Penicillin-binding proteins 4 and 5 of Haemophilus influenzae are involved in cell wall incorporation

Abstract Permeabilized cells of Haemophilus influenzae incorporate wall precursors into murein material in an ampicillin-sensitive reaction. In resistant transformants that contain the low antibiotic affinity penicillin-binding proteins (PBPs) 4 and 5, the sensitivity of this incorporation reaction to ampicillin is proportionally lower, suggesting a catalytic role for these proteins in wall synthesis. We conclude that, analogous to the reaction in Escherichia coli , PBPs 4 and 5 of H. influenzae have transpeptidase activity.     

Regeneration of Triticum aestivum apical explants after microinjection of germ line progenitor cells with DNA

A highly efficient method of regenerating fertile, phenotypically normal plants from shoot apex cultures of T. aestivum was developed. The hypodermal layer (L2) of the vegetative apex containing germ line precursor cells could be located with bright field microscopy and targeted for microinjection. Fluorescently labelled dextrans were used as markers to develop a microinjection procedure which did not disrupt nuclear or cytoplasmic structure. This procedure was used to inject plasmid DNA into L2 cells. Capillary microinjection did not shear the plasmid DNA and delivery of DNA was confirmed by polymerase chain reaction analysis of DNA isolated from injected apices. The significance of these findings in relation to the development of cereal transformation systems will be discussed.     

Scale sensitivity of synthetic long‐term vegetation time series derived through overlay of short‐term field records

Questions: Is change in cover of dominant species driving the velocity of succession or is it species turnover (1)? Is the length of the time‐step chosen in sampling affecting our recognition of the long‐term rate of change (2)1 Location: 74 permanent plots located in the Swiss National Park, SE Switzerland, ca. 1900 m a.s.l. Methods: We superimpose several time‐series from permanent plots to one single series solely based on compositional dissimilarity. As shown earlier (Wildi & Schütz 2000) this results in a synthetic series covering about 400 to 650 yr length. Continuous power transformation of cover‐percentage scores is used to test if the dominance or the presence‐absence of species is governing secondary succession from pasture to forest. The effect of time step length is tested by sub‐samples of the time series. Results: Altering the weight of presence‐absence versus dominance of species affects the emerging time frame, while altering time step length is uncritical. Where species turnover is fast, different performance scales yield similar results. When cover change in dominant species prevails, the solutions vary considerably. Ordinations reveal that the synthetic time series seek for shortest paths of the temporal pattern whereas in the real system longer lasting alternatives exist. Conclusions: Superimposing time series differs from the classical space‐for‐time substitution approach. It is a computation‐based method to investigate temporal patterns of hundreds of years fitting between direct monitoring (usually limited to decades) and the analysis of proxy‐data (for time spans of thousands of years and more).     

Advances in alternative DNA delivery techniques

This review describes recent advances in alternative DNA-delivery techniques with particular emphasis on silicon carbide fibers, intact tissue electroporation, electrophoresis and microinjection. The advantages/disadvantages of each method along with a historical overview and theory of practice are discussed.     

Autogenous growth factor production by reticuloendotheliosis virus-transformed hematopoietic cells

Reticuloendotheliosis virus strain T (REV-T)-transformed cells gave rise spontaneously to variants which secrete a factor that forms a distinct visible ring of precipitation (halo) surrounding colonies grown in soft agar. An Mr 15,000 protein was produced at higher levels by halo variants than by nonhalo-producing cells. An assay designed to detect the formation of precipitates enabled purification of an Mr 15,000 protein, p15, from serum-free medium conditioned by the growth of REV-T-transformed hematopoietic cells. Fractions enriched in p15 permitted the growth of REV-T-transformed cells under conditions where they normally failed to proliferate.     

Sensitivity of the cell cycle to TGFβ1 does not correlate with transformation of a rat liver epithelial cell line

The effects of TGF1 on cell cycle events in a rat liver derived epithelial cell line (BL9) and in two in vitro transformants of this line were studied by flow cytometry. Using either ethidium bromide staining or the incorporation of bromodeoxyuridine to evaluate DNA synthesis it was shown that TGF1 prevented the entry of G0/G1 phase BL9 cells into S phase. TGF1 did not exert its inhibitory effect(s) on DNA synthesis by the modulation of early events in the cell cycle. The tumorigenic transformed BL9 cell lines gave contrasting responses to the effects of TGF1. DNA synthesis in a BL9 cell line derived by transfection with an active N-ras oncogene was unaffected by TFG1 and thus appeared refractory to its growth controlling effects. On the other hand cells from a BL9 cell line derived by in vitro transformation with activated aflatoxin B₁ retained their sensitivity to the effects of TGF1. Thus the loss of the inhibitory effect of TGF1 on DNA synthesis is not obligatory for the malignant transformation of rat liver epithelial cells.Abbreviations TGF1 transforming growth factor 1 - BSA bovine serum albumin - FBS foetal bovine serum - BrdUrd bromodeoxyuridine - PI propidium iodide - PBS phosphate buffered saline     

A free-radical hypothesis for the instability and evolution of genotype and phenotypein vitro

It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., anin vitro evolution, which includes crisis, spontaneous transformation (immortalization), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by thein vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause ofin vitro evolution. We propose an oxyradical hypothesis to explain each of the stages ofin vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.ORR, CDER, FDA Mod-1, Room 2023, 8301 Muirkirk Road, Laurel MD 20708, USA     

The isolation and restriction mapping of a miniplasmid from the Actinomycete Nocardia corallina

Abstract A variety of plasmids has been identified as covalently closed circular and linear DNA in certain Actinomycetes, such as Streptomyces . This paper describes the first isolation and characterisation of a plasmid from the genus Nocardia . The plasmid pKU100 isolated from Nocardia corallina is a cccDNA molecule, 2.7 kb in length. This plasmid has been mapped with a wide variety of restriction enzymes and contains a number of unique restriction sites making it suitable for development as a cloning vector.     

Regeneration in Hydrozoa: distal versus proximal transformation in Hydractinia

Summary Polyps of mature colonies of Hydractinia echinata obey the rule of distal transformation by regenerating heads but not stolons. However, this rule is not valid for young polyps as these regenerate stolons from proximal cut ends. Also, small cell aggregates and even small fragments excised from full-grown polyps are capable of stolon formation. Aggregates produced from dissociated cells undergo either distal or proximal transformation depending on their size, speed of head regeneration in the donor used for dissociation and the positional derivation of the cells. The latent capability of stolon formation is released under conditions that cause loss of morphogens and depletion of their sources. However, internal regulative processes can also lead to gradual proximal transformation: regenerating segments of polyps sometimes form heads at both ends and the distal pattern is duplicated. Subsequently, all sets of proximal structures, including stolons, are intercalated. In contrast to distal transformation, proximal transformation is a process the velocity of which declines with the age and size of the cell community.