Penetration of Bdellovibrio bacteriovorus into Host Cells

Electron microscopy reveals that, in Bdellovibrio infection, after the formation of a passage pore in the host cell wall, the differentiated parasite penetration pole is associated with the host protoplast. This firm contact persists throughout the parasite penetration and after this process is completed. In penetrated hosts this contact is also apparent by phase microscopy. The association between the walls of the parasite and the host at the passage pore, on the other hand, is transient. Bdellovibrio do not penetrate hosts whose protoplast and cell walls are separated by plasmolysis, or in which the membrane-wall relationship is affected by low turgor pressure. It is concluded, therefore, that for penetration to occur it is essential that the host protoplast be within reach of the parasite, so that a firm contact can be established between them. A penetration mechanism is proposed that is effected by forces generated by fluxes of water and solutes due to structural changes in the infected host envelope. These forces cause a differential expansion of the host protoplast and cell wall and their separation from each other around the entry site, while the parasite remains firmly anchored to the host protoplast. Consequently, the parasite ends up enclosed in the expanded host periplasm. The actual entry, therefore, is a passive act of the parasite.     

Developing an Extended Genomic Engineering Approach Based on Recombineering to Knock-in Heterologous Genes to <Emphasis Type="Italic">Escherichia coli</Emphasis> Genome

Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage λ (λ Red) and flippase–flippase recognition targets (FLP–FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P _trc and P _araB ), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol) under various genetic backgrounds (i.e., HB101 and DH5α). The expression of the inserted foreign genes was subjected to regulation using appropriate inducers [isopropyl β-d-1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive genomic engineering of E. coli, but also paves an effective way to “tailor” plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic engineering.     

Determination of electric current distributions in animals and humans exposed to a uniform 60-Hz high-intensity electric field

The thermographic method for determining specific absorption rate (SAR) in animals and models of tissues or bodies exposed to electromagnetic fields was applied to the problem of quantifying the current distribution in homogeneous bodies of arbitrary shape exposed to 60-Hz electric fields. The 60-Hz field exposures were simulated by exposing scale models of high electrical conductivity to 57.3-MHz VHF fields of high strength in a large 3.66 × 3.66 × 2.44-m TE₁₀₁ mode resonant cavity. After exposure periods of 2–30 s, the models were quickly disassembled so that the temperature distribution (maximum value up to 7 °C) along internal cross-sectional planes of the model could be recorded thermographically. The SAR, W′, calculated from the temperature changes at any point in the scale model was used to determine the SAR, W, for a full-scale model exposed to a 60-Hz electric field of the same strength by the relation W = (60/ f² · (σ′/σ) · W′ where f′ is the model exposure frequency, σ′ is the conductivity of the scale model at the VHF exposure frequency, and σ is the conductivity of the full-scale subject at 60 Hz. The SAR was used to calculate either the electric field strength or the current density for the full-scale subject. The models were used to simulate the exposure of the full-scale subject located either in free space or in contact with a conducting ground plane. Measurements made on a number of spheroidal models with axial ratios from 1 to 10 and conductivity from 1 to 10 s/m agreed well with theoretical predictions. Maximum current densities of 200 nA/cm² predicted in the ankles of man models and 50 nA/cm² predicted in the legs of pig models exposed to 60-Hz fields at 1kV/m agreed well with independent measurements on full-scale models.     

Composition and Metabolism of Gangliosides in Rat Peripheral Nervous System During Development

Abstract: Ganglioside composition of rat trigeminal nerve was studied during development in order to understand the changes that occur as a result of cellular differentiation in the nerve. The ganglioside composition of the trigeminal nerve was entirely different from that of brain. The major gangliosides in adult trigeminal nerve were GM₃, GD₃, and LM₁ (sialosyl-lactoneotetraosylceramide or sialosylparagloboside). The structure of LM₁ and other gangliosides was established by enzymatic degradation and by analysis of the products of acid hydrolysis. At 2 days after birth, when the Schwann cells were immature, GM₃ and GD₃ were the major gangliosides in the nerve, 50 and 18 mol %, respectively. As the nerve developed and Schwann cells proliferated and myelinated the axons, the mol % of GM₃ and GD₃ reduced and that of LM₁ steadily increased. Polysialogangliosides did not change drastically with nerve development. The rate of deposition of LM₁ in the nerve with age was very similar to that of myelin marker lipids, cerebrosides, and sulfatides; thus, deposition appears to be localized mainly in the rat nerve myelin. LM₁ also had long-chain fatty acids 22:0 and 24:0, which are not usually found in CNS gangliosides. The ganglioside pattern of the rat trigeminal nerve was very similar to that of rat sciatic nerve, but was different from that of rabbit and chicken sciatic nerve. The activity of the two key enzymes involved in the metabolism of GM₃, viz., CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase and UDP-N-acetylgalactosamine:GM₃-N-acetylgalactosaminyltransferase, was also studied during development of the nerve and brain. The developmental profiles of both enzymes were consistent with the amounts of GM₃ present in the nerve.     

DNA fingerprinting of human cell lines using PCR amplification of fragment length polymorphisms

Summary Methods for monitoring cell line identification and authentication include species-specific immunofluorescence, isoenzyme phenotyping, chromosome analysis, and DNA fingerprinting. Most previous studies of DNA fingerprinting of cell lines have used restriction fragment length polymorphism analysis. In this study, we examined the utility of an alternative and simpler method of cell line DNA fingerprinting—polymerase chain reaction (PCR) amplification of fragment length polymorphisms. Fourteen human cell lines previously found by other methods to be either related or disparate were subjected to DNA fingerprinting by PCR amplification of selected fragment length polymorphism loci. Cell identification patterns by this method were concordant with those obtained by isoenzyme phenotyping and restriction fragment length polymorphism-DNA fingerprinting, and were reproducible within and between assays on different DNA extracts of the same cell line. High precision was achieved with electrophoretic separation of amplified DNA products on high resolution agarose or polyacrylamide gels, and with fragment length polymorphism (FLP) loci-specific “allelic ladders” to identify individual FLP alleles. Determination of the composite fingerprint of a cell line at six appropriately chosen fragment length polymorphism loci should achieve a minimum discrimination power of 0.999. The ability of PCR-based fragment length polymorphism DNA fingerprinting to precisely and accurately identify the alleles of different human cell lines at multiple polymorphic fragment length polymorphism loci demonstrates the feasibility of developing a cell line DNA fingerprint reference database as a powerful additional tool for future cell line identification and authentication.     

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth _vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogenor prolactin-dependent growth. Primary tumors still displayed a constitutional expression of α-, β-, and γ-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in α- and β-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of α-, β-, and γ-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10–14 days. The accumulation of β-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance β-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.     

Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae

A 40-kD protein kinase C (PKC)epsilon related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18. The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKC epsilon specific antibody showed that the 40-kD species, and not native PKC epsilon (90 kD) associated with the cytokeratins. Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKC epsilon, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. A peptide pseudosubstrate specific for PKC epsilon inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates. Tryptic peptide map analysis of the cytokeratins that were phosphorylated by purified rat brain PKC epsilon or as immunoprecipitates by the associated kinase showed similar phosphopeptides. Furthermore, PKC epsilon immunoreactive species and CK8/18 colocalized using immunofluorescent double staining. We propose that a kinase related to the catalytic fragment of PKC epsilon physically associates with and phosphorylates cytokeratins 8 and 18.