Altitudinal changes in the incidence of crassulacean acid metabolism in vascular epiphytes and related life forms in Papua New Guinea

Summary The occurrence of Crassulacean acid metabolism (CAM), as judged from ¹³C values, was investigated in epiphytes and some related plant species at a series of sites covering the approximate altitudinal range of epiphytes in Papua New Guinea. Comprehensive collections were made at each site and the occurrence of water storage tissue and blade thickness was also determined. Some 26% of epiphytic orchids from a lowland rainforest (2–300 m.a.s.l) showed ¹³C values typical of obligate CAM and possessed leaves thicker than 1 mm. A second group of orchids, mostly with succulent leaves, possessed intermediate ¹³C values between -23 and -26% and accounted for 25% of the total species number. Some species of this group may exhibit weak CAM or be facultative CAM plants. The remainder of the lowland rainforest species appeared to be C₃ plants with ¹³C values between -28 and -35%. and generally possessed thin leaves. Obligate CAM species of orchids from a lower montane rainforest (1175 m.a.s.l) comprised 26% of the species total and mostly possessed thick leaves. The remainder of the species were generally thin-leaved with ¹³C values between -26 and -35%. largely indicative of C₃ photosynthesis. Orchids with intermediate ¹³C values were not found in the lower montane rainforest. Obligate CAM appeared to be lacking in highland epiphytes from an upper montane rainforest and subalpine rainforest (2600–3600 m.a.s.l). However the fern, Microsorium cromwellii had a ¹³C value of -21.28%. suggesting some measure of CAM activity. Other highland ferns and orchids showed more negative °¹³C values, up to-33%., typical of C₃ photosynthesis. The highland epiphytic orchids possessed a greater mean leaf thickness than their lowland C₃ counterparts due to the frequent occurrence of water storage tissue located on the adaxial side of the leaf. It is suggested that low daytime temperatures in the highland microhabitats is a major factor in explaining the absence of CAM. The increased frequency of water storage tissue in highland epiphytes may be an adaptation to periodic water stress events in the dry season and/or an adaptation to increased levels of UV light in the tropicalpine environment.     

A comparison of the radiation-induced mutation at the HGPRT locus in V79 and BHK cells

Mutation response at the HGPRT locus has been compared in two differing cell lines: V79/4, an aneuploid Chinese-hamster fibroblast line with a complement of 20 chromosomes, and BHK21-C13, a diploid Syrian-hamster fibroblast line with a complement of 44 chromosomes. The data presented show that BHK is slightly more radiosensitive than V79/4; however, the toxicity curves and expression times are similar for both cell lines. If radiosensitivity is taken into account, a common line can be drawn for radiation mutagenesis. We conclude from the data that radiation-induced mutagenesis is broadly equivalent in the two cell lines examined, and is not dependent on the chromosome complement.     

In vitro teratogenicity of ethylenethiourea in the rat

We have demonstrated that ethylenethiourea (ETU) is a potent teratogen to the rat embryo developing in vitro. Sprague Dawley rat embryos were explanted on gestation day 10 and cultured for 48 hours in the presence of 40-200 micrograms/ml ETU. This resulted in a dose-related inhibition of growth and differentiation as assessed by crown-rump length, protein and DNA content, and somite number and in an increase in the frequency of abnormalities. A variety of anomalies was produced, including fluid accumulation in the brain (hydrocephalus), decreased mandibular size, decreased telencephalon size, abnormal dorsiflexion, as well as subectodermal blisters on the tail and limb buds and maxilla. Frank malformations have been observed at these same sites--hydrocephalus, brachygnathia, kyphosis, limb and tail defects, cleft palate--in the term fetus in vivo. The presence of abnormal fluid accumulation in the embryos--distended neural tube and subectodermal blisters--suggesting that the osmotic environment of the embryo had been altered by ETU exposure. Osmolality of the exocoelomic fluid (ECF) surrounding the embryo was measured after 48 hours of exposure to a concentration of ETU that caused nearly a 100% incidence of subectodermal blisters. ECF osmolality was found to be significantly lower than that of control embryos. Lowered osmolality would cause water to move out of the ECF, presumably causing the observed fluid accumulation in the embryo. It is speculated that altered osmotic balance and localized edema in the embryo are contributory steps in the formation of defects after ETU exposure.     

Development and keratinization of the epidermis in the common lizard, Anolis carolinensis

Epithelial-mesenchymal interactions play important roles in the development of the vertebrate integument with its diverse appendages. As a result of these interactions, specific morphogenetic events occur which result in the formation of distinct epidermal appendages. Following the early morphogenetic events involving cell proliferation and movement, other developmental events such as stratification, histotypic differentiation, and terminal cytodifferentiation occur in the epidermis. Using the common lizard Anolis carolinensis, we are seeking to obtain a better understanding of the relationship between the various developmental events and the expression of alpha and beta keratins, with the aim of eventually understanding the mechanisms by which tissue-specific keratinization patterns are established in the integument. As a first step, we have used immunoblot analyses and indirect immunofluorescence procedures with antisera specific for either alpha or beta keratins to determine the temporal and spatial appearance of these keratins at specific developmental stages. We have found that: 1) There are relatively low molecular weight alpha keratin polypeptides present in the epidermis early in development as morphogenesis is taking place. 2) After morphogenesis occurs and histogenesis is well under way, the alpha keratins which characterize the adult epidermis appear. 3) Only alpha keratins are found in the basal cells of all regions of the epidermis. 4) beta keratins are found only in the suprabasal layers of well-developed scales and show region-specific distribution in overlapping scales.     

High-resolution 1H NMR study of the solution structure of alamethicin

A 1H NMR study of the peptide alamethicin, which forms voltage-gated ion channels in membranes, is described. The molecule was studied in methanol as a function of temperature and pH. A complete assignment of the spectra is given, including several stereospecific assignments. Alamethicin was found to have a structure substantially similar to the crystal although, in solution, the C-terminal dipeptide adopts a somewhat extended conformation. The overall conformation was insensitive to the ionization of the side chain of the only ionizable group, Glu-18.     

Solution conformation of the branch points of N-linked glycans: synthetic model compounds for tri'-antennary and tetraantennary glycans

The solution conformation of model compounds for the tri'-antennary and tetraantennary (six-arm) branch point of N-linked glycans has been determined through the use of chemical shift, relaxation, and nuclear Overhauser enhancement data. The object was to establish the conformation about the glycosidic linkages in the N-linked substructure GlcNAc(beta 1,6) [GlcNAc(beta 1,2)] Man(alpha)- by estimation of values for the appropriate glycosidic torsional angles. The GlcNAc(beta 1,6) linkage in a trisaccharide model compound was found to be constrained to a narrow rotameric subpopulation about the substituted Man C5-C6 bond (omega = -60 degrees) and a narrow range of possible phi - psi values. Free rotation about the Man C5-C6 bond was obstructed by unfavorable steric interactions between the GlcNAc(beta 1,6) and GlcNAc(beta 1,2) residues. A phi, psi value of 55 degrees, 190 degrees was found to be consistent with the NMR data for the GlcNAc(beta 1,6) linkage. However, the value of psi appears to be "virtual" in that the molecule is in equilibrium between two different values (90 degrees and 252 degrees). For the GlcNAc(beta 1,2) linkage, complete agreement between all the observed NMR parameters and all the calculated ensemble average values could only be obtained with a set of potential energy functions which included hydrogen bonding. Other choices of potentials yielded calculated values that disagreed with at least two of the observed quantities. As a result, we infer that an interresidue hydrogen bond is formed, and we find it to be between the GlcNAc(beta 1,2) ring oxygen and the Man C3 hydroxyl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Virtual and solution conformations of oligosaccharides

The possibility that observed nuclear Overhauser enhancements and bulk longitudinal relaxation times, parameters measured by 1H NMR and often employed in determining the preferred solution conformation of biologically important molecules, are the result of averaging over many conformational states is quantitatively evaluated. Of particular interest was to ascertain whether certain 1H NMR determined conformations are "virtual" in nature; i.e., the fraction of the population of molecules actually found at any time within the subset of conformational space defined as the "solution conformation" is vanishingly small. A statistical mechanics approach was utilized to calculate an ensemble average relaxation matrix from which (NOE)'s and (T1)'s are calculated. Model glycosidic linkages in four oligosaccharides were studied. The solution conformation at any glycosidic linkage is properly represented by a normalized, Boltzmann distribution of conformers generated from an appropriate potential energy surface. The nature of the resultant population distributions is such that 50% of the molecular population is found within 1% of available microstates, while 99% of the molecular population occupies about 10% of the ensemble microstates, a number roughly equal to that sterically allowed. From this analysis we conclude that in many cases quantitative interpretation of NMR relaxation data, which attempts to define a single set of allowable torsion angle values consistent with the observed data, will lead to solution conformations that are either virtual or reflect torsion angle values possessed by a minority of the molecular population. On the other hand, calculation of ensemble average NMR relaxation data yields values in agreement with experimental results. Observed values of NMR relaxation data are the result of the complex interdependence of the population distribution and NOE (or T1) surfaces in conformational space. In conformational analyses, NMR data can therefore be used to test different population distributions calculated from empirical potential energy functions.     

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.