Inhibition of myeloid differentiation by inhibitors of ADP-ribosylation

Inhibitors of ADP-ribosylation inhibited the myeloid differentiation of murine myelomonocytic leukemia, WEHI-3BD+ cells induced by granulocyte colony-stimulating factor. Benzamide, at 2.0 mM, inhibited 50% of the WEHI-3BD+ cell differentiation but had no significant effect on the proliferation. However, benzylaminododecylguanine hydrochloride and p-methoxylbenzylaminodecamethylene guanidine sulfate at 2.0 and 2.2 microM, respectively, inhibited 50% of proliferation but had no effect at all on differentiation. The differential effects of inhibitors provide a model to study the role of ADP-ribosylation in myeloid differentiation.     

Stimulation of the protein synthetic process by adenosine 3':5'-monophosphate and hexose phosphates in gel-filtered rabbit reticulocyte lysates.

The addition of 0.167 to 4.0 mM cAMP to gel-filtered rabbit reticulocyte lysates stimulates the initial rate and the extent of polypeptide synthesis. The stimulation is at the initiation step of polypeptide synthesis as measured by the (i) increased dipeptide, methionyl-valine, accumulation in the presence of the specific initiation inhibitor, pactamycin, and (ii) increased formation of the 40 S and 80 S initiation complex when gel-filtered lysates are incubated with [35S]Met-tRNAFMet. Furthermore, a synergistic stimulation of protein synthesis is observed when cAMP and hexose phosphates (which alone elicit a 1.8-fold stimulation of protein synthesis) are added simultaneously to gel-filtered rabbit reticulocyte lysates. These results indicate that cAMP and hexose phosphates are both essential to maintain the high rate of initiation.     

Analysis of Escherichia coli K12 F factor transfer genes: traQ, trbA, and trbB

The genes that encode the transfer properties of plasmid F, the fertility factor of Escherichia coli K12, are known to be clustered over a large, 33.3-kb segment of F DNA. As the central segment of the transfer region has not previously been well characterized, we constructed a detailed restriction map of the large F EcoRI DNA fragment, fl, and isolated a series of plasmid derivatives that carry various overlapping segments of this F tra operon DNA. We also analyzed the protein products of those clones that carried DNA segments extending over the region between traF and traH. This region was known to include traQ, a gene required for efficient conversion of the direct product of traA to the 7000-Da pilin polypeptide. We identified the traQ product as a polypeptide that migrates as a 12,500-Da protein on sodium dodecyl sulfate-polyacrylamide gels. We also detected the products of two other new genes that we have named trbA and trbB. These polypeptides migrate with apparent molecular weights of 14,200 and 18,400, respectively. Analysis of plasmid deletion derivatives that we constructed in vitro shows that these genes map in the order traF trbA traQ trbB traH. The presence of a plasmid carrying a small 0.43-kb fragment that expressed only the 12,500 traQ product caused the traA product of a co-resident compatible plasmid to be converted to the 7000-Da pilin polypeptide, demonstrating that TraQ is the only tra operon product required for this step of F-pilin biosynthesis.     

Localization of mitochondria in plant cells by vital staining with rhodamine 123

The positively-charged fluorescent dye rhodamine 123 (r-123) specifically stains mitochondria in living plant protoplasts, suspensionculture cells, and root hairs. This dye functions as a vital stain and permits visualization of the localization, distribution and movement of the mitochondria. Dehydration of root hairs caused mitochondria to aggregate into clumps. Mitochondria were either homogenous or heterogeneous and were frequently seen to accumulate in the perinuclear regions of suspension-culture cells but not in those of protoplasts or root-hair cells. Dinitrophenol and high concentrations of ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid and KCl immediately eliminated fluorescence in r-123-stained mitochondria whereas ionomycin enhanced it. Treatment of seedlings with r-123 resulted in differential brightness of fluorescence in different tissues. Meristematic tissues, such as root and shoot tips, exhibited the brightest fluorescence. The cytotoxicity of r-123 in both germinating seedlings and suspension-culture cells was low. The specificity, sensitivity and low toxicity of r-123 should make it a useful tool in experiments designed to examine agents and conditions which affect the location, the physiological status or the viability of mitochondria.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - DAPI 46-diamidino-2-phenylindole - r-123 rhodamine 123