cDNA cloning of an mRNA encoding a sulfur-rich 10 kDa prolamin polypeptide in rice seeds

Using a rice maturing seed pUC9 expression library, we isolated a cDNA clone corresponding to 10 kDa sulfurrich prolamin by immunoscreening. A longer cDNA clone was obtained from a gtll library by plaque hybridization using this ³²P-labeled cDNA as a probe. A polypeptide sequence composed of 134 amino acids was deduced from the nucleotide sequence. A 24 amino acid signal peptide was assigned by computer calculation for the membrane spanning region and Edman sequencing of the purified mature polypeptide. Remarkably, 20% of methionine and 10% of cysteine were found in the mature polypeptide as well as high contents of glutamine, and hydrophobic amino acids. Part of the amino acid sequence was homologous with a conserved cysteine-rich region found in other plant prolamins. Two repeats of amino acid sequence were found in the polypeptide.     

Purification and properties of an endonuclease endogenous to rat-liver nuclei

An endonuclease endogenous to rat-liver nuclei has been purified by a series of chromatographic procedures and finally by isoelectric focusing (IEF) electrophoresis. The nuclease fraction prepared by the IEF electrophoresis (IEF fraction) showed a pI value of 5.7 and migrated as a single band to a molecular weight position of 46,000 on an SDS-polyacrylamide gel. The activity for single-stranded DNA was enhanced by 10 mM MgCl2 and/or by 5-15 mM MgCl2 in the presence of 2 mM CaCl2 (an optimum pH, 7.0), but was lowered by CaCl2 alone and inhibited strongly by ZnCl2 or MnCl2. The activity for duplex DNA was rather low, although an optimum condition was 10 mM MgCl2. In fact, even under this condition, the activity was about 40% lower than that for single-stranded DNA. Moreover, the IEF fraction formed single-strand nicks much more rapidly than double-strand cuts in pBR322 DNA, and preferentially produced deoxyadenosine 5'-monophosphate termini in the DNA. In addition, RNAase activity was also detected in this fraction.     

Expression of goat alpha-lactalbumin in Escherichia coli and its refolding to biologically active protein

A cDNA encoding the mature region of goat alpha-lactalbumin and the 3'-non-coding region was fused to cDNA of the N-terminal half of porcine adenylate kinase which had been placed under the control of the tac promoter in an expression vector in Escherichia coli. In addition, a methionine codon was inserted between the two cDNAs. When the plasmid carried the full-length 3'-non-coding region, little accumulation of the fused protein was observed. However, the deletion of two-thirds of the 3'-non-coding region produced significant expression of the fused protein in E. coli strain JM105. Since goat alpha-lactalbumin contains no methionine residue, the mature goat alpha-lactalbumin was isolated by CNBr digestion of the fused insoluble protein and refolded using thioredoxin. The homogeneous and biologically active goat alpha-lactalbumin was purified by Ca2+ ion-dependent hydrophobic chromatography.     

Purification of epidermal plasminogen activator inhibitor

A plasminogen activator inhibitor was purified from human cornified cell extract by DEAE-Sepharose, Sephacryl S-200, and high-performance liquid chromatographies on hydroxyapatite HPHT and anion-exchanger Mono Q at pH 7.2 and 8.0. The purified inhibitor showed Mr 43,000 and pI 5.2 50% inhibition of fibrinolytic activity (1.5 IU) of urokinase and tissue-type plasminogen activator was attained by 0.60 ng and 11.0 ng purified inhibitor, respectively. Synthetic substrate assay demonstrated slow tight-binding inhibition to both urokinase and tissue-type plasminogen activator. The inhibitor did not inactivate plasmin, thrombin, glandular kallikrein or trypsin.     

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.     

Time courses of cell electroporation as revealed by submicrosecond imaging of transmembrane potential.

Changes in the membrane conductance of sea urchin eggs, during the course of electroporation, were investigated over the time range of 0.5 microsecond to 1 ms by imaging the transmembrane potential at a submicrosecond resolution with the voltage-sensitive fluorescent dye RH292. When a rectangular electric pulse of moderate intensity was applied across an egg, a position-dependent potential developed synchronously with the pulse, as theory predicts for a cell with an insulating membrane. From the rise and fall times, the membrane capacitance of unfertilized eggs was estimated to be 0.95 microF/cm2 and the intracellular conductance 220 omega.cm. Under an electric pulse of much higher intensity, the rise of the induced potential stopped at a certain level and then slowly decreased on the microsecond time scale. This saturation and subsequent reversal of the potential development was ascribed to the introduction of finite membrane conductance, or permeabilization of the membrane, by the action of the intense pulse (electroporation). Detailed analysis indicated the following: already at 0.5 microsecond in the rectangular electric pulse, the two sides of the egg facing the positive and negative electrodes were porated and gave a high membrane conductance in the order of 1 S/cm2; the conductance on the positive side appeared higher. Thereafter, the conductance increased steadily, reaching the order of 10 S/cm2 by 1 ms. This increase was faster on the negative-electrode side; by 1 ms the conductance on the negative side was more than twice that on the positive side. The recovery of the porated membrane after the pulse treatment was assessed from the membrane conductance estimated in a second electric pulse of a small amplitude. At least two recovery processes were distinguished, one with a time constant of 7 microseconds and the other 0.5 ms, at the end of which the membrane conductance was already < 0.1 S/cm2.     

Chemical characterization, synthesis and distribution of proteinase inhibitor in newborn rat epidermis

A protein solubilized in Tris-HCl/saline buffer from keratinized cells of newborn rat epidermis exhibited inhibitor activity to papain and ficin, but not to trypsin, cathepsin D and pepsin. This protein was purified from keratinized cells as well as nonkeratinized and germinative cells by means of IgG affinity chromatography. The inhibitors extracted from all cell layers were immunologically identical and had a molecular weight of approximately 12,500 +/- 500. Since amino acid analysis showed that the inhibitor contains about 35 residues of glycine per mol, [3H]glycine was used to investigate synthesis of the protein. The inhibitor from nonkeratinized and germinative cells was radioactively labeled by 2 h after injection and appeared in keratinized cells by 48 h after injection. Indirect immunofluorescence microscopy demonstrated in situ distribution of the protein in the entire epidermis, and the protein localized by the plasma membrane in granular cells and diffusely in keratinized cells was shown to be insoluble in Tris-HCl saline buffer. The results indicate that a thiol-proteinase inhibitor is synthesized in epidermal cells during keratinization and is retained as part of the cytoplasmic structure     

Strain difference in expression of the adult-type polycystic kidney disease gene, pcy, in the mouse

DBA/2FG-pcy and C57BL/6FG-pcy congenic strains were established by transferring the polycystic kidney disease gene, pcy, to DBA/2 and C57BL/6 mice. We carried out pathological and hematological examinations of these strains at 4, 8, 16 and 30 weeks of age. In DBA/2FG-pcy mice more than 8 weeks of age, macroscopic renal cysts were observed on the surface of both kidneys. Their kidneys weight was significantly greater than in DBA/2 mice at all ages examined. Microscopic renal cysts were evenly distributed at 4, 8 and 16 weeks of age. At 30 weeks of age, the kidneys were filled with numerous polycysts. In C57BL/6FG-pcy mice, no macroscopic renal cysts were found until the animals were 30 weeks old, and the weight of their kidneys was greater than in B6 mice of the same age. From 8 weeks of age on, a limited number of microscopic renal cysts was observed, and many renal cysts were found adjacent to the enlarged Bowman's capsules. With age, the red blood cell count and hematocrit level decreased while the platelet count increased in both strains, with greater changes occurring in DBA/2FG-pcy mice than in C57 BL/6FG-pcy mice. These findings demonstrate that polycystic kidney disease exhibits strain differences in animals with a DBA/2 and C57BL/6 background. Our results suggest that phenotypic expression of the pcy gene in the mouse depends on genetic background, and that variations in the severity of human polycystic kidney disease may be explained, at least in part, by individual differences in genetic background.     

Cellular localization of thiol-proteinase inhibitor in the epidermis of the newborn rat

Summary In cichlid, poecilid and centrarchid fishes luteinizing hormone releasing hormone (LHRH)-immunoreactive neurons are found in a cell group (nucleus olfactoretinalis) located at the transition between the ventral telencephalon and olfactory bulb. Processes of these neurons project to the contralateral retina, traveling along the border between the internal plexiform and internal nuclear layer, and probably terminating on amacrine or bipolar cells. Horseradish peroxidase (HRP) injected into the eye or optic nerve is transported retrogradely in the optic nerve to the contralateral nucleus olfactoretinalis where neuronal perikarya are labeled. Labeled processes leave this nucleus in a rostral direction and terminate in the olfactory bulb. The nucleus olfactoretinalis is present only in fishes, such as cichlids, poecilids and centrarchids, in which the olfactory bulbs border directly the telencephalic hemispheres. In cyprinid, silurid and notopterid fishes, in which the olfactory bulbs lie beneath the olfactory epithelium and are connected to the telencephalon via olfactory stalks, the nucleus olfactoretinalis or a comparable arrangement of LHRH-immunoreactive neurons is lacking. After retrograde transport of HRP in the optic nerve of these fishes no labeling of neurons in the telencephalon occurred. It is proposed that the nucleus olfactoretinalis anatomically and functionally interconnects and integrates parts of the olfactory and optic systems.