Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in Drosophila, Caenorhabditis elegans, and mammals. One subfamily composed of l(2)35Aa is essential in Drosophila

The completed fruit fly genome was found to contain up to 15 putative UDP-N-acetyl-alpha-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) genes. Phylogenetic analysis of the putative catalytic domains of the large GalNAc-transferase enzyme families of Drosophila melanogaster (13 available), Caenorhabditis elegans (9 genes), and mammals (12 genes) indicated that distinct subfamilies of orthologous genes are conserved in each species. In support of this hypothesis, we provide evidence that distinctive functional properties of Drosophila and human GalNAc-transferase isoforms were exhibited by evolutionarily conserved members of two subfamilies (dGalNAc-T1 (l(2)35Aa) and GalNAc-T11; dGalNAc-T2 (CG6394) and GalNAc-T7). dGalNAc-T1 and novel human GalNAc-T11 were shown to encode functional GalNAc-transferases with the same polypeptide acceptor substrate specificity, and dGalNAc-T2 was shown to encode a GalNAc-transferase with similar GalNAc glycopeptide substrate specificity as GalNAc-T7. Previous data suggested that the putative GalNAc-transferase encoded by l(2)35Aa had a lethal phenotype (Flores, C., and Engels, W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2964-2969), and this was substantiated by sequencing of three lethal alleles l(2)35Aa(HG8), l(2)35Aa(SF12), and l(2)35Aa(SF32). The finding that subfamilies of GalNAc-transferases with distinct catalytic functions are evolutionarily conserved stresses that GalNAc-transferase isoforms may serve unique biological functions rather than providing functional redundancy, and this is further supported by the lethal phenotype of l(2)35Aa.     

Rapid aggregation and assembly in aqueous solution of A<Emphasis Type="Italic">β</Emphasis> (25–35) peptide

The highly toxic Aβ(25–35) is a peculiar peptide that differs from all the other commonly studied β-amyloid peptides because of its extremely rapid aggregation properties and enhanced neurotoxicity. We investigated Aβ(25–35) aggregation in H₂O at pH 3.0 and at pH 7.4 by means of in-solution analyses. Adopting UV spectroscopy, Congo red spectrophotometry and thioflavin T fluorimetry, we were able to quantify, in water, the very fast assembling time necessary for Aβ(25–35) to form stable insoluble aggregates and their ability to seed or not seed fibril growth. Our quantitative results, which confirm a very rapid assembly leading to stable insoluble aggregates of Aβ(25–35) only when incubated at pH 7.4, might be helpful for designing novel aggregation inhibitors and to shed light on the in vivo environment in which fibril formation takes place.     

Synthesis of orthogonally protected (<Emphasis Type="Italic">3R</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)- and (<Emphasis Type="Italic">3S</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)-4,5-diamino-3-hydroxypentanoic acids

Summary.  The paper describes two methods of the synthesis of ethyl (3R,4S)- and (3S,4S)-4-[(benzyloxycarbonyl)amino]-5-[(tert-butyloxycarbonyl)amino]-3-hydroxypentanoates, useful for the syntheses of edeine analogs. Differently N-protected (S)-2,3-diaminopropanoic acid was used as a substrate in both procedures. The absolute configuration of newly generated asymmetric carbon atoms C-3 in β-hydroxy-γ,δ-diamino products was assigned by means of ¹H NMR spectroscopy after their transformation into corresponding piperidin-2-ones. Received May 24, 2002 Accepted October 10, 2002 Published online December 18, 2002 Acknowledgment The authors are indebted to the Faculty of Chemistry, Technical University of Gdańsk for financial support. Authors' address: Zbigniew Czajgucki, M. Sc., Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Technical University of Gdańsk, 11/12 Narutowicza St., 80-952 Gdańsk, Poland, Fax +48 58 347 11 44, E-mail: zmczaj@wp.pl     

Rapid and repetitive plant regeneration of <Emphasis Type="Italic">Aralia elata</Emphasis> Seem. via somatic embryogenesis

In this work, we established a rapid and repetitive plant regeneration system for Aralia elata Seem. via primary and secondary somatic embryogenesis. Primary somatic embryogenesis was induced using leaf disks, petiole, and root segments, individually cultured for 5 weeks on Schenk and Hildebrandt (SH) (1972) medium with 0–5.0 mg/l indolebutyric acid (IBA). Our investigation demonstrated that optimal IBA concentrations of 3.0, 2.0, and 0.3 mg/l resulted in 100% somatic embryogenesis rates and averages of 11.3, 10.0, and 8.6 somatic embryos per explant for leaf disks, petiole, and root segments, respectively. The primary somatic embryos were used to conduct secondary somatic embryogenesis and the following treatments, in a gradient series, were examined: 0.3–4.0 mg/l IBA, 10–70 g/l sucrose and 0.2–3.0 mg/l abscisic acid (ABA). The results indicated that IBA was more effective than sucrose and ABA, and 3.0 mg/l IBA was the most suitable concentration for secondary somatic embryogenesis. Histological preparations indicated a multi-cellular origin of secondary somatic embryos and different morphological developmental stages during secondary somatic embryogenesis. Primary and secondary somatic embryos germinated readily and developed into normal plantlets after 2 weeks in woody plant medium (WPM, Lloyd and McCown 1980) with 20 g/l sucrose. At 4–5 cm in length, plantlets were transferred to soil (1:1 v/v of peat moss and sand) and the survival rate was 89% after 4 weeks under greenhouse conditions. This system provides a viable contribution to A. elata gene transformation, breeding and regeneration.     

Peat-Based Inoculum of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> Supplemented with Xanthan Gum

The addition of xanthan to high water retention capacity peat (HWRC) inoculants did not show differences on the survival of Bradyrhizobium japonicum E109. In low water retention capacity peats (LWRC) however, xanthan increased the survival of B.japonicum significantly. Xanthan showed the best effect at 0.1 g/l for B. japonicum, in contrast to Sinorhizobium fredii USDA205 where the concentrations evaluated (0–1.0 g/l) did not affected significantly its survival. Nevertheless, when the symbiotic performance on soybean was evaluated, the presence of 0.1 g xanthan/l increased the nodule number for both strains.     

<Emphasis Type="Italic">CbLEA</Emphasis>, a Novel<Emphasis Type="Italic">LEA</Emphasis> Gene from<Emphasis Type="Italic">Chorispora bungeana</Emphasis>, Confers Cold Tolerance in Transgenic Tobacco

A novel late embryogenesis abundant (LEA) gene (AY804193), namedCbLEA, has now been isolated fromChorispora bungeana. This rare alpine subnival plant can survive sudden snowstorms and low temperatures. The full-lengthCbLEA is 842 bp, with an open reading frame encoding 169 ami no acids. The putative molecular weight ofCbLEA protein is 17.9 kDa, with an estimatedpl of 6.45. To investigate the functioning of thisCbLEA protein in cold-stress tolerance,CbLEA was introduced into tobacco under the control of the CaMV35S promoter. Second-generation (R₁) transgenic tobacco plants exhibited significantly increased tolerance to cold. These transgenics maintained lower malondialdehyde (MDA) contents and electrolyte leakage (EL) but their relative water content (RWC) was significantly higher compared with non-transgenic plants under chilling stress. Further experimental results showed that non-transgenic plants had severe freezing damage after exposure to -2°C for 1 h, whereas the transgenics suffered only slight injury under the same conditions. Moreover, survival was longer in the latter genotype at that temperature. The extent of increased cold tolerance was positive correlated with the level ofCbLEA protein accumulation, and was also reflected by the delayed development of damage symptoms. This indicates thatCbLEA is an excellent stress tolerance gene, and holds considerable potential as a new molecular tool for engineering improved plant genetics.     

Toxicity of a <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>-like strain against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Bacillus thuringiensis (Bt) Berliner is a promising agent for microbial control of agriculturally and medically important insects. This study aimed at searching for Bt strains encoding Cry proteins that act more efficiently against fall armyworm. Thirty Bt strains were isolated from soil samples in Pernambuco State and evaluated through bioassays. Among these, strain I4A7 was the most efficient against the fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), and thus it was characterized by biochemical sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and molecular (polymerase chain reaction (PCR) and sequencing reaction) methods. The protein pattern of this strain on a SDS–PAGE was similar to that of B. thuringiensis israelensis (Bti). Moreover, I4A7 cry DNA sequence showed high identity (99–100%) to genes cry4Aa, 4Ba, 10Aa, 11Aa, cyt1Aa and cyt2B from Bti. The toxicity of the newly isolated Bti-like strain upon S. frugiperda should be considered as this strain might be used in combination with other Bt strains, such as B. thuringiensis var. kurstaki (Btk). Handling Editor: Helen Roy.     

Isolation of <Emphasis Type="Italic">madA</Emphasis> homologs in <Emphasis Type="Italic">Pilobolus crystallinus</Emphasis>

Pilobolus crystallinus shows unique photoresponses at various growing stages. cDNAs for putative photoreceptors were cloned from this fungus. Three genes named pcmada1, pcmada2, and pcmada3 were identified from the PCR fragments, and amplified with degenerated primers for the LOV domain, which is conserved in many blue-light receptors. Deduced amino acid sequences for PCMADA1, PCMADA2, and PCMADA3 had one light-oxygen-voltage (LOV)-sensing and two PER-ARNT-SIM (PAS) domains. A zinc finger DNA-binding motif was conserved in the C-terminals of PCMADA1 and PCMADA3. However, PCMADA2 lacked the zinc finger motif. Expression of pcmada1 was suppressed by blue light whereas that of pcmada3 was promoted by blue-light irradiation.     

Effects of constant light on circadian rhythmicity in mice lacking functional <Emphasis Type="Italic">cry</Emphasis> genes: dissimilar from <Emphasis Type="Italic">per</Emphasis> mutants

Mutations in each of the genes mPer1, mPer2, mCry1 and mCry2 separately cause deviations from the wild type circadian system. Differences between these mutant strains have inspired the hypothesis that the duality of circadian genes (two mPer and two mCry genes involved) is related to the existence of two components in the circadian oscillator (Daan et al., J Biol Rhythms 16:105–116, 2001). We tested the predictions from this theory that the circadian period (τ) lengthens under constant illumination (LL) in mCry1 and mPer1 mutant mice, while it shortens in mCry2 and mPer2 mutants. mCry1 ^−/− and mCry2 ^−/− knockout mice both consistently increased τ with increasing light intensity, as did wild type mice. With increasing illumination, rhythmicity is reduced in mCry1, mCry2 and mPer1, but not in mPer2 deficient mice. Results for mPer mutant mice are in agreement with data reported on these strains earlier by Steinlechner et al. (J Biol Rhythms 17:202–209, 2002), and also with the predictions from the model. The increase in cycle length of the circadian system by light in the mCry2 deficient mice violates the predictions. The model is thereby rejected: the mCry genes do not play a differential role, although the opposite responses of mPer mutants to light remain consistent with a functional Evening–Morning differentiation.     

<Emphasis Type="Italic">In Silico</Emphasis> sequence analysis and molecular modeling of the three-dimensional structure of DAHP synthase from <Emphasis Type="Italic">Pseudomonas fragi</Emphasis>

The shikimate pathway is involved in production of aromatic amino acids in microorganisms and plants. The enzymes of this biosynthetic pathway are a potential target for the design of antimicrobial compounds and herbicides. 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHPS) catalyzes the first step of the pathway. The gene encoding DAHPS was cloned and sequenced from Pseudomonas fragi, the bacterium responsible for spoilage of milk, dairy products and meat. Amino acid sequence deduced from the nucleotide sequence revealed that P. fragi DAHPS (Pf-DAHPS) consists of 448 amino acids with calculated molecular weight of ∼50 kDa and isoelectric point of 5.81. Primary sequence analysis of Pf-DAHPS shows that it has more than 84% identity with DAHPS of other Pseudomonas species, 46% identity with Mycobacterium tuberculosis DAHPS (Mt-DAHPS), the type II DAHPS and less than 11% sequence identity with the type I DAHPS. The three-dimensional structure of Pf-DAHPS was predicted by homology modeling based on the crystal structure of Mt-DAHPS. Pf-DAHPS model contains a (β/α)₈ TIM barrel structure. Sequence alignment, phylogenetic analysis and 3D structure model classifies Pf-DAHPS as a type II DAHPS. Sequence analysis revealed the presence of DAHPS signature motif DxxHxN in Pf-DAHPS. Highly conserved sequence motif RxxxxxxKPRT(S/T) and xGxR present in type II DAHPS were also identified in Pf-DAHPS sequence. High sequence homology of DAHPS within Pseudomonas species points to the option of designing a broad spectrum drug for the genus. Pf-DAHPS 3D model provides molecular insights that may be beneficial in rationale inhibitor design for developing effective food preservative against P. fragi.     

Detecting <Emphasis Type="Italic">Suaeda salsa</Emphasis> L<Emphasis Type="Italic">.</Emphasis> chlorophyll fluorescence response to salinity stress by using hyperspectral reflectance

Measurements of chlorophyll fluorescence and hyperspectral reflectance were used to detect salinity stress in Suaeda salsa L., beach of Dongtai, Jiangsu Province, China. Three experimental sites were used in our study, which belong to low salinity, middle salinity and high salinity. The results showed that leaf chlorophyll fluorescence changed along salinity gradient. To select the sensitive hyperspectral ranges of leaf chlorophyll fluorescence, the correlationship between leaf chlorophyll fluorescence and hyperspectral reflectance was regressed and analyzed. Statistical results indicated that the 680 and 935 nm were the most sensitive hyperspectral bands for estimating leaf chlorophyll fluorescence. Then, 11 relative hyperspectral indices were selected based on the sensitive bands and previous literature. (R ₆₈₀ − R ₉₃₅)/(R ₆₈₀ + R ₉₃₅) and R ₆₈₀/R ₉₃₅ have higher correlationship coefficient (R) and lower root mean square error, may be used for detecting chlorophyll fluorescence, such as F _o, F _m, F _v/F _m, qP, and ΦPSII, while NPQ may be detected by (R ₇₈₀ − R ₇₁₀)/(R ₇₈₀ − R ₆₈₀). These results suggest that chlorophyll fluorescence of halophyte response to salinity stress could be identified by remote sensing.     

The viability of the first generation roach (<Emphasis Type="Italic">Rutilus rutilus</Emphasis> L.), bream (<Emphasis Type="Italic">Abramis brama</Emphasis> L.), and blue bream (<Emphasis Type="Italic">Abramis ballerus</Emphasis> L.) hybrids at early stages of development

The viability of bream, roach, and blue bream F ₁hybrids at early stages of their development is analyzed. Viability is controlled according to parameters of the survival rate at stages from fertilized eggs to yearlings. During embryogenesis, significant stages (blastula—gastrula and hatching) are revealed by the amount of losses. The viability of hybrids of the first generation (compared to pure species) from the hatching stage and in the subsequent development constantly increases. At the stage of fingerlings, the viability of F ₁hybrids significantly exceeds that of pure species, which points to the heterozygous effect according to the parameters of hybrid survival for the first generation, which is absent in embryogenesis.     

The <Emphasis Type="Italic">Nramp1</Emphasis>AA genotype confers susceptibility to <Emphasis Type="Italic">Brucella abortus</Emphasis> in water buffalo

The 3′ untranslated region of the water buffalo Nramp1 (natural resistance-associated macrophage protein 1) gene contains two alleles (Nramp1A and Nramp1B), as detected by the denaturing gradient gel electrophoresis (DGGE) technique. The Nramp1BB genotype is associated with resistance of water buffalo to the intracellular pathogen Brucella abortus. This article provides evidence that the Nramp1AA genotype is associated with susceptibility to the same pathogen. Susceptibility or resistance of water buffalo to B. abortus was established by agglutination, complement fixation, and skin tests. The Nramp1 genotype was established by DGGE analysis. The association between the Nramp1AA genotype and susceptibility to B. abortus was demonstrated in two independent population samples (152 cases and 281 controls; 87 cases and 124 controls, respectively). Macrophages from Nramp1AA subjects displayed a lower Nramp1 mRNA level when compared with macrophages from Nramp1BB subjects. Also, monocytes and macrophages from Nramp1AA subjects displayed a higher number of viable intracellular bacteria in comparison with monocytes and macrophages from Nramp1BB animals, providing biological significance to the results from association studies.     

Somatic embryogenesis from zygotic embryos and shoot-tips of the Chilean tree <Emphasis Type="Italic">Gomortega keule</Emphasis>

The endangered Chilean tree Gomortega keule (Mol.) Baillon produces edible fruit, making it a potential crop. However, its cultivation from seed or cuttings is extremely difficult. This paper reports the induction of somatic embryogenesis and the initiation of liquid cultures in this species. Callus was induced from zygotic embryos and field-collected shoots. Somatic embryogenesis on zygotic embryos occurred at a low frequency. Induction of somatic embryogenesis was accomplished on micropropagated shoots after 6.5 months on semi-solid Murashige and Skoog (MS) medium with 30 g/l sucrose, 1.0 mg/l 2,4-dichlorophenoxyacetic acid and 1.0 mg/l 6-(γ,γ-dimethylallylamino) purine (2iP). Liquid cultures of compact callus and small aggregates were obtained and showed optimum proliferation in MS medium with 20 g/l sucrose, 0.01 mg/l α-naphthaleneacetic acid and 0.1 mg/l 2iP. The proliferation of friable embryogenic callus was observed in liquid medium and will allow the propagation of selected genotypes of this tree on a large scale. Genetic variation in two embryogenic genotypes cultured in vitro was not detected in an assessment using microsatellites; this approach is suitable for tracing genotypes.     

Enhanced production and characterization of a β-glucosidase from <Emphasis Type="Italic">Bacillus halodurans</Emphasis> expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A putative β-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-β-D-1-thiogalactopyranoside, the enzyme expressed at ∼40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A ₆₀₀ 12 in auto-inducing M9NG medium, β-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the β-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45°C and 8.0, respectively, under the assay conditions. K _m and k _cat against p-nitrophenyl-β-D-glucopyranoside were 4 mM and 0.75 sec⁻¹, respectively. To our knowledge, this is the first report of high-level expression and characterization of a β-glucosidase from B. halodurans.     

Ectopic expression of two MADS box genes from orchid (<Emphasis Type="Italic">Oncidium</Emphasis> Gower Ramsey) and lily (<Emphasis Type="Italic">Lilium longiflorum)</Emphasis> alters flower transition and formation in <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis>

Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine (BAP) and α-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis with high efficiency of transgenic plants (15%) in culture of 11 weeks’ duration. Further ectopic expression of two MADS box genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation.