Band 38, Heft 1-2

A more detailed description o f the small-angle-X-ray camera without slithigh-error is given together with a calculation o f the beam condition and its influence on different scattering functions. Also the proceeding to measure molecular weights is described and some scattering curves are figured as an example of the equipments efficiency.

The yeast Candida boidinii ATCC 32195 produces a cell-bound red pigment during growth on 1% ethanol in the presence of 0.05% monofluoracetate. Its isolation and purification are described. The structure was elucidated by chemical degradation and physical methods, mainly mass and nuclear magnetic resonance spectroscopy.

From methanolic extracts of dried leaves of Vitis vinifera 3 ellagitannins were isolated: brevilagin 1, 1,3-digalloyl-4,6-dehydrohexahydroxydiphenoylglucose (=vitilagin), 3,4-digalloyl-1,6-de- hydrohexahydroxydiphenoylglucose (= iso-vitilagin).

The structure of 2-methoxystypandrone, a new naphthoquinone from the stem bark of Rhamnus fallax B., was elucidated by UV, IR, 1 H-NMR, 13 C-NMR, MS and chemical degradation as 6-Acetyl-5-hydroxy-2-methoxy-7-methyl-1,4-naphthoquinone. This is the first described occurrence of a naphthoquinone in a Rhamnus species. Free alaternin, 2-hydroxyemodin, was found for the second time in plant material.

A cell-free system from developing seeds of red radish (Raphanus sativus L. var. sativus) catalyzed the transfer of the sinapoyl moiety of 1-O-sinapoyl-β-ᴅ-glucose to choline to form O-sinapoylcholine (sinapine). A wide range of proportionality with respect to incubation time and protein concentration was found in the determination of this enzymatic activity using a high performance liquid chromatographic (HPLC) method. The transacylase activity showed an absolute acceptor specificity towards choline and a high hydroxycinnamoyl donor specificity with 1-sinapoylglucose. Maxim|al rate of sinapine formation was found to be around pH 7.0 in 0.08 M potassium phosphate buffer. The apparent K m values were 0.30 mм for 1-sinapoylglucose and 7.64 mм for choline. Change in extractable transacylase activities from different stages of seed development correlated well with the in vivo accumulation kinetics of sinapine. Maximal activity around 36 pkat seed -1 and 129μkat kg -1 protein was extracted from dark-green immature seeds, which are in the process of rapid sinapine accumulation. About 20% of this activity still has been found in dark-brown mature seeds. This enzymatic acitivity refers to an enzyme which can be classified as 1-sinapoylglucose: choline sinapoyltransferase (SCT).

The triazole fungicide etaconazole (CGA64 251) interferes with the ergosterol biosynthesis in Ustilago maydis by inhibiting the C-14 demethylation of the sterol nucleus. During the late log growth phase of U. maydis a novel endogenous sterol metabolite (14α-methyl-ergosta-8,24(28)- dien-3β,6α-diol) was discovered and analyzed, which accumulates under the influence of the fungicide. The structure of this metabolite points to a hydroxylation-dehydration mechanism for the introduction of the double bond at C-5 during the ergosterol biosynthesis.

Chlamydomonas reinhardii 104 mutant strain cells contain many NAD(P)H-cytochrome c reductase activities unrelated to nitrate reduction in addition to an NAD(P)H-cytochrome c reductase related to NAD(P)H-nitrate reductase, which have molecular weights from 37000 to 124000. By combining chromatographic procedures and preparative isoelectric focusing, three major fractions of NAD(P)H-cytochrome c reductase were isolated with pi at 7.1, 5.5 and 5.05, respectively. Of these fractions, only the last one was identified as the diaphorase subunit of the NAD(P)H-nitrate reductase by its ability to reconstitute in vitro the nitrate reductase native complex upon complementation with active terminal nitrate reductase of 305 mutant of C. reinhardii.

The effect of 5 silatranes RSi(OCH 2 CH 2 ) 3 N, where R = C 2 H s O; C 6 H 5 ; ClCH 2 and RSi[OCH(CH 3 )CH 2 ] 3 N, where R = C 6 H 5 , ClCH 2 and tetraethoxysilane were studied on the growth, photosynthetic oxygen evolution and dark respiration of the marine centric diatom Cyclotella cryptica. Both silica-free and Si(OH) 4 supplemented media were used. Only 1-ethoxy- silatrane was hydrolyzed by the diatom, giving sufficient quantities of Si(OH) 4 to maintain growth without exogenous Si(OH) 4 . In this case the diatoms multiplied by a factor of 18 within 48 h. With silica in the medium, 1-phenylsilatrane, 1-phenyl-3,7,10-trimethylsilatrane, 1-chlormethylsilatrane and 1-chlorm ethyl-3,7,10-trimethylsilatrane exhibited only slightly inhibitory activity. In contrast, however, tetraethoxysilane (5 · 10 -4 mol/1) inhibited growth almost completely. Respiration was unaffected by all the silatranes tested, but net photosynthetic oxygen evolution was reduced by approx. 50% by 1-chlormethylsilatrane and 1-chlormethyl- 3,7,10-trimethylsilatrane and almost completely by 1-phenylsilatrane and 1-phenyl-3,7,10-tri- methylsilatrane. In the absence of silica as nutrient in the medium, the silatranes hardly affected dark respiration within 24 h. Conversely, the same samples showed no photosynthetic oxygen evolution after 24 h, and 24 h later again a negative O 2 balance was produced in the light. Different silatranes produced significant differences in the final oxygen balance.

Spheroplast membranes of a thermophilic cyanobacterium Synechococcus sp. have been treated with the detergent lauryldim ethylam ine oxide (LDAO). The resulting extracts show (1) light-induced O 2 evolution with artificial electron acceptors, (2) four-fold enhancement of the O 2 evolution relative to chlorophyll, (3) parallel increase of both the molar ratios of PS 2/C hl and cyt b 559 /Chl in the extract, (4) dissociation of the auxiliary pigment phycocyanin upon treatment with LDAO, but still tight association of allophycocyanin to the photosystem 2 preparation.

By mono esterification of 3′(2′)-hydroxyl residues of adenine nucleotides with various carboxylic acids a series of nucleotide analogs is available including fluorescent and photoaffinity labels. Their chemical synthesis is described. The equilibrium between 2′ and 3′ esters is determined by NMR spectroscopy, stability of the esters and their tendency of acyl migration is discussed. The interaction of the ADP derivatives with the chloroplast ATP synthesizing system is investigated. Actually, the analogs are typical energy transfer inhibitors, strongly inhibiting photophosphorylation and concomitant coupled electron transport (c i50 values ranging from 0.3 to 85 hm). On the basis of inhibitory activities of analogs bearing varying 3′-(2′)-substituents, structure-activity relationships are discussed. The inhibitory properties of the employed ADP analogs are based on their specific interaction with the catalytic ADP binding site of CF, and their extremely slow phosphorylation on the enzyme (rate 0.25% or less compared to ADP phosphorylation). Inhibition is competitive to ADP but non-competitive with regard to P i . It is specific for the ADP derivatives, whereas the corresponding ATP analogs are only weak inhibitors in phosphorylation and the AMP derivatives are completely inactive. In light-triggered ATP hydrolysis, however, the ATP analogs exhibit an even stronger competitive inhibition than the ADP derivatives. The results suggest that a conformational change of ATPase takes place when the chloroplasts are transferred from energized to de-energized conditions which greatly affects the properties of the active site with respect to nucleotide binding.

When Euglena gracilis is dark adapted for 10 min or more, oxygen evolution as the consequence of short (5 μsec) saturating light flashes does not show the picture of a damped oscillation with a periodicity of 4, as known from the literature. The overall picture of this flash pattern is given by the fact that O 2 -evolution in the first two flashes is practically zero and rises from there onward in a continuous manner to the steady state with barely any visible oscillation at all. However, a second flash sequence fired one to two minutes after this first sequence induces an oxygen evolution pattern which is barely distinguishable from the well known usual Chlorella vulgaris pattern. The phenomenon is not influenced by changes in the oxygen tension nor do additions of chemicals like CCCP, sodium azide, or reducing agents like hydroxylamine or hydrogen peroxide substantially alter the described behavior. Deactivation experiments give the overall impression that the deactivation of the S-states is slower than with Chlorella. Hydroxylamine strongly accelerates the deactivation. The analysis of the S-state distribution in a four and five state Kok-model suggests that dark adapted Euglena is in a more reduced condition than dark adapted Chlorella. It looks as if dark adapted Euglena were in a condition which would correspond to 60 percent S -1 , 30 percent S 0 and 10 percent S 1 . The experimental flash sequence of such dark adapted cells fits best a synthetic sequence when the misses are in the region of 20-25 percent, with double hitting playing practically no role at all (the first two flashes are zero!). The impression that dark adapted Euglena starts its oxygen evolution from a more reduced state is strengthened by the analysis of room temperature fluorescence induction (Kautsky effect). It can be shown that the fluorescence induction curve of Euglena corresponds to that of Chlorella cells provided the latter have been briefly treated with a strong reductant such as sodium dithionite.

The nucleoside antibiotic formycin, 7-amino-3-(β-ᴅ-ribofuranosyl)pyrazolo(4,3-d)pyrimidine, a structural analogue of adenosine, is deaminated about 10-fold faster by adenosine deaminase than adenosine itself, and is therefore a superior substrate for both routine assays and kinetic studies with the purified enzyme. The luminescence properties of formycin have been profited from to develop a fluorimetric assay for adenosine deaminase which is considerably more sensitive than the spectrophotometric procedure widely employed with adenosine as substrate. Examples are presented of its application to routine assays of adenosine deaminase levels in cellular extracts, as well as to kinetic studies with the purified enzyme, including the properties of some pyrazolopyrimidine and purine substrates and inhibitors.

Using T-lymphocyte and granulocyte colony assays with truly proliferating cells the effects of the polyamine spermine and of other naturally-occurring inhibitors of cell proliferation have been differentiated. It has been confirmed that spermine, in the presence of fetal calf serum, is a potent inhibitor of cell proliferation. This inhibition could be reversed by the addition of either 3- hydroxybenzyl-oxyamine or 4-bromo-3-hydroxybenzyl-oxyamine, both of which are inhibitors of the polyamine oxidase. In comparison, fractions isolated from calf thymus were shown to inhibit lymphocyte, but not granulocyte colony growth, indicating their tissue specificity and lymphocyte chalone activity. Further this inhibition was not reversed by polyamine oxidase inhibitors demonstrating that polyamines were not the inhibitory principles in this preparation.

Effect of organic solvents on enzymatic hydrolysis of celluloses was studied. It was found that toluene and xylene can accelerate the rate of the enzymatic hydrolysis reaction of filter paper and cellulose powder. The addition of N,N-dimethylformamide, acetone, and alcohols was negative effect for the enzymatic hydrolysis of solid celluloses. In the addition of alcohols and glycols, the relative glucose yields increased with increasing the carbon number of their solvents. The addition of organic solvents on the enzymatic hydrolysis of celluloses was discussed with swelling effect. It was found that the glucose yield increased as the dipole moment of the solvents decreased.

Time-resolved fluorescence studies of hematoporphyrin in aqueous solution and in different organic solvents are presented. The| observation of two exponential components in the fluorescence decay in aqueous solution reveals the presence of a monomeric and aggregated (probably dimeric) form of the molecule, as confirmed by absorption and fluorescence measurements. The slow component (~ 15 ns in aqueous solution) and the fast one (~ 3.8 ns in aqueous solution) are attributed to monomers and dimers, respectively. Higher aggregated species of hematoporphyrin are also present, which are essentially devoid of fluorescence properties.

The nature of the ferric high spin iron complex located at g = 4.3 has been investigated by means of electron spin resonance spectroscopy. It could be shown that the iron is bound to two histidines, three ascorbic acids, and one bicarbonate. This agrees well with previous findings according to which the ligand field of iron is composed mainly of oxygen and nitrogen atoms. Another low-field signal located at g = 9.5 appears always concomitantly with the g = 4.3 signal. It should be due, therefore, to a transition between the two sublevels of the low-lying Kramers doublet in one principal direction.

Two alternative methods for the attachment of monomethoxypolyethyleneglycols (PEG) to proteins are proposed; they are based upon the replacement of the hydroxy terminal function of PEG to carboxylate followed by its activation with dicyclohexylcarbodiimide and N-hydroxy-succinimide. The methods, which give more homogeneous product than that employing trichloro-s-triazine as coupling reagent, may also be used for the modification of essential -SH containing enzymes. The attachment of PEG activated via esters was tested with several model proteins and the influence of the extent of modification i. on the biological activity of various enzymes, ii. on the binding capacity for albumin and iii. on the clearance time in rats using superoxide dismutase as model tracer was evaluated. It was also demonstrated that the extent of PEG attachment varies greatly according to the different proteins used.

Poly(ribonucleic acids), poly A, poly C and poly U, were irradiated in O 2 -free dilute aqueous solution at pH 7 to 8 with single pulses (50 to 200 ns) of 16MeV electrons. With the aid of Rayleigh light scattering measurements main-chain scission, induced by OH radicals, was observed with the three polynucleotides. From the time dependence of the decrease of the light scattering intensity (LSI), the existence of two modes of decrease was inferred, indicating at least two different chemical mechanisms were operative in main-chain degradation. Evidence for the assignment of the slow mode of LSI decrease to the lifetime of a free radical was obtained from quenching experiments with cysteamine. It is noteworthy, that the extent and the lifetime of LSI decrease are not the same for the three polynucleotides. The differences indicate the influence of the chemical nature of the bases on main-chain scission. Consequently, it is concluded that OH attack at carbons in 1′ and/or 2′ position of the ribose moiety contributes essentially to the degradation mechanism.

Upon digestion with a streptococcal extracellular nuclease, yeast RNA yielded acid-insoluble core having increased carrier activity for streptolysin S. The carrier activity was found in minor fractions of the core which were eluted from a DEAE-cellulose column at higher salt concentrations. Upon gel filtration through a Sephadex G-75 column, the effective component (Fr. I) was eluted earlier than bulk oligonucleotides (Fr. II). Nucleotide composition (in mol %) of Fr. I was AMP: 21.8; GMP: 55.1; CMP: 8.2; UMP: 14.9, whereas that of Fr. II was AMP: 38.0; GMP: 33.1; CMP: 8.0; UMP: 20.9. Chromatographic patterns of SLS complex induced by Fr. I were similar to those of the toxin formed in the presence of active fraction prepared from RNase I core. Hemolytic activity of the latter complex was, like the former, unaffected by streptococcal nuclease treatment. The carrier activity of DNA digested with the nuclease was also investigated.

The mode of growth of several tissues in Gryllus bimaculatus was investigated during postembryonic development by cytophotometric methods. In contrast to the situation in holo- metabolous insects, the tissues growing by endomitosis reach only moderate levels of polyploidy. In this case the growth of tissues is achieved by mitotic divisions of small cells with subsequent polyploidization. The time courses of DNA synthesis were measured within the 3rd and, for comparison, the 8th larval instar by incorporation of labelled thymidine followed by autoradiography. Hemocytes, cells of the regeneration crypts of the midgut, gonads and nervous tissue showed a continuous incorporation rate; by contrast, DNA synthesis in other tissues was confined to a given time within the moulting cycle. The changes in moulting hormone titre of the 3rd larval instar were investigated. The quantities of ecdysone and 20-OH-ecdysone were estimated by radioimmunoassay, and the hormones were identified by high pressure liquid chromatography (HPLC). The titre changes in time with at least 2 distinct maxima. The DNA synthesis periods are correlated with the hormone peaks, that of epidermis and tracheae with the first peak, and that of pylorus, ileum, rectum, Malpighian tubules and fat body with the second. DNA synthesis in prothoracic glands and oenocytes exhibits a time course that is the inverse of the hormone-secretion time course. The question whether moulting hormones have an influence on DNA synthesis is discussed.

Chromatin undergoes structural changes in dependence on the ionic strength of monovalent cations. At low ionic strength an extended chain of nucleosomes is apparent while with increasing ionic strength more compact structures are formed. Soluble chromatin was prepared from rat liver and fractionated by gel chromatography. Quasielastic light scattering experiments on chromatin were done with monovalent salt concentrations ranging from 3-150 mм. Using this method translational diffusion coefficients have been derived. These appeared to be nearly independent of monovalent salt concentrations, indicating that the hydrodynamic radius of chromatin molecules did not change. On the other hand, sedimentation coefficients were increasing according to an exponential relation. Taken together, both findings reveal a rising of the molar mass with increasing ionic strength. On the contrary, chromatin prepared in physiological salt apparently disintegrates by lowering the ionic strength. Furthermore, it could be demonstrated by earlier small angle X-ray scattering studies that the diameter of the higher order chromatin fiber remained constant at approximately 32 nm even if these structures had been reconstituted from smaller pieces starting at low ionic strength. Thus, isolated chromatin fibers are capable of self-assembling to regular quaternary structures, even if the DNA does not form a continuous strand.

In subcutaneous calcinoses induced in rats by topical calciphylaxis and topical calcergy, the ultrastructural aspects related to the evolution of calcified deposits were previously described. In the present study by means of X-ray diffraction, infrared spectrometry and electron spin resonance, different biophysical analyses are performed on the mineral substance deposited during development of calciphylaxis and calcergy. A rapid evolution of the calcium phosphate deposits into hydroxyapatite was noticed along the first 20 days of the calcinoses; from then, there is no important modification even at the later stages, and the characteristics of the mineral substance are mostly similar to that of bone tissue. The concentration of trace elements such as Mg 2+ and Fe 2+ , is found higher in cutaneous calcinoses than in bone tissue.

In vitro cultures of neural retina, obtained after dispersion and trypsinization of tissue frag ments, were composed of 3 morphologically distinct types o f neural cells, as demonstrated by sil­ver impregnation. They resem bled ganglion or receptor cells, horizontal or amacrine cells, and bi­polar cells of the intact retina. Pigment epithelium was cultured without trypsinization. Both kinds o f techniques may prove helpful for long term experiments in neurobiology and neurovirology.

The frond exudates of the two gymnogrammoid ferns, Notholaena peninsular is and N. pallens, consist mainly of a diterpene, accompanied by small amounts of flavonoid aglycones. The diterpenes from both sources are shown to be identical. Their structure has been established by spectroscopic methods as (-)-kaur-16-ene-19-oic acid. This result is confirmed by direct comparison with an authentic sample.

Gossypetin 7-methyl ether 3-rutinoside has been characterized as a yellow flower pigment in Ruta graveolens and in five other Ruta species. By contrast, in four Haplophyllum species, the 7-glucoside of gossypetin 3′-methyl ether provides the yellow coloration in the flower. Although carotenoid accompanies the yellow flavonol in R. graveolens, there is no evidence of UV patterning and the petals are uniformly UV absorbing.

Antimicrobial activity of N,N′-bis(decylmethyl)-α,ω-alkanediamine dioxides determined on Staphylococcus aureus, Escherichia coli, and Candida albicans is presented as minimal inhibitory concentration (MIC). The effect of the length of linking alkylene chain on this activity has been followed.

Hematoxylin, Fluorescence, Fluorochrom es Treatments o f chicken blood and Ehrlich ascites tumor smears with hematoxylin solutions give a fluorescence reaction in chromatin, basophilic cytoplasm and leukocyte granules. In these structures the fluorescence emission in­ creases upon dye aging and prolonged staining times. We present a preliminar spectral analysis suggesting the possibility to employ hematoxylin as a fluorochrome.

An iterative algorithm for constructing the optimal phylogenetic tree from a given set o f dissimilarity data is described. The procedure is applied for illustrative purposes an a data set com piled by Fitch and Margoliash.