Female-specific expression of a hexamerin gene in larvae of an autogenous mosquito.

Fourth-instar larvae of the autogenous mosquito, Aedes atropalpus, synthesize three hexamerins or hexameric storage proteins which are distinguished by different methionine and aromatic amino-acid contents. One protein, Hexamerin-1.2 (AatHex-1.2) is only found in female larvae and pupae. In order to investigate the molecular basis for this sex-specific accumulation, we have cloned and sequenced the cDNA encoding AatHex-1.2 and isolated and sequenced over 1 kb of the 5' flanking region of the AatHex-1.2 gene. The AatHex-1.2 transcript encodes a 81.6-kDa hexamerin subunit which contains 19.8% phenylalanine, tyrosine and tryptophan and 8.6% methionine residues. The single-copy AatHex-1.2 gene consists of three exons and two small introns located at its 5' end. A 2.3-kb AatHex-1.2 mRNA accumulates only in female larvae and pupae and is expressed at very low levels in adult female mosquitoes. The temporal expression profile of this transcript is typical of other mosquito hexamerin genes, with rapid disappearance of the mRNA shortly after pupation. Hence this is the first observation of exclusively female-specific gene activity during preadult development of an insect. In the 5' flanking region of the AatHex-1.2 gene, we identified putative binding sites for transcription factors, such as GATA, C/EBP and Doublesex, typically involved in fat body- and female-specific gene activity in Diptera. These findings suggest that mechanisms for sex-specific transcription in the fat body may be well conserved between flies and mosquitoes.

Bucindolol, a nonselective beta 1- and beta 2-adrenergic receptor antagonist, decreases beta-adrenergic receptor density in cultured embryonic chick cardiac myocyte membranes.

Bucindolol and carvedilol, nonselective beta1- and beta2-adrenergic receptor antagonists, have been widely used in clinical therapeutic trials of congestive heart failure. The aim of the current study was to investigate long-term effects of bucindolol or carvedilol on beta-adrenergic receptor protein and gene expression in cardiac myocytes. Embryonic chick cardiac myocytes were cultured and incubated with bucindolol (1 microM), carvedilol (1 microM), or norepinephrine (1 microM) for 24 h. 125I-iodocyanopindolol binding assays demonstrated that incubation with norepinephrine or bucindolol, but not carvedilol, significantly decreased beta-adrenergic receptor density in crude membranes prepared from the myocytes. Neither bucindolol nor carvedilol significantly stimulated adenylyl cyclase activity in membranes from drug-untreated cells. Unlike by norepinephrine, the receptor density reduction by bucindolol incubation was not accompanied by a change in beta1-adrenergic receptor messenger RNA abundance. A decrease in membrane beta-adrenergic receptor density without a change in cognate messenger RNA abundance was also observed in hamster DDT1 MF2 cell line incubated with bucindolol (1 microM, 24 h). We conclude that incubation with bucindolol, but not carvedilol, results in true reduction of beta-adrenergic receptor density in chick cardiac myocyte membranes by mechanisms that are distinct from those responsible for receptor density reduction by the agonist norepinephrine.

Expression of aerobactin genes by Shigella flexneri during extracellular and intracellular growth.

The expression of the Shigella flexneri chromosomal aerobactin genes during growth of the bacterium within tissue culture cells was assayed. During intracellular growth, aerobactin promoter activity was repressed relative to the level observed in bacteria grown extracellularly, even when the bacteria had been starved for iron prior to infection. Similarly, the level of one of the proteins encoded by this operon, the aerobactin outer membrane receptor, Iut, was reduced in the intracellular environment. These studies indicate that the aerobactin system is not highly expressed by bacteria within host cells, suggesting that siderophore-independent iron acquisition systems can provide essential iron during intracellular multiplication.

Identification of Mycobacterium tuberculosis and M. avium complex directly from smear-positive sputum specimens and BACTEC 12B cultures by high-performance liquid chromatography with fluorescence detection and computer-driven pattern recognition models.

A high-performance liquid chromatography method that utilized fluorescence detection (HPLC-FL) of mycolic acid 6,7-dimethoxycoumarin esters was developed to identify Mycobacterium tuberculosis (MTB) and M. avium complex (MAC) directly from fluorochrome stain smear-positive sputum specimens and young BACTEC 12B cultures. HPLC-FL chromatograms from a training set that included 202 smear-positive clinical sputum specimens and 343 mycobacterial cultures were used to construct a calibrated peak-naming table and computer-based pattern recognition models for MTB and MAC. Pattern recognition model performance was measured with an evaluation set of samples that included 251 smear-positive clinical sputum specimens and 167 BACTEC 12B cultures. Evaluation sputum specimens were culture positive for MTB (n = 132) and MAC (n = 48). With evaluation sputa, the MTB and MAC models were 56.8 and 33.3% sensitive, respectively. Evaluation set BACTEC 12B cultures were culture positive for MTB (n = 97) and MAC (n = 53). The sensitivities of the MTB and MAC models for identification of BACTEC 12B cultures were 99.0 and 94.3%, respectively. The specificity of both models was 100% for both types of evaluation samples. The average times from BACTEC 12B inoculation to cell harvest were 10.2 and 7.4 days for MTB and MAC, respectively. HPLC-FL can identify MTB and MAC in 1 day from many smear-positive sputa. Rapid and sensitive identification of MTB and MAC from young BACTEC 12B cultures was achieved.

Differential protein expression by Shigella flexneri in intracellular and extracellular environments.

Shigellae were intrinsically radiolabeled with [35S]methionine either extracellularly or while multiplying within infected HeLa cell monolayers. A complex pattern of suppression and induction of proteins was observed. Proteins of approximately 97, 62, 58, 50, 25, and 18 kilodaltons (kDa) were induced in Shigella flexneri isolated from infected monolayers. Proteins of 100, 85, 70, 64, and 55 kDa were suppressed under the same conditions but were seen in cells labeled in the tissue culture medium alone. Protein expression during the stages of attachment, invasion, and intracellular multiplication was examined by pulse-labeling. The 58-kDa protein was induced only during invasion, and the 62- and 25-kDa proteins were induced only during intracellular multiplication. Shift into a minimal medium with ion concentrations and pH mimicking intracellular conditions and endosomal pH resulted in the induction of the 97- and 58-kDa proteins, and reduction of the intracellular-like medium with 2-mercaptoethanol resulted in the induction of the 97-, 50-, and 25-kDa proteins and suppression of the 55-kDa protein. Radioimmunoprecipitations of shigellae grown in vitro and in vivo revealed differential expression of immunogenic proteins. Proteins corresponding in size to IpaB (62 kDa), IpaC (42 kDa), and IpaD (38 kDa) were lost during intracellular multiplication, whereas another protein corresponding to IpaA (80 kDa) was found to increase under the same conditions.

Influence of iron on growth, morphology, outer membrane protein composition, and synthesis of siderophores in Campylobacter jejuni.

Three human isolates of Campylobacter jejuni were grown in a biphasic culture medium with and without the addition of a synthetic chelator to induce iron limitation. Cells grown in low-iron medium exhibited slower growth rates and altered cellular morphology. Increased numbers of longer, more filamentous forms were seen in Gram-stained smears. Three proteins, with apparent Mrs of 82,000, 76,000, and 74,000, were consistently present in the outer membrane of cells grown in low-iron medium. At least one of these proteins (76,000 to 74,000) was exposed on the cell surface. A bioassay was used to look for the production of siderophores by these and other strains of C. jejuni. Seven of 26 strains tested produced detectable amounts of siderophores. Growing strains at 42 degrees C failed to suppress siderophore synthesis or to alter the outer membrane protein profiles of iron-starved cells. The ability of three strains to utilize exogenously supplied siderophores for growth in low-iron medium was also examined. All three strains were able to utilize enterochelin and ferrichrome, but none utilized aerobactin, rhodotorulic acid, or desferrioxamine B. The effect of iron on the virulence of C. jejuni for 11-day-old chicken embryos inoculated via the chorioallantoic membrane was also determined.

The chicken embryo as a model for campylobacter invasion: comparative virulence of human isolates of Campylobacter jejuni and Campylobacter coli.

Eleven-day-old chicken embryos were used to compare the relative virulence of minimally passaged human isolates of Campylobacter jejuni and Campylobacter coli. Graded doses of bacteria were inoculated onto the chorioallantoic membrane, and 50% lethal doses were calculated at 72 h postinfection. Strains varied markedly in their ability to invade the chorioallantoic membrane and kill the embryos. The 50% lethal doses varied by about 6 logs for 25 strains of C. jejuni, and by 2 logs for 5 strains of C. coli. Although both outbred and inbred embryos were employed in the study, the latter were found to be more susceptible to infection with most strains. All isolates were screened for plasmid DNA, but there was no apparent relationship between plasmid content and virulence of strains for the embryos. Neither could virulence be associated with the production of siderophores by the strains. The ability of selected strains of C. jejuni to invade the liver of embryos was also studied. The number of campylobacters culturable from the liver was found to be inversely related to the 50% lethal dose of the strain. By inoculating 11-day-old embryos intravenously, it was possible to demonstrate that a strain of C. jejuni which was poorly virulent after chorioallantoic inoculation was relatively noninvasive. Invasiveness alone, however, could not fully account for the lethality of two highly virulent strains of C. jejuni administered by the intravenous route. Finally, there was no correlation between motility and virulence in this model system.