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Biomedical subjects

K Akagawa

Publications and source records attributed to K Akagawa.

At least 73 records · Page 4Linked to original sources
PubMed1991

Nuclear membrane antigen specific to nerve and muscle tissues.

A monoclonal antibody, 2F7, raised against a nuclear protein subfraction recognized the nuclear membrane of nervous and muscular tissues of guinea pig, rat and rabbit, but no other tissue was stained. In the nervous system, both neurons and glial cells were labelled. An electron microscopic immunohistochemical study demonstrated that 2F7 antibody labelled the inner surface of the nuclear membrane. Western blot analysis on the nuclear envelope fraction containing nuclear lamina revealed that this antibody reacted to two minor component proteins of 80 and 82KDa. These 2F7 antigens expressed preferentially in the nervous and muscular tissues were distinct from the major nuclear envelope proteins reported so far and might be related to neuronal or muscular tissue-specific functions.

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PubMed1991

Protection of mice from lethal endotoxemia by use of an ornithine-containing lipid or a serine-containing lipid.

The effects of an ornithine-containing lipid [alpha-N-(3-acyloxyacyl)-ornithine (Orn-L)] or a serine-containing lipid [alpha-N-(3-acyloxyacyl)-serine (Ser-L)] from Flavobacterium meningosepticum on lethal endotoxemia in mice were examined. When 500 micrograms of Orn-L was intravenously administered 1 h before intravenous administration of a lethal dose of endotoxin, none of the mice died. The protective effect of Ser-L was weaker than that of Orn-L. Light and electron microscopic studies demonstrated that necrosis of hepatocytes caused by endotoxin was prevented by pretreatment with Orn-L. Furthermore, Kupffer cells were activated morphologically 1 h after the administration of Orn-L or Ser-L, and the liposomes of the lipoamino acids were incorporated into phagolysosomes in activated Kupffer cells. The activity of tumor necrosis factor in sera of endotoxin-treated mice was decreased markedly by pretreatment of mice with Orn-L. In vitro, the lipoamino acids suppressed endotoxin-induced tumor necrosis factor generation but did not suppress tumor necrosis factor generation induced by zymosan and whole cells of Staphylococcus aureus. These results suggested that Orn-L and Ser-L can be used as specific blocking agents against endotoxin. The blocking mechanism may be antagonistic, because of the structural similarities between the lipoamino acids and endotoxin lipid A.

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PubMed1990

Presence of light-responding neurons in the reaggregate cultures of rat retinae.

Since histological studies on retinal reaggregates have given evidence of lamination similar to that in the intact retina, the presence of retinal function, i.e. light response, in vitro has been expected as well. Therefore, neurons in the outermost layers of reaggregates, mostly consisting of amacrine cells, were studied with cell-attached recording. About one fifth of the neurons in that layer showed spontaneous discharges. Interestingly, the frequency of spontaneous discharges of some neurons increased with light stimulation but decreased to the basal level under dark conditions. They were reproducible and lasted more than 10 min in the best case. These results suggest that functional connections between photoreceptors which are exclusively present in the central region and amacrine cells in the outermost layer might be reconstituted in reaggregate cultures.

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PubMed1990

Calcium- and voltage-dependent potassium channel in the rat retinal amacrine cells identified in vitro using a cell type-specific monoclonal antibody.

Single-channel currents were studied from identified amacrine cells of rat retinal monolayer cultures. Cells were immunohistochemically identified using an amacrine cell-specific monoclonal antibody, HPC-1. Single-channel currents with the following properties were observed. (1) The mean opening duration was voltage dependent, tending to increase on hyperpolarization. (2) The reversal potential was almost identical to the calculated EK derived from the potassium concentration gradient. (3) The conductance was 110 pS at a low extracellular potassium concentration. (4) Currents were blocked by TEA. (5) Mean opening duration was also dependent on the calcium concentration of the intracellular side. These properties were consistent with the voltage and calcium-dependent potassium channels of a large conductance. Our results also demonstrated that cell-type-specific monoclonal antibodies together with electrophysiological recordings offer a very useful method to investigate the physiological properties of distinct neuronal types in mixed cell cultures.

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PubMed1990

A monoclonal antibody 5E5 recognizes an intranuclear antigen selectively present in a subpopulation of the neurons.

Monoclonal antibody 5E5 labeled the nuclear antigen of the neurons in the guinea pig and rat central nervous systems including the cerebrum, cerebellum, spinal cord and retina. This antibody could discriminate neurons even among the same cell class. In in vitro study, only 10% of dividing PC12 cells was labeled with this antibody. An electron microscopic immunohistochemical study also revealed that this antibody selectively labeled heterochromatins in the neurons. Although we could not obtain any positive result by an immunoblot study, the antigenicity was remarkably diminished by the DNase I or S1 nuclease treatment on the tissue sections whereas RNase and trypsin was ineffective. These results suggested that this antigen might be a single-stranded DNA-protein complex resistant to proteolytic procedures, and possibly related to cell function or state of differentiation.

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PubMed1989

Macrophage activation by an ornithine-containing lipid or a serine-containing lipid.

alpha-N-(3-Acyloxyacyl)-ornithine (or -serine) is the structure of lipoamino acids obtained by us previously from some gram-negative bacteria (Y. Kawai and I. Yano, Eur. J. Biochem. 136:531-538, 1983; Y. Kawai, I. Yano, and K. Kaneda, Eur. J. Biochem. 171:73-80, 1988; Y. Kawai, I. Yano, K. Kaneda, and E. Yabuuchi, Eur. J. Biochem. 175:633-641, 1988). The 3-acyloxyacylamide structure is present in both the lipoamino acids and lipid A of lipopolysaccharide (endotoxin). The efficacy of lipoamino acids (an ornithine-containing lipid and a serine-containing lipid) in activating C3H/HeSlc mouse peritoneal exudate macrophages was compared with that of bacterial lipopolysaccharide, because the two types of substances were expected to exhibit similar biological activities and physiological functions on the basis of their structural similarities. Actually, the lipoamino acids, as well as lipopolysaccharide, strongly activated the macrophages to generate the immunoregulatory substances prostaglandin E2 and interleukin-1, but their effect on the induction of L929 cell cytolytic factor (a possible tumor necrosis factor), another immunoregulatory substance, was weaker than that of lipopolysaccharide. The effect of lipoamino acids on the cytotoxicity of macrophages for EL-4 leukemia cells was very weak. However, all of these activities, as far as tested, were strongly enhanced by synergistic action with gamma interferon. Only the serine-containing lipid killed both C3H/HeSlc and C3H/HeJ macrophages to almost the same degree as endotoxin killed C3H/HeSlc macrophages. On the other hand, lethal toxicity for mice was not found with either the ornithine-containing lipid or the serine-containing lipid, even when 7 mg of compound was injected into a mouse. These studies suggest that the lipoamino acids are nontoxic characteristic immunoactivators.

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PubMed1987

Histiotypic organization and cell differentiation in rat retinal reaggregate cultures.

Reaggregate cultures have been formed from cell suspensions of neonatal rat retinas. Histological sections of the reaggregates showed evidence of lamination with central rosettes formed around a lumen, a clear neuropil layer and an outer cellular layer. Each of the major retinal cell types, except ganglion cells, could be positively identified using cell type-specific antibodies to label cryostat sections. Many of these were found to occupy positions within the reaggregates similar to those found in the intact retina. Electron microscopic observations showed abundant immature and mature synaptic endings within the neuropil layer, including a number of ribbon synapses. Examination of the rosettes showed an arrangement of Müller glia and photoreceptors that closely resembled that of the intact retina. Within the lumen of rosettes, photoreceptors were found to contain stacks of disc-like membranes bounded by a plasma membrane, analogous to immature outer segments. The photoreceptors within rosettes also underwent molecular differentiation and expressed an outer segment specific marker. The findings suggest that retinal cells have intrinsic properties that allow them to organize themselves into a correctly laminated structure and that particular cell interactions are necessary for continued differentiation of at least rod photoreceptors and Müller cells.

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PubMed1987

Identification and characterisation of cell types accumulating GABA in rat retinal cultures using cell type specific monoclonal antibodies.

Monolayer and reaggregate cultures have been established from neonatal rat retina. After 7 days in culture, 60 nM [3H]gamma-aminobutyric acid (GABA) was used to identify cells with a high affinity uptake mechanism for GABA. Approximately 80% of the process-bearing cells were found to be labelled. These cells were identified as amacrine cells by double-labelling experiments combining [3H]GABA uptake with immunocytochemical labelling with monoclonal antibody HPC-1 which in retina is specific for amacrine cells. The ability of cultures to synthesize GABA from glutamate was investigated at various times. Little synthesis was observed during the first few days in culture. This lag was followed by an increase in the amount of synthesis until 3 weeks of culture. When clumps and reaggregate cultures of retinal cells were examined by [3H]GABA uptake, a time-dependent redistribution of labelled cells was observed. After 20 h in culture, GABA-positive cells were distributed over the whole cell mass. Over the next few days, the labelled cells became more common on the outer edge of the aggregates and less common in inner regions. By 7 days of culture, no labelled cell bodies were found on the inside of the aggregates, although such cells could be labelled by [3H]D-aspartate. The results provide positive identification of a subclass of retinal cells in culture, and show that at least one aspect of retinal histogenesis is not dependent upon extra-retinal tissues or the position imposed by the temporal order of retinal cell birth.

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PubMed1987

Selective localization of glycine-accumulating cells in reaggregate culture of rat retina.

Uptake of [3H]glycine into the cells either in monolayer or reaggregate cultures of retina from two-day-old rat pups was studied. The glycine-accumulating cells (glycine cells) had short processes with several branches. Only 5% of process-bearing cells were labelled in the monolayer cultures. The major cell type, previously identified as photoreceptor cells, was unlabelled. In reaggregate cultures, the glycine cells were localized mainly in the outermost layer of the reaggregate. But the proportion of positive cells among all the cells in that layer was not so large. Although the cell type of the glycine cells has not yet been unambiguously identified, these results demonstrate a possible example of selective sorting out of a group of biochemically distinct cells from a cell mixture.

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PubMed1986

Identification and characterization of cell types in monolayer cultures of rat retina using monoclonal antibodies.

This study describes the identification and differentiation of neonatal rat retinal cells in monolayer cultures. A panel of monoclonal antibodies was used as a molecular probe of both cell type and developmental stage. Previously described cell-type specific monoclonal antibodies were used to label rod photoreceptors, horizontal cells, amacrine cells or ganglion cells. Two new antibodies that react with rat retina are described. The first, RET-G7, reacts with a cytoplasmic antigen of Muller glia, astrocytes and some horizontal cells. The second, RET-B2, reacts with bipolar cells and photoreceptor inner segments. Two main findings are presented. The first is that each of the major subclasses of retinal neurons have been unambiguously identified in these cultures. The morphology of some subclasses was very characteristic. All photoreceptors, as defined by reactivity with antibody RET-P1, were small spherical cells with one or fewer processes. Horizontal cells, as defined by reactivity with antibody B-1, were large with a characteristic multipolar network of processes. Bipolar and amacrine cells, on the other hand, were of similar size and could only be distinguished on the basis of immunocytochemical labeling. The second finding is that while RET-B2 antigen appeared on bipolar and photoreceptor cells after about 5 days in culture, several Muller cell and photoreceptor antigens were not expressed in monolayer cultures. The results suggest that the expression of some molecules in culture is the result of properties intrinsic to the cells whereas expression of others depends upon extrinsic factors or cell interactions that may not be present in monolayer cultures.

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PubMed1985

Enhancement of O2- generation and tumoricidal activity of murine macrophages by a monosaccharide precursor of Escherichia coli lipid A.

The effects of a monosaccharide precursor of Escherichia coli lipid A (lipid X) on murine macrophages were studied. Lipid X is a diacylglucosamine 1-phosphate bearing beta-hydroxymyristoyl groups at positions 2 and 3. Lipid X, as well as lipopolysaccharide and lipid A, enhanced O2- generation in mouse peritoneal macrophages and a macrophage-like cell line, J774.1, and further induced the tumor-cytotoxic activity of peritoneal macrophages. Elimination of a 1-phosphate or 3-O-beta-hydroxymyristoyl groups are essential for the elevated O2- generation and induction of tumoricidal activity due to lipid X.

Animals↗
PubMed1985

A marker of early amacrine cell development in rat retina.

A monoclonal antibody, HPC-1, labels only amacrine and displaced amacrine cells in adult rat retina. Reactivity with displaced amacrine cells was demonstrated by double-label immunofluorescence with HPC-1 and ganglion cell-specific reagents. HPC-1 antibody reacts with a polypeptide of apparent molecular weight 35,000. HPC-1 antibody labels migrating amacrine cells in late embryonic retinas. The results define cell-specific gene expression in relation to migration of one subclass of CNS neurons.

Animals↗
PubMed1985

Macrophage activation by monosaccharide precursors of Escherichia coli lipid A.

Certain Escherichia coli mutants defective in phosphatidylglycerol biosynthesis accumulate two novel glycolipids, designated X and Y. Lipid X is a diacylglucosamine 1-phosphate bearing beta-hydroxymyristoyl groups at positions 2 and 3, and lipid Y has the same structure as X, except for the additional presence of a palmitoyl moiety on the N-linked beta-hydroxymyristate. We have examined the activities of X, Y, and several related compounds as activators of macrophages. Both X and Y induce morphological changes (spreading), prostaglandin E2 synthesis, and killing of tumor cells by mouse peritoneal macrophages in vitro, properties with which lipopolysaccharide and lipid A are also endowed. Both glycolipids have similar effects on the macrophage-like mouse cell line J774.1. Selective removal from lipid X of either the ester-linked beta-hydroxymyristate at position 3 or the phosphate at position 1 abolishes activity. Our results show that the monosaccharides X and Y retain some of the properties of intact lipopolysaccharide and lipid A with respect to macrophage activation. Because the structures of X and Y are defined, our findings should facilitate the elucidation of the molecular mechanism of macrophage activation by lipid A.

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PubMed1984

Partial purification and properties of the inhibitors of Na, K-ATPase and ouabain-binding in bovine central nervous system.

Endogenous inhibitors of Na,K-ATPase and ouabain-binding were partially purified from bovine central nervous system, and some of their properties were studied. They were eluted as low-molecular-weight fractions by gel filtration. They could be adsorbed by both Amberlite IR 120 and Amberlite IRA 400 at acidic and basic pH, respectively, indicating that they could act as both anions and cations at different pH. These inhibitors of ouabain-binding appeared to affect specific binding of ouabin, and Scatchard plot analysis showed that the inhibition was competitive, suggesting that they could bind to the same site as ouabain, presumably to Na,K-ATPase itself. The inhibitory activities were heat stable, but charring inactivated them completely.

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