Endopeptidase Activity of Phage λ-Endolysin

JACOB and Fuerst^1,2 demonstrated the presence of a bacteriolytic enzyme (λ-endolysin) in the induced cultures of lysogenic Escherichia coli K12 (λ). The enzyme was later identified as the product of gene R; of phage λ³ which is involved in bacterial lysis at the end of a latent period. The enzyme is apt to form spheroplast-like structures in E. coli² and one would therefore expect its substrate to be murein.     

Regeneration of fertile transgenic indica (group 1) rice plants following microprojectile transformation of embryogenic suspension culture cells

Regenerable embryogenic suspensions of elite Indica (group 1) rice varieties IR24, IR64, IR72 and an advanced Indica rice breeding line IR57311-95-2-3 were established within 6–8 weeks from 3–4 week old calli derived from mature seeds. Transgenic rice plants were obtained by introducing a plasmid carrying genes encoding hygromycin phosphotransferase (hph, conferring resistance to hygromycin B) and ß-glucuronidase (uidA), both driven by the CaMV 35S promoter, via particle bombardment of embryogenic suspensions. The effect of osmotic conditioning on transformation was evaluated. Regenerated plants were resistant to hygromycin B and expressed the uidA (GUS) gene. The growth of mother plants (R₀) was normal and seeds were produced. Southern blot analysis of R₀ and R₁ plants showed that hygromycin resistant plants contained intact hph genes that were inherited in a Mendelian fashion. A protocol for a simple, efficient, repeatable, genotype- and environment-independent Indica rice transformation system is described.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - kb kilobase - GUS ß-glucuronidase - hph hygromycin B phosphotransferase     

人粒细胞集落刺激因子（hG－CSF）cDNA3′－UTR对其表达的影响

利用人粒细胞集落刺激因子（ｈＧ－ＣＳＦ）ｃＤＮＡ３′端非翻译区（３′－ＵＴＲ）中存在的ＤｒａⅠ酶切位点,通过部分酶切与完全酶切,删除３′－ＵＴＲ不同长度,构建了四种ｈＧ－ＣＳＦｃＤＮＡ瞬时重组表达质粒。转染ＣＯＳ－７细胞后,生物活性测定结果提示,ｈＧ－ＣＳＦｃＤＮＡ３′－ＵＴＲ对其表达起负调控作用,其关键性序列位于紧接终止密码子ＴＧＡ下游的６５ｂｐ范围内,３′－ＵＴＲ对ｈＧ－ＣＳＦｃＤＮＡ表达的影响与转录水平的差别有一定关系。     

Structure determination of N-linked oligosaccharides engineered at the CH1 domain of humanized LL2

Two humanized antibody mutants, hLL2HCN1 and hLL2HCN5, engineeredwith CH₁ domain-appended carbohydrates (CHOs) were generatedto facilitate site-specific conjugation of radionudides andanti-cancer drugs to antibodies. Such site-specific conjugationmay minimize the incidence of immunoreactlvity perturbationas is often observed with random conjugation. Since the compositionsand structures of CHOs are important in determining the chemistry,efficiency, and extent of conjugation, the sequences of theCH₁-appended CHOs were determined by exoglycosidase digestionsand fluorophore-assisted CHO electrophoresis (FACE). The CHOspecies attached at HCN1 and HCN5 sites in hLL2HCN1 and IJLL2HCN5,respectively, were distinct from each other, heterogeneous,and extensively processed. All of these CHOs were corefucosylatedcomplex-type oligosaccharides and contained Gal (galactose)and GlcNAc (N-acetylglucosamine) residues in the outer branches.Some of the outer branches were composed of Gal     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Oleosin KD 18 on the surface of oil bodies in maize. Genomic and cDNA sequences and the deduced protein structure

Oleosins are newly discovered, abundant, and small Mr hydrophobic proteins localized on the surface of oil bodies in diverse seeds. So far, most of the studies have been on the general characteristics of the proteins, and only one protein (maize KD 16) has been studied using a cDNA clone containing an incomplete coding sequence. Here, we report the sequences of a genomic clone and a cDNA clone of a new maize oleosin (KD 18). There is no intron in the gene. The 5'-flanking region contains potential regulatory elements including RY repeats, CACA consensus, and CATC boxes, which are presumably involved in the specific expression of the proteins in maturing seeds. The deduced amino acid sequence was analyzed for secondary structures. We suggest that KD 18 of 187-amino acid residues contains three major structural domains: a largely hydrophilic domain at the N terminus, a hydrophobic hairpin alpha-helical domain at the center, and an amphipathic alpha-helix domain at the C terminus. These structural domains are very similar to those of oleosin KD 16. However, the KD 18 and KD 16 amino acid sequences as well as nucleotide sequences are highly similar only at the central domain (72 and 71%, respectively). The similarities are highest at the loop region of the alpha-helical hairpin. These results suggest that KD 18 and KD 16 are isoforms, encoded by genes derived from a common ancestor gene. We propose that the hairpin domain acts as an indispensible internal signal for intracellular trafficking of oleosins during protein synthesis as well as an anchor for oleosins on the oil bodies. The other two domains can undergo relatively massive amino acid substitutions without impairing the structure/function of the oleosins or have evolved to generate oleosins having different functions.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

Single-cell transcriptomic atlas of primate cardiopulmonary aging

Aging is a major risk factor for many diseases,especially in highly prevalent cardiopulmonary comorbidities and infectious diseases including Coronavirus Diseas...