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Biomedical subjects

F W Wagner

Publications and source records attributed to F W Wagner.

At least 37 records · Page 2Linked to original sources
PubMed1986

High-performance liquid chromatographic separation of leghemoglobins from soybean root nodules.

A crude fraction of soybean nodule ferri-leghemoglobin was absorbed onto a commercial DEAE HPLC column at pH 8.0, and resolved into eight isoprotein fractions. The identity of the leghemoglobins were determined by their order of elution from the DEAE column and by isoelectric focusing, using isoprotein standards isolated by conventional procedures. The three isoproteins of the c complex, c1, c2, c3, were not resolved. Unexpected heme containing proteins eluted just after leghemoglobin a and the c complex. These components possessed proteins similar to leghemoglobin a and the c complex, respectively, as judged by isoelectric focusing and absorbance spectra of the ferri and ferrous forms. The components designated leghemoglobin a' and leghemoglobin c' were also differentiated from leghemoglobin a and c by reverse-phase HPLC in a C18 column. Amounts of protein for the DEAE HPLC column ranged from 10 micrograms to 20 mg and sample volumes ranged from 2 to 250 microliters. The time required for chromatography varied depending on the gradient used, but never exceeded 40 min for samples up to 5 mg protein or 120 min for samples containing 5 to 20 mg protein. Due to the sensitivity of detection at 403 nm and leghemoglobins being the predominant chromophore at that wavelength, it was possible to quantitate levels of individual leghemoglobins in samples extracted from single nodules (ca. 15 to 65 mg fresh weight tissue). Quantitation was performed by interfacing the spectrophotometer output (10 mV) to a microcomputer and using commercially available software.

Chromatography, High Pressure Liquid↗
PubMed1986

Bacteroids Are Stable during Dark-Induced Senescence of Soybean Root Nodules.

Physiological and biochemical markers of metabolic competence were assayed in bacteroids isolated from root nodules of control, dark-stressed, and recovered plants of Glycine max Merr. cv ;Woodworth.' Nitrogenase-dependent acetylene reduction by the whole plant decreased to 8% of control rates after 4 days of dark stress and could not be detected in plants dark stressed for 8 days. However, in bacteroids isolated anaerobically, almost 50% of initial acetylene reduction activity remained after 4 days of dark stress but was totally lost after 8 days of dark stress. Bacteroid acetylene reduction activity recovered faster than whole plant acetylene reduction activity when plants were dark stressed for 8 days and returned to a normal light regimen. Significant changes were not measured in bacteroid respiration, protein content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles, or in bacteroid proteolytic activity throughout the experiment. Immunoblots of bacteroid extracts revealed the presence of nitrogenase component II in control, 4-day dark-stressed, and 8-day dark-stressed plants that were allowed to recover under a normal light regimen, but not in 8-day dark-stressed plants. Our data indicate that dark stress does not greatly affect bacteroid metabolism or induce bacteroid senescence.

Journal Article↗
PubMed1985

Spectral and kinetic studies of metal-substituted Aeromonas aminopeptidase: nonidentical, interacting metal-binding sites.

Apoenzyme prepared by removal of the 2 mol of Zn2+/mol from Aeromonas aminopeptidase is inactive. Addition of Zn2+ reactivates it completely, and reconstitution with Co2+, Ni2+, or Cu2+ results in a 5.0-, 9.8-, and 10-fold more active enzyme than native aminopeptidase, respectively. Equilibrium dialysis and spectral titration experiments with Co2+ confirm the stoichiometry of 2 mol of metal/mol. The addition of only 1 mol of metal/mol completely restores activity characteristic of the particular metal. Interaction between the two sites, however, causes hyperactivation; thus, addition of 1 mol of Zn2+/mol subsequent to 1 mol of Co2+, Ni2+, or Cu2+ per mole increases activity 3.2-, 42-, or 59-fold, respectively. The cobalt absorption spectrum has a peak of 527 nm with a molar absorptivity of 53 M-1 cm-1 for 1 mol of cobalt/mol, which increases to 82 M-1 cm-1 for a second cobalt atom and is unchanged by further addition of Co2+. Circular dichroic (CD) and magnetic CD spectra indicate that the first Co2+ binding site is tetrahedral-like and that the second is octahedral-like. Stoichiometric quantities of 1-butylboronic acid, a transition-state analogue inhibitor of the enzyme [Baker, J. O., & Prescott, J. M. (1983) Biochemistry 22, 5322], profoundly affects absorption, CD, and MCD spectra, but n-valeramide, a substrate analogue inhibitor, has no effect. These findings suggest that the tetrahedral-like site is catalytic and the other octahedral-like site is regulatory or structural.

Aeromonas↗
PubMed1984

Physical and enzymatic properties of a class III isozyme of human liver alcohol dehydrogenase: chi-ADH.

chi-Alcohol dehydrogenase (chi-ADH), a class III isozyme characterized by its anodic electrophoretic mobility and lack of inhibition by 4-methylpyrazole, has been isolated from human liver and purified to homogeneity in a reducing medium. chi-ADH resembles other human liver ADH isozymes of classes I and II with respect to its molecular weight, dimeric structure, stoichiometry of zinc and NADH binding, and pH optima for the oxidation of alcohols. This homodimer exhibits subtle differences in its absorption spectrum and amino acid composition relative to those of other human isozymes but differs markedly from their specificity toward alcohols and aldehydes. chi-ADH oxidizes ethanol very poorly. The reaction is bimolecular, and an apparent Km cannot be discerned up to 2.3 M ethanol. The enzyme is inactive toward methanol, ethylene glycol, digitoxigenin, digoxigenin, and gitoxigenin , but alcohols with carbon chain lengths greater than four are oxidized rapidly with Km values decreasing with increasing carbon chain length. Taken jointly, the composition, structure, and enzymatic properties of the ADH isozymes purified and studied so far strongly imply that their metabolic roles, yet to be discovered, will give a new perspective to ethanol metabolism and pathology.

Alcohol Dehydrogenase↗
PubMed1984

Efficacy of methylprednisolone in acute spinal cord injury.

A multicenter double-blind randomized trial was conducted to examine the efficacy of a high dose of methylprednisolone (1,000-mg bolus and daily thereafter for ten days) compared with a standard dose (100-mg bolus and daily thereafter for ten days) in 330 patients with acute spinal cord injury. No difference in neurological recovery of motor function or pinprick and light touch sensation was observed between the two treatment groups six weeks and six months after injury. The lack of a treatment effect was independent of the severity of the initial lesion or the time from injury to starting treatment. Although not statistically significant, early case fatality was greater in the high-dose protocol (relative risk of 3.1 and 1.9, less than or equal to 14 and 15 to 28 days after injury, respectively) but not from 29 to 210 days after injury. Wound infections of both trauma and operative sites were more prevalent in the high-dose regimen (relative risk of 3.6).

Acute Disease↗
PubMed1983

One hundred fold increased activity of Aeromonas aminopeptidase by sequential substitutions with Ni(II) or Cu(II) followed by zinc.

Full substitution of Cu(II) or Ni(II) for the two g-atom zinc in Aeromonas aminopeptidase hyperactivates the enzyme 6.5 and 25 fold respectively. Even greater enhancements of activity can be achieved with mixed metal substitutions. Thus, apoenzyme reactivated by first adding one g-atom zinc followed by one g-atom of either Cu(II) or Ni(II) is 15 and 22 times more active than the native enzyme. Reversing the order, i.e. by first adding either one g-atom Cu(II) or Ni(II) followed by one g-atom zinc, activates the enzyme nearly 100 fold. The order of metal addition is critical and suggests the existence of two non-identical metal sites, each with a different function.

Aeromonas↗
PubMed1983

Kinetic properties of human liver alcohol dehydrogenase: oxidation of alcohols by class I isoenzymes.

Class I isoenzymes of alcohol dehydrogenase (ADH) were isolated by chromatography of human liver homogenates on DEAE-cellulose, 4-[3-[N-(6-aminocaproyl)-amino]propyl]pyrazole--Sepharose and CM-cellulose. Eight isoenzymes of different subunit composition (alpha gamma 2, gamma 2 gamma 2, alpha gamma 1, alpha beta 1, beta 1 gamma 2, gamma 1 gamma 1, beta 1 gamma 1, and beta 1 beta 1) were purified, and their activities were measured at pH 10.0 by using ethanol, ethylene glycol, methanol, benzyl alcohol, octanol, cyclohexanol, and 16-hydroxyhexadecanoic acid as substrates. Values of Km and kcat for all the isoenzymes, except beta 1 beta 1-ADH, were similar for the oxidation of ethanol but varied markedly for other alcohols. The kcat values for beta 1 beta 1-ADH were invariant (approximately 10 min-1) and much lower (5-15-fold) than those for any other class I isoenzyme studied. Km values for methanol and ethylene glycol were from 5- to 100-fold greater than those for ethanol, depending on the isoenzyme, while those for benzyl alcohol, octanol, and 16-hydroxyhexadecanoic acid were usually 100-1000-fold lower than those for ethanol. The homodimer beta 1 beta 1 had the lowest kcat/Km value for all alcohols studied except methanol and ethylene glycol; kcat values were relatively constant for all isoenzymes acting on all alcohols, and, hence, specificity was manifested principally in the value of Km. Values of Km and kcat/Km revealed for all enzymes examined that the short chain alcohols are the poorest while alcohols with bulky substituents are much better substrates. The experimental values of the kinetic parameters for heterodimers deviate from the calculated average of those of their parent homodimers and, hence, cannot be predicted from the behavior of the latter. Thus, the specificities of both the hetero- and homodimeric isoenzymes of ADH toward a given substrate are characteristics of each. Ethanol proved to be one of the "poorest" substrates examined for all class I isoenzymes which are the predominant forms of the human enzyme. On the basis of kinetic criteria, none of the isoenzymes of class I studied oxidized ethanol in a manner that would indicate an enzymatic preference for that alcohol.

Alcohol Dehydrogenase↗
PubMed1983

Reversible dark-induced senescence of soybean root nodules.

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 12 to 16 days. Acetylene reduction activity returned to normal, and both total soluble protein and leghemoglobin were resynthesized while protease activity against azocasein decreased to the level of control nodules. The nodule population that had turned green after 8 days in the dark exhibited a progressive increase in red color starting nearest the exterior of the nodule, and after 16 days of recovery nodules were indistinguishable from control nodules maintained under a normal photoperiod.

Journal Article↗
PubMed1983

Proteolytic Activity in Soybean Root Nodules : Activity in Host Cell Cytosol and Bacteroids throughout Physiological Development and Senescence.

Root nodules were harvested from chamber-grown soybean (Glycine max L. Merrill 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 bacteroid extracts was not observed. These results are consistent with the view that soybean nodule bacteroids are capable of redifferentiation into free-living bacteria upon deterioration of the legume-rhizobia symbiosis.

Journal Article↗
PubMed1983

An index to measure the healing potential of ischaemic ulcers using Thallium 201.

Prediction of healing of ulcers in ischaemic limbs can preclude unnecessary treatment for ulcers that cannot heal. Non-invasive methods are of marked value as the ischaemic limb is susceptible to further ulceration from local skin penetration. Relative hyperemia of the ulcer was measured by scintillation count over the ulcer and at points 2.5 cm from the edge of the ulcer. Relative hyperemia was determined by dividing the count per unit area of the ulcer by the counts per unit area of the surrounding tissue. All ulcers with a relative hyperemia over 1.5 healed.

Humans↗
PubMed1983

Prediction of leg viability and amputation level by fluorescein uptake.

There is a continuous search for more reliable, locally non-invasive tests for prediction of leg viability and for the selection of amputation levels in dysvascular lower limbs. Refinement of the dermofluorometer by addition of a shielded probe and fibreoptic bundles has reduced the bulk of the instrument and permitted rapid testing. Excitation light is carried to the tissues and emitted fluorescence is returned to a photo multiplier. The Index of Fluorescein Uptake is 20-30 units in control areas. Healing is constant above 15 units, inconstant from 5 to 15 units, and absent below 5 units. Reliability has been virtually 10% in preliminary tests.

Amputation, Surgical↗
PubMed1983

Ambulation levels of bilateral lower-extremity amputees. Analysis of one hundred and three cases.

One hundred and three bilateral lower-extremity amputees were evaluated to determine their eventual ambulation level. Of thirty-eight bilateral above-the-knee amputees, two with traumatic amputation were prosthetically rehabilitated, while none of the thirty-five with dysvascular amputation were so rehabilitated. Twenty-two of the dysvascular above-the-knee amputees were wheelchair ambulators and thirteen were bedridden. Prosthetic rehabilitation has been successful for traumatic bilateral above-the-knee amputees but has never been successful for our dysvascular bilateral above-the-knee amputees. The goal for dysvascular bilateral above-the-knee amputees is wheelchair ambulation. Of twenty-one patients with combinations of above-the-knee and below-the-knee amputations, five were prosthetically rehabilitated, including four dysvascular amputees; ten were wheelchair ambulators; and six were bedridden. Of forty-four patients with bilateral below-the-knee amputation, thirty-five were prosthetically rehabilitated and the remaining nine were wheelchair ambulators. Since the success rate for prosthetic rehabilitation is higher for amputees with combination above-the-knee and below-the-knee amputation than for those with bilateral above-the-knee amputation, and again increases for those with bilateral below-the-knee amputation, the significance of preserving the knee joint, even a single knee, cannot be overemphasized.

Activities of Daily Living↗
PubMed1981

Purification and characterization of multiplication-stimulating activity (MSA) carrier protein.

The rat liver cell line, BRL-3A, is known to produce a family of polypeptides referred to as multiplication-stimulating-activity (MSA). Serum-free conditioned medium from this cell line is a rich source for the purification of these somatomedin-like molecules. Somatomedins in serum, as well as MSA produced by BRL-3A cells in culture, exist primarily as a high molecular weight complex bound to specific carrier proteins. This study describes the purification of the MSA carrier protein (MCP) from conditioned medium using affinity chromatographic procedures. The purified carrier protein is shown to specifically bind labeled MSA and generates a complex with an apparent molecular weight of 60,000-70,000 daltons. Characterization of the carrier protein indicates that it consists of two different noncovalently linked protein chains with apparent molecular weights of 30,000 and 31,500 daltons. The availability of a pure carrier protein should provide a unique opportunity to investigate the functional significance of the carrier protein in the biological activity of the somatomedins.

Animals↗
PubMed1981

Peptidohydrolases of Soybean Root Nodules : IDENTIFICATION, SEPARATION, AND PARTIAL CHARACTERIZATION OF ENZYMES FROM BACTEROID-FREE EXTRACTS.

Nodule extracts prepared from Glycine max var Woodworth possessed endopeptidase, aminopeptidase, and carboxypeptidase activities. 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-l-Leu-beta-naphthylamide, while the second hydrolyzed n-benzoyl-l-Arg-beta-naphthylamine (Bz-l-Arg-betaNA), n-benzoyl-l-Arg-p-nitroanilide (Bz-l-Arg-pNA), and azocasein. The third endopeptidase hydrolyzed Bz-l-Arg-betaNA, Bz-l-Arg-pNA, and hemoglobin. Fractions of these enzymes extracted from electrophoresis gels were shown to have pH optima from 7.5 to 9.8. All of the endopeptidases were completely inhibited by diisopropylphosphorofluoridate, demonstrating that they were serine proteases.Aminopeptidase activity was measured using amino acyl-beta-naphthylamides. Electrophoresis of nodule extracts at pH 6.8 resolved the aminopeptidase activity of nodule extracts into at least four fractions based on mobility and on activities toward amino acyl-beta-naphthylamides. The major activity of two of the aminopeptidases was directed toward l-Leu- and l-Met-beta-naphthylamide, while the other two aminopeptidases exhibited broader specificity and were capable of hydrolyzing a large number of amino acyl-beta-naphthylamides. Two of the aminopeptidases extracted from electrophoresis gels were classified as thiol type enzymes, and all four aminopeptidases had neutral to basic pH optima.

Journal Article↗
PubMed1981

Partial and total calcanectomy: a review of thirty-one consecutive cases over a ten-year period.

From 1968 to 1978 we performed twenty partial and eleven total calcanectomies in twenty-nine patients, eighteen of whom were diabetics with ulcerations of the heel. All but two had osteomyelitis of the calcaneus. Eight of the patients had an injury of the spine. The remaining patients had conditions in which sensation in the heel was absent or decreased. In about one-half of the diabetic patients, immediate failure of the procedure led to amputation. Primary wound-healing occurred in only four of the eighteen diabetics, but in the non-diabetic patients ten of the thirteen heels showed primary healing. Late failure also occurred in three diabetic patients. The overall rate of failure in the diabetics was 65%.

Adolescent↗