Postoperative decrease of regional volumetric bone mineral density measured by quantitative computed tomography after lumbar fusion surgery in adjacent vertebrae.

We investigated the effect of posterior lumbar fusion surgery on the regional volumetric bone mineral density (vBMD) measured by quantitative computed tomography. Surgery negatively affected the regional vBMD in adjacent levels. Interbody fusion was independently associated with vBMD decline and preoperative epidural steroid injections (ESIs) were associated with less postoperative vBMD decline. INTRODUCTION: Few studies investigate postoperative BMD changes after lumbar fusion surgery utilizing quantitative computed tomography (QCT). Additionally, it remains unclear what preoperative and operative factors contribute to postoperative BMD changes. The purpose of this study is to investigate the effect of lumbar fusion surgery on regional volumetric bone mineral density (vBMD) in adjacent vertebrae and to identify potential modifiers for postoperative BMD change. METHODS: The data of patients undergoing posterior lumbar fusion with available pre- and postoperative CTs were reviewed. The postoperative changes in vBMD in the vertebrae one or two levels above the upper instrumented vertebra (UIV+1, UIV+2) and one level below the lower instrumented vertebra (LIV+1) were analyzed. As potential contributing factors, history of ESI, and the presence of interbody fusion, as well as various demographic/surgical factors, were included. RESULTS: A total of 90 patients were included in the study analysis. Mean age (±SD) was 62.1 ± 11.7. Volumetric BMD (±SD) in UIV+1 was 115.4 ± 36.9 mg/cm3 preoperatively. The percent vBMD change in UIV+1 was - 10.5 ± 12.9% (p < 0.001). UIV+2 and LIV+1 vBMD changes showed similar trends. After adjusting with the interval between surgery and the secondary CT, non-Caucasian race, ESI, and interbody fusion were independent contributors to postoperative BMD change in UIV+1. CONCLUSIONS: Posterior lumbar fusion surgery negatively affected the regional vBMDs in adjacent levels. Interbody fusion was independently associated with vBMD decline. Preoperative ESIs were associated with less postoperative vBMD decline, which was most likely a result of a preoperative decrease in vBMD due to ESIs.

Spinal interneurons play a minor role in generating ongoing renal sympathetic nerve activity in spinally intact rats.

The purpose of the present study was to determine whether spinal interneurons play a role in the regulation of sympathetic activity in spinally intact rats. In acutely spinally transected rats, we have described a population of spinal interneurons that, by virtue of correlations between their ongoing firing rates and the magnitude of ongoing renal sympathetic nerve activity (RSNA), are candidates for generators of sympathetic activity. Further evidence for a sympathetic role for these neurons comes from our observation that cervical spinal stimulation that reduces RSNA also reduces their discharge rates. In chloralose-anesthetized, spinally intact and spinally transected rats, we recorded ongoing RSNA and the ongoing activities of T(10) dorsal horn and intermediate zone interneurons, and we determined the incidence of sympathetically related neurons in these rats by cross-correlating their activities with RSNA. The incidence of correlated neurons was much smaller in spinally intact than in spinally transected rats. We stimulated the dorsolateral, C(2-3) spinal cord before and after acute C(1) spinal transection. Dorsolateral cervical stimulation in spinally transected rats reduced both RSNA and the activities of most T(10) interneurons, but stimulation in spinally intact rats increased RSNA while still reducing the activities of most T(10) interneurons. Both the low incidence of sympathetically correlated spinal neurons in intact rats and the dissociation between the effects of cervical stimulation on RSNA and the discharge rates of spinal interneurons argue against these neurons playing a major role in regulating sympathetic activity in intact rats.

Do some plant responses to cytokinins involve the cyanide-resistant respiratory pathway?

A disengagement of the cyanide-resistant, alternative respiratory pathway in soybean (Glycine max (L.) Merr.) callus tissue was observed prior to the start of deoxyisoflavone production stimulated by addition of the cytokinin benzyladenine. To test whether this loss of alternativepathway activity was part of the response to cytokinin, inhibitors of the alternative pathway were assayed for their ability to elicit cytokinin-like responses. Salicylhydroxamic acid (SHAM) was found to produce a deoxyisoflavone difference spectrum similar to that observed following treatment of the callus tissue with benzyladenine, while propyl gallate (PG) was without effect. Both SHAM and PG were further tested for cytokinin-like activity in other bioassays. In two anti-senescence bioassays using leaf tissue (of Avena sativa L. and Xanthium pensylvanicum Wallr.) and in the Cucumis sativus L. bioassay which measures stimulation of weight gain by excised cotyledons, both SHAM and PG were effective "cytokinins" at 1 mM and 0.1 mM, respectively. In two other bioassays (betacyanin formation in Amaranthus caudatus L. seedlings and the soybean-callus celldivision assay), SHAM appeared to be toxic. These results substantiate the suggestion that effects on the alternative pathway may play a role in some cytokinin responses and further raise the question of what should be considered a true cytokinin response.

Possible Regulatory Roles of Cytokinins : NADH Oxidation by Peroxidase and a Copper Interaction.

Apparently free-base cytokinins can interact with cupric ions in a specific manner. Oxidation of NADH by a horseradish peroxidase system was strongly promoted by such cytokinins provided cupric ions were present. Oxidation was promoted by 5 micromolar kinetin, zeatin, 6-benzylaminopurine (BA), or 6-(Delta(2)-isopentenylamino)purine (2iP) but not by adenine, 6-methylaminopurine or 6,6-dimethylaminopurine. The 6-methylaminopurine promoted oxidation at 500 micromolar but adenine and 6,6-dimethylaminopurine did not. Activity of the free-base purines correlated well with their activity in cell-division assays. However, addition of methoxymethyl-, cyclohexyl-, or tetrahydropyranyl- at N-9 of BA or of ribosyl- at N-9 of BA, 2iP, kinetin, or zeatin eliminated activity in the peroxidase system. In a nonenzymic system containing cupric ions, all of the bases, including adenine, inhibited the Cu(2+) -stimulated oxidation of ascorbic acid. As in the peroxidase system, the N-9 derivatives were inactive. The cytokinin promotion of NADH oxidation by peroxidase may result from an interaction of the hormones with copper, with peroxidase conferring a specificity similar to the cytokinin specificity observed in growth and development.

Cytokinin modification of mitochondrial function.

6-Benzylaminopurine, 6-(Delta(2)-isopentenylamino)purine, 6-furfurylaminopurine, rotenone, and antimycin A inhibited oxidation of NADH by mitochondrial sonicates or submitochondrial particles (but not by intact mitochondria) from pea (Pisum sativum L., cult. Alaska) stems and mung bean (Vigna radiata L. Wilczak) hypocotyls. The above purine cytokinins can interfere with electron transport from NADH to the cytochrome system in the inner mitochondrial membrane. Adenine did not inhibit oxidation by sonicated mitochondria, and zeatin was almost ineffective. Zeatin scarcely inhibited state 3 malate respiration by intact mitochondria, but the O-formyl and O-n-propionyl esters of zeatin and the O-acetyl ester of 2-chlorozeatin were more active. Perhaps zeatin is ineffective because it does not get into the inner membranes of the isolated mitochondria, whereas the esters and other cytokinins mentioned above do. N-4-(2-chloropyridyl)-N'-Phenylurea, which has cytokinin-like effects on plant growth and development, inhibited NADH oxidation by sonicated mitochondria. It also inhibited malate, succinate, and NADH oxidation by intact mitochondria; in contrast, the latter two oxidations were not decreased by purine cytokinins.The benzyl, isopentenyl, and furfuryl aminopurines inhibited malate (but not succinate) oxidation by rat liver mitochondria, if ADP was present. In the absence of ADP, these cytokinins promoted succinate (but not malate) oxidation. Zeatin and adenine had slight, if any, effect in either situation.

Hormonal control of tobacco crown gall tumor morphology.

The endogenous levels of auxin and cytokinin in teratoma and unorganized tobacco (Nicotiana tabacum L. var Wisconsin #38) crown gall tumor tissues were determined. Teratoma tissues contain levels of auxin and cytokinin favorable for shoot formation, whereas unorganized tumors contain levels of auxin that suppress shoot formation. This conclusion is based upon the observation that when levels of auxin and cytokinin similar to those found in a teratoma were added to the growth medium of nontumorous tobacco tissue, shoot formation resulted; when levels similar to those found in unorganized tumors were added, the normal tissue grew as unorganized callus.

Cytokinin inhibition of respiration in mitochondria from six plant species.

The influence of 6-benzylaminopurine (BAP) on the respiration by mitochondria from bush bean (Phaseolus vulgaris L.), mung bean (P. aureus Roxburgh), soybean [Glycine max (L.) Merrill], maize (Zea mays L.), pea (Pisum sativum L.), and wheat (Triticum aestivum L.) was examined. BAP, a synthetic cytokinin, consistently inhibited oxygen uptake by mitochondria from all species when malate was used as the substrate. The decrease in respiration was especially evident in the presence of ADP or an uncoupler of oxidative phosphorylation. 6-Isopentenylaminopurine and 6-furfurylaminopurine also inhibited malate oxidation, but zeatin and adenine did not. In certain instances, BAP reduced succinate and NADH oxidation. With succinate as the substrate and with antimycin A present, inhibition by BAP paralleled that caused by salicylhydroxamic acid, an inhibitor of alternative respiration. A suggested scheme features a cytokinin-inhibited point located between NADH dehydrogenase and cytochrome b of the electron transport system. Electrons from the NADH generated by malate oxidation are assumed to flow through this point, with electrons from externally supplied or cytosolic NADH and succinate doing so only under certain conditions such as when alternative respiration is occurring. Cytokinin effects on respiration and perhaps on other phenomena may be mediated by this mechanism.

Cytokinin inhibition of respiration by cells and mitochondria of soybean, Glycine max (L.) Merrill.

Cells of a soybean tissue strain, suspended in an aerated liquid medium, caused disappearance of p-coumaric acid from the medium and oxidation of guaiacol, benzidine, pyrogallol, L-dihydroxyphenylalanine and L-epinephrine. Both the disappearance and the oxidations were inhibited by 6-benzylaminopurine (BAP) at a concentration of 0.5 mM. BAP at other concentrations either promoted or inhibited oxidation of epinephrine in precisely the pattern reported earlier for the disappearance of coumarate; therefore, the disappearance of coumarate probably involves its oxidation. The effectiveness of other cytokinins in inhibiting the oxidation was studied.At 0.5 mM, and perhaps even at 0.5 µM, some of the several cytokinins tested inhibited oxygen consumption by the soybean cells. This inhibition, which did not require any of the above metabolizable compounds, was especially marked in the presence of cyanide, azide or Antimycin A, and was detectable in 10 min or less. Either Antimycin A or salicylhydroxamic acid alone promoted O2 consumption but together they were quite inhibitory. The soybean cells apparently have an alternate respiratory pathway and cytokinins may influence its operation.Several cytokinins at 0.5 mM, and perhaps at 0.5 µM, also inhibited oxygen consumption by mitochondrial preparations from the soybean cells, the inhibition being evident in about 20 s. The consumption required a substrate such as malate, succinate or NADH. Cytokinins and related compounds varied in effectiveness as follows: BAP and 6-isopentenyla-minopurine ≥ 9-tetrahydropyranyl-BAP > kinetin, ribosyl-isopentenylaminopurine, 9-methyl-BAP and 9-methoxymethyl-BAP > 6,6-dimethylaminopurine and zeatin (slight activity) > 6-methylaminopurine, nicotinamide and adenine (ineffective). To a great extent this order parallels the order of effectiveness of the compounds in causing cell division. Mitochondria, therefore, may contain a site for an important cytokinin action.

Cytokinin modification of metabolism of p-coumaric acid by a cell suspension of soybean (Glycine max (L.) Merrill).

Cells of a soybean tissue strain suspended in an aerated liquid medium caused the disappearance of p-coumaric acid from the medium. The rate of disappearance was modified by cytokinins. When the coumarate and the cytokinin were added to the medium simultaneously, disappearance was increased if the cytokinin was used in the concentration range from 0.05 to 50 µM; higher concentrations inhibited the disappearance. If, however, the cytokinin was added at the beginning of the shaking period (for aeration) and the coumarate added 1 h later, the results were more complex. With this procedure, cytokinins at concentrations from 0.0005 to about 1 µM inhibited, at 50 µM they promoted, and at higher concentrations they inhibited the coumarate disappearance. The promotion was elicited by zeatin, ribosylzeatin, kinetin, 6-benzylaminopurine (BAP), by BAP substituted at the 9-position by methyl, methoxymethyl, cyclohexyl or tetrahydropyran-2-yl groups, by adenine with the amino group substituted by methyl, dimethyl, n-propyl, n-pentyl or n-hexyl groups, by 1,3-diphenylurea and nicotinamide, all at about 50 µM. Adenine and benzimidazole were not effective. The promotion was detected in as little as 12 min. The delayed inhibitory effect required the presence of the cytokinin during the 1 h of shaking before the coumarate was added. This effect was elicited by zeatin, ribosylzeatin, kinetin, BAP, the aforementioned 9-substituted-BAP compounds, 9-glucosyl-BAP, 7-glucosyl-BAP, and 6-isopentenylaminopurine and its ribonucleoside. It was not caused by adenine, cis-ribosylzeatin, diphenylurea, benzimidazole, 6-methylaminopurine, 6,6-dimethylaminopurine or nicotinamide. The chemical specificity for this effect was much the same as that known for promotion of cell division in the soybean tissue.

Glucosyl Zeatin and Glucosyl Ribosylzeatin from Vinca rosea L. Crown Gall Tumor Tissue.

Recently detected but unidentified cytokinin activity in crown gall tumor tissue from Vinca rosea L. grown on media containing sources of reduced nitrogen has now been attributed to two adenine-type cytokinins. These compounds are glucopyranosyl derivatives of zeatin and ribosylzeatin. The substitution in each case is on the isopentenyl chain of the parent compound. Neither of these compounds had activity in the soybean callus bioassay at concentrations lower than 1 nm whereas zeatin had activity at 0.1 nm.

Cytokinins in Vinca rosea L. Crown Gall Tumor Tissue as Influenced by Compounds Containing Reduced Nitrogen.

Several compounds containing reduced nitrogen markedly increased the yields of cell-division compounds extractable from an A6 Vinca rosea L. crown gall tumor tissue. Casein hydrolysate, several amino acids, and ammonium salts were effective. Both trans-zeatin and ribosyl-trans-zeatin were substantially increased in total amount per culture and in concentration. These two compounds have been identified by several criteria including mass spectra. The reduced nitrogen treatments also caused the appearance of a cytokinin not previously detected in this tissue; it has not yet been identified. The tumor tissue rapidly absorbed [8-(14)C]adenine from a liquid medium. Within 1 hour, the tissue converted some of the adenine to zeatin and ribosylzeatin, and greater degrees of conversion occurred in 2-, 4-, and 8-hour periods. The tissue grown on a medium containing ammonium chloride accumulated considerably greater quantities of the two cytokinins made from the labeled adenine during each incubation period.

Cell-division factors from Vinca rosca L. crown gall tumor tissue.

A cell-division factor has been precipitated from extracts of cultured Vinca rosea L. crown gall tumor tissue by using the mercuric acetate procedure previously employed by Wood and colleagues to obtain their "cytokinesin I." On the basis of its mass spectrum, ultraviolet light absorbancy spectra, solubilities, chromatographic migration values, and growth activity, the factor is ribosyl-trans-zeatin, that is, 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-9-beta-D-ribofuranosylpurine. Ribosylzeatin has now been isolated from tumor tissue by four experimental techniques; any possibility that it is an artifact seems to have been eliminated. Contrary to the report by Wood and colleagues, synthetic ribosylzeatin is precipitated from an aqueous solution by mercuric acetate, provided the complete precipitation procedure is utilized. These facts and others discussed strongly support our suggestion that ribosylzeatin was present in the preparation ("cytokinesin I") examined by Wood and colleagues in several biological assays. The reasons advanced by Wood and others for rejecting this suggestion have been found either not to be pertinent to the question or to have insufficient experimental bases.

Revised Methods for Purification of Ribosyl-trans-zeatin from Vinca rosea L. Crown Gall Tumor Tissue.

Ribosyl-trans-zeatin has been purified from Vinca rosea L. crown gall tumor tissue by using two new sequences of isolation procedures. Identification of the compound has been established by mass spectrometry, ultraviolet absorbancy spectra, chromatographic values, and growth activity. The isolation sequences eliminate the exposures to pH extremes and the strong cation-exchange resin used in the purification reported earlier. The initial extraction procedures have been designed so as to avoid enzymatic alteration or production of active material and to prevent the inclusion in the extracts of nucleic acids which might serve as soures of the small active compounds. The production of ribosylzeatin by the tumor tissue is confirmed and the validity of isolation steps such as the use of cation exchangers is supported.

Detection and identification of cytokinins produced by mycorrhizal fungi.

Several fungi including six species of the genus Rhizopogon, 22 species of Hebeloma and one of Agaricus have been screened for production of cytokinins. The screening was done by culturing cytokinin-requiring soybean callus tissue alongside the fungus on a medium lacking a cytokinin supply. Growth of the soybean callus indicated production of cytokinins by the fungus. Of the fungi tested, only R. ochraceorubens A. H. Smith gave off sufficient cytokinin to be detected. Although a number of mycorrhizal species are now known to make and give off cytokinins, an even larger number apparently do not do so under the conditions of screening employed. An unidentified ectendotrophic species definitely gave off trans-zeatin, which has been crystallized, and probably trans-ribosylzeatin. Suillus punctipes (Pk.) Sing. apparently produced the same two cytokinins.

Ribosyl-trans-Zeatin, A Major Cytokinin Produced by Crown Gall Tumor Tissue.

A cell division factor in crystalline form has been isolated from ethanolic extracts of cultured Vinca rosea L. crown gall tumor tissue. The crystalline material shows the melting point, mass spectrum, ultraviolet-light absorbancy spectra, solubilities, chromatographic migration values, ion-exchange behavior, and growth activities expected of ribosyl-trans-zeatin, that is, 6-(4-hydroxyl-3-methyl-trans-2-butenylamino)-9-beta-D- ribofuranosylpurine. This compound, which appears to be a major cell-division factor in the extracts, has also been crystallized from liquid media in which the tumor tissue had been growing and apparently was the only active compound in such media.

Effects of cytokinins on the respiration of soybean callus tissue.

A technique which incorporates a brief blending step to disperse callus tissue into small clumps of cells was developed, and the effects of cytokinins on respiration of soybean (Glycine max [L.] Merrill var. Acme) callus tissue prepared in this way were studied. Adenine alone did not affect respiration, but kinetin and zeatin showed effects correlating with their reported effects on growth of this tissue; after about 3 hours both hormones promoted respiration at concentrations which promote growth, while kinetin, but not zeatin, also exhibited inhibition at higher concentrations. Studies with 2,4-dinitrophenol led to the suggestion that although the respiration of this tissue is largely under the control of ATP levels, kinetin does not exert its control on respiration through effects on ATP levels or oxidative phosphorylation during the monitoring period. Further inhibitor and substrate studies provided evidence that the promotion of respiration by kinetin results from an increase in substrate entering the tricarboxylic acid cycle, perhaps by an effect on pyruvate metabolism.The inhibition of respiration by high concentrations of kinetin is partially due to effects on oxidative phosphorylation or ATP utilization, but 70% of the inhibition cannot be attributed to this.

Modification of the cytokinin promotion of deoxyisoflavone synthesis in soybean tissue.

Soybean tissue incubated in liquid media formed daidzin in response to cytokinin when the media contained 0.1 m sucrose but formed another, unidentified compound when the media contained 0.6 m sucrose or mannitol. The cytokinin effect in either setup was detectable only after a lag period of several hours. Addition of possible precursors of the compounds being synthesized or of related compounds did not alter the lag period to any great extent. Trans-cinnamic acid did perhaps shorten it a little as did preincubation of the tissue in the basal medium before the addition of kinetin. Several inhibitors of RNA synthesis substantially reduced the production of daidzin and the other compound. Protein synthesis inhibitors were also effective except that cycloheximide and puromycin actually promoted synthesis of the unknown compound while inhibiting that of daidzin. The results do not give a clear impression as to how the cytokinins are involved in the synthesis of the compounds, but are at least suggestive that cytokinin is involved in RNA or protein synthesis.

Cytokinins from a variant strain of cultured soybean cells.

A strain of soybean cells capable of growing on a tissue culture medium lacking a cytokinin produced at least 3 compounds active in the soybean cytokinin assay. The characteristics of these compounds were consistent with their being zeatin in the free form, zeatin ribonucleoside and zeatin ribonucleotide. Although the conversion from a cytokinin dependent to independent condition in this strain parallels the change of normal cells to crown gall tumor state in terms of the capacity to synthesize cell division substances, the soybean factors are distinct from the nicotinamide derivatives reported for tumor cells of Vinca.

6-(gamma,gamma-Dimethylallylamino)Purine As A Precursor of Zeatin.

A 6-(gamma,gamma-dimethylallylamino) purine-like compound was found in the culture medium of Rhizopogon roseolus, which had been shown earlier to synthesize zeatin. The role of 6-(gamma,gamma-dimethylallylamino) purine as a precursor of zeatin was studied. Rhizopogon was furnished with 6-(gamma,gamma-dimethylallylamino) purine-8-(14)C. Cochromatography, oxidation studies with potassium permanganate, and bromination indicated that labeled zeatin ribonucleoside was isolated from the medium. The fungus also incorporated labeled adenine, hypoxanthine, and 4-amino-5-imidazole carboxamide into zeatin ribonucleoside.

Control of deoxyisoflavone synthesis in soybean tissue.

1. Callus tissues of Glycine max, cv. Acme, on solid or in liquid media quickly responded to cytokinins by synthesizing two compounds which appear to be glycosides of the deoxyisoflavone, daidzein. Auxin also was necessary for the effect. 2. After a lag, the response could be detected in 24 h or less by reading ultraviolet optical densities of simple acidic or ethanolic extracts of the tissues or of the liquid media and suspended tissues. 3. The effects of different levels or kinds of auxins and cytokinins were parallel to the known growth responses. 4. When 2,4-dichlorophenoxyacetic acid was used, addition of a cytokinin was not required for deoxyisoflavone synthesis. 5. Malonic acid promoted deoxyisoflavone synthesis but only in the presence of an auxin. Addition of a cytokinin was not necessary for this promotion. Citric acid also was effective but acetic, pyruvic, α-ketoglutaric, succinic, malic and fumaric acids were not. 6. The influence by cytokinins and auxins on deoxyisoflavone synthesis offers a system usable for assay of the growth substances and for study of their modes of action.