Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines

Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.     

Effects of previous intensive agricultural management on microorganisms and the biodiversity of soil fauna

In this study effects of previous intensive management on microorganisms and soil fauna were investigated. It was hypothesized that a former but now abandoned intensive management practice still causes negative effects years after conversion into conventional management.Two agricultural fields were investigated. Until 1990 one field (preINT) had been managed with the intensive crop hops (Humulus lupulus), whereas the second one (preCON) had been under crop rotation. In consequence the main difference between the two fields was the high copper load of preINT. In the period during this study both fields were cultivated the same way (1991: winter wheat, 1992: spring barley).Microbial biomass, species numbers and abundances of collembola as well as of earthworms were much lower in preINT in the first year of investigation. In 1992, earthworm biomass was still extremely low, whereas the microbial biomass and total collembola had reached the values of preCON. Between the collembola populations there were still distinct differences in species abundances. In preCON,Folsomia quadrioculata, Onychiurus armatus, and theMesaphorura krausbaueri group each made up about 1/4 of the total population. In preINT nearly 60% of the population consisted of theMesaphorura krausbaueri group, followed byIsotoma notabilis andOnychiurus armatus.Folsomia quadrioculata andIsotomiella minor in preINT exhibited extremely small abundances during the whole investigation period. Thus, in comparative field studies their abundances could serve as indicators for copper contamination.Supplementary microcosm studies had shown that severe reductions of earthworm numbers, especially with simultaneous high mesofauna abundances, will be accompanied by increased mineralization rates. Therefore changes in soil fauna composition caused by copper contamination could have pronounced effects on mineralization rates and nutrient fluxes.     

Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general.     

Activation of aldo-keto reductase family member 1B14 (AKR1B14) by bile acids: Activation mechanism and bile acid-binding site

Aldo-keto reductase (AKR) 1B14, a rat ortholog of mouse androgen-dependent vas deferens protein (AKR1B7), is involved in the synthesis of prostaglandin F_2α and detoxification of 4-oxononenal formed by lipid peroxidation. The NADPH-linked reductase activity of AKR1B14 was activated by various bile acids. Although the activation was increased by decreasing pH from 9.0 to 6.0, the concentrations giving maximum stimulation (2- to 18-fold) were 0.2-6.0 μM for bile acids at pH 7.4. Kinetic analyses of the activation by glycochenodeoxycholic acid in the forward and reverse reactions, together with fluorescence changes and protection against 4-oxononenal-induced inactivation by bile acid, indicate that the bile acid binds to the enzyme and its coenzyme binary complex as a non-essential activator. The bile acid binding to AKR1B14 mainly accelerates the NADP⁺ dissociation, the rate-limited step of the enzyme reaction. AKR1B7 was also activated by bile acids, but the activation was low and independent of pH. The mutagenesis of His269 and Leu267 of AKR1B14 into the corresponding residues (Arg and Pro, respectively) of AKR1B7 resulted in low and pH-independent activation by bile acids. The results, together with the docking of the bile acid in the recently determined crystal structure of AKR1B14, identify the bile acid-binding site of which His269 plays a key role in significant activation through its electrostatic interaction with the carboxyl group of bile acid, facilitating the release of NADP⁺.     

Reevaluation of colorimetric iron determination methods commonly used in geomicrobiology

The ferrozine and phenanthroline colorimetric assays are commonly applied for the determination of ferrous and total iron concentrations in geomicrobiological studies. However, accuracy of both methods depends on slight changes in their protocols, on the investigated iron species, and on geochemical variations in sample conditions. Therefore, we tested the performance of both methods using Fe(II)((aq)), Fe(III)((aq)), mixed valence solutions, synthetic goethite, ferrihydrite, and pyrite, as well as microbially-formed magnetite and a mixture of goethite and magnetite. The results were compared to concentrations determined with aqua regia dissolution and inductively coupled plasma atomic emission spectroscopy (ICP-AES). Iron dissolution prior to the photometric assays included dissolution in 1M or 6M HCl, at 21 or 60°C, and oxic or anoxic conditions. Results indicated a good reproducibility of quantitative total iron determinations by the ferrozine and phenanthroline assays for easily soluble iron forms such as Fe(II)((aq)), Fe(III)((aq)), mixed valence solutions, and ferrihydrite. The ferrozine test underestimated total iron contents of some of these samples after dissolution in 1M HCl by 10 to 13%, whereas phenanthroline matched the results determined by ICP-AES with a deviation of 5%. Total iron concentrations after dissolution in 1M HCl of highly crystalline oxides such as magnetite, a mixture of goethite and magnetite, and goethite were underestimated by up to 95% with both methods. When dissolving these minerals in 6M HCl at 60°C, the ferrozine method was more reliable for total iron content with an accuracy of ±5%, related to values determined with ICP-AES. Phenanthroline was more reliable for the determination of total pyritic iron as well as ferrous iron after incubation in 1M HCl at 21°C in the Fe(II)((aq)) sample with a recovery of 98%. Low ferrous iron concentrations of less than 0.5mM were overestimated in a Fe(III) background by up to 150% by both methods. Heating of mineral samples in 6M HCl increased their solubility and susceptibility for both photometric assays which is a need for total iron determination of highly crystalline minerals. However, heating also rendered a subsequent reliable determination of ferrous iron impossible due to fast abiotic oxidation. Due to the low solubility of highly crystalline samples, the determination of total iron is solely possible after dissolution in 6M HCl at 60°C which on the other hand makes determination of ferrous iron impossible. The recommended procedure for ferrous iron determination is therefore incubation at 21°C in 6M HCl, centrifugation, and subsequent measurement of ferrous iron in the supernatant. The different procedures were tested during growth of G. sulfurreducens on synthetic ferrihydrite. Here, the phenanthroline test was more accurate compared to the ferrozine test. However, the latter provided easy handling and seemed preferable for larger amounts of samples.     

Carbohydrate levels among winter wheat cultivars varying in freezing tolerance and snow mold resistance during autumn and winter

The LT₅₀ values and soluble carbohydrate levels in wheat ( Triticum aestivum L.) crowns and leaves were monitored throughout autumn and winter in cultivars varying in freezing tolerance and snow mold resistance during 1993/1994 and 1994/1995 in the field at Sapporo, Japan. During the first stage of hardening, from sowing to mid‐November, the pattern of accumulation of mono‐ and disaccharides was similar for all cultivars. During the second stage of cold hardening, from mid‐November to mid‐December, the greatest accumulation of mono‐ and disaccharides, without a corresponding increase in fructan, was observed among the freezing‐tolerant cultivars; and levels of simple saccharides rapidly decreased under snow cover. Conversely, levels of mono‐ and disaccharides in snow mold‐resistant cultivars were less than 70% of those in freezing‐tolerant cultivars before snow cover and maintained low levels throughout winter, while polysaccharide levels in snow mold‐resistant cultivars were about 120% of those in freezing‐tolerant cultivars in December. Sugar metabolism during the winter was examined using 18 cultivars in 1994/1995. LT₅₀ values were correlated to the greatest extent with total mono‐ and disaccharide and fructan content among wheat cultivars excluding snow mold‐resistant cultivars in December. Snow mold‐resistant cultivars tended to metabolize carbohydrates more slowly until the end of the snow cover period. This result suggested that the enzymatic metabolism of the synthesis of sugars and the conversion of fructan to cryoprotective sugars in response to low temperatures, especially subzero ones, might be different between the two contrasting types in resistance to winter stress.     

Genetic,morphological, and virulence characterization of the entomopathogenic fungus Verticillium lecanii

In order to clarify relationships among genetic diversity, virulence, and other characteristics of conidia, 46 isolates of Verticillium lecanii from various hosts and geographical locations were examined. The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of ribosomal DNA (rDNA), mitochondrial small subunit rDNA (mt-SrDNA) and beta-tubulin were analyzed by PCR-RFLP. PCR-single stranded conformational polymorphism (SSCP) was performed on regions of the mitochondrial large subunit rDNA, mt-SrDNA, beta-tubulin and histone 4. There were no relationships among the results of RFLP, SSCP, isolation source, and location. However, amplified product size of IGS did have relationships with conidia size and sporulation. Six isolates with 4.0-kb IGS products had large conidia dimensions, and yielded low numbers of conidia compared with other isolates. Three out of the six isolates were high virulence (over 90%) against green peach aphids. Furthermore, double-stranded RNA (dsRNA) was detected in 22 out of 35 V. lecanii isolates and related with the amplicon sizes of IGS, though not with virulence or isolation location. Isolates containing dsRNA were divided into six distinct types based on banding pattern. These data demonstrate the level of genetic diversity of V. lecanii, and suggest relations among the genetic properties and conidial morphology.     

Alternative Inactivated Poliovirus Vaccines Adjuvanted with Quillaja brasiliensis or Quil-A Saponins Are Equally Effective in Inducing Specific Immune Responses

Inactivated polio vaccines (IPV) have an important role at the final stages of poliomyelitis eradication programs, reducing the risks associated with the use of attenuated polio vaccine (OPV). An affordable option to enhance vaccine immunogenicity and reduce costs of IPV may be the use of an effective and renewable adjuvant. In the present study, the adjuvant activity of aqueous extract (AE) and saponin fraction QB-90 from Quillaja brasiliensis using poliovirus antigen as model were analyzed and compared to a preparation adjuvanted with Quil-A, a well-known saponin-based commercial adjuvant. Experimental vaccines were prepared with viral antigen plus saline (control), Quil-A (50 µg), AE (400 µg) or QB-90 (50 µg). Sera from inoculated mice were collected at days 0, 28, 42 and 56 post-inoculation of the first dose of vaccine. Serum levels of specific IgG, IgG1 and IgG2a were significantly enhanced by AE, QB-90 and Quil-A compared to control group on day 56. The magnitude of enhancement was statistically equivalent for QB-90 and Quil-A. The cellular response was evaluated through DTH and analysis of IFN-γ and IL-2 mRNA levels using in vitro reestimulated splenocytes. Results indicated that AE and QB-90 were capable of stimulating the generation of Th1 cells against the administered antigen to the same extent as Quil-A. Mucosal immune response was enhanced by the vaccine adjuvanted with QB-90 as demonstrated by increases of specific IgA titers in bile, feces and vaginal washings, yielding comparable or higher titers than Quil-A. The results obtained indicate that saponins from Q. brasiliensis are potent adjuvants of specific cellular and humoral immune responses and represent a viable option to Quil-A.