Chloroplast division in cultured cells of the hornwortAnthoceros punctatus

Using cultured cells of the hornwortAnthoceros punctatus, the change in the relative chloroplast DNA content in each stage of chloroplast division was investigated to clarify the relationship between the division cycle of a chloroplast and a cell nucleus. Samples of cultured cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and then observed with an epifluorescence microscope and a chromosome image analyzing system (CHIAS). A chloropiast in cultured cells duplicated DNA with an increase in size. When a chloroplast began to divide, it was constricted in the middle, taking a dumbbell shape, and then divided into two daughter chloroplasts. In cultured cells of this species, the pattern of quantitative change of chloroplast DNA, that is, the DNA replication pattern of chloroplasts, corresponded to that of cell nuclear DNA in mitosis.     

The effects of hemorrhagic shock on thyrotropin-releasing hormone and its receptors in discrete regions of rat brain

The effects of hemorrhagic shock on thyrotropin-releasing hormone (TRH) levels and its receptors were studied in different regions of the rat brain. Rats were bled for 30 min from the left femoral artery, and their mean arterial pressure was kept at 40 mmHg for the following hour. The rats were killed by decapitation. Rat brains were immediately removed and dissected into 7 regions. Hemorrhagic shock decreased TRH significantly in the frontal cortex, septum, hippocampus, and hindbrain but TRH was not changed in the striatum, hypothalamus, and midbrain. Hemorrhagic shock significantly decreased TRH receptor binding in the septum and hindbrain. Scatchard analysis of saturation isotherms of specific TRH binding showed that the decreased specific TRH binding in the hindbrain resulted not from an increase of the dissociation constant (Kd), but from a decrease in the maximum number of binding sites (Bmax). In the septum, the decrease in specific binding was due both to a decrease in Bmax and an increase in Kd. The findings indicate that TRH plays a role in the physiological response to hemorrhagic shock.     

Stable transformation of cultured cells of the liverwortMarchantia polymorpha by particle bombardment

Suspension-cultured cells (A-18 line) of the liverwortMarchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene (hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×10⁶ cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines.     

L cells expressing DQ molecules of the DR3 and DR4 haplotypes: reactivity patterns with mAbs

cDNAs coding for the HLA class II DR and DQ and chains of the diabetogenic haplotypes DR3 and DR4 were introduced into a mammalian expression vector and transfected into L-cell mouse fibroblasts to produce cells expressing individual human class II molecules. Stable L transfectants were generated expressing each of the DR or DQ isotypes of the cis-encoded and chains of the DR3 or DR4 haplotypes, as well as the trans-encoded and chains of the DQ molecules of the two haplotypes. However, isotype mismatched combinations (DR/DQ or DQ/DR) did not result in any stable transfectants. The stable DQ L-cell transfectants obtained, along with homozygous B-cell lines expressing the DQ2 and DQ8 specificities, were tested against a large panel of twentyone anti-HLA class II monoclonal antibodies (mAbs). Their unusual reactivity patterns are described including the failure of most pan-DQ mAbs to react with all DQ expressing L-cell transfectants. Interestingly, some mAbs react with certain heterodimers expressed on B-LCL but fail to recognize the same heterodimers expressed on the transfectants. This is suggestive of minor structural modifications that class II molecules undergo depending on the cells they are expressed on.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U07848. The name DQB1 ^* 0202 was officially assigned by the WHO Nomenclature Committee in April 1994. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1992), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report     

Phosphorylation of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) by Proline-Directed Protein Kinases and Its Dephosphorylation

Abstract: Excitatory amino acid (EAA) neurotransmitters may play a role in the pathophysiology of traumatic injury to the CNS. Although NMDA receptor antagonists have been reported to have therapeutic efficacy in animal models of brain injury, these compounds may have unacceptable toxicity for clinical use. One alternative approach is to inhibit the release of EAAs following traumatic injury. The present study examined the effects of administration of a novel sodium channel blocker and EAA release inhibitor, BW1003C87, or the NMDA receptor-associated ion channel blocker magnesium chloride on cerebral edema formation following experimental brain injury in the rat. Animals (n = 33) were subjected to fluid percussion brain injury of moderate severity (2.3 atm) over the left parietal cortex. Fifteen minutes after injury, the animals received a constant infusion of BW1003C87 (10 mg/kg, i.v.), magnesium chloride (300 µmol/kg, i.v.), or saline over 15 min (2.75 ml/kg/15 min). In all animals, regional tissue water content in brain was assessed at 48 h after injury, using the wet weight/dry weight technique. In saline-treated control animals, fluid percussion brain injury produced significant regional brain edema in injured left parietal cortex ( p < 0.001), the cortical area adjacent to the site of maximal injury ( p < 0.001), left hippocampus ( p < 0.001), and left thalamus ( p = 0.02) at 48 h after brain injury. Administration of BW1003C87 15 min postinjury significantly reduced focal brain edema in the cortical area adjacent to the site of maximal injury ( p < 0.02) and left hippocampus ( p < 0.01), whereas magnesium chloride attenuated edema in left hippocampus ( p = 0.02). These results suggest that excitatory neurotransmission may play an important role in the pathogenesis of posttraumatic brain edema and that pre- or post-synaptic blockade of glutamate receptor systems may attenuate part of the deleterious sequelae of traumatic brain injury.     

Carcinogenesis in accessory sex organs of rats induced by 3,2′-dimethyl-4-aminobiphenyl: response to androgen deprivation

 It is generally accepted that early human prostate cancers reveal higher androgen dependency than do advanced ones. In the present study, we examined whether the animal model of prostate cancer has already lost androgen dependency at the early stages of carcinogenesis. At experimental week 46, androgen deprivation was induced in rats and the incidences of atypical hyperplasia and cancer were examined in the ventral, dorsolateral prostate, coagulating glands, and seminal vesicles. Androgen deprivation significantly lowered the incidence of atypical hyperplasia in all four organs. As for the incidence of cancer, no significant differences were observed in the coagulating glands and seminal vesicles. Regarding atypical hyperplasia, androgen deprivation significantly decreased the proliferative cell nuclear antigen labeling index in the coagulating gland and seminal vesicles. The presence of cancer was also decreased in the coagulating gland but not in the seminal vesicles. With control group specimens, more intense staining of androgen receptor was observed in atypical hyperplasias than in cancers. Compared with the atypical hyperplasias, the cancers revealed low androgen dependency at the early stages of carcinogenesis. The cancers in the seminal vesicles also revealed higher androgen independency than did those in the coagulating gland. Accepted: 6 May 1997     

Involvement of L-Type-Like Amino Acid Transporters in S-Nitrosocysteine-Stimulated Noradrenaline Release in the Rat Hippocampus

Abstract: Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S-nitroso-l -cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [³H]NA release from prelabeled hippocampal slices in a dose-dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na⁺-containing Tyrode's buffer, SNC-stimulated [³H]NA release was inhibited 30% by the coaddition of l -leucine. In the Na⁺-free, choline-containing buffer, SNC-stimulated [³H]NA release, which was similar to that in the Na⁺-containing buffer, was inhibited markedly by l -leucine, l -alanine, l -methionine, l -phenylalanine, and l -tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of l -leucine was stronger than that of d -leucine. A specific inhibitor of the L-type amino acid transporter, 2-aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), inhibited the effects of SNC on [³H]NA release in the Na⁺-free buffer. Uptake of l -[³H]leucine into the slices in the Na⁺-free buffer was inhibited by SNC, BCH, and l -phenylalanine, but not by l -lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by l -leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 µM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L-type-like amino acid transporter, at least in Na⁺-free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP-independent manner.     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

The Structure and Characterization of the Surface Protein Layer of a Comamonas-Like Organism

The regular surface layer of a strain of a Comamonas-like organism was examined by electron microscopy. The surface layer protein was easily extracted from the cell surface by a 2.5 M solution of lithium chloride. The protein subunit has a molecular size of 32,000 daltons, but usually forms a large aggregate of more than 1,200,000 daltons. In the extract it formed a regular array of p4 symmetry and was observed to be intimately associated with fragments of lipopolysaccharide. The size of a subunit determined by the negative staining method and the image processing method measured 5.2 × 6.4 nm (width and length), was arranged in a cobblestone-like pattern, and was located in a lattice space measuring 13.0 nm square.     

The Purification of a GroEL-Like Stress Protein from Aerobically Adapted Campylobacter jejuni

From plate cultures of Campylobacter jejuni grown in room air a particulate protein of 62 kDa was isolated by ion-exchange chromatography. The protein had a square shape from the side view but when viewed from the top it had a star-shaped structure. The molecular size of the whole particle determined by gel filtration was 850 kDa which suggested the presence of 14 subunits of 62 kDa in each particle. The N-terminal 37 amino residues showed more than 80% homology with the sequence of these heat shock protein (HSP) 60 homologs of Chlamydia trachomatis, Helicobacter pylori, and Escherichia coli (GroEL). This protein is immunologically cross-reactive with the antiserum for the 60-kDa HSP of Yersinia enterocolitica. Production of the 62-kDa protein increased under heat stress and growth in an aerobic atmospheric environment. From these observations we concluded that the 62-kDa protein is a Campylobacter stress protein (Cj62) which belongs to the HSP 60 family.