Effect of sulfolipid antibodies on the macromolecular synthesis of Mycobacterium smegmatis ATCC607

Abstract The biosynthesis of DNA, RNA, proteins and lipids in the presence of antiserum to sulfolipids was investigated by studying the incorporation of radiolabelled precursors like ³H-thymidine ¹⁴C-uracil, ¹⁴C-leucine and ¹⁴C-Acetate into their respective macromolecules. Antiserum to sulfolipids had a inhibitory effect on the biosynthesis of all these components. Antiserum also exhibited a growth inhibitory effects as compared to normal serum.     

Gibberellic acid promotes growth and cell wall synthesis in Avena internodes regardless of the orientation of cell expansion

Segments cut from the next-to-last (peduncular-1) internode of Avena sativa L. cv. Victory (oat) shoots elongate as much as 10-fold in response to gibberellic acid (GA₃). The objective of the present investigation was to differentiate the effects of GA₃ on growth from its effects on wall synthesis (measured gravimetrically and through the incorporation of [¹⁴C]-glucose) by using several cell wall synthesis inhibitors with widely varying mechanisms of action. Four compounds, viz. monensin, cycloheximide, lanthanum, and galactose. caused (1) relatively little inhibition of either cell wall synthesis or elongation in segments without GA₃, (2) roughly proportionate, dose-dependent inhibition of elongation and wall synthesis in GA₃-treated segments and (3) generally greater inhibition of GA₃-promoted uptake of radioactivity than of wall incorporation or elongation. Two other compounds, colchicine and 2,6-dichlorobenzonitrile (DCB). (1) inhibited GA₃-induced elongation considerably more than cell wall synthesis and (2) caused swelling (radial expansion). especially of GA₃-treated segments. DCB-treated internodal cells apparently compensated for inhibited cellulose synthesis by greater synthesis of matrix polysaccharide (beginning between 3 and 6 h). While normal cellulose synthesis was not required for short-term (up to 6 h) GA₃-induced elongation or for long-term hormone-promoted radial expansion, it was required for sustained GA₃-induced elongation. These results indicate that GA₃-promoted cell wall loosening (manifested as radial expansion) and cell wall synthesis in Avena internodes occur at least partially independently of any hormonal effect on the orientation of microtubules and microfibrils.     

Effects of gibberellic acid on patterns of carbohydrate distribution and acid invertase activity in Phaseolus vulgaris

The application of gibberellic acid (GA₃,10 μ M ) as a root drench to 16-day-old plants of Phaseolus vulgaris L. cv. Masterpiece stimulated growth of the stem internodes and reduced root growth. GA₃ treatment did not affect the export of ¹⁴C from a primary leaf to which [¹⁴C]-sucrose was applied, but greatly increased upward translocation to the elongation region of the stem at the expense of transport to the hypocotyl and root system. The observed changes in the patterns of growth and [¹⁴C]-labelled assimilate distribution were correlated with an increase in the specific activity of acid invertase in the elongating stem internodes and a decrease in invertase activity in the hypocotyl and root. Sucrose concentration in the elongating internodes fell substantially after treatment with GA₃ while the concentration of hexose sugars increased. We suggest that by stimulating acid invertase synthesis in the elongating internodes, GA₃ acts to establish a more favourable sucrose gradient between these sinks and source leaves. Under source-limiting conditions this, in turn, will lead to a reduced rate of assimilate translocation to competing sinks in the root system.     

Biological activity, identification and quantification of gibberellins in seedlings of Norway spruce (Picea abies) grown under different photoperiods

Short photoperiod induces growth cessation in seedlings of Norway spruce ( Picea abies (L.] Karst.). Application of different gibberellins (GAS) to seedlings growing under a short photoperiod show that GA₉ and GA₂₀ can not induce growth. In contrast application of GA, and GA₄ induced shoot elongation. The results indicate that 3β-hydroxylation of GA₉ to GA₄ and of GA₂₀ to GA₁ is under photoperiodic control. To confirm that conclusion, both qualitative and quantitative analyses of endogenous GAs were performed. GA₁, GA₃, GA₄, GA₇, GA₉, GA₁₂, GA₁₅, GA₁₅, GA₂₀, GA₂₉, GA₃₄ and GA₅₁ were identified by combined gas chromatography-mass spectrometry in shoots of Norway spruce seedlings. The effect of photoperiod on GA levels was determined by using deuterated and ¹⁴C-labelled GAs as intermal standards. In short days, the amounts of GA₉, GA₄ and GA₁ are less than in plants grown in continuous light. There is no significant difference in the amounts of GA₃, GA₁₂, and GA₂₀ between the different photoperiods. The lack of accumulation of GA₉ and GA₂₀ under short days is discussed.     

Stimulation of shoot elongation in Salix pentandra by gibberellin A9; activity appears to be dependent upon hydroxylation to GAl via GA20

Cessation of shoot elongation in seedlings of Salix pentandra L. is induced by short photoperiod. Gibbereliin A₉ (GA₉) applied either to the apical bud or injected into a mature leaf, induced shoot elongation under a short photoperiod of 12 h, and GA₉ could completely substitute for a transfer to a long photoperiod. When [³H]GA₉ or [²H₂]GA₉ was injected into a leaf, no [³H]GA₉ was detected in the elongating apex and only traces of [³H]GA₉ were found in the shoot above the treated leaf. By the use of gas chromatography-mass spectrometry (GC-MS), [²H₂]GA₂₀ was identified as the main metabolite of [²H₂]GA₉ in both the shoot and the treated leaf. In addition, [²H₂]GA₁ and [²H₂]GA₂₉ were also identified as metabolites of [²H₂]GA₉. These results are consistent with the hypothesis that exogenous GA, promotes shoot elongation in Salix through its metabolism to GA₂₀ and GA,.     

Effect of gibberellic acid on growth and carbohydrate metabolism of developing tubers of potato (Solanum tuberosum)

Potato plants (Solanum tuberosum L. cv. Ostara) were grown in aerated water culture in a controlled environment. When the tubers had reached a diameter of 1–3 cm. ¹⁴C-labelled or unlabelled gibberellic acid (GA₃) was applied to the surface of the stolons at points approximately 1 crn from the developing tubers, and treatment continued for 10 days. - Significant quantities of GA₃ moved into tuber tissue within 2–4 days of hormone application. This influx of GA₃ was accompanied by a marked reduction in both the activity of ADPG-pyrophospharylase and the ratio ADPG-pyrophosphorylase/starch phosphorylase and an increase in the activity of UDPG-pyrophosphorylase. Starch phosphorylase activity initially increased slightly but then fell, whereas the activity of starch synthase remained constant throughout the experiment. The soluble sugar composition of the tubers changed qualitatively towards a pattern characteristic of growing stolon tips prior to tuber initiation, but there was no clear evidence of net starch degradation. Changes in the activities of the enzymes were observed prior to noticeable effects of the hormone on tuber growth rate or the development of new stolons at the tuber eyes. - GA₃- treated tubers imported more ¹⁴C from labelled photosynthate than expected on the basis of growth rate. However, the capacity to convert solub#e-¹⁴C to ethaTiol-insoluble-¹⁴C (predominantly starch) was reduced in comparison with non-treated tubers. - The observed changes in carbohydrate composition and enzyme activities indicate that GA₃ induces a drastic change in potato tuber metabolism towards a pattern characteristic for the termination of the storage process.     

Internode length in Pisum. IV. The effect of the Le gene on gibberellin metabolism

The highly active, polar gibberellin-like substance found in the apical region of shoots of tall (genotype Le ) peas ( Pisum sativum L.) is shown by combined gas chromatography-mass spectrometry (GC/MS) to be GA₁. This substance is either absent or present at only low levels in dwarf ( le ) plants. Multiple ion monitoring (MIM) tentatively suggests that GA₈ may also be present in shoot tissue of tall peas. Gibberellin A₁ is the first 3 β-hydroxylated gibberellin positively identified in peas, and its presence in shoot tissue demonstrates the organ specificity of gibberellin production since GA₁ has not been detected in developing seeds. Application of GA₁ can mask the Le/le gene difference. However, whilst Le plants respond equally to GA₂₀ and GA₁, le plants respond only weakly to GA₂₀, the major biologically active gibberellin found in dwarf peas. These results suggest that the Le gene controls the production of a 3 β-hydroxylase capable of converting GA₂₀ to GA₁. Further support for this view comes from feeds of [³H] GA₂₀ to Le and le plants. Plants with Le metabolise [³H] GA₂₀ to three major products whilst le plants produce only one major product after the same time. The metabolite common to Le and le plants co-chromatographs with GA₂₉. The additional two metabolites in Le peas co-chromatograph with GA₁ and GA₈.     

Hormone directed sucrose transport during fruit set induced by gibberellins in Pisum sativum

A new system has been developed to study hormone-directed transport in intact plants during parthenocarpic fruit set induced by gibberellins. Gibberellic acid (GA₃) and gibberellin A₁ (GA₁) applied to unpollinated ovaries of pea ( Pisum sativum L. cv. Alaska) promoted sucrose transport from the leaf to the site of hormone application. In vivo experiments showed an early (30 min) accumulation of [¹⁴C]-sucrose in ovaries of pea stimulated by gibberellins. This activation of sucrose transport appears to be mediated by gibberellins (GA₁, GA₃), increasing both loading of phloem with sucrose in the leaf (source) and sucrose unloading in the ovary (sink). The ability of pea tissue segments to take up sucrose in vitro was not affected by the hormonal treatment.     

A chlorate-hypersensitive, high nitrate/chlorate uptake mutant of Arabidopsis thaliana

N-ethylmaleimide (NEM) Lit 10-100 μ M led to a strong inhibition of the auxin-induced elongation growth of colcoptile segments, while fusicoccin-enhanced growth was not affected. Growth inhibition occurred only if NEM and auxin were allowed to act simultaneously. Preincubation of plant segments with NEM in the absence of auxin caused no inhibition of a subsequent growth stimulation by auxin, whenever NEM was removed before the application of IAA. However, preincubation with NEM plus auxin led to a remaining growth inhibition, which could not be reversed by a second auxin incubation in the absence of NEM. Fusicoccin added to NEM- plus auxin-treated segments was able to restore growth. It is suggested that auxin causes the unmasking of essential SH-groups of a protein to which NEM links covalently. thus inhibiting the growth process. This assumption was further supported by labeling experiments wish [¹⁴C]-NEM using membranes of maize ( Zea mays L. cv. Inraplus) coleoptiles. Two membrane fractions (S₂= 480-1900 g; S₄= 4300-15000 g) revealed a significantly higher [¹⁴C]-NEM labeling in the presence of auxin (2,4-diehlorophe-noxyacctic acid compared to 2,6 dichlorophenoxyacetic acid). This effect disappeared when the membranes were previously washed with EGTA [ethyleneglycolbis-(β-aminoethylether)-N,N,Nr',N'-tetraacetic acid]. The auxin-induced sensitization of coleoptilc segments against thiol-reagents and the auxin-induced expression of SH-groups of proteins of isolated membranes from coleoptiles arc suggested to be events involved in the primary action of auxins.     

Internode length in Pisum. Estimation of GA1 levels in genotypes Le, le and led

The levels of GA₁, 3-epiGA₁ and GA₈ in genotypes Le, le and le^d of Pisum sativum L. were determined by gas chromatography-selected ion monitoring (GC-SIM) after feeds of [³H, ¹³C]-GA₂₀ to each genotype. The levels of endogenous and [¹³C]-labelled metabolites were determined by reverse isotope dilution with unlabelled GA₁, 3-epiGA₁ and GA₈. The results demonstrate a quantitative relationship between the level of GA₁ and the extent of elongation both on a per plant and a per g fresh weight basis. These results are consistent with previous findings in peas and other species possessing a predominant early 13-hydroxylation pathway for GA biosynthesis.
The levels of 3-epiGA₁ also decreased in the genotypic sequence Le, le, le^d although not as rapidly as for the level of GA₁. This may suggest that the alleles at the le locus also influence the formation of 3-epiGA₁.     

Internode length in Lathyrus odoratus. Effects of mutants l and lb on gibberellin metabolism and levels

After the application of [¹³C³H]-gibberellin A₂₀ to wild-type (tall) sweet peas ( Lathyrus odoratus L.) labelled gibberellin A₁ (GA₁), GA₈, GA₂₉ and 2-epiGA₂₉ were identified as major metabolities by gas chromatography-mass spectrometry after high performance liquid chromatography. By contrast in genetically comparable dwarf ( II ) plants only labelled GA₂₉ and 2-epiGA₂₉ were produced in significant amounts, although evidence was obtained for trace amounts of labelled GA₁ and GA₈. The apical portions of dwarf plants contained 8–10 times less GA₁ than those of tall plants but at least as much GA₂₀ (measured using di-deuterated internal standards). In conjunction with previous data these results strongly indicate that in genotype ll internode length is reduced and leaf growth altered by a reduction in GA1 levels attributable to a partial block in the 3β-hydroxylation of GA₂₀ to GA₁.
In contrast to dwarf plants, semidwarf plants (genotype lblb ) contained more GA₁ in the apical portion than wild-type counterparts. This is consistent with the suggestion that lb alters some aspect of GA sensitivity.     

An acylcyclohexadione retardant inhibits gibberellin A1 metabolism, thereby nullifying phytochrome-modulation of cowpea epicotyl explants

The regulation by phytochrome of stem elongation in light-grown plants depends on gibberellins (GAs). To investigate whether this is mediated by a change in GA metabolism, the effect of the GA biosynthesis inhibitor LAB 198 999 (an acylcyclohexadione derivative) on the end-of-day far-red (FR) response in cowpea ( Vigna sinensis L.) epicotyl explants has been investigated. Growth of epicotyl explants of light-grown seedlings was enhanced when treated with far-red light before incubation in the dark (end-of-day FR effect). Low doses of LAB 198 999 (0.05 and 0.5 μg explant⁻¹) reduced the effect of FR, whereas 5 to 50 μg explant⁻¹ stimulated elongation of both red light (R)- and FR-treated epicotyl explants while nullifying the differences between R and FR treatments. In paclobutrazol-treated epicotyl explants, FR enhanced the response to applied GA₁ and GA₂₀, whereas LAB 198 999 increased the activity of GA₁ and decreased that of GA₂₀, [³H]Gibberellin A₁, injected into the basal part of the epicotyl, was transported and metabolized mainly to [³H]GA₈ in the apical 20 mm of the epicotyl. The conversion of [³H]GA₁ to [³H]GA₈ was dramatically reduced by both end-of-day FR treatments and LAB 198 999 applications. In addition, both treatments enhanced epicotyl elongation. It is proposed that the regulation of cowpea epicotyl growth by phytocrome is mediated, at least partially, by modifying GA₁ degradation.     

Growth and development of Brassica genotypes differing in endogenous gibberellin content. II. Gibberellin content, growth analyses and cell size

Three rapid cycling Brassica rapa genotypes were grown in greenhouse conditions to investigate the possible relationships between endogenous gibberellin (GA) content and shoot growth. Endogenous GA₁ GA₃ and GA₂₀ were extracted from stem samples harvested at 3 weekly intervals and analyzed by gas chromatography-mass spectrometry with selected ion monitoring, using [²H₂]-GA₁ and [²H₂]-GA₂₀ as quantitative internal standards. During the first 2 weeks, GA levels of the dwarf, rosette ( ros ), averaged 36% of levels in normal plants (on a per stem basis). Levels in the tall mutant, elongated internode (ein) , were consistently higher, averaging 305% of levels in normal plants.
Differences in shoot height across the genotypes resulted from varying internode length which resulted from epidermal cell length and number being increased in ein and decreased in ros relative to the normal genotype. The exogenous application of GA₃ to normal plants increased cell length while the application of paclobutrazol (PP333), a triazole plant growth retardant, reduced cell size. Thus, exogenous GA manipulations mimicked the influence of the mutant genes ros and ein. The dwarf, ros , had reduced shoot dry weights and relative growth rates compared to the other genotypes. Total dry weights were similar in ein and the normal genotype but stem weights were increased in ein , compensating for decreased leaf weights. Thus, the gibberellin-deficiency of ros resulted in generally reduced shoot growth. The overproduction of endogenous GA by ein did not result in enhanced shoot growth but rather a specific enhancement of internode elongation and stem growth at the expense of leaf size.     

EFFECTS OF THYROXINE AND PROLACTIN ON THE RATES OF PROTEIN SYNTHESIS IN THE THIGH BONES OF THE TADPOLE OF RANA CATESBEIANA

In thigh bones isolated from a Rana catesbeiana tadpole which has been kept in a 5 × 10⁻⁸ M thyroxine solution for several days, the rate of ¹⁴C-leucine incorporation into protein becomes higher than that in the thigh bones of control animals. Intraperitoneal injection of prolactin also results in an increase in the rate of ¹⁴C-leucine incorporation into protein in the thigh bones at a rate very similar to that in thyroxine-treated animals. In the thigh bones of the thyroxine-treated tadpoles, the rate of ¹⁴C-proline incorporation into protein is markedly higher than that of control animals. Prolactin treatment of the tadpoles also causes an increase in the rate of ¹⁴C-proline incorporation, but the rate is lower than that found in thyroxine-treated animals. The injection of prolactin into thyroxine-treated tadpoles fails to cause further increase in the rates of incorporation of these amino acids into protein. In the thigh bones of tadpoles at the climax of metamorphosis, prolactin injection does not cause any increase in the rates of ¹⁴C-labeled proline and leucine incorporation, whereas both rates become slightly higher in the thigh bones of thyroxine-treated tadpoles at this stage. The thigh bones probably become insensitive to prolactin when they are exposed to thyroxine.     

Arachidonic Acid Turnover and Phospholipase A2 Activity in Neuronal Growth Cones

Abstract: We analyzed de novo synthesis and local turnover of phospholipids in the growing neuron and the isolated nerve growth cone. The metabolism of phosphatidylinositol (PI) was studied with regard to the incorporation of saturated and unsaturated fatty acids and inositol. A comparison of de novo phospholipid synthesis in the intact neuron (whole brain, cell cultures) versus local turnover in isolated growth cone particles (GCPs) from fetal rat brain revealed different incorporation patterns and, in particular, high arachidonic acid (AA) turnover in PI of GCPs. These observations, together with elevated levels of free AA (2.5% of total AA content) in GCPs, demonstrate the predominance of acylation/deacylation in the sn -2 position of PI. GCP phospholipase A₂ (PLA₂) activity was demonstrated using [^3H]-or [¹⁴C]AA-phosphatidylcholine (PC) or -PI as the substrate in vitro and GCPs or a cytosolic GCP extract as the source of enzyme. In contrast to PC, which is hydrolyzed very slowly, PI is a very good GCP PLA₂ substrate. PLA₂ activity is much higher in GCPs than that of phospholipase C, as demonstrated by the comparison of AA and inositol turnover, by the low levels of 1,2-diacylglycerol generated by GCPs, and by the resistance of AA release to treatment of GCPs with RHC-80267, a specific inhibitor of diacylglycerol lipase. The predominance of PLA₂ activity in GCPs raises questions regarding its regulation and the functional roles of PI metabolites, especially lysocompounds, in growth cones.     

Hormonal control of cambial activity and vessel differentiation in Quercus robur

Cambial activity and vessel differentiation of the Quercus robur stem were investigated in relation to concentration of growth regulators and sucrose, seasonal changes in the sensitivity of cambial cells, and axial polarity of the stem. Basipetal efflux of natural auxin was measured in the oak stem cambial region. IAA, GA₃, kinetin and sucrose affected cambial activity and/or initiation of vessel differentiation differently, depending upon concentration. Depending upon the season, kinetin increased or reduced the stimulation of cambial activity caused by IAA and GA₃, but it did not affect the differentiation of vessels. Supply of sucrose in higher concentrations reduced the number of differentiated vessels but did not decrease the stimulation of cambial divisions.Unlike stimulation of cambial activity by GA₃, auxin stimulation of cambial divisions and differentiation of vessels were highly dependent upon stem polarity, 2,3,5-triiodobenzoic acid (TIBA) inhibited formation of vessels, but not cambial activity. The oscillations in basipetal efflux of natural auxin from the cambial stem region of successive 6 mm long sections substantiate the hypothesis that the histogenesis of xylem tissue in ring-porous species is under control of the vectoriat field that is associated with oscillatory phenomena in polar auxin transport.     

Vitamin D3 affects growth and Ca2+ uptake by Phaseolus vulgaris roots cultured in vitro

Vitamin D₃ at low concentration (10⁻⁹ M) inhibited the growth of Phaseolus vulgaris L. (cv. Contrancha) roots in vitro as measured by elongation (14 h) and [³H]-leucine incorporation into protein (2 h), and increased their labelling with ⁴⁵Ca²⁺ (2 h). Cycloheximide and puromycin (50 u.M) blocked vitamin D₃ stimulation of root ⁴⁵Ca²⁺ labelling, indicating that it is mediated by de novo protein synthesis. The calcium ionophore X-537A (10⁻⁵JW) induced similar changes both in root elongation and ⁴⁵Ca²⁺ uptake (14 h). This may indicate that the inhibitory effects of the sterol on root growth are mediated by changes in Ca²⁺ fluxes. However, this interpretation should be further strengthened by additional studies as the ionophore may have acted on root growth, affecting physiological processes other than Ca²⁺ transport.     

Accumulation of a 31-kDa glycoprotein in association with the expression of embryogenic potential by spinach callus in culture

Calli grown from segments of spinach ( Spinacia oleracea L.) root in the presence of gibberellic acid (GA₃) plus auxin, differentiated to yield somatic embryos after transfer to a medium without growth regulators, while calli formed in the absence of GA₃ failed to generate any embryos. We extracted proteins from the two types of callus and analysed them by polyacrylamide gel electrophoresis. Compared with the proteins from calli formed on medium that contained only naphthaleneacetic acid (NAA) as a growth regulator, the proteins from calli grown in the presence of GA₃ included appreciably higher levels of a 31-kDa basic protein (pI = 8.8). The protein resembled type I ribosome-inactivating proteins (EC 3.2.2.22) in terms of molecular mass, isoelectric point, sequence of amino-terminal amino acids and extent of glycosylation. The 31-kDa protein was barely detectable in extracts of various tissues from seedlings. Thus, it is possible that an increase in the relative level of this protein might be associated with the expression of embryogenic potential expressed by spinach callus.