Torque generation in the flagellar motor of Escherichia coli: evidence of a direct role for FliG but not for FliM or FliN.

Among the many proteins needed for assembly and function of bacterial flagella, FliG, FliM, and FliN have attracted special attention because mutant phenotypes suggest that they are needed not only for flagellar assembly but also for torque generation and for controlling the direction of motor rotation. A role for these proteins in torque generation is suggested by the existence of mutations in each of them that produce the Mot- (or paralyzed) phenotype, in which flagella are assembled and appear normal but do not rotate. The presumption is that Mot- defects cause paralysis by specifically disrupting functions essential for torque generation, while preserving the features of a protein needed for flagellar assembly. Here, we present evidence that the reported mot mutations in fliM and fliN do not disrupt torque-generating functions specifically but, instead, affect the incorporation of proteins into the flagellum. The fliM and fliN mutants are immotile at normal expression levels but become motile when the mutant proteins and/or other, evidently interacting flagellar proteins are overexpressed. In contrast, many of the reported fliG mot mutations abolish motility at all expression levels, while permitting flagellar assembly, and thus appear to disrupt torque generation specifically. These mutations are clustered in a segment of about 100 residues at the carboxyl terminus of FliG. A slightly larger carboxyl-terminal segment of 126 residues accumulates in the cells when expressed alone and thus probably constitutes a stable, independently folded domain. We suggest that the carboxyl-terminal domain of FliG functions specifically in torque generation, forming the rotor portion of the site of energy transduction in the flagellar motor.     

A cellular cofactor facilitates efficient 3CD cleavage of the poliovirus P1 precursor.

The production of poliovirus capsid proteins from a capsid protein precursor (P1) is mediated by virus-encoded proteinase 3CD and involves a complicated set of proteinase-substrate interactions. In addition to substrate and enzymatic determinants required for this interaction, we describe a cellular cofactor, which facilitates 3CD recognition of the P1 precursor. Cellular cofactor activity is 3CD dependent and salt dependent. Our analysis shows that proteolytic cleavage of the P1 precursor at the VP0/VP3 cleavage site exhibits a greater dependency on the cellular cofactor than cleavage at the VP3/VP1 site. Such a greater dependency on cellular cofactor activity can be relieved (in part) by the substitution of an Ala residue for the Pro residue at the -4 position of the VP0/VP3 cleavage site. However, mutant viruses containing Pro-to-Ala substitutions at the -4 position of the VP0/VP3 site exhibit defects in viral growth.     

Randomised controlled trial of oxytocin alone versus oxytocin and ergometrine in active management of third stage of labour.

OBJECTIVE--To compare intramuscular oxytocin alone and intramuscular oxytocin with ergometrine (Syntometrine) for their effect in reducing the risk of postpartum haemorrhage when both are used as part of the active management of the third stage of labour. DESIGN--Double blind, randomised controlled trial. SETTING--Two metropolitan teaching hospitals in Perth, Western Australia. SUBJECTS--All women who expected a vaginal birth during the period of the trial. Informed consent was obtained. MAIN OUTCOME MEASURES--Postpartum haemorrhage, nausea, vomiting, and increased blood pressure. RESULTS--3497 women were randomly allocated to receive oxytocin-ergometrine (n = 1730) or oxytocin (n = 1753). Rates of postpartum haemorrhage (> or = 500 ml or > or = 1000 ml) were similar in both arms (odds ratio 0.90 (0.82); 95% confidence interval 0.75 to 1.07 (0.59 to 1.14) at 500 ml (1000 ml) threshold). The use of oxytocin-ergometrine was associated with nausea, vomiting, and increased blood pressure. CONCLUSIONS--There are few advantages but several disadvantages for the routine use of oxytoxinergometrine when prophylactic active management of the third stage of labour is practised. Further investigation of dose-response for oxytocin may be warranted.     

An Improved Method for Preparing the Chromosomes of Pines and other Gymnosperms

A simple technic is described to produce well spread gymnosperm chromosomes. Root tip meristems are digested with a pectinase:cellulase mixture to produce a cell suspension which then is squashed to yield flat, well spread chromosome complements that can be stained or used for in situ hybridization.     

Detection of extracellular proteinase of Pseudomonas fragi by enzyme-linked immunosorbent assay

A double-antibody-sandwich, enzyme-linked immunosorbent assay was developed to detect an extracellular proteinase produced by Pseudomonas fragi. The method was capable of detecting 4 g/ml of the proteinase in spiked samples of buffer and broth and 4.2 g/ml in a broth culture of the organism. The assay detected the presence of proteinase at bacterial densities of approximately 10⁴ cfu/ml, which develop after incubation for 15 h at 25°C in a broth medium. All assays could be completed within 7 h. This assay is of value in plotting proteolytic expression in relation to the growth cycle of Ps. fragi in broth culture and may be of value, with development, in other more complex milieux.     

SATELLITE-MONITORED MOVEMENTS AND DIVE BEHAVIOR OF A BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) IN TAMPA BAY, FLORIDA

An adult, female bottlenose dolphin ( Tursiops trucncatus ) was radio tagged and monitored via satellite-based Argos receivers for 25 d from 28 June to 23 July 1990, in Tampa Bay, Florida. A total of 794 transmissions were obtained during 106 satellite passes. A mean of 3.9 (SE = 0.24) locations/day were determined by Service Argos and showed the animal remained in the bay, usually close to the southeastern shore. The dolphin moved at least 581 km at a minimum mean speed of 1.2 (SE = 0.1) km/h. Data from 63, 922 dives were recorded. The animal spent an average of 87.1 (SE = 0.6)% of the time submerged, with a mean dive duration of 25.8 (SE = 0.5) sec. Mean dive duration differed significantly between four periods of the day, as did the mean percent of time spent submerged. During the early morning the animal spent more time at the surface, averaged shorter dives, and was submerged less than other times of day. This is the first study to demonstrate die1 dive cycles in a bottlenose dolphin. Four months after tag loss, the dolphin was photographed with no evidence of necrosis or disfigurement of the dorsal fin. Satellite telemetry was demonstrated as an effective means of documenting the movements and dive behavior of a small inshore cetacean.     

Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor

S. cerevisiae kex2 mutants are defective for the production of two biologically active secreted peptides: killer toxin and the mating pheromone, alpha-factor. Both molecules are excised from larger precursor polypeptides. In normal cells, the alpha-factor precursor is core-glycosylated and proteolytically processed intracellularly. In kex2 mutants, however, prepro-alpha-factor is not proteolytically cleaved and is secreted in a highly glycosylated form. All kex2 mutants examined (three independent alleles) lack a Zn++-sensitive membrane-associated endopeptidase with specificity for cleaving on the carboxyl side of a pair of basic residues. Absence of this activity cosegregates with the other phenotypes of a kex2 lesion in genetic crosses. The normal KEX2 gene was isolated by complementation of three of the phenotypes conferred by the kex2-1 mutation. The cloned DNA, either on a multicopy plasmid or integrated into the genome, restores both enzymatic activity in vitro and the normal pattern of proteolytic processing and glycosylation of prepro-alpha-factor in vivo. Gene dosage effects suggest that KEX2 is the structural gene for the endopeptidase.     

High ethanol productivities using small Ca-alginate beads of immobilized cells of Zymomonas mobilis

Summary Zymomonas mobilis cells were immobilized into small 1 mm diameter beads of Ca-alginate in order to minimize mass transfer limitations and maximize immobilized cell activity. A combination of small bead size with a high cell concentration of 58 g dry wt. cell per lit. bead volume resulted in high ethanol productivities using a newly designed packed bed bioreactor system. Steady-state dilution rates ranging from 0.4 h^-1 to 3.9 h^-1 were run resulting in a maximum productivity of 102 g ethanol/l/h for an inlet substrate concentration of 100 g glu/l and 87% conversion. The bioreactor was run continuously at a fixed dilution rate for 384 h and short intermittent treatment of the beads with CaCl₂ temporarily increased ethanol productivity to a maximum of 116 g ethanol/l/h.