Immunochemical Studies Research Articles

Comparative Characterization of the Lipopolysaccharides of Different Pseudomonas fluorescens Biovar I Strains

From the biomass of five Pseudomonas fluorescensbiovar I strains, including the P. fluorescenstype strain IMV 4125 (ATCC 13525), lipopolysaccharides (LPS) were isolated (by extraction with a phenol–water mixture followed by repeated ultracentrifugation), as well as individual structural components of the LPS macromolecule: lipid A, the core oligosaccharide, and O-specific polysaccharide (O-PS). 3-Hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, hexadecanoic, octadecanoic, hexadecenoic, and octadecenoic fatty acids were present in lipid A of the LPS of all the strains studied. Glucosamine, ethanolamine, and phosphoethanolamine were revealed in the lipid A hydrophilic part of all of the strains. Glucose, rhamnose, mannoze, glucosamine, galactosamine, KDO, a trace amount of heptoses, ethanolamine, phosphoethanolamine, alanine, and phosphorus were identified as the main core components. Interstrain differences in the core oligosaccharide composition were revealed. Structural analysis showed that the O-PS of the type strain, as distinct from that of other strains, is heterogeneous and contains two types of repetitive units, including (1) three L-rhamnose residues (L-Rha), one 3-acetamide-3,6-dideoxy-D-galactose residue (D-Fuc3NAc) as a branching substitute of the L-rhamnan chain and (2) three L-Rha residues and two branching D-Fuc3NAc residues. The type strain is also serologically distinct from other biovar I strains due to the LPS O-chain structure, which is similar to those of the strains of the species Pseudomonas syringae, including the type strain. The data of structural analysis agree well with the results of immunochemical studies of LPS.

Antigenic properties of human glycophorins--an update.

Glycophorins are complex heavily glycosylated antigens carrying peptidic and glycopeptidic epitopes. Detailed immunochemical studies showed that GPA/GPB and GPC/GPD molecules have defined sites which are particularly immunogenic. These sites include N-terminal portions of all glycophorins, internal fragments of their extracellular domains, and cytoplasmic tails. The extracellular epitopes involve directly oligosaccharide chains (e.g. blood group M- and N-related epitopes, or N-terminal epitopes of GPC) or have peptidic character, shown by the reaction of respective antibodies with synthetic peptides. Peptidic eitopes are independent of glycosylation, or are variably affected by adjacent O-glycans which may mask the epitopes or may be required for a proper exposure of an antibody binding site. Several low incidence epitopes are present on variant glycophorin molecules. Among anti-glycophorin antibodies there are the 'bispecific' ones, or antibodies recognizing an epitope formed by an interaction of two proteins (Wr(b)). Alltogether, the glycophorins serve as convenient model antigens for studying Ag-Ab interaction and a role of O-glycosylation in protein antigenic properties. Moreover, well defined specificty of monoclonal anti-glycophorin antibodies makes them more precise tools in serological investigation and identification of normal and variant antigens. Last but not least, elucidation of antigenic properties of glycophorins is important for identification and characterization of human anti-glycophorin antibodies, which in some cases create medical problems at transfusion or pregnancy.

The expression of CD70 and CD80 by gene-modified tumor cells induces an antitumor response depending on the MHC status.

The expression of costimulatory molecules such as CD70 or CD80 by gene-modified tumor cells has been shown to enhance the antitumor immune response based mainly on T lymphocytes. However, most human tumors show defects of major histocompatibility complex (MHC) expression, preventing them from being recognized by MHC-restricted T cells. To investigate if coexpression of CD70 and CD80 costimulatory molecules induces comparable antitumor responses in low and high MHC-expressing tumor cells, we used two low immunogenic murine tumor models, the B16.F10 melanoma and the TS/A mammary adenocarcinoma cell lines expressing, respectively, low and high levels of MHC class I molecules. Transfection of both CD70 and CD80 genes resulted in an increased capacity of gene-modified tumor cells to costimulate in vitro the proliferation and cytokine production of optimally activated lymphoid cells. Coexpression of CD70 and CD80 by the two tumor cell lines, TS/A and B16.F10, resulted in both cases in partial regression of subcutaneous tumors. Immunochemical analysis and studies in nude mice showed that, even in the B16.F10 model, T cells had a significant role in the antitumor response induced by combining CD70 and CD80. However, rejection of the CD70/CD80-transfected tumor cells appeared more effective in the MHC class I high TS/A model, leading to a protection against parental tumor cells. B16.F10 and TS/A transfectants were then tested with fibroblasts genetically modified to secrete interleukin-12 (IL-12) as a therapeutic vaccine in mice bearing parental tumors. In the two models tested, the injections of irradiated IL-12 and CD70/CD80 gene-modified cells generated an antitumor response to established tumors leading to the slowing down of the tumor growth rate. Although the mechanisms remain to be defined, these findings suggest that the combination of several immuno-modulatory molecules could provide additional strategies for cancer immuno-gene therapy, even for MHC expression-deficient tumors.

Elucidation of the primary structure of NB1-glycoprotein (HNA-2a antigen)

The human granulocyte antigen HNA-2a is expressed on a subpopulation of granulocytes in 97% of Caucasians. Alloimmunization against this antigen can cause alloimmune neonatal neutropenia, transfusion-related acute lung injury and immune neutropenia after bone marrow transplantation. Immunochemical studies demonstrated that NB1 allogenic determinants reside on a GPI-linked glycoprotein with apparent molecular weight of 56–64 kDa. To elucidate the primary structure of NB1 protein, we isolated NB1 protein using NB1-specific mab 7D8 by affinity chromatography. Identity of the purified glycoprotein was confirmed by immunoblot using a panel of seven human and two monoclonal NB1-specific antibodies. Determination of molecular mass using MALDI yielded a 50·6 kDa glycoprotein. After removal of N-linked carbohydrates, the molecular mass was reduced to 43·1 kDa. N-terminal amino acid sequencing by Edman degradation yielded a 20 aa-peptide. Rapid amplification of cDNA ends derived from an HPA-2a (+) individual using primers based on N-terminal amino acid sequence resulted in a 1614-bp cDNA, consisting of a 27-bp 5′ untranslated region, a 1311-bp open reading frame and a 276-bp 3′ untranslated region. The 1311 bp open reading frame encoded for 437 amino acids, of which the first 21 were found to form the signalpeptide. The identity of this transkript was confirmed by the expression of full lengh NB1 cDNA (bp 22–1394) in COS-7 cells.

Detection of immunoreactive napsin A in human urine

Human napsin A is an aspartic proteinase highly expressed in kidney and lung. To elucidate whether napsin A is excreted in the urine we have performed an immunochemical study using anti-napsin A polyclonal antibody. As a result an immunoreactive band at approx. 38 kDa was detected which corresponds to the molecular mass of recombinant active human napsin A. A deglycosylation study showed that excreted napsin A is N-glycosylated on apparently all of the three potential glycosylation sites. Immunoreactive napsin A was also observed in urine from patients with a transplanted kidney whose kidney function appeared half to fully normal. On the other hand, no or very low immunostaining was detected in samples from patients with diseased kidneys. The urinary excretion pattern correlates well with the enzymatic activity of napsin A. These data show that human napsin A is excreted as functional proteinase in the urine. Furthermore, immunochemical studies suggest a relation between urinary excretion of napsin A and renal function. More specifically, lack of urinary excretion of napsin A could potentially serve as a tool for the detection of kidney dysfunction.

Plasmodium falciparum histidine-rich protein 1 associates with the band 3 binding domain of ankyrin in the infected red cell membrane

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5′ repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.

Sildenafil augments pelvic nerve-mediated female genital sexual arousal in the anesthetized rabbit.

The NO-cGMP pathway has been implicated in clitoral and vaginal smooth muscle relaxation based on previous immunochemical, biochemical and physiologic studies. There are limited data from in vivo studies demonstrating enhancement of the genital sexual arousal response by pharmacologic agents influencing the NO-cGMP pathway. The goal of this study was to investigate if sildenafil, a phosphodiesterase type-5 inhibitor, facilitated female genital sexual arousal in an animal model in response to pelvic nerve stimulation (PNS). Using female New Zealand White rabbits, we measured the following parameters before, during and after PNS at 4, 16, and 32 Hz: a) hemoglobin concentration and oxygen saturation in female genital (vaginal, labial, clitoral) tissues by laser oximetry; b) clitoral blood flow by laser Doppler flowmetry; c) vaginal luminal pressure by a balloon catheter pressure transducer; d) vaginal lubrication by tampon. Sildenafil was administered intravenously (0.21 microg/kg, 0.42 microg/kg, 2.1 microg/kg) to achieve a systemic concentration of 5, 10 and 50 nM, respectively. After 20 minutes, physiologic measurements were repeated. Sildenafil (50 nM) caused a significant increase in genital oxyhemoglobin concentration and a significant decrease in genital deoxyhemoglobin concentration. Sildenafil also increased the duration of response following PNS, relative to genital hemoglobin concentration and mean clitoral blood flow. Sildenafil caused a decrease in vaginal luminal pressure and resulted in an increase in vaginal lubrication. These data indicate that the NO-cGMP pathway is involved in the physiologic mechanism of female genital arousal and that sildenafil facilitates this response in an in vivo animal model.

Aromatic components of two ferric enterobactin binding sites in Escherichia coli FepA.

Ferric enterobactin is a catecholate siderophore that binds with high affinity (Kd approximately 10-10 M) to the Escherichia coli outer membrane protein FepA. We studied the involvement of aromatic amino acids in its uptake by determining the binding affinities, kinetics and transport properties of site-directed mutants. We replaced seven aromatic residues (Y260, Y272, Y285, Y289, W297, Y309 and F329) in the central part of FepA primary structure with alanine, individually and in double combinations, and determined the ability of the mutant proteins to interact with ferric enterobactin and the protein toxins colicins B and D. All the constructs showed normal expression and localization. Among single mutants, Y260A and F329A were most detrimental, reducing the affinity between FepA and ferric enterobactin 100- and 10-fold respectively. Double substitutions involving Y260, Y272 and F329 impaired (100- to 2500-fold) adsorption of the iron chelate more strongly. For Y260A and Y272A, the drop in adsorption affinity caused commensurate decreases in transport efficiency, suggesting that the target residues primarily act in ligand binding. F329A, like R316A, showed greater impairment of transport than binding, intimating mechanistic involvement during ligand internalization. Furthermore, immunochemical studies localized F329 in the FepA ligand binding site. The mutagenesis results suggested the existence of dual ligand binding sites in the FepA vestibule, and measurements of the rate of ferric enterobactin adsorption to fluoresceinated FepA mutant proteins confirmed this conclusion. The initial, outermost site contains aromatic residues and probably functions through hydrophobic interactions, whereas the secondary site exists deeper in the vestibule, contains both charged and aromatic residues and probably acts through hydrophobic and electrostatic bonds.

Blood group A and B antigens are strongly expressed on platelets of some individuals

It is widely thought that expression of ABH antigens on platelets is insufficient to materially affect the survival of ABH-incompatible platelets in transfusion recipients, but anecdotal reports of poor survival of A and B mismatched platelets suggest that this is not always the case. The A and B antigen expression on platelets of 100 group A(1) and group B blood donors was measured, and 7% and 4%, respectively, had platelets whose A and B antigen levels consistently exceeded the mean plus 2 SD. On the basis of flow cytometric and statistical analysis, donors whose platelets contained higher than normal levels of A antigen were subdivided into 2 groups, designated Type I and Type II ("high expressers"). Serum A(1)- and B-glycosyltransferase levels of A and B high expressers were significantly higher than those of group A(1) and B individuals with normal expression. H antigen levels were low on the red cells of high expressers, indicating that the anomaly affects other cell lineages. Immunochemical studies demonstrated high levels of A antigen on various glycoproteins (GPs) from high-expresser platelets, especially GPIIb and PECAM (CD31). The A(1) Type II high-expresser phenotype was inherited as an autosomal dominant trait in one family. The sequences of exons 5, 6, and 7 of the A(1)-transferase gene of one Type II A(1) high expresser and exon 7 from 3 other genes were identical to the reported normal sequences. Further studies are needed to define the molecular basis for the high-expresser trait and to characterize its clinical implications. (Blood. 2000;96:1574-1581)

Blood group A and B antigens are strongly expressed on platelets of some individuals

AbstractIt is widely thought that expression of ABH antigens on platelets is insufficient to materially affect the survival of ABH-incompatible platelets in transfusion recipients, but anecdotal reports of poor survival of A and B mismatched platelets suggest that this is not always the case. The A and B antigen expression on platelets of 100 group A1 and group B blood donors was measured, and 7% and 4%, respectively, had platelets whose A and B antigen levels consistently exceeded the mean plus 2 SD. On the basis of flow cytometric and statistical analysis, donors whose platelets contained higher than normal levels of A antigen were subdivided into 2 groups, designated Type I and Type II (“high expressers”). Serum A1- and B-glycosyltransferase levels of A and B high expressers were significantly higher than those of group A1 and B individuals with normal expression. H antigen levels were low on the red cells of high expressers, indicating that the anomaly affects other cell lineages. Immunochemical studies demonstrated high levels of A antigen on various glycoproteins (GPs) from high-expresser platelets, especially GPIIb and PECAM (CD31). The A1 Type II high-expresser phenotype was inherited as an autosomal dominant trait in one family. The sequences of exons 5, 6, and 7 of the A1-transferase gene of one Type II A1 high expresser and exon 7 from 3 other genes were identical to the reported normal sequences. Further studies are needed to define the molecular basis for the high-expresser trait and to characterize its clinical implications.

Immunogenicity and immunolocalization of the 22.6 kDa antigen of Schistosoma japonicum.

The 22.6 kDa tegumental-associated antigens of Schistosoma mansoni (Sm22.6) and Schistosoma japonicum (Sj22.6) are of recognized interest in schistosomiasis vaccine development, although no direct vaccination/challenge experiments using either Sm22.6 or Sj22.6 had been previously described. We report that Escherichia coli-expressed reSj22.6 failed to protect mice or water buffaloes against subsequent challenge with S. japonicum cercariae. This was despite the fact that specific IgG (buffaloes) and IgG and IgE (CBA mice) antibodies were generated against the 22.6 kDa molecule, observations consistent with some of our earlier findings. We could find no evidence from immunolocalization studies that Sj22.6 is expressed or exposed on the surface of the adult parasite since it appears to be restricted to the apical cytoplasm of the tegument and is not associated with the apical or basal membrane or any membrane-bound structures in the apical cytoplasm. Nevertheless, Sj22.6 must be released to the immune system during the course of infection because specific anti-Sj22.6 IgG antibodies were present in the sera of nonvaccinated but challenged mice. We conclude that it may be necessary to produce reSj22.6 in a more relevant expression system, such as baculovirus, to further establish its vaccine potential and that detailed immunochemical and immunolocalization studies of early developmental stages may be necessary to determine how Sj22.6 is released or shed in S. japonicum infections.

Development of Highly Sensitive Ru-Chelate Based ECL Immunoassay 2: Electrochemical and Immunochemical Studies on Homogeneous and Heterogeneous ECL Excitation

Studies on the electrochemical and immunochemical reaction kinetics of the heterogeneous type of ECL excitations were made comparatively with homogeneous types of ECL excitations by measuring human IgG (hIgG) using an antibody labeled with a ruthenium(II) tris(bipyridyl) (Ru-chelate) as the luminophore (Ru-Ab). Solid-phase sandwich-type immunoassays were carried out on the surface of magnetic micro-beads (MB) with a diameter of 4.5 µm. In the ECL measurement, two types of ECL excitation methods were compared. One was a homogeneous ECL excitation, where the reacted MB together with non-reacted Ru-Ab were excited in a suspending phase without any bind/free (B/F) separation procedure. The other was a heterogeneous one where the reacted MB were excited over the electrode after being collected by a magnet following the B/F separation to remove the non-reacted Ru-Ab. In electrochemical studies, it was revealed that the Ru-Ab reacted with hIgG decreased the ECL emission efficiency. The decreasing ratio was inversely correlated with the cubic root of the luminophore molecular weight. In homogenous ECL excitation for the reacted matrix containing both the reacted MB and the non-reacted Ab, however, a reverse correlating dose response curve appeared only in the area beyond the antigen-antibody equivalent point, the so-called antigen excess zone; as a result the S/N ratio of the ECL signal was as small as only 1.3. In contrast, the heterogeneous ECL excitation for the reacted MB, with the non-reacted Ru-Ab removed by B/F separation, demonstrated 1000 or more times the S/N ratio in the area before the antigen-antibody equivalent point. Thus, this heterogeneous ECL excitation with B/F separation improved the detection sensitivity dramatically up to 1000 or more times higher than that of the homogeneous ECL excitation. Consequently, the sensitivity of heterogeneous ECLIA could be competitive with that of the conventional chemiluminescence immunoassay.

Deficiencies of human complement component C4A and C4B and heterozygosity in length variants of RP-C4-CYP21-TNX (RCCX) modules in caucasians. The load of RCCX genetic diversity on major histocompatibility complex-associated disease.

The complement component C4 genes located in the major histocompatibility complex (MHC) class III region exhibit an unusually complex pattern of variations in gene number, gene size, and nucleotide polymorphism. Duplication or deletion of a C4 gene always concurs with its neighboring genes serine/threonine nuclear protein kinase RP, steroid 21-hydroxylase (CYP21), and tenascin (TNX), which together form a genetic unit termed the RCCX module. A detailed molecular genetic analysis of C4A and C4B and RCCX modular arrangements was correlated with immunochemical studies of C4A and C4B protein polymorphism in 150 normal Caucasians. The results show that bimodular RCCX has a frequency of 69%, whereas monomodular and trimodular RCCX structures account for 17.0 and 14.0%, respectively. Three quarters of C4 genes harbor the endogenous retrovirus HERV-K(C4). Partial deficiencies of C4A and C4B, primarily due to gene deletions and homoexpression of C4A proteins, have a combined frequency of 31.6%. This is probably the most common variation of gene dosage and gene size in human genomes. The seven RCCX physical variants create a great repertoire of haplotypes and diploid combinations, and a heterozygosity frequency of 69.4%. This phenomenon promotes the exchange of genetic information among RCCX constituents that is important in homogenizing the structural and functional diversities of C4A and C4B proteins. However, such length variants may cause unequal, interchromosomal crossovers leading to MHC-associated diseases. An analyses of the RCCX structures in 22 salt-losing, congenital adrenal hyperplasia patients revealed a significant increase in the monomodular structure with a long C4 gene linked to the pseudogene CYP21A, and bimodular structures with two CYP21A, which are likely generated by recombinations between heterozygous RCCX length variants.

Sialoblastoma of the submandibular gland: report of a case and review of the literature

Sialoblastomas are rare perinatal epithelial salivary tumors, with only 22 reported cases in the literature. While they have been reported to occur predominantly in the parotid gland, we present one case of sialoblastoma of submandibular gland origin. The surgical management of this patient is discussed. Histopathologic examination, including immunochemical, ultrastructural and cytogenetic studies, was performed. Pertinent literature is reviewed.

A novel spore peptidoglycan hydrolase of Bacillus cereus: biochemical characterization and nucleotide sequence of the corresponding gene, sleL.

The exudate of germinated spores of B. cereus IFO 13597 in 0.15 M KCl-50 mM potassium phosphate (pH 7.0) contained a spore-lytic enzyme which has substrate specificity for fragmented spore cortex from wild-type organisms (cortical-fragment-lytic enzyme [CFLE]), in addition to a previously characterized germination-specific hydrolase which acts on intact spore cortex (spore cortex-lytic enzyme [SCLE]) (R. Moriyama, S. Kudoh, S. Miyata, S. Nonobe, A. Hattori, and S. Makino, J. Bacteriol. 178:5330-5332, 1996). CFLE was not capable of degrading isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid delta-lactam. This suggests that CFLE cooperates with SCLE in cortex hydrolysis during germination. CFLE was purified in an active form and identified as a 48-kDa protein which functions as an N-acetylglucosaminidase. Immunochemical studies suggested that the mature enzyme is localized on a rather peripheral region of the dormant spore, probably the exterior of the cortex layer. A gene encoding the enzyme, sleL, was cloned in Escherichia coli, and the nucleotide sequence was determined. The gene encodes a protein of 430 amino acids with a deduced molecular weight of 48,136. The N-terminal region contains a repeated motif common to several peptidoglycan binding proteins. Inspection of the data banks showed no similarity of CFLE with N-acetylglucosaminidases found so far, suggesting that CFLE is a novel type of N-acetylglucosaminidase. The B. subtilis genome sequence contains genes, yaaH and ydhD, which encode putative proteins showing similarity to SleL.

Atrazine induction of cytochrome P450 in Chironomus tentans larvae

Cytochrome P450-dependent aldrin epoxidation was characterized in third instar larvae of the aquatic midge, Chironomus tentans. Optimal in vitro assay conditions for the epoxidase were pH 7.6 and 31°C. Activity was linear up to 40 min of incubation time and 0.5 mg microsomal protein per incubation. The activity was concentrated in the microsomal fraction of whole body homogenates and was NADPH-dependent. The effect of atrazine exposure on aldrin epoxidase was measured to determine if this herbicide induces cytochrome P450-dependent activity. Comparisons of control and atrazine-exposed midges indicated increased epoxidase activity as a result of atrazine exposure, and a 45 kDa protein of increased intensity was observed after SDS-PAGE of microsomal protein. The molecular weight of this protein was similar in size to cytochrome P450 enzymes reported for other insects. Heme staining of SDS-PAGE gels and immunochemical studies using a Drosophila melanogaster anti-P450 polyclonal antiserum, further support the cytochrome P450 nature of this inducible 45 kDa protein.

Formation of 20-Hydroxyeicosatetraenoic Acid, a Vasoactive and Natriuretic Eicosanoid, in Human Kidney: ROLE OF CYP4F2 AND CYP4A11

20-hydroxyeicosatetraenoic acid (20-HETE), an omega-hydroxylated arachidonic acid (AA) metabolite, elicits specific effects on kidney vascular and tubular function that, in turn, influence blood pressure control. The human kidney's capacity to convert AA to 20-HETE is unclear, however, as is the underlying P450 catalyst. Microsomes from human kidney cortex were found to convert AA to a single major product, namely 20-HETE, but failed to catalyze AA epoxygenation and midchain hydroxylation. Despite the monophasic nature of renal AA omega-hydroxylation kinetics, immunochemical studies revealed participation of two P450s, CYP4F2 and CYP4A11, since antibodies to these enzymes inhibited 20-HETE formation by 65. 9 +/- 17 and 32.5 +/- 14%, respectively. Western blotting confirmed abundant expression of these CYP4 proteins in human kidney and revealed that other AA-oxidizing P450s, including CYP2C8, CYP2C9, and CYP2E1, were not expressed. Immunocytochemistry showed CYP4F2 and CYP4A11 expression in only the S2 and S3 segments of proximal tubules in cortex and outer medulla. Our results demonstrate that CYP4F2 and CYP4A11 underlie conversion of AA to 20-HETE, a natriuretic and vasoactive eicosanoid, in human kidney. Considering their proximal tubular localization, these P450 enzymes may partake in pivotal renal functions, including the regulation of salt and water balance, and arterial blood pressure itself.

Transduction of Human GAD67 cDNA into Immortalized Striatal Cell Lines Using an Epstein–Barr Virus-Based Plasmid Vector Increases GABA Content

The M213-20 and M213-1L cell lines were immortalized from rat striatum using the tsA58 allele of the SV40 large T antigen, contain the GAD enzyme, and produce GABA (Giordano et al., 1994, Exp. Neurol.124:395–400). Cell lines that produce large amounts of GABA may be useful for transplantation into the brain in conditions such as Huntington's disease or epilepsy, where localized application of GABA may be of therapeutic value. We have explored the potential use of the pREP10 plasmid vector, which replicates episomally, to increase GAD expression and GABA production in M213-20 and M213-1L cells. Human GAD67 cDNA was transfected into M213-20 and M213-1L, and subclones were isolated with hygromycin selection. Immunochemical studies showed increased GAD67 expression compared to the parent M213-20 and M213-1L cell lines. Staining for the EBNA antigen and Southern blots demonstrated that the pREP10 plasmid was stably maintained in the cells for at least 12–15 months in culture. Several clones were isolated in which GABA concentrations were increased by as much as 4-fold (M213-1L) or 44-fold (M213-20) compared to the parent cell lines or 12-fold (M213-1L) and 94-fold (M213-20) greater than rat striatal tissue (1.678±0.4 μmol/g prot). The ability of these cells to continue to produce large amounts of GABA while being maintained in culture for extended periods suggests that similar methods might be used with human cell lines to produce cells that can be transplanted into the brain to deliver GABA for therapeutic purposes.

Hatching of an estuarine crab,Sesarma haematocheir: From disappearance of the inner (e3) layer to rupture of the egg case

Hatching of decapod crustaceans is characterized by the sudden rupture of the egg case. This study focused on the following two issues regarding the hatching mechanism of the estuarine terrestrial crab Sesarma haematocheir: (1) dissolution of the egg case, and (2) the site where the egg case breaks. The egg case comprises three layers: the outer two (E1 and E2) layers and the inner (E3) thin layer (0.2 microm in thickness). The outer layers showed no morphological changes upon hatching, but the inner layer (E3) was markedly digested. The digestion of this layer would enable the embryo to absorb ambient water via reverse peristalsis of the intestine, resulting in an increase of the volume. The egg case always ruptured perpendicular to the longitudinal axis of the embryo. In addition, breakage of the egg case occurred at the dorsal thorax of the embryo. The three major organs positioned at this area were (1) a sharp projection (dorsal spine), (2) an assemblage of muscles, and (3) a pair of secretory glands, each of which was about 30 microm in diameter. The dorsal projection is soft before hatching, and it is clear that the egg case does not break with the posterior expansion of this projection. The rupture instead appears to be caused by the expansion of the muscles arranged perpendicular to the body axis. In addition, some (unknown) factor might weaken the egg case just before hatching. The secretory glands may be a kind of rosette gland, but the role that this gland plays at hatching is not known. As a duct comes out from the center and enters the dorsal projection, some active substance may be released at the tip of this projection. However, immunochemical studies are not consistent with this substance being an ovigerous hair stripping substance (OHSS).

A chimeric lectin formed from Bauhinia purpurea lectin and Lens culinaris lectin recognizes a unique carbohydrate structure.

Lectins are carbohydrate-binding proteins widely used in biochemical, immunochemical, and histochemical studies. Bauhinia purpurea lectin (BPA) is a leguminous lectin with an affinity for galactose and lactose. Nine amino acids, DTWPNTEWS, corresponding to the amino acid sequence from aspartic acid-135 to serine-143 in the primary structure of BPA were replaced with the corresponding amino acid residues from the mannose-binding Lens culinaris lectin (LCA), and the chimeric lectin obtained was expressed in Escherichia coli cells. The carbohydrate-binding specificity of the recombinant chimeric lectin was investigated in detail by comparing the elution profiles of various glycopeptides and oligosaccharides with defined carbohydate structures from immobilized lectin columns. Glycopeptides carrying three constitutive carbohydrate sequences of Galbeta1-3GalNAc-Ser/Thr and a complex-type biantennary glycopeptide, which show a high affinity for BPA or LCA, were shown to have no affinity for the chimeric lectin. In contrast, hybrid-type and high mannose-type glycopeptides with a Manalpha1-6(Manalpha1-3)Manalpha1-6Man sequence were found to have a moderate affinity for the chimeric lectin. This result demonstrates that a novel type of lectin with a unique carbohydrate-binding specificity can be constructed from BPA by substituting several amino acid residues in its metal-binding region with other amino acid residues. Additional lectin(s) with distinctly different carbohydrate-binding specificities will provide a powerful tool for many studies.