Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster

For insertional mutagenesis of Pseudomonas aeruginosa, a derivative of the kanamycin-resistance (KmR) transposon Tn5 was constructed (Tn5-751) that carried the trimethoprim-resistance (TpR) determinant from plasmid R751 as an additional marker. Double selection for KmR and TpR avoided the isolation of spontaneous aminoglycoside-resistant mutants which occur at high frequencies in P. aeruginosa. As a delivery system for the recombinant transposon, plasmid pME305, a derivative of the broad-host-range plasma RP1, proved effective; pME305 is temperature-sensitive at 43 degrees C for maintenance in Escherichia coli and P. aeruginosa and deleted for IS21 and the KmR and primase genes. In matings with an E. coli donor carrying pME9(= pME305::Tn5-751), transposon insertion mutants of P. aeruginosa PAO were recovered at approx. 5 X 10(-7)/donor at 43 degrees C. Among Tn5-751 insertional mutants 0.9% were auxotrophs. A thr::Tn5-751 mutation near the recA-like locus rec-102 is useful for the construction of recombination-deficient strains. Several arc::Tn5-751 mutants could be isolated that were defective in anaerobic utilization of arginine as an energy source. From three of these mutants the arc gene region was cloned into an E. coli vector plasmid. Since Tn5-751 has a single EcoRI site between the TpR and KmR genes, EcoRI-generated fragments carrying either resistance determinant plus adjacent chromosomal DNA could be selected separately in E. coli. Thus, a restriction map of the arc region was constructed and verified by hybridization experiments. The arc genes were tightly clustered, confirming earlier genetic evidence.     

Regulatory effects of steroids on the pituitary response to LH-RH.

The effects of continuous intravenous administration of luteinizing hormone-release hormone in conjunction with physiological or pharmacolog ical doses of estradiol (E2), progesterone (P), 17alphaOH-progesterone (17-P), 20alphaOH-progesterone (20-P), chlormadinone acetate (CA), testosterone (T) and 5alpha-dihydrotestosterone (5-DHT) on pituitary response was evaluated in normal and postmenopausal women, normal men, and 1 patient with Klinefelter's syndrome. An all subjects, except the Klinefelter's syndrome patient, 132 mcg/hour/6 hours of E2 blocked the pituitary response to LH-RH, although a dose of 6.6 mcg/hour/6 hours was not markedly effective. None of the progestational steroids blocked the pituitary response to LH-RH. However, the dual administration of CA with E2 antagonized the pituitary response in normal and postmenopausal women. 600 mcg/hour/6 hours of T partially inhibited the pituitary resp onse in men, but had little effect on postmenopausal women and the Klinefelter's syndrome patient. The pituitary response to LH-RH was not inhibited by 5-DHT. The results indicate that sex steroids can modulate the hypothalamic-pituitary axis, though estradiol by itself does not appear to increase the pituitary response to LH-RH since it is not solely responsible for the pituitary responsiveness to LH-RH at the mid-luteal phase.     

Characterization of chloroplast division using the Arabidopsis mutant arc5.

arc5 is a chloroplast division mutant of Arabidopsis thaliana. To identify the role of ARC5 in the chloroplast replication process we have followed the changes in arc5 chloroplasts during their perturbed division. ARC5 does not affect proplastid division but functions at a later stage in chloroplast development. Chloroplasts in developing mesophyll cells of arc5 leaves do not increase in number and all of the chloroplasts in mature leaf cells show a central constriction. Young arc5 chloroplasts are capable of initiating the division process but fail to complete daughter-plastid separation. Wild-type plastids increase in number to a mean of 121 after completing the division process, but in the mutant arc5 the approximately 13 plastids per cell are still centrally constricted but much enlarged. As the arc5 chloroplasts expand and elongate without dividing, the internal thylakoid membrane structure becomes flexed into an undulating ribbon. We conclude that the ARC5 gene is necessary for the completion of the last stage of chloroplast division when the narrow isthmus breaks, causing the separation of the daughter plastids.     

A difference in optomotor behaviour of two Antarctic nototheniid fishes is correlated with the presence of a choroid<Emphasis Type="Italic"> rete mirabile</Emphasis> and Root effect

The hypothesis that fish vision is influenced by the presence of a choroid rete mirabile (an oxygen-concentrating apparatus in the eye) and the Root effect was tested using the optomotor response in two nototheniid species from Antarctica. Pagothenia borchgrevinki, an active cryopelagic zooplanktivore, does not exhibit a major Root effect and the rete is absent. In contrast, the sluggish, benthic nototheniid, Trematomus bernacchii, possesses both a rete and a significant Root effect. Whole-eye ocular PO2 was higher in T. bernacchii (18.6 kPa) than in P. borchgrevinki (18.1 kPa) and the optomotor response of T. bernacchii was positively influenced by subtended angle on a moving background whereas P. borchgrevinki did not respond to any subtended angle (0–600 min of arc). The optomotor response of T. bernacchii may therefore have been facilitated by elevated ocular PO2 due to the presence of the choroid rete and the Root effect.     

A health centre E.C.G. services: its use and abuse.

An average of 10-5 E.C.G.s were recorded weekly in a health centre used by 32 general practitioners serving a population of almost 65,000. The main indication for an E.C.G. was chest pain (73%). 47% of the E.C.G.s were abnormal. A change in clinical diagnosis occurred in 28% of cases and in patient management in 16%. A significant number of these changes were unwarranted, however. It is recommended that the E.C.G.s should be recorded by suitably trained nurses and reported by a specially trained general practitioner. Further education of general practitioners in the clinical use of the E.C.G. is required.     

Analysis of apolipoprotein E5 gene from a patient with hyperlipoproteinemia

We have isolated and analyzed apolipoprotein E5 gene from a patient with hyperlipoproteinemia. Apolipoprotein E5 is a variant of apolipoprotein E with two additional units of positive charge and smaller apparent molecular weight than apolipoprotein E3, which is the major isoform of apolipoprotein E. The heterozygous gene of apolipoprotein E5/3 from the patient was cloned into lambda phage. The cloned apolipoprotein E genes were subcloned into a murine retrovirus shuttle vector and were expressed. One out of four clones expressed apolipoprotein E5. The analysis of the nucleotide sequence of the exons and exon-intron boundary regions has shown a G to A substitution in the 18th nucleotide from the 5'-end of the third exon. This single base substitution changes the amino acid residue Glu to Lys at the third position from the amino-terminus of the mature protein, and gives two additional units of positive charge to the molecule.     

Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa.

A mutant of Pseudomonas aeruginosa was characterized which could not grow anaerobically with nitrate as the terminal electron acceptor or with arginine as the sole energy source. In this anr mutant, nitrate reductase and arginine deiminase were not induced by oxygen limitation. The anr mutation was mapped in the 60-min region of the P. aeruginosa chromosome. A 1.3-kb chromosomal fragment from P. aeruginosa complemented the anr mutation and also restored anaerobic growth of an Escherichia coli fnr deletion mutant on nitrate medium, indicating that the 1.3-kb fragment specifies an FNR-like regulatory protein. The arcDABC operon, which encodes the arginine deiminase pathway enzymes of P. aeruginosa, was rendered virtually noninducible by a deletion or an insertion in the -40 region of the arc promoter. This -40 sequence (TTGAC....ATCAG) strongly resembled the consensus FNR-binding site (TTGAT....ATCAA) of E. coli. The cloned arc operon was expressed at low levels in E. coli; nevertheless, some FNR-dependent anaerobic induction could be observed. An FNR-dependent E. coli promoter containing the consensus FNR-binding site was expressed well in P. aeruginosa and was regulated by oxygen limitation. These findings suggest that P. aeruginosa and E. coli have similar mechanisms of anaerobic control.     

马口鱼和宽鳍(鱼巤)鳃上发现寄生桡足类一新种(杯口水蚤目,鳋科)(英文)

在湖北省丹江口水库的马口鱼和宽鳍(鱼巤)的鳃上发现的寄生桡足类1新种丹江鳋。形态观察的结果表明,丹江鳋与鳋属的其他种类有很多共同特征,尤其是与博氏鳋和膨大鳋。与其它所有鳋属种类不同的是丹江鳋的第2触肢内缘光滑无刺,及其楔形第2节的外缘呈明显的弧状。以核糖体内转录间隔区(ITS)为标记进行系统进化分析,结果表明,与膨大鳋相比,博氏鳋与丹江鳋的亲缘关系更近些。无论是形态还是系统进化分析的结果都支持丹江鳋为鳋属1个独立的新种。     

马口鱼和宽鳍鱲鳃上发现寄生桡足类一新种(杯口水蚤目,鳋科)

在湖北省丹江口水库的马口鱼和宽鳍鱲的鳃上发现的寄生桡足类1新种丹江鳋.形态观察的结果表明,丹江鳋与鳋属的其他种类有很多共同特征,尤其是与博氏鳋和膨大鳋.与其它所有鳋属种类不同的是丹江鳋的第2触肢内缘光滑无刺,及其楔形第2节的外缘呈明显的弧状.以核糖体内转录间隔区(ITS)为标记进行系统进化分析,结果表明,与膨大鳋相比,博氏鳋与丹江鳋的亲缘关系更近些.无论是形态还是系统进化分析的结果都支持丹江鳋为鳋属1个独立的新种.     

Complement receptor 1 (CD35) on human reticulocytes: normal expression in systemic lupus erythematosus and HIV-infected patients.

The low levels of complement receptor 1 (CR1) on erythrocytes in autoimmune diseases and AIDS may be due to accelerated loss in the circulation, or to a diminished expression of CR1 on the red cell lineage. Therefore, we analyzed the expression of CR1 on reticulocytes (R) vs erythrocytes (E). Healthy subjects had a significant higher CR1 number per cell on R (919 +/- 99 CR1/cell) than on E (279 +/- 30 CR1/cell, n = 23), which corresponded to a 3. 5- +/- 1.3-fold loss of CR1. This intravascular loss was confirmed by FACS analysis, which showed that all R expressed CR1, whereas a large fraction of E was negative. The systemic lupus erythematosus (SLE), HIV-infected, and cold hemolytic Ab disease (CHAD) patients had a CR1 number on R identical to the healthy subjects, contrasting with a lower CR1 on their E. The data indicated a significantly higher loss of CR1 in the three diseases, i.e., 7.0- +/- 3.8-, 6.1- +/- 2.9-, and 9.6- +/- 5.6-fold, respectively. The intravascular loss was best exemplified in a patient with factor I deficiency whose CR1 dropped from 520 CR1/R to 28 CR1/E, i.e., 18.6-fold loss. In one SLE patient and in the factor I-deficient patient, the FACS data were consistent with a loss of CR1 already on some R. In conclusion, CR1 is lost progressively from normal E during in vivo aging so that old E are almost devoid of CR1. The low CR1 of RBC in autoimmune diseases and HIV-infection is due to a loss occurring in the circulation by an active process that remains to be defined.     

A Genetic Analysis of Chloroplast Division and Expansion in Arabidopsis thaliana

A nuclear recessive mutant of Arabidopsis thaliana, arc5, has been isolated in which there is no significant increase in chloroplast number during leaf mesophyll cell expansion and in which there are only 13 chloroplasts per mesophyll cell compared with 121 in wild-type cells. Mature arc5 chloroplasts in fully expanded mesophyll cells are 6-fold larger than in wild-type cells. A large proportion of arc5 chloroplasts also show some degree of central constriction, suggesting that the mutation has prevented the completion of the chloroplast division process. To examine the interaction of arc loci, a double mutant was constructed between arc1, a mutant possessing many small chloroplasts, and arc5. A second double mutant was also constructed between arc3, a previously discovered mutant also possessing few large chloroplasts per cell, and arc1. Analysis of these double mutants shows that chloroplast number per mesophyll cell is greater when arc5 and arc3 mutations are expressed in the arc1 background than when expressed alone. The cell-specific nature of arc mutants was also analyzed. The phenotypic traits characteristic of arc3 and arc5 are a reduction in chloroplast number and an increase in chloroplast size in mesophyll cells: these changes are also observed in reduced form in the epidermal and guard cell chloroplasts of arc3 and arc5 plants. Analysis of parenchyma sheath cell chloroplasts suggests that in leaves of arc1 plants the normal developmental distinction between mesophyll and parenchyma sheath chloroplasts is perturbed. The relevance of these findings to the analysis of the control of chloroplast division in mesophyll cells is discussed.     

笑弧在面部宽窄不同的个体中对微笑美学的影响

目的:研究面部宽窄不同女性的笑弧对微笑美学的影响。方法:选取女性正位微笑像1张,应用Adobe Photoshop CS5软件对笑弧和面部宽窄进行修改,由正畸医生和非专业人士对照片的微笑魅力值进行评判。结果:笑弧和面部宽窄程度在魅力微笑的判别中无交互作用(P0.05)。两组对理想的笑弧、略窄的面型和适宜的面型的魅力微笑判别分值较高(P0.05)。非专业人群中,在三种笑弧中,除了很窄的面型,魅力微笑的分值均随着面型的增宽降低(P0.05)。专业人群中,对于平坦的笑弧,除了很宽的面型,魅力微笑的分值随着面型的增宽而增加(P0.05);对于理想的笑弧,魅力微笑的分值随着面型的过宽或过窄而降低,但仍高于其他两种笑弧(P0.05);对于弯曲的笑弧,除了很窄的面型,魅力微笑的分值随着面型的增宽而降低(P0.05)。结论:笑弧和面部宽窄对魅力微笑的判别具有一定影响。不同人群对笑弧及面部宽窄与微笑美学的认识也存在差异,在正畸治疗中应根据患者面部宽窄合理的选择治疗方案。     

The long-lived fusogenic state induced in erythrocyte ghosts by electric pulses is not laterally mobile.

The long-lived fusogenic state induced in spherical-shaped erythrocyte ghosts by electric field pulses (Sowers, A.E. 1984. J. Cell Biol. 99:1989-1996; Sowers, A.E. 1986. J. Cell Biol. 102:1358-1362) was studied in terms of how the fusion yield depended on both (a) the location where membrane-membrane contact took place with respect to the orientation of the electric pulse and (b) the time interval between the pulse treatment and membrane-membrane contact. Fusion yields were greater for membrane-membrane contact locations closer to where the pulse-induced transmembrane voltage was expected to be greatest and showed a time interval-dependent accelerating decay. The portion of the membrane that became fusogenic included the area up to a latitude of approximately 38 degrees of arc towards the equators of the membranes. A time interval-dependent increase or decrease in rate of decay in the fusion yield for membrane-membrane contacts induced closer to the equator of the membranes did not occur showing that the pulse-induced fusogenic state is immobile in the early 5-45-s interval after induction and has a rate of decay, which does not permit long time interval changes in lateral position to be measured.     

Possible Association between Triphasic E.E.G. Waves and Disorder of Dopaminergic Systems

In a patient probably suffering from Jakob-Creutzfeldt disease of the Jones-Nevin type and in another with a cerebral infarction treatment with amantadine (Symmetrel) 100 mg twice a day caused repetitive triphasic waves to disappear from the E.E.G. in each case. In view of these findings and the fact that some other conditions in which repetitive triphasic E.E.G. waves are a clinical feature greatly improve on treatment with levodopa, the suggestion is made that these waves result from a disorder of the dopaminergic system.     

The distinctive roles of five different ARC genes in the chloroplast division process in Arabidopsis

ARC (accumulation and replication of chloroplasts) genes control different aspects of the chloroplast division process in higher plants. In order to establish the hierarchy of the ARC genes in the chloroplast division process and to provide evidence for their specific roles, double mutants were constructed between arc11, arc6, arc5, arc3 and arc1 in all combinations and phenotypically analysed. arc11 is a new nuclear recessive mutant with 29 chloroplasts compared with 120 in wild type. All the phenotypes of the double mutants are unambiguous. ARC1 down-regulates proplastid division but is on a separate pathway from ARC3, ARC5, ARC6 and ARC11. ARC6 initiates both proplastid and chloroplast division. ARC3 controls the rate of chloroplast expansion and ARC11 the central positioning of the final division plane in chloroplast division. ARC5 facilitates separation of the two daughter chloroplasts. ARC5 maps to chromosome 3 and ARC11 and ARC6 map approximately 60 cM apart on chromosome 5.     

Specificity of amphiphilic anionic peptides for fusion of phospholipid vesicles.

We have synthesized five amphiphilic anionic peptides derived from E5 peptide [Murata, M., Takahashi, S., Kagiwada, S., Suzuki, A., Ohnishi, S. 1992. Biochemistry 31:1986-1992. E5NN and E5CC are duplications of the N-terminal and the C-terminal halves of E5, respectively, and E5CN is an inversion of the N- and the C-terminal halves. E5P contains a Pro residue in the center of E5 and E8 has 8 Glu residues and 9 Leu residues. We studied fusion of dioleoylphosphatidylcholine (DOPC) large unilamellar vesicles assayed by fluorescent probes. The peptides formed alpha-helical structure with different degrees; E5NN, E5CN, and E8 with high helical content and E5CC and E5P with low helical content. These peptides bound to DOPC vesicles at acidic pH in proportion to the helical content of peptide. The peptides caused leakage of DOPC vesicles which increased with decreasing pH. The leakage was also proportional to the helicity of peptide. Highly helical peptides E5NN, E5CN, and E8 caused hemolysis at acidic pH but not at neutral pH. The fusion activity was also dependent on the helicity of peptides. In fusion induced by an equimolar mixture of E5 analogues and K5 at neutral pH, E8, E5NN, and E5CN were most active but E5CC did not cause fusion. In fusion induced by E5-analogue peptides alone, E5CN was active at acidic pH but not at neutral pH. Other peptides did not cause fusion. Amphiphilic peptides also appear to require other factors to cause fusion.     

Bacillus naphthovorans sp. nov. from oil-contaminated tropical marine sediments and its role in naphthalene biodegradation

. A Bacillus sp., designated as strain MN-003, was isolated as the dominant cultivatable naphthalene-degrading organism from oil-contaminated tropical marine sediments. Strain MN-003 is strictly aerobic, rod-shaped, Gram-positive, catalase positive, oxidase negative, and forms endospores. Strain MN-003 grew at salinities ranging from 0.28 to 7.00% and temperatures ranging from 15 to 41°C. Phylogenetic analyses reveal that strain MN-003 is most similar to Bacillus sp. VAN14, with a 16S rRNA sequence identity of 97.9%. Based on taxonomic and 16S rRNA data, strain MN-003 was named Bacillus naphthovorans sp. nov. When grown with naphthalene as sole carbon source, strain MN-003 had a maximal specific growth rate (µmax) of 0.32ǂ.03 h^–1, and a half-saturation constant (Ks) of 22.3dž.2 µM. A batch study of the tropical marine sediments enriched with naphthalene showed that cells of the Bacillus genus grew to become dominant members of the microbial community. The bacilli comprised 39.5Lj.5% of the microbial fraction after 20 days of enrichment.     

Stereotactic radiosurgery utilizing a linear accelerator

The authors have developed a radiosurgical technique based on multiple arc irradiations. The target is fixed to the rotational isocenter of a Varian 4 MV linear accelerator. The first irradiation is carried out while the radiating source is rotating on a 100-140 degrees arc. The patient is then rotated around a vertical axis passing through the target, and arc irradiations are repeated in different angular positions. By this technique it is possible to obtain very steep dose gradients at the borders of the target volume. High doses are usually delivered in two shots. 47 patients have been treated so far in a clinical trial that started in November 1982. The paper deals with the preliminary results (more than 6 months' follow-up) obtained in patients affected by nonresectable brain tumors and AVMs.