In Vitro Effect of Rifampin on Mycobacteria

Rifampin inhibited 20 strains of Mycobacterium tuberculosis in concentrations of 0.005 to 0.02 mug/ml in 7H-9 broth with Tween 80 and killed all or nearly all of the inoculum in four to eight times greater concentrations. In the same medium without Tween 80, as well as on 7H-10 agar, about 16 to 64 times these amounts were required to produce the same effect. Rifampin was also active against M. kansasii and some of the nonchromogenic mycobacteria. The incidence of mycobacterial cells resistant to rifampin within the cultures studied was in the range of one to four per 10(8) to 10(9) colony-forming units with concentrations of 4 to 125 mug of rifampin per ml. Only one of the Battey cultures and that of M. fortuitum yielded cells resistant to rifampin at 125 mug/ml but not at 500 mug/ml. The same strains yielded more than double that number of organisms resistant to streptomycin and up to 100 times more organisms resistant to isoniazid. All three drugs stopped the growth or reduced the mycobacterial population in growing cultures after contact for 24 to 48 hr. Complete inhibition of growth was produced by rifampin at 1.0 mug/ml in an average of 6 days and by streptomycin at 5.0 mug/ml in 3 days. After an average contact of 10.7 days with rifampin, five of seven strains resumed growth and all strains began regrowth after exposure to streptomycin for 9.4 days. The marked susceptibility of M. tuberculosis and of atypical mycobacteria to rifampin in vitro and the relatively low incidence of resistant mutants suggests that this agent may have clinical usefulness in the treatment of tuberculosis and some other mycobacterioses.     

Cephalexin and Cephaloglycin Activity In Vitro and Absorption and Urinary Excretion of Single Oral Doses in Normal Young Adults

A large number of recently isolated bacterial pathogens were tested for susceptibility to cephalexin and cephaloglycin by the replica inoculating method. Strains of group A hemolytic streptococci, viridans (alpha and gamma) streptococci, pneumococci, gonococci, meningococci, and penicillin G-sensitive Staphylococcus aureus were all moderately to highly susceptible to both of these cephalosporin analogues, nearly all of the strains being two to eight (median four) times more susceptible to cephaloglycin than to cephalexin. The penicillin G-resistant, penicillinase-producing strains of S. aureus varied in their susceptibility; many were moderately resistant to both analogues, particularly to cephalexin. Strains of enterococci, Haemophilus influenzae, and most of the common gram-negative bacilli were moderately to highly resistant. Reducing the size of the inoculum had variable effects on inhibition by these drugs, depending on the species or strain. The activity of cephalexin was very little affected by pH of the medium within the clinical range or by incubation at 37 C in broth for up to 24 hr. In contrast, cephaloglycin in broth deteriorated rapidly at 37 C, and its activity was markedly reduced in alkaline medium. Both cephalexin and cephaloglycin were rapidly absorbed and excreted into the urine after single oral doses of 500 mg. Much higher levels were achieved and sustained with the former. Absorption of both analogues was delayed when taken with food, and the levels in the serum were significantly higher and better sustained when probenecid was also given. Very high concentrations of cephalexin were excreted into the urine during the first 4 hr, and the levels were still high in the 4- to 8-hr collection. The concentrations of cephaloglycin in the urine at these times were much lower. An average of 80 to 93% of the dose of cephalexin and 25 to 30% of the cephaloglycin were accounted for as active drug in the urine collected in 8 hr. Both analogues were well tolerated.     

Tryptic fragments of the Escherichia coli DNA gyrase A protein

Treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two large fragments which are stable to further digestion. The molecular masses of these fragments are 64 and 33 kDa, and they are shown to be derived from the N terminus and the C terminus of the A protein, respectively. These fragments could represent structural and/or functional domains within the A subunit of DNA gyrase. The trypsin-cleaved A protein (A'), in combination with the B subunit of gyrase, can support ATP-dependent supercoiling of relaxed DNA and other reactions of DNA gyrase. The isolated 64-kDa fragment will also catalyse DNA supercoiling in the presence of the B protein, but the 33-kDa fragment shows no enzymic activities. We conclude that the N-terminal 64-kDa fragment represents the DNA breakage/reunion domain of the A protein, while the 33-kDa fragment may contribute to the stability of the gyrase-DNA complex.     

Measurement of strength and loading variables on the knee during Alpine skiing

The study focusses on the prevention of knee injuries during snow skiing. In order to develop a technology of knee injury prevention, both the strength and loading on the knee during skiing activity must be known. This paper reports measurements of variables influencing both knee strength and loading of the joint. The strength variables measured included the degree of activity in six muscles crossing the knee, the knee flexion angle, and the axial load (i.e. weight bearing) transmitted to the knee. Transducers included surface electrodes to monitor electromyogram signals indicating the degree of muscle activity and a goniometer to measure both hip and knee flexion angles. The complete loading on the knee was derived from a dynamometer which measured the six load components at the boot-dynamometer interface. The transducer data were acquired and stored by a compact, battery powered digital data acquisition-controller system. Three male subjects of similar physical size (nominal was 1.8 m and 75 kg) and skiing ability (advanced intermediate to expert) were tested under similar conditions. Each subject skied a total of four slalom runs--one snowplow and three parallel. The total time of each test was 21 s. Example data plots from different types of runs are presented and discussed. Based on observations from the data, necessary performance features for ski bindings offering improved protection from knee ligamentous injuries are defined.     

Mitochondrial gene mutation: the ageing process and degenerative diseases

Polymerase chain reaction (PCR) amplification was carried out on total DNA from a range of autopsy tissues from deceased human subjects with no known mitochondrial disease, aged from birth (80 minutes) to 87 years. We report the finding of an age-related 5 kb deletion in the mitochondrial genomes of these subjects. The deletion occurs between nucleotide positions 8470 and 13459 of the mitochondrial genome, and is flanked by a 13 bp direct repeat. All tissues from adult subjects showed the presence of mitochondrial DNA molecules with the deletion after a 30 cycle PCR amplification; by contrast the deletion was not similarly detected in any of the infant tissues analysed. However, the occurrence of the deletion was detected in the infant tissues after 60 PCR cycles of MtDNA amplification. It is concluded that such deletions are not necessarily associated with particular mitochondrial diseases but occur naturally, and with increasing frequency with age. A consequence of the accumulation of this deletion could be a progressive decrease with age of bioenergetic capacity which in turn could influence the rate of ageing and predispose to age-associated degenerative diseases.     

Ventilatory effects of substance P, vasoactive intestinal peptide, and nitroprusside in humans.

Animal studies suggest that the neuropeptides, substance P and vasoactive intestinal peptide (VIP), may influence carotid body chemoreceptor activity and that substance P may take part in the carotid body response to hypoxia. The effects of these peptides on resting ventilation and on ventilatory responses to hypoxia and to hypercapnia have been investigated in six normal humans. Infusions of substance P (1 pmol.kg-1.min-1) and of VIP (6 pmol.kg-1.min-1) were compared with placebo and with nitroprusside (5 micrograms.kg-1.min-1) as a control for the hypotensive action of the peptides. Both peptides caused significantly less hypotension than nitroprusside. Substance P and nitroprusside caused significantly greater increases in ventilation and in the hypoxic ventilatory response than VIP. No changes were seen in hypercapnic sensitivity. The stimulation of ventilation and the differential effects on ventilatory chemosensitivity that accompanied hypotension are consistent either with stimulation of carotid body chemoreceptor activity or with an interaction with peripheral chemoreceptor input to the respiratory center, as is seen in animals. The similar cardiovascular but different ventilatory effects of the peptides suggest that substance P may also stimulate the carotid body in a manner independent of the effect of hypotension. This is consistent with a role of substance P in the hypoxic ventilatory response in humans.     

The nature of inhibition of DNA gyrase by the coumarins and the cyclothialidines revealed by X-ray crystallography.

This study describes the first crystal structures of a complex between a DNA topoisomerase and a drug. We present the structures of a 24 kDa N-terminal fragment of the Escherichia coli DNA gyrase B protein in complexes with two different inhibitors of the ATPase activity of DNA gyrase, namely the coumarin antibiotic, novobiocin, and GR122222X, a member of the cyclothialidine family. These structures are compared with the crystal structure of the complex with an ATP analogue, adenylyl-beta-gamma-imidodiphosphate (ADPNP). The likely mechanism, by which mutant gyrase B proteins become resistant to inhibition by novobiocin are discussed in light of these comparisons. The three ligands are quite dissimilar in chemical structure and bind to the protein in very different ways, but their binding is competitive because of a small degree of overlap of their binding sites. These crystal structures consequently describe a chemically well characterized ligand binding surface and provide useful information to assist in the design of novel ligands.