Mechanisms of TonB-catalyzed iron transport through the enteric bacterial cell envelope

The recent solution of enteric bacterial porin structure, and new insights into the mechanism by which outer membrane receptor proteins recognize and internalize specific ligands, advocates the re-evaluation of TonB-dependent transport physiology. In this minireview we discuss the potential structural features of siderophore receptors and TonB, and use this analysis to evaluate both existing and new models of energy and signal transduction from the inner membrane to the outer membrane of gram-negative bacteria.     

Epidermal growth factor receptors lose ligand binding ability as WI-38 cells progress from short-term to long-term quiescence

WI-38 cells, density arrested for short periods of time, can be stimulated to re-enter the cell cycle by epidermal growth factor (EGF) alone. However, cells density arrested for longer periods have a prolonged prereplicative phase when serum stimulated and cannot be stimulated by EGF alone. Radio-ligand binding studies performed on WI-38 cells showed that actively growing cells bind [¹²⁵I]EGF at relatively low levels that increase to a maximum as the cells become contact inhibited. As the cells enter a state of deeper quiescence, EGF binding falls to one-third to one-fifth the short-term growth arrested levels, remaining constant thereafter. The EGF-receptor complexes internalize more slowly in long-term growth arrested cells, and the rate of ligand association to the receptor is lower than short-term growth arrested cells. The amount of EGF receptor protein in lysates of equal numbers of both short- and long-term quiescent cells remains the same. These results suggest that the failure of long-term growth arrested cells to respond to EGF is not due to dramatic changes in the amount of receptor protein during prolonged quiescence but more likely to an alteration in the ability of these receptors to bind ligand and/or activate the EGF signal transduction pathway. © 1993 Wiley-Liss, Inc.     

Cell Cycle Arrest of Proliferating Neuronal Cells by Serum Deprivation Can Result in Either Apoptosis or Differentiation

Abstract: Apoptotic cell death plays a critical role in the development of the nervous system. The death of mature nondividing neurons that fail to receive appropriate input from the target field has been extensively studied. However, the mechanisms mediating the extensive cell death occurring in areas of the developing brain where proliferating neuroblasts differentiate into mature nondividing neurons have not been analyzed. We show here that the cell cycle arrest of a proliferating cell of neuronal origin by removal of serum results in either apoptotic cell death or differentiation to a mature nondividing neuronal cell. The proportion of cells undergoing death or differentiation is influenced in opposite directions by treatment of the cells with cyclic AMP and retinoic acid. This suggests that following the withdrawal of signals stimulating neuroblast cell division, neuronal cells either can cease to suppress a constitutive suicide pathway and hence die by apoptosis or, alternatively, can differentiate into a mature neuronal cell. Regulation of the balance between apoptosis and neuronal differentiation could therefore play a critical role in controlling the numbers of mature neurons that form.     

A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.     

Effects of stripe rust on the evolution of genetically diverse wheat populations

Summary Eighteen populations, composed of four wheat (Triticum aestivum) varieties that were originally mixed together at equal frequencies, were grown for one-to-three generations at two locations. In addition, pure stands of the four varieties were grown in each year. Populations were either exposed to two stripe rust (Puccinia striiformis) races, protected from stripe rust, or exposed to alternating years of diseased and disease-free conditions. Regression of the logit of a variety's frequency versus generation number was used to calculate the relative fitness of each variety in each population. These analyses suggest that the relative fitnesses of the wheat varieties were affected by disease and geographic location and were constant over time. However, frequency-changes of varieties in the mixtures were negatively correlated with their planting frequencies (0.0001 < P < 0.085 in 14 out of 16 cases), suggesting that fitnesses were frequency-dependent in both the presence and absence of disease. We hypothesize that failure to detect frequency-dependence of fitness in the logit analyses was due to a limited number of generations and a limited range of initial variety frequencies. This is supported by data from longer-term studies in the literature that provide evidence for frequency-dependence of fitness in plant mixtures. Analyses of currently available field data suggest that stable equilibria may be a more likely outcome for mixtures of varieties that are more closely related and/or more uniformly adapted to the environment in which they are grown.Paper No. 9820 of the journal series of the Oregon Agricultural Experiment Station.     

On the mechanism of a 60-Hz electric field induced growth reduction of mammalian cellsin vitro

Data on 60-Hz electric field (EF) induced reduction in growth rate of plant roots have strongly supported the hypothesis that the effect is related to an EF-induced transmembrane potential (V ⁱ _m). An investigation was undertaken to determine if this hypothesis is also applicable to 60-Hz EF-induced reductions in growth rate of mammalian cells in vitro. Human lymphoblastic (RPMI 1788) and human carcinoma (HeLa) cells were selected for study, the former having a relatively small diameter (11.2 m), and the latter having a relatively large diameter (15.4 tm). The 60-Hz EFs ranged from 430–1200 V/m in the culture medium. The growth rate of RPMI 1788 cells after 4-days was depressed by about 42% at a 60-Hz EF of 1000–1200 V/m with a response threshold occurring at 950 V/m; theV ⁱ _m at the response threshold was 8 mV There was no 60-Hz EF-induced effect on HeLa cell growth rate of aV ⁱ _m of 8 mV (60-Hz EF=700 V/m); a statistically significant effect was achieved atV ⁱ _m of 11 mV (950 V/m). The data support the hypothesis that above a threshold 60-Hz EF,V ⁱ _m acts as the initial signal leading to growth rate reductions.     

1061. Lunaria annua subsp. pachyrrhiza (Borb as) Maire & Petitm.,: Cruciferae (Brassicaceae)

Lunaria annua is overviewed and its two subspecies, subsp. annua and subsp. pachyrrhiza, discussed, the latter described. Two cultivars of subsp. pachyrrhiza, ‘Corfu Blue’ and ‘Mistras’ are illustrated and described. Details of cultivation are also included.     

Embedding of Neural Tissue in Agarose or Glyoxyl Agarose for Vibratome Sectioning

Agarose was used to embed the brain or spinal cord of lampreys or rats before cutting vibratome sections. Agarose embedding was compatible with immunocytochemistry or the use of horseradish peroxidase as a neuroanatomical tracer. Concentrated agarose with high intrinsic gel strength was optimal for embedding glutaraldehyde fixed neural tissue. A quick procedure was to blot tissue and embed in 5% (w/v] Sigma type I-A or Litex type LSL agarose at 45-55 C dissolved in 50 mM neutral-pH TFUS buffer before cutting 50-100 μm vibratome sections. An alternative procedure that improved retention of tissue sections in the agarose was to rinse the tissue in H₂0, blot and embed in 5% (w/v] Sigma type I-A or Litex type LSL agarose at 45-55 C dissolved in H₂0, then equilibrate the block overnight in buffer. Phosphate buffer prevented complete dissolving of agarose. Tissue could be covalently linked to the embedding matrix using a novel aldehyde-derived agarose (NuFix® FMC BioProducts). Slices of spinal cord from neonatal rats could be cut after embedding in 5% FMC Seaprep® agarose in rat Ringer's at 23-26 C.     

Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae

Abstract An internal fragment from each of the penicillinebinding protein (PBP) 1A, 2B and 2X genes of Streptococcus pneumoniae , which included the region encoding the active-site serine residue, was replaced by a fragment encoding spectinomycin resistance. The resulting constructs were tested for their ability to transform S. pneumoniae strain R6 to spectinomycin resistance. Spectinomycin-resistant transformants could not be obtained using either the inactivated PBP 2X or 2B genes, suggesting that deletion of either of these genes was a lethal event, but they were readily obtained using the inactivated PBP 1A gene. Analysis using the polymerase chain reaction confirmed that the latter transformants had replaced their chromosomal copy of the PBP 1A gene with the inactivated copy of the gene. Deletion of the PBP 1A gene was therefore tolerated under laboratory conditions and appeared to have little effect on growth or susceptibility to benzylpenicillin.