Wheat germ agglutinin binding sites on human urothelial cells of different grades of transformation

¹²⁵I-Wheat germ agglutinin (WGA) binding parameters of human urothelial cell lines of different grades of transformation (TGrll and TGrlll) were compared. The values of association constant (K_a) and the number of binding sites/cell for HCV29 (TGrll) cell line were about 3×10⁶M^–1 and over 4×10⁷, respectively. Two TGrlll cell lines, HCV29T and Hu549 revealed lower values for K_a, and considerably higher numbers of binding sites/cell (about 3×10⁸ and 2×10⁸, respectively). Binding of¹²⁵I-WGA to total cellular proteins resolved by SDS-PAGE and transferred to nitrocellulose showed multiple diffused bands in the range of 58–180 kDa. Some of these bands were characteristic for TGrll cells (124 kDa) or TGrlll cells (135 and 148 kDa).Abbreviations TGr transformation grade - WGA wheat germ agglutinin - sWGA succinylated wheat germ agglutinin - GlcNAc N-acetyl-d-glucosamine - BSA bovine serum albumin - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Accumulation of the early histone messenger RNAs during the development of Strongylocentrotus purpuratus

The developmental profile of amounts of the mRNAs for the early histones was determined. Sea urchin embryos (Strongylocentrotus purpuratus) were labeled with ³H nucleoside, and the RNA was extracted and fractionated by electrophoresis on polyacrylamide gels. Radioactivity in the resolved mRNA bands was determined and converted to molar quantities by knowledge of the precursor pool-specific activity. Between the 16- and the 200-cell stages 7–10 × 10⁶ molecules of each core histone mRNA, and 2.5 × 10⁵ molecules of H1 mRNA accumulate. During the subsequent few hours of cleavage the accumulated mRNAs disappear with a half-life of 1.5–2 hr. It is argued that mRNA half-life may be regulated during cleavage.     

Vasa and the germ line lineage in a colonial urochordate

Germ cell sequestering in Animalia is enlightened by either, launching true germ line along epigenetic or preformistic modes of development, or by somatic embryogenesis, where no true germ line is set aside. The research on germ line-somatic tissue segregation is of special relevancy to colonial organisms like botryllid ascidians that reconstruct, on a weekly basis, completely new sets of male and female gonads in newly formed somatic tissues. By sequencing and evaluating expression patterns of BS-Vasa, the Botryllus schlosseri orthologue of Vasa, in sexually mature and asexual colonies during blastogenesis, we have demonstrated that the BS-Vasa mRNA and protein are not expressed exclusively in germ cell lineages, but appeared in cells repeatedly emerging de novo in the colony, independently of its sexual state. In addition, we recorded an immediate Vasa response to cellular stress (UV irradiation) indicating additional functions to its germ line assignments. To confirm germ lineage exclusivity, we examined the expression of three more stem cell markers (BS-Pl10, Bl-piwi and Oct4). Vasa co-expression with Pl10 and Oct4 was detected in germ line derivatives and with Bl-piwi in somatic tissues. Presumptive primordial germ cells (PGC-like cells), that are Vasa⁺/Pl10⁺/Oct4⁺ and 6-12 μm in diameter, were first detected in wrapped-tail embryos, in oozooids, in sexual/asexual colonies, within a newly identified PGC niche termed as ‘budlet niche’, and in circulating blood borne cells, indicating epigenetic embryogenesis. Alternatively, BS-Vasa co-expression with piwi orthologue, an omnipresent bona fide stemness flag, in non germ line cell populations, may indicate germ cell neogenesis (somatic embryogenesis) in B. schlosseri. Both alternatives are not necessarily mutually exclusive.     

Analysis of TET Expression/Activity and 5mC Oxidation during Normal and Malignant Germ Cell Development

During mammalian development the fertilized zygote and primordial germ cells lose their DNA methylation within one cell cycle leading to the concept of active DNA demethylation. Recent studies identified the TET hydroxylases as key enzymes responsible for active DNA demethylation, catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. Further oxidation and activation of the base excision repair mechanism leads to replacement of a modified cytosine by an unmodified one. In this study, we analyzed the expression/activity of TET1-3 and screened for the presence of 5mC oxidation products in adult human testis and in germ cell cancers. By analyzing human testis sections, we show that levels of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are decreasing as spermatogenesis proceeds, while 5-methylcytosine levels remain constant. These data indicate that during spermatogenesis active DNA demethylation becomes downregulated leading to a conservation of the methylation marks in mature sperm. We demonstrate that all carcinoma in situ and the majority of seminomas are hypomethylated and hypohydroxymethylated compared to non-seminomas. Interestingly, 5-formylcytosine and 5-carboxylcytosine were detectable in all germ cell cancer entities analyzed, but levels did not correlate to the 5-methylcytosine or 5-hydroxymethylcytosine status. A meta-analysis of gene expression data of germ cell cancer tissues and corresponding cell lines demonstrates high expression of TET1 and the DNA glycosylase TDG, suggesting that germ cell cancers utilize the oxidation pathway for active DNA demethylation. During xenograft experiments, where seminoma-like TCam-2 cells transit to an embryonal carcinoma-like state DNMT3B and DNMT3L where strongly upregulated, which correlated to increasing 5-methylcytosine levels. Additionally, 5-hydroxymethylcytosine levels were elevated, demonstrating that de novo methylation and active demethylation accompanies this transition process. Finally, mutations of IDH1 (IDH1 ^R132) and IDH2 (IDH2 ^R172) leading to production of the TET inhibiting oncometabolite 2-hydroxyglutarate in germ cell cancer cell lines were not detected.     

Pseudomonas aeruginosa UCBPP-PA14 a useful bacterium capable of lysing Microcystis aeruginosa cells and degrading microcystins

To identify a useful bacterium capable of controlling both Microcystis aeruginosa and microcystins (MCs), 30 strains of Pseudomonas were screened. Two of them (Pseudomonas aeruginosa UCBPP-PA14 and Pseudomonas putida KCCM 10464) could cause significant lysis of M. aeruginosa. PA14 exhibited higher degradation activity against microcystins than KCCM 10464, and hence, it was selected as the bacterium for further analysis. Following its introduction into M. aeruginosa culture (10⁵ cells mL^-1) at densities of 10⁷, 10⁵, and 10³ PA14 cells mL^-1, higher initial inoculations of PA14 removed correspondingly more M. aeruginosa cells (100%, 100%, and 92% at 15, 30, and 10?days, respectively) and degraded microcystin (extracellular MCs: 83.7%, 77.7%, and 51.6% at 30?days; total MCs: 91.0%, 86.9%, and 61.6% at 30?days, respectively). However, the activity of PA14 diminished when its density decreased to less than 10⁶ cells mL^-1. At three initial algal densities (10⁶, 10⁵, and 10³ cells mL^-1), PA14 at a density of 10⁵ cells mL^-1 easily and quickly removed algal cells (100%, 100%, and 97.3% at 8, 16, and 30?days, respectively). Host range assays showed that at lower initial PA14 inoculation (10⁵ cells mL^-1), the algicidal activity of PA14 was effective species-specifically on M. aeruginosa, while at higher initial inoculation (10⁷ cells mL^-1), a wider algicidal range regardless of the general taxonomical relationships was observed. These results indicate that inoculation with 10⁵ Pseudomonas aeruginosa PA14 cells mL^-1 into developing natural algal blooms can remove both M. aeruginosa and MCs without causing problems for other algae species.     

An extracellular matrix protein prevents cytokinesis failure and aneuploidy in the C. elegans germline

Interactions between extracellular matrix (ECM) proteins and their transmembrane receptors mediate cytoskeletal reorganization and corresponding changes in cell shape during cell migration, adhesion, differentiation and polarization. Cytokinesis is the final step in cell division as cells employ a contractile ring composed of actin and myosin to partition one cell into two. Cells undergo dramatic changes in cell shape during the division process, creating new membrane and forming an extracellular invagination called the cleavage furrow. However, existing models of cytokinesis include no role for the ECM. In a recent paper, we demonstrate that depletion of a large secreted protein, hemicentin, results in membrane destabilization, cleavage furrow retraction and cytokinesis failure in C. elegans germ cells and in preimplantation mouse embryos.Here, we demonstrate that cytokinesis failure produces tetraploid intermediate cells with multipolar spindles, providing a potential explanation for the large number of aneuploid progeny observed among C. elegans hemicentin mutant hermaphrodites.Key words: aneuploidy, cytokinesis, extracellular matrix, C. elegans, cleavage furrow, hemicentin, tetraploid intermediateThe karyotype of C. elegans has five autosomes and one or two X chromosomes in males and hermaphrodites, respectively. The majority of self-progeny produced by wild-type hermaphrodites are hermaphrodites (∼99.8%), while rare meiotic nondisjunction of the X chromosome produces nullo-X gametes and 0.2% males. Mutations in over 30 genes result in a 10–150-fold increase in the frequency of males among hermaphrodite self-progeny, due to increases in defects in X chromosome segregation.¹ The majority of these ‘him’ (high incidence of males) loci are genes that encode proteins associated with the intracellular machinery of meiotic chromosome segregation.^2,3 Unique among him genes, the him-4 locus encodes hemicentin, a large, highly conserved component of the extracellular matrix (ECM).⁴ In addition to defects in germline chromosome segregation, him-4 mutants have pleiotropic defects in somatic cell adhesion and migration.^1,4 The extracellular distribution of hemicentin at cell junctions that are defective in him-4 mutants dovetails with current models of cell adhesion and migration.⁵ However, it leaves unexplained several questions about how a secreted ECM component promotes correct chromosome segregation in the C. elegans germline.C. elegans hermaphrodite gonads are composed of two U-shaped tubes, and gametogenesis proceeds sequentially from the distal to the proximal end of each tube. Germ cells in C. elegans have incomplete cleavage furrows that connect them to a central cytoplasmic core, allowing distal cells to act as “nurses” while allowing more mature proximal oocytes to fill with bulk cytoplasm.^6–8 Several genetic and cytogenetic observations suggest a mitotic rather than a meiotic origin for germline chromosome segregation defects observed in the absence of hemicentin.⁴ For example, jackpots of male progeny from individual hermaphrodites and nullisomy of primary meiocytes in him-4 mutants suggest a defect in a mitotic germline stem cell rather than in a post-mitotic process. Our recent work describing hemicentin localization at the cleavage furrows of dividing cells in the early mouse embryo and C. elegans germline, in addition to membrane destabilization, cleavage furrow retraction and cytokinesis failure in the absence of hemicentin, suggests that hemicentin has an evolutionarily conserved role in stabilizing and preventing retraction of nascent cleavage furrows.⁹Aneuploid cells are frequently observed in, and may be associated with the generation of, human tumor cells. Recent work from several laboratories suggests that cytokinesis failure is one of several mechanisms whereby tumor cells generate tetraploid intermediates that result in the production of aneuploid daughter cells in subsequent cell divisions. One proposed mechanism for the generation of aneuploid daughter cells from a tetraploid intermediate is thought to involve multipolar mitotic spindles that result in asymmetric mitoses.^10–13To determine whether a similar mechanism might be responsible for the aneuploidy observed in the absence of hemicentin, him-4 (rh319) animals were examined for multipolar mitotic spindles. A significant fraction (14%) of mitotic germ cells have multipolar spindles that are not observed in a wild-type background (Fig. 1 and Fig. 1).Open in a separate windowFigure 1Multinucleate germ cells and multipolar germ cells observed in the mitotic zone of him-4 mutant hermaphrodites. (A) PH::RFP and histone::GFP in the mitotic region of wild-type (left) and him-4 (rh319) hermaphrodite gonads. Large numbers of multinucleate cells are observed among mitotic germ cells in mutant gonads (arrows). (B) PH::RFP and tubulin::GFP in the mitotic region of wild-type (left) and him-4 (rh319) hermaphrodite gonads. A significant fraction (14^–16.

Table 1

Severity and types of defective germ cells in him-4 gonads

RNA synthesised during oogenesis and early embryogenesis in an insect egg (Euscelis plebejus)

Summary RNA labelled during oogenesis or early embryogenesis was isolated from eggs of the leaf hopperEuscelis plebejus. The polyadenylated RNA fraction deposited during early oogenesis accounted for approximately 2.7% of the total RNA content of the newly laid egg. This fraction differed significantly in molecular weight (15–32 S) from poly(A)-containing RNA synthesised between early cleavage and early germ anlage stages (4–20S). Locally injected³H-uridine spread through the egg within approximately 3 h. A considerable fraction (25–35%) of label injected as³H-uridine during early cleavage was recovered in DNA at subsequent stages (10–20 h later); labelled RNA was not found prior to the cellular blastoderm stage. When the yolk-endoplasm was separated from the blastoderm cells, only the latter contained demonstrable amounts of RNA synthesised by the embryo. Of the precursor incorporated into embryonic RNA, approximately 10% was found in the polyadenylated fraction at the early blastoderm stage, but only 3% at the early germ anlage stage. No differences in size distribution of polyadenylated RNA were evident between anterior and posterior halves of the early germ anlage stage.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

Polyspermic fertilization of sea urchin eggs treated with protease inhibitors: Localization of sperm receptor sites at the egg surface

The normal elevation of the fertilization membrane and the establishment of the block to polyspermy are retarded in Arbacia punctulata eggs by specific protease inhibitors, soybean trypsin inhibitor (SBTI), leupeptin, and antipain. Ultrastructural observations show that the vitelline layer remains attached to the plasma membrane of fertilized SBTI treated eggs at numerous sites (cortical projections). Quantitive morphometric analysis indicates that the vitelline layer elevates from about 65% of the surface of SBTI treated eggs during the first 3 min post insemination. However, the vulnerability of SBTI treated eggs to refertilization (polyspermy) only declined during the subsequent gradual detachment of the vitelline layer from the cortical projections over the next 15 min. Antipain and leupeptin (10^?5 to 10^?3M) also promoted polyspermy in Arbacia eggs by a process of refertilization extending for a 10- to 15-min period after the initial monospermic insemination. Normal cleavage and development was obtained when eggs were placed in leupeptin and antipain (10^?3M) after the fertilization membrane had elevated. The data indicate that the normal secretory function (or functions) of the cortical granule protease in establishing the block to polyspermy is retarded by these protease inhibitors, and that the vitelline layer is transformed into a mechanical barrier to prevent penetration by supernumerary sperm during its detachment from the plasma membrane of the egg. Furthermore, the vitelline layer in unfertilized eggs appears to be a mosaic structure, with sperm receptor sites localized in regions of the egg's surface, which give rise to cortical projections in the presence of SBTI.     

A clonal analysis of the roles of somatic cells and germ line during oogenesis in Drosophila

In order to test whether particular female sterile mutations block functions which normally occur in somatic or germ line derivatives, clones homozygous for each mutation were X-ray induced in heterozygous females. Using the germ line-dependent egg marker, fs(1)K10, it was possible to identify the eggs derived from clones which had been induced in the germ line. Mutations were classified as germ line dependent when these eggs also showed the phenotype associated with the female sterile mutation. Two mutations which caused early abnormalities in oogenesis (fs(1)116, fs(1)1304) were shown to affect germ cells, whereas two mutations which caused egg retention (fs(1)462, fs(1)1001) were somatically dependent. A mutation altering egg dimensions without affecting egg volume (short egg) was also shown to depend on somatic cells in the ovary. With one exception (fs(1)K4), mutations which caused production of fragile, collapsed eggs (fs(1)180, fs(1)473, fs(1)384, and fs(1)1163) were somatically dependent. Patches of mutant fs(1)384 morphology were found in the chorions of the eggs not derived from germ line clones. These patches are interpreted as being caused by homozygous clones in the somatically derived follicle cell epithelium and suggest that fs(1)384 affects processes occurring in these cells during the synthesis of the egg coverings.     

Possibility of membrane reception of neurotransmitter in sea urchin early embryos

1. Methiodide derivatives of serotonin blockers—inmecarb and K.Yur-14 which poorly penetrate the cells specifically affect the pattern of cleavage division in half-embryos of Paracentrotus lividus and Scaphechinus mirabilis.2. Specific [³H]8-OH-DPAT binding under conditions strictly limiting penetration of the ligand into the cells of Strongylocentrotus intermedius was also shown (K_d ∼ 3 × 10⁻¹⁰M for a site with the greatest affinity).3. On the basis of the data obtained it is concluded that neurotransmitter-specific membrane receptors may be present in sea urchin embryos during cleavage divisions. The “protosynapse” hypothesis is proposed which suggests the existence of a specific structure responsible for early blastomere interaction involving transmitters.     

BAX-mediated cell death affects early germ cell loss and incidence of testicular teratomas in Dnd1 mice

A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1^Ter/Ter) results in a significant early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129Sv/J background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost or what factors directly control the strain-specific phenotype variation. To determine the mechanism underlying early PGC loss we crossed Dnd1^Ter/Ter embryos to a Bax-null background and found that germ cells were partially rescued. Surprisingly, on a mixed genetic background, rescued male germ cells also generated fully developed teratomas at a high rate. Double-mutant females on a mixed background did not develop teratomas, but were fertile and produced viable off-spring. However, when Dnd1^Ter/Ter XX germ cells developed in a testicular environment they gave rise to the same neoplastic clusters as mutant XY germ cells in a testis. We conclude that BAX-mediated apoptosis plays a role in early germ cell loss and protects from testicular teratoma formation on a mixed genetic background.     

Rates of histone synthesis during early development of Rana pipiens

Newly synthesized histones have been extracted from Rana pipiens oocytes or cleaving embryos previously injected with [³H]lysine or [³H]arginine. The radioactive proteins were fractionated by cation-exchange chromatography and electrophoresis on acid/urea or SDS-polyacrylamide gels; histones were identified by coelectrophoresis with authentic markers. From percentage total incorporation in the putative histones, and absolute rates of lysine or arginine incorporation, rates of histone synthesis were estimated. Rates of histone synthesis in two-cell embryos were at least 10-fold higher than in maturing oocytes. Between the two-cell and blastula stages, the rate increased an additional threefold, from about 1200 pg hr^?1 per embryo to about 4500 pg hr^?1 per embryo. While all histone classes are synthesized during cleavage, synthesis of the various classes is not coordinated; histones are not synthesized in the same relative proportions at which they are found in blastula chromatin. The synthesis of histone H4 in particular is barely detectable during cleavage. This, and other observations, suggested the existence of cytoplasmic histone pools. In approaching the possible existence of histone pools, the amount of H4 present in oocytes was determined. Oocytes contain about 74 ng of H4, an amount sufficient to allow development to the blastula stage. These data are compared to those reported by others on histone synthesis during cleavage in Xenopus.     

Analysis of the myogenic lineage in chick embryos: I. Studies on the terminal cell division

An antibody prepared against the MM isozyme of creatine phosphokinase (M-CK) stained multinucleated myotubes and post-mitotic mononucleated myoblasts in mass cultures of myogenic cells taken from the breast muscles of 11-day chick embryos. No cycling cells bound the antibody. Single cells isolated either directly from the embryo or from mass cultures were seeded at clonal density and allowed to undergo one division. The resulting pairs of cells were stained with the antibody and were scored as (a) both members of the pair M-CK⁺; (b) both M-CK^?; or (c) mixed (one M-CK⁺ and one M-CK^?). No mixed pairs were observed. Conditioned medium did not induce all myogenic pairs to differentiate and growth medium did not keep myogenic pairs in the cell cycle. About 10% of clonal pairs established from 10 h cultures were M-CK⁺, while about 27% of pairs established from 30 h mass cultures were M-CK⁺. These results indicate that (1) the myogenic lineage ends in a symmetrical division whose products are two post-mitotic M-CK⁺ cells; (2) the expression of the muscle phenotype is not determined exclusively by the environment; (3) the terminal cells are the product of an intrinsic program or cell lineage in which only the last cells can synthesize muscle-specific proteins.     

NEW MEMBRANE FORMATION DURING CYTOKINESIS IN NORMAL AND CYTOCHALASIN B-TREATED EGGS OF XENOPUS LAEVIS : II. Electrophysiological Observations

The electrical membrane potential (E_m) and electrical membrane resistance (R_m) were measured continuously during the first cleavage of Xenopus eggs, using intracellular microelectrodes. A sharp hyperpolarization of E_m and decrease in R_m can be observed from 6 to 8 min after the onset of cleavage. This moment coincides with the onset of the insertion of new membrane (Bluemink and de Laat, 1973) leading to the formation of the interblastomeric membrane during normal cleavage. Removal of the vitelline membrane or exposure to cytochalasin B (CCB) leads to exposure of the entire surface area of the membrane newly formed during cleavage. These conditions allow for a direct measurement of the permeability properties of the new membrane. It was found that under these conditions E_m reaches values about 3 times more negative and R_m reaches values about 1.5–3 times smaller than during normal cleavage. The extent of reduction of R_m can be correlated with the surface area of the newly formed membrane. We conclude that the new membrane has different ionic permeability properties than the pre-existing membrane (most probably a relatively high permeability for K⁺ ions). Its mean specific resistance is 1–2 kΩ·cm², as against 74 kΩ·cm² for the pre-existing membrane. No influence of CCB on the permeability properties of the pre-existing or new membrane could be detected.     

Evidence for the origin of the unoccupied oestrogen receptor in nuclei of a human breast-cancer cell line (MCF-7)

The origin of the unoccupied nuclear oestrogen receptor (R_n) was studied. Three working hypotheses were investigated. (a) R_n is a dissociation product of the oestrogen occupied nuclear receptor (ER_n). (b) ER_n is only partially occupied, so that additional binding may occur at 0°C (the temperature at which oestradiol saturates unoccupied sites). (c) R_n is derived from the penetration of unoccupied cytoplasmic receptor (R_c) into the nucleus. The MCF-7 cell line was used as a model in the present investigation. The amount of unoccupied receptors was measured by saturation with 7.5nm-[³H]oestradiol at 0°C, whereas the occupied receptors were measured by exchange at 30°C. The cells at preconfluency were exposed to a medium fortified with 10nm-[³H]oestradiol for 1h, washed and cultured up to 5 days in fresh growth medium. The distribution of oestradiol receptors was determined before exposure and during the following 5 days. After 1h exposure only ER_n was found in the nuclear fraction. Thereafter ER_n declined continuously so that on day 5 it approached 15% of its value measured 1h after exposure. Although after 3 days about 80% of ER_n disappeared no R_n appeared, which contradicts hypotheses (a) and (b). On day 4 R_n and R_c appeared simultaneously. The appearance of R_n and R_c was not prevented by culturing the cells in an oestrogen-free medium, supporting hypothesis (c). Exposure of cells to increasing concentration of [³H]oestradiol (0.1–10nm) for 1h resulted in a parallel increase in ER_n without increasing the amount of unoccupied binding sites, which contradicts hypothesis (b). The present study supports the hypothesis (c), i.e., R_c may also penetrate the nucleus without binding to oestradiol.     

Some peculiarities of the outer membrane composition of marine Gram-negative bacterium Chryseobacterium indoltheticum CIP 103168T

Some peculiarities of the lipid composition of the outer membrane of the marine Gram-negative bacterium Chryseobacterium indoltheticum CIP 103168^T have been studied. It is found that C. indoltheticum cells do not contain phosphatidylglycerol. Lipopolysaccharide could not be extracted by conventional methods; therefore, lipid A was isolated by hydrolysis of whole bacterial cells with 10% acetic acid with subsequent treatment with a chloroform: methanol mixture. The isolated lipid A has an unusual monosaccharide structure and constitutes 1-phosphate-D-glucosamine acylated with (R)-3-hydroxy-15-methylhexadecanoic and (R)-3-hydroxy-13-methyltetradecanoic acids at C2 and C3 atoms, respectively. The C. indoltheticum cells are shown to have a capsule; its polysaccharide apparently fulfils the LPS function.     

The Ser176 of T4 endonuclease IV is crucial for the restricted and polarized dC-specific cleavage of single-stranded DNA implicated in restriction of dC-containing DNA in host Escherichia coli

Endonuclease (Endo) IV encoded by denB of bacteriophage T4 is an enzyme that cleaves single-stranded (ss) DNA in a dC-specific manner. Also the growth of dC-substituted T4 phage and host Escherichia coli cells is inhibited by denB expression presumably because of the inhibitory effect on replication of dC-containing DNA. Recently, we have demonstrated that an efficient cleavage by Endo IV occurs exclusively at the 5′-proximal dC (dC₁) within a hexameric or an extended sequence consisting of dC residues at the 5′-proximal and the 3′-proximal positions (dCs tract), in which a third dC residue within the tract affects the polarized cleavage and cleavage rate. Here we isolate and characterize two denB mutants, denB(W88R) and denB(S176N). Both mutant alleles have lost the detrimental effect on the host cell. Endo IV(W88R) shows no enzymatic activity (<0.4% of that of wild-type Endo IV). On the other hand, Endo IV(S176N) retains cleavage activity (17.5% of that of wild-type Endo IV), but has lost the polarized and restricted cleavage of a dCs tract, indicating that the Ser¹⁷⁶ residue of Endo IV is implicated in the polarized cleavage of a dCs tract which brings about a detrimental effect on the replication of dC-containing DNA.     

The ontogeny of germ plasm during oogenesis in Drosophila

Primordial germ cells can be induced at both the anterior and ventral region of the Drosophila egg by transplanted posterior polar plasm. Two questions arise from these results: (1) Is fertilization required for germ plasm to be functional, and (2) at what stage during oogenesis does the posterior polar plasm become established as a germ-cell determinant?Polar plasm from unfertilized eggs and from oocytes at stage 10 to 14 of Drosophila melanogaster was implanted into the anterior region of cleavage embryos. Some injected embryos were analyzed at the ultrastructural level during blastoderm formation. Polar plasm from unfertilized eggs and from oocytes of stages 13 and 14 was found to be integrated into several anterior cells that resembled morphologically normal pole cells. The formation of such cells, however, could not be detected in embryos injected with polar plasm from oogenetic stages 10 to 12. Experimentally induced pole cells proved to be capable of differentiating into functional germ cells when cycled through the germ line of genetically different host embryos. About 5% of the flies developing from these embryos produced progeny that originated from the induced pole cells. Germ-line mosaicism in those flies also could be detected histochemically in their gonads. No germ cells were recovered with polar plasm transplants from oogenetic stages 10 to 12.The results show that posterior polar plasm of the unfertilized egg is functional in germ-cell determination, and that prior to egg maturation this cytoplasm has already acquired its determinative ability. This is the first demonstration that specific developmental information stored in the cytoplasm can be traced back to a particular region of the oocyte.     

Long-term surveillance of sulfate-reducing bacteria in highly saline industrial wastewater evaporation ponds

Abundance and seasonal dynamics of sulfate-reducing bacteria (SRB), in general, and of extreme halophilic SRB (belonging to Desulfocella halophila) in particular, were examined in highly saline industrial wastewater evaporation ponds over a forty one month period. Industrial wastewater was sampled and the presence of SRB was determined by quantitative real-time PCR (qPCR) with a set of primers designed to amplify the dissimilatory sulfite reductase (dsrA) gene. SRB displayed higher abundance during the summer (10⁶–10⁸ targets ml^-1) and lower abundance from the autumn-spring (10³–10⁵ targets ml^-1). However, addition of concentrated dissolved organic matter into the evaporation ponds during winter immediately resulted in a proliferation of SRB, despite the lower wastewater temperature (12–14°C). These results indicate that the qPCR approach can be used for rapid measurement of SRB to provide valuable information about the abundance of SRB in harsh environments, such as highly saline industrial wastewaters. Low level of H₂S has been maintained over five years, which indicates a possible inhibition of SRB activity, following artificial salination (≈16% w/v of NaCl) of wastewater evaporation ponds, despite SRB reproduction being detected by qPCR.