In vitro phagocytosis and intracellular survival of Campylobacter jejuni strain 2964 in mononuclear phagocytes were studied. The following three types of mononuclear phagocytes were used: a J774G8 peritoneal macrophage line derived from BALB/c mice, resident BALB/c peritoneal macrophages, and human peripheral blood monocytes. When C. jejuni and mononuclear phagocytes were combined at a ratio of 75:1, light microscopy, fluorescent microscopy, and electron microscopy all indicated that C. jejuni cells were readily phagocytized. The majority of C. jejuni cells were spirals immediately following ingestion and were rapidly converted to the coccal form within 4 to 8 h. Conversion from the spiral form to the coccal form was complete in the presence of phagocytes within 96 h. In control preparations without phagocytes, conversion began after 24 h and was complete after 48 h. The extent of phagocytosis over time was determined by observing Giemsa-stained preparations and counting the number of intracellular bacterial colony-forming units after removal of extracellular C. jejuni. Human monocytes ingested C. jejuni more rapidly and vigorously than murine macrophages. Intracellular survival of C. jejuni was examined by measuring the number of C. jejuni colony-forming units associated with phagocytes after phagocytosis for 2 h and removal of extracellular bacteria. C. jejuni survived intracellularly for up to 6 to 7 days.

Two phosphofructokinase genes have been described previously in Entamoeba histolytica. The product of the larger of the two genes codes for a 60-kDa protein that has been described previously as a pyrophosphate (PP i )-dependent enzyme, and the product of the second, coding for a 48-kDa protein, has been previously reported to be a PP i -dependent enzyme with extremely low specific activity. Here it is found that the 48-kDa protein is not a PP i -dependent enzyme but a highly active ATP-requiring enzyme (k cat ‫؍‬ 250 s ؊1 ) that binds the cosubstrate fructose 6-phosphate (Fru-6-P) with relatively low affinity. This enzyme exists in concentration-and ATP-dependent tetrameric active and dimeric inactive states. Activation is achieved in the presence of nucleoside triphosphates, ADP, and PP i , but not by AMP, P i , or the second substrate Fru-6-P. Activation by ATP is facilitated by conditions of molecular crowding. Divalent cations are not required, and no phosphoryl transfer occurs during activation. Kinetics of the activated enzyme show cooperativity with Fru-6-P (Fru-6-P 0.5 ‫؍‬ 3.8 mM) and inhibition by high ATP and phosphoenolpyruvate. The enzyme is active without prior activation in extracts of E. histolytica. The level of mRNA, the amount of enzyme protein, and the enzyme activity of the 48-kDa enzyme are about one-tenth that of the 60-kDa enzyme in extracts of E. histolytica trophozoites.Entamoeba histolytica along with a number of other parasitic protists utilizes an unusual form of phosphofructo-1-kinase (PFK) 1 in a central step in carbohydrate metabolism. This form of PFK employs inorganic pyrophosphate (PP i ) as a phosphoryl donor. Two genes for PP i -PFK have been described in E. histolytica (1-3) with a sequence identity between the two proteins of 17%. The sequence of the larger gene, which codes for a protein of ϳ60 kDa, has greater identity to the more phylogenetically advanced plant PP i -PFKs than it does to bacterial PP i -PFKs. The cDNA of this gene has been expressed in Escherichia coli and was found to have kinetic properties that were identical to those of the enzyme isolated from E. histolytica (3).

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This review will concentrate on certain aspects of the nucleic acids of Entamoeba histolytica. Utilization and synthesis of purines and pyrimidines will initially be briefly discussed, e.g. salvage vs. de novo pathways, uptake studies and recognition of at least 4 transport loci. Data will be presented which show that the distribution and synthesis of RNA (to a lesser extent DNA) in the nucleus is basically the opposite one finds in other eukaryotes, viz. most RNA (ribosomal?) is synthesized (or accumulates) in the peripheral chromatin (functional equivalent of nucleolus?). The DNA is distributed and synthesized primarily throughout the nucleus. It is usually so dispersed that it will not stain with e.g. the standard Feulgen technique, unless the DNA condenses around the endosome (not a nucleolar equivalent) prior to nuclear division. Isolation of rRNA was difficult due, in part, to potent and difficult to inhibit RNase(s), some of which are apparently intimately bound to ribosomal subunits. The 25S (1.3 kDa), 17S (0.8 kDa) and 5S rRNA were recovered after isolation with a high salt SDS-DEP technique. This is the only procedure which enables us to obtain high yields of 25S rRNA; guanidine or guanidinium which permits isolation of intact functional mRNA results in isolation of small amounts of 25S RNA relative to 17S RNA. The 25S RNA is "nicked" (apparently during nuclear processing) and dissociates readily into 17S (0.7 kDa) and 16S (0.6 kDa) species, and a more rigidly bound 5.8S species. A small amount of "unnicked" 25S RNA was recovered with guanidine.(ABSTRACT TRUNCATED AT 250 WORDS)