Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes

Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectale group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 x 10(10) cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 x 10(10) cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 x 10(7) to 7 x 10(8) per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.     

Atypical multicentric reticulohistiocytosis with paraproteinemia

Two women had multiple subcutaneous nodules that showed features of multicentric reticulohistiocytosis (MR). Neither had joint symptoms. Both had a raised erythrocyte sedimentation rate, an immunoglobulin G paraproteinemia, and raised levels of nonhepatic serum alkaline phosphatase. The skin lesions have been followed up, using light and electron microscopy, immunoperoxidase, and histochemical methods. The material in the giant cells stained positively for gamma heavy chain determinants: the light chain type in each case was that of the paraprotein. An attempt to reproduce the skin lesions in one patient by intradermal injection of her paraprotein failed.     

Isolation of a spontaneous CHO amino acid transport mutant by a combination of tritium suicide and replica plating

A spontaneous transport mutant of Chinese hamster ovary cells, CHY-1, was isolated by a combination of [3H]proline suicide and replica plating. The mutant took up less tritium than the parent, resulting in a lower killing rate during storage. Transport by four separate amino acid transport systems (A, ASC, L, Ly+) was examined. The CHY-1 mutant exhibited normal uptake via the ASC, L, and Ly+ systems. By contrast, uptake of the most specific substrate of the A system, 2-(methylamino)-isobutyric acid, was significantly reduced at low, but not high, concentrations, due to a 3.5-fold increase in Km and a 1.5-fold increase in Vmax. Taken together, these data suggest that the CHY-1 mutation may be in the structural gene coding for the A transport protein. The tritium suicide procedure is discussed, and general equations are derived to predict the maximum storage time for the survival of one mutant cell and the optimum size of the cell population for maximum mutant enrichment.