Isolating substrates for an engineered alpha-lytic protease by phage display

Panning of a substrate phage library with an -lytic protease mutant showed that substrate phage display can be used to isolate sequences with improved protease sensitivity even for proteases of relatively broad specificity. Two panning experiments were performed with an engineered -lytic protease mutant known to have a preference for cleavage after His or Met residues. Both experiments led to the isolation of protease-sensitive phage containing linker sequences in which His and Met residues were enriched compared with the initial library. Despite the relatively hydrophobic substrate binding site of the enzyme, the predominant protease-sensitive sequence isolated from the second library panning had the sequence Asp-Ser-Thr-Met. Kinetic studies showed that this sequence was cleaved up to 4.5-fold faster than rationally designed positive controls. Protease-resistant phage particles were also selected and characterized, with the finding that Gly and Pro appeared frequently at the putative P₄ positions, whereas Asp dominated the putative P₁ position.     

Epitope Mapping of Human Alpha-Fetoprotein

The epitope structure of human alpha-fetoprotein (AFP) was studied using more than 50 monoclonal antibodies (MAB) to human AFP. These MAB obtained from various world laboratories of the TD-2 AFP Workshops of the International Society for Oncodevelopmental Biology and Medicine (ISOBM-1996-1998-2000) were analyzed by competitive immunoaffinity electrochromatography (IAE) on nitrocellulose membranes (NCM). Five types of interaction of the AFP–MAB complex with the MAB fixed on NCM were found: 1) complete neutralization; 2) partial neutralization; 3) unidirectional neutralization; 4) enhanced binding; 5) lack of interaction. By IAE, 51 MAB were found to recognize 23 different epitopes in the AFP molecule. Based on these findings, an epitope map of AFP was designed which consists of eight epitope clusters and eight individual epitopes. The epitope location is considered with respect to the conformational state of the AFP molecule. Possible causes of the five types of interaction found on neutralization are discussed.     

Systemic IL-1 and adjuvant treatment of an experimental tumor

In this investigation, systemic administration of interleukin-1 (IL-1) and local adjuvant therapy were shown to modify immunological parameters associated with the lymphatics draining the site of experimental tumor inoculation. These immunological parameters were shown to be modified early (within 7 days) following tumor inoculation and within the time period of IL-1 administration. IL-1 induced a marked increase in the number of lymphocytes within the brachial and axillary lymph nodes associated with the tumor inoculation site. This increase was characterized by an overall augmentation in the number of CD8⁺ and CD4⁺ lymphocytes.In vitro, these lymph node cells showed enhanced proliferation in response to interleukin-2 (IL-2) when compared to non-IL-1 treated animals, and were capable of mounting a potentially greater cytotoxic response for both NK sensitive and NK resistant tumor targets. Without IL-1 administration, temporal and sequential lymph node cellular changes were observed, but were diminished and delayed when compared to the IL-1 treated animals. By adoptive transfer of tumor resistance, lymph node cells from IL-1 treated animals were demonstrated to be tumor-protectivein vivo. These results demonstrate that systemic IL-1 induces regional changes in the lymphatics of mice undergoing primary tumor challenge with adjuvant therapy and that these changes result in tumor protection for the host.     

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin

Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Glycosylated α-chymotrypsin as a catalyst for kyotorphin synthesis in water-organic media

-Chymotrypsin was covalently modified with cellobiose by chemical means. After adsorption on to a porous polyamide support, both the native and the glycosylated immobilized derivatives were used to synthesize a kyotorphin derivative (N-benzoyl-l-tyrosyl-l-argininamide) in acetonitrile/water. Glycosylated chymotrypsin gave a 125% increase in product formation (750 nmol mg^–1 catalyst in 3 h) at 60% (v/v) acetonitrile/water. Maximal protective effect of this glycosylation process was at 70% (v/v) acetonitrile/water, at which concentration the half-life of the glycosylated enzyme was 20-times longer than that of the native form (52 min and 2.8 min, respectively).     

Supramolecular interactions mediated thermal stabilization for α-amylase modified with a β-cyclodextrin-carboxymethylcellulose polymer

-Amylase from Bacillus subtilis was modified with a -cyclodextrin-carboxymethylcellulose polymer and retained 90% of its initial activity. Its thermostability was enhanced from 68 °C to 82.5 °C over 10 min incubation and the resistance to inactivation at 75 °C was increased 5-fold. The influence of supramolecular associations polymer-protein on enzyme thermostabilization was demonstrated.     

The Kavar(D) dominant female-sterile mutations of Drosophila reveal a role for the maternally provided alpha-tubulin4 isoform in cleavage spindle maintenance and elongation

The dominant-negative female-sterile Kavar^D mutations and their revertant kavar^r alleles identify the Tubulin67C gene of Drosophila melanogaster, which codes for the maternally provided -tubulin⁴ isoform. The mutations result in the formation of monopolar, collapsed spindles (each with two nearby centrosomes, a tassel of microtubules and overcondensed chromosomes), thus revealing a novel function for -tubulin⁴ in spindle maintenance and elongation. Molecular features of the two Kavar^D alleles and a kavar^null allele are described and models for their actions are discussed.     

Activation of JNK/SAPK in Primary Glial Cultures: II. Differential Activation of Kinase Isoforms Corresponds to Their Differential Expression

Recently, we reported on the activation of c-Jun N-terminal kinase (JNK) in primary glial cells noting certain differences in the patterns of kinase activation in astrocytes and oligodendrocytes (Zhang et al., J Neurosci Res 46:114–121;1996). In this extended study, we have examined the activation and expression levels of JNK1 and JNK2 isoforms in different glial cell types including the two in vitro-defined astroglial subtypes (type-1 and type-2), oligodendrocytes and microglia. An in-gel kinase assay of cell extracts and JNK-immunoprecipitates revealed the activation of both JNK1 and JNK2 in type-1 astrocytes in response to TNF, and in microglia, in response to TNF and bacterial lipopolysaccharide. The strong activation of the two JNK isoforms in type-1 astrocytes and microglia contrasted with a predominant activation of JNK1 over JNK2 in type-2 astrocytes and oligodendrocytes, the two glial subtypes sharing a common lineage. Immunoblot and immunocytochemical analyses using isoform-speciflc antibodies showed a differential expression of the two isoforms in different glial cells thereby accounting for their observed differential activation.     

Terminal glycosylation and disease: Influence on cancer and cystic fibrosis

Terminal glycosylation has been a recurring theme of the laboratory. In cystic fibrosis (CF), decreased sialic acid and increased fucosyl residues in 1,3 position to antennary N -acetyl glucosamine is the CF glycosylation phenotype. The glycosylation phenotype is reversed by transfection of CF airway cells with wtCFTR. In neuronal cells, polymers of 2,8sialyl residues are prominent in oligodendrocytes and human neuroblastoma. These findings are discussed in relationship to early studies in our laboratories and those of other investigators. The potential extension of these concepts to future clinical therapeutics is presented.     

Importance of monocytes/macrophages and fibroblasts for healing of micronecroses in porcine myocardium

In porcine heart, embolization of small coronary arteries with microspheres in 25 m in diameter induces collateral capillary vessel growth by angiogenesis in and around focal necrosis. By histological analysis the inflammatory infiltrates in this porcine tissue were characterized by numerous monocytes/macrophages and fibroblasts as well as neutrophils and numerous capillaries, some in mitosis. The aim of the present study, therefore, was to clarify the role of monocytes/macrophages and fibroblasts in angiogenesis and in repair in ischemic porcine myocardium. Using a human acidic fibroblast growth factor (aFGF) cDNA probe forin situ hybridisation labeling for aFGF mRNA was seen in monocytes and macrophages only, beginning at day 1, with a maximum at 3 and 7 days, and minimal labeling at 4 weeks. We have also shown, with a specific antibody and fluorescence microscopy, that tumur necrosis factor alpha (TNF) follows the same time sequence and that it is produced by monocytes/macrophages. The number of capillaries in infiltrates at 3 and 7 days as revealed by the lectin Dolichus Biflorus Agglutinin was high and declined at 4 weeks.In situ hybridisation using a rat cDNA probe for fibronectin showed the increased production of fibronectin mRNA in fibroblasts. To describe the expression of fibronectin and the collagens I, III, VI immunohistochemistry was used. A comparison showed that fibroblasts produced fibronectin mRNA starting at day 3, but the protein was only maximally expressed at day 7 and 4 weeks. Collagen I, III, VI expression was highest at 1–4 weeks. Conclusion: monocytes and macrophages produce the growth factors aFGF and TNF which seem to be important for angiogenesis in the ischemic myocardium. Fibroblasts, while they produce fibronectin and collagen, exert their major function in repair and scar formation, but may take also part in angiogenesis.     

BCL-2 in prostate cancer: a minireview

Prostate cancer progression and the development of androgen-independent prostate cancer have been largely related to a number of genetic abnormality that affect not only the androgen receptor but also crucial molecules involved in the regulation of survival or apoptotic pathways. One of these molecules, the pro-survival protein BCL-2, has been associated with the development of androgen-independent prostate cancer due to its high levels of expression in androgen-independent tumors in advanced stages of the pathology. The upregulation of BCL-2 after androgen ablation in prostate carcinoma cell lines and in a castrated-male rat model further established a connection between BCL-2 expression and prostate cancer progression. This review focuses on the experimental evidence that associates BCL-2 expression with prostate carcinogenesis and cancer progression, and analyzes the evidence that links the phosphatidylinositol 3-kinase (PI 3-kinase)/nuclear factor kappa B (NF-B) survival pathway with the upregulation of BCL-2. The way in which hormone ablation influences this survival pathway and the potential application of novel therapeutic strategies to overcome this anti-apoptotic mechanism is examined.     

Relationship between boron content and antioxidant compounds in Citrus leaves taken from fields with different water source

Boron contamination in the agricultural lands is an important problem for Western Turkey, which has rich boron deposits. This study was carried out in Nazilli regions upon orange (Citrus sinensis L. Osbeck) plants irrigated with relatively high boron laden channel waters (LCI) and with well waters (LWI) which contain lower amounts of boron. The leaves of the plants irrigated with channel water were found to contain twice the amount of boron compared with those irrigated with well waters. Boron content of leaves in both groups were approximately ten times the boron content in the soil on which they are grown. In the leaves of the plants irrigated with channel waters there were approximately 50% chlorophyll loss and higher chlorophyll a/b and caretonoid/chlorophyll ratios. In the excessive boron containing leaves was found higher soluble protein and carbohydrate contents, but lower determined free proline value. In plants that irrigated with high boron laden channel water significantly lower -tocopherol content and two fold higher ascorbate concentration were determined. The lower activities of catalase and glutathione reductase enzymes and higher total superoxide dismutase activity were measured in high boron content leaves. The retardation of growth due to boron toxicity can be attributed to the chlorophyll loss and inhibition of the carbohydratemetabolism. Boron at toxic level may cause the cell membrane lipids to be damaged by the free radicals by decreasing the -tocopherol levels. The increase in the ascorbate concentration may have a vital role in the protection of the inner cell structures against the boron toxicity.     

Reminiscences,collaborations and reflections

This is a personal account by a semi old-timer who completed his official term as a professor of plant biochemistry at Nagoya University in Japan in 1992. My university student life began soon after the World War II (1948). I shared the hardships of many in my age group, in that life was difficult during my college years. I was fortunate to have the opportunity of studying in the USA on a Fulbright scholarship first at Purdue University (1955–1956), and then at the University of California, Berkeley (1956–1957). My graduate study and postdoctoral training in the new world were vitally refreshing and stimulating, which gave me the impetus for becoming a natural scientist associated with academic institutions. Consciously and subconsciously I was impressed by the friendly and liberal atmosphere surrounding young students as well as senior scholars in the United States. But more importantly, I was inspired by the critical and competitive minds prevailing among these people.The appointment as a biochemist at the International Rice Research Institute (IRRI) in the Philippines (1962–1964) was the real start of my professional career. The work was continued upon my return to Nagoya to become a staff member of the Research Institute for Biochemical Regulation (1964–1992). Throughout the years, my major research interest has covered photosynthesis as a whole, involving photosynthetic CO₂-fixation (RuBisCO), carbohydrate metabolism, e.g. starch biosynthesis and breakdown (-amylase), and metabolic regulation, which are interrelated in the basic metabolism of plant cells.I shall briefly describe in this article highlights from my studies and discoveries made and I shall also discuss their possible significance in plant metabolism, with the hope that it does not contradict my sense of humility: They are (a) discovery of ADPG in plants and its role in starch biosynthesis; (b) structure-function relationship of RuBisCO proteins, in particular on heterologous recombination of their subunits of plant-type enzyme molecules derived from the prokaryotic photosynthetic bacteria; (c) molecular evolution of RuBisCO genes; (d) mode of actions (formation, intracellular transport and secretion) of rice seed -amylase and its structural characteristics (distinctive glycosylation), and (e) DNA methylation and regulatory mechanism of photosynthesis gene expression in plastids (amyloplasts). In each step of my research, I shared joy, excitement, disappointment, and agony with my colleagues, an experience that may be common to all researchers. Although it is now becoming well recognized among the scientific community in Japan, I want to point out that interaction of multinational scientific minds in the laboratory produces a vital and creative atmosphere for performance of successful research. I experienced and realized this important fact in my earlier days in the USA and the Philippines. Inasmuch as I believe that this is the most crucial element for any research laboratory to possess, I fondly remember the friendships gained with numerous overseas visitors and collaborators who have contributed immensely to our work.Written at the invitation of Govindjee.     

α-tocopherol inhibits oxidative stress induced by cholestanetriol and 25-hydroxycholesterol in porcine ovarian granulosa cells

The cytotoxicity of oxysterols including 7-ketocholesterol, -epoxide, cholestanetriol and 25-hydroxycholesterol and the possible protecting effect of -tocopherol on cholestanetriol and 25-hydroxycholesterol-induced cytotoxicity were investigated in primary cultures of porcine ovarian granulosa cells. Cell viability as determined by % trypan blue staining and mitochondrial function as determined using 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) reduction were decreased significantly after 24 h exposure to 2.5 M -epoxide, cholestanetriol and 25-hydroxycholesterol. 7-ketocholesterol (2.5 M) did not affect cell viability or mitochondrial function under the same culture conditions. The specific activities of catalase and superoxide dismutase, two antioxidant defense enzymes were increased significantly (p < 0.01) following 24 h exposure to 2.5 M concentrations of cholestanetriol while only superoxide dismutase was increased in 25-hydroxycholesterol-treated cells (p < 0.001). Specific activity of glutathione peroxidase was unchanged relative to control cells. Levels of thiobarbituric acid reactive substances remained unchanged after exposure to 7-ketocholesterol, -epoxide, cholestanetriol, 25-hydroxycholesterol and cholesterol. Administration of 1 M -tocopherol to the culture medium significantly improved cell viability and restored both superoxide dismutase and catalase activities to control levels in cholestanetriol -treated cells and only superoxide dismutase in 25-hydroxycholesterol-treated cells. These studies suggest that the cytotoxic nature of physiologically relevant concentrations of cholestanetriol and 25-hydroxycholesterol in granulosa cells is in part due to oxidative stress, but it may be reduced in the presence of a-tocopherol.     

Distribution of Fucoidan Hydrolases and Some Glycosidases among Marine Invertebrates

Thirty-three species of marine invertebrates from the Sea of Japan were analyzed for contents of fucoidan hydrolases and some glycosidases. Fucoidan hydrolase activity was assessed by examining the effect of animal tissue extracts on fucoidans from the two brown seaweeds Laminaria cichorioides and Fucus evanescens, which have different structural characteristics. The activity of glycosidases (-glucosidase, -galactosidase, -fucosidase, and -mannosidase) was determined using p-nitrophenyl derivatives of sugars as substrates. It was found that glycosidases and fucoidan hydrolases of different specificities are fairly widely distributed among marine invertebrates. Mollusks and some species of echinoderms and arthropods showed the highest enzymatic activity. This research will enable us to choose organisms for the separation and study of fucoidan hydrolases and glycosidases, which may be useful in determining the structure of fucoidans.     

Na,K-ATPase: Isoform structure,function, and expression

An interesting feature of the Na,K-ATPase is the multiplicity of and isoforms. Three isoforms exist for the subunit, 1, 2, and 3, as well for the subunit, 1, 2, and 3. The functional significance of these isoforms is unknown, but they are expressed in a tissue- and developmental-specific manner. For example, all three isoforms of the subunit are present in the brain, while only 1 is present in kidney and lung, and 2 represents the major isoform in skeletal muscle. Therefore, it is possible that each of these isoforms confers different properties on the Na,K-ATPase which allows effective coupling to the physiological process for which it provides energy in the form of an ion gradient. It is also possible that the multiple isoforms are the result of gene triplication and that each isoform exhibits similar enzymatic properties. In this case, the expression of the triplicated genes would be individually regulated to provide the appropriate amount of Na,K-ATPase to the particular tissue and at specific times of development. While differences are observed in such parameters as Na⁺ affinity and sensitivity to cardiac glycosides, it is not known if these properties play a functional role within the cell.Site-directed mutagenesis has identified amino acid residues in the first extracellular region of the subunit as major determinants in the differential sensitivity to cardiac glycosides. Similar studies have failed to identify residues in the second extracellular region involved in cardiac glycoside inhibition. Further analysis of the enzymatic properties of the enzyme, understanding the regulated expression of the genes, and structure-function studies utilizing site-directed mutagenesis should provide new insights into the enzymatic and physiological roles of the various subunit isoforms of the Na,K-ATPase.     

A single residual replacement improves the folding and stability of recombinant cassava hydroxynitrile lyase in E. coil

Substitution of Ser113 for Gly113 in the cap domain of hydroxynitrile lyase from Manihot esculenta (MeHNL) was performed by site-directed mutagenesis to improve its self-generated folding and stability under denaturation conditions. The yield of the recombinant mutant HNL1 (mut-HNL1), which had higher specific activity than the wild type HNL0 (wt-HNL0), was increased by 2 to 3-fold. Thermostability of MeHNL was also enhanced, probably due to an increase in content of the -strand secondary structure according to CD analysis. Our data in this report suggest that Ser113 significantly contributes to the in vivo folding and stability of MeHNL and demonstrates an economic advantage of mut-HNL1 over the wt-HNL0.