Nonlocal interactions stabilize compact folding intermediates in reduced unfolded bovine pancreatic trypsin inhibitor.

To further our understanding of the protein folding process, it is desirable to examine the structural intermediates (equilibrium and kinetic) that are populated between the statistical coil state and the folded molecule. X-ray crystallography and NMR structural studies are unable to determine long-range distances in proteins under denaturing solution conditions. Nonradiative (F?rster) energy transfer, however, has been shown to be a spectroscopic ruler for the measurement of distance distributions and diffusion between selected sites in proteins under a range of different solution conditions. The distributions of distances between a donor probe at the N-terminal residue and an acceptor attached to one of the four lysine residues (15, 26, 41, 46) of reduced and unfolded (in 6 M guanidine hydrochloride and 20 mM dithiothreitol) bovine pancreatic trypsin inhibitor (BPTI) were measured as a function of temperature. Even in strong denaturant and reducing agent, BPTI does not exist as a statistical coil polypeptide. It appears that nonlocal (long-range) interactions are already beginning to "fold" the protein toward a more compact, native conformation. As the temperature is increased under these conditions, hydrophobic interactions lead to an even more compact structure consistent with the predictions of phase diagrams for globular proteins.     

Efficient generation of chimaeric mice using embryonic stem cells after long-term culture in the presence of ciliary neurotrophic factor

The aim of our study was to evaluate whether ciliary neurotrophic factor (CNTF) can substitute for leukaemia inhibitory factor (LIF) in maintaining pluripotential embryonic stem (ES) cells in culture. Two subclones of D3 ES cells were used to assess cell proliferation and differentiation in the presence of CNTF, LIF or Buffalo rat liver (BRL) cell-conditioned medium, or in the absence of exogenous differentiation inhibiting factors. ES cells maintained in medium supplemented with CNTF for up to four weeks were injected into blastocysts to investigate theirin vivo pluripotency in terms of chimaera formation. CNTF inhibited ES cell differentiation in a dose-dependent manner. The most effective concentration was 10 ng CNTF per ml of medium. The effects of CNTF on ES cell differentiation and proliferation were comparable to those of LIF at the same concentration. BRL cell-conditioned medium was less effective at preventing ES cell differentiation but induced their proliferation very markedly. Both ES cell clones efficiently formed chimaeras after long-term culture with CNTF as the only differentiation inhibiting agent. The ability of these ES cells to colonize the germ-line is the ultimate proof that CNTF can preserve the pluripotency of ES cells.     

Actions and interactions of growth hormone and insulin-like growth factor-II: body and organ growth of transgenic mice

To characterize long-term actions and interactions of growth hormone (GH) and insulin-like growth factor-II (IGF-II) on postnatal body and organ growth, hemizygous phosphoenolpyruvate carboxykinase (PEPCK)-human IGF-II transgenic mice were crossed with hemizygous PEPCK-bovine GH transgenic mice. The latter are characterized by two-fold increased serum levels of IGF-I and exhibit markedly increased body, skeletal and organ growth. Four different genetic groups were obtained: mice harbouring the IGF-II transgene (I), the bGH transgene (B), or both transgenes (IB), and non- transgenic controls (C). These groups of mice have previously been studied for circulating IGF-I levels (Wolf et al., 1995a), whereas the present study deals with body and organ growth. Growth curves (week 3 to 12) were estimated by regression with linear and quadratic components of age on body weight and exhibited significantly (p < 0.001) greater linear coefficients in B and IB than in I and C mice. The linear coefficients of male I and C mice were significantly (p < 0.001) greater than those of their female counterparts, whereas this sex-related difference was absent in the bGH transgenic groups. The weights of internal organs as well as the weights of abdominal fat, skin and carcass were recorded from 3.5- to 8- month-old mice. In addition, organ weight-to-body weight-ratios (relative organ weights) were calculated. Except for the weight of abdominal fat, absolute organ weights were as a rule significantly greater in B and IB than in I and C mice. IGF-II overproduction as a tendency increased the weights of kidneys, adrenal glands, pancreas and uterus both in the absence and presence of the bGH transgene. Analysis of relative organ weights demonstrated significant (p < 0.05) effects of elevated IGF- II on the relative growth of kidneys (males and females) and adrenal glands (females), confirming our previous report on organ growth of PEPCK-IGF-II transgenic mice. In females, IGF-II and GH overproduction were additive in stimulating the growth of spleen and uterus, providing evidence for tissue-specific postnatal growth promoting effects by IGF-II in the presence of elevated IGF-I     

Proferrioxamine synthesis in Erwinia amylovora in response to precursor or hydroxylysine feeding: metabolic profiling with liquid chromatography-electrospray mass spectrometry

Metabolic profiling by capillary liquid chromatography-electrospray mass spectrometry was used to monitor shifts in the proferrioxamine profiles of Erwinia amylovora in response to externally supplied potential proferrioxamine precursors, selected stable-isotope-labeled precursors and atypical precursors. Based on the qualitative and quantitative shifts in the proferrioxamine profiles, lysine and arginine are unambiguous, and agmatine, ornithine, diaminobutyric acid and the corresponding C_3–5 diamines are highly likely precursors for proferrioxamine biosynthesis in E. amylovora. 5-Hydroxylysine (Hyl), a recently discovered growth inhibitor for E. amylovora, suppresses proferrioxamine production. The Hyl-induced growth inhibition can be reversed by basic amino acids. The basic amino acids also partly restore proferrioxamine synthesis.Part 12 in the series Metabolites of Erwinia, for Parts 10 and 11 see Feistner (1994d) and Feistner (1995b), respectively. Presented, in part, at ALEX '93. San Francisco. October 5–7. 1993, and at the 42nd ASMS Conference. Chicago. May 29–June 3, 1994.     

Salivary gland of the tick vector (R. appendiculatus) of East Coast fever. I. Ultrastructure of the type III acinus

The brown ear tick Rhiplcephalus appendiculatus is the vector for East Coast fever, a disease that seriously limits livestock production in East Africa. The sporozoites of the infectious agent Theileria parva develop in the tick salivary gland. This paper describes the organization of the type III acinus of the gland and establishes unambiguous ultrastructural criteria for identification of the three secretory cell types: the d-cell, e-cell and f-cell. These observations are basic to exploration of possible cell-type specificity of the invading theileria and other aspects of host-parasite relations.     

Tissue specific variation of C-glycosylflavone patterns in oat leaves as influenced by the environment

A comparison is made between the flavone patterns accumulating in epidermal tissues and in the mesophyll of oat primary leaves grown in a phytotron and under field conditions. In developing leaves cultivated under standard conditions, varying patterns of two vitexin-derived O-rhamnosides and of one isovitexin O-arabinoside are produced in the basal region as the result of basal meristem activity. These patterns are tissue specific and differ quantitatively in the epidermis and the mesophyll. During the course of subsequent growth and differentiation, this pattern is constant as the compounds are moved upwards due to basipetal leaf growth. In comparison, the flavone patterns generated in the basal section of leaves grown in the field do not vary significantly. There is the additional accumulation of isoorientin O-arabinoside. Again flavone patterns are tissue specific, but in contrast to standard growth they are modified characteristically in those leaf tissues which are already morphologically differentiated. It is possible that the isovitexin moiety of the O-arabinoside is oxidized to the corresponding isoorientin derivative in the mesophyll. Moreover, field-grown leaves show a two-fold increase in flavone content in each leaf epidermis and a six-fold increase in the mesophyll when compared to the corresponding tissues of phytotron-grown leaves.     

Specific inhibition of outgrowth of Bacillus subtilis spores by novobiocin.

Spores of a Bacillus subtilis mutant temperature sensitive in deoxyribonucleic acid (DNA) replication proceeded through outgrowth at the nonpermissive temperature to the same extent as the wild-type parent spores. In contrast, the DNA synthesis inhibitor novobiocin completely prevented spore outgrowth while displaying a marginal effect on logarithmic growth during one generation time. Inhibition of outgrowth by novobiocin occurred in the absence of DNA replication, as demonstrated in an experiment with spores of the temperature-sensitive DNA synthesis mutant at the restrictive temperature. Novobiocin inhibited the initial rate of ribonucleic acid synthesis to the same extent in germinated spores and in exponentially growing cells. A novobiocin-resistant mutant underwent normal outgrowth in the presence of novobiocin. Therefore, novobiocin inhibition was independent of its effect on chromosome replication per se.