1H n.m.r. studies of insulin. Assignment of resonances and properties of tyrosine residues.

The assignment of the aromatic 1H n.m.r. resonances of the four tyrosine residues of bovine 2-zinc insulin is reported, based on double resonance techniques, use of Hahn spin echo pulse sequences and examination of specific derivatives nitrated at tyrosines A14 and A19 as well as des-(B26-B30)-insulin. Titration curves of the four tyrosine residues show that residues A14 and B16 have normal pK' values of 10.3-10.6 in solution, consistent with their accessibility to solvent in monomer and dimer in the crystal. Tyrosine residues A19 and B26 have pK' values of 11.4 and exhibit other features in their titration curves that are consistent with limited accessibility to solvent and a nonpolar environment. The meta protons of residues B16 and B26 both observe the titration of a nearby tyrosine residue, probably A19. Interpretation of the n.m.r. data obtained in solution is consistent with the crystallographic data for the monomer and dimer obtained on insulin crystals [Blundell, Dodson, Hodgkin & Mercola (1972) Adv. Protein Chem. 26, 279-402].     

Andropause ou P.A.D.A.M.

Recently the word P.A.D.A.M. has been proposed to replace the term Andropause (Partiel Andrologen Deficiency in Aging Males). This implies that most if not all problems of the aging male are due to testosterone déficiency, which is not at all true when one considers the results of endocrine investigations in that group. So many other sociological and relational causes are involved that the term P.A.D.A.M. is clearly a misnomer.     

1H-n.m.r. and c.d. studies of haem orientational disorder in sperm-whale myoglobin and human haemoglobin.

1H-n.m.r. and c.d. studies on sperm-whale myoglobin show that the c.d. signal in the Soret region is inversely and linearly related to the proportion of minor isomer present. An alternative method, 'pH jump', is described for inducing orientational disorder in sperm-whale myoglobin without recourse to reconstitution. 1H-n.m.r. studies on human haemoglobin A indicate little heterogeneity in freshly isolated haemoglobin A, but the effect is enhanced in freeze-dried Sigma haemoglobin A.     

Ligand binding to heme proteins. VI. Interconversion of taxonomic substates in carbonmonoxymyoglobin.

The kinetic properties of the three taxonomic A substates of sperm whale carbonmonoxy myoglobin in 75% glycerol/buffer are studied by flash photolysis with monitoring in the infrared stretch bands of bound CO at nu(A0) approximately 1967 cm-1, nu(A1) approximately 1947 cm-1, and nu(A3) approximately 1929 cm-1 between 60 and 300 K. Below 160 K the photodissociated CO rebinds from the heme pocket, no interconversion among the A substates is observed, and rebinding in each A substate is nonexponential in time and described by a different temperature-independent distribution of enthalpy barriers with a different preexponential. Measurements in the electronic bands, e.g., the Soret, contain contributions of all three A substates and can, therefore, be only approximately modeled with a single enthalpy distribution and a single preexponential. The bond formation step at the heme is fastest for the A0 substate, intermediate for the A1 substate, and slowest for A3. Rebinding between 200 and 300 K displays several processes, including geminate rebinding, rebinding after ligand escape to the solvent, and interconversion among the A substates. Different kinetics are measured in each of the A bands for times shorter than the characteristic time of fluctuations among the A substates. At longer times, fluctuational averaging yields the same kinetics in all three A substates. The interconversion rates between A1 and A3 are determined from the time when the scaled kinetic traces of the two substates merge. Fluctuations between A1 and A3 are much faster than those between A0 and either A1 or A3, so A1 and A3 appear as one kinetic species in the exchange with A0. The maximum-entropy method is used to extract the distribution of rate coefficients for the interconversion process A0 <--> A1 + A3 from the flash photolysis data. The temperature dependencies of the A substate interconversion processes are fitted with a non-Arrhenius expression similar to that used to describe relaxation processes in glasses. At 300 K the interconversion time for A0 <--> A1 + A3 is 10 microseconds, and extrapolation yields approximately 1 ns for A1 <--> A3. The pronounced kinetic differences imply different structural rearrangements. Crystallographic data support this conclusion: They show that formation of the A0 substate involves a major change of the protein structure; the distal histidine rotates about the C(alpha)-C(beta) bond, and its imidazole sidechain swings out of the heme pocket into the solvent, whereas it remains in the heme pocket in the A1 <--> A3 interconversion. The fast A1 <--> A3 exchange is inconsistent with structural models that involve differences in the protonation between A1 and A3.     

A Bryophyte flora of Cornwall. II. Sphagna

Abstract

The taxonomy of Anomobryum julaceum and allied species with axillary bulbils in Europe and Asia is reviewed. A. concinnatum is regarded as a distinct species, occurring in W. and C. Europe, SW., N. and C. Asia and N. America. A. bavaricum has often been confused with A. concinnatum, but differs in its more numerous, small, reddish bulbils and in leaf shape; it is known only from the European Alps. The Asian A. nitidum also has numerous small reddish bulbils but it differs from A. bavaricum in leaf shape and bulbil form. A lectotype is designated for A. nitidum, of which A. gemmigerum and other nominal taxa are regarded as synonyms. Information is presented on geographical ranges and habitats of the four valid species.     

A comparative morphological observations of Actinidia chinensis Planch. var. chinensis and A. chinensis Planch. var. hispida C. F. Liang

A comparative observations of the morphology on the stem, winter bud, fruit, pollen grains of A. chinensis Planch. var. chinensis and A. chinensis Planch. var. hispida C. F. Liang have been made and the obvious differences in these aspects are obvious. In stem tissue culture, the frequency of calli induced and plantlets produced of A. chinensis Planch. var. hispida is also higher than that of A. chinensis Planch. chinensis. For this reason we suggest to raise A. chinensis Planch. var. hispida as a newspecies.     

Control of the initiation of DNA replication in Escherichia coli. II. Function of the dnaA product.

Summary General growth parameters and the kinetics of DNA replication have been determined in merogenotes carrying different combinations of the dnaA⁺ and the dnaA5 allele. The strain which is homozygous diploid for dnaA5 is different from all other combinations in cell volume, DNA per mass ratio, number of replication points per chromosome, and polymerization rate of DNA. From this we deduce that the dnaA product is a positively acting regulatory protein in initiation.In an appendix we show that in combinations between the dnaA5 and dnaA204 alleles the phenotype of dnaA5 is dominant.     

Genetic differentiation of U.S.S.R. house mice: electrophoretic study of proteins

Protein electrophoresis at 24 loci was used to characterize house mice from 56 localities in the U.S.S.R., concentrating on samples from Moldavia to Primorye (extreme south-east of the U.S.S.R.). Mus -2A is the most widespread form, extending over the European part of the U.S.S.R., Middle Asia and Siberia as far east as the Pacific Ocean. In Moldavia the group is sympatric with Mus-iB . It is found with Mus -4A in Transcaucasus, where it may hybridize with Mus -1. In Primorye Mus -2A and M. raddei have a wide zone of hybridization with Mus -2C.     

A replication-defective variant of Moloney murine leukemia virus. I. Biological characterization.

We have studied the virus produced by a clone, termed 8A, that was isolated from a culture of murine sarcoma virus-transformed mouse cells after superinfection with Moloney murine leukemia virus (MuLV-M). Clone 8A produced high levels of type C virus particles, but only a low titer of infectious murine sarcoma virus and almost no infectious MuLV. When fresh cultures of mouse cells were infected with undiluted clone 8A culture fluids, they released no detectable pogeny virus for several weeks after infection. Fully infectious MuLV was then produced in these cultures. This virus was indistinguishable from MuLV-M by nucleic acid hybridization tests and in its insensitivity to Fv-1 restriction. It also induced thymic lymphomas in BALB/c mice. To explain these results, we propose that cone 8A is infected with a replication-defective variant of MuLV-M. Particles produced by clone 8A, containing this defective genome, can establish an infection in fresh cells but cannot produce progency virus at detectable levels. Several weeks after infection, the defect in the viral genome is corrected by back-mutation or by recombination with endogenous viral genomes, resulting in the formation of fully infectious progeny MuLV. The progeny MuLV'S that arose in two different experiments were found to be genetically different from each other. This is consistent with the hypothesis that, in each experiment, the progeny virus is formed clone 8A cells and assayed for infectivity by the calcium phosphate transfection technique. No detectable MuLV was produced by cells treated with this DNA. This finding, along with positive results obtained in control experiments, indicates that clone 8A cells do not contain a normal MuLV provirus.     

Cis proline mutants of ribonuclease A. I. Thermal stability.

A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.     

Culture studies of Acrochaete leptochaete comb. nov. and A. wittrockii comb. nov. (Chaetophoraceae, Chlorophyceae)

A culture study of the plants known as Phaeophila leptochaete (Huber) Nielsen and P. wittrockii (Wille) Nielsen proved that these are distinct species. Both have the Acrochaete type of hairs, and therefore, the new combinations Acrochaete leptochaete and A. wittrockii are introduced. Several isolates of both species were started from plants collected at different places in Europe.
A. leptochaete in culture was mainly characterised by having two or more (up to six) pyrenoids in many of its cells, while in A. wittrockii there invariably was only one. The swarmers formed by A. leptochaete had two or four flagella. Several hairs from one basal swelling were sometimes observed in one of the isolates. Another isolate often formed a hair on the germinated zoospore. The isolates of A. wittrockii had a variable morphology, from unicellular plants to large pseudoparenchymatous cell masses. Different kinds of swarmers were observed; zoospores with three flagella seemed typical, but ones with two or four flagella were also observed. Small, pale biflagellate swarmers were assumed to be gametes. One of the isolates of A. wittrockii differed from the rest as the only swarmer type observed was zoospores with two flagella. Chlorophilum ephemerum is considered identical to A. wittrockii. Observations on hair formation in A. repens suggested that the hair structure represents a separate cell.     

Cis proline mutants of ribonuclease A. II. Elimination of the slow-folding forms by mutation.

Ribonuclease A is known to form an equilibrium mixture of fast-folding (UF) and slow-folding (US) species. Rapid unfolding to UF is then followed by a reaction in the unfolded state, which produces a mixture of UF, USII, USI, and possibly also minor populations of other US species. The two cis proline residues, P93 and P114, are logical candidates for producing the major US species after unfolding, by slow cis <==> trans isomerization. Much work has been done in the past on testing this proposal, but the results have been controversial. Site-directed mutagenesis is used here. Four single mutants, P93A, P93S, P114A, and P114G, and also the double mutant P93A, P114G have been made and tested for the formation of US species after unfolding. The single mutants P114G and P114A still show slow isomerization reactions after unfolding that produce US species; thus, Pro 114 is not required for the formation of at least one of the major US species of ribonuclease A. Both the refolding kinetics and the isomerization kinetics after unfolding of the Pro 93 single mutants are unexpectedly complex, possibly because the substituted amino acid forms a cis peptide bond, which should undergo cis --> trans isomerization after unfolding. The kinetics of peptide bond isomerization are not understood at present and the Pro 93 single mutants cannot be used yet to investigate the role of Pro 93 in forming the US species of ribonuclease A. The double mutant P93A, P114G shows single exponential kinetics measured by CD, and it shows no evidence of isomerization after unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of a G.T.A triplet in an intramolecular DNA triplex.

A 32-base DNA oligonucleotide has been studied by one- and two-dimensional 1H NMR spectroscopy and is shown to form a stable, pyr.pur.pyr, intramolecular triple helical structure, with a four C loop and a TATA loop connecting the Watson-Crick- and Hoogsteen-paired strands, respectively. This triplex contains five T.A.T base triplets, two C+.G.C base triplets, and an unusual G.T.A base triplet which disrupts the pyr.pur.pyr motif. The G.T.A triplet consists of a Watson-Crick T.A base pair, with the T situated in the "purine strand" and the A situated in the "pyrimidine strand" and a G situated in the Hoogsteen-base-paired "pyrimidine strand" hydrogen bonded to the T. The base-pairing structure of the G.T.A triplet has been investigated and has been found to involve a single hydrogen bond from the guanine amino group to the O4 carbonyl of the thymine, leaving the guanine imino proton free. The specific amino proton involved in the hydrogen bond is the H2(2) proton. This orients the guanine such that its sugar is near the thymine methyl group. The guanine sugar adopts an N-type (C3'-endo) sugar pucker in this triplet. The stability of the G.T.A triplet within pyr.pur.pyr triplexes is discussed.     

Amphirosellinia gen. nov. and a new species of Entoleuca

The new genus Amphirosellinia is erected to include five xylariaceous fungi with erumpent or immersed perithecioid stromata. Amphirosellinia fushanensis, A. nigrospora and A. tennesseensis are newly described, whereas A. evansii and A. quercina are new combinations. Synnematous, geniculosporium-like anamorphs are known for A. fushanensis, A. nigrospora, A. tennesseensis and A. evansii; the anamorph of the latter species was produced on natural substratum, whereas those of the former three species were produced in culture. Dichotomous keys are presented for the Amphirosellinia species and for some genera that might be confused with Amphirosellinia. Entoleuca ellisii also is described as new. It readily can be separated from the known species in the genus by its smaller ascospore size range and short ascospore germ slit.     

Taxonomy and Morphology of Axonchium (Nematoda: Belondiroidea), and a description of A. thornei n. sp.

Useful diagnostic characters in the nematode genus A xonchium include: lip shape, styler length, shape of the esophageal constriction, presence or absence of spiral musculature in the esophageal sheath, proportion of the esophageal length occupied by the esophageal expansion, length and shape of cardia, shape of the vulva and vaginal cuticularization, development of the anterior gonad, shape of the posterior uterus, subcuticle thickness at mid-body, tail shape, number and arrangement of supplements and caudal pores, and body measurements. A. thornei n. sp. is separated from A. choristum by its thinner subcuticle at mid-body, number of supplements, and shorter spicules, from A. solitare by presence of males, and from both species by the female tail shape and shorter stylet. A. saccatum is synonymized with A. gossypii and A. nitidum is synonymized with A. bulbosum. A. leptocephalum, A. Iongicollis, A. magnicollis, and A. tenuicollis are made species inquirendae. A key to 25 species of Axonchium is given.     

Interaction of membrane aminophospholipids of E. coli with fluorodinitrobenzene and trinitrobenzenesulfonate.

E. coli cells were reacted with TNBS in bicarbonate-NaCl buffer, pH 8.5 (buffer A) and in phosphate-NaCl buffer, pH 7.0 (buffer B). In buffer A, DNP-GPE is the major product when FDNB is used. DNP-PE and DNP-LPE are formed in lesser amounts. Phospholipase A activity is high in buffer A. When TNBS is used, the labeling of the lipid components is less than with FDNB and more TNP-PE is formed relative to TNP-GPE. This data suggests that the phospholipases which are located primarily on the outer L-membrane of the cell wall act to a lesser extent on TNP-PE than on DNP-PE. E. coli cells were prelabeled with TNBS and FDNB in buffer A, washed and incubated in buffer A. The endogenous labeled DNP-PE gradually decreased with time with a concomitant increase in DNP-LPE and DNP-GPE due to phospholipase A activity. In contrast, the endogenous labeled TNP-PE also decreased with time as did the endogenous labeled TNP-LPE but a new orange lipid was produced. This lipid is believed to be a derivative of TNP-PE in which one of the nitro groups has been reduced to an amino group by nitroreductase. E. coli cells were prelabeled with TNBS and FDNB in buffer A, washed and incubated in buffer B. Under these conditions with both TNBS and FDNB there is an increase in TNP-PE and DNP-PE with a concomitant decrease in TNP-LPE, TNP-GPE, DNP-LPE and DNP-GPE. These results show that at neutral pH acylation occurs to regenerate TNP-PE and DNP-PE. E. coli cells were incubated with exogenous DNP-GPE or TNP-GPE in buffer A. The DNP-GPE and TNP-GPE were rapidly hydrolyzed by a phosphodiesterase to DNP-ethanolamine and TNP-ethanolamine. An orange derivative was formed which was provisionally identified as a derivative of DNP-ethanolamine or TNP-ethanolamine in which a nitro group has been reduced to an amino group by nitroreductase. The phospholipases and acylating enzymes present in the cell wall of E. coli are active on the dinitrophenyl and trinitrophenyl derivatives of PE and LPE and may act in concert to model and repair the plasma membrane.     

Polymorphism of a tumor ce-l population and selection processes. IV. The effect of dibunol and nitrosourea on the variability of Ehrlich-I. Ch. Ph. ascitic strain tumor cells]

A study was made of the effect of dibunol and methyl-N-nitrosourea (MNU) on two tumor cell subpopulations of the Ehrlich-I. Ch. Ph. ascites strain, one of which is characterized with A + B + 2C and A + D + 2C--markers and the other one--with A1 + A2 + 2B + D + C markers. Dibunol that belongs to the class of inhibitors of free-radical processes was shown to bring about changes in cell subpopulations, the mode of changes depending on the dose and regime of treatment. The effect of MNU on the population resulted predominantly in the accumulation of cells with various chromosome aberrations. At early stages of tumor progression, aberrations were more pronounced in cells with marker chromosome "A" than in the cells with 44 chromosomes and markers A1 + A2 + 2B + D + C.     

The orchidology of H. G. Jones

A bibliography of the numerous, scattered orchid publications of the late H. G. Jones of Bridgetown, Barbados is presented. The new taxa and combinations proposed by Jones are listed. A search for his personal herbarium, which is believed to be non-existent, is discussed.     

Assembly and secretion of fibrinogen. Degradation of individual chains.

Hep G2 cells produce surplus A alpha and gamma fibrinogen chains. These excess chains, which are not secreted, exist primarily as free gamma chains and as an A alpha-gamma complex. We have determined the intracellular location and the degradative fate of these polypeptides by treatment with endoglycosidase-H and by inhibiting lysosomal enzyme activity, using NH4Cl, chloroquine, and leupeptin. Free gamma chain and the gamma component of A alpha-gamma are both cleaved by endoglycosidase-H, indicating that the gamma chains accumulate in a pre-Golgi compartment. Lysosomal enzyme inhibitors did not affect the disappearance of free gamma chains but inhibited A alpha-gamma by 50%, suggesting that A alpha-gamma is degraded in lysosomes. The degradative fate of individual chains was determined in transfected COS cells which express but do not secrete single chains. Leupeptin did not affect B beta chain degradation, had very little affect on gamma chain, but markedly inhibited A alpha chain degradation. Antibody to immunoglobulin heavy chain-binding protein (GRP 78) co-immunoprecipitated B beta but not A alpha or gamma chains. Preferential binding of heavy chain-binding protein to B beta was also noted in Hep G2 cells and in chicken hepatocytes. Taken together these studies indicate that B beta and gamma chains are degraded in the endoplasmic reticulum, but only B beta is bound to BiP. By contrast A alpha chains and the A alpha-gamma complex undergo lysosomal degradation.