The chemical stimulus essential for growth by increase in cell number

Summary The development of this study has been a succession of steps each of which rests on the preceding. It falls naturally into three distinct stages. The first is that ofIdentification Here it was found that the lead precipitate present in the meristematic region of root tips grown in Pb-containing culture solutions is a combination of lead with sulfhydryl. In such tips mitosis but not growth by increase in cell size is inhibited. Also it was found that sulfhydryl is concentrated in the meristematic region of normal roots. Therefore the hypothesis was developed that growth by increase in cell number is specifically factored by -SH. The next stage was theTesting of Extracts Here it was found that acid extracts of the meristem of root tips accelerated root length growth when controlled by acid extracts of the next distal portion, while alkaline extracts similarly controlled showed no such activity. This proved that the root region of highest sulfhydryl concentration and mitotic activity contains a naturally occurring acid-stable, alkali-labile substance stimulative of root growth in length. These findings are thus physiologically and chemically consistent with the hypothesis. The next stage was theTesting of Synthetic Compounds Here the action of a variety of sulfhydryl compounds on mitosis in root tips and reproduction rate in Paramecium was studied, using the same compounds minus the sulfur moiety as controls. It was found that the -SH group stimulates cell division in both plants and animals. Cell size growth is not stimulated. Thus, through identification and testing of the identified group in natural and synthetic compounds, the conclusion is arrived at that Sulfhydryl is the essential stimulus to growth by increase in cell number.Credit is due to MissElizabeth Justice and MissJane Anderson whose conscientious and painstaking efforts made this study possible.     

Protein reactions with methyl and ethyl vinyl sulfones

Disulfide bonds of bovine serum albumin and wool were reduced byn-tributylphosphine to sulfhydryl groups that were then modified by methyl or ethyl vinyl sulfone in a nucleophilic addition reaction toS-(-ethylsulfonylmethyl)-l-cysteine andS(-ethylsulfonylethyl)-l-cysteine, respectively. The reductive alkylation was carried out either simultaneously, with both the reducing and alkylating agents present in the reaction mixture, or sequentially, with the reduced proteins first isolated before alkylation. Amino acid analysis studies showed that authentic, syntheticS-(-ethylsulfonylethyl)-l-cysteine eluted as a well-resolved peak after serine but that the peak associated with the corresponding methyl derivative overlapped the corresponding peak due to threonine. The extent of alkylation of the sulfhydryl groups of cysteine, -NH₂ of lysine, and NH groups of the imidazole ring of histidine was also measured by amino acid analysis. The results show that alkyl vinyl sulfones have a strong chemical affinity for protein functional groups.     

Influence des parasites oophages sur la régulation des populations deCassida deflorata Suff

Summary The layings ofCassida deflorata Suffr. are parasited in southern France byMymaridae (Anaphoidea sp. andFulmekiella ovata soyka),Trichogrammatidae (Monorthochaeta nigra Blood.) andEulophidae (Foersterella flavipes Foerst. andTetrastichus rhosaces Walk.). In the Alpes-Maritimes, two successive generations ofMymaridae andTrichogrammatidae seem to attack the layings ofC. deflorata. In Corsica, parasitism byMymaridae andF. flavipes is very frequent and may be the essential regulating factor of the populations ofCassida. In the other regions that have been studied, although the large number ofCassida imagos appearing in spring show that mortality during the aestivation and hibernation is not the primary factor, it does not seem that the limitation of the populations is essentially caused by the activity of oophagous insects.      

Microorganisms of thePleuropneumonia group (family ofMycoplasmataceae) in man

Summary Sixty one strains of pleuropneumonia-like organisms (P.P.L.O.) have been isolated in man: 25 from the discharge of patients with urogenital disorders, 13 from women who were pregnant or who had borne a child 6 weeks previously. In women the frequency of positive cultures increased with the disappearance of Lactobacilli. Twenty three strains were cultivated from the buccal mucosa of 27 healthy subjects. Classification by means of cultural appearance and biochemical behaviour allowed of differentiation into 4 groups, three of which were in conformity with three species ofMycoplasma described byEdward andFreundt. The only difference as compared with this latter classification is that with our methodM. fermentans andM. salivarius regularl gave clearcut hemolysis, which was not found byEdward. Two urogenital strains which grew well on not specially enriched media could not be identified.     

The effect of 2-chloro, 6-(trichloromethyl) pyridine on the chemoautotrophic metabolism of nitrifying bacteria

Studies were conducted to elucidate the mechanism of action of 2-chloro-6-(trichloromethyl)pyridine or Technical N-SERVE on the nitrification process brought about byNitrosomonas europaea. The growth ofNitrosomonas was completely inhibited in the presence of 0.2 ppm N-SERVE while 1.0 ppm of the chemical was effective in the complete inhibition of ammonia oxidation by fresh cell suspensions. Cells stored at 4 C for a period of three days required somewhat higher concentrations (1.5 ppm) of N-SERVE for the complete inhibition of their ammonia oxidizing ability while the cytochrome oxidase of these cells was inhibited to the extent of 65 to 70 percent in the presence of a corresponding amount of N-SERVE. A 45 – 70 percent reversal of the inhibition of ammonia oxidation caused by N-SERVE was obtained by the addition of 6×10^–4 M Cu⁺⁺. An equivalent concentration of Cu⁺⁺ was also effective for the complete reversal of the inhibition of cytochrome oxidase present in whole cells.Hydroxylamine oxidation by intactNitrosomonas cells was not affected by levels of N-SERVE ranging from 1 – 3 ppm. The cytochrome oxidase effective in hydroxylamine oxidation and present in cell-free extracts was not inhibited by even 100 ppm N-SERVE. Likewise, the hydroxylamine activating enzyme hydroxylamine cytochromec reductase was also not inhibited by such levels of the chemical. Raising the concentration to 170 ppm N-SERVE, however, caused a 90 percent inhibition of the enzyme.Although a 5×10^–6 M concentration of allylthiourea completely inhibited ammonia oxidation byNitrosomonas cells, concentrations up to 10^–3 M of this compound did not affect the cytochrome oxidase activity of whole cells or cell-free extracts. The inhibition of ammonia oxidation caused by 5×10^–6 M allythiourea, unlike the inhibition by N-SERVE, could not be reversed by the addition of 6×10^–4 M Cu⁺⁺.Evidence is presented that the action of N-SERVE is on that component of cytochrome oxidase which is involved in ammonia oxidation.     

Characterization of raffinose synthase from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L. var. Nipponbare)

The putative raffinose synthase gene from rice was cloned and expressed in Escherichia coli. The enzyme displayed an optimum activity at 45°C and pH 7.0, and a sulfhydryl group was required for its activity. The enzyme was specific for galactinol and p-nitrophenyl-α-d-galactoside as galactosyl donors, and sucrose, lactose, 4−β-galactobiose, N-acetyl-d-lactosamine, trehalose and lacto-N-biose were recognized as galactosyl acceptors.     

A new genus of theActinoplanaceae: Dactylosporangium,gen. nov.

Summary Two aerobic mesophilic species of a new genus belonging to the familyActinoplanaceae are described under the nameDactylosporangium (D. aurantiacum strainD/748 type species andD. thailandensis strainD/449). The new genus is characterized by the production of finger shaped sporangia emerging directly from the vegetative mycelium.The motile sporangiospores, three to four in number are arranged in a single straight row inside the sporangium.The genusActinoplanes of the familyActinoplanaceae was described in 1950 byCouch and is characterized by the bacteria-like, flagellated spores formed in sporangia. Other members of the familyActinoplanaceae have been studied byKarling (1954),Rothwell (1957) andCross et al. (1963) in the United States, byGaertner (1955) in Germany, byVan Brummelen andWent (1957) in Holland, byNonomura andOhara (1960) in Japan, byTaig et al. (1962),Tsyganov et al. (1963), andKoniev et al. (1965) in Russia. Except for the organisms studied byKarling and byRothwell, which undoubtedly belonged to theActinoplanes but were not studied in pure culture, the organisms studied by most of the other authors belonged to the genusStreptosporangium.Three new genera having motile spores were described more recently:Ampullariella andSpirillospora described byCouch (1963, 1964), andPlanomonospora byThiemann et al. (1967b).     

Regulation of aspartokinase activity in non-fermentative,marine eubacteria

Summary Cell-free extracts of gram-negative, non-fermentative, marine eubacteria were assayed for aspartokinase activity. The organisms tested included polarly flagellated species and groups which had GC contents in their DNAs of 46 to 64 moles % (Alteromonas, Pseudomonas) as well as species which had peritrichous flagellation and moles % GC contents of 53 to 68 (Alcaligenes). The results of these studies suggested that in all the strains tested, aspartokinase activity was catalyzed by a single enzyme. On the basis of the effect ofl-threonine,l-lysine,l-methionine, andl-isoleucine on activity, five different types of aspartokinases (designated I through V) were delineated. In aspartokinase types I through IV,l-threonine andl-lysine inhibited activity by means of a concerted feedback inhibition; in type V, activity was inhibited byl-threonine but unaffected byl-lysine. In types I, III, and IV,l-threonine andl-lysine alone were inhibitory, while in type II these effectors had virtually no effect on activity when tested singly. Three distinct responses were observed in the presence of two other end products of the aspartate pathway,l-methionine andl-isoleucine. In types I and II, these two amino acids usually stimulated activity and overcame the inhibition byl-threonine andl-lysine; in types IV and V,l-methionine andl-isoleucine had no effect; and in type III these amino acids inhibited activity. The results of this study indicate that the aspartokinases of a number of species and groups of marine bacteria have similarities and differences which should be of use in making future taxonomic groupings.     

Purification and characterization of dihydroorotase fromPseudomonas putida

Dihydroorotase was purified to homogeneity fromPseudomonas putida. The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative molecular mass of 41 kDa. The enzyme only hydrolyzed dihydro-l-orotate and its methyl ester, and the reactions were reversible. The apparentK _m andV _max values for dihydro-l-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 μmol min⁻¹ mg⁻¹, respectively; and those forN-carbamoyl-dl-aspartate (at pH 6.0) were 2.2 mM and 68 μmol min⁻¹ mg⁻¹, respectively. The enzyme was inhibited by metal ion chelators and activated by Zn²⁺. However, excessive Zn²⁺ was inhibitory. The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited byN-carbamoylamino acids such asN-carbamoylglycine, with aK _i value of 2.7 mM. The enzyme was also inhibited noncompetitively by pyrimidine-metabolism intermediates such as dihydrouracil and orotate, with aK _i value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays a role in the control of pyrimidine biosynthesis.     

Facultative parasites and host respiration. I. Healthy tissue adjacent to the site of infection

Summary The respiration, as measured by oxygen uptake, was higher for healthy tissue adjacent to soft rots of apple (var. Bramley) caused byPenicillium expansum Link andThom. andBotrytis cinerea Fr. and of potato tuber (var. Arran Banner) caused byErwinia aroideae Townsend, than tissue at a more distant site. The respiration of tissue adjacent to a physiological rot caused by bruising was not affected. It was concluded that there is diffusion of a substance(s) from the site of infection which causes an increase in the rate of respiration of the adjacent tissue.This work formed part of a Ph. D. thesis submitted to the University of London, July 1961.     

Regulation of homoserine dehydrogenase inAzotobacter species

The regulation of homoserine dehydrogenase activity was studied in nineAzotobacter strains belonging to five different species. In all the species the enzyme is subject to feedback inhibition byl-threonine andl-isoleucine, the first being much more active as inhibitor. The inhibition byl-threonine is noncompetitive with respect to NADPH and of mixed type with respect to aspartate-Β-semialdehyde; the inhibition byl-isoleucine is noncompetitive with respect to both substrates. The synthesis of homoserine dehydrogenase inAzotobacter chroococcum I.P. is somewhat repressed by 1mm l-methionine and 5mm l-isoleucine. In all the strains examined either NADPH or NADH can serve as cofactors for this activity, though the ratio of activity with the two pyridine nucleotides (NADPH/NADH) shows higher values (3.3–3.8) in the speciesmacrocytogenes andinsignis than in thechroococcum, beijerinckii andvinelandii group (1.5–1.6). The pattern of control of this enzyme in the genusAzotobacter is discussed in relation to other bacterial homoserine dehydrogenases. We are grateful to Dr. G. N. Cohen, Service de Physiologie Microbienne, Institut Pasteur, Paris, for helpful discussions and encouragements.     

Dominance of iminopeptidase activity in the human oral bacteriumTreponema denticola ATCC 35405

Treponema denticola ATCC 35405, a human oral spirochete associated with periodontal disease, was shown to contain three enzymes (I, II, and III) with proline iminopeptidase activity. II and III were considered to be true iminopeptidases, whereas enzyme I was found to be a benzoylarginine peptidase with iminopeptidase activity. Enzyme III, the dominant proline iminopeptidase ofT. denticola in terms of its activity towardN-l-prolyl-2-naphthylamine, was considered to be a sulfhydryl peptidase: 0.167 M p-chloromercuribenzoic acid totally inactivated the enzyme, and 1.0 mM dithiothreitol restored 92% of activity. The activity of this enzyme was not affected by metal chelators. Chemical modification of enzyme III suggests that tyrosyl (or histidyl) and carboxyl groups may be necessary for its activity. The hydrolysis ofN-l-prolyl-2-naphthylamine was found to be very characteristic ofT. denticola ATCC 35405; out of 24 differentN-l-aminoacyl-2-naphthylamines tested, only the proline derivative was hydrolyzed at a high rate. The substrate specificity of the enzymes discovered indicates that they may be important for the nutrition ofT. denticola. The iminopeptidase activity may be related to the pathogenicity of this organism in periodontal disease.     

Cytochemical demonstration of increased adenosine triphosphatase activity in lymphocytes activated in vitro by phytohaemagglutinin or by the mixed lymphocyte reaction

Summary Cytochemical investigations of ATPase activity were performed on lymphocytes isolated from peripheral blood and activatedin vitro by phytohaemagglutinin or by the two-way mixed lymphocyte reaction. Uncultured lymphocytes showed very little activity localized in small granules. The activity increased markedly during transformation. In fully transformed and actively proliferating cells, the ATPase activity was intense and localized in a crescentic perinuclear area of cytoplasm which was pale-staining and vesicular in Giemsa-stained preparations. In mitotic cells, the activity was in discrete granules or elongated structures suggestive of mitochondria, scattered throughout the cytoplasm. The ATPase activity had a pH optimum of 8.5 to 9.5 and was strongly inhibited at pH 7.5. The activity was stimulated by Ca²⁺ and Mg²⁺ and was inhibited byp-chloromercuribenzoate but not by oligomycin, which appeared to enhance the reaction. Lead nitrate at a concentration of 3mm did not inhibit the reaction.     

Purification and partial characterization ofd-(−)-lactate dehydrogenase fromLactobacillus helveticus CNRZ 32

Summary d-(–)-Lactate dehydrogenase (LDH) was purified to homogeneity from a cell-free extract ofLactobacillus helveticus CNRZ 32. The native enzyme was determined to have a molecular weight of 152 000 and consisted of four identical subunits of 38 000. This enzyme was NAD dependent fructose 1,6-diphosphate (FDP) and ATP independent. It was most active on pyruvate followed by -hydroxypyruvate as substrates. TheK _m values for pyruvate andd-(–)-lactate were 0.64 and 68.42 mM respectively, indicating that the enzyme has a higher affinity for pyruvate. The enzyme activity was completely inhibited byp-chloromercuribenzoate (1 mM) and partially by iodoacetate, suggesting the involvement of the sulfhydryl group (-SH) in catalysis. Optima for activity by the purified enzyme were pH 4.0 and 50–60°C. Limited inhibition ofd-(–)-LDH was observed with several divalent cations. Additionally, HgCl₂ was observed to strongly inhibit enzyme activity. The purified enzyme was not affected by dithiothreitol or any of the metal chelating agents examined.     

Purification and some properties of the extracellular protease ofBacillus pumilus

Supernatant of a culture ofBacillus pumilus D 78 was precipitated with ethanol and chromatographed on DEAE- and CM-cellulose to isolate and purify a neutral protease with fibrinolytic and caseinolytic activity. Analysis by ultracentrifugation and immunoelectrophoresis indicate the homogeneity of the purified enzyme with the sedimentation constant s_20,w equal to 2.3. The fibrinolytic activity had a lower heat stability and was also more sensitive to pH higher than 8.0. The caseinolytic activity was stable over a wide range of pH (4.5 to 11.0). The enzyme binds acid dyes and is inhibited by Cu²⁺, Zn²⁺, Ca²⁺ and Fe³⁺, as well as byL-cysteine and KCN at a concentration of 10mM. Likewise, EDTA andp-chloromercuribenzoate show an inhibitory effect.     

Effects of chemical modification of amino and sulfhydryl groups on KATP channel function and sulfonylurea binding in CRI-G1 insulin-secreting cells

The effects of several group-specific chemical reagents were examined upon the activity of the ATP-sensitive potassium (K_ATP) channel in the CRI-G1 insulin-secreting cell line. Agents which interact with the sulfhydryl moiety (including 1 mM N-ethylmaleimide (NEM), 1 mM 5,5-dithio-bis-(2-nitrobenzoic acid) (DNTB) and 1 mm o-iodobenzoate) produced an irreversible inhibition of K_ATP channel activity when applied to the intracellular surface of excised inside-out patches. This inhibition was substantially reduced when attempts were made to eliminate Mg²⁺ from the intracellular compartment. ATP 50 m and 100 m tolbutamide were each shown to protect against the effects of these reagents. The membrane impermeable DNTB was significantly less effective when applied to the external surface of outside-out patches. Agents which interact with peptide terminal amine groups and amino groups of lysine [1 mm methyl acetimidate and 1 mm trinitrobenzene sulfonic acid (TNBS)] and also the guanido group of arginine (1 mm methyl glyoxal) produced a Mg²⁺-dependent irreversible inhibition of K_ATP channel activity which could be prevented by ATP but not tolbutamide. The irreversible activation of the K_ATP channel produced by the proteolytic enzyme trypsin was prevented only when methyl glyoxal and methyl acetimidate were used in combination to inhibit channel activity. Radioligand binding studies showed that the binding of ³H glibenclamide was unaffected by any of the above agents with the exception of TNBS which completely inhibited binding with a EC₅₀ of 307 ±6 m.These results provide evidence for the presence of essential sulfhydryl (possibly cysteine), and basic amino acid (possibly lysine and arginine) residues associated with the normal functioning of the K_ATP channel. Furthermore, we believe that the sulfhydryl group in question is situated at the internal surface of the membrane, possibly near to the channel pore.K.L. is a Wellcome Prize Student. This work was supported by the Wellcome Trust, MRC and BDA.     

Restricted cell/cell communication in the shoot apex ofSilene coeli-rosa during the transition to flowering is associated with a high mitotic index rather than with evocation

Summary Plants ofSilene coeli-rosa given 5 or more long days (LDs) flowered, even when the LDs were followed by 48 hours of darkness before their return to short days (SDs). The mitotic indices of shoot apices from induced plants shortly after induction were significantly higher than the indices of shoot apices from vegetative plants. Two major mitotic peaks were observed in the shoot apices of plants given 7 long days (LDs) on day 8. One coincided with that reported byFrancis andLyndon (1979).Cell to cell movement was tested in the shoot apices of vegetative and LD treated plants using probes with a molecular size of 749 daltons (fluorescein-hexaglycine) and 847 daltons (fluorescein-leucyl diglutamyl leucine). These probes showed some movement in the shoot apices of both short day (SD) and LD treated plants, but fluorescein-leucyl diglutamyl leucine was immobile in the induced apices of 7 LD plants on day 8 at time intervals which coincided with major mitotic activity in the shoot apex. Symplasmic restriction in the shoot apex was also observed in plants given 8 LDs (i.e., plants not returned to SDs on day 7).In plants that were placed in 48 hours of darkness after the 7 LD treatment or in plants given 5 LDs, there was no strong peak in the mitotic index, even though all these LD treatments resulted in 100% flowering. In such plants no symplasmic restriction was found in the shoot. Thus the symplasmic restriction on day 8 of 7 LD plants is associated with the high mitotic index, but neither of these phenomena is an essential part of the evocation process.Abbreviations F(Glu)₂ L-glutamylglutamic acid conjugated to fluorescein isothiocyanate isomer I (F-) - F(Gly)₆ F-hexaglycine - FLGGL F-leucyl-diglutamyl-leucine - F(PPG)₅ F-the pentamer (propyl-propyl glycine) - LD long day - LDs long days - SD short day - SDs short days     

Interchromosomal effects of inversions on crossover rate in maize

Summary The influence of heterozygous inversions on heterologous chromosome recombination has been studied in maize.Inv 2a causes an increase in crossing-over rate ofYg-Sh region of chromosome9: the reverse is true for the more proximal regionBz-Wx. The coincidence indexes for the traitYg-Sh-Bz is decreased, while the same index is increased for the regionSh-Bz-Wz. Inv 9a is accompanied by an high recombination rate in the distal region of chromsome2 Lg-Gl, while in the regionGl-V the effect is relatively less pronounced.The results seem to exclude mitotic recombination events, but are less suitable for supporting other specific interpretations. However, taking into considerationDrosophila experiments, our data are not conflicting with the physiological hypothesis proposed byRamel, which, at present, appears to explain most of the experimental results, although some difficulty arises in considering the cases of decreased recombination rate.The possibility of modifying, by genetical means, the recombination potential is of significance in some stage of plant breeding, at least in some species.With 2 Figures in the Text     

Proline production via the arginine biosynthetic pathway: transfer of regulatory mutations of arginine biosynthesis into a proline-producing strain ofSerratia marcescens

Summary Proline production via a part of the arginine biosynthetic pathway was examined. About 20 mg/ml ofl-proline was produced by using arginine biosynthetic enzymes. Accordingly, three mutations of arginine biosynthesis, namely, derepression of arginine biosynthetic enzymes (assigned byargR2), feedback inhibition-resistant N-acetylglutamate synthase (assigned byargA2) and defectiveness in N-acetylornithine aminotransferase (assigned byargD ^–) were introduced by three transductional crosses into a proline-producing strain which produced about 55 mg/ml ofl-proline. The constructed strain produced 62 mg/ml ofl-proline, although about 10 mg/ml ofl-arginine and 1 mg/ml of N-acetylglutamate--semialdehyde were produced as by-products.     

Saccharomyces Marxianus Hansen

III Conclusion and Summary Zygosaccharomyces Marxianus andSaccharomyces macedoniensis belong to the same species. This species is met with in the haplophase (Z. Marxianus) as well as in the diplophase (S. macedoniensis). It was possible to bring this yeast from the haplophase into the diplophase and vice versa. By keeping this yeast during long times on maltagar it showed a tendency to change from the haplophase into the diplophase, but not into the opposite direction.It seems quite possible thatHansen, who did not describe a conjugation in this yeast, had met with the diplophase.It has been once more emphasized — at whichWinge andLaustsen and alsoLindegren andLindegren have pointed —, that the genusZygosaccharomyces is no valid genus.The yeast studied here belongs to the genusSaccharomyces and must be designated with the original name given to it byHansen:Saccharomyces Marxianus.For the sake of completeness it is mentioned here that also an imperfect stage ofS. Marxianus has been describedviz., Candida macedoniensis (A. Castellani) Berkhout (I).Saccharomyces fragrans Beijerinck has to be considered as its synonym.