Growth phase-associated changes in protein expression in Mycobacterium smegmatis identify a new low molecular weight heat shock protein.

De novo protein synthesis and the heat-shock response during different stages of bacterial culture of Mycobacterium smegmatis LR222 were investigated. A discontinuance in the increase in number of colony forming units occurred at mid-exponential phase of growth. This coincided with a plateau in the ATP content of the culture, a reduction in the synthesis of exponential phase proteins (58, 30.5, and 20 kDa), a transitory synthesis of a 32 kDa protein and the induction of stationary-phase proteins (48, 46, 31, 25, and 20 kDa). The response to heat shock showed a growth-phase dependency, with the highest fold-induction of the 75 kDa (DnaK) protein occurring during the transitory cessation in the increase in CFU, while the greatest increase of the 95 kDa, 66 kDa (GroEL), and approximately 17 kDa (a doublet) proteins occurred during stationary phase. The approximately 17 kDa doublet was resolved into four polypeptides by two-dimensional electrophoresis. Mass spectrometric analysis of the sequence of one polypeptide (named Hsp17-2, 16.8 kDa) revealed significant homology to a conserved, 16.2 kDa, hypothetical protein of unknown function in Mycobacterium tuberculosis H37Rv. The increased synthesis of Hsp17-2 in response to heat shock suggests that it may represent a new low molecular weight heat shock protein.

Production of TNF alpha by LPS-stimulated murine, rat and human blood and its pharmacological modulation.

Tumour necrosis factor alpha (TNF alpha) has been reported to play a key role in the pathogenesis of sepsis and chronic inflammatory diseases, including rheumatoid arthritis and atherosclerosis, suggesting that agents which inhibit TNF alpha production may have therapeutic utility for the treatment of such conditions. Production of TNF alpha by LPS (lipopolysaccharide)-stimulated murine, rat and human heparinized blood was investigated. LPS (1-100 micrograms/ml) caused a similar concentration- and time-dependent stimulation of TNF alpha production by rat and human blood, achieving levels of 750-5000 U/ml (L929 bioassay) at 6 h. In contrast, TNF alpha production by LPS-stimulated murine blood was poor and variable (0-150 U/ml). Dexamethasone and pentoxifylline caused a concentration-dependent inhibition of TNF alpha production by LPS-stimulated human and rat blood with IC50s of 0.26 +/- 0.05 and 73.0 +/- 26.4 microM for human and 5.7 +/- 1.8 nM and 20.6 +/- 8.0 microM for rat blood, respectively. Therefore, LPS-stimulated rat and human, but not murine, blood are suitable systems for the detection and evaluation of inhibitors of TNF alpha production.