On the media improving freeze-sectioning of plant material

Using a semiconductor freezing microtome, there is no difficulty in getting an ample supply of quite suitable sections of Ca formol fixed root tips ofVicia faba, applying Holt’s sirup or plain 0.88 M sucrose as media for keeping the material, getting the sections and treating them after sectioning, but the protoplasts shrink and often fall out from the cells. No improvement in this respect was revealed using — at different concentrations — sucrose and arabic gum alone. The changes in the proportions of the ingredients of the sirup were also ineffective. A series of experiments were therefore performed with plain sucrose, but none of the factors tested was efficient enough to improve the results as compared with the standard procedure, the gradual transfer being the only exception. Since this makes the procedure more laborious and time consuming, certain other media were tested, 0.88 M ethanol and 0.88 M dimethylsulf-oxide yielding the best results. Attempts are being made to understand some points of the present experience in connection with the results of other authors and on the basis of general theory respectively.     

On the interaction of growth retardants with IAA and kinetin

In the pea test a highly positive response to the treatment with IAA reversed to a negative one or became 5 to 6 times weaker when CCC was applied together with IAA. In cultivating pea seedlings, following their decapitation, for two days in a 0.25 per cent CCC solution and then in water, growth of their cotyledonous axillaries (cotylaries) were inhibited. This inhibitive action of CCC could be made ineffective when the seedlings, following two-days’ cultivation in the CCC solution, were grown further in kinetin solutions (0.37–3 mg per 1). Cotylaries of decapitated pea seedlings, when grown in kinetin solutions were inhibited. With kinetin solutions of 6–12 mg/l a strong inhibition also occured in the growth of roots at the apical parts of which spherical swellings were developing. The CCC supplied to the roots of intact etiolated pea seedlings is translocated acropetally into the stem at a rate of about 5 cm per hour. Decapitation of the plant causes retardation of this transport, yet a coat of 0.00001–1% IAA or kinetin paste produces acceleration of the stream. Existence of an antagonism between CCC and IAA, demonstrated earlier, was found holding true also for B-9 (N, N-dimethyl-aminesuccinamic acid) and IAA, as the inhibitive action of B-9, 0.06% solution on the growth of lettuce hypocotyls was reduced to a highly significant degree when the plants were supplied with B-9 together with IAA at a concentration of 10 mg/l.     

Antibacterially active substances

Synthesis of two hydroxy-derivatives of nalidixic acid as a result of microbial transformation was demonstrated in certain species of the genusAspergillus. Aspergillus alliaceus produced 7-hydroxy-nalidixic acid andAspergillus niger 6-hydroxy-nalidixic acid. It was demonstrated that the antibacterial activity of both hydroxy-derivatives (tested inEscherichia coli) was lower than that of the initial nalidixis acid.     

The inactivation ofBacillus subtilis transforming DNA by mutagenic agents

The inactivation of three genetic markers inBacillus subtilis transforming DNA by UV rays, nitrous acid and hydroxylamine was studied. The inactivation of markers by UV and HNO₂ was marker specific, whereas the hydroxylamine inactivates the markers approximately to the same degree.     

Untersuchung der eiweißspindeln des kakteen-X-Virus mit fluoreszierenden Antikörpern

A?koliv na zá kladě mnoha pokus? se p? edpokládalo, ?e tzv. bÍlkovinná v?etena v buňkách tzn. buně?né inkluse X-viru kaktus? (Ca XV), jsou slo?ena z ?etních prodlou?ených ?ásti Ca XV, p?esto to dosud nebylo proká zá no. Proto jsme se pokusili pomocÍ fluoreskujÍcÍch protilátek doká ?at, ?e bilkovinná v?etena jsou skute?ně agregáty virových ?ástic. V těto práci jsme pouzili tzv. nep?Ímé metody. Nejprve jsme p? sobili na buňky obsahujÍci tato v?etena homologiokým antisé rem proti Ca XV, zÍskanym imunizacÍ králÍk? a teprve potom jsme buňky vlo?ili do roztoku fluoreskujicÍch protilátek proti králicimu γglobulinu. BÍlkovinná v?etena svitila potom ve fluorescen?nÍm mikroskopu silně ?lutozeleně (bylopou?ito fluoresceinisothiocyaná tu). Tato fluorescence ná m uká zala, ?e nastala pozitivnÍ reakce a ?e bÍlkovianá v?etena jsou slo?ena z virových ?ástic. ?etné kontrolnÍ pokusy potvrdily ná? základnÍ pokus.     

Factors affecting the accuracy of chlorophylla andb determination by means of their paper chromatographic separation and colorimetric measurement in eluates

Quantitative determination of chlorophyll a and β can be made by paper chromatography of acetone extracts of plant material with colorimetric measurement of the eluates from the separated zones. From the suitable solvent systems which give adequate separation of the pigments at a distance of 20 cm. from the start,Hager's mixture (1955) separates the chlorophylls better than the toluene-isopropanol (400: 1 v/v.) mixture, which, however, is better for the separation of carotenoids. Twice the amount of chlorophyll is separated on Whatman 31 ET paper, equally well and with the same time of development, as on Whatman No. 3 paper, on which it is possible to separate a maximum of about 15 μg of chlorophyll pigments per 1 em. start length. Losses on elution are, however, higher on using Whatman 31 ET paper. In plants with a high chlorophyllase activity, the error of determining chlorophyll a andb is greatly reduced if the leaves are placed for 1 min. in boiling water before extraction. For elution of chlorophylla andb from paper it is better to use anhydrous acetone, for chlorophyllides 80% acetone. A comparison of the procedure investigated with the method of two-wave length spectrophotometric measurement of crude acetone extracts showed that in view of the average 10% loss, the chromatographic method is hardly suitable for determining the absolute amounts of chlorophylla andb, although the relation (a/b) can be determined with similar precision by both methods. Moreover, in view of the greater amount of work involved the chromatographic method can only be recommended for confirming the results of spectrophotometrie determination. Quantitative determination of chlorophylls from the area of the spot or from the "R_F" value can only be of an informative character.     

Effect of paclobutrazol onin vitro formation of potato microtubers and their sprouting after storage

Paclobutrazol, the gibberellin biosynthesis inhibitor, acceleratedin vitro tuber initiation of potato cv. Rema and increased the uniformity of tuberization period. However, the high concentrations (10–1000 mg l^?1) of this retardant, strongly decreased mass and/or number of microtubers. The microtubers were harvested and stored in darkness (22±2°C) for 250 d. After this period both sprouting and growth of sprouts were affected by previous paclobutrazol treatment.     

Application of enzyme flow microcalorimetry to the study of microkinetic properties of immobilized biocatalyst

Summary Flow microcalorimeter was used for the study of microkinetic properties of Escherichia coli cells enriched with the penicillin G acylase activity immobilized in calcium pectate gel. The experimental kinetic data were obtained by measurement of the thermometric signal in the microcalorimetric column with immobilized enzyme and described by the introduced mathematical model involving the mass transfer and reaction kinetic phenomena.     

Drug resistance inYarrowia lipolytica

Six strains ofYarrowia lipolytica tested here were resistant to 10–20 g erythromycin or chloramphenicol per L glycerol-agar medium. Cells tolerating 4 g chloramphenicol per L were very rare and reverted rapidly to the highest resistance. In analogy with Ery^R mutants ofKluyveromyces lactis, our strains did not grow at 36°C but did not lose their viability at that temperature. Two levels of resistance were found with oligomycin and antimycin A,i.e. 10 and 3 mg/L in the former and 10 and 2 mg/L in the latter. The higher resistance levels segregated mitotically and were, therefore, controlled extrachromosomally. The lower resistance levels showed very frequent changes from sensitivity to resistance that prevented the genetic analysis of this resistance. An almost continuous range of tolerance to <5–400 μg mucidin per L was found in populations of the strains analyzed. Newly formed Muc^R cells were established only in the presence of the antibiotic. Pure cultures of Muc^R cells showed an extremely high instability caused by their lower viability and very low growth rate in the absence of mucidin. No loss of resistance to antimycin A was found, although Ant^R cells revealed similar negative selection. Mutability Muc^S»Muc^R and Muc^R»Muc^S was higher in Ant^R cells than in Ant^S ones.     

Apoptosis and nutrition: Involvement of amino acid transport system in repression of hybridoma cell death

Mouse hybridoma cells cultured on the verge of starvation-induced apoptosis, i.e. in a medium diluted with saline, proved to serve as a sensitive screening system for apoptosis-suppressing activity of nutrient medium components. Conventional amino acid mixtures were found to suppress the starvation-induced apoptosis, whereas a vitamin mixture was ineffective. (Frank F (1995) Biotechnol. Bioeng. 45: 86–90). Recent experiments showed that suppression of apoptosis, and concurrent resumption of growth, could be achieved by addition of single substances at millimolar concentrations. The set of active substances included certain coded L-amino acids (glycine, alanine, serine, threonine, proline, asparagine, glutamine, histidine), non-coded amino acids (-alanine, taurine, 4-aminobutyric acid), and a non-metabolizable analogue (2-aminoisobutyric acid). This finding shows that some amino acids do not act solely as nutrients, but also as specific signal molecules. The specificity of the effect points to the involvement of adaptively regulated amino acid transport systems A and N in maintaining the balance between triggering and suppression of starvation-induced apoptosis.