Spider toxin (JSTX) on the glutamate synapse

A new neurotoxin (JSTX) was separated from spider (Nephila clavata, Joro spider) venom. JSTX irreversibly suppressed the excitatory postsynaptic potential (EPSP) and the glutamate potential in the lobster neuromuscular junction with high degree of specificity. The threshold concentration for suppressing EPSPs corresponds to a small fraction of the toxin in a venom gland, roughly estimated as low as 10(-10) M/l. 10(-10) M/l. In the giant synapse of squid stellate ganglion JSTX suppressed EPSPs without affecting the antidromic response. Glutamate-induced membrane depolarization was blocked by JSTX. In mammalian brain slice preparation, JSTX suppressed the orthodromic spike response but failed to affect on the antidromic spike in the hippocampal pyramidal neuron of CA1 and CA3 region. The above results strongly support the view that the squid giant synapse and synapses in the hippocampal pyramidal neuron are mediated by glutamate.     

Isolation of an autonomously replicating sequence (ARS) from satellite DNA of Drosophila melanogaster

Summary Autonomously replicating sequences (ARSs) were cloned from the 1.688 satellite DNA of D. melanogaster using YIp5, consisting of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^– yeast strain as the recipient. Three out of six clones contained an ARS and the average frequncy of the occurrence of ARS was thus calculated to be approximately one per 14 kbp of the satellite DNA. A 500 bp ARS fragment (BgHS500) was obtained from one of the resultant clones (pYDS57). BgHS500 does not hybridize with the major repeating unit (370 bp) but it does with the minor unique sequence of the satellite. The sequence of BgHS500 was determined and found to be rich in AT and to contain the sequence, 5AAAACATAAAA3, a sequence common to yeast ARSs. However, a smaller fragment (150 bp) isolated from BgHS500 and containing the 11 bp sequence did not exhibit the characteristics of an ARS. The average copy number in the transformants of pBgHS500, a recombinant molecule of BgHS500 and YIp5, ranged from 0.05–0.5, while that of the parent plasmid, pYDS57, was about 2–10. On the basis of these results, it is postulated that the sequence 5AAAACATAAAA3 may possibly consiitute the core of ARSs and certain other sequences may also be necessary to insure that the ARS consistently undergoes at least one complete replication in each cell cycle. The role of ARSs in the genome of D. melanogaster is discussed.     

Establishment of a tumor-specific immunotherapy model utilizing TNP-reactive helper T cell activity and its application to the autochthonous tumor system

Preinduction of potent hapten-reactive helper T cell activity and subsequent immunization with hapten-coupled syngeneic tumor cells result in enhanced induction of tumor-specific immunity through T-T cell collaboration between anti-hapten helper T cells and tumor-specific effector T cells. On the basis of this augmenting mechanism, a tumor-specific immunotherapy protocol was established in which a growing tumor regresses by utilizing a potent trinitrophenyl (TNP)-helper T cell activity. C3H/He mice were allowed to generate the amplified (more potent) TNP-helper T cell activity by skin painting with trinitrochlorobenzene (TNCB) after pretreatment with cyclophosphamide. Five weeks later, the mice were inoculated intradermally with syngeneic transplantable X5563 tumor cells. When TNCB was injected into X5563 tumor mass, an appreciable number of growing tumors, in the only group of C3H/He mice in which the amplified TNP-helper T cell activity had been generated were observed to regress (regressor mice). These regressor mice were shown to have acquired tumor-specific T cell-mediated immunity. Such immunity was more potent than that acquired in mice whose tumor was simply removed by surgical resection. These results indicate that in situ TNP haptenation of the tumor cells in TNP-primed mice can induce the enhanced tumor-specific immunity leading to the regression of a growing tumor. Most importantly, the present study further investigates the applicability of this TNP immunotherapy protocol to an autochthonous tumor system. The results demonstrate that an appreciable percent of growing methylcholanthrene-induced autochthonous tumors regressed by the above TNP immunotherapy protocol. Thus, the present model provides an effective maneuver for tumor-specific immunotherapy in syngeneic transplantable as well as autochthonous tumor systems.     

Photoaffinity labeling with dihydropyridine derivatives of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain

The characteristics of photoaffinity labeling with the calcium agonist [3H]Bay K 8644 (Bay) and the calcium antagonists [3H]nitrendipine (Nit) and (+)PN200-110 (PN) of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain were investigated. In all these crude membranes, [3H](+)PN (20 nM) was mainly photoincorporated into one protein band with a molecular weight of 30,000 - 41,000 Da. It was also incorporated into some other bands of all these crude membranes. The photoincorporation of [3H](+)PN into these crude membranes was inhibited by the presence of 20 microM unlabeled (+)PN. The photoincorporation of [3H](+)PN into these crude membranes depended on its dose and on the time of UV irradiation. No incorporation of [3H](+)PN was observed in the absence of UV irradiation. The incorporation was not affected by the presence of 1 mM CaCl2 and/or 0.15 M NaCl, but was significantly decreased by 20 microM (+)PN and slightly decreased by 20 microM (-)PN, 20 microM Bay, 1 mM diltiazem, or 1 mM verapamil. Namely, enantiomers of PN caused various extents of stereoselective inhibition of photoaffinity labeling by [3H](+)PN of specific protein bands in these crude membranes. [3H]Nit was photoincorporated into these crude membranes in the same way as [3H](+)PN, but [3H]Bay was not photoincorporated. However, 20 microM unlabeled Nit did not consistently inhibit photoaffinity labeling with [3H]Nit. These findings suggested that measurement of photoaffinity of crude membranes from rat skeletal, cardiac, and uterine muscles and whole brain with [3H](+)PN by UV irradiation is a useful method for investigating the characteristics of the voltage-dependent calcium channels that are affected by 1,4-dihydropyridine derivatives.     

Cetacean relaxin. Isolation and sequence of relaxins from Balaenoptera acutorostrata and Balaenoptera edeni

The tendency toward extremely high variability among relaxins derived from purportedly closely related species has come to an abrupt end with the discovery of quasi-porcine relaxin in the minke whale (Balaenoptera acutorostrata) and the Bryde's whale (Balaenoptera edeni). An aqueous abstract of the corpora lutea of the two baleen whales contained significant amounts of relaxin-like activity as determined by a mouse bioassay and by cross-reactivity with anti-pig relaxin antibodies. The activity could be isolated and purified to homogeneity. Sequence analysis revealed that both whale relaxins differed from each other by about 3 residues, whereas the relaxin of B. edeni differed at only one position from that of pig relaxin. The similarity appears to include even the chain length heterogeneity observed at the C-terminal end of the B chain in porcine relaxin which is produced by a peculiar mode of connecting peptide removal from the pro-hormone. This finding may well represent one of the better documented challenges to the current paradigm of molecular evolution.     

Partial purification and characterization of membrane-associated diacylglycerol kinase of Drosophila heads.

A membrane-associated diacylglycerol kinase of Drosophila heads was purified to near homogeneity from the KCl extract of Drosophila heads. The purification procedure involved chromatography on Q-Sepharose, ammonium sulfate fractionation, Superose 12, hydroxyapatite and ATP-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of fractions after the ATP-agarose column chromatography showed that only a 115 kDa protein correlated well with the enzyme activity. The apparent Km values of partially purified DG kinase were 220 microM for ATP and 540 microM for diolein, respectively. The activity of the DG kinase was inhibited by deoxycholate and was not activated by Ca2+.     

Haemophilia B (sixth edition): a database of point mutations and short additions and deletions.

The sixth edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1380 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on factor IX activity, factor IX antigen in circulation and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.     

Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector

Replication-defective recombinant adenovirus, Adex4SRLacZL, was used as a vector for transferring exogenous genes in mouse zona pellucida-free eggs at the pronuclear stage. The vector contained the E. coli LacZ reporter gene under the control of the SRα promoter (SV40 early promoter-fused HTLV-I LTR), and the expression of the reporter gene was examined during preimplantation development in culture. Histochemical staining of the embryos for β-galactosidase activity showed that the exogenous LacZ gene as expressed in 98% of the embryos at the morula-blastocyst stages. As in the microinjection method, the exogenous genes could be pursued from the 2-cell stage. Neither apparent morphological changes nor cytotoxic effects were observed. Both the percentages of embryos expressing reporter genes and the rate of development to the blastocyst stage were higher in the adenovirus vector-treated embryos than in the microinjected ones. These results suggest that the adenovirus vector system is a useful tool in investigating the genetic control of early mammalian development. © 1995 wiley-Liss, Inc.     

Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene, second edition.

A large number of different mutations in the factor VIII (F8) gene have been identified as a cause of haemophilia A. This compilation lists known single base-pair substitutions, deletions and insertions in the F8 gene and reviews the status of the inversional events which account for a substantial proportion of mutations causing severe haemophilia A.