The inactivation of beta-galactosidase (E. coli) by the carbodiimide reaction

When beta-galactosidase reacted with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC), activity was lost. The inhibitor, isopropyl-beta-D-galactopyranoside (IPTG), decreased inactivation. Of 3 nucleophiles tested, incorporation was only decreased in the protected (IPTG added) enzyme when sulfanilic acid was the nucleophile but HPLC profiles of tryptic peptides were identical in protected and unprotected enzyme (except for magnitude). There were also no differences (except for magnitude) of HPLC profiles after 10 and 90 min of reaction and between active (soluble) and inactive (precipitated) enzyme. The data indicate that inactivation is not caused by reaction with a specific active site group. Inactivation probably occurs when a combination of groups are reacted.     

A sequence motif found in a Drosophila heterochromatin protein is conserved in animals and plants.

Modifiers of position-effect-variegation in Drosophila encode proteins that are thought to modify chromatin, rendering it heritably changed in its expressibility. In an attempt to identify similar modifier genes in other species we have utilized a known sequence homology, termed chromo box, between a suppressor of position-effect-variegation, Heterochromatin protein 1 (HP1), and a repressor of homeotic genes, Polycomb (Pc). A PCR generated probe encompassing the HP1 chromo box was used to clone full-length murine cDNAs that contain conserved chromo box motifs. Sequence comparisons, in situ hybridization experiments, and RNA Northern blot analysis suggest that the murine and human sequences presented in this report are homologues of the Drosophila HP1 gene. Chromo box sequences can also be detected in other animal species, and in plants, predicting a strongly conserved structural role for the peptide encoded by this sequence. We propose that epigenetic (yet heritable) changes in gene expressibility, characteristic of chromosomal imprinting phenomena, can largely be explained by the action of such modifier genes. The evolutionary conservation of the chromo box motif now enables the isolation and study of putative modifier genes in those animal and plant species where chromosomal imprinting has been described.     

Selectivity in metabolic cooperation between cultured mammalian cells

Selective communication between cultured mammalian cells was detected as selectivity in metabolic cooperation. Whilst the majority of the cell types examined (human skin fibroblast, PC13, G3, Don, PyY) showed metabolic cooperation at almost all (>95%) of their homotypic cell-to-cell contacts, they did not necessarily show cooperation at such a high proportion of their heterotypic contacts. Less than 10% of G3/human fibroblast contacts, and usually less than 30% of G3/PC13 contacts were observed to be positive for metabolic cooperation. L cells differed from these other cell types in that they formed permeable junctions at a greater proportion of their heterotypic cell-to-cell contacts (contacts between L and PyY cells) than their homotypic contacts. We question why it was that the contacts between any two poorly-compatible cell types were positive for metabolic cooperation in only a small proportion of cases. We could find no indication that this phenomenon was attributable to heterogeneity within the cell stocks. Time course studies upon PC13 and G3 cells showed that the proportion of cooperation-positive contacts was not constant but that it continued to increase over many hours of co-culture. In comparing the homotypic and heterotypic interactions of these cell types, selectivity manifested as a difference in the rate of appearance of permeable junctions. We discuss possible explanations for these findings.     

The differentiation of teratocarcinoma stem cells is marked by the types of collagen which are synthesized.

A cloned line of undifferentiated teratocarcinoma cells (OC15S1) was either maintained as a homogeneous embryonal carcinoma (EC) cell population or was cultured under conditions where the cells differentiated into endoderm-like (END) cells. In this study we examine the synthesis of collagen in both EC and END cells. Cell cultures were incubated with tritiated proline and lysine, and the radioactive collagen secreted into the medium was extracted and purified or immunoprecipitated by antibodies to type IV collagen (Adamson and Ayers, 1979). Radioactive collagens were identified by electrophoretic mobility, by sensitivity to collagenase and to reduction, by insensitivity to pepsin, by cyanogen bromide peptides, and by aminoacid analyses of 3-hydroxyproline, 4-hydroxyproline and proline. OC15S1 EC cells were found to synthesize several collagenous polypeptides, of which 60–70% of the radioactivity was like that of basement membrane (type IV) collagen. Type I-like collagen was the main collagenous product of END cells, but a minor product of EC cells. We concluded that type IV collagen synthesis was suppressed during the differentiation of EC cells to END, while type I-like synthesis was increased. Similarly, other EC cell lines produced mainly type IV-like collagen polypeptides (PC13, F9, PSA1), and following the formation of END cells, two lines produced mainly type I-like collagen polypeptides (PC13, C145b). The type of endoderm formed on embryoid bodies, however, presents an alternate route of differentiation, since immunoperoxidase tests showed that it was synthesizing significant amounts of type IV collagen. We discuss the significance of these findings in relation to a similar change which occurs during normal development.     

Effect of intravenous dextran 70 and pneumatic leg compression on incidence of postoperative pulmonary embolism.

The incidence of pulmonary embolism and deep vein thrombosis was measured in 50 matched pairs of patients undergoing common surgical procedures with preoperative and postoperative ventilation-perfusion lung scans and the fibrinogen uptake test. One patient in each pair was treated with intravenous dextran 70 and pneumatic leggings. The incidence of pulmonary embolism among the treated patients was significantly reduced from 24% to 8%, but the incidence of deep vein thrombosis was not significantly reduced (34% to 24%).     

Gastrointestinal torsion in a channel catfish (Ictalurus punctatus)

A case of gastrointestinal torsion with dilatation in a farm-raised channel catfish (Ictalurus punctatus) was examined at the Thad Cochran National Warmwater Aquaculture Center (Stoneville, Mississippi, USA). The affected fish was a gravid female broodfish, which displayed pale gills and a markedly distended abdomen. Internal examination revealed that the gastrointestinal tract and ovaries were rotated around each other four times in a counterclockwise direction as viewed in right lateral recumbency. The catfish had a markedly distended gastrointestinal tract, pale liver, hypoplastic spleen, hypoplastic swim bladder, and high volume of ascitic fluid. Blood analysis indicated multiple abnormalities, including severe anemia and metabolic acidosis. The etiology of the torsion was uncertain; however, the presence of a hypoplastic swim bladder most likely allowed for increased movement of the gastrointestinal tract and ovaries. When examining cases of abdominal distention in fish, gastrointestinal torsion can be considered among the differential diagnoses.     

Recent, independent and anthropogenic origins of Trypanosoma cruzi hybrids

The single celled eukaryote Trypanosoma cruzi, a parasite transmitted by numerous species of triatomine bug in the Americas, causes Chagas disease in humans. T. cruzi generally reproduces asexually and appears to have a clonal population structure. However, two of the six major circulating genetic lineages, TcV and TcVI, are TcII-TcIII inter-lineage hybrids that are frequently isolated from humans in regions where chronic Chagas disease is particularly severe. Nevertheless, a prevalent view is that hybridisation events in T. cruzi were evolutionarily ancient and that active recombination is of little epidemiological importance. We analysed genotypes of hybrid and non-hybrid T. cruzi strains for markers representing three distinct evolutionary rates: nuclear GPI sequences (n?=?88), mitochondrial COII-ND1 sequences (n?=?107) and 28 polymorphic microsatellite loci (n?=?35). Using Maximum Likelihood and Bayesian phylogenetic approaches we dated key evolutionary events in the T. cruzi clade including the emergence of hybrid lineages TcV and TcVI, which we estimated to have occurred within the last 60,000 years. We also found evidence for recent genetic exchange between TcIII and TcIV and between TcI and TcIV. These findings show that evolution of novel recombinants remains a potential epidemiological risk. The clearly distinguishable microsatellite genotypes of TcV and TcVI were highly heterozygous and displayed minimal intra-lineage diversity indicative of even earlier origins than sequence-based estimates. Natural hybrid genotypes resembled typical meiotic F1 progeny, however, evidence for mitochondrial introgression, absence of haploid forms and previous experimental crosses indicate that sexual reproduction in T. cruzi may involve alternatives to canonical meiosis. Overall, the data support two independent hybridisation events between TcII and TcIII and a recent, rapid spread of the hybrid progeny in domestic transmission cycles concomitant with, or as a result of, disruption of natural transmission cycles by human activities.