Identification and characterization of a gene cluster involved in nitrate transport in the cyanobacterium Synechococcus sp. PCC7942

Summary The nrtA gene, which has been proposed to be involved in nitrate transport of Synechococcus sp. PCC7942 (Anacystis nidulans R2), was mapped at 3.9 kb upstream of the nitrate reductase gene, narB. Three closely linked genes (designated nrtB, nrtC, and nrtD), which encode proteins of 279, 659, and 274 amino acids, respectively, were found between the nrtA and narB genes. NrtB is a hydrophobic protein having structural similarity to the integral membrane components of bacterial transport systems that are dependent on periplasmic substrate-binding proteins. The N-terminal portion of NrtC (amino acid residues 1–254) and NrtD are 58% identical to each other in their amino acid sequences, and resemble the ATP-binding components of binding protein-dependent transport systems. The C-terminal portion of NrtC is 30% identical to NrtA. Mutants constructed by interrupting each of nrtB and nrtC were unable to grow on nitrate, and the nrtD mutant required high concentration of nitrate for growth. The rate of nitrate-dependent O₂ evolution (photosynthetic O₂ evolution coupled to nitrate reduction) in wild-type cells measured in the presence of l-methionine d,l-sulfoximine and glycolaldehyde showed a dual-phase relationship with nitrate concentration. It followed saturation kinetics up to 10 mM nitrate (the concentration required for half-saturation = 1 M), and the reaction rate then increased above the saturation level of the first phase as the nitrate concentration increased. The high-affinity phase of nitrate-dependent O₂ evolution was absent in the nrtD mutant. The results suggest that there are two independent mechanisms of nitrate uptake and that the nrtB-nrtC-nrtD cluster encodes a high-affinity nitrate transport system.     

cDNA Cloning and Expression of the Plastidic Copper/Zinc-Superoxide Dismutase from Spinach (Spinacia oleracea L.) Leaves

A cDNA clone for copper/zinc-superoxide dismutase (Cu/Zn-SOD)was isolated from spinach (Spinacia oleracea L.) leaves. Itsnucleotide sequence showed that it codes for a precursor polypeptideof 222 amino acids, including the NH₂-terminal 68-residue extensionwhich corresponds to a plastidic transit peptide. Northern hybridization,using plastidic and cytosolic Cu/Zn-SOD cDNAs as the probes,revealed that these two genes are differentially expressed inthe roots and leaves of spinach. ¹Present address: Department of Biochemistry and Microbiology,Cook College, Rutgers University New Brunswick, NJ 08903-0231,U.S.A.     

Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine

Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H₂O₂. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD.     

Additive Induction of Egr-1 (zif/268) mRNA Expression in Neuroblastoma × Glioma Hybrid NG108-15 Cells via Cholinergic Muscarinic, α2-Adrenergic, and Bradykinin Receptors

Abstract: Administration of carbachol, noradrenaline, and bradykinin induced Egr-1 mRNA expression within 1 h in mouse neuroblastoma × rat gliomahybrid NG108–15 cells. With specific receptor antagonists, the Egr-1 inductions by carbachol and noradrenaline were shown to be mediated via cholinergic muscarinic and α₂-adrenergic receptors, respectively. At their saturation levels for Egr-1 induction, the two agonists had additive effects when added together, but no prolongation of the effect on Egr-1 induction was observed. Addition of carbachol or noradrenaline 6 h after primary stimulation with carbachol or noradrenaline did not result in secondary Egr-1 induction, probably because of receptor desensitization. On the other hand, bradykinin consistently had an additive effect on Egr-1 induction, irrespective of the time of its addition, suggesting that the signal pathways for Egr-1 induction by carbachol or noradrenaline and by bradykinin are different. Treatment of cells with pertussis toxin or cholera toxin strongly inhibited Egr-1 induction by carbachol or noradrenaline but only partially inhibited the induction by bradykinin. Thus, the signals transduced in NG108–15 cells by different neurotransmitter receptors appear to have different effects on Egr-1 induction, depending on the times of stimulation and the combinations of receptors stimulated.     

Histological studies on male sterility of hybrids between laboratory and wild mouse strains

In this study the cellular mechanisms of male sterility in F1 hybrids (BNF1) between BALB/c and wild-derived M.MUS-NJL (NJL) was investigated. Cell proliferation and differentiation in the sterile testis were examined by bromodeoxyuridine-labeling and use of germ cell stage-specific antibodies. In BNF1 testes, spermatogonia actively proliferated with a seminiferous epithelial cycle, and were retained in the basal layer of the tubules. However, preleptotene, leptotene and zygotene spermatocytes moved to the adluminal region. Immunohistological data with germ cell stage-specific antibodies indicated the presence of few, if any, pachytene spermatocytes in BNF1 testes. Thus, spermatogenesis seemed to be blocked at the zygotene stage. For examination of germ cell-Sertoli cell interactions, testes of aggregation chimeras between BNF1 and C3H/HeN were analyzed immunohistologically with C3H-specific antibody. Results showed that spermatogenesis of C3H-germ cells was normal, even when these cells in contact with BNF1-Sertoli cells. Differentiation of BNF1-germ cells progressed from zygotene to pachytene stage spermatocytes when these cells were surrounded by C3H-Sertoli cells, but never proceeded beyond the pachytene stage. These observations suggest that at least two different cellular factors may be involved in spermatogenesis, one acting in the germ cells and the other mediated by Sertoli cells. Furthermore, mating experiments revealed that the degree of spermatogenesis varied in different F1 hybrids, and that the major sterility factor was closely linked to the T -locus on chromosome 17.     

Effects of body and head positions on bilateral difference in tympanic temperatures

We have examined the nonparallel changes in tampanic membrane temperatures (T _ty) from the two ears in response to various changes in body and head positions. Upon assuming a lateral recumbent position, the T _ty on the lower side increased while that on the upper side decreased. Pressure application over a wide area of the lateral chest only caused inconsistent and obscure asymmetric changes in T _ty. A lateral flexion of the head with the subject sitting upright and a rotation of the head to the side in a supine position induced an increase in the T _ty on the lower side compared to that on the upper side. The temperature and blood flow of the forehead often decreased on the lower side and increased on the upper side, although such responses were not always concomitant with the asymmetric changes in T _ty. A dorsal flexion of the head with the subject in a reclining position caused a slight increase in the T _ty, whereas raising the head upright induced a slight decrease in them. Two additional experiments were carried out with single photon emission computed tomography using ^99mTc-hexamethylpropyleneamine oxime as tracer, and a slight, relative decrease in counts was noted in the right hemisphere during rotation of the head to the right. These results would strongly suggest that unilateral increases and decreases in T _ty could have been caused by one-sided decreases and increases, respectively, in blood flow to the brain and/or the tympanic membrane, induced by a vasomotor reflex involving vestibular stimulation.     

A new snailfish of the genus Careproctus (Cottoidei: Liparidae) from the Pacific coast of southern Japan

Ichthyological Research - A new snailfish, Careproctus tomiyamai, is described on the basis of four specimens collected from Suruga Bay, Tosa Bay, and the Hyuga-nada Sea, southern Japan...     

Chemical synthesis of insulin-like peptide 1 and its potential role in vitellogenesis of the kuruma prawn Marsupenaeus japonicus

The insulin superfamily comprises a group of peptides with diverse physiological functions and is conserved across the animal kingdom. Insulin-like peptides (ILPs) of crustaceans are classified into four major types: insulin, relaxin, gonadulin, and androgenic gland hormone (AGH)/insulin-like androgenic gland factor (IAG). Of these, the physiological functions of AGH/IAG have been clarified to be the regulation of male sex differentiation, but those of the other types have not been uncovered. In this study, we chemically synthesized Maj-ILP1, an ILP identified in the ovary of the kuruma prawn Marsupenaeus japonicus, using a combination of solid-phase peptide synthesis and regioselective disulfide bond formation reactions. As the circular dichroism spectral pattern of synthetic Maj-ILP1 is typical of other ILPs reported thus far, the synthetic peptide likely possessed the proper conformation. Functional analysis using ex vivo tissue incubation revealed that Maj-ILP1 significantly increased the expression of the yolk protein genes Maj-Vg1 and Maj-Vg2 in the hepatopancreas and Maj-Vg1 in the ovary of adolescent prawns. This is the first report on the synthesis of a crustacean ILP other than IAGs and also shows the positive relationship between the reproductive process and female-dominant ILP.