PKC phosphorylation disrupts gap junctional communication at G0/S phase in clone 9 cells

Transduction of extracellular signals through the membrane involves both the lipid and protein moiety. Phosphatidylserine participates to these processes as a cofactor for protein kinase C activity and thus the existence of a regulatory mechanism for its synthesis ought to be expected. In plasma membranes from rat cerebral cortex, the activity of serine base exchange enzyme, that is mainly responsible for phosphatidylserine synthesis in mammalian tissues, was reduced by the addition to the incubation mixture of AlF4- or GTP-g-S, known activators of G proteins, whereas ATP was almost uneffective. GTP-g-S inhibited the enzyme activity only at relatively high concentration (> 0.5 mM). When the synthesis of phosphatidylserine in the same cerebral area was investigated by measuring the incorporation of labelled serine into the phospholipid in the homogenate buffered at pH 7.6, ATP had an inhibitory effect as GTP-g-S and AlF4-. Heparin activated both serine base exchange enzyme in plasma membranes and phosphatidylserine synthesis.The preincubation of plasma membranes in the buffer without any other addition at 37øC for 15 min reduced by 30% serine base exchange enzyme activity. The remaining activity responded to the addition of GTP-g-S but was insensitive to 5 mM AlF4-, a concentration that inhibited by 60% the enzyme assayed without preincubation.These results indicate the existence of different regulatory mechanisms, involving ATP and G proteins, possibly acting on different enzymes responsible for the synthesis of phosphatidylserine. Since previous studies have shown that hypoxia increases the synthesis of this phospholipid in brain slices or homogenate (Mozzi et al. Mol Cell Biochem 126: 101-107, 1993), it is possible that hypoxia may interfere with at least one of these mechanisms. This hypothesis is supported by the observation that in hypoxic homogenate 20 mM AlF4- was not able to reduce the synthesis of phosphatidylserine as in normoxic samples. A similar difference between oxygenated and hypoxic samples, concerning their response to AlF4-, was observed when the incorporation of ethanolamine into phosphatidylethanolamine was studied. The incorporation of choline into phosphatidilcholine was, on the contrary, inhibited at a similar extent in both experimental conditions.     

Computational Sequence Analysis of the Tissue Inhibitor of Metalloproteinase Family

The tissue inhibitor of metalloproteinase (TIMP) family regulates extracellular matrix turnover and tissue remodeling by forming tight-binding inhibitory complexes with matrix metalloproteinases (MMPs). MMPs and TIMPs have been implicated in many normal and pathological processes, such as morphogenesis, development, angiogenesis, and cancer metastasis. This minireview provides information that would aid in classification of the TIMP family and in understanding the similarities and differences among TIMP members according to the physical data, primary structure, and homology values. Calculations of molecular weight, isoelectric point values, and molar extinction coefficients are reported. This study also compares sequence similarities and differences among the TIMP members through calculations of homology within their individual loop regions and the mature region of the molecule. Lastly, this report examines structure–function relationships of TIMPs. Thorough knowledge of TIMP primary and tertiary structure would facilitate the uncovering of the molecular mechanisms underlying metalloproteinase, inhibitory activities and biological functions of TIMPs.     

Random sequence analysis of genomic DNA of a hyperthermophile: Aquifex pyrophilus

Aquifex pyrophilus is one of the hyperthermophilic bacteria that can grow at temperatures up to 95°C. To obtain information about its genomic structure, random sequencing was performed on plasmid libraries containing 0.5–2 kb genomic DNA fragments of A. pyrophilus. Comparison of the obtained sequence tags with known proteins revealed that 123 tags showed strong similarity to previously identifed proteins in the PIR or Genebank databases. These included three proteases, two amino acid racemases, and three enzymes utilizing oxygen as substrate. Although the GC ratio of the genome is about 40%, the codon usage of A. pyrophilus showed biased occurrence of G and C at the third position of codons, especially those for amino acids such as asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, and tyrosine. A higher ratio of positively charged amino acids in A. pyrophilus proteins as compared with proteins from mesophiles suggested that Aquifex proteins might contain increased ion-pair interaction that could help to maintain heat stability. Received: March 1, 1997 / Accepted: May 9, 1997     

Activation of human progelatinase A by collagenase and matrilysin: Activation of procollagenase by matrilysin

Proteolytic and nonproteolytic methods were used to investigate the mechanism(s) by which human fibroblast progelatinase A and fibroblast-type procollagenase can be activated. Both collagenase and matrilysin were able to activate progelatinase A, resulting in an amino terminus in gelatinase A of Tyr.⁸¹ The cleavage occurred distal to Cys⁷³ within the sequence of PR_CGNPDVAN⁸⁰-Y⁸¹NFFPRKP. While several nonproteolytic reagents were tested, only the heavy metal Hg() andp-chloromercuribenzoate (PCMB) were able to induce activation of progelatinase A and resulted in the conversion of the latent 72-kDa gelatinase A to an active form of about 64.5 kDa. Matrilysin was also able to activate procollagenase and resulted in an amino terminus in collagenase of Phe.⁸¹ These results suggest that fibroblast-type collagenase and matrilysin may be physiologically relevant activators of progelatinase A; the maintenance of latency and the process of activation for progelatinase A may occur through the cysteine-switch mechanism, and the proteolytic activation of procollagenase by matrilysin resulted in the same amino terminus as produced by stromelysin-1.Abbreviations APMA p-aminophenylmercuric acetate - Bistris [bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane] - CAPS 3-(cyclohexylamino)-1-propane sulfonic acid - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - GSSG oxidized glutathione - HFC human fibroblast-type collagenase, MMP-1 - HFG human fibroblast gelatinase A/72-kDa type IV collagenase, MMP-2 - HFS human fibroblast stromelysin-1, MMP-3 - MMP matrix metalloproteinase - MT-MMP membrane-type matrix metalloproteinase, MMP-14 - NEM N-ethylmaleimide - PCMB p-chloromercuribenzoate - PMA phenylmercuric acetate - PMC phenylmercuric acid - PMSF phenylmethanesulfonyl fluoride - PTH phenylthiohydantoin - RTT rat tail tendon - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tes [tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid] - TIMP tissue inhibitor of metalloproteinase - Tris tris(hydroxymethyl)aminomethane - Tricine N-[tris(hydroxymethyl)methyl]glycine     

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein from the Chinese oak Silkmoth, Antheraea pernyi.

In our research to identify gene involved in the cuticle protein, we cloned a novel cuticle protein gene, ApCP13, from the Chinese oak silkmoth, Antheraea pernyi, larvae cDNA library. The ApCP13 gene encodes a 120 amino acid polypeptide with a predicted molecular mass of 13 kDa and a pI of 4.01, and is intron-less gene. The ApCP13 contained a type-specific consensus sequence identifiable in other insect cuticle proteins and the deduced amino acid sequence of the ApCP13 cDNA is most homologous to another wild silkmoth, A. yamamai CP12 (86% protein sequence identity), followed by Bombyx mori LCP18 (35% protein sequence identity). Northern blot analysis revealed that the ApCP13 showed the epidermis-specific expression. This is the first report of cuticle protein gene in the wild silkmoth, A. pernyi.     

Optimization of phage λ promoter strength for synthetic small regulatory RNA-based metabolic engineering

Synthetic small regulatory RNAs (sRNAs) are gene-silencing tools that can be used to tune gene expression in prokaryotes. A recent study by our group proposed rational design principles, introduced a regulatory system that may be used to implement synthetic sRNAs, and showed their utility in metabolic engineering. The regulatory system employed the strong phage λ P_R promoter to tightly control synthetic sRNA production. Here, we fine-tuned the strength of the P_R promoter via mutagenesis in order to optimize the level of synthetic sRNAs while maintaining the ability of the promoter to be regulated by CI proteins. Five mutant promoters of different strengths, ranging from 24 to 87% of that of the wild-type P_R promoter, were identified and confirmed to be repressed by CI proteins. A mutated promoter with only 40% of the original strength still produced enough synthetic sRNAs to inhibit the translation of the target mRNA to ~10% of the original level. As a practical application, we tested our promoters as drivers for a synthetic anti-murE sRNA, which was used to adjust the production of cadaverine. As the promoter strength decreased, the cadaverine titer first increased and then dropped. A mutated promoter with 39% of the original strength achieved the improved cadaverine titer of 2.15 g/L. The mutant promoters developed in this study should prove useful for tuning the expression levels of synthetic sRNAs for metabolic engineering.     

Assembly of the capsid protein of red-spotted grouper nervous necrosis virus during purification,and role of calcium ions in chromatography

Currently virus-like particles (VLPs) are receiving much attention as platforms for next generation vaccines. However, chromatography-based methods for purifying VLPs remain challenging. Unlike traditional methods using density gradient for purifying VLPs, there have been few advances in explaining how assembled particles can be obtained by chromatography. Nervous necrosis virus (NNV) infects over 30 species of fish and leads to large economic losses in the farmed fish industry. Previously we developed a heparin chromatography-based method for purifying red-spotted grouper NNV (RGNNV) VLPs. However it is unclear how the assembled RGNNV VLPs are obtained by this method. It is known that assembly of NNV capsid proteins depends on calcium ions. In the present study, we found that the yield of purified RGNNV capsid protein in heparin chromatography was enhanced when calcium ions were present during binding. Also, it appears that the capsid protein of RGNNV undergoes partial disassembly and reassembly during sample preparation prior to heparin chromatography and the protein finally undergoes assembly during the chromatography. Therefore, our results indicated that heparin-binding affinity of RGNNV capsid protein is linked to its ability for VLP formation. The assembly of RGNNV capsid proteins recombinantly produced is a good model for explaining VLP formation during chromatography-based purification processes.     

Origins of recently re‐established and newly discovered populations of the endangered butterfly Shijimiaeoides divinus (Lepidoptera: Lycaenidae) in Oita Prefecture,Japan

The endangered butterfly Shijimiaeoides divinus was believed to have been extirpated from Oita Prefecture, Kyushu, Japan, but was rediscovered in Taketa in recent years. This population is considered to have re‐established as a result of natural dispersal from Kumamoto, a neighboring prefecture located to the west of Oita. Furthermore, another population was recently found in Yufu, Oita Prefecture, which is an area where the species had never been recorded. To elucidate the origins of these two populations newly found from Oita Prefecture, their DNA sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene were compared with those of other S. divinus populations from Kumamoto Prefecture, Honshu and Korea. The results supported the hypothesis that the Taketa population originated from Kumamoto Prefecture. However, it was not clear whether this population originated from the natural dispersal or deliberate release of individuals. It was also found that the Yufu population was not established by the deliberate release of individuals from Honshu or Korea; however, it remained unclear whether the population of S. divinus was native to Yufu, or originated from other localities in Kyushu.