Lobeline production by hairy root culture of Lobelia inflata L.

Hairy roots were obtained following inoculation of the stems of Lobelia inflata L. with Agrobacterium rhizogenes strain ATCC 15834. These hairy roots contained agropine and mannopine. In addition, lobeline was detected by HPLC and confirmed by mass spectrometry. Various media were tested for the growth of hairy roots as well as for the content of lobeline in hairy roots. The growth rate of hairy roots cultured in Nitsch and Nitsch's medium was approximately one third of those cultured in other media. The lobeline content of hairy roots (18–54 g/g dry weight) cultured in these media was the same order of magnitude compared with that of roots of L. inflata (24 g/g dry weight) cultivated in pots. The hairy roots cultured in Nitsch and Nitsch's medium were morphologically different from those cultured in other media.Abbreviations MS medium Murashige and Skoog's medium - 1/2 MS medium one-half strength of the standard Murashige and Skoog's medium - B5 medium Gamborg's B5 medium - NN medium Nitsch and Nitsch's medium - FW fresh weight - DW dry weight     

Chloroplast DNA topoisomerase I from cauliflower

An ATP-independent DNA topoisomerase has been isolated from chloroplasts of cauliflower leaves (Brassica oleracea var. botrytis) through DEAE-cellulose, AF-blue Toyopearl, and hydroxyapatite column chromatography. The sedimentation coefficient and Stokes radius of this enzyme are 3.6S and 3.6 nm, respectively, and the molecular weight of native enzyme is estimated to be 54,000. This enzyme changes the linking number in steps of one. The enzyme activity is stimulated by MgCl2, and this enzyme shows optimum activity at 30 degrees C in the range of 3 mM MgCl2 + 100 mM KCl-10 mM MgCl2 + 50 mM KCl. The enzyme activity was reduced remarkably by N-ethylmaleimide, indicating that a free sulfhydryl group is important for the activity; heparin and ellipticine also reduced the activity. Both cauliflower chloroplast topoisomerase and spinach chloroplast topoisomerase can relax positive supercoils as well as negative supercoils. From these properties, cauliflower chloroplast topoisomerase can be classified as a eukaryotic type I DNA topoisomerase.     

Three short fragments of Rts1 DNA are responsible for the temperature-sensitive growth phenotype (Tsg) of host bacteria.

Rts1 is a multiphenotype drug resistance factor, and one of its phenotypes is temperature-sensitive growth (Tsg) of host bacteria. A 3.65-kb fragment from Rts1 DNA was shown to cause the Tsg phenotype in host cells. This tsg fragment was split by a restriction enzyme, HincII, into four fragments. Two of these fragments were called HincII-S (short) and HincII-L (long), respectively. Each of these two fragments conferred the Tsg phenotype, indicating that, in fact, these two independent regions were responsible for the Tsg phenotype. The HincII-S 783-bp and HincII-L 1,479-bp fragments were sequenced. The region in the HincII-S fragment to which the Tsg phenotype was attributed was narrowed to a 146-bp (nucleotides 1 to 146) fragment by various restriction enzyme digestions. Further digestion of the 146-bp fragment with Bal 31 suggested that the 116-bp (nucleotides 9 to 124) fragment is the minimum sequence required for Tsg. On the other hand, in the HincII-L fragment, a fragment of 249 bp (nucleotides 1210 to 1458) and a fragment of 321 bp (nucleotides 1942 to 2262) contained separate temperature-sensitive growth activity. None of three tsg fragments contained open reading frames. The 249-bp fragment had very weak Tsg activity, while the 321-bp fragment had no Tsg activity. On the other hand, when these two fragments were together in the pUC19 vector, they exhibited very strong Tsg activity equivalent to that of the original 1,479-bp fragment. In addition, two of the 249-bp fragments gave similar, strong Tsg activity. The HincII-L 1,479-bp fragment contained an open reading frame for kanamycin resistance which was found between nucleotides 1423 and 2238. This kanamycin resistance gene sequence was different from that of the reported kanamycin resistance gene of Tn903 at 12 positions which were deduced to change seven amino acids.     

Salmonid olfactory system-specific protein (N24) exhibits glutathione S-transferase class pi-like structure

A salmonid olfactory system-specific protein (N24) that has been identified in lacustrine sockeye salmon (Oncorhynchus nerka) was characterized by biochemical and molecular biological techniques. N24 is a homodimer, and the intact molecular mass is estimated as approximately 43.3 kDa by gel filtration. Furthermore, N24 was located only in the cytosolic fraction of the olfactory tissues as determined by subcellular fractionation. cDNA encoding the lacustrine sockeye salmon N24 was isolated and sequenced. This cDNA contained a coding region encoding 216 amino acid residues and the molecular mass of this protein is calculated to be 242,224.77. The protein and nucleotide sequencing demonstrates the existence of a remarkable homology between N24 and glutathione S-transferase (GST; EC 2.5.1.18) class pi enzymes. Northern analysis showed that N24 mRNA with a length of 950 bases is expressed in lacustrine sockeye salmon olfactory epithelium. Olfactory receptor cells showed strong hybridization signals for N24 mRNA in the olfactory epithelium. N24 demonstrated glutathione binding activity in affinity-purified GST column experiments. The present study describes for the first time cDNA cloning of GST in fish olfactory epithelium.     

Dynamin Isoforms Decode Action Potential Firing for Synaptic Vesicle Recycling

Presynaptic nerve terminals must maintain stable neurotransmission via synaptic vesicle membrane recycling despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neuronal activity to vesicle trafficking is unknown. Here, we combined genetic knockdown and direct physiological measurements of synaptic transmission from paired neurons to show that three isoforms of dynamin, an essential endocytic protein, work individually to match vesicle reuse pathways, having distinct rate and time constants with physiological AP frequencies. Dynamin 3 resupplied the readily releasable pool with slow kinetics independently of the AP frequency but acted quickly, within 20 ms of the incoming AP. Under high-frequency firing, dynamin 1 regulated recycling to the readily releasable pool with fast kinetics in a slower time window of greater than 50 ms. Dynamin 2 displayed a hybrid response between the other isoforms. Collectively, our findings show how dynamin isoforms select appropriate vesicle reuse pathways associated with specific neuronal firing patterns.     

A homozygous mutation in the tight-junction protein JAM3 causes hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts

The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans.     

Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation.

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.     

Molecular cloning and immunohistochemical localization of ubiquitin C-Terminal hydrolase expressed in testis of a teleost,the Nile Tilapia,Oreochromis niloticus

We previously produced four monoclonal antibodies to testicular proteins of a teleost, the Nile tilapia. One of the monoclonal antibodies, TAT(Testicular Antigen of Tilapia)-10, recognizes a Mr=27,000 protein (27 kD protein), which is present in A and early B type spermatogonia, spermatids, and spermatozoa in testis. In order to clarify the function of this protein, molecular cloning was conducted. The cDNA for the 27 kD protein contains a complete open reading frame encoding 220 amino acid residues. The predicted amino acid sequence of the 27 kD protein was homologous to those of the ubiquitin carboxy-terminal hydrolases (UCH) reported in mammals. The measurement of the ubiquitin-releasing activity of the recombinant 27 kD protein revealed that the protein is the active form of UCH. Northern blot analysis showed that the UCH mRNA was expressed in ovary and brain in addition to the testis. Immunohistochemical study showed that, in brain, UCH was localized especially on the olfactory organ including the olfactory bulb and olfactory epithelium in olfactory rosetta, suggesting the involvement of the protein in chemoreceptive function. In the Tilapia ovary, UCH localized especially in pre-vitellogenic oocytes, suggesting that the enzyme activity could be important in oocyte growth. This is the first report for the cDNA cloning and cellular localization of UCH in fish. J. Exp. Zool. 293:368-383, 2002.     

Development of a genomic in situ hybridization method using Technovit 7100 sections of early wheat embryo

It is difficult to observe the behavior of chromosomes in early wheat embryos because they are wrapped in several cell layers of the ovary. Here we conducted genomic in situ hybridization on sections of ovary embedded in Technovit 7100, a resinous compound suitable for in situ hybridization of mRNA in sectioned tissues. With this resin it is possible to make thin sections with high resolution, no autofluorescence, and good water permeability. These features enable histochemical study using fluorescence microscopy. We established the most suitable conditions for the denaturation of target DNA embedded in Technovit resin, and performed GISH on them. Using this method, we identified Leymus mollis chromosomes in the young ovary of F₁ hybrids between wheat and L. mollis. Furthermore, we observed the behavior of maize chromosomes in early wheat × maize hybrid embryos.