Isolation and characterization of a potentially virulent species Entamoeba nuttalli from captive Japanese macaques.

We have recently proposed revival of the name Entamoeba nuttalli Castellani, 1908 for a virulent amoeba (P19-061405 strain) isolated from a rhesus monkey (Macaca mulatta) and located phylogenetically between E. histolytica and E. dispar. In this study, E. nuttalli was isolated from feces of captive Japanese macaques (M. fuscata) in an open-air corral in Japan. The sequence of the 18S rRNA gene in the isolates differed from the P19-061405 strain in 2 nucleotide positions, but was identical to the EHMfas1 strain isolated previously from a cynomolgus monkey (M. fascicularis). One of the E. nuttalli isolates from Japanese macaques, named the NASA6 strain, was axenized and cloned. In isoenzyme analysis, the mobilities of hexokinase and phosphate glucose isomerase in the NASA6 strain were identical to those in the P19-061405 and EHMfas1 strains, but the mobility of phosphoglucomutase was different. These results were supported by gene analyses of these enzymes. Inoculation of NASA6 strain trophozoites into the liver of hamsters led to formation of an amoebic liver abscess. The liver lesions were characterized by extensive necrosis associated with inflammatory reactions. These results demonstrate that the NASA6 strain is potentially virulent and that E. nuttalli should be recognized as a common parasite in macaques.

Ultrastructure of placental hyperplasia in mice: comparison of placental phenotypes with three different etiologies.

Hyperplastic placentas have been reported in several experimental mouse models, including animals produced by somatic cell nuclear transfer, by inter(sub)species hybridization, and by somatic cytoplasm introduction to oocytes followed by intracytoplasmic sperm injection. Of great interest are the gross and histological features common to these placental phenotypes--despite their quite different etiologies--such as the enlargement of the spongiotrophoblast layers. To find morphological clues to the pathways leading to these similar placental phenotypes, we analyzed the ultrastructure of the three different types of hyperplastic placenta. Most cells affected were of trophoblast origin and their subcellular ultrastructural lesions were common to the three groups, e.g., a heavy accumulation of cytoplasmic vacuoles in the trophoblastic cells composing the labyrinthine wall and an increased volume of spongiotrophoblastic cells with extraordinarily dilatated rough endoplasmic reticulum. Although the numbers of trophoblastic glycogen cells were greatly increased, they maintained their normal ultrastructural morphology, including a heavy glycogen deposition throughout the cytoplasm. The fetal endothelium and small vessels were nearly intact. Our ultrastructural study suggests that these three types of placental hyperplasias, with different etiologies, may have common pathological pathways, which probably exclusively affect the development of certain cell types of the trophoblastic lineage during mouse placentation.

Anabolic steroids increase exercise tolerance.

The influence of an anabolic androgenic steroid (AAS) on thymidine and amino acid uptake in rat hindlimb skeletal muscles during 14 days after a single exhaustive bout of weight lifting was determined. Adult male rats were divided randomly into Control or Steroid groups. Nandrolone decanoate was administered to the Steroid group 1 wk before the exercise bout. [3H]thymidine and [14C]leucine labeling were used to determine the serial changes in cellular mitotic activity, amino acid uptake, and myosin synthesis. Serum creatine kinase (CK) activity, used as a measure of muscle damage, increased 30 and 60 min after exercise in both groups. The total amount of weight lifted was higher, whereas CK levels were lower in Steroid than in Control rats. [3H]thymidine uptake peaked 2 days after exercise in both groups and was 90% higher in Control than in Steroid rats, reflecting a higher level of muscle damage. [14C]leucine uptake was approximately 80% higher at rest and recovered 33% faster postexercise in Steroid than in Control rats. In a separate group of rats, the in situ isometric mechanical properties of the plantaris muscle were determined. The only significant difference was a higher fatigue resistance in the Steroid compared with the Control group. Combined, these results indicate that AAS treatment 1) ameliorates CK efflux and the uptake of [3H]thymidine and enhances the rate of protein synthesis during recovery after a bout of weight lifting, all being consistent with there being less muscle damage, and 2) enhances in vivo work capacity and the in situ fatigue resistance of a primary plantarflexor muscle.

EAT/mcl-1 expression in the human embryonal carcinoma cells undergoing differentiation or apoptosis.

Differentiation and apoptosis are precisely regulated events in early embryogenesis. Retinoic acid-induced differentiation in the embryonal carcinoma (EC) cell line NCR-G3 triggers concurrent induction of apoptosis. Using this system, which serves as a model of early embryogenesis, the expression of various bcl-2-related genes was analyzed as these genes display either positive or negative regulatory effects on apoptosis. EAT/mcl-1, an antiapoptotic bcl-2-related gene and immediate early gene, was dramatically expressed at an early stage of NCR-G3 differentiation. Bcl-xL, another antiapoptotic gene, was induced at a middle stage of differentiation and then gradually decreased to basal level. Expression of Bax, a proapoptotic molecule, was detected at a high level and remained relatively constant. Meanwhile, Bcl-2 and Bcl-xS were below detectable levels throughout the various stages of differentiation. As the balance of bcl-2 genes is a crucial regulatory step in apoptosis, the results suggest that EAT and Bax likely regulate apoptosis in the early stages of differentiation. In later stages of differentiation, down-regulation of EAT was found to coincide with a gradual increase in apoptosis of NCR-G3 cells. Furthermore, use of the monoclonal antibody (3A2) specific to EAT revealed that EAT is localized to the outer mitochondrial membrane in human EC cells. In addition, EAT immunoreactivity was not detected in apoptotic NCR-G3 cells while it was observed in nearly all viable cells. The findings suggest that rapid induction of EAT may prevent NCR-G3 cells from undergoing apoptosis, thereby supporting viability at the early stage of differentiation.

[Clinical spectrum and management of holoprosencephaly].

To study the phenotypic spectrum and management of holoprosencephaly (HPE), we reviewed the findings of eight children with HPE from 3 to 10 years of age, who underwent intervention programs and rehabilitation at our center. One patient had alobar HPE, three semilobar HPE, and four lobar HPE. All patients had postnatal growth retardation, and seven showed a decreased BMI (< 25% tile). All patients had severe developmental delay and mental retardation (DQ < 40), showing no obvious correlation between their severity and the type of HPE. Neurologically seven patients had spasticity (3 spastic quadriplegia, 2 spastic diplegia, 2 mixed-type), except one patient with a 7q deletion [46,XY,del(7) (q35)] who had generalized hypotonia. Seven had variable types of seizures. All patients had feeding difficulties and were assessed by speech-language therapists. Four patients required tube feeding, four had gastroesophageal reflux disease. Recurrent respiratory tract infection was common. Three patients had abnormal serum sodium concentration (1 diabetes insipidus, 1 idiopathic hypernatremia, 1 hyponatremia). No family history of HPE was elicited. In conclusion, patients with HPE should be followed up closely for complications such as feeding difficulty, malnutrition, seizures, spasticity, infection, and osmoreceptor-hypothalamus-hypophyseal axis abnormalities.

Limited myogenic response to a single bout of weight-lifting exercise in old rats.

The purpose of the present study was to compare the myogenic response of hindlimb muscles in young (14-20 wk of age) and old (>120 wk of age) rats with a single exhaustive bout of heavy resistance weight lifting. [(3)H]thymidine and [(14)C]leucine labeling were monitored for up to 2 wk after the exercise bout to estimate serial changes in mitotic activity and the level of amino acid uptake and myosin synthesis. Histological, histochemical, and immunohistochemical [anti-5-bromo-2'-deoxyuridine and myogenic determination genes (MyoD)] analyses of whole muscles and analysis of muscle-specific gene expression (MyoD) using Western blotting and RT-PCR were performed. Old rats showed significant muscle atrophy and a lower exercise capacity than young rats. Exercise-induced muscle damage, as assessed in histological sections, and increases in serum creatine kinase activity were evident in both young and old exercised groups. Mitotic activity was increased in young, but not old, rats 2 days after exercise. There was a biphasic increase in [(14)C]leucine uptake during the 14 days postexercise (peaks at 1-4 and 10 days) in young rats: only the first peak was observed in old rats. There was a lower uptake of [(14)C]leucine in the myosin fraction and an impaired expression of MyoD at the protein (immunohistochemistry and Western blotting) and mRNA (RT-PCR) levels in old rats throughout the postexercise period. These results demonstrate a reduced reparative capability of muscle in response to a single bout of exercise in old compared with young rats.

Rat lens epithelial cell damage and cataract formation induced by immunological response to bovine lens membrane protein

Purpose: To evaluate the correlation of cataractogenesis and immune mechanisms, we investigated the rat lens morphologically and immunologically.Methods and Results: Wistar rats were divided into three groups: Group A was immunized with bovine-lens membrane protein (B-LMP) and adjuvant, Group B was immunized with adjuvant only, and Group C was not given any treatment as a control. Titer levels of anti-B-LMP antibody and anti-rat-LMP antibody were elevated and posterior subcapsular cataract was developed in Group A. In flat preparations, a noncellular part in the lens epithelium was observed in all members of Group A. In this noncellular part, a lens capsule protruding into the lens epithelial cell layer was observed by light microscopy.Conclusion: These data suggest that lens epithelial cells may be damaged by immune response, causing the development of cataract.

[Rat lens epithelial cell damage and cataract formation induced by immunological response to bovine lens membrane protein].

PURPOSE: To evaluate the correlation of cataractogenesis and immune mechanisms, we investigated the rat lens morphologically and immunologically. METHODS AND RESULTS: Wistar rats were divided into three groups: Group A was immunized with bovine-lens membrane protein (B-LMP) and adjuvant, Group B was immunized with adjuvant only, and Group C was not given any treatment as a control. Titer levels of anti-B-LMP antibody and anti-Rat-LMP antibody were elevated and posterior subcapsular cataract was developed in Group A. In flat preparations, a noncellular part in the lens epithelium was observed in all members of Group A. In this noncellular part, a lens capsule protruding into the lens epithelial cell layer was observed by light microscopy. CONCLUSION: These data suggest that lens epithelial cells may be damaged by immune response, causing the development of cataract.

Induction of glomerular lesions in the kidneys of mice infected with vero toxin-producing Escherichia coli by lipopolysaccharide injection.

Lipopolysaccharide was injected into germ-free mice after they had been infected with Vero toxin-producing Escherichia coli. Microscopic examination of the kidneys of these mice showed an increased number of mesangial cells and a vacancy in the glomerular capillary lumen. A significant elevation in the expression level of interferon (IFN)-gamma in the kidney may have played a key role in the induction of glomerular lesions, because the administration of neutralizing antibody to IFN-gamma markedly alleviated such lesions.

Overexpression of a minor component of myelin basic protein isoform (17.2 kDa) can restore myelinogenesis in transgenic shiverer mice.

Shiverer (shi) mice, which are neurologically mutant, lack a large portion of the gene for the myelin basic proteins (MBPs), have virtually no myelin in their central nervous system (CNS), and shiver, undergo seizures, and die early. At least five types of MBPs (21.5, 18.5, 17.3, 17.2 and 14.0 kDa) are known to be generated through alternative splicing from a single MBP gene. We have produced transgenic shi mice carrying a cDNA encoding mouse 14-kDa MBP isoform, the most abundant form of MBPs, under control of a mouse MBP gene promoter, and showed that expression of the 14-kDa MBP can restore CNS myelination. To test whether the 17.2-kDa MBP isoform, one of the minor components of MBPs, can also elicit myelination in homozygous shi mutants, we produced seven independent transgenic shi mice carrying cDNA encoding the mouse 17.2-kDa MBP isoform, and the transcription of which was driven by a mouse MBP gene promoter. The axons in the cerebellum of one transgenic line, which exhibited the highest expression of transgene-derived mRNA ( approximately 50% of the level of total MBP mRNA in the normal mouse brain), were myelinated. This mouse exhibited nearly normal behavior. These findings indicate that the 17.2-kDa MBP isoform, even when the only 17.2-kDa MBP isoform is present, has the ability to elicit CNS myelination in transgenic shi mice. This transgenic strategy will be useful for elucidating the role of each type of MBP isoform in CNS myelinogenesis.

Morphological and biochemical evidence of muscle hyperplasia following weight-lifting exercise in rats.

We used a rat model of weight lifting to examine the serial biochemical and morphological changes following muscle fiber hyperplasia during 14 days of exercise. [3H]thymidine and [14C]leucine labeling were used to determine the serial changes in cellular mitotic activity and the level of amino acid uptake and myosin synthesis. Morphological changes were assessed with light and transmission electron microscopy, whereas proliferation of cells was evaluated immunohistochemically with 5-bromo-2'-deoxyuridine (BrdU). The intensity of the exercise and degree of muscle damage were monitored by serum creatine kinase (CK) activity. Damaged fibers were sparsely distributed, and a significant CK leakage was observed 30-60 min after exercise. Anti-BrdU-positive cells were observed in damaged fibers and at the periphery of undamaged fibers. Changes typical of muscle regeneration were observed; however, the formation of new fibers in the interstitial space was also evident. The mitotic activity also changed and reflected the appearance of anti-BrdU-positive cells and activated satellite cells. Amino acid uptake increased during the first week of exercise, probably reflecting muscle hypertrophy and synthesis of other noncontractile related proteins. The uptake also increased during the second week, probably due to hyperplasia, a finding also supported by electron microscopy. Our results suggest that one bout of weight-lifting exercise in untrained rats induced muscle hyperplasia following regeneration. The process of muscle hyperplasia was activated by muscle fiber damage in our model.

A case of angiosarcoma of the breast.

This is a case report of a 20-year-old woman who had primary angiosarcoma of the left breast, with metastases to the spleen and ovary. Eight months after detecting a mass in her breast, she underwent mastectomy with biopsy of the ipsilateral axillary lymph nodes, splenectomy and bilateral oophorectomy. Five months after the operation, the patient succumbed to lung metastases. Angiosarcoma of the breast is a rare condition with a poor prognosis, and there are no established chemotherapeutic regimens as yet. Immunohistochemical staining for endoglin, known to be expressed mainly on the surface of endothelial cells, was positive. This suggests the possibility of treating angiosarcoma with anti-endoglin monoclonal antibodies.

Appearance of complex branched muscle fibers is associated with a shift to slow muscle characteristics.

To examine the effects of numerous complex branched fibers (CBF) on whole muscle contractile properties, we established a model of myopathic muscles containing a large number of CBF by repeated local injection of bupivacaine hydrochloride (Marcaine) into the plantaris (PLA) muscle. Marcaine injections were administered once weekly for 10 weeks into the right PLA muscles of Wistar male rats. The in situ contractile properties of Marcaine-injected PLA muscles (I-PLA) were examined under urethane anesthesia, and compared with the contralateral (control) PLA muscle (C-PLA). Numerical and morphological examination using the modified nitric acid fiber digestion method and scanning electron microscopy showed that Marcaine resulted in an 8-fold increase in the number of branched fibers in the I-PLA muscles and about 70% of these fibers were CBF. The latter were composed of ten or more muscle fibers fused together along with many thin and thick, long and short daughter branches. The time to peak tension of twitch and tetanus, and 1/2 relaxation time were significantly longer in I-PLA muscles, representing a shift to slow muscle characteristics. However, the total area of slow fibers/muscle cross-sectional area was similar in I-PLA and C-PLA muscles. Aggregates of same-type fibers (slow fibers) with small and large diameters were observed, reflecting an expected cross-sectional property of CBF. Our results suggest that the appearance of several CBF in a muscle is associated with a shift towards slower contractile properties in the affected muscle.

Characteristics of compensatory hypertrophied muscle in the rat: I. Electron microscopic and immunohistochemical studies.

BACKGROUND: Complex branched muscle fibers are frequently observed in the muscles of mdx mutant mice and/or in damaged muscles. To investigate whether the complex branched fibers were present in the compensatory hypertrophied muscles of rats, we examined the morphological changes in these muscles. METHODS: We examined the hypertrophied plantaris (PLA) muscle of the Wistar male rats, prepared by surgical ablation of synergistic muscles. The muscle was examined using three-dimensional analysis with scanning electron microscopy, immunohistochemical detection of proliferating cells using 5-bromo-2'-deoxyuridine (BrdU) and histological and histochemical characterization. Studies were performed at 48 hours, 2, 4, 6, 10, and 15 weeks after surgical preparation. RESULTS: The muscle hypertrophy ratio (muscle weight relative to the contralateral intact control side), gradually increased from 2 to 10 weeks, and the peak value (48.6%) occurred at the 10th week. The total number of fibers did not change significantly at any time interval. However, the number of branched muscle fibers increased significantly (P < 0.05) after 6 weeks, and accounted for about 2.5% of the total fibers at the 15th week. Most branched fibers showed complex features resembling the "anastomosing syncytial reticulum" described in myopathic animals. The fibers were observed mainly in the middle and distal portions of the PLA muscle. The proportion and distribution of proliferating cells in the entire PLA muscle corresponded with the distribution of the complex branched fibers. These results were also observed in muscle tissues prepared for histological and histochemical examination. CONCLUSIONS: The presence of a large proportion of complex branched fibers in a limited segment of the compensatory hypertrophied muscle suggests that this hypertrophy model represents a pathological and/or pathophysiological hypertrophy model rather than a normal physiological process.

A Japanese patient with X-linked alpha-thalassemia/mental retardation syndrome: an additional case report.

X-linked alpha-thalassemia/mental retardation syndrome (ATR-X) is characterized by severe mental retardation, wide range of minor abnormalities, and association with an unusual form of alpha-thalassemia. Fifty patients in Caucasian origin have been reported. This is the second report of the syndrome demonstrated in Oriental patients.

Mechanism of membrane damage by El Tor hemolysin of Vibrio cholerae O1.

El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cholerae O1 spontaneously assembled oligomeric aggregates on the membranes of rabbit erythrocyte ghosts and liposomes. Membrane-associated oligomers were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting into two to nine bands with apparent molecular masses of 170 to 350 kDa. ETH assembled oligomers on a liposomal membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone. Cholesterol could be replaced with diosgenin or ergosterol but not with 5alpha-cholestane-3-one, suggesting that sterol is essential for the oligomerization. The treatment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium. Because dextrin 20 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysis, this hemolysis is likely to be caused by pore formation on the membrane. The pore size(s) estimated from osmotic protection assays was in the range of 1.2 to 1.6 nm. The pore formed on a rabbit erythrocyte membrane was confirmed morphologically by electron microscopy. Thus, we provide evidence that ETH damages the target by the assembly of hemolysin oligomers and pore formation on the membrane.

The defective secretion of a naturally occurring alpha-1-antichymotrypsin variant with a frameshift mutation.

A newly found variant alpha-1-antichymotrypsin (ACT), ACT Isehara-2, has a deletion of two bases (AA) at codon 391 near the carboxyl terminus. This frameshift mutation caused a change in the amino acid sequence and generated 10 extra amino acids (408 amino acids total) [Tsuda, M., Sei, Y., Matsumoto, M., Kamiguchi, H., Yamamoto, Y., Shinohara, Y., Igarashi, T. & Yamamura, M. (1992) Hum. Genet. 91. 467-468]. The serum ACT levels in three unrelated heterozygotes with this mutant ACT gene were 37% 49% and 54% that of the normal individuals. To examine the reduced serum levels, the normal ACT and the mutant ACT created by site-directed mutagenesis were transfected into COS-7 cells for comparison. The value for the retention rate (intracellular ACT/total ACT) was apparently higher in the cells expressing mutant ACT Isehara-2 than those bearing the normal gene. In the pulse-chase experiments, the secretion of the synthesized mutant ACT into the medium was not observed, whereas the normal ACT was mostly secreted as a 64-kDa form. The endoglycosidase H digestion and an electron microscopic analysis indicated that the retained mutant ACT was present in the endoplasmic reticulum. These results provide the biochemical basis for the decreased serum ACT level of individuals with ACT Isehara-2, and suggest the importance of the carboxyl-terminal region for its secretion.

Hepatic cytokeratin changes in obstructive jaundice: before and after biliary drainage.

We investigated the changes in hepatic cytokeratins in obstructive jaundice by immunohistochemistry. The results can be summarized as follows: 1) In accordance with the progression of the jaundice, the lobular and cellular distribution of cytokeratin reactivity in hepatocytes expanded. 2) Cytokeratin reactivity in obstructive jaundice was improved by removal of the bile duct obstruction (decompression), but this decompressive effect deteriorated in the case of prolonged jaundice. 3) It is suggested that the specific lobular and cellular distribution and/or the changes in cytokeratin aggregation might be of value in determining the stage and in predicting the prognosis of obstructive jaundice.