Activated gelatinase A is reportedly associated with tumor spread. We identified novel matrix metalloproteinases that localize on the cell surface and mediate the activation of progelatinase A. Thus, these progelatinase A activators were named membrane-type matrix metalloproteinase-1 and -2 (MT-MMP-1 and -2, respectively). MT-MMP-1 is overexpressed in malignant tumor tissues, including lung and stomach carcinomas that contain activated gelatinase A. This suggests that MT-MMP-1 is associated with the activation of progelatinase A in these tumor tissues. The expression of MT-MMP-1 also induced binding of gelatinase A to the cell surface by functioning as a receptor. The cell surface localization of proteinases has advantages over pericellular proteolysis. MT-MMP-1 and its family may play a central role in the cell surface localization and activation of progelatinase A and via this mechanism, tumor cell use exogenous progelatinase A to mediate the proteolysis associated with invasion and metastasis.

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A key step in homologous recombination is the formation of a heteroduplex joint between double-stranded DNA and single-stranded DNA by the homologous pairing and strandexchange, and this step is also important in recombinational repair of damaged DNA in various organisms. The homologous pairing and the strand-exchange are promoted in vivo and in vitro by RecA protein of Escherichia coli or its homologues of bacteria, virus, and lower and higher eukaryotes. A central question on the mechanism of homologous recombination is how RecA protein (and its homologues) recognizes homologous sequences between single-stranded DNA and double-stranded DNA. Recent studies suggest that RecA protein promotes homologous recognition between these DNA molecules by the formation of a transient and additional pairing of identical sequences via non-Watson-Crick interactions to the Watson-Crick-type duplex DNA, and that RecA protein uses three non-equivalent DNA-strand-binding sites in this reaction.

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Recombinant human eukaryotic initiation factor-4E (eIF-4E), purified by m⁷GTP-Sepharose 4B affinity chromatography, was used for crystallization. After concentration of the eIF-4E protein (7mg/ml), the solution was subjected to crystallization by the hanging-drop method. Transparent needle crystals complexed with m⁷GTP were obtained from 50mM 2-(N-morpholino)ethanesulfonic acid-KOH buffer (pH 6.5) containing 25% (w/v) polyethylene glycol 6000 and 0.2M (NH₄)₂SO₄. The crystals belong to tetragonal space group P4₁ or P4₃, of Z=4, with unit-cell dimensions of a=89.26, b=89.26, and c=38.51 Å, and diffract beyond 2.1 Å resolution. The V_m value was calculated to be 3.07 ų/Da, which indicates a solvent content of 59.9%.

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The three-dimensional structure of a complex of cinnamycin, a 19-amino acid residue immunopotentiator peptide, and lysophosphatidylethanolamine was determined by ¹H-NMR. The complex was cylindrical in shape, 11 Å in diameter and 26 Å in length, excluding the acyl chain of the phospholipid. The peptide had a hydrophobic pocket surrounded by residues Phe-7 through Ala (S)-14 to bind to the head group of the ligand. Fitting of the head group to the hydrophobic pocket was so good that other than a glycerophosphoethanolamine head group would be unable to fit the pocket. The goodness of the fitting is compatible with the strict specificity of ligand binding of the peptide.

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Interferon stimulated gene factor 3 (ISGF3) is a trimeric transcription factor activated on treatment of cells with interferon-α and β (type I IFNs). Upon stimulation, the regulatory subunits, p84/91 and p113, present in the cytoplasm are phosphorylated at specific tyrosine residues and assemble with the DNA binding subunit, ISGF3γ, into the active ISGF3 in the nucleus. Thus, ISGF3 plays a primary role in the transmission of a signal from the cell surface to the nucleus. In this report, we describe the cloning of a mouse eDNA encoding a polypeptide homologous to human ISGF3γ. Comparison of the deduced amino acid sequences revealed the middle region was significantly different between mouse and man. The mouse cDNA was shown to encode a functional ISGF3 subunit by means of an in vitro reconstitution assay. Furthermore, the locus of the ISGF3γ gene, designated as Isgf3g, was mapped to distal mouse chromosome 14 by linkage analysis using an intersubspecific backcross typing panel.

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A 220-bp fragment of PTK7 cDNA was previously cloned from normal human melanocyte RNAs by means of the reverse transcription-polymerase chain reaction [Lee, S. -T., Strunk, K. M., and Spritz, R. A. (1993) Oncogene 8, 3403-3410]. We now report the cloning of the human full-length PTK7 cDNA and its characterization. The 1, 070-amino acid PTK7 polypeptide deduced from the cDNA sequence constitutes receptor protein tyrosine kinase (RPTK), but has several unusual residues in some of the highly conserved tyrosine kinase motifs. PTK7 mRNA was expressed at the highest level in a human erythroleukemia cell line among tested samples, and at relatively high levels in liver, lung, pancreas, kidney, placenta, and melanocytes. Human PTK7 is 72% identical to chick KLG, suggesting that PTK7 is homologous or possibly orthologous to chick KLG, and that these represent a new subfamily of RPTKs.

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Male Wistar rats show typical age-related variations in the distribution of high-density lipoprotein subfractions that include an increase in HDL1 and a decrease in HDL2 proportion. The role of liver in these variations was evaluated by studying the lipoprotein and bile secretions from perfused livers of 14±1 and 3.5±0.5 month old Wistar rats (adult and young animals, respectively). The lipid content of lipoproteins secreted from adult livers was higher in HDL2 fraction and lower in VLDL fraction. The lipid output did not show significant age-related variations in the case of HDL1 fraction. However, the lipoproteins secreted from adult livers contained a higher proportion of phospholipids, and a lower proportion of triacylglycerols in comparison with lipoproteins secreted by young livers. Therefore, the molar ratio of core to surface lipids was lower in lipoproteins secreted by adult livers. Adult livers showed a reduction in bile flow by about 37% with a significantly higher phospholipid secretion. These findings suggest that both the hepatic metabolism of glycerophospholipids and their repartition between plasma and bile compartments are affected by aging process. In conclusion, present data show that the age-related increase in plasma HDL1 proportion, previously observed in this rat strain in vivo, are not due to a higher liver secretion of these particles. Conversely, liver appears to have a major role in the age-related VLDL increase and in the variations of phospholipid lipoprotein secretion.

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The metabolism of diacetyl (2, 3-butanedione), acetoin (3-hydroxy-2-butanone), and 2, 3-butanediol, which are metabolites of acetaldehyde was quantitatively investigated using rat liver homogenate, liver perfusion, and in vivo experiments. Diacetyl and acetoin were reduced to 2, 3-butanediol in these experiments, but acetoin and 2, 3-butanediol were scarcely oxidized to diacetyl, indicating that the reduction reaction to 2, 3-butanediol from diacetyl occurs actively in rat liver. The formation of acetoin from diacetyl required either NADH or NADPH as a reductant, while the reduction of acetoin to 2, 3-butanediol required NADH. Acetoin and 2, 3-butanediol were more readily accumulated than diacetyl in brain tissue.

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A transcription and translation coupled reticulocyte lysate system was established for rapid screening of antisense oligodeoxyribonucleotides (ODNs) to determine which are most effective for mRNA translation-arrest. A plasmid containing the target cDNA under the control of the T7 (or SP6) promoter was added to the lysate system in the presence of the T7 (or SP6) RNA polymerase, RNase H, and the antisense ODN under test. Transcription and translation were accomplished in a one-tube reaction. Translation-arrest caused by antisense ODN was evaluated in terms of the amounts of de novo-synthesized, [³⁵S]-methionine or [³⁵S] cysteine labeled target protein measured by gel electrophoresis and autoradiography. The properties of this system and optimal reaction conditions for use in antisense ODN screening were determined. Our method is simpler and more rapid than other in vitro screening methods.

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Cytosolic NADPH-dependent ubiquinone reductase (NADPH-UQ reductase) accounted for about 68% of the total ubiquinone (UQ) reductase activity in rat liver homogenate [Takahashi, T. et al. (1995) Biochem. J. 309, 883-890]. We investigated the effects of various factors on this enzyme activity in rat liver cytosol with the aim of elucidating its physiological roles. The NADPH-UQ reductase in rat liver cytosol catalyzed the reduction of UQ to UQH₂ with concomitant oxidation of equimolar NADPH. The optimal pH was around 7.4, and the optimal temperatures were about 28°C for NADH and about 37°C for NADPH. NADH, deamino NADH, and deamino NADPH were much less active hydrogen donors than NADPH, whereas reduced nicotinamide mononucleotide, ascorbate, erythorbate, reduced glutathione, and cysteine were inactive. As the hydrogen acceptor, UQ-9 had the highest V_max/K_m among the long-chain UQ homologues tested. FAD and FMN stimulated the activity. Anionic detergents, Mg²⁺ and Sr²⁺ also enhanced the activity. Rotenone, malonic acid, antimycin A, and KCN, which inhibit mitochondrial and microsomal electron transfer enzymes, superoxide dismutase, and acetylated cytochrome c had no effect on the NADPH-UQ reductase activity. These results indicated that the NADPH-UQ reductase in rat liver cytosol is a flavoprotein that reduces UQ-10 by a two-electron reduction mechanism DT-diaphorase [EC 1. 6. 99. 2].

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Chondrocytes derived from rat xiphoid cartilage dedifferentiated into fibroblast-like cells as the number of passages of the cells in culture increased. During in vitro dedifferentiation the growth of the cells was markedly suppressed. We had proposed previously that C-type natriuretic peptide (CNP) might be a potent antimitogenic factor for chondrocytes, and TGF-β1 induced a marked increase in CNP secretion of chondrocytes. Therefore, we investigated the expression of CNP, B-type natriuretic peptide receptor (NPR-B or GC-B), and TGF-β1 in this process. Radioimmunoassay and RNase protection analyses revealed passage-associated increase in CNP-like immunoreactivity and in levels of NPR-B mRNA, respectively. Northern blot analyses showed that the level of TGF-β1 mRNA decreased with increasing passage number. These results suggest that the expression of CNP and NPR-B might be involved in in vitro dedifferentiation of chondrocytes and TGF-β1 does not affect the increasing level of CNP during in vitro dedifferentiation.

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Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) of the post-synaptic density (PSD) was solubilized and activated 4- to 5-fold by limited α-chymotrypsin digestion with prior autophosphorylation of the kinase. The enzyme was also activated by trypsin and μ-calpain, a Ca²⁺-dependent protease. The active catalytic fragment was purified to homogeneity using gel filtration and ion exchange chromatography. The purified active fragment was completely Ca²⁺/calmodulin-independent and exists as a monomer. Kinetic studies with the purified fragment revealed similar K_m values for ATP and synthetic peptide substrate, and an about 8-fold increment in V_max, compared with native PSD CaM kinase II.

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Effects of basic glycoside antibiotic aculeximycin (ACM) on the oxidative phosphorylation of rat-liver mitochondria were examined. ACM was shown to be a potent uncoupler of the oxidative phosphorylation. To cause the same extent of respiration release, higher concentration of ACM was required in phosphate (P_i ) -free medium than in P_i medium. During the uncoupling caused by ACM in P_i medium, large amplitude swelling and oxidation of intramitochondrial NAD (P) H occurred, indicating that ACM remarkably enhances permeability of the inner mitochondrial membrane. The P_i uptake via P_i/H⁺ symporter was shown to play an important, but not essential, role in the uncoupling by ACM, indicating the increase in membrane permeability is mostly due to acceleration of P_i/H⁺ influx through P_i /H⁺ symporter activated by ACM. ACM is the first naturally occurring antibiotic, to our knowledge, which activates P_i/H⁺ symporter. However, since the inhibition of P_i/H⁺ symporter by N-ethylmaleimide did not completely abolish the uncoupling activity of ACM, and ACM induced the uncoupling even in P_i-free medium, an increase in the membrane permeability for other ions, such as Na⁺ and K⁺, due to a different action mechanism has also to be considered. On the other hand, positively charged amine local anesthetics, like dibucaine, prevented the uncoupling activity by ACM in both P_i and P_i-free medium. The uncoupling activity of N-diacetylated ACM lacking free amino groups was ca. 1/120th that of ACM, indicating that positively charged amino groups are important for the uncoupling activity. It is suggested that some specific interactions between positively charged amino groups of ACM and the binding site, which is probably negatively charged, are triggers that affect the permeability of the inner mitochondrial membrane. Amine local anesthetics may mask the negative charge of the binding site, thereby interfering with ACM binding.

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Nine monoclonal mouse anti-human aldolase C antibodies, mAbs A4, A8, B4, B7, B8, C1, D9, E10, and H1, were isolated and characterized. These mAbs fall substantially into four groups according to their reactivity with antigens. (i) Human aldolase C-specific mAbs (B8, D9, and H1). (ii) Type C aldolase-specific mAbs (B4 and E10). (iii) Ubiquitous mAbs, which react with vertebrate aldolases irrespective of type of isozyme and species (A4 and B7). (iv) Sub-ubiquitous mAbs, which are closely similar to the ubiquitous mAbs but differ slightly in terms of antigenic specificity (A8 and C1). Aldolase C-specific mAbs B8, H1, B4, and E10, but not D9, have their epitopes on a region within amino acid positions 79-193 of antigens, where the type-C isozyme group-specific sequence-3 (IGS-3) is situated. In contrast, ubiquitous mAbs A4 and B7 and sub-ubiquitous mAb A8 may have their epitopes on the commonly conserved regions of the three isozyme groups. The epitope of sub-ubiquitous mAb C1 appears to be on the IGS-2/3 but this is yet to be resolved. These nine mAbs can be classified into two groups based on the mode of epitope recognition, which was determined by ELISA, immunoblotting, and immunoprecipitation assays: (i) primary sequenceepitope mAbs such as B4, E10, and B7; and (ii) conformation-epitope mAbs (B8, D9, H1, A4, A8, and C1). Among these mAbs, aldolase C-specific mAbs H1 and E10 appear to be useful as probes for detection of conformational change around the type-C IGS-3 motif of human aldolase C because, when assessed by immunoprecipitation assay, mAb H1 reacts only with human aldolase C but not with CA250 and CA306, while mAb E10 reacts with CA250 and CA306 but not with aldolase C, even though these antigens have a common type-C IGS-3 motif. Similarly, the ubiquitous mAb B7 should serve as a probe for general use to detect vertebrate aldolases irrespective of isozyme groups and species.

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The roles of adult worker honeybees change with age; young workers nurse brood by secreting bee milk (royal jelly), and older workers forage for nectar and pollen and process nectar into honey. The electrophoretic profile of worker hypopharyngeal-gland proteins changes with age and/or role. Immunoblotting analysis using affinity-purified antibodies against three major proteins (50, 56, and 64 kDa) of the nurse-bee gland showed that they are synthesized selectively and secreted as bee-milk proteins. Immunofluorescence study showed that the proteins condense in the duct after secretion from acini. However, a major 70-kDa protein synthesized specifically in the forager-bee hypopharyngeal gland was identified as an α-glucosidase. Therefore, the hypopharyngeal gland seems to have two distinct states differentiated by synthesizing of different major proteins depending on the age-dependent role change.

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To elucidate the mechanisms underlying cell type-specific expression of the growth hormone (GH) and prolactin (PRL) genes, we used rat pituitary-derived cell lines producing exclusively GH (GC cells) or PRL (235 cells), and examined the following: expression of transcription factors essential for GH and/or PRL gene expression; promoter/enhancer activity of the GH and PRL genes transiently introduced by transfection; and chromatin structures of the GH and PRL genes. Even in PRL-nonproducing GC cells, the PRL promoter/enhancer was more active than the GH promoter, and the transcription factors, Pit-1 and estrogen receptor (ER), essential for PRL gene expression were functional. The PRL promoter/enhancer of GC cells was normal. On DNase I sensitivity analysis of the chromatin structure, two hypersensitive sites were detected in PRL gene chromatin of PRL-producing 235 cells but none in that of GC cells. It thus follows that the major reason for absence of the expression of the endogenous PRL gene in GC cells is neither the lack of transcription factors necessary for PRL gene expression nor an anomaly of the PRL gene itself, but that the chromatin structure of the PRL gene differs in PRL-nonproducing and -producing cells. It was shown in this study that neither Pit-1 nor ER is required for conversion of the structure of PRL gene chromatin to a DNase I-hypersensitive state.

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The polymerase chain reaction was used to amplify and clone a fragment of a novel fucosyltransferase cDNA from mouse embryonal carcinoma F9 cells. Southern blot analysis of mouse genomic DNA with the cloned cDNA fragment revealed that there is a single copy of the fucosyltransferase gene per haploid genome. A 5 kb EcoRI restriction fragment of the genomic DNA hybridized with the cDNA has been cloned and a 1, 697bp fragment containing the sequence of the cDNA was sequenced. The nucleotide sequence and the deduced amino acid sequence of the clone showed a highest degree of homology with those of human α-1, 3 fucosyltransferase IV, i.e. 77.1 and 75.5% identity, respectively. Consistent with the sequence similarity, the transfection of a 1, 544bp fragment under the control of the cytomegarovirus enhancer and β actin promoter into COS-1 cells confers α-1, 3 fucosyltransferase activity on the cells, resulting in cell surface expression of SSEA-1.

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BTEB is a GC-binding protein that regulates the transcription of genes with a single GC-box or tandemly repeated GC-boxes in the promoter. The DNA-binding domain of BTEB consists of three contiguous Cys₂-His₂ zinc finger motifs and short segments adjacent to their N- and C-terminal sides [Kobayashi et al. (1995) J. Biochem. 117, 91-95]. The truncated BTEB (residues 120 to 244) containing the DNA-binding domain was expressed in Escherichia coli and purified to homogeneity under denaturing conditions. DNAbinding activity of the BTEB was regenerated by refolding in the presence of Zn²⁺. The efficiency in regeneration was 70±10%, and the dissociation constant (K_d) of the DNAcomplex was 4±2nM. Co²⁺ also regenerated the DNA-binding affinity of BTEB, albeit with less efficiency than Zn²⁺. Co-BTEB showed a slightly lower affinity to the specific DNA than Zn-BTEB. Refolding in the presence of Cd²⁺ resulted in an extremely low efficiency in regeneration of the DNA-binding activity. Zn-BTEB is in a monomer state at concentrations lower than 0.5μM, and forms a dimer in the concentration range of about 10 to 200μM.

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Rhodotorula minuta, a red yeast, produces a cytochrome P450, tentatively named P450rm, catalyzing the formation of isobutene from isovalerate. We found that P450rm interacted with benzoate and the dissociation constant of P450rm for benzoate was 36μM. A reconstituted system that consisted of purified P450rm and cytochrome P450 reductase catalyzed the 4-hydroxylation of benzoate in addition to the formation of isobutene; the turnover rate was _??_40nmol/min/nmol P450rm. The P450rm-monooxygenase system was specific for benzoate and did not catalyze hydroxylation of other aromatic carboxylates. Since only a benzoate 4-hydroxylase that requires tetrahydropteridine has been isolated to date, P450rm appears to be the first isolated cytochrome P450 that acts as a benzoate 4-hydroxylase. The P450rm-monooxygenase system in microsomes of R. minuta might function in the degradation of L-phenylalanine on the pathway to β-ketoadipate.

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Rae-1 cDNA is one of the retinoic acid (RA)-inducible cDNA clones in mouse embryonal carcinoma F9 cells. Rae-1 mRNAs were detected in mouse early embryos, but not in various tissues of adult mice. RAE-1 protein apparently consists of 253 amino acids and is likely to be a glycoprotein consisting of a leader sequence, an extracellular domain, a serine, threonine, proline-rich domain, and a transmembrane domain. Interestingly, it has a weak, but significant homology with major histocompatibility complex (MHC) class I molecules and was immunocytochemically identified as a cell surface protein. By determining partial nucleotide sequences of 17 Rae-1 cDNAs isolated from the RA-induced F9 cells, at least three different kinds of Rae-1 cDNAs were identified and were named Rae-1α, Rae-1β, and Rae-1γ cDNAs, respectively. As the overall nucleotide sequence homology among these three cDNAs was about 98%, they constitute a novel gene family which is likely to be involved in early mammalian embryogenesis.

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Activities of protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) in autoimmune MRL/MpJ-lpr/lpr mice (lpr mice) were measured and compared with the activities in the tissues from MRL/MpJ-+/+ mice (+/+ mice) as the control. In the spleen and liver, PTK activities in cytosol and membrane fractions were about 1.7- and 1.3-fold, respectively, higher in lpr mice than +/+ mice. PTP activities in cytosol and membrane fractions from lpr mice were 1.7- and 1.3-fold, respectively, higher in spleen, and 2.5- and 1.3-fold, respectively, higher in liver compared with those of the controls. These results demonstrate that the mutation of lpr gene resulted in elevation of PTK and PTP activities. Then, we measured the amounts and activities of SH-PTP1, a cytosolic PTP playing a crucial role in intracellular signaling from Fas antigen. The amounts of SH-PTP1 were about 4-fold larger in thymus, spleen, and lymphnodes than in liver, but there was no marked difference in the amounts between lpr and +/+ mice. On the other hand, activity of SH-PTP1 was definitely lower in lpr spleen and lymphnodes than +/+ spleen, but several times higher in lpr liver than +/+ liver. Tyrosine phosphorylation levels of SH-PTP1 in spleen of lpr and +/+ mice were similar. However, in liver, it was less phosphorylated in lpr than in +/+ mice. This hypophosphorylation might cause the activation of SH-PTP1 activity in lpr liver.

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Our previous studies have shown that some human cancer cell lines produce pancreatic trypsinogen, plasminogen, and tissue-type kallikrein. To understand the regulatory mechanism of these proteinases, serine proteinase inhibitors secreted by human glioblastoma cell line T98G were analyzed by gelatin reverse zymography with trypsin. The serum-free conditioned medium of T98G cells showed more than ten trypsin inhibitor bands ranging from 16 to 150 kDa in the reverse zymography. Major trypsin inhibitors were purified by trypsin-affinity chromatography. Analysis of their N-terminal amino acid sequences demonstrated that the purified inhibitors were identical to the secreted forms of amyloid protein precursors (APPs), tissue factor pathway inhibitor (TFPI), placental protein 5 (PP5)/TFPI-2, and secretory leukocyte proteinase inhibitor (SLPI). In addition, a novel 25-kDa trypsin-binding protein, tentatively named p25TI, was identified. p25TI showed weak inhibitory activity against trypsin in reverse zymography as compared with the other inhibitors. The secretion of multiple forms of serine proteinase inhibitors by human cancer cells raises the possibility that they might be involved in the abnormal growth of cancer cells.

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The constitutive expression of hepatic fatty acid hydroxylase was examined in both sexes of ddY mice by measuring the activities of lauric acid ω-hydroxylase (LAH). The activity of male mice was significantly higher than that of female mice. Such a sex difference of hepatic LAH activity was not observed in other strains of mice, including BALB/c and C57BL/6. To examine whether decreased total P450 activities caused low LAH activity levels in female ddY mice, ethoxycoumarin O-deethylase activity, which is exhibited by many P450s, was measured in both sexes of mice. This activity had no sex difference. The developmental regulation of hepatic fatty acid hydroxylase was then examined by making consecutive measurements of LAH activity in ddY mice. The activity is the same in immature male and female mice, but is differentiated in the sexually mature state. Furthermore, in male mice, orchiectomy caused a dramatic decrease in hepatic LAH activity and the activity was restored by testosterone treatment to the level of the intact animal. In female mice, ovariectomy and estradiol treatment had no effect on the activity, but testosterone treatment caused an increase in the activity. The above data are consistent with the constitutive expression of CYP4A-related proteins measured by using anti-rat CYP4A1 polyclonal antibody. Anti-CYP4A1 antibody inhibited LAH activity, but not lauric acid (ω-1)-hydroxylase activity. These results suggest that some factors associated with male sex hormone are involved in the regulation of hepatic fatty acid ω-hydroxylase in ddY mice.

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We previously reported a new type of lectin, p33/41 (annexin IV), which was isolated from a bovine tissue extract [Kojima, K. et al. (1992) J. Biol. Chem. 267, 20536-20539]. When the expression of p33/41 (annexin IV) was surveyed in the lysates of 39 human tumor cell lines by SDS-PAGE, followed by Western blot analysis with polyclonal anti-bovine p33/41 and monoclonal anti-annexin IV (Z016, Zymed) antibodies, 21 cell lines were found to be reactive with the polyclonal antibody, whereas all 39 cell lines were stained with Z016. These results together with those obtained with standard proteins, annexins IV and V, suggested that the monoclonal antibody, Z016, recognizes annexin V, but not p33/41 (annexin IV). Therefore, we performed cDNA cloning of human p33/41 (annexin IV) to prepare a recombinant protein and raised monoclonal antibodies against the protein. Northern blot analysis with the cDNA as a probe showed that a human colon cancer cell line, HT29, contains p33/41 (annexin IV) mRNA of two sizes, 2.0 and 3.0kb. The two monoclonal antibodies, AS11 and AS17, against the recombinant protein generated were useful for flow cytometric analysis, ELISA, Western blot analysis and immunoprecipitation. Flow cytometric analysis with AS17 showed that p33/41 (annexin IV) is located in the cytoplasm of HT29 cells, but not on the cell surface. However, one of the cell surface proteins first labeled with biotin and then solubilized with a detergent was immunoprecipitated with AS17. The results suggest the existence of a membrane spanning form of p33/41 (annexin IV).

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Incubation of Sarcophaga fat bodies in buffered insect saline resulted in simultaneous activation of multiple defense protein genes [Sugiyama, H. and Natori, S. (1991) Eur. J. Biochem. 200, 495-500]. In this study, we demonstrated that the addition of both adenosine and homocysteine thiolactone to this medium abolished activation of these defense protein genes. As S-adenosyl-homocysteine, a potent inhibitor of the transmethylation reaction, is produced in the fat body under these conditions, methylation of a certain fat body protein seems to be a prerequisite for the selective activation of insect defense protein genes. Activation of the HSP70 gene was not interfered with S-adenosyl-homocysteine.

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Hemoglobins of rats switch from an embryonic to an adult type during fetal development. However, very little is known about the structures and molecular species of hemoglobins occurring in the fetal life of rats. In the present study we isolated five embryonic hemoglobins, designated El, E2, E3, E4, and E5, from the blood of rat fetuses on day 14 of gestation by ion exchange chromatography. Reverse-phase high performance liquid chromatography revealed that these hemoglobins each consist of two kinds of globins: E1 (¹¹α:ε1), E2 (¹α:ε1), E3 (ζ:ε1), E4 (¹α:ε3), and E5 (ζ:ε3), respectively. The complete amino acid sequences of the ζ, ε1, and ε3 globins were determined. The ζ globin showed characteristic features common in α-type embryonic globins of known species in that the N-terminus is blocked and the amino acid at position 38 is Gin. ε1 and ε3 are β-type embryonic globins, sharing 73.7% amino acid homology. Interestingly, they are more similar to the corresponding mouse β-type embryonic globins, y and z, respectively, than to each other, implying that these globins have evolved orthologously from common ancestral proteins. It was also shown that the ζ, ε1, and ε3 globins are almost completely replaced by the adult type α and β globins in the blood of rat fetuses by day 18 of gestation.

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A novel and efficient method for analyzing sugar-lectin interaction using affinity electrophoresis (AEP) is described. Polyacrylamide gels covalently conjugated with 2-acetamido-2-deoxy-n-glucopyranose (GlcNAc) residues were successfully prepared by radical copolymerization of highly reactive 3-(N-acryloylamino) propyl 2-acetamido-2-deoxy-β-D-glucopyranoside with acrylamide in the presence of N, N'-methylenebisacrylamide (BIS). When the glycogels carrying various densities of GlcNAc branches were employed for polyacrylamide gel electrophoresis (PAGE) of lectins, the mobilities of wheat germ agglutinin (WGA) were specifically reduced by increasing the concentrations of the GlcNAc residues in gels, although concanavalin A (Con A) showed no significant change in the mobility. It was also demonstrated that the association constant of WGA with immobilized GIcNAc residue can be determined by combined use of this stable glycogel and an automated gel-scanning system associated with fluorometric spectroscopy. The association constant of WGA with the GlcNAc moiety was estimated to be 1.24×10⁴M^-1.

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A human blood type A hemagglutinating activity was detected in albumin gland extracts of Epiphragmophora trenquelleonis snail separated by GalNAc-agarose affinity chromatography, of which two N-acetyl-D-galactosamine-binding lectins in the extracts were ETL1 was displaced from the affinity column with 1mM GalNAc, and ETL2 with 20mM GalNAc. Both lectins agglutinated specifically human blood type A and AB erythrocytes, but not type B and O erythrocytes. Gel filtration chromatography gave a native molecular weight of about 59 kDa for ETL1 and about 54 kDa for ETL2. On SDS-PAGE under nonreducing conditions, ETL1 showed two protein subunits of about 29 and 27 kDa, while ETL2 showed three protein subunits of about 27, 24, and 22 kDa. On SDS-PAGE under reducing conditions, both lectins showed four protein subunits of 17, 16, 12, and 11 kDa. By Western blot analyses developed with biotin-labeled lectins, N-linked oligosaccharides were detected in the 17- and 16-kDa protein subunits of ETL1 and ETL2, and in the 12-kDa protein subunit of ETL2. O-linked oligosaccharides were detected only in the 11-kDa protein subunit of ETL1 and ETL2. On isoelectric focusing both lectins exhibited microheterogeneity: ETLI focused as three protein bands with pls in the range of 5.6-6.0, while ETL2 focused as four protein bands with pls in the range of 6.8-7.4. We suggest that native ETL1 and ETL2 are glycoprotein complexes with molecular weights of 59-54 kDa, composed of two 29-22-kDa nonreduced protein subunits held together by noncovalent hydrophobic interactions. Each of the nonreduced protein subunits seems to be composed of two 17-11-kDa reduced protein subunits held together by interchain disulfide linkages. The main differences between ETL1 and ETL2 could be due to different posttranslational modifications or to the relative contribution of one or more of their protein subunits.

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Since it was considered that an active immunization against ganglioside Gfpt1 (IV²Fuc-, II³NeuAc-Gg₄Cer) expressed by human small cell lung cancer cells may be beneficial in the treatment of this neoplasm in humans, an optimal mode of vaccination in model mice was investigated. A novel Gfpt1-muramyldipeptide conjugate (Gfpt1-MDP) was synthesized. Its ganglioside carbohydrate-directed immunogenicity in mice as measured by serum antibody titers was comparable to that of the previously described Gfptl-keyhole limpet hemocyanin conjugate (Gfpt1-KLH). Similar immunogenicity was displayed by free Gfpt1 in muramyldipeptide-phosphoethanolamine-containing phosphatidyl-choline, -serine (PC, PS) liposomes. Immunization with Gfpt1-vaccines in the presence of monophosphoryllipid A (MPL), in general, raised titers of anti-Gfpt1 antibodies effectively. Immunization with PC, PS-liposomes containing unconjugated Gfpt1 and MPL stimulated the highest titers observed, thereby effectively preventing tumor growth in Balbc nu/nu-mice challenged with human small cell lung cancer cells. However, there was a strong crossreactÌon of these and most other sera with the structurally related and widely distributed ganglioside Gtetl (II³NeuAc-Gg₄Cer). Only immunization with Gfpt1-KLH conjugate in the presence of MPL stimulated selectively high anti-Gfpt1 antibody titers showing comparably low crossreactivity to ganglioside Gtet1.

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