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Biomedical subjects

M Aikawa

Publications and source records attributed to M Aikawa.

At least 145 records · Page 8Linked to original sources
PubMed1994

Use of beta-1,3-glucan-specific antibody to study the cyst wall of Pneumocystis carinii and effects of pneumocandin B0 analog L-733,560.

A new class of promising antipneumocystis agents, cyclic lipopeptide pneumocandin analogs, has been shown to effectively prevent Pneumocystis carinii cyst development in murine models. These compounds are believed to inhibit the biosynthesis of beta-1,3-glucan, a major constituent of the cell walls of various pathogenic fungi. However, all evidence of the presence of this polymer in P. carinii cysts is based on indirect methods. To address this, highly specific rabbit polyclonal antiserum was raised against a laminariheptaose-human transferrin hapten conjugate. This antiserum was used to demonstrate the presence of beta-1,3-glucan in alkaline extracts of P. carinii-infected rat lung tissue and to quantitate the degree of infection in this tissue as laminarin equivalents. The antiserum was also used to localize beta-1,3-glucan in P. carinii-infected rat lung tissue at the transmission electron microscopic level by immunogold labeling. High concentrations of beta-1,3-glucan were present in the electron-lucent layer of the P. carinii cyst wall, but beta-1,3-glucan was absent from intracystic bodies and trophozoites. Ultrastructural evaluation of lung tissue from P. carinii-infected rats treated with the pneumocandin analog L-733,560 demonstrated that the few cysts which remained are deformed, lack the translucent layer of the cyst wall, and contain minimal amounts of beta-1,3-glucan.

Amino Acid Sequence↗
PubMed1994

Anatomical observations on the musculi suprahyoidei and apparatus hyoideus with special reference to the musculus jugulohyoideus and os stylohyoideum of the mouse, hamster and rat.

The musculi suprahyoidei and apparatus hyoideus were investigated using a combination of gross dissection and histological techniques in the mouse, hamster and rat. The new findings with regard to these are as follows: (1) the musculus jugulohyoideus, which runs from the processus jugularis to the os stylohyoideum, exists in the mouse and hamster, but is absent in the rat; (2) the m. jugulohyoideus is innervated with the ramus jugulohyoideus from the nervus facialis; (3) the os stylohyoideum and the os tympanohyoideum exist on the lateral surface of the bulla tympanica in the mouse, hamster and rat, and (4) four kinds of muscle (m. stylohyoideus, m. styloglossus, m. stylopharyngeus, m. jugulohyoideus) attach to the os stylohyoideum in the mouse and hamster; however, no muscles attach to the os stylohyoideum in the rat.

Animals↗
PubMed1994

Motor unit size of M. stylohyoideus and M. stapedius in the mouse.

To elucidate the relation between the caliber of myelinated nerve fibers (MNFs) and motor unit size (i.e., number of muscle fibers per motor unit), motor unit size was measured for the mouse m. stylohyoideus, innervated predominantly by extra large MNFs, and compared with that of the mouse m. stapedius, innervated predominantly by large MNFs. All muscle fibers and MNFs were counted morphologically, and mean motor unit size was calculated for each muscle. The results showed that the average of 12 mean motor unit sizes for the m. stylohyoideus (35.8 +/- 8.2), innervated predominantly by extra large MNFs, was greater than that for the m. stapedius (6.5 +/- 1.3), innervated predominantly by large MNFs.

Animals↗
PubMed1994

gamma delta T cells play a crucial role in the expression of 65,000 MW heat-shock protein in mice immunized with Toxoplasma antigen.

Toxoplasma gondii is an obligate intracellular protozoan parasite and cellular immunity plays a crucial role in protection against infection with this pathogen. When mice are immunized with Toxoplasma homogenate, they readily acquire resistance against infection with a lethal dose of a low virulence Beverley strain of T. gondii. We have reported previously that expression of 65,000 MW heat-shock protein (hsp 65) in host macrophages closely correlates with protective potentials of hosts, while this protein is not expressed in Toxoplasma themselves. In this study, we examined the mechanism of expression of hsp 65 in mice immunized with Toxoplasma homogenate. Heat-shock protein was detected in peritoneal macrophages of BALB/c mice immunized 7 days previously by electroblot assay with a specific monoclonal antibody (mAb) for microbial hsp 65. Furthermore, an immunogold ultracytochemistry assay demonstrated that this protein was expressed on the cell surface of peritoneal macrophages in immune mice. This expression was not induced in those of immune athymic nude mice and SCID mice. Treatment of BALB/c mice with anti-Thy-1.2 mAb 1 day before immunization led to an almost complete loss of the expression of hsp 65. To determine the subsets of T cells responsible for induction of this protein, mice were depleted of gamma delta T cells, alpha beta T cells, CD4+ T cells or CD8+ T cells by treating with corresponding antibodies before immunization. From these experiments, gamma delta T cells were shown to be essential for the expression of hsp 65, although CD4+ alpha beta T cells also contributed to some extent. Thus, gamma delta T cells appear to play an important role in protective immunity against infection with T. gondii through mediating the expression of hsp 65 in host macrophages.

Animals↗
PubMed1993

Transmission-blocking activity of a chitinase inhibitor and activation of malarial parasite chitinase by mosquito protease.

During development in the mosquito midgut, malarial parasites must traverse a chitin-containing peritrophic matrix (PM) that forms around the food bolus. Previously Huber et al. [Huber, M., Cabib, E. & Miller, L. H. (1991) Proc. Natl. Acad. Sci. USA 88, 2807-2810] reported that the parasite secretes a protein with chitinase activity, and they suggested that parasite chitinase (EC 3.2.1.14) plays an important role in the parasite's egress from the blood meal. We found that allosamidin, a specific inhibitor of chitinase, completely blocked oocyst development in vivo and thus blocked malaria parasite transmission. Addition of exogenous chitinase to the blood meal prevented the PM from forming and reversed the transmission-blocking activity of allosamidin. Using exogenous chitinase, we also found that the PM does not limit the number of parasites that develop into oocysts, suggesting that the parasite produces sufficient quantities of chitinase to penetrate this potential barrier. In addition, we found that treatment of parasite chitinase with a diisopropyl fluorophosphate-sensitive trypsinlike protease from the mosquito midgut or endoproteinase Lys-C increased its enzymatic activity. These results suggest that malaria parasite has evolved an intricate mechanism to adapt to the PM and the protease-rich environment of the mosquito midgut.

Acetylglucosamine↗
PubMed1993

Pfs2400 can mediate antibody-dependent malaria transmission inhibition and may be the Plasmodium falciparum 11.1 gene product.

Monoclonal antibodies (mAb) have been raised against Plasmodium falciparum gametocyte stage protein extracts, in an effort to identify novel parasite antigens that might mediate malaria transmission-blocking immunity. mAb 1A1 identified Pfs2400, a sexual stage-specific antigen of greater than 2 megadaltons, that is associated with the outer leaflet of the parasitophorous vacuole membrane in mature circulating gametocyte-infected red blood cells. Upon induction of gametogenesis, Pfs2400 partitions between the gamete plasmalemma and the degenerating erythrocyte membrane. The antigen is no longer detectable in the fully emerged gamete. mAb 1A1 dramatically reduces the number of oocysts formed in P. falciparum gametocyte-fed mosquitoes. The cognate antigen is probably the product of the Pf11.1 gene (Scherf et al. 1988. EMBO [Eur. Mol. Biol. Organ.]J. 7:1129) on the basis that a peptide composed of two copies of the degenerate nine amino acid repeat sequence in the Pf11.1 protein, can inhibit binding of mAb1A1 to the native antigen. The mechanism of transmission inhibition mediated by the Pfs2400 is discussed.

Amino Acid Sequence↗
PubMed1993

Morphological changes of clefts in Plasmodium-infected erythrocytes under adverse conditions.

Blood infected with human or rodent malaria parasites, Plasmodium falciparum or Plasmodium berghei, was exposed to higher pH, higher PO2, and lower temperature than those used in standard cultivation conditions. Parasitized blood was incubated for 20, 25, and 30 min with RPMI 1640 medium, 10% (vol/vol) serum, pH 8.0, at 20 degrees C in the air, conditions which are ultimately lethal to the asexual stages of malarial parasites. Markedly dilated clefts were observed in the cytoplasm of the malaria-infected erythrocytes so treated. These clefts can take up colloidal gold particles and macromolecules such as Protein A, rhodamine-dextran, and lucifer yellow-dextran. Such dilated clefts were not seen in the cytoplasm of infected erythrocytes that were incubated under normal cultivation conditions before fixation. These had slender clefts of the usual sort that did not take up colloidal gold particles and macromolecules.

Animals↗
PubMed1993

Babesia divergens: characterization of a 17-kDa merozoite membrane protein.

Large amounts of viable merozoites were purified from in vitro cultures of Babesia divergens by a two-step sieving procedure. A monoclonal antibody produced against B. divergens merozoites, mAb DG7, stained the merozoite plasma membrane and an intra-parasitic linear organelle in indirect immunofluorescence. Immunogold labeling in electron microscopy demonstrated that the antigen recognized by mAb DG7 was localized just beneath the merozoite plasma membrane. Immunoprecipitations of metabolically labeled ([35S]methionine) B. divergens antigens showed that the epitope recognized by mAb DG7 was present on a 17-kDa polypeptide (Bd17) and was shared in all B. divergens geographical isolates tested so far. Bd17 was always present in the in vitro culture supernatants of all these isolates. Furthermore, Triton X-114 phase separation of babesial antigens demonstrated the hydrophilic character of Bd17 which suggests that it is an extrinsic protein present on the cytosol side of the parasite membrane. When added to the culture medium, mAb DG7, purified from ascite fluids, drastically altered the growth of the parasite with concentrations inhibiting 50% of development (IC50) ranging between 16.6 and 26.1 micrograms/ml).

Animals↗
PubMed1993

Expression of members of the heat-shock protein 70 family in the exoerythrocytic stages of Plasmodium berghei and Plasmodium falciparum.

Exoerythrocytic stages of Plasmodium berghei cultured in HepG2-A16 hepatoma cells and those of P. falciparum in human hepatocytes transplanted under the kidney capsule of CB-17/ICr scid/scid mice were used to evaluate expression of heat-shock-related stress proteins. Although undetectable in the sporozoites, the expression of proteins similar in sequence of a heat-shock protein of 70 kDa and a glucose-regulated protein of 78 kDa was markedly induced in the hepatic stages of malaria parasites. Expression of these proteins in the exoerythrocytic stages of the malaria parasite warrants a systematic evaluation of their potential role in eliciting cellular immune responses directed against infected hepatocytes.

Animals↗
PubMed1993

Cloning and characterization of a Plasmodium falciparum gene encoding a novel high-molecular weight host membrane-associated protein, PfEMP3.

The rat monoclonal antibody, mAb 12C11, reacts with numerous proteins from mature asexual stages of Plasmodium falciparum. The largest is 315 kDa and is designated PfEMP3. A lambda gt11 expression library, generated from genomic DNA of Malayan Camp strain parasites, was screened with mAb 12C11. One positive clone, lambda 12.1.3, contained a 1.4-kb fragment in frame with the beta-galactosidase gene of lambda gt11. The deduced 455-amino acid sequence is a novel, highly charged sequence encoding two 15-amino acid repeats at the N-terminus followed by 27 repeats of 13 amino acids. The last 59 C-terminal residues are non-repetitive. Two in-frame stop codons at the 3' end of the DNA suggests that this DNA fragment encodes the C-terminus of the protein. Southern blotting with the cloned fragment identified two copies of this fragment per haploid genome in knob-positive, parasitized erythrocytes (K+PE). Both DNA fragments are absent from K - PE. Northern blotting of trophozoite-stage PE total RNA revealed mRNAs of 10, 4.4 and 2 kb in K+PE, but no hybridization with K - PE. Immune sera were elicited against the lambda 12.1.3 beta-galactosidase fusion protein and peptides generated from the predicted lambda 12.1.3 amino acid sequence. These sera and mAb 12C11 reacted specifically with PfEMP3 in Western blots of mature K+PE but not with K - PE. Rat and mouse sera against the recombinant protein produced an immunofluorescence pattern in fixed mature K+PE almost identical to the pattern produced by a monoclonal antibody against the knob-associated protein, Histidine Rich Protein 1. The same antibodies were immunofluorescence negative with fixed K - PE. Mouse antibodies against the recombinant protein reacted on immunoelectron microscopy with the erythrocyte membrane of K+PE, labeling knobs as well as the membrane between knobs. In contrast, a mAb against Histidine Rich Protein 1 reacted only under the electron dense material of knobs. We conclude that the lambda 12.1.3 clone encodes the C-terminal portion of the 315 kD PfEMP3 antigen and that PfEMP3 may be involved in knob formation or other perturbations of the erythrocyte membrane.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and localization of an abundant novel protein of Plasmodium berghei.

Screening of Plasmodium berghei genomic libraries using DNA insert corresponding to the 3' half of P. falciparum 70-kDa heat shock protein gene identified several abundant clones which represent a novel gene in the parasite. The complete sequence was obtained using an approach based on inverse polymerase chain reaction. Analysis of the deduced amino acid sequence revealed the presence of 19 imperfect repeats of the sequence Gly-Gly-Met-Pro toward the carboxy terminus. Except for the similar sequence repeated seven times in the malarial 70-kDa heat shock protein, the sequence of the cloned gene product is very different. Moreover, the sequence also revealed acidic and basic domains in the protein which are more than 60% similar in sequence to functional domains present in numerous DNA binding transcription factors. A 56-kDa protein was identified by immunoprecipitation from labeled P. berghei extract using antisera raised in mice against gene products expressed in Escherichia coli. The protein is present in all the different life cycle stages of the parasites as revealed by immunoelectron microscopy.

Amino Acid Sequence↗
PubMed1993

Ductus arteriosus. Advanced differentiation of smooth muscle cells demonstrated by myosin heavy chain isoform expression in rabbits.

BACKGROUND: The closure of the ductus arteriosus (DA) is one of the most striking cardiovascular events that occur at birth. It has been attributed to oxygenation and intrinsic prostaglandins. However, selective constriction of DA suggests the presence of highly specialized contractile mechanisms in DA. We previously reported that smooth muscle myosin heavy chain isoforms, SM1 and SM2, are molecular markers for smooth muscle differentiation because of their unique expression pattern during vascular development. SM1 and SM2 are generated from a single gene through developmentally regulated alternative RNA splicing; SM1 is expressed in almost all stages of differentiation of the vascular smooth muscles, but SM2 is found only after birth. METHODS AND RESULTS: Immunohistochemistry was performed to study the expression of the different types of myosin heavy chain isoforms in DA of fetal and neonatal rabbits. Electron microscopic examinations were also carried out to demonstrate ultrastructural characteristics of ductus muscles. We found that SM2 is expressed before birth in the medial layer of DA, indicating advanced differentiation of smooth muscle cells in DA. The exact location of immunoreactivity for SM2 was in the smooth muscle cell of the medial layer of DA. Immunoreactivity for SM1, however, was not different for DA and adjacent great arteries. Transmission electron microscopy demonstrated greater amounts of myofilaments in medial smooth muscles of DA than those of aorta. CONCLUSIONS: These results indicate that smooth muscles in DA are more differentiated than those in other arteries, which may be one of the cellular mechanisms responsible for the unique closure of DA at birth.

Actin Cytoskeleton↗
PubMed1993

Human smooth muscle myosin heavy chain isoforms as molecular markers for vascular development and atherosclerosis.

Smooth muscle myosin heavy chains (MHCs) exist in multiple isoforms. Rabbit smooth muscles contain at least three types of MHC isoforms: SM1 (204 kD), SM2 (200 kD), and SMemb (200 kD). SM1 and SM2 are specific to smooth muscles, but SMemb is a nonmuscle-type MHC abundantly expressed in the embryonic aorta. We recently reported that these three MHC isoforms are differentially expressed in rabbit during normal vascular development and in experimental arteriosclerosis and atherosclerosis. The purpose of this study was to clarify whether expression of human smooth muscle MHC isoforms is regulated in developing arteries and in atherosclerotic lesions. To accomplish this, we have isolated and characterized three cDNA clones from human smooth muscle: SMHC94 (SM1), SMHC93 (SM2), and HSME6 (SMemb). The expression of SM2 mRNA in the fetal aorta was significantly lower as compared with SM1 mRNA, but the ratio of SM2 to SM1 mRNA was increased after birth. SMemb mRNA in the aorta was decreased after birth but appeared to be increased in the aged. To further examine the MHC expression at the histological level, we have developed three antibodies against human SM1, SM2, and SMemb using the isoform-specific sequences of the carboxyl terminal end. Immunohistologically, SM1 was constitutively positive from the fetal stage to adulthood in the apparently normal media of the aorta and coronary arteries, whereas SM2 was negative in fetal arteries of the early gestational stage. In human, unlike rabbit, aorta or coronary arteries, SMemb was detected even in the adult. However, smaller-sized arteries, like the vasa vasorum of the aorta or intramyocardial coronary arterioles, were negative for SMemb. Diffuse intimal thickening in the major coronary arteries was found to be composed of smooth muscles, reacting equally to three antibodies for MHC isoforms, but reactivities with anti-SM2 antibody were reduced with aging. With progression of atherosclerosis, intimal smooth muscles diminished the expression of not only SM2 but also SM1, whereas alpha-smooth muscle actin was well preserved. We conclude from these results that smooth muscle MHC isoforms are important molecular markers for studying human vascular smooth muscle cell differentiation as well as the cellular mechanisms of atherosclerosis.

Aging↗
PubMed1993

Immunogenicity of the Plasmodium falciparum asexual blood-stage synthetic peptide vaccine SPf66.

The immunogenicity of the cocktail vaccine SPf66 against Plasmodium falciparum was investigated in rabbits, monkeys, and human volunteers. This polymerized peptide vaccine incorporates a portion of each of the 35-kD, 55-kD, and 83-kD blood-stage proteins, linked by an amino acid sequence reproducing one repeat from the circumsporozoite protein of P. falciparum. Results from this study show that vaccination with this vaccine molecule elicited high titers of antibodies to SPf66 and its constituents, but these antibodies did not correlate with regular or sensitive indirect fluorescent antibody (IFA) titers to blood-stage malaria parasites. However, rabbits injected with individual peptides produced antibodies with low affinity to indefinite parasite structures by immunoelectron microscopy, and rabbits injected with SPf66 had high antibody titers against the peptide (NANP)40. Consequently, these anti-SPf66 serum samples recognized P. falciparum sporozoites in the IFA. Such reactivity was not observed in monkeys or human volunteers vaccinated with SPf66. In addition, SPf66 components were recognized by antibodies induced by natural infection in humans and by laboratory-monitored infections in Aotus monkeys. These results suggest that this vaccine candidate merits further developmental work to better define immune response elicited by the copolymer to the parasite.

Adolescent↗
PubMed1993

Differential sequestration of parasitized erythrocytes in the cerebrum and cerebellum in human cerebral malaria.

Sequestration of parasitized red blood cells (PRBC) in the microvessels results in impairment of microcirculation with organ dysfunction in complicated human Plasmodium falciparum malaria. In cerebral malaria patients, the percentage of small blood vessels with PRBC sequestration is higher in the brain than in other organs. The clinical severity of cerebral malaria depends on the level of PRBC sequestration in the brain. In our study, postmortem samples from cerebrum and cerebellum of 16 patients who died of P. falciparum malaria were examined and compared using light microscopy. In the cerebellum, the percentage of microvessels with PRBC sequestration was higher than that in the cerebrum. The difference in sequestration rates between cerebrum and cerebellum is statistically significant (P < 0.05). There is a higher degree of vascularity in the cerebellum (7 vessels/mm2) than in the cerebrum (5 vessels/mm2), and the difference is also statistically significant (P < 0.025). Perivascular hemorrhages also occur more frequently in the cerebellum than in the cerebrum. The results of this study, which show that differential sequestration of PRBC occurs in the microvessels of the cerebrum and cerebellum, explain the varied neurologic manifestations that result from cerebral and cerebellar dysfunction in human cerebral malaria. This study also reveals the necessity of postmortem histologic examination of the cerebellum in every suspected case of cerebral malaria.

Brain↗
PubMed1993

A primate model for severe human malaria with cerebral involvement: Plasmodium coatneyi-infected Macaca fuscata.

To develop an animal model for severe human malaria, we carried out clinical and pathologic observations of Japanese monkeys (Macaca fuscata) infected with Plasmodium coatneyi. Two monkeys, eight and nine months of age, were used in this experiment. After inoculation with the parasite, both monkeys developed a fulminating acute infection with high parasitemia (20-28.2%) and became moribund with typical signs of severe malaria. In the splenectomized Japanese monkey, sequestered infected erythrocytes blocked brain capillaries. Electron microscopic studies on brain tissues revealed electron-dense knobs protruding from the membrane of infected erythrocytes that formed focal junctions with the cerebral capillary endothelial cells. These findings were remarkably similar to those seen in human cases. Prominent sequestration of the infected erythrocytes was uniformly distributed in capillaries of the lungs and heart. The nonsplenectomized Japanese monkey developed acute anemia with a packed cell volume of 6%, but blockage of brain capillaries was minimal. However, sequestered, infected erythrocytes were evident in capillaries of the heart and lungs of this animal. Our study showed that the Japanese monkey is highly susceptible to P. coatneyi infection and that this system provides a model for the study of severe human malaria.

Animals↗
PubMed1993

Morphologic effects of artemisinin in Plasmodium falciparum.

Ultrastructural changes induced in Plasmodium falciparum by artemisinin were studied in vitro. Electron microscopic autoradiography was performed on infected erythrocytes that were exposed in vitro to 3H-dihydroartemisinin and 14C-artemisinin. These drugs consistently were located in food vacuoles and mitochondria. Two hours after administration, changes were observed in parasite mitochondria, rough endoplasmic reticulum, and nuclear envelope. At four hours, in addition to the earlier changes, nuclear membranes and, to a lesser extent, some plasma membranes formed myelin figures. In addition, there was a disappearance of ribosomes, and a destruction of food vacuole membranes. These changes may lead to the total disorganization of the parasites. Approximately 30% of the parasites manifested these alterations.

Animals↗