Cloning of AAC(3) and AAD(2″) genes fromAcinetobacter: differential expression in the host strain

The mechanism of resistance to gentamicin and tobramycin in a clinical isolate ofAcinetobacter baumannii, in which aminoglycoside-modifying enzymes were not detected, was investigated. For increase of the resistance gene product, DNA prepared from theA. baumannii isolate was cloned into pUC18 and introduced intoEscherichia coli by transformation. Gentamicin-resistant transformants were screened for aminoglycoside-modifying enzymes. This approach identified two genes encoding AAC(3) and AAD(2) activity, respectively. To determine whether both genes are expressed in the hostAcinetobacter strain, we extracted total cellular RNA from this strain, and Northern blots were hybridized with the cloned AAC(3) and AAD(2) structural genes. mRNA transcribed from the AAC(3) gene alone was detected. This shows that cloning a functional resistance gene is not sufficient in itself to investigate mechanisms of resistance in bacterial strains without detectable aminoglycoside-modifying activity. Furthermore, this study suggests a potential limitation of antibiotic resistance gene probes for studying mechanisms of resistance.     

The Effect of Freezing Rate on the Survival of Cryopreserved African Sharptooth Catfish (Clarias gariepinus) Spermatozoa

Cryoprotective agents were evaluated to find the optimal concentration of the cryoprotectant and most suitable combination of solution and cryoprotectant. A cryoprotective agent composed of 4% glucose and 9% glycerol yielded the best results. It was established that the optimal freezing rate is dependent on the composition of the cryoprotective agent. Maximal survival of catfish spermatozoa (60%) occurs at 5°C min^-1 and faster and slower freezing rates result in poor survival or no survival at all. Incorporation of an isothermal holding period into the freezing rate led to remarkable increase (20-30%) in sperm survival when Me₂SO was present in the cryoprotective agent. Cryoprotective agents containing glucose also showed improved survival when a three phase freezing rate was used. These results lead to the conclusion that the presence of an isothermal holding period in the freezing rate is beneficial for the cryoprotective action of Me₂SO and glucose.     

Cloning and expression in Escherichia coli of a recA homologue from Mycobacterium tuberculosis.

A 3.8 kb PstI fragment of Mycobacterium tuberculosis was cloned in a recA-deleted Escherichia coli by selecting transformants with increased EMS resistance. The cloned fragment restored homologous recombination in Hfr crosses and conferred resistance to long wave (302 nm) but not short wave (254 nm) UV light. E. coli containing the 3.8 kb PstI fragment produced a 38-40 kDa protein which cross-reacted with antibodies raised against the E. coli RecA protein. The cloned DNA thus probably encodes a RecA homologue.     

Production of sterigmatocystin by Aspergillus versicolor and Bipolaris sorokiniana on semisynthetic liquid and solid media.

Higher yields of sterigmatocystin were obtained with Aspergillus versicolor than with Bipolaris sorokiniana both in liquid and on solid media. The optimum temperature for sterigmatocystin production by A. versicolor was 27 to 29 degrees C and 23 degrees C for B. sorokiniana. In liquid shake cultures, production of sterigmatocystin by B. sorokiniana was negligible, whereas maximal production by A. versicolor was 210 mg/liter. On solid substrates, the highest yields (8 g/kg) were obtained with A. versicolor on still cultures of whole corn supplemented with Soytone.     

The breeding biology of the Scimitarbilled Woodhoopoe

Steyn, P. 1999. The breeding biology of the Scimitarbilled Woodhoopoe. Ostrich 70 (3&4): 173–178.

The breeding biology of the Scimitarbilled Woodhoopoe Rhinopomastus cyanomelas was studied at two sites in Zimbabwe over a 13-year period from 1964–1977. The pairs were resident, remained together throughout the year, and inspected their nest sites occasionally during the non-breeding season. The breeding season extended from August to December with a marked September/October peak. Eggs were laid at daily intervals. Clutch size averaged 2.9 (range 2–4). Incubation began either with the penultimate or last laid egg. During the 13–14 day incubation period the female left the nest only occasionally each day and was reliant on the male for food. This pattern continued for four days after the chicks hatch. Thereafter she started to forage and gradually increased her contribution to chick provisioning until she overtook that of the male. With one exception, he never fed the chicks directly and delivered the food to the female. The nestlings were brooded overnight for the first two weeks. The anti-predator response of the young included a malodorous brown exudation from the preen gland and unpleasant liquid excreta. The nestling period was 21–24 days and the young left the vicinity of the nest with their parents and did not return to roost in it. Twelve breeding cycles were monitored and 76% of eggs laid (n=37) produced fledged young. Second broods were raised in the same nest on two occasions after successful rearing of the first, presumably by the same pair, but the birds were not individually marked. There was no evidence of helpers at the nest.     

Novel method for the proteomic investigation of a dairy-associated Bacillus cereus biofilm

The biofilm proteome of a dairy-associated Bacillus cereus strain (B. cereus 5) was investigated. Biofilm biomass of sufficient concentration for 2D-PAGE was obtained by growing the culture in the presence of glass wool. B. cereus 5 readily attached to the glass wool and biofilms formed within 18 h. The biofilm proteome of whole-cell proteins revealed that 10 proteins were synthesized as a result of surface attachment of which four were unique to the biofilm profile. Seven proteins appeared to be absent in the biofilm profile. The altered proteomes indicated that changes took place in the regulation of protein expression when B. cereus 5 cells attached to surfaces.     

The discovery of highly selective erbB2 (Her2) inhibitors for the treatment of cancer

The synthesis and biological evaluation of potent and selective inhibitors of the erbB2 kinase is presented. Based on the 4-anilinoquinazoline chemotype, the syntheses of several new series of erbB2 inhibitors are described with quinazoline and pyrido[4,3-d]pyrimidine cores. The vast majority of these compounds are found to be >100x selective over the closely related EGFR kinase. Two lead compounds are further shown to have low clearance and moderate bioavailability in rat.     

Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium tuberculosis Growth and Infectivity

Ergothioneine (EGT) is synthesized in mycobacteria, but limited knowledge exists regarding its synthesis, physiological role, and regulation. We have identified Rv3701c from Mycobacterium tuberculosis to encode for EgtD, a required histidine methyltransferase that catalyzes first biosynthesis step in EGT biosynthesis. EgtD was found to be phosphorylated by the serine/threonine protein kinase PknD. PknD phosphorylates EgtD both in vitro and in a cell-based system on Thr²¹³. The phosphomimetic (T213E) but not the phosphoablative (T213A) mutant of EgtD failed to restore EGT synthesis in a ΔegtD mutant. The findings together with observed elevated levels of EGT in a pknD transposon mutant during in vitro growth suggests that EgtD phosphorylation by PknD negatively regulates EGT biosynthesis. We further showed that EGT is required in a nutrient-starved model of persistence and is needed for long term infection of murine macrophages.