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Biomedical subjects

B Akerman

Publications and source records attributed to B Akerman.

At least 19 recordsLinked to original sources
PubMed2002

Interaction of cyanine dyes with nucleic acids: XXVI. Intercalation of the trimethine cyanine dye cyan 2 into double-stranded DNA: study by spectral luminescence methods.

The interaction between double-stranded (ds) DNA and the cyanine dye Cyan 2 has been studied with spectral luminescence methods. Binding constant values have been determined by fluorescence titration and dye distribution in the two-phase system ethyl acetate-water (3.6 x 10(4) and 1.5 x 10(4) M(-1), respectively). Cyan 2 exhibits a small specificity for guanine-cytosine (GC) sequences in total DNA and synthetic polydeoxynucleotides poly(dA/dT) and poly(dGdC/dGdC). The DNA complexes with Cyan 2 are stable at high-ionic strength solution when NaCl is added. The dye molecule complexed with DNA is apparently shielded from the anionic quencher--iodide ion. The negative linear dichroism of the visible absorption band of aligned Cyan 2-DNA complexes indicates that the bound dye lies almost perpendicularly to the DNA helix axis. The linear dichroism of the absorption band at 260 nm suggests a considerable change in the DNA B-form. The results are consistent with an intercalative binding interaction between Cyan 2 and ds DNA.

Carbocyanines↗
PubMed2001

17-beta-estradiol induces an inhibitor of active caspases.

We have shown previously that caspase-6 activity is lethal to human neurons (LeBlanc et al., 1999; Zhang et al., 2000). Here we find that 17-beta-estradiol but not 17-alpha-estradiol prevents caspase-6-mediated neuronal cell death. 17-beta-estradiol-treated neuronal extracts directly inhibit recombinant active caspase-6, caspase-3, caspase-7, and caspase-8 in vitro. We conclude that 17-beta-estradiol induces a caspase inhibitory factor (CIF) that is preventing neuronal apoptosis. The induction of CIF occurs within 10 min of 17-beta-estradiol exposure to neurons, does not require de novo protein synthesis, and involves mitogen-activated protein kinase activation. The effect is antagonized by the estrogen receptor antagonist tamoxifen. In contrast, 17-beta-estradiol does not induce CIF or prevent caspase-mediated cell death in cultured astrocytes. CIF does not act through oxidation of the caspase active site. CIF activity copurifies with proteins of between 12 and 14 kDa in size. Our results indicate that 17-beta-estradiol induces an inhibitor of active caspases through a receptor-mediated nongenomic pathway and provide an additional mechanism for the neuroprotective action of 17-beta-estradiol that is likely highly relevant to the understanding of the role of estrogen against Alzheimer's disease.

Apoptosis↗
PubMed2001

Photobleaching of asymmetric cyanines used for fluorescence imaging of single DNA molecules.

The photobleaching of the cyanine dyes YO and YOYO has been investigated for both free and DNA-bound dyes, using absorption and fluorescence spectroscopy coupled with fluorescence microscopy. For the free dyes, the nature of the reactive species involved in the photodegradation process is different for the monomer and the dimer, as shown by scavenger studies. For DNA-bound dyes, photoinduced fading of the visible absorption band occurs by different pathways depending on the drug binding mode and can be attenuated by appropriate scavengers. However, none of these scavengers were found to have any significant effect on the photobleaching of dye fluorescence. It appears that the reduction of fluorescence intensity comes from a quenching of the dye fluorescence by modified DNA bases, possibly 8-oxo-7,8-dihydro-2'-deoxyguanosine.

Animals↗
PubMed2000

Electroinjection of colloid particles and biopolymers into single unilamellar liposomes and cells for bioanalytical applications.

A combined electroporation and pressure-driven microinjection method for efficient loading of biopolymers and colloidal particles into single-cell-sized unilamellar liposomes was developed. Single liposomes were positioned between a approximately 2-microm tip diameter solute-filled glass micropipet, equipped with a Pt electrode, and a 5-microm-diameter carbon fiber electrode. A transient, 1-10 ms, rectangular waveform dc voltage pulse (10-40 V/cm) was applied between the electrodes, thus focusing the electric field over the liposome. Dielectric membrane breakdown induced by the applied voltage pulse caused the micropipet tip to enter the liposome and a small volume (typically 50-500 x 10(-15) L) of fluorescein, YOYO-intercalated T7-phage DNA, 100-nm-diameter unilamellar liposomes, or fluorescent latex spheres could be injected into the intraliposomal compartment. We also demonstrate initiation of a chemical intercalation reaction between T2-phage DNA and YOYO-1 by dual injection into a single giant unilamellar liposome. The method was also successfully applied for loading of single cultured cells.

Biopolymers↗
PubMed2000

Enhanced capacity for electrophoretic capture of plasmid DNA by agarase treatment of agarose gels.

Agarase was used investigate the effect of increasing the number of polymer ends on the electrophoretic trapping of circular DNA in agarose gels. The electric field strength required to trap circular DNA was found to be the same in control and treated gels, indicating the treatment did not result in longer traps. Loading experiments indicated that treated gels had a significantly higher capacity for the open circular DNA. Electrophoretic mobility measurements using pulsed fields indicated a higher density of active traps for treated gels compared to controls. Linear dichroism experiments showed that impalement occurred by a fast and a slow process that had characteristic time constants in the one and tens of seconds ranges, respectively. The open circular DNA was more efficiently impaled in the treated gel compared to the control. The considerably higher efficiency of trapping indicated that agarase treatment increased the concentration of traps substantially.

DNA↗
PubMed1998

Effects of supercoiling in electrophoretic trapping of circular DNA in polyacrylamide gels.

Electrophoretic velocity and orientation have been used to study the electric-field-induced trapping of supercoiled and relaxed circular DNA (2926 and 5386 bp) in polyacrylamide gels (5% T, 3.3% C) at 7.5-22.5 V/cm, using as controls linear molecules of either the same contour length or the same radius of gyration. The circle-specific trapping is reversible. From the duration of the reverse pulse needed to detrap the molecules, the average trap depth is estimated to be 90 A, which is consistent with the molecular charge and the field strengths needed to keep molecules trapped. Trapped circles exhibit a strong field alignment compared to the linear form, and there is a good correlation between the enhanced field alignment for the circles and the onset of trapping in both constant and pulsed fields. The circles do not exhibit the orientation overshoot response to a field pulse seen with linear DNA, and the rate of orientation growth scales as E(-2+/-0.1) with the field, as opposed to E(-1.1+/-0.1) for the linear form. These results show that the linear form migrates by cyclic reptation, whereas the circles most likely are trapped by impalement on gel fibers. This proposal is supported by very similar velocity and orientation behavior of circular DNA in agarose gels, where impalement has been deemed more likely because of stiffer gel fibers. The trapping efficiency is sensitive to DNA topology, as expected for impalement. In polyacrylamide the supercoiled form (superhelical density sigma = -0.05) has a two- to fourfold lower probability of trapping than the corresponding relaxed species, whereas in agarose gels the supercoiled form is not trapped at all. These results are consistent with existing data on the average holes in the plectonemic supercoiled structures and the fiber thicknesses in the two gel types. On the basis of the topology effect, it is argued that impalement during pulsed-field electrophoresis in polyacrylamide gels may be useful for the separation of more intricate DNA structures such as knots. The results also indicate that linear dichroism on field-aligned molecules can be used to measure the supercoiling angle, if relaxed DNA circles are used as controls for the global degree of orientation.

Acrylic Resins↗
PubMed1997

Effects of local changes in the helix flexibility on electrophoretic migration of DNA in agarose gel.

We present a study of how kinks, flexible bends, and flexible joints in the DNA helix, induced by binding cis-diamminedichloroplatinum(II) (cis-DDP), transdiamminedichloroplatinum(II) (trans-DDP), and chlorodiethylenetriammineplatinum(II) (dien-Pt) to the DNA, affect the electrophoretic migration of DNA in agarose gels. For long DNA the conformation oscillates between extended and compact states during the migration, as for native DNA. The presence of flexible joints decreases both the length of time and the step length of the cycles, but in a compensatory manner so that there is no net effect on the mobility. This demonstrates that in some cases mobility alone cannot detect pertubations in the DNA helix. Kinks and flexible bends reduce the mobility because they both lead to longer time periods of the cycles. With kinks the reduction is strongest at low fields because at high fields the kinks are straightened out; the steps thus become even longer than for native DNA. The results suggest that a combination of mobility and orientation measurements on reptating DNA can be used for distinguishing different kinds of structural alterations in the DNA.

Bacteriophage lambda↗
PubMed1996

Single- and double-strand photocleavage of DNA by YO, YOYO and TOTO.

Photocleavage of dsDNA by the fluorescent DNA stains oxazole yellow (YO), its dimer YOYO) and the dimer TOTO of thiazole orange (TO) has been investigated as a function of binding ratio. On visible illumination, both YO and YOYO cause single-strand cleavage, with an efficiency that varies with the dye/DNA binding ratio in a manner which can be rationalized in terms of free dye being an inefficient photocleavage reagent and externally bound dye being more efficient than intercalated dye. Moreover, the photocleavage mechanism changes with binding mode. Photocleavage by externally bound dye is, at least partly, oxygen dependent with scavenger studies implicating singlet oxygen as the activated oxygen intermediate. Photocleavage by intercalated dye is essentially oxygen-independent but can be inhibited by moderate concentrations of beta- mercaptoethanol--direct attack on the phosphoribose backbone is a possible mechanism. TOTO causes single-strand cleavage approximately five times less efficiently than YOYO. No direct double-strand breaks (dsb) are detected with YO or YOYO, but in both cases single-strand breaks (ssb) are observed to accumulate to eventually produce double-strand cleavage. With intercalated YO the accumulation occurs in a manner consistent with random generation of strand lesions, while with bisintercalated YOYO the yield of double-strand cleavage (per ssb) is 5-fold higher. A contributing factor is the slow dissociation of the bis-intercalated dimer, which allows for repeated strand-attack at the same binding site, but the observation that the dsb/ssb yield is considerably lower for externally bound than for bis-intercalated YOYO at low dye/DNA ratios indicates that the binding geometry and/or the cleavage mechanism are also important for the high dsb-efficiency. In fact, double-strand cleavage yields with bis-intercalated YOYO are higher than those predicted by simple models, implying a greater than statistical probability for a second cleavage event to occur adjacent to the first (i.e. to be induced by the same YOYO molecule). With TOTO the efficiency of the ssb-accumulation is comparable to that observed with YOYO.

Bacteriophage phi X 174↗
PubMed1996

Cyclic migration of DNA in gels: DNA stretching and electrophoretic mobility.

Available data from spectroscopic and microscopy studies of electrophoretic orientation of long DNA (above 40 kbp) in agarose gels is analyzed on the basis of the fact that the migration in constant fields is cyclic in nature. Defining a cycle period as the time between two consecutive compact states, a simple model is used to obtain data on the average time period (< T >) and the step length (< L >) of the migration cycle from spectroscopic measurements of the dynamics of helix orientation and center-of-mass velocity. Furthermore, the degree of orientation is used to analyze tube-orientation and DNA stretching contributions to < L > and < T >. Finally, the average electrophoretic velocity v = < L >/< T > is analyzed in terms of < L > and < T > for different DNA sizes (Lc), field strengths (E), and gel concentrations (A). The main results of the analysis are: (i) the increase and saturation of the electrophoretic mobility with increasing E is mainly governed by < L > via the degree of DNA stretching, (ii) DNA molecules of different sizes migrate with the same velocity because < L > and < T > both increase approximately linearly with Lc, and (iii) migration in a denser gel is slower mainly because < T > increases, while the step length is approximately constant. Assuming the charge Q of DNA is the same as in free solution, these results suggest that the reason the fundamental reptation equation for the electrophoretic mobility mu = (Q/zeta) < (hx/Lt)2 > also applies in the presence of strong fluctuations in the tube length Lt, and end-to-end distance hx, is that the friction coefficient zeta for motion along the tube is lower the more stretched the DNA is.

DNA↗
PubMed1995

Double bands in DNA gel electrophoresis caused by bis-intercalating dyes.

Many bis-intercalating dyes used for fluorescence detection of DNA in electrophoresis have been reported to give band-splitting and band-broadening, which results in poor resolution and a decreased detection sensitivity. We have studied the dimeric dye YOYO-1, and to some extent also TOTO-1 and EthD-1, and found that in complex with DNA these dyes give rise to two components with different electrophoretic mobilities. Electrophoresis experiments and spectroscopic measurements on the two components show that they differ in that the DNA molecules have different amounts of dye bound. Our results exclude that the extra bands are caused by intermolecular cross-linking. Incubation of the samples for increasing times before electrophoresis makes the bands move closer and closer to each other as the dye molecules become more homogeneously distributed among the DNA molecules. Finally, the two bands merge into one at an intermediate position. This equilibration process is extremely slow at room temperature (days), and is therefore not a practical method to eliminate band-splitting in routine analysis. However, we find that if the temperature is raised to 50 degrees C, the dye-DNA complexes equilibrate completely in only 2 h.

Bacteriophage T7↗
PubMed1995

Isolation of a cDNA encoding human holocarboxylase synthetase by functional complementation of a biotin auxotroph of Escherichia coli.

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of the four biotin-dependent carboxylases in human cells. Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS is targeted to the mitochondria and cytoplasm. We isolated 21 human HCS cDNA clones, in four size classes of 2.0-4.0 kb, by complementation of an Escherichia coli birA mutant defective in biotin ligase. Expression of the cDNA clones promoted biotinylation of the bacterial biotinyl carboxyl carrier protein as well as a carboxyl-terminal fragment of the alpha subunit of human propionyl-CoA carboxylase expressed from a plasmid. The open reading frame encodes a predicted protein of 726 aa and M(r) 80,759. Northern blot analysis revealed the presence of a 5.8-kb major species and 4.0-, 4.5-, and 8.5-kb minor species of poly(A)+ RNA in human tissues. Human HCS shows specific regions of homology with the BirA protein of E. coli and the presumptive biotin ligase of Paracoccus denitrificans. Several forms of HCS mRNA are generated by alternative splicing, and as a result, two mRNA molecules bear different putative translation initiation sites. A sequence upstream of the first translation initiation site encodes a peptide structurally similar to mitochondrial presequences, but it lacks an in-frame ATG codon to direct its translation. We anticipate that alternative splicing most likely mediates the mitochondrial versus cytoplasmic expression, although the elements required for directing the enzyme to the mitochondria remain to be confirmed.

Amino Acid Sequence↗
PubMed1994

Local analgesic and vascular effects of intradermal ropivacaine and bupivacaine in various concentrations with and without addition of adrenaline in man.

Ropivacaine, a new long-acting amino-amide local anaesthetic agent, and bupivacaine, in various concentrations with or without addition of adrenaline, were tested in a randomized, double-blind study using intradermal wheals. Ten non-smoking, healthy, young male volunteers participated. In series I plain solutions of ropivacaine (0.25%, 0.5%, 0.75% and 1%) and bupivacaine (0.25%, 0.5% and 0.75%) were injected intradermally and in series II the same concentrations, with the addition of adrenaline 5 ug.ml-1 (1:200,000), were used. The same volunteers took part in both series, with an interval of at least three weeks between the experiments. Saline was included as control in both series. Pin-pricking was used to assess the dermal analgesia. Plain solutions of ropivacaine produced significantly longer durations of dermal analgesia than did plain solutions of bupivacaine, in all tested concentrations. A significant increase in duration was seen for both local anaesthetics when adding adrenaline. Local vascular effects at the injected areas were determined by visual inspection (nil, pink, pale). Local blanching (pale) was significantly more frequent for plain solutions of ropivacaine, in all tested concentrations. Local redness (pink) was significantly more frequent with plain bupivacaine, in a dose-dependent relation. An initial redness was frequently observed for both local anaesthetics containing adrenaline, followed by blanching at most sites.

Adult↗
PubMed1994

On the relative potency of amino-amide local anaesthetics in vivo.

With the aim of comparing the analgesic effectiveness of lidocaine, prilocaine, bupivacaine and etidocaine in vivo, a study of the relationships between dose and duration of infraorbital nerve block (IONB) of various intensities (IONB degrees 3-10) was performed in the rat. With increasing doses longer durations of action were obtained. Further analyses were performed using multiple regression analysis. The log (dose)-duration lines for bupivacaine and etidocaine were found to be linear, whereas those for lidocaine in degree 3 and degree 10 IONB and for prilocaine in degree 10 IONB were so only after omission of some data. The only comparisons yielding no deviation from parallelism of log (dose)-duration lines were etidocaine vs. lidocaine at IONB degree 10 and lidocaine vs. prilocaine and bupivacaine vs. etidocaine at IONB degree 3. The difference between these agents with respect to their duration of action at all dose levels amounted to 11 +/- 3 minutes (M +/- s.e.m.) for etidocaine vs. lidocaine (IONB degree 10), 27 +/- 4 min for prilocaine vs. lidocaine and 54 +/- 5 min for bupivacaine vs. etidocaine (IONB degree 3). For all other comparisons the log (dose)-duration lines deviated from parallelism, i.e. differences between agents with respect to their duration of action were found to be dose-dependent. The slopes of the log (dose)-duration lines were found to correlate closely to the log (partition coefficient) and log (protein binding) of the investigated agents.

Anesthetics, Local↗
PubMed1993

The effects of venous blood, silicone gel and prilocaine on capsular contraction around silicone implants studied with a new method in the rat.

The pressure exerted by wound tissue on a subcutaneously implanted silicone expander was studied in rats. A significant increase in pressure was noted four and eight weeks postoperatively when venous blood was instilled into the cavity around the expander, but this increase was significantly reduced at eight weeks if 1.0 ml of prilocaine 5 mg/ml with adrenaline 4 micrograms/ml was instilled at the same time as the blood. Instillation of silicone gel outside the expander tended to increase the pressure eight weeks postoperatively, but there was no increase when silicone gel was given together with prilocaine and adrenaline. The next step is to investigate whether prilocaine with adrenaline instilled around an implanted mammary prosthesis in humans is not only capable of reducing pain and bleeding but can also inhibit capsular contracture.

Animals↗
PubMed1989

Orientational dynamics of T2 DNA during agarose gel electrophoresis: influence of gel concentration and electric field strength.

The understanding, on a molecular level, of the mechanisms responsible for the improved separation in DNA gel electrophoresis when using modulated electric fields requires detailed information about conformational distribution and dynamics in the DNA/gel system. The orientational order due to electrophoretic migration ("electrophoretic orientation") is an interesting piece of information in this context that can be obtained through linear dichroism spectroscopy [M. Jonsson, B. Akerman, and B. Nordén, (1988) Biopolymers 27, 381-414]. The technique permits measurement of the orientation factor S of DNA (S = 1 corresponds to perfect orientation) within an electrophoretic zone in the gel during the electrophoresis. It is reported that the degree of orientation of T2 DNA [170 kilo base pairs (kpb)] is considerable (S = 0.17 in 1% agarose at 10 V/cm) compared to relatively modest orientations of short fragments found earlier (for 23-kbp DNA, S = 0.03 in 1% agarose at 10 V/cm), showing that large DNA coils are substantially deformed during the migration. Growth and relaxation dynamics of the orientational order of the T2 DNA are also reported, as functions of gel concentration (0.3-2%), electric field strength (0-40 V/cm), and pulse characteristics. The rise profile of the DNA orientation, when applying a constant field, is a nonmonotonic function that displays a pronounced overshoot, followed by a minor undershoot, before it reaches steady-state orientation (after 12 s in 1% agarose, 9 V/cm). The orientational relaxation in absence of field shows a multiexponential decay in a time region of some 10 s, when most of the DNA anisotropy has disappeared. A surprising phenomenon is a memory over minutes of the DNA/gel system to previous pulses: with two consecutive rectangular pulses (of the same polarity), the orientational overshoot and undershoot as a response to the second pulse are significantly reduced compared to the first pulse. The time required to recover 90% of their amplitudes is typically 1200 s (1% agarose, 9 V/cm), which may be compared to the time required to relax 90% of the DNA orientation, which is only 6 s. The major part of the over- and undershoot recovery is thus a reorganization of a system in which DNA is already randomly oriented. The different response amplitudes and relaxation times, including the amplitude and recovery time of the overshoot, of the orientational order of DNA in the electrophoretic gel have been studied as functions of gel concentration and field strength. The results are discussed against relevant theories of polymer dynamics.

Chemical Phenomena↗