Biochemistry, Biophysics, and Structural Biology Commons^™

Improved Selective Medium For The Isolation Of Treponema Hyodysenteriae, Robert A. Kunkle, J. M. Kinyon

School of Veterinary and Biomedical Sciences: Faculty Publications

An agar medium with improved selection for Treponema hyodysenteriae was developed. Cultures of T. hyodysenteriae and T. innocens, feces from 11 clinically normal pigs, and colonic contents from 6 pigs with gross lesions consistent with swine dysentery were diluted in phosphate-buffered saline and plated on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) with 5% citrated bovine blood (TSA), TSA with 400 ,ug of spectinomycin per ml (TSA-S400), TSA-S400 with 25 ,ug each of colistin and vancomycin per ml, and TSA with 5% pig feces extract and five antimicrobial agents (spiramycin, rifampin, vancomycin, colistin, and spectinomycin) (BJ). Viable numbers …

Nucleotide Sequence Of The Fadr Gene, A Multifunctional Regulator Of Fatty Acid Metabolism In Escherichia Coli, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

The Escherichia coli fadR gene is a multifunctional regulator of fatty acid

and acetate metabolism. In the present work the nucleotide sequence of the

1.3 kb DNA fragment which encodes FadR has been determined. The coding

sequence of the fadR gene is 714 nucleotides long and is preceded by a typical

E. coli ribosome binding site and is followed by a sequence predicted to be

sufficient for factor-independent chain termination. Primer extension experiments

demonstrated that the transcription of the fadR gene initiates with an

adenine nucleotide 33 nucleotides upstream from the predicted start of

translation. The derived fadR peptide has …

Reduced Co2/O2 Specificity Of Ribulose-Bisphosphate Carboxylase/Oxygenase In A Temperature-Sensitive Chloroplast Mutant Of Chlamydomonas, Zhixiang Chen, Chris J. Chastain, Souhail R. Al-Abed, Raymond Chollet, Robert J. Spreitzer

Department of Biochemistry: Faculty Publications

The Chlamydomonas reinhardtii chloroplast mutant 68-4PP is phenotypically indistinguishable from wild type at 25°C but fails to grow photosynthetically at 35°C. It had about 30% of the wild-type level of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) holoenzyme and carboxylase activity when grown at 25°C, but less than 15% when grown at 35°C. Pulse-labeling with ³⁵S showed that the decrease in enzyme level at the restrictive temperature was not a result of reduced synthesis of enzyme subunits. The CO₂/O₂ specificity factor (V_cK_o/V_oK_c, where V_c and V_o are V_{max …}

The Binding Of Host-Selective Toxin Analogs To Mitochondria From Normal And 'Texas' Male Sterile Cytoplasm Maize, Kurt A. Frantzen, J. M. Daly, Herman W. Knoche

Department of Biochemistry: Faculty Publications

Tritium-labeled toxin analogs were prepared by reduction with NaB³H₄ of either the toxin from Helminthosporium maydis race T or a toxin component from Phyllosticta maydis. These reduced analogs had high radiochemical specific activities, high biological activities, and plant specificities identical to the native toxins. A filtration assay was developed to test the binding of these labeled analogs to isolated- mitochondria. Binding was not energy dependent nor was there measurable matrical uptake. The analogs were shown to be lipophilic, a characteristic which gave rise to considerable nondisplaceable binding. Under conditions limiting nondisplaceable binding, -the displaceable binding was …

In Vitro Phosphorylation Of Maize Leaf Phosphoenolpyruvate Carboxylase, Raymond J. A. Budde, Raymond Chollet

Department of Biochemistry: Faculty Publications

Autoradiography of total soluble maize (Zea mays) leaf proteins incubated with ³²P-labeled adenylates and separated by denaturing electrophoresis revealed that many polypeptides were phosphorylated in vitro by endogenous protein kinase(s). The most intense band was at 94 to 100 kilodaltons and was observed when using either [γ -32P]ATP or [β-³²P]ADP as the phosphate donor. This band was comprised of the subunits of both pyruvate, Pi dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPCase). PPDK activity was previously shown to be dark/light-regulated via a novel ADP-dependent phosphorylation/Pi-dependent dephosphorylation of a threonyl residue. The identity of the acidstable 94 …

Formation Of Influenza Virus Particles Lacking Hemagglutinin On The Viral Envelope, Asit K. Pattnaik, Donald J. Brown, Debi P. Nayak

School of Veterinary and Biomedical Sciences: Faculty Publications

We investigated the intraceUular block in the transport of hemagglutinin (HA) and the role of HA in virus particle formation by using temperature-sensitive (Is) mutants (1s134 and 1s61S) of inOuenza virus AlWSN/33. We found that at the nonpermissive temperature (39.5°C), the exit of ts HA from the rough endoplasmic reticulum to the Golgi complex was blocked and that no additional block was apparent in either the exit from the Golgi complex or post-Golgi complex transport. When MDBK ceUs were infected with these mutant viruses, they produced noninfectious virus particles at 39.5°C. The efficiency of particle formation at 39.5°C was essentiaUy …

Nonsense Mutations In The Chlamydomonas Chloroplast Gene That Codes For The Large Subunit Of Ribulosebisphosphate Carboxylase/Oxygenase, Robert J. Spreitzer, Michel Goldschmidt-Clermont, Michele Rahire, Jean-David Rochiax

Department of Biochemistry: Faculty Publications

The Chlamydomonas reinhardtii chloroplast mutants 18-SB and 18-7G lack both the chloroplast-encoded large subunit and nuclear-encoded small subunit of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). A chloroplast intergenic-suppression model has been postulated to account for the genetic instability of 18-5B revertants. Here, we have determined the molecular basis of the 18-SB and 18-7G mutants. They contain nonsense mutations close to the 3' and 5' ends of their large-subunit genes, respectively. Puls-chase experiments revealed that the 18-SB mutant produces a truncated large subunit that is unstable. In connection with previous experiments, this work identifies nonsense suppression in the chloroplast. Small …

Cloning And Characterization Of A Gene (Fadr) Involved In Regulation Of Fatty Acid Metabolism In Escherichia Coli, Concetta C. Dirusso, William D. Nunn

Department of Biochemistry: Faculty Publications

The regulatory gene fadR has been previously characterized by classical genetic means as a diffusible protein which exerts negative control over fatty acid degradation and acetate metabolism. fadR has also been implicated in the regulation of unsaturated fatty acid biosynthesis. To facilitate the identification of the product of the fadR gene and to study the mechanism by which this multifunctional regulatory gene exerts its control, we cloned a segment of DNA containing the fadR gene in the phage vector λL47. Subsequent subcloning of a segment of the chromosomal DNA from the λfadR⁺ phage into various plasmid vectors resulted in …

Reduction Of Ferric Leghemoglobin In Soybean Root Nodules, Keuk-Ki Lee, Robert V. Klucas

Department of Biochemistry: Faculty Publications

Reduction of ferric leghemoglobin to ferrous leghemoglobin in soybean nodules (Glycine max [L.] Meff. cv Woodworth) was studied using a spectrophotometer equipped with an in-cell space diffuse reflectance accessory. Nodule slices prepared and scanned under nitrogen gas showed a ferrous leghemoglobin absorption spectrum. Nodule slices equilibrated with 100% O₂ or air exhibited two absorption bands characteristic of oxygenated leghemoglobin. The addition of CO shifted those bands to CO leghemoglobin absorption bands. Potassium ferricyanide was not effective in oxidizing ferrous to feffic leghemoglobin in nodule slices. However, ferric leghemoglobin was formed by treating the nodule slices with hydroxylamine, and …

The Role Of Recognition In Plant Disease, J. M. Daly

Department of Biochemistry: Faculty Publications

As noted by Sequeira (110,111), the term recognition has become very popular in the literature of host-pathogen interactions over the last ten years. Despite recent facile usage, its meaning is not always clear to me. Recognition is defined by Clarke & Knox (16) as the "initial event in cell-cell communication that elicits a defined biochemical, physiological, or morphological response." This requires the not-so-easy task of defining "initial event" and "communica­ tion" with precise chemical or physical mechanisms. For pathologists, Se­ queira (110) has provided a more restricted meaning: "An early specific event that triggers a rapid, overt response by the …

Identification Of Four Complementary Rna Species In Akabane Virus-Infected Cells, Asit K. Pattnaik, G. Abraham

School of Veterinary and Biomedical Sciences: Faculty Publications

The analysis of RNA extracted from purified Akabane virus demonstrated the presence of three size classes of single-stranded RNAs with sedimentation coefficients of 31S (large, L), 26S (medium, M), and 13S (small, S). Molecular weights of these RNA species were estimated to be 2.15 x 10⁶,1.5 X 10⁶, and 0.48 x 10⁶ for the L, M, and S RNAs, respectively. Hybridization analysis involving viral genomic RNA and RNA from virus-infected cells resulted in the identification of four virus-specific cRNA species in infected cells. These cRNAs were found to be nonpolyadenylated by their inability to bind …

Nickel: A Micronutrient Element For Hydrogen-Dependent Growth Of Rhizobium Japonicum And For Expression Of Urease Activity In Soybean Leaves, Robert V. Klucas, F. Joe Hanus, Sterling A. Russell, Harold J. Evans

Department of Biochemistry: Faculty Publications

Soybean plants and Rhizobium japonicum 122 DES, a hydrogen uptake-positive strain, were cultured in media purified to remove Ni. Supplemental Ni had no significant effect on the dry matter or total N content of plants. However, the addition of Ni to both nitrate-grown and symbiotically grown plants resulted in a 7- to 10-fold increase in urease activity (urea amidohydrolase, EC 3.5.1.5) in leaves and significantly increased the hydrogenase activity (EC 1.18.3.1) in isolated nodule bacteroids. When cultured under chemolithotrophic conditions, free-living R. japonicum required Ni for growth and for the expression of hydrogenase activity. Hydrogenase activity was minimal or not …

Reversible Dark-Induced Senescence Of Soybean Root Nodules, N. E. Pfeiffer, Nasir S.A. Malik, Fred W. Wagner

Department of Biochemistry: Faculty Publications

Nodule senescence was induced in intact soybean Glycine max. (L.) Merr., cv Woodworth plants by an 8-day dark treatment. Dark-induced senescence resulted in the complete loss of acetylene reduction activity, a 67% loss of total soluble protein, and an almost complete loss in total leghemoglobin of nodule extracts. Isoelectric focusing gels demonstrated a preferential loss of certain proteins, which was correlated with an increase in endoprotease specific activity toward azocasein. Nodules were completely green after the 8-day dark treatment. If plants were returned to a normal photoperiod after 8 days in the dark, nodules recovered from the dark treatment in …

Proteolytic Activity In Soybean Root Nodules: Activity In Host Cell Cytosol And Bacteroids Throughout Physiological Development And Senescence, Nancy E. Pfeiffer, Cecilia M. Torres, Fred W. Wagner

Department of Biochemistry: Faculty Publications

Root nodules were harvested from chamber-grown soybean (Glycine max L. Merril cv Woodworth) plants throughout development. Apparent nitrogenase activity (acetylene reduction) peaked before seeds began to develop, but a significant amount of activity remained as the seeds matured. Nodule senescence was defined as the period in which residual nitrogenase activity was lost. During this time, soluble protein and leghemoglobin levels in the host cell cytosol decreased, and proteolytic activity against azocasein increased. Degradative changes were not detected in bacteroids during nodule senescence. Total soluble bacteroid protein per gram of nodule remained constant, and an increase in proteolytic activity in …

Peptidohydrolases Of Soybean Root Nodules, Nasir S. A. Malik, N. E. Pfeiffer, D. R. Williams, Fred W. Wagner

Department of Biochemistry: Faculty Publications

Nodule extracts prepared from Glycine max var Woodworth possessed endopeptidase, aminoptidase, and carboxypeptidase actiities. Three distinct endopeptidase activities could be resolved by disc-gel electrophoresis at pH 8.8. According to their order of increasing electrophoretic mobility, the first of these enzymes hydrolyzed azocasein and n-benzoyl-LLeu-β-naphthylamide, while the second hydrolyzed n-benzoyl-L-Arg-/8- naphthylamine (Bz-L-Arg-/βNA), n-benzoyl-L-Arg-β-nitroanilide (Bz-L-Arg-βNA), and azocasein. The third endopeptidase hydrolyzed Bz-L-Arg-βNA, Bz-L-Arg-βNA, and hemoglobin. Fractions of these enzymes extracted from electrophoresis gels were shown to have pH optima from 7.5 to 9.8. AU of the endopeptidases were completely inhibited by diisopropylphosphorofluoridate, demonstrating that they were serine proteases.

Photoelectric Properties And Detection Of The Aromatic Carcinogens Benzo[A]Pyrene And Dimethylbenzanthracene, William A. Houle, Hugh M. Brown, O. Hayes Griffith

Department of Biochemistry: Faculty Publications

The absolute photoelectron quantum yield spectra for benzo[ajpyrene and dimethylbenzanthracene are presented in the wavelength range 180-230 nm. These polycyclic aromatic carcinogens have photoelectron quantum yields of approximately 2 X 10^-3 electrons per incident photon at 180 nm. The quantum yields fall off quickly and monotonically at wavelengths longer than 210 nm (5.9 eV). Threshold values for benzo[a]pyrene and dimethylbenzanthracene are 5.25 ± 0.06 eV and 5.27 ± 0.04 eV, respectively. The photoelectron quantum yields of benzo[a]pyrene and dimethylben-zanthracene are several orders of magnitude greater than typical components of biological membranes (amino acids, phospholipids, and polysaccharides). Preliminary micrographs of …

An Immunochemical Study Of The Combining Sites Of The Second Lectin Isolated From Bandeiraea Simplicifolia (Bs Ii), Charles Wood, E. A. Kabat, S. Ebisu, Irwin J. Goldstein

Nebraska Center for Virology: Faculty Publications

Two lectins with different binding specificities have been isolated from extracts of seeds of Bandeiraea simplicifolia. The first, Bandeiraea lectin I [11] was specific for terminal non-reducing αDGalactosyl residues. It reacted with B substances from human ovarian cysts and with several galactomannans to form precipitin lines in agar gels. Polysaccharides with terminal αDGalactosyl residues, such as larch galactan, did not react. The lectin agglutinated B erythrocytes strongly but also reacted to a lower titre with A₁ and very weakly with A₂ erythrocytes [15, 28] indicating that terminal non-reducing αDGalNAc [24] can …

Recollections, George W. Beadle

Department of Biochemistry: Faculty Publications

In these exciting times when elementary and high schools teach modern biology, including many of the intricacies of biochemical genetics, the long slow process by which our present knowledge in this area was gained is not often fully appreciated. A third of a century elapsed before Mendel’s work was "rediscovered" and properly appreciated. Archibald E. Garrod’s (1) prophetic appreciation of the relation genetics and biochemistry, beginning soon after the so-called rediscovery of Mendel, lay fallow for more than forty years despite the fact that he published widely and relatively voluminously. As late as a quarter of a century after the …

Characterization Of A Fatty Acid Synthetase From Corynebacterium Diphtheriae, Herman W. Knoche, Kirston E. Koths

Department of Biochemistry: Faculty Publications

A fatty acid synthetase from Corynebacterium diphtheriae has been purified to a specific activity of 450 nmoles of malonyl coenzyme A incorporated per min per mg. The enzyme is optimally active in 0.5 M phosphate buffer. C. diphtheriae appears to be the most primitive organism having a multienzyme complex for fatty acid synthesis.

Palmityl Coenzyme A Inhibition Of Fatty Acid Synthesis, Herman Knoche, Theodore W. Esders, Kirston Koths, Konrad Bloch

Department of Biochemistry: Faculty Publications

The effects of acyl-CoA derivatives (C₈ to C₂₀) on the activity

of the fatty acid synthetases from yeast and Corynebacterium

diphtheriae have been examined. Both enzyme

systems are inhibited by the longer chain acyl thioesters

(C₁₆ to C₂₀) and protected against this inhibition by bovine

serum albumin (BSA). Identical relief from acyl-CoA inhibition

is provided by the 6-0-methylglucose-containing

lipopolysaccharide (MGLP), from Mycobacterium phlei. It

is shown that MGLP forms a stable complex with palmitylCoA.

This interaction accounts for the BSA-like effects of

the polysaccharide. BSA and MGLP have two further effects

on the fatty …

The Structure Of An Ornithine-Containing Lipid From Thiobacillus Thiooxidans, Herman W. Knoche, Jessup M. Shively

Department of Biochemistry: Faculty Publications

The structure of an ornithine-containing lipid from Thio-bacillus thiooxidans

has been elucidated. Methanolysis of

the lipid released methyl cis-ll,lZ-methylene-Z-hydroxyoctadecanoate.

Acid hydrolysis of the residue yielded ornithine

and a mixture of fatty acids, the major components of

which were 3-hydroxyhexadecanoic and 2-hexadecenoic

acids. Identification of the 3-hydroxy fatty acid was based

on the thin layer chromatographic mobilities of the acid, its

methyl ester, the methyl ether, and acetate derivatives of its

methyl ester, on the equivalent chain lengths of the derivatives

of the acid and the acid obtained by oxidation of the

natural acid with permanganate, and on mass spectral studies. …

Form And Level Of Coumarin In Deer's Tongue, Trilisa Odoratissima, Francis A. Haskins, H. J. Gorz, R. C. Leffel

Department of Agronomy and Horticulture: Faculty Publications

Fresh leaves of deer’s tongue contain large quantities (more than 10% of the dry weight, in some cases) of o-hydroxycinnamic acid (o-HCA). Both cis- and trans-o-HCA are present, and both isomers exist in the fresh tissue predominantly as glucosides. Cured deer’s tongue leaves contain relatively high levels of coumarin and lower amounts of o-HCA glucosides. It is probable that during the curing process cis-o-HCA glucoside is hydrolyzed by an endogenous β-glucosidase, and that the liberated cis-o-HCA lactonizes spontaneously to form coumarin.

The Identification Of Cis-Ii, I2-Methylene-2-Hydroxyoctadecanoic Acid From Thiobacillus Thiooxidans, H. W. Knoche, J. M. Shively

Department of Biochemistry: Faculty Publications

A polar fatty acid has been observed as a component of an

ornithine-containing lipid of Thiobacillus thiooxidans. A

comparison of thin layer chromatographic mobilities of reference

compounds to those of the natural acid and its derivatives

suggested that the acid was a 2-hydroxy fatty acid.

The presence of a cyclopropane function in the acid was

indicated by l4e-Iabeling experiments and infrared spectroscopy.

Mass spectrometry of the methyl ester and the acetylated

methyl ester of the natural acid provided a molecular

weight for the acid. Equivalent chain lengths were determined

for the natural acid, the acid obtained by oxidative

decarboxylation of …

Carbohydrate And Lipid Metabolism During Germination Of Uredospores Of Puccinia Graminis Tritici, J. M. Daly, H. W. Knoche, M. V. Wiese

Department of Biochemistry: Faculty Publications

Uredospores of Puccinia gramintis (Pers.) tritici (Eriks. and Henn.) were uniformly labeled with ¹⁴C by permitting the host (Triticuml aestivum L.) to carry out photosynthesis in ¹⁴CO₂ during the process of spore production by the obligate parasite. The use of ¹⁴C labeled spores provided advantages in a study of the utilization of endogenous substrates at frequent intervals with small amounts of spores under conditions conducive to germination.

Genetic And Biochemical Aspects Of O-Hydroxycinnamic Acid Synthesis In Melilotus Alba, Herman J. Gorz, Francis A. Haskins, A. Kleinhofs

Department of Agronomy and Horticulture: Faculty Publications

In sweetclover (Melilotus alba Desr.) the cis- and trans-isomers of o-hydroxycinnamic acid occur primarily as the respective, β-D-glucosides. Available evidence indicates that these glucosides are formed via the following pathway: phenylalanine (formed from shikimic acid) → trans-cinnamic acid → o-coumaric acid (trans-o-hydroxycinnamic acid) → o-coumaryl glucoside (trans-β-D-glucosyl-o-hydroxycinnamic acid) → coumarinyl glucoside (cis-β-D-glucosyl-o-hydroxycinnamic acid). In tissues that are disrupted, coumarinyl glucoside is rapidly hydrolyzed by the action of endogenous β-glucosidase to yield coumarinic acid, which lactonizes spontaneously to form coumarin. The cu …

Viral Infections Of Domestic Animals, George A. Young

School of Veterinary and Biomedical Sciences: Faculty Publications

There is a strong tendency in current research on mammalian virus diseases to concentrate on virus activity at a cellular or subcellular level. This trend is summarized adequately by Enders in relation to viral replication as follows: ','The large and ever-increasing volume of published experimental work on viral replication strikingly reveals the central position of this phenomenon in contemporary virological research. There are good reasons, both biological and practical, underlying this intensive effort to understand, i n detail, how a virus particle, without energy transforming apparatus of its own, manages to utilize the metabolic equipment of the cell to produce …

Worm Eggs: Cost You Money, E.F. Frolik, E.W. Janike

School of Veterinary and Biomedical Sciences: Faculty Publications

Contents

One Million Eggs Daily.......... 3

Damaging Trip......... 4

Worm Remedies......... 5

Modern Worm Remedies......... 6

A Control Program.......... 8

Concerning The Site Of Nitrogen Absorption In Rats Fed Autoclaved Or Raw Soybean Oil Mea, Raymond Borchers

Department of Biochemistry: Faculty Publications

Carroll, Hensley, and Graham (1) have concluded that much of the nitrogen absorption in rats fed raw soybean oil meal must take place in the cecum. This conclusion was reached from data showing that the apparent digestibility of raw soybean nitrogen in the terminal 20% of the small intestine was 32.65%, whereas in the feces the value was 76.96%. Values reported, for apparent digestibility of heated soybean nitrogen were 78.66% and 81.78%, respectively. This observation presented a notable advance in explaining the lower nutritive value of raw soybeans compared with autoclaved soybeans. It therefore seemed advisable to repeat this work …

A Metabolic Relationship Between The Aromatic Amino Acids, Joseph F. Nyc, Francis A. Haskins, Herschel K. Mitchell

Department of Agronomy and Horticulture: Faculty Publications

The present work involves a further investigation of a Neurospora mutant, C-86, previously mentioned by Lein, Mitchell and Houlahan (1) as one that can utilize anthranilic acid, indole, tryptophan, kynurenine, 3-hydroxyanthranilic acid, and nicotinic acid as supplements for growth. Mutant C-86, when crossed to a “wild-type” strain, was found to differ from this wild type strain, with respect to tryptophan biosynthesis, by a mutation at a single locus. The evidence presented suggests the possibility of a common precursor to the aromatic amino acids.

Riboflavin Production By Molds, George L. Peltier, Raymond Borchers

Department of Biochemistry: Faculty Publications

Very little information is at hand regarding the ability of molds to synthesize riboflavin. Few citations pertaining directly to riboflavin production by true

molds are available. Pontovich (1943) found as much as 2 mg riboflavin per g

of Aspergillus flavus mycelium. Tanner et at. (1945) determined the quantity of

riboflavin in the submerged fermentation media of Penicillium chrysogenum.

The highest value found was 1. 36 mg per ml. The primary purpose of this study

was to screen several hundred isolates, recently obtained from soil, crop residues,

and composts, for their ability to produce riboflavin on a wheat bran substrate.