Influence of RET/PTC1 and RET/PTC3 oncoproteins in radiation-induced papillary thyroid carcinomas on amounts of cytoskeletal protein species

Radiation-induced human papillary thyroid carcinomas (PTCs) show a high prevalence of fusions of the RET proto-oncogene to heterologous genes H4 (RET/PTC1) and ELE1 (RET/PTC3), respectively. In contrast to the normal membrane-bound RET protein, aberrant RET fusion proteins are constitutively active oncogenic cytosolic proteins that can lead to malignant transformation of thyroid epithelia. To detect specific tumor-associated protein changes that reflect the effect of RET/PTC fusion proteins, we analyzed normal thyroid tissues, thyroid tumors of the RET/PTC1 and RET/PTC3 type and their respective lymph node metastases by a combination of high-resolution two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-mass spectrometry. PTCs without RET rearrangements served as controls. Several cytoskeletal protein species showed quantitative changes in tumors and lymph node metastases harboring RET/PTC1 or RET/PTC3. We observed prominent C-terminal actin fragments assumedly generated by protease cleavages induced due to enhanced amounts of the active actin-binding protein cofilin-1. In addition, three truncated vimentin species, one of which was proven to be headless, were shown to be highly abundant in tumors and metastases of both RET/PTC types. The observed protein changes are closely connected with the constitutive activation of RET-rearranged oncoproteins and reflect the importance to elucidate disease-related typical signatures on the protein species level.     

Xyloglucan−pectin linkages are formed intra-protoplasmically,contribute to wall-assembly,and remain stable in the cell wall

We tested two hypotheses for the mechanism by which xyloglucan–pectin covalent bonds are formed in Arabidopsis cell cultures. Hypothesis 1 proposed hetero-transglycosylation, with xyloglucan as donor substrate and a rhamnogalacturonan-I (RG-I) side-chain as acceptor. We looked for enzyme activities that catalyse this reaction using α-(1→5)-l-[³H]arabino- or β-(1→4)-d-[³H]galacto-oligosaccharides as model acceptor substrates. The ³H-oligosaccharides were supplied (with or without added xyloglucans) to living Arabidopsis cell-cultures, permeabilised cells, cell-free extracts, or four authentic XTHs. No hetero-transglycosylation occurred. Therefore, we cannot support hypothesis 1. Hypothesis 2 proposed that some xyloglucan is manufactured de novo as a side-chain on RG-I. To test this, we pulse-labelled Arabidopsis cell-cultures with [³H]arabinose and monitored the radiolabelling of anionic (pectin-bonded) xyloglucan, which was resolved from free xyloglucan by ion-exchange chromatography. [³H]Xyloglucan–pectin complexes were detectable <4 min after [³H]arabinose feeding, which is shorter than the transit-time for polysaccharide secretion, indicating that xyloglucan–pectin bonds were formed intra-protoplasmically. Thereafter, the proportion of the wall-bound [³H]xyloglucan that was anionic remained almost constant at ∼50% for ≥6 days, showing that the xyloglucan–pectin bond was stable in vivo. Some [³H]xyloglucan was rapidly sloughed into the medium instead of becoming wall-bound. Only ∼30% of the sloughed [³H]xyloglucan was anionic, indicating that bonding to pectin promoted the integration of xyloglucan into the wall. We conclude that ∼50% of xyloglucan in cultured Arabidopsis cells is synthesised on a pectic primer, then secreted into the apoplast, where the xyloglucan–pectin bonds are stable and the pectic moiety aids wall-assembly.     

A Genome-Wide Association Study Identifies Potential Susceptibility Loci for Hirschsprung Disease

Hirschsprung disease (HSCR) is a congenital and heterogeneous disorder characterized by the absence of intramural nervous plexuses along variable lengths of the hindgut. Although RET is a well-established risk factor, a recent genome-wide association study (GWAS) of HSCR has identified NRG1 as an additional susceptibility locus. To discover additional risk loci, we performed a GWAS of 123 sporadic HSCR patients and 432 unaffected controls using a large-scale platform with coverage of over 1 million polymorphic markers. The result was that our study replicated the findings of RET-CSGALNACT2-RASGEF1A genomic region (_rawP = 5.69×10⁻¹⁹ before a Bonferroni correction; _corrP = 4.31×10⁻¹³ after a Bonferroni correction) and NRG1 as susceptibility loci. In addition, this study identified SLC6A20 (_adjP = 2.71×10⁻⁶), RORA (_adjP = 1.26×10⁻⁵), and ABCC9 (_adjP = 1.86×10⁻⁵) as new potential susceptibility loci under adjusting the already known loci on the RET-CSGALNACT2-RASGEF1A and NRG1 regions, although none of the SNPs in these genes passed the Bonferroni correction. In further subgroup analysis, the RET-CSGALNACT2-RASGEF1A genomic region was observed to have different significance levels among subgroups: short-segment (S-HSCR, _corrP = 1.71×10⁻⁵), long-segment (L-HSCR, _corrP = 6.66×10⁻⁴), and total colonic aganglionosis (TCA, _corrP>0.05). This differential pattern in the significance level suggests that other genomic loci or mechanisms may affect the length of aganglionosis in HSCR subgroups during enteric nervous system (ENS) development. Although functional evaluations are needed, our findings might facilitate improved understanding of the mechanisms of HSCR pathogenesis.     

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[³H]glucosamine, [³⁵S]sulfate, and L-[¹⁴C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[³H]glucosamine, but not of [³⁵S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[¹⁴C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect on D-[³H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[³H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures (∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.     

Bisphenol A at a human exposed level can promote epithelial-mesenchymal transition in papillary thyroid carcinoma harbouring BRAFV600E mutation

Bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical, alters the function of endocrine system and enhances the susceptibility to tumorigenesis in several hormone-dependent tumours as thyroid carcinoma. About 50% of papillary thyroid cancers (PTC), the most common type of thyroid malignancy, harbours the BRAF^V600E mutation. This study aimed to investigate a potential combined effect of BPA exposure and BRAF^V600E mutation on epithelial-mesenchymal transition (EMT) in PTC. Firstly, the level of BPA in plasma, the evaluation of BRAF^V600E mutation and the level of EMT-related proteins in PTC samples were individually determined. Additionally, the migration, invasion, colony formation capacity and the expression of EMT-related proteins after exposure to BPA were precisely analysed in vitro thyroid cells genetically modified by the introduction of BRAF^V600E mutation. Moreover, ERK-Cox2 signalling pathway was also introduced to explore the possible mechanism in PTC development. As expected, whether the clinical investigation or cultured thyroid cells demonstrated that BPA at a concentration compatible with human exposed levels (10^-7 M) synergized with the BRAF^V600E mutation promoted EMT via the activation of ERK-Cox2 signalling pathway. Our findings offer some evidence that BPA as an environmental risk factor can facilitate the progression of PTC harbouring BRAF^V600E mutation.     

Molecular Analysis of Structural Abnormalities in Papillary Thyroid Carcinoma Genome

Rearrangements of the protooncogene RET (RET/PTC) and somatic mutations of the gene BRAF are the most common events in the etiopathogenesis of papillary thyroid carcinoma (PTC). The rates of RET/PTC rearrangements and BRAF mutations in different nodular formations of the thyroid gland (TG) have been estimated. Comparative expression analysis of the extracellular (EC) and tyrosine kinase (TK) domains of RET has shown that 14% (12 out of 85) of PTC cases are RET/PTC-positive, including one RFP/RET-, two RET/PTC3-, and seven RET/PTC1-positive tumors, as well as two unidentified chimeric RET/PTC oncogenes. The standard T1796A transversion in the gene BRAF has been found in 60% (55 out of 91) PTC cases with the use of amplification refractory mutation system–polymerase chain reaction (ARMS–PCR). Somatic mutation G1753A and deletion del1800_1811 have been identified in PTC for the first time. The absence of the BRAF mutations in RET/PTC-positive tumors allows these two genetic defects to be regarded as alternative mechanisms of the RAS–RAF–MEK–ERK mitogen-activated protein (MAP) kinase cascade activation in PTCs. In none of the three follicular thyroid carcinomas (FTCs), 11 follicular thyroid adenomas (FTAs), and 13 nodular goiters have either BRAF mutations or RET/PTC rearrangements been found. Thus, the RET/PTC chimeric oncogenes and BRAF somatic mutations are specific markers of PTC and can be used for the preoperative diagnosis of these tumors.     

Transport, degradation and cell wall-integration of XXFGol, a growth-regulating nonasaccharide of xyloglucan, in pea stems

When [glucitol-³H]XXFGol (a NaB³H₄-reduced xyloglucan nonasaccharide) was applied to excised shoots of pea (Pisum sativum L. cv. Progress) at the base of the epicotyl, it inhibited growth in the elongation zone, 4–5 cm distal. Experiments were conducted to discover whether such ³H-oligosaccharides are translocated intact over this distance, or whether an intercellular second messenger would have to be postulated. After 24 h, ³H from [glucitol-³H]XXFGol and [glucitol-³H]XXXGol showed U-shaped distributions, with most ³H at the base and apex of the stem. Radioactivity from [fucosyl-³H]XXFG and [xylosyl-³H]XXFG also moved acropetally, but did not concentrate at the apex, possibly owing to removal from the transpiration stream of fucose and xylose formed by partial hydrolysis of XXFG en route. When 10⁻⁷ M [glucitol-³H]XXFGol was supplied, about 14 fmol ·  seedling^–1 of apparently intact [³H]XXFGol was extractable from the elongation zone after 24 h. Larger amounts of degradation products were extractable (including free [³H]glucitol) and some wall-bound ³H-hemicellulose was present (presumably formed by the oligosaccharides acting as acceptor substrates for transglycosylation). We conclude that biologically active oligosaccharides of xyloglucan can move through the stem acropetally and that they are maintained at low steady-state concentrations by both hydrolysis and transglycosylation. Received: 1 April 1997 / Accepted: 28 May 1997     

Biologically based risk estimation for radiation-induced CML

Chronic myeloid leukemia (CML) invites biologically based radiation risk modeling because CML is simultaneously well-understood, homogeneous and prevalent. CML is known to be caused by a translocation involving the ABL and BCR genes, almost all CML patients have the BCR-ABL translocation, and CML is prevalent enough that its induction is unequivocally detected among Hiroshima A-bomb survivors. In a previous paper, a linear-quadratic-exponential (LQE) dose- response model was used to estimate the lifetime excess risk of CML in the limit of low doses of γ-rays, R _γ. This estimate assumed that BCR-ABL translocation dose- response curves in stem cells for both neutrons and γ-rays, differ only by a common proportionality constant from dicentric aberration dose-response curves in lymphocytes. In the present paper we challenge this assumption by predicting the BCR-ABL dose response. The predictions are based on the biophysical theory of dual radiation action (TDRA) as it applies to recent BCR-to-ABL distance data in G₀ human lymphocytes; this data shows BCR and ABL geometric distributions that are not uniform and not independent, with close association of the two genes in some cells. The analysis speaks against the previous proportionality assumption. We compute 11 plausible LQE estimates of R _γ, 2 based on the proportionality assumption and 9 based on TDRA predictions. For each estimate of R _γ we also compute an associated estimate of the number of CML target cells, N; the biological basis of the LQE model allows us to form such estimates. Consistency between N and hematological considerations provides a plausibility check of the risk estimates. Within the group of estimates investigated, the most plausible lifetime excess risk estimates tend to lie near R _γ=0.01 Gy^–1, substantially higher than risk estimates based on the proportionality assumption. Received: 10 August 2000 / Accepted: 20 December 2000     

Physical properties of two types of calcium stores and SERCAs in human platelets

The aim of this work was to obtain experimental data depending on the properties of calcium stores and SERCAs, to analyse these data in terms of the models based on simulation of the cellular compartments as communicating vessels, and to relate this way the data to the above properties. The main characteristics of the stores and corresponding SERCAs have been estimated. Calcium content in the DTS is ∼1.5 × 10⁶ ions per cell, that in the acidic stores, ∼0.64 × 10⁶ ions per cell. The rate constant of background calcium leakage from the DTS is ∼0.0033 s⁻¹, that from the acidic stores, ∼0.1 s⁻¹. The background activity of SERCA2b is ∼0.22 × 10⁶ s⁻¹ ions per cell, that of SERCA3, ∼2.5 × 10⁶ s⁻¹ ions per cell. The properties of both calcium stores and the SERCAs and the characteristics found might be related to physiological or pathological state of the cells.     

Loci controlling lymphocyte production of interferon γ after alloantigen stimulation in vitro and their co-localization with genes controlling lymphocyte infiltration of tumors and tumor susceptibility

Low infiltration of lymphocytes into cancers is associated with poor prognosis, but the reasons why some patients exhibit a low and others a high infiltration of tumors are unknown. Previously we mapped four loci (Lynf1–Lynf4) controlling lymphocyte infiltration of mouse lung tumors. These loci do not encode any of the molecules that are involved in traffic of lymphocytes. Here we report a genetic relationship between these loci and the control of production of IFNγ in allogeneic mixed lymphocyte cultures (MLC). We found that IFNγ production by lymphocytes of O20/A mice is lower than by lymphocytes of OcB-9/Dem mice (both H2 ^pz ) stimulated in MLC by irradiated splenocytes of C57BL/10SnPh (H2 ^b ) or BALB/cHeA (H2 ^d ) mice, or by ConA. IFNγ production in MLCs of individual (O20 × OcB-9)F₂ mice stimulated by irradiated C57BL/10 splenocytes and genotyped for microsatellite markers revealed four IFNγ-controlling loci (Cypr4-Cypr7), each of which is closely linked with one of the four Lynf loci and with a cluster of susceptibility genes for different tumors. This suggests that inherited differences in certain lymphocyte responses may modify their propensity to infiltrate tumors and their capacity to affect tumor growth.     

Mathematical interpretation of dose-response curves

The dose-response of an individual organism can be described by a step functions if the organism survives when the dose is below a certain lethal level and dies when this level is exceeded. If, in a population of organism, the lethal dose for an individual has a unimodal distribution, the latter's properties will determine the shape of the population's response in the following manner. If the distribution is symmetric the dose-response curve has a symmetric sigmoid shape when plotted on linear coordinates. The location of the inflection point and the curve's slope around it are determined by the distribution's mode and variance. When the distribution is skewed, the dose-response curve has an asymmetric sigmoid shape which becomes reminiscent of an exponential decay when the distribution is strongly skewed to the right. The population's dose-response curve can be constructed by integration of the step changes over the distribution range. The step function representing the dose-response of an individual organism can be approximate by a Fermi function, and the distribution of an lethal doses can be represented by the Weibull distribution function. When the two functions are combined, the resulting dose-response of the populationS(X)), which is the fraction of survivors after exposure to a doseX, is given by:S(X)=∫ ₀ ¹ [1/{+exp{(X-X _c (φ))/a _i ]}]dω whereX _c (ω)={(1/b)[-ln(1-ω)]}^(1/n),n andb being the constants of the Weibull distribution anda _i an arbitrarily small number, i.e.a _i ≪[X−X _c (ϕ)], whose actual magnitude is of little significance. This model can be used to determine the underlying distributions of experimental dose-response relationship. It was applied to published survival data of microorganisms exposed to pulsed electric field, X-ray radiation and ozone to show that the different observed shapes of the dose-response curve, and shifts between them, can be expressed in terms of the correponding distribution parameters, namely the mode, variance and skewness.     

Comparative characterization of H2 production by the conventional Mo nitrogenase and the alternative “iron-only” nitrogenase of Rhodobacter capsulatus hup - mutants

 Cell suspensions of uptake-hydrogenase-deficient (hup ^-) mutants of a wild-type (B10S) and a nifHDK deletion strain of Rhodobacter capsulatus were used comparatively to characterize the conventional, Mo-containing and the alternative, “iron-only” nitrogenase of this organism by determining the H₂ production and acetylene reduction activities under argon and dinitrogen atmospheres. A comparison with the corresponding hup ⁺ strains revealed that the hup ^- mutation did not affect the nitrogenase activity and specificity within the acetylene-reduction assay, but caused a significant increase in H₂ production, which was more prominent in the case of the ΔnifHDK strain. The ΔnifHDK hup ^- cells, grown in Mo-depleted medium and, thus, expressing the alternative nitrogenase system, were more than ten times less active in the acetylene-reduction assay but exhibited H₂ production rates equivalent to about 60% of the rates obtained with B10S hup ^- cells after growth in a medium containing 10 μM MoO^- ₄. When these conditions were applied, the B10S strain only expressed the Mo nitrogenase. Under an argon atmosphere containing about 5.5% (v/v) acetylene and under a dinitrogen atmosphere, ΔnifHDK hup ^- cells produced H₂ at even higher rates than B10S hup ^- cells. The implications of our findings on a possible biotechnological H₂ production and on the mechanism of nitrogenase catalysis are considered. Received: 24 February 1996/Received revision: 15 May 1996/Accepted: 19 May 1996     

Radioactive contamination of the marine environment: Uptake and distribution of3H inDunaliella bioculata

The marine flagellateDunaliella bioculata, which is easily cultivated under laboratory conditions, is a suitable organism for assessing the importance of the radioactive contamination by³H bound to organic molecules. We have studied the uptake of the following tritiated precursors: thymidine-methyl-³H, adenine-2-³H, uridine-5-³H, l-leucine-4-³H, glycine-2-³H, l-arginine-3.4-³H, 1-aspartic acid-2. 3-³H, 1-phenylalanine-2.3-³H, D-glucose-2-³H and D-glucose-6-³H. Under the experimental conditions (2000 lux; incubation time 30 min), all tritiated molecules are taken up byD. bioculata. Their intracellular concentration may reach that of the external medium. However, leucine and adenine accumulate in the algae: their respective concentrations are 10 and 100 times higher than in the culture medium. The molecular distribution of³H has been studied by various biochemical techniques and by sieve chromatography on sepharose 4B. It has been found that more l-leucine-4-³H is incorporated into acid and acetone soluble substances than into proteins. Adenine-2-³H is mainly incorporated into macromolecules of biological significance (RNA, DNA). CsCl gradient centrifugation has shown that the total DNA ofDunaliella is constituted by a major (ϖ=1.707 g/cm³) and by a minor (ϖ=1.693 g/cm³) component. Fellow of the Commission of the European Communities     

Subpopulations of cancer stem cells found in papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) is the most common form of thyroid cancer and while it has a generally good prognosis, tumor recurrence remains a major clinical challenge. Studying laboratory cell lines as well as clinical specimens indicate that PTC may follow the cancer stem cell (CSC) model. However, CSC characteristics relevant in PTC initiation and progression remain largely unknown. Here we studied a population of sphere-growing tumor cells isolated from primary cultures of clinical PTC. These sphere-growing cells consisted of aldehyde dehydrogenase positive (ALDH⁺) and ALDH negative (ALDH^-) cell subpopulations and demonstrated a hierarchical pattern of cell division. Using combinations of selective depletion, specific inhibition and cell sorting, we found that both subpopulations of the sphere cells were able to self-renew and initiate xenograft tumors independently, and fulfilled the definition of CSC. Importantly, when the subpopulations functioned together, the cancer-initiation efficiency and the xenograft tumor progression were significantly enhanced compared to either subpopulation alone. These data revealed crucial roles of ALDH^- CSC in PTC biology and suggested that CSC subpopulations function cooperatively to control PTC initiation and progression. Together, our study indicates that CSC subpopulations isolated from clinical specimens offer unprecedented opportunities for investigating PTC pathogenesis and developing effective therapies.     

Association of RET Genetic Polymorphisms and Haplotypes with Papillary Thyroid Carcinoma in the Portuguese Population: A Case-Control Study

Thyroid cancer has a multifactorial aetiology resulting from the interaction of genetic and environmental factors. Several low penetrance susceptibility genes have been identified but their effects often vary between different populations. Somatic point mutations and translocations of the REarranged during Transfection (RET) proto-oncogene are frequently found in thyroid cancer. The aim of this case-control study was to determine the effect of four well known RET single nucleotide polymorphisms (SNPs) on the risk for differentiated thyroid carcinoma. A total of 545 Portuguese patients and 543 controls were genotyped by PCR and restriction enzyme analysis, for the following SNPs: G691S (exon 11, rs1799939 G/A), L769L (exon 13, rs1800861 T/G), S836S (exon 14, rs1800862 C/T), and S904S (exon 15, rs1800863 C/G). The minor allele of S836S was overrepresented in patients with papillary thyroid carcinoma (PTC) when compared to controls (OR 1.57; 95% CI 1.05–2.35; p = 0.026). The GGTC haplotype was also overrepresented in PTC (OR 2.51; 95% CI 1.07–5.91; p = 0.029). No associations were found in follicular thyroid carcinoma (FTC). Multivariate logistic regression analysis showed no differences regarding gender, age at diagnosis, lymph node or distant metastasis. However, a near significant overrepresentation of the minor alleles of G691S and S904S was found in patients with tumours greater than 10 mm of diameter at diagnosis. These data suggest that the RET S836S polymorphism in exon 14 and the GGTC haplotype are risk factors for PTC, but not FTC, and that the G691S/S904S polymorphisms might be associated with tumour behaviour.     

Changes in steady state on introduction of a Lactobacillus contaminant to a continuous culture ethanol fermentation

Lactobacillus paracasei was introduced as a contaminant into a multistage continuous culture ethanol fermentation system at ratios of 1:100, 1:1, and 70:1 with Saccharomyces cerevisiae, but failed to overtake the yeast. None of the inoculation ratios allowed L. paracasei to affect S. cerevisiae in the first fermentor in the multistage system. S. cerevisiae remained constant at ∼3×10⁷ CFU/ml regardless of the bacterial inoculation level, and even at the 70:1 inoculation ratio, glucose, ethanol, and lactic acid concentrations did not change from the steady-state concentrations seen before bacterial inoculation. However, L. paracasei decreased steadily from its initial inoculation level of ∼2.2×10⁹ CFU/ml and stabilized at 3.7×10⁵ CFU/ml after 10 days of steady-state operation. Both organisms then persisted in the multistage system at an approximate L. paracasei/S. cerevisiae ratio of 1:100 which confirms that, in continuous fuel ethanol production, it would be difficult to eliminate this bacterium. Only when the pH was controlled at 6.0 in fermentor 1 (F1) were changes seen which would affect the multistage system. Ethanol concentration then decreased by 44% after 4 days of pH-controlled operation. This coincided with an increase in L. paracasei to >10¹⁰ CFU/ml, and a 4× increase in lactic acid concentration to 20 g/l. When the clarified contents from other fermentors (F2–F5) in the multistage system were used as growth media, L. paracasei was not able to grow in batch culture. This indicated that the first fermentor in the multistage system was the only fermentor capable of supporting the growth of L. paracasei under the described conditions. Journal of Industrial Microbiology & Biotechnology (2001) 27, 39–45. Received 26 February 2001/ Accepted in revised form 29 May 2001     

Shooting of sporidium by "gun" cells in <Emphasis Type="Italic">Haptoglossa heterospora</Emphasis> and <Emphasis Type="Italic">H. zoospora</Emphasis> and secondary zoospore formation in <Emphasis Type="Italic">H. zoospora</Emphasis>

Haptoglossa spp. (Lagenidiales, Oomycetes) have been known to parasitize microscopic animals by means of a "gun" cell that shoots an infection cell, named the sporidium, into the body of the animal. A thallus grown from the sporidium changes into a zoosporangium at maturation to produce a number of zoospores that encyst after a swarming period, and the resulting cysts germinate to produce gun cells. In Haptoglossa zoospora, endoparasitic in nematodes, the cysts of primary zoospores that swam for about 5 min did not develop gun cells but produced secondary zoospores that swam for about 3 h. After encystment of the secondary zoospores, each secondary cyst germinated to produce a gun cell. In the present study, the secondary zoospores of the genus Haptoglossa could be recorded with a videocassette recorder for the first time. The videocassette recording also revealed the infection of a nematodes by H. zoospora and H. heterospora to be composed of two steps of injection of a sporidium by the gun cell, in which the gun cell came in contact with the cuticle of a nematode and produced a spherical adhesorium on the tip of the cell in 0.07–0.1 s in both species. The adhesorium was ∼2 μm in H. zoospora and ∼4 μm in H. heterospora. When the adhesorium infiated to full size, it shot the sporidium into the nematode's body in 0.5–0.65 s and in 0.2–0.5 (or rarely 1.0) s in H. zoospora and H. heterospora, respectively. After shooting, the empty gun cell with an empty cyst case was separated from the cuticle immediately in both species. Received: October 3, 2001 / Accepted: December 13, 2001     

Synthesis of novel selenium containing sulfa drugs and their antibacterial activities

Synthesis of 3-[4-(N-substituted sulfamoyl)phenyl]-3,4-dihydro-4-oxo-7,9-dimethylpyri-do[3′,2′:4,5]selenolo[3,2-d]pyrimidines,7-[4-(N-substituted sulfamoyl)phenyl]-7,8-dihydro-8-oxo-3,4-diphenylpyrimido[4′,5′:4,5]selenolo [2,3-c]pyridazines and 1-[4-(N-substituted sulfamoyl)phenyl]-1,11-dihydro 11-oxo-4-methylpyrimido[4′,5′:4,5]selenolo[2,3-b]quinolines is reported. 4-Amino-N-pyrimidine-2-ylbenzene sulfonamide (a), 4-amino-N-(2,6-dimethylpyrimidin-4-yl)benzene sulfonamide (b), N-[(4-aminophenyl)sulfonyl] acetamide (c) with N-ethoxymethyleneamino of selenolo pyridine, selenolo pyridazine and selenolo quinoline derivatives respectively were obtained starting from 1-amino-N ⁴-substituted sulfanilamides. Spectroscopic data (IR, ¹H NMR, ¹³C NMR and Mass spectral) confirmed the structure of the newly synthesized compounds. Substituted pyrimidines, pyridazines and quinolines were screened for antibacterial activity against gram-positive and gram-negative bacteria. Selenolo derivative of N-[(4-aminophenyl)sulfonyl] acetamide (substitutent of sulfacetamide c) showed strong bactericidal effect against all the tested organisms. Selenolo[3,2-d]pyrimidin (substitutent a) showed a good bactericidal effect against Serratia marcescens, Staphylococcus aureus and Escherichia coli. Compounds selenolo[2,3-c]pyridazine (substitutent b), selenolo[2,3-b]quinoline(substitutents c)) exhibited a moderate bactericidal effect against Serratia marcescens. None of the synthesized seleno pyridazines has a considerable antimicrobial activity against the tested organisms. The minimum inhibitory concentration (MIC) of the most active compound-3-[4-(N-acetyl sulfamoyl)phenyl]-3,4-dihydro-4-oxo-7,9-dimethylpyrido[3′,2′:4,5]selenolo [3,2-d]pyrimidine was 10 mg ml⁻¹.     

The synthesis of <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine heteroaromatic <Emphasis Type="Italic">N</Emphasis>-β-glycosides under phase transfer conditions: Part III. 1,2,4-triazolin-3-one glucosaminides

Reactions of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl chloride with thiosubstituted 5-thio-4-phenyl-Δ²-l,2,4-triazolin-3-ones were studied in solid alkali-organic solvent and aqueous alkali-organic solvent catalytic phase transfer systems. The major products of glucosaminylation were the appropriate N-β-glucosaminides. The effects of reaction conditions on the product yield and composition were studied with the reaction α-D-glucosaminyl chloride with 5-methylthio-4-phenyl-Δ²-l,2,4-triazolin-3-one. Optimal conditions of interphase glycosylation were found. The formation of N-l,2-trans-glycosidic bond was proved by ¹H NMR and IR data as well as by comparison with the published spectral data for glycosides of similar structures.