Interactive cytogenetic effects on rat bone-marrow due to chronic ingestion of 2,5,2',5' and 3,4,3',4' PCBs.

Rats who were fed low doses of single PCBs, either 2,5,2',5' or 3,4,3',4', did not demonstrate any chromosome breakage or mitotic changes in their bone marrow cells. However, there was a significant increase in chromosome damage observed in bone marrow cells of rats ingesting 10 ppm 2,5,2',5' plus 0.1 ppm 3,3,3',4' in combination. It is suggested that this PCB combination, previously found to cause superadditive chromosome damage in vitro, is also capable of causing chromosome damage in vivo, but these effects do not compromise cell proliferation because the mitotic index is not depressed.     

A simple radioimmunoassay for 3',5', cyclic adenosine monophosphate.

A radioimmunoassay for 3′,5′ cAMP has been developed in which [³H]3′,5′ cAMP is the radioligand. Antibody-bound and free fractions are separated with dextran-coated charcoal. The sensitivity of the assay is 0.03 pmoles and antiserum specificity is 7 orders with respect to other adenine nucleotides. Samples are prepared by ethanol precipitation. Tissue levels of 3′,5′ cAMP are comparable to those reported by others.     

Human exonuclease I is required for 5' and 3' mismatch repair.

We have partially purified a human activity that restores mismatch-dependent, bi-directional excision to a human nuclear extract fraction depleted for one or more mismatch repair excision activities. Human EXOI co-purifies with the excision activity, and the purified activity can be replaced by near homogeneous recombinant hEXOI. Despite the reported 5' to 3' hydrolytic polarity of this activity, hEXOI participates in mismatch-provoked excision directed by a strand break located either 5' or 3' to the mispair. When the strand break that directs repair is located 3' to the mispair, hEXOI- and mismatch-dependent gap formation in excision-depleted extracts requires both hMutSalpha and hMutLalpha. However, excision directed by a 5' strand break requires hMutSalpha but can occur in absence of hMutLalpha. In systems comprised of pure components, the 5' to 3' hydrolytic activity of hEXOI is activated by hMutSalpha in a mismatch-dependent manner. These observations indicate a hydrolytic function for hEXOI in 5'-heteroduplex correction. The involvement of hEXOI in 3'-heteroduplex repair suggests that it has a regulatory/structural role in assembly of the 3'-excision complex or that the protein possesses a cryptic 3' to 5' hydrolytic activity.     

Rapid detection of Salmonella from poultry meat products using the '1. 2. Test®'

The 1.2 Test® is a new method of Salmonella detection and it was tested in naturally contaminated poultry meat products. The method, first tested on 48 samples using only previous pre-enrichment phase (experiment 1). gave 30% sensitivity and 91% specificity compared with a classical method. A second study was conducted using the 1.2 Test® following three different cultural procedures ((i) pre-enrichment, (ii) enrichment, and (iii) a combination of pre-enrichment and enrichment) (experiment 2). The three cultural procedures, before inoculation of the 1.2 Test®, gave 73, 73, and 93% sensitivity respectively and all of them were 100% specific. Moreover, the 1.2 Test® gave results very similar to those obtained with the 'enrichment serology' of Sperber and Deibel. It appears that both the preenrichment and enrichment steps are necessary in order to obtain reliable results from the 1.2 Test®, even with raw and 'highly' contaminated products.     

Evidence for a 5' leads to 3' direction of hydrolysis by a 5' mononucleotide-producing exoribonuclease from Saccharomyces cerevisiae.

[³H]rRNA labeled at the 5′ terminus with ³²P and [³H]rRNA labeled at the 3′ end with [¹⁴C] (pA)_n have been degraded at 0° with a highly purified exoribonuclease from $\text{Saccharomyces cerevisiae}$. The results show that with the [³²P, ³H] substrate, the ³²P label is rendered acid-soluble at a much faster rate than the ³H label. Both acid-soluble labels are found in 5′ mononucleotide. With the [¹⁴C, ³H]rRNA, the ³H label is hydrolyzed at a faster rate than the ¹⁴C label. The exoribonuclease hydrolyzes in the 5′ → 3′ direction.     

Radioimmunoassay of cytidine 3',5' monophosphate (cCMP). I. Development of the assay.

A method is presented for the production of reagents for a radioimmunoassay for cCMP. cCMP was succinylated at the 2′0 position with [1,4 ¹⁴C] succinic anhydride, and the monosuccinyl cCMP coupled to Keyhole limpet hemocyanin and injected into rabbits. Antibodies to cCMP were produced that showed minimal crossreactivity with other cyclic nucleotides. Monosuccinyl cCMP was coupled to tyrosine methyl ester, then labeled with ¹²⁵I, and used as the radiolabeled ligand in the immunoassay of cCMP. By use of this assay, the concentration of cCMP in various tissues of rat and guinea pig have been determined.     

The interaction of 3,3',4',5-tetrachlorosalicylanilide with phosphatidylcholine bilayers.

1. The interaction of the germicide 3,3',4',5-tetrachlorosalicylanilide (T4CS) with vesicles and dispersions of egg phosphatidylcholine has been studied by gel permeation chromatography, electron microscopy, electron spin resonance spin labelling and ion permeability measurements. 2. Incorporation of T4CS into vesicles of egg phosphatidylcholine gives rise to a large increase in the permeability rate of the paramagnetic cation N,N-dimethyl-N-(1'-oxyl-2',2',6',6'-tetramethyl-4'-piperidyl)-2-hydroxyethylammonium chloride through the lipid bilayer but has no significant effect on the vesicle sizes as measured by gel permeation chromatography or electron microscopy. 3. ESR studies using a spin-labelled fatty acid have demonstrated the presence of two different environments for the spin label when T4CS is incorporated into phosphatidylcholine bilayers. These two environments are identified as (a) highly ordered areas of the bilayer, rich in T4CS and (b) areas with very similar ordering to that in pure egg phosphatidylcholine. 4. The effectiveness of very low concentrations of the germicide in increasing vesicle permeability is explained in terms of its clustering to give rigid patches, rich in T4CS, rather than being evenly distributed throughout the bilayer. It is proposed that the increased ion permeability arises from leakage at the interfaces between the rigid and flexible regions of the lipid bilayer. 5. Comparisons between the effective levels of T4CS in phosphatidylcholine vesicles and its minimum inhibitory concentration with a Gram-positive bacterium confirm the validity of phospholipid vesicles as a model for studies of germicidal activity.     

Inhibition of ribosomal peptidyltransferase with cytidyl-3' leads to 5'-[2'(3')-O-L-phenylalanyl]-L-adenosine.

The title compound 1e, obtained by chemical synthesis, is an inhibitor of E. coli ribosomal peptidyltransferase. A 50% inhibition of peptidyltransferase-catalyzed N-Ac-Phe-puromycin formation at puromycin concentration 1 × 10^?4 M with 70 S ribosome-poly U-N-Ac[¹⁴C-Phe-tRNA complex occurred at 5 × 10^?4 M of 1e. In contrast, the parent compound 2′(3′)-O-L-phenylalanine-L-adenosine (1b) is a much weaker inhibitor causing only 5% inhibition at 1 × 10^?3 M. Alkaline hydrolysis of compound 1e to cytidylyl-3′→5′-L-adenosine (1c) results in a greatly diminished inhibition which, however, exceeds that of 1b by a factor of two. The inhibition of peptidyltransferase with 1e can be reversed by puromycin. The latter effect levels off at 40% inhibition.