Restriction analysis of the Streptomyces glaucescens genome by agarose gel electrophoresis

A method for the analysis of total DNA of Streptomyces glaucescens is described. The relevant steps are (a) extraction and purification of DNA, (b) restriction of DNA samples with type II restriction enzymes, (c) one dimensional separation of restriction fragments by agarose gel electrophoresis. A typical banding pattern was obtained for each wild type strain, independant of growth conditions or age of the culture. Mutant strains exhibited in most cases the same banding pattern as the parent wild type strain. Only in one specific mutant class a fragment of about 9 megadalton was missing.     

The natural history of the helicoidal occlusal plane and its evolution in early Homo

In modern man the pitch of the occlusal plane may vary along the tooth-row. When anterior cheek-teeth show a plane sloping upward palatally, whilst that on posterior cheek-teeth slopes upward buccally, there results a twisted or helicoidal occlusal plane (Ackermann). Several hypotheses have been proposed for the structural basis of the helicoidal occlusal plane. Campbell's proposal ('25) has gained widest acceptance, namely that the helicoid results from anteroposterior differences in upper and lower alveolar arch width. In the early 1960s, while studying the Olduvai hominids assigned to Homo habilis, the author noted changing occlusal slopes along the tooth-row and a slight helicoid, although these featues had not been noted in other early hominids. Subsequently, Wallace showed a total absence of the helicoid from South African australopithecines, and its presence in Swartkrans Homo, SK 45 and SK 80. Recent studies confirm the presence of the helicoid in all available specimens of H. habilis, including Stw 53 found at Sterkfontein in 1976. Hence, this trait may distinguish between Australopithecus and early Homo. Measurements of the maxillary arch widths have shown that, whereas in Australopithecus arch widths increase to a maximum at M3, in early Homo maxillary arch widths are greatest at M2. The decline in posterior maxillary arch width is part of a general reduction of that region. Thus despite striking elongation of premolars and M1 in early Homo, M2 and M3 are mesiodistally abbreviated. It is hypothesized that the onset of posterior arch reduction, with the appearance of a helicoid, was a structural and functional concomitant of the transition from the presumed australopithecine ancestor to H. habilis.     

Stability of a model beta-sheet in water.

We have used molecular dynamics simulations to determine the stability in water of a model beta-sheet formed by two alanine dipeptide molecules with two intermolecular hydrogen bonds in the closely spaced antiparallel arrangement. In this paper we describe our computations of the binding free energy of the model sheet and a portion of the free energy surface as a function of a reaction co-ordinate for sheet formation. We used the free energy surface to identify stable conformations along the reaction co-ordinate. To determine whether or not the model sheet with two hydrogen bonds is more stable than a single amide hydrogen bond in water, we compared the results of the present calculations to results from our earlier study of linear hydrogen bond formation between two formamide molecules (the formamide "dimer"). The free energy surfaces for the sheet and formamide dimer each have two minima corresponding to locally stable hydrogen-bonded and solvent-separated configurations. The binding free energies of the model sheet and the formamide dimer are -5.5 and -0.34 kcal/mol, respectively. Thus, the model sheet with two hydrogen bonds is quite stable while the simple amide hydrogen bond is only marginally stable. To understand the relative stabilities of the model sheet and formamide dimer in terms of solute-solute and solute-water interactions, we decomposed the free energy differences between hydrogen-bonded and solvent-separated conformations into energetic and entropic contributions. The changes in the peptide-peptide energy and the entropy are roughly twice as large for the sheet as they are for the formamide dimer. The magnitude of the peptide-water energy difference for the sheet is less than twice (by about 3.5 kcal/mol) that for the formamide dimer, and this accounts for the stability of the sheet. The presence of the side-chains and/or blocking groups apparently prevents the amide groups in the sheet from being solvated as favorably in the separated arrangement as in the formamide dimer, where the amide groups are completely exposed to the solvent.     

Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa

Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5–2 m) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.Abbreviations BrdU 5-bromodeoxyuridine - DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether) - N,N,N,N tetraacetic acid We thank Ann Cork for technical assistance, Professor B.E.S. Gunning (Australian National University) and Drs. A.R. Hardham (A.N.U.) and R.E. Williamson (A.N.U.) for intellectual and material support, Dr D. McCurdy (A.N.U.) for the purified anti-actin antibody, and Professor B. Stone (La Trobe University, Melbourne, Australia) for generously providing the anti-callose antibody. We also thank the Electron Microscopy Unit of A.N.U. for the use of facilities. L.C.F. gratefully acknowledges financial support from the National Sciences and Engineering Research Council of Canada.     

Thermodynamics and mechanism of alpha helix initiation in alanine and valine peptides

We used molecular dynamics simulations to study the folding/unfolding of one of turn of an alpha helix in Ac-(Ala)3-NHMe and Ac-(Val)3-NHMe. Using specialized sampling techniques, we computed free energy surfaces as functions of a conformational coordinate that corresponds to alpha helices at small values and to extended conformations at large values. Analysis of the peptide conformations populated during the simulations showed that alpha helices, reverse turns, and extended conformations correspond to minima on the free energy surfaces of both peptides. The free energy difference between alpha helix and extended conformations, determined from the equilibrium constants for helix unfolding, is approximately -1 kcal/mol for Ac-(Ala)3-NHMe and -5 kcal/mol for Ac-(Val)3-NHMe. The mechanism observed in our simulations, which includes reverse turns as important intermediates along the helix folding/unfolding pathway, is consistent with a mechanism proposed previously. Our results predict that both peptides (but especially the Ala peptide) have a much larger equilibrium constant for helix initiation than is predicted by the helix-coil transition theory with the host-guest parameters. We also predict a much greater difference in the equilibrium constants than the theory predicts. Insofar as helix initiation is concerned, our results suggest that the large difference between the helical propensities of Ala and Val cannot be explained by simple concepts such as side-chain rotamer restriction or unfavorable steric interactions. Rather, the origin of the difference appears to be quite complicated because it involves subtle differences in the solvation of the two peptides. The two peptides have similar turn-extended equilibria but very different helix-turn equilibria, and the difference in helical propensities reflects the fact that the helix-turn equilibrium strongly favors the turns in Ac-(Val)3-NHMe, while it favors the helices in Ac-(Ala)3-NHMe. We also computed thermodynamic decompositions of the free energy surfaces, and these revealed that the helix-turn equilibria are vastly different primarily because the changes in peptide-water interactions that accompany helix-to-turn conformational changes are qualitatively different for the two peptides.     

Methylprednisolone restores sensitivity to beta-adrenergic agonists in Basenji-Greyhound dogs.

This study investigated the effect of chronic methylprednisolone treatment on the ability of albuterol and aminophylline to inhibit methacholine-induced airway constriction in Basenji-Greyhound (BG) dogs in vivo. Pulmonary responsiveness to methacholine was measured in five untreated BG dogs and in the same dogs pretreated with albuterol or aminophylline (which has been shown in this model to release endogenous catecholamines). Each dog was studied before, during, and after daily subcutaneous methylprednisolone for 6 wk. Changes in pulmonary resistance and dynamic compliance with methacholine aerosol challenge were measured. Neither baseline pulmonary function nor pulmonary responsiveness to aerosolized methacholine was significantly altered by albuterol, aminophylline, or chronic methylprednisolone administration alone. However, pretreatment with albuterol or aminophylline significantly attenuated airway responses to methacholine in BG dogs chronically receiving methylprednisolone. Because the reduced sensitivity to albuterol and aminophylline was restored by chronic methylprednisolone treatment, we conclude that at least part of the beneficial effects of corticosteroids on airways in BG dogs is through modulation of beta-adrenergic function.     

Properties of the foot inhibitor from hydra

Summary A substance was isolated from crude extracts of hydra that inhibits foot regeneration. This substance, the foot inhibitor, has a molecular weight of 500 daltons. It is a hydrophilic molecule, slightly basic in character and it has no peptide bonds. The pruified substance acts specifically and at concentrations lower than 10^–7 M. At this low concentration only foot and not head regeneration is inhibited. Hydra are sensitive to purified foot inhibitor between the second and eight hour after initiation of foot regeneration by cutting. In normal animals the foot inhibitor is most likely produced by nerve cells. A substance with similar biological and physico-chemical properties is found in other coelenterates.     

A new species of Eugenia (Myrtaceae) from Venezuela

Eugenia mcvaughii is described from a forest remnant of the Jardin Botánico of Caracas, Venezuela. Found also in the hills adjacent to Caracas, it may be considered as an endemic species of the Interior Coastal Cordillera.     

Heavy metal-nucleoside interactions. Binding of methylmercury(II) to inosine and catalysis of the isotopic exchange of the C-8 hydrogen studied by 1-H nuclear magnetic resonance and raman difference spectrophotometry.

Raman difference spectrophotometry reveals that CH3HgII binds quantitatively to N(1) of inosine at pH 8, substituting for the proton. When N(1) is saturated, binding occurs at a second site. Measurements of the 1-H nuclear magnetic resonance spectra of both inosine and of CH3Hg-II are in agreement with the N(1) binding and indicate that the second site for mercuriation is N(7). This second binding reaction is observed to increase the rate of exchange of the C(8) hydrogen with solvent, consistent with results observed for alkylation at N(7). Coordination of the electrophilic CH3Hg-II to N(7) increases the acidity of H(8), facilitating OHminus--catalyzed proton abstraction and reprotonation by themedium. For comparison, the reaction of CH3Hg-II with [8-2-H]inosine has been studied. Displacement of the N(1) hydrogen upon mercuriation of inosine causes a significant electron delocalization into the ring, increasing the basicity of N(7), and accounting for the synergic effect in metal binding observed originally by Simpson. In contrast, 1-methylinosine interacts only slightly with CH3Hg-II at pH 8. Coordination appears to be at N(7), since H(8) again is observed to exchange rapidly with solvent protons. In acidic solution, pH less than 2, binding to inosine is almost quantitative and exclusively to N(7). The behavior of CH3Hg-II is compared with that of Pt(II) and with Ni(II), Co(II), AND Zn(II). A brief comparison is made among ultraviolet absorption spectrophotometry, nuclear magnetic resonance (NMR), and Raman difference spectrophotometry for studying reactions of nucleosides and nucleotides.     

Evidence for Geographic Isolation and Signs of Endemism within a Protistan Morphospecies

The possible existence of endemism among microorganisms resulting from and preserved by geographic isolation is one of the most controversial topics in microbial ecology. We isolated 31 strains of “Spumella-like” flagellates from remote sampling sites from all continents, including Antarctica. These and another 23 isolates from a former study were characterized morphologically and by small-subunit rRNA gene sequence analysis and tested for the maximum temperature tolerance. Only a minority of the Spumella morpho- and phylotypes from the geographically isolated Antarctic continent follow the worldwide trend of a linear correlation between ambient (air) temperature during strain isolation and heat tolerance of the isolates. A high percentage of the Antarctic isolates, but none of the isolates from locations on all other continents, were obligate psychrophilic, although some of the latter were isolated at low ambient temperatures. The drastic deviation of Antarctic representatives of Spumella from the global trend of temperature adaptation of this morphospecies provides strong evidence for geographic transport restriction of a microorganism; i.e., Antarctic protistan communities are less influenced by transport of protists to and from the Antarctic continent than by local adaptation, a subtle form of endemism.