Cytochemical markers of bladder carcinogenesis

Summary Enzyme cytochemical and immunocytochemical techniques at the light and electron microscope levels were used to study the distribution of potential markers of chemical transformation in rodent bladders. In rat tumours induced byin vivo treatment with methylnitrosourea, alkaline phosphatase localization was normal on the external surface of the plasma membranes of some cells but abnormal in others where reaction product was seen only on intracellular membranes. 5-Nucleotidase localization was abnormal in all cells, being seen on endoplasmic reticulum and nuclear membranes only, while in normal bladders only ectoenzyme localization was seen. Heterogeneity of alkaline phosphatase and 5-nucleotidase localization was seen on the plasma membranes of these tumours after 15 days in organ culture. Some cells produced enzyme and others did not; in other cells only parts of the membrane reacted heavily, while other regions were negative.In transformed cell cultures and tumours of mouse bladder derived byin vitro treatment of explants with dimethylbenz (a) anthracene, a bimodal pattern of alkaline phosphatase localization was seen. Cells had either normal ectoenzyme reaction product or abnormal intracellular membrane reaction product. 5-Nucleotidase and ADPase were lost after transformation while cAMP-phosphodiesterase was retained as an ectoenzyme. Mg.ATPase and a cAMP-independent, calcium-insensitive protein phosphatase were induced in transformed cell cultures. An epithelial antigen was detected in the cytoplasm of both normal and transformed cells associated with reticular cytoplasmic ground substance, plasma membrane vesicles and cytoskeletal elements.     

A phaseolus mutation results in a reduced level of lectin mRNA

Summary The molecular basis of a lectin gene mutation in the Phaseolus vulgaris cultivar Pinto was investigated. Rocket immunoelectrophoresis studies showed that seed lectin is reduced approximately 40-fold in comparison to the normal Phaseolus vulgaris cultivar Contender. DNA gel blot studies using a lectin cDNA probe suggested that the Pinto and Contender varieties contain similar numbers of lectin genes, although qualitative differences were observed in the gel blot banding patterns. Hybridization of the lectin cDNA probe to gel blots containing normal and mutant embryo mRNAs produced a 1 kb mRNA band in both mRNA populations. However, the amount of lectin mRNA was reduced approximately tenfold in the mutant Pinto cultivar. Together, these findings suggest that the reduced seed lectin level is due, in part, to a reduction in seed lectin mRNA.Abbreviations mRNA messenger RNA - cDNA complementary DNA - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - BSA bovine serum albumin - kd kilodaltons - kb kilobases     

An ultraviolet light induced bacteriophage inBeneckea gazogenes

An ultraviolet light induced prophage has been discovered in the red pigmented marine vibrioBeneckea gazogenes. Two spontaneously derived pigment mutants, one forming pink colonies and one lacking pigment and forming white colonies, were also irradiated. The presence of pigment was not related to phage induction; uv-induced cell lysis occurred in wildtype and mutant strains at the same dosages. Lysis was not prevented or retarded by exposure after irradiation to visible light indicating the phenomenon was not photoreactivable. Electron micrographs of the T-likeB. gazogenes phage are shown.A second beneckea was isolated from the anaerobic zone of cyanobacterial mats growing in the hypersaline environment of Laguna Mormona, Baja California. The Baja beneckea does not harbor a uv inducible prophage and is resistant to theB. gazogenes phage under all conditions tested.     

Antitumor effect of recombinant tumor necrosis factor-α against murine sarcomas at visceral sites: tumor size influences the response to therapy

Summary We examined the antitumor efficacy of rTNF- administration on established tumor at two visceral sites, lungs and liver. Treatment of B6 mice harboring multiple (>100 foci of 0.5 mm diameter) 10-day pulmonary macrometastases from the MCA-106 sarcoma, with dosages of rTNF- (5–10 g, single dose i. v.) that caused hemorrhagic necrosis and regression of a 6 mm MCA-106 s. c. tumor, had no impact on the number (or size) of lung nodules. Similarly, rTNF- failed to show an antitumor effect in B6 mice with advanced day 8 or 10 multiple (>100 foci of 0.5 mm diameter) hepatic metastases at single i. v. doses up to 20 g, as measured by either enumeration of residual liver nodules or survival. B6 mice injected s. c. with MCA-106 sarcoma and treated with rTNF- as a single i. v. dose on day 0, 3, 5, or 7 experienced marked tumor regression only after the day 7 rTNF- injection, when the tumor had achieved a size of 5–6 mm in diameter. Since tumor size appeared important for rTNF- susceptibility in vivo, we next induced a single hepatic tumor of the MCA-106 sarcoma by the direct injection of cells into the left lobe of the liver and treated these mice at day 10 when the nodule had achieved a size of 5–6 mm in diameter. Increasing doses of rTNF- (up to 8 g) given as a single i. v. injection resulted in increasingly greater reductions in hepatic tumor as well as significant survival benefit of the treated mice. Sites of regressing hepatic tumor exhibited central necrosis accompanied by polymorphonuclear leukocytes and lymphocytes. Collectively, these results show that rTNF- administration can mediate a significant antitumor effect on visceral tumor and suggest that tumor size is an important factor in rTNF- susceptibility not only for tumors growing at s. c. sites but also for those established at visceral sites.Howard Hughes Medical Institute Research Scholar Abbreviations used: rTNF-, recombinant tumor necrosis factor-; B6 mice, C57BL/6 mice; MCA, 3-methylcholanthrene; HBSS, Hanks' balanced salt solution; LAK, lymphokine-activated killer; rIL-2, recombinant interleukin-2     

Application of the restriction landmark genomic scanning (RLGS) method to rice cultivars as a new fingerprinting technique

The restriction landmark genomic scanning (RLGS) method was applied to rice, using two Japanese cultivars, Nipponbare and Koshihikari, and a Chinese landrace, LiuZhou'Bao'Ya'Zao'. More than 3000 landmarks were detected as spots on the individual autoradiograms of each cultivar. Nipponbare and LiuZhou' Bao'Ya'Zao' showed apparently different RLGS profiles, from which the genetic similarity (GS) between them was estimated as 0.344. Although the two Japanese cultivars, Nipponbare and Koshihikari showed quite similar RLGS profiles, they were easily distinguished on the basis of the presence or absence of specific spots; the GS value between them was calculated as 0.980. The RLGS method is shown to be a powerful fingerprinting technique, especially for the classification and identification of cultivars in rice and probably in other crops as well.     

Does an animal peptide:N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein?

Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide:N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994)J Biol Chem 269, 17611–18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g.K _m=114 µm for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase; the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man1 3(Man1 6)Man, but not by mannose and -methyl-d-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition proteinin vivo. Such dual properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases — PNGase A and PNGase F, respectively.Abbreviations GLC gas liquid chromatography - GlcNAc-Asn 2-acetamido-1--(l-aspartamido)-1,2-dideoxy-d-glucose - BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Man d-mannose; triomannose, Man1 3(Man1 6)Man; - MES 2-(N-morphorino)ethanesulfonic acid - NeuAc N-acetyl-neuraminic acid - PNGase peptide:N ⁴-(N-accetyl-glucosaminyl)asparagine amidase (peptide:N-glycanase,EC 3.5.1.52) - PNP p-nitrophenyl     

Climatic adaptability of populations ofDiplotaxis erucoides D.C. (Brassicaceae) from Sicily,based on leaf morphology,leaf anatomy and δ13 C studies

The morphological and anatomical variability ofDiplotaxis erucoides populations from Sicily was investigated. Populations growing during the summer months exhibit distinct xeromorphic features. Leaf area is strongly reduced and leaf thickness is increased when compared with winter populations. Cell size, as well as cell arrangement and mesophyll cell surface area differ significantly between summer and winter populations. Leaf thickness is almost three times higher in summer populations andA (cell)/A, i.e. the mesophyll cell surface area per unit leaf area changes from about 16 for winter populations to almost 52 for summer populations. These differences are partly due to differences in intercellular volume and partly due to alterations in mesophyll cell sizes. The organic materal of the summer populations exhibits ¹³C values in the order of –27%. to –28%., while the corresponding values for the winter populations are in the order of –31%. to –33%.. Analysis ofD. erucoides populations from the transition period revealed intermediate ¹³C values. Anatomical variations such as reductions or increases ofA (cells)/A and changes of intercellular volume correlate with the corresponding ¹³C data. The ¹³C data are discussed in conjunction with the differences in leaf anatomy.     

The contribution of pigment transitions to sensitivity changes in the barnacle photoreceptor and the correlation with the prolonged depolarizing afterpotential

A conditioning light can cause a decrease (adaptation) or an increase (facilitation) in the sensitivity of barnacle photoreceptors, as measured by the amplitude of the late receptor potential (LRP). We show that a net transfer of visual pigment from the rhodopsin (R) to the metarhodopsin (M) state induces a large facilitation whereas the reverse transfer results in a much smaller facilitation or even an adaptation. These effects were not due to the response to the conditioning light but to the pigment reactions. When the conditioning light did not alter the pigment population (i.e., M M, R R) it was followed by an intermediate degree of facilitation. These conclusions are correct for cells which have relatively low sensitivity. In sensitive cells, all pigment transitions produce adaptation.LRP facilitation and the prolonged depolarizing afterpotential (PDA) show several common characteristics with respect to pigment transitions: 1.Their magnitude increases with the amount of pigment transferred from R to M. 2. Both are depressed by the M R transition. 3. Their production is impeded by the M R transition. 4. The PDA itself is facilitated by the R M transition and this facilitation decays with a time course comparable to that of LRP facilitation. These results suggest that there may be an underlying process common to LRP facilitation and PDA.     

Study on liquid-holding recovery in DEB-inactivated rad3 mutant of Saccharomyces cerevisiae

Summary Maximal liquid-holding recovery (LHR) of the DEB-treated rad3 mutant occurs at 30° C in buffer supplemented with glucose. Addition of cycloheximide (CHX) to the buffer, the increase in cell density above 2 × 10⁷/ml as well as lowering of temperature during liquid holding (LH) below 27° C decrease considerably the cell capacity for recovery. LHR does not take place at 5° C. No measurable DNA synthesis or degradation occurs in cells held in buffer alone, while addition of 0.02% glucose results in incorporation of radioactivity into DNA both of DEB-treated and control cells. Similarly, protein synthesis was observed only in cultures held in buffer supplemented with glucose. Cells transfered to growth medium directly after treatment complete one round of DNA replication and at least one division cycle, but further DNA replication and cell division are inhibited. Cells placed in growth medium after 5 days LH show an increased rate of DNA replication and cell division. Completion of the first posttreatment round of DNA replication in growth medium abolishes ishes the cell capacity for LHR. DEB treatment results in abnormal cell division of the rad3 mutant, giving colonies consisting of several cells, usually abnormal in shape, held together by common cell walls.     

DoesAgapetus fuscipes cultivate algae in its case?

The presence of algae within the cases ofAgapetus fuscipes was investigated. Cases recognised as dirty or clean with the naked eye had more and less algal growth, respectively. Larvae in the former survived significantly longer when starved in the laboratory. It is suggested that the presence of algae within the cases would be of ecological advantage during periods of flood.     

Repressor defective mutants, virulent mutants and adsorption resistance of lysogenic cells in case of phage kappa of Serration

Summary Thermoconditional clearplaque mutants (c_t) of phage have been isolated, which as prophages can be induced by exposure of the 30°C-grown lysogenic bacteria to 42°C. The thermoinduction of the lysogenic cells needs protein synthesis as shown by the inhibitory effect of chloramphenicol. The c_t-mutations are located in clearplaque region III of the genetic map of . Thus this region contains information for the prophage repressor.-lysogenic cells are adsorption resistant against -phages except the cells with mutant prophages of clearplaque region I (l_I mutants). Virulent (v) mutants which can grow in l_I-lysogenic cells appear after direct plating of extracellularly uv irradiated phage with l_I-lysogenic indicator bacteria. Surprisingly all these mutants show a more or less reduced plaque forming ability at 37°C compared with 30°C. For one of the v-mutants the maximum thermosensitive phase in the latent period was checked and found to be very shortly before lysis.     

Analysis of hybrids obtained by rare-mating of Saccharomyces strains

Summary Rare-mating of closely related Saccharomyces cerevisiae and S. diastaticus strains led to the formation of different hybrids. Mating-type switching and chromosome losses could be observed by means of classical genetic analysis and pulsed field gel electrophoresis of intact chromosomes. The latter was facilitated by extensive chromosome length polymorphism in both strains. When crossing the two haploid strains S. cerevisiae 41 and S. diastaticus ATCC 28339 , two different types of hybrids occurred. Both types showed complete addition of both parental genomes, one a-status and the other -status. The -status could be explained by assuming a transient premutational lesion in MAT . Usually lesions are repaired after a mating event and the -mating type is restored. When crossing a diploid S. diastaticus strain, isogenic to the one previously mentioned, with the haploid S. cerevisiae strain, three different types of hybrids could be distinguished regarding their mating-types. It was possible to prove that the haploid S. diastaticus strain ATCC 28339 is disomic and the diploid hybrid, named 41ATCC-b, is trisomic for chromosome I. This could be shown by means of electrophoretic karyotyping of the hybrid and of the four single-spore cultures from one ascus of the hybrid.     

New cases of somatic conversion (paramutation) in tomato (Lycopersicon esculentum Mill.)

Summary The genetic behaviour of three chlorophyll variegated F₁ tomato plants, derived from irradiated gametophytes, was analyzed over several generations of selfing and backcrossing. The results suggest that irradiation has put genes, different in all three mutants, into a labile state, n*, remaining so after fertilization. This state had the power of converting the associated wild allele N into a deficient form.Somatic conversion was soon followed, in Plant C₁₁ always and nearly always in Plant C₁₂, by stabilization of both alleles in a conversion-inactive recessive state, genetically similar, and stable except in special conditions.In the other type, found seldom in C₁₂, always in C₆, the n* state was permanent and transmissible. Conversion occurred with a certain frequency determined by developmental and genotypic influences, and the converted allele also acquired conversion power, so that gametes from an N n* plant were of three kinds: N, n* and n*. This process corresponds to paramutation (Brink 1958).Results were compared and contrasted with other published data.Contribution nr. 748 of the Biology Division of Euratom.     

The herbicide-resistant D1 mutant L275F of Chlamydomonas reinhardtii fails to show the bicarbonate-reversible formate effect on chlorophyll a fluorescence transients

Herbicide-resistant mutants of the eukaryotic green alga Chlamydomonas reinhardtii, that are altered in specific amino acids in their D-1 protein, show differential bicarbonate-reversible formate effects. These results suggest the involvement of D1 protein in the bicarbonate effect. A 25 mM formate treatment of mixotrophically or photoautotrophically grown wild type cells results in a slower rise of chlorophyll a fluorescence transient followed by a dramatically slowed decline during measurements in continuous light. These effects are fully reversed upon addition of 10 mM bicarbonate. The mutant BR-202 [L275F] is, however, highly insensitive to 25 mM formate suggesting that a significant change in formate (bicarbonate) binding has occurred in helix V of the D1 protein near histidine involved in Fe binding. With the exception of DCMU-4 [S264A], which is considerably more sensitive to formate than the wild type, five other different [V219I, A25IV, F255Y, G256D and cell-wall deficient CW-15] mutants display a relatively similar response to formate as wild type. Absence of formate effect on a PS II-lacking [FuD-7] mutant confirms the sole involvement of PS II in the bicarbonate effect.     

Studies on the ultraviolet-sensitive mutants of blue-green alga Synechocystis aquatilis Sanv.

Summary Several mutants of the unicellular blue-green alga Synechocystis aquatilis Sanv. were isolated. They differed from the wild type by the levels of sensitivity to ultraviolet (UV) irradiation. The most sensitive mutant is unable to carry out photoreactivation and shows increased resistance to mitomycin C, N-methyl-N-nitro-N-nitrosoguanidine and methyl methanesulfonate. This strain shows an enhanced rate of spontaneous and UV-induced mutagenesis. Another UV-sensitive mutant with normal level of X-ray sensitivity is characterized by a decreased mutability. The three other UV-sensitive mutants show simultaneous decrease of resistance to X-ray and alkylating agents. The existence of these cross-sensitive mutants indicates that a repair mechanism may operate in blue-green algae similar to dark repair systems of bacteria and yeast.     

Lethal and mutagenic effects of nitrosoguanidine on synchronizedChlamydomonas

Summary The lethal and mutagenic effects of 5 g/ml N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were maximal during the nuclear S-period of synchronously grownChlamydomonas reinhardtii. This was revealed by a 50% drop in survival and a 50- to 100-fold increase in the recovery of slow-growth mutants (up to 40% of the survivors) which were first recognized as small colonies on agar medium. Partial characterization of these isolates revealed about 50% to be stable on subculture, and several were demonstrated to be either acetate-dependent, dark-lethal (light-dependent), or acetate-sensitive mutants. There was no significant increase of lethality or of slow-growth mutants correlated with treatment during the chloroplast DNA replication phase of the cell-cycle.The results of genetic analysis with 13 mutants induced during the nuclear S-period were consistent with their nuclear origin. These analyses were hampered by the high proportion of lethality among the progeny of most crosses.It is concluded that the enhanced mutant induction among nuclear S-phase cells may indicate preferential mutagenesis of replication fork DNA and induction of multiple-closely-linked mutations, as in some bacteria. Consequently, forC. reinhardtii, caution should be exercised in drawing relationships between abnormal behavioral and biochemical phenotypes in MNNG-induced mutants.     

Maximisation of reproductive success by European orchidaceae under conditions of infrequent pollination

Summary The pollination biology of a group of European orchids (Dactylorhiza, Ophrys, Orchis, Platanthera, Goodyera, andSerapias species) are investigated, and their anthecological characteristics considered in relation to natural levels of reproductive success. Pollen ovule (P O) ratios of the European orchids surveyed range from 10 1 (Goodyera repens) to 24 1 (Platanthera chlorantha). Average pollen-load ovule ratios are consistently lower than P O ratios. Naturally occurring pollen loads range from 1 massula to >1 pollinium. Even the smallest pollen load is sufficient to stimulate embryogenesis in experimentally pollinatedDactylorhiza purpurella flowers, although more seeds are set with larger loads. Pollen tubes grow rapidly through the stylar canal and into the top of the ovary within 2 or 3 days of pollination, and grow down either side of the 3 parietal ridges in the ovary. Fertilisation occurs throughout the length of the ovary but its distribution is non-random, especially when pollen loads are limiting, with more seeds being set at the stylar end. All species ofDactylorhiza, Ophrys, andOrchis studied are highly self-compatible. In the absence of pollination,Ophrys andOrchis flowers remain open and fertile for at least 3 weeks. Pollinated flowers remain receptive to further pollinations for at least 8 days. Some fruits can even be obtained on selfing 20-day-old unpollinatedOrchis morio flowers. Excised pollinia retain germinability for a long time, up to 51 days inDactylorhiza purpurella. The arrival of pollen on the stigma hastens floral senescence, but post-pollination changes are relatively slow when compared with those reported for tropical orchid species. It is concluded that characteristics of the pollination biology of European orchids act to maximise reproductive success by (1) prolonging the opportunity for effective pollen deposition both pre- and post-pollination, (2) increasing the likelihood of widespread dispersal, (3) reducing pollen wastage, and (4) increasing seed quality by promoting some pollen competition. As most European orchids are xenogamous and require pollen to arrive on the stigma before seed can be set, reproductive maximisation is of particular adaptive advantage because many of them are infrequently visited by insects so that the probability of successful pollination can be very low.Abbreviations P O pollen ovule ratio - PL O pollen-load ovule ratio - FCR fluorochromatic reaction test     

The kinetics and feedback inhibition of cytidine 5′-triphosphate synthetase in wild-type and mutant Chinese hamster cells

The kinetics and cytidine 5-triphosphate (CTP) feedback inhibition of CTP synthetase in wild-type and four mutants of Chinese hamster V79 cells have been studied. The enzymes of the wild type and three of the four mutants exhibited positive cooperativity with the substrate uridine 5-triphosphate (UTP). Three of the mutants had K _{m app} and S ₅₀ valuves distinctly greater than those of the wild type, while the fourth mutant had values similar to those of the wild type. all four mutants exhibited resistance to CTP feedback inhibition, while the wild type was sensitive to such inhibition. It is postulated that a single mutational event in each mutant had caused a concomitant change of the enzyme in its binding both to the substrate UTP and to the end-product CTP.This work was supported by Grant GM 20608 from the U.S. Public Health Service.     

An HNCO-based Pulse Scheme for the Measurement of 13Cα-1Hα One-bond Dipolar couplings in 15N, 13C Labeled Proteins

A triple resonance pulse scheme is presented for recording 13C-1H one-bond dipolar couplings in 15N, 13C labeled proteins. HNCO correlation maps are generated where the carbonyl chemical shift is modulated by the 13C-1H coupling, with the two doublet components separated into individual data sets. The experiment makes use of recently described methodology whereby the protein of interest is dissolved in a dilute solution of bicelles which orient above a critical temperature, thus permitting measurement of significant couplings (Tjandra and Bax, 1997a). An application to the protein ubiquitin is described.     

Screening for naturally occurring apolipoprotein A-I variants: apo A-I(ΔK107) is associated with low HDL-cholesterol levels in men but not in women

Isoelectric focussing (IEF) in carrier ampholyte-generated pH gradients and hybrid isoelectric focussing (HIEF) in immobilized pH gradients under nondenaturing conditions were used in parallel to screen 5,500 plasma samples for naturally occurring variants of apolipoprotein A-I (apo A-I). The following defects were identified in four unrelated subjects heterozygous for apo A-I variants: apo A-I(K107)(2 ×), apo A-I(K107M)(1 ×), and apo A-I(E41R)(1 ×). The later variant is a novel finding. Family studies did not reveal any association of apo A-I(K107M) and apo A-I(E41R) with dyslipidemia, but identified several heterozygotes for apo A-I(K107) who had low levels of high density lipoprotein (HDL)cholesterol. Therefore, and since the apo A-I(K107) is the most frequent apo A-I variant in Germany (1 5,000) we evaluated our data and that reported from 11 families with 32 heterozygous carriers and 30 unaffected controls. This analysis revealed that apo A-I(K107) is associated with lower HDL-cholesterol (-30%) and higher triglycerides (+ 48%) in men but not in women as compared with unaffected family members as well as with controls from the Prospective Cardiovascular Münster (PROCAM) study. Moreover, 11 of 15 male apo AI(K107) heterozygotes but only 2 of 17 female apo AI(K107) heterozygotes had HDL-cholesterol levels below the 20th percentile of sex-matched controls from the PROCAM study. We conclude that heterozygosity for apo A-I(K107) decreases HDL-cholesterol and increases triglycerides in men but not in women.