel small molecule, functionally selective CCR5 agonist, 2,2-dichloro-1-(triphenylphosphonio)vinyl formamide perchlorate (YM-370749), down-modulates CCR5 from the cell surface without inducing a chemotactic response and inhibits HIV-1 replication. In molecular docking studies of YM-370749 and a three-dimensional model of CCR5 based on the rhodopsin crystal structure as well as binding and functional studies using various CCR5 mutants, the amino acid residues necessary for interaction with YM-370749 were marked. These results provide a structural basis for understanding the activation mechanism of CCR5 and for designing functionally selective agonists as a novel class of anti-HIV-1 agents.

Animals↗
PubMedDate not supplied

The DNA-instability test as a specific marker of malignancy and its application to detect cancer clones in borderline malignancy.

Recent progress in cytogenetic and biochemical mutation assay technologies has enabled us to detect single gene alterations and gross chromosomal rearrangements, and it became clear that all cancer cells are genetically unstable. In order to detect the genome-wide instability of cancer cells, a new simple method, the DNA-instability test, was developed. The methods to detect genomic instability so far reported have only demonstrated the presence of qualitative and quantitative alterations in certain specific genomic loci. In contrast to these commonly used methods to reveal the genomic instability at certain specific DNA regions, the newly introduced DNA-instability test revealed the presence of physical DNA-instability in the entire DNA molecule of a cancer cell nucleus as revealed by increased liability to denature upon HCl hydrolysis or formamide exposure. When this test was applied to borderline malignancies, cancer clones were detected in all cases at an early-stage of cancer progression. We proposed a new concept of "procancer" clones to define those cancer clones with "functional atypia" showing positivities for various cancer markers, as well as DNA-instability testing, but showing no remarkable ordinary "morphological atypia" which is commonly used as the basis of histopathological diagnosis of malignancy.

Animals↗
PubMed2004

Dextran from Leuconostoc mesenteroides augments immunostimulatory effects by the introduction of phosphate groups.

The immunological effects of phosphorylated dextran (in which phosphate groups were chemically introduced) on murine splenocytes were examined. When dextran produced by Leuconostoc mesenteroides was phosphorylated by a reaction with polyphosphoric acid in formamide solution for 48 h, the degree of phosphorylation of dextran was maximal. The highest phosphorus content (1.7%, wt/wt) was observed in 40 kDa of dextran. The mitogenic response of murine splenocytes was enhanced by the phosphorylated dextran, but its activity was not related to its molecular weight. A strong response was detected at a concentration of 10 to 500 microg/ml, and the highest activity was obtained 48 h after stimulation. Phosphorylated dextran was characterized as a B-cell-specific mitogen. The expressions of CD86 on CD8alpha- CD11c- and CD8alpha- CD11c+ cells were augmented by phosphorylated dextran. The levels of mRNA expression of gamma interferon and interleukin-10 on murine splenocytes were also increased by the stimulation. These results demonstrate that dextran exerts immunostimulation by the introduction of phosphate groups.

Animals↗
PubMed1975

Charcoal sampling method for determining the concentration of styrene in air.

The vapors of styrene (vinylbenzene) were trapped on charcoal in a glass tube. Air from the worker's breathing zone was sucked through the tube by a battery-operated, modified, MSA personal sampling pump. The sampler and pumping system were worn by the worker. The styrene were eluted with dimethyl formamide and estimated by gas chromatography. The range and sensitivity of the method when sampling with a flow rate of 0.2 1/min during 1 h were 5 to 1,500 ppm, and during 4 h they were 2 to 400 ppm. The recovery (accuracy) was over 90%. The precision for the method, in terms of relative standard deviation, was 8.4%.

Air Pollutants↗
PubMed1992

Cloning of the 5' flanking region of the murine laminin B1 gene by genomic PCR.

Induction of the murine laminin B1 gene in F9 cells occurs 24-48 hours after the retinoic acid (RA) addition. In order to reveal the mechanism of the late induction of the laminin B1 gene, it is necessary to understand fully the promoter structure of it. We report here that the promoter region of the laminin B1 was rapidly isolated utilizing the genomic PCR technique. MgCl2, formamide concentration, and annealing temperature were optimized for PCR. The result showed that MgCl2 concentration profoundly affects the efficiency in amplifying the specific DNA. The annealing temperature (51 degrees C-63 degrees C) did not significantly affect the yield. Under the optimal conditions, about 50 ng of the specific DNA was obtained from 1 microgram of total genomic DNA after 20 cycles of amplification, indicating that approximately 2 x 10(5) fold specific amplification had occurred. Southern blot analysis and sequence data proved that the amplified DNA fragment contained the promoter region of the laminin B1 gene.

Animals↗
PubMed1976

Isolation of rat liver albumin messenger RNA.

Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.

Albumins↗
PubMed1998

Synthesis of biologically active bis-[4-amino-5-mercapto-1,2,4- triazol-3yl]-alkanes and their derivatives.

The preparation of bis-(4-amino-5-mercapto-1,2,4-triazol-3yl)alkanes is described. These bis-(amino-mercapto-triazolyl) alkanes are characterized by analytical and spectral data. They are also characterized by the preparation of the Schiff bases by condensing them with various benzaldehydes and nitro furfural diacetate in dimethyl formamide-ethanol medium in the presence of concentrated sulphuric acid. The structures of Schiff bases are assigned based on analytical and spectral data. The newly synthesized compounds are screened for their antibacterial properties against Gram-positive and Gram-negative bacteria. Most of them showed significant activity.

Anti-Bacterial Agents↗
PubMed1999

[Rapid detection of non-deletional alpha-thalassemia mutations by PCR-LIS-SSCP].

OBJECTIVE: To establish a rapid and convenient single strand conformation polymorphism (SSCP) analysis method for detecting the point mutation of non-deletional alpha-thalassemia. METHODS: The 543bp DNA fragment spanning the hot spot region mainly responsible for non-deletional alpha-thalassemia was nested amplified using the selective amplification of alpha2 globin gene as a template and was denatured with low ionic strength(LIS) solution followed by SSCP analysis. RESULTS: LIS buffer was more efficient for ssDNA formation than formamide buffer was and the formation of ssDNA was very stable. In addition to a normal electrophoresis pattern, at least three SSCP profiles can be detected by the present method when the DNA samples bearing non-deletional genes of Hb H disease were screened. Confirmed by DNA sequencing analysis, the DNAs represented these profiles have turned out to be the different three mutants, i.e., the alpha&supCS; mutation, the alpha &supQS; mutation and the alpha&supWestmead; mutation, respectively. Only 3 hours were needed to complete the electrophoresis procedure of this method. CONCLUSION: PCR-LIS-SSCP can be used as a tool in rapid screening for the alterations in human alpha-globin gene.

Humans↗
PubMed1999

[Rapid detection of non-deletional alpha-thalassemia mutations by PCR-LIS-SSCP]

OBJECTIVE: To establish a rapid and convenient single strand conformation polymorphism (SSCP) analysis method for detecting the point mutation of non-deletional alpha-thalassemia. METHODS: The 543bp DNA fragment spanning the hot spot region mainly responsible for non-deletional alpha-thalassemia was nested amplified using the selective amplification of alpha2 globin gene as a template and was denatured with low ionic strength(LIS) solution followed by SSCP analysis. RESULTS: LIS buffer was more efficient for ssDNA formation than formamide buffer was and the formation of ssDNA was very stable. In addition to a normal electrophoresis pattern, at least three SSCP profiles can be detected by the present method when the DNA samples bearing non-deletional genes of Hb H disease were screened. Confirmed by DNA sequencing analysis, the DNAs represented these profiles have turned out to be the different three mutants, i.e., the alphaCS mutation, the alpha QS mutation and the alphaWestmead mutation, respectively. Only 3 hours were needed to complete the electrophoresis procedure of this method. CONCLUSION: PCR-LIS-SSCP can be used as a tool in rapid screening for the alterations in human alpha-globin gene.

Journal Article↗
PubMed1976

Alkali-labile regions and strand breaks in DNA from cells treated with daunorubicin.

Lymphocyte cells (P-288) in culture were exposed to daunorubicin (Dm) and analyzed for DNA damage by means of alkaline sucrose and neutral (formamide) sucrose gradients. In neutral gradients, the sedimentation rate of DNA from cells treated with 25 X 10(-8) M Dm for brief periods up to 60 min is the same as that of the DNA from control cells (i.e., drug-treated for "zero" time). In contrast, extensive DNA damage is already apparent in alkaline gradients after treatment of the cells for only 30 min. After treatment for 2 hr, single-strand damage is evident in both types of gradient. These results demonstrate the introduction of alkali-labile regions in the DNA of cells treated for 45 or 60 min with Dm. The DNA damage is concurrent with inhibition of thymidine incorporation, and both may occur at threshold levels without affecting subsequent growth of the P-288 cells after short exposures to low concentrations of the drug.

Animals↗
PubMedDate not supplied

Meso-substituted cationic porphyrins interact with dsDNA and exhibit different localization patterns in radiation-induced fibrosarcoma cells.

Meso-substituted cationic porphyrins were examined for binding dsDNA. Subcellular localization time studies used Confocal Laser Scanning Microscopy of radiation-induced fibrosarcoma (RIF) cells incubated with porphyrins. Binding studies revealed a reversible interaction between porphyrin and dsDNA that is a function of DNA shape. Binding was inhibited at high salt concentrations, and enhanced by heat and DNA denaturants such as dimethyl formamide (DMF). Trans dicationic porphyrin required more stringent binding conditions than cis dicationic and tetracationic porphyrins. Phenol extraction of porphyrin from the DNA-porphyrin complex demonstrates that cationic porphyrins do not damage dsDNA at high concentrations. Localization studies within a 24-hour range reveal different distribution patterns. Metal chelates of tetracationic porphyrin exhibited a cytoplasmic localization with the exception of the zinc chelate. Localization of other metal chelates appears to be redistributed to lysosomes and mitochondria between 3 and 6 hours post-incubation. HPPH used in PDT clinical trials localizes to the cytoplasmic compartment.

Animals↗
PubMed1999

[Blood-group genotyping by constant denaturing gel electrophoresis (CDGE)].

Constant Denaturing Gel Electrophoresis (CDGE) was used as an analytical method of the genetic markers. Even a single base change in a fragment amplified by PCR was detected exactly by CDGE. The computational simulation of CDGE gave the calculation whether a single base change in a fragment amplified by PCR could be detected by CDGE or not. In this report, genotyping of three blood groups, MN, Duffy and Kidd, and Gc system is described. The regions reflecting allelic differences of each system were amplified from genomic DNAs. The concentration of denaturants (urea and formamide) in CDGE gels was decided with the computational simulation as follows: 15% for MN, 27% for Duffy, 24% for Kidd, 30% for Gc. CDGE was run in TBE buffer at 60 degrees C, 100 V constant voltage. PCR amplified fragments with 1-3 base changes were separated clearly in each gel. By staining the gels with ethidium bromide, the genotype of each system was determined. The genotyping of system by CDGE can avoid mistakes in the conventional method, which requires complicated and multiple troublesome operations. Analysis of PCR amplified fragments by CDGE will make a beneficial contribution to medico-legal practice.

Blood Group Antigens↗
PubMedDate not supplied

[Molecular analysis and prenatal diagnosis of beta-thalassemia: about our experience in central Tunisia].

Beta-thalassemia, by its high frequency and its heterogeneity, constitutes a real problem of health in Tunisia. Prenatal diagnosis by DNA analysis represents the only reality for couples at risk. The denaturant gradient (urea and formamide) on polyacrylamide gel electrophoresis has been performed in our laboratory, using psoralen as chemical clamps. This method is simple, reliable, safe, rapid, without radioactivity and has a reasonable cost (chemical clamps). Even if it needs an informatic modelization in other laboratories, this method seems to be adapted to our economic and work conditions and to the molecular heterogeneity of the Tunisian beta-thalassemia. We present the results of an epidemiological molecular study on 75 patients with beta-thalassemia and the results of ten prenatal diagnosis. The molecular lesions codon 39 (C-T) and IVS1 nt2 (T-G) are the most frequent in our study. This technical approach provides genetic counselling for at risk families by offering prenatal diagnosis (reducing as possible the cost and the delay of the result) after prealable family study and identification of the mutation(s).

Codon↗
PubMed2000

Identification of medically important Aspergillus species by single strand conformational polymorphism (SSCP) of the PCR-amplified intergenic spacer region.

The amplified 5.8S RNA coding DNA with the neighbouring internal transcribed spacers ITS I and ITS II (ITS I--5.8S rDNA--ITS II) of 27 culture collection strains of Aspergillus fumigatus, Aspergillus flavus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus were investigated by single strand conformational polymorphism (SSCP) analysis. All strains showed a polymerase gel electrophoresis (PCR) product of 0.6 kb. Separation of DNA single strands of the PCR product in an acrylamide-bisacrylamide gel containing formamide SSCP resulted in individual patterns for each of the species. A minor variability within the species A. fumigatus and A. flavus did not affect the correct species identification. The results were confirmed when investigating 55 wild strains from patients and the environment. It is concluded that the analysis of the amplified ITS I--5.8S rDNA--ITS II region by SSCP allows the differentiation of the medically most relevant aspergilli. As the method does not require morphologically fully developed fungal colonies, it yields species diagnosis faster than the conventional macroscopic and microscopic identification.

Aspergillosis↗
PubMed1975

Size of the polyadenylic acid region of newly synthesized globin messenger ribonucleic acid.

The size of the polyadenylic acid region of newly synthesized globin mRNA was determined on mRNA isolated from nucleated erythroid spleen cells of anemic mice. The globin mRNA was purified by a combination of affinity chromatography on oligodeoxythymidylate-cellulose (oligo-(dT)-cellulose) and sucrose density gradient centrifugation. The purified RNA was shown to be globin mRNA by virtue of its ability to direct the synthesis of mouse alpha- and beta-globin chains in a cell-free system and by the presence of two bands migrating identically with authentic mouse alpha- and beta-globin mRNA when subjected to electrophoresis in polyacrylamide gels in the presence of formamide. When labeled for 1 hour with [3H]adenosine, the newly synthesized radioactive mRNA also migrated as two bands in these gels but they moved slower than the main bands suggesting that they have higher molecular weights. The polyadenylic acid region of the mRNA was isolated from the T1 and pancreatic RNase digestion mixture by acrylamide sodium dodecyl sulfate gel electrophoresis. The polyadenylic acid region was found to contain approximately 150 adenylate residues. As it is known that globin mRNA isolated from reticulocytes contains only 40 to 60 residues, it follows that at least 150 adenylic acid residues are added to the globin mRNA soon after its synthesis and that some of these are removed during the subsequent maturation of the erythroid cell.

Adenine Nucleotides↗