A simple and efficient method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded plasmid DNA.

A method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded DNA without the necessity for phenotypic selection is described. Plasmids denatured with alkali and purified by adsorption to and elution from nitrocellulose have single-stranded regions where primers can hybridize and serve as templates for a T7 DNA polymerase-catalyzed synthesis of complementary mutant DNA strands. When this procedure was carried out such that the original nonmutant strand contained uracil [method of Kunkel, Proc. Natl. Acad. Sci. USA 82(1985)488-492], mutation frequencies of between 30% and 40% were obtained. The technique has been used to generate mutant genes in plasmids of a wide variety of sizes. The largest plasmid manipulated and successfully mutagenized was 22 kb. The method is rapid and efficient and is not dependent upon either f1 phage vectors or the presence of restriction sites in the vicinity of the sequence targeted for mutation.     

Ionic strength dependence of the binding of methylene blue to chromatin and calf thymus DNA.

The binding of the intercalating dye methylene blue (MB) to chromatin and to free DNA has been studied as a function of ionic strength at very low binding ratios (1 MB/400 DNA bases) using absorption spectroscopy. With increasing salt concentration MB is displaced from chromatin to a higher extent than from DNA. The free energy change for MB binding to chromatin is found to be approximately 5 kJ/mole lower than for binding to DNA. This difference can be explained by the reduced number of high affinity binding sites in chromatin due to the presence of histone proteins. The difference in binding energy is virtually independent of the degree of chromatin condensation and also of the valence of counter ions, suggesting that neither the affinity for, nor the number of intercalation sites in the linker DNA is markedly changed upon the salt-induced condensation. The unaffected thermodynamics of the linker binding suggests that factors such as DNA superhelicity and the electrostatic influence from the chromatosomes remain unchanged during chromatin condensation.     

Cloning and characterization of two plasmids from Bacillus thuringiensis in Bacillus subtilis

Bacillus thuringiensis subspecies israliensis plasmids pTX14-1 and pTX14-3 were cloned and analyzed by Southern blot hybridization for their replication mechanism in Bacillus subtilis. The cloning of pTX14-1 into the replicon deficient vector pBOE335 showed the usual characteristics of single-stranded DNA plasmids, i.e., it generated circular single-stranded DNA and high molecular weight (HMW) multimers. The other plasmid, pTX14-3, behaved differently; it generated neither single-stranded DNA nor HMW multimers. Treatment with rifampicin did not result in the accumulation of single-stranded DNA. However, deletion of an EcoRI-PstI fragment resulted in the accumulation of both single-stranded DNA and HMW multimers. From various deletion derivatives, we have mapped the minus origin and the locus responsible for suppression of HMW multimer formation. Full activity of the minus origin and of the locus suppressing HMW formation was only observed on the native replicon, indicating a coupling to the plus strand synthesis.     

P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines

MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However, several exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported. In many of these, a correlation between P-gp expresson and transport properties can not be established. Co-amplification of genes adjacent to the mdr1 gene, mutations [122], splicing of mdr1 RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased efflux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict - but do not exclude - that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux. Furthermore, large amounts of P-gp in the plasma membrane altering the ultrastructure and generalized changes, such as increases or decreases in membrane fluidity, alterations in lipid composition, changes in transmembrane pH gradient and membrane potential have been described in MDR cell lines and could account for some of the findings.     

Compliance with diphtheria,tetanus, and pertussis immunisation in Bangladesh: factors identifying high risk groups.

OBJECTIVE--To evaluate factors associated with non-compliance with having second vaccination against diphtheria, tetanus, and pertussis in a treatment centre in Dhaka to determine which children were most at risk of not completing immunisation. DESIGN--Cohort study of infants given first dose of the vaccine and followed up six weeks later to ascertain compliance with having second dose. Factors associated with non-compliance were evaluated. SETTING--Dhaka treatment centre of the International Centre for Diarrhoeal Disease Research, Bangladesh. SUBJECTS--136 unimmunised children aged 6 weeks to 23 months who lived within reach of the treatment centre. At time of the six week follow up 16 of the children could not be traced and seven had died. INTERVENTIONS--All children received their first dose of the vaccine. In each case health education workers had informed the mother about the value of immunisation, and she was given clear instructions to bring the child back after four weeks for the second dose. MAIN OUTCOME MEASURE--Rate of non-compliance with advice to return child for second vaccination. RESULTS--46 of 113 children (41%) received the second dose of the vaccine. Factors most closely associated with mothers'' failure to comply with the second dose were lack of education and low income. Children whose mothers knew most about immunisation at first interview were more likely to have their second dose. CONCLUSIONS--Preventive health care services such as immunisation are appropriately offered in treatment centres, but compliance among children varies with socioeconomic status and mother''s education. Further research should be aimed at ways to make health education more effective among uneducated parents.     

Calcitonin gene-related peptide, neurokinin A and substance P: effects on nociception and neurogenic inflammation in human skin and temporal muscle.

Calcitonin gene-related peptide (CGRP) was injected alone and in combination with substance P (SP) or neurokinin A (NKA) into the forearm skin and temporal muscle of human volunteers. In the skin, 50 pmol of CGRP induced a wheal response and a delayed erythema. No pain was recorded. No interaction between CGRP and SP or NKA was observed. In the temporal muscle, 200 pmol of CGRP alone did not induce pain or tenderness but, in combination with SP or NKA, CGRP elicited a significant pain sensation. It is concluded that CGRP may be involved in neurogenic inflammation and that only SP, of the three peptides present in nociceptive C fibers, seems to be of major importance in relation to cutaneous nociception. Simultaneous neurogenic release of CGRP and other neuropeptides in skeletal muscle may induce myofascial pain.     

Chromosome abnormalities found among 34910 newborn children: results from a 13-year incidence study in Århus,Denmark

Summary As part of a larger prospective study of the influence of environmental factors on pregnancy, birth and the fetus, chromosome examinations have been made in 34910 newborn children in Århus over a 13-year period. Klinefelter's syndrome was found in 1 per 576 boys, XYY in 1 per 851 boys, triple-X in 1 per 947 girls and Turner's syndrome in 1 per 1893 girls. Other sex chromosome aberrations were found in 1 per 11637 children. The total incidence of sex chromosome abnormalities was 1 per 426 children or 2.34 per 1000. The most frequent autosomal abnormalities were that of Down's syndrome with 1 per 592 children, and reciprocal translocations with 1 per 712 children. The total incidence of autosomal abnormalities was 1 per 164 children. Chromosome abnormalities were found in 276 liveborn children and in 19 fetuses, who were aborted after prenatal chromosome examination. The combined incidence of sex chromosomal and autosomal abnormalities was 1 per 118 children or 8.45 per 1000 children.     

Human proteins IEF 58 and 57a are associated with the Golgi apparatus

A mouse monoclonal antibody (mAB 22-II-D8B) raised against lysed transformed human amnion cells (AMA) has been characterized. The mAB decorated the Golgi apparatus in growing and quiescent cultured monolayer cells (fibroblasts and epithelial cells) of various species as determined by double immunofluorescence labeling and colocalization with galactosyltransferase antibodies. It reacted with the acidic human proteins IEF 58 (Mr = 29,000) and 57a, respectively (Mr = 30,000) (HeLa protein catalogue number; [(1982) Clin. Chem. 28, 766]), Golgi staining was also observed in BS-C-1 cells microinjected with mAB 22-II-D8B suggesting that the epitopes recognized by the antibody are most likely located on the cytoplasmic face of the membranes. The precise localization of the antigens to the various cisternae of the Golgi apparatus could not be demonstrated by immunogold cytochemistry on ultrathin cryosections due to either weak reactivity of the antibody or low concentration of the antigens. Immunofluorescence staining with mAB 22-II-D8B of lymphoid human Molt-4 cells and some human tissues failed to reveal any significant staining even though these expressed high levels of both IEF 58 and 57a. These results are taken to imply that the epitopes recognized by mAB 22-II-D8B may be masked in some cell types.     

The apparent target size of rat brain benzodiazepine receptor, acetylcholinesterase, and pyruvate kinase is highly influenced by experimental conditions

Radiation inactivation is a method to determine the apparent target size of molecules. In this report we examined whether radiation inactivation of various enzymes and brain receptors is influenced by the preparation of samples preceding irradiation. The apparent target sizes of endogenous acetylcholinesterase and pyruvate kinase from rat brain and from rabbit muscle and benzodiazepine receptor from rat brain were investigated in some detail. In addition the target sizes of alcohol dehydrogenase (from yeast and horse liver), beta-galactosidase (from Escherichia coli), lactate dehydrogenase (endogenous from rat brain), and 5-HT2 receptors, acetylcholine muscarine receptors, and [35S] butyl bicyclophosphorothionate tertiary binding sites from rat brain were determined. The results show that apparent target sizes are highly influenced by the procedure applied for sample preparation before irradiation. The data indicate that irradiation of frozen whole tissue as opposed to lyophilized tissue or frozen tissue homogenates will estimate the smallest and most relevant functional target size of a receptor or an enzyme.