Encephalitozoon-Like Organisms in Patients with Alveolar Hydatid Disease: Cell Culture, Ultrastructure, Histoimmunochemical Localization and Seroprevalence

ABSTRACT. We found Encephalitozoon -like organisms in an in vitro culture of a human liver lesion which was due to larval Echinococcus multilocularis. The organisms developed in the same fashion as an Encephalitozoon cunculi. The spores that developed in parasitophorous vacuoles were 2.0–2.6 × 1.1–1.5 μm: each contained a single nucleus and 4–5 polar tubule coils, closely resembling E. cuniculi in its ultrastructure. Mature spores were collected from the supernatants by the use of Percoll centrifugation resulting in the banding of the spores on continuous gradients. We prepared three sorts of spores which were different in buoyant density in 0.15 M NaCl: 1.05–1.07 g/ml spores, 1.12 g/ml spores, and spores of over 1.14 g/ml. Polyclonal antibodies to a pool of each spore preparation were produced in a rabbit and applied to the detection of microsporidian antigen in situ. The histoimmunoperoxidase (HIP) procedure was used to detect the microsporidian antigen in echinococcal liver lesions from patients with alveolar hydatid disease (AHD). Ten echinococcal liver lesions from different AHD patients were examined and four were found to be positive in the HIP test. The Percoll-separated spores were also used as an antigen to detect for antibodies in the sera from the patients with AHD by Western blotting. Antibodies were detected in 62 (52%) of the 119 AHD patients and in only 8 (5%) of the 159 normal healthy individuals.     

EFFECT OF CENTRIFUGATION ON THE DEVELOPMENT AND TIMING OF PREMITOTIC POSITIONING OF THE NUCLEUS IN ADIANTUM PROTONEMATA

When single-celled protonemata of Adiantum capillus-veneris L. were centrifuged immediately before transferring to darkness from continuous irradiation with red light, their nuclei were displaced basipetally. Both filamentous and branched protonemata were obtained. The stronger the centrifugal acceleration, the more frequently the branched protonemata were induced.
The effect of centrifugation at 1,300 x g for 15 min on nuclear displacement was different at different stages of the cell cycle. In early G₁ phase, the nucleus was easily displaced by centrifugation, but quickly returned to the original position after centrifugation. In late G₁ phase, the nucleus was displaced, but after centrifugation it never came back to the original position. In late G₂ and M phases, the nucleus was no longer displaced by the centrifugation. Premitotic positioning of the nucleus in cytokinesis took place about 5 hr before cell plate formation in all centrifugal treatments described above.     

Localization of Thermoreceptor Systems that Induce Step-Up and Step-Down Thermophobic Responses and Switching in the Dominance of These Systems in Blepharisma

The distribution of thermoreceptor systems that initiate step-up and step-down thermophobic responses in bisected cells of Blepharisma was examined. Anterior cell fragments responded by ciliary reversal to a step-down in temperature and by repression of spontaneous ciliary reversal to a step-up. Posterior fragments responded by ciliary reversal to a step-up in thermal stimulation and by repression of spontaneous ciliary reversal in response to step-down stimulation. Results indicate that two kinds of thermoreceptor systems exist in the anterior half of each cell; one is responsible for ciliary reversal induced by step-down stimulation, and the other is responsible for repression of the ciliary reversal caused by step-up thermal stimulation. Likewise, there are also two kinds of thermoreceptor systems in the posterior half of the cell; one is responsible for ciliary reversal in response to a step-up in temperature, and the other is responsible for the repression of ciliary reversal on a step-down in thermal stimulation. Below about 27°C, intact cells showed ciliary reversal only when a step-down in thermal stimulation occurred, while above about 27°C cells only responded to a step-up in thermal stimulation. At about 27°C there was a switch in the dominant response from the anterior to the posterior half of an individual cell.     

Why 11-cis-Retinal?

The C₂₀ diterpenoid compound retinal is the chromophore of thevisual pigments the rhodopsins, and the pigments present inHalobacterium halobium, namely, bacteriorhodopsin (proton pump),halorhodopsin (chloride pump), and the sensory rhodopsins (phototaxisreceptor). In all cases, they are bound covalently to the receptorprotein by a protonated Schiff base. However, in rhodopsins,the retinal is the 11-cis isomer, whereas in H. halobium pigmentsit is the all-trans isomer. Why did Nature choose retinal asthe chromophore, and why 11-cis in some cases and all-transin other cases? Also why is the chromophore a protonated Schiffbase? These points are addressed after giving an outline ofthe current status of the various photoreceptor pigments     

Purification of Acetylcholinesterase from Sea Urchin (Hemicentrotus pulcherrimus) Embryos by Affinity Chromatography

Only one form of acetylcholinesterase (AchE) was detected in Hemicentrotus pulcherrimus embryos. In H. pulcherrimus embryos as well as in the other sea urchin embryos, AchE activity begins to increase rapidly after gastrula stage.
Purification of AchE from plutei has been carried out by the procedure including affinity chromatography. Purified AchE had the activity 14,600 times higher than that of homogenate, and the final yield of AchE was 8%. The enzyme seems to be electrophoretically homogeneous, and has a molecular weight of 3 × 10⁵ as determined by Sepharose CL–6B column chromatography.     

Nucleotide Sequences of DNA Fragments of Encephalitozoon cuniculi Amplified by Polymerase Chain Reaction with Primers Regarded as Specific for Echinococcus

Encephalitozoon -like spores were separated from a human echinococcal liver lesion, which was caused by Echinococcus multilocularis. They were found to fall into the species Encephalitozoon cuniculi , which was shown to have En. cunniculi specific DNA by way of polymerase chain reaction (PCR). We also used PCR to genetically discriminate between the En. cuniculi spores and the Ec. multilocularis larvae. Two primer sets, known to be specific for Echinococcus , were examined. These primers were expected to work normally when the two quite different DNA preparations were tested as templates, i.e. only Echinococcus DNA could give a positive signal in the PCR tests. However, it was found that the two Echinococcus -specific primer sets could amplify not only EC. multilocularis DNA, but also En. cuniculi spore DNA. We then tried to determine the order of nucleotides in the Echinococcus -specific primers-amplified En. cuniculi PCR products and compared the determined sequences with those of Ec. multilocularis. The results clearly indicated that sequencing made little difference between En. cuniculi and Ec. multilocularis.     

PHOTOCONTROL OF THE ORIENTATION OF CELL DIVISION IN ADIANTUM. I. EFFECTS OF THE DARK AND RED PERIODS IN THE APICAL CELL OF GAMETOPHYTES

When protonemata of Adiantum capillus-veneris L. which had been grown filamentously under continuous red light were transferred to continuous white light, the apical cell divided transversely twice, but the 3rd division was longitudinal. An intervening period of darkness lasting from 0 to 90 hr either between the 1st and the 2nd cell division or between the 2nd and the 3rd one did not affect the number of protonemata in which the 3rd cell division was longitudinal. The insertion of red light instead of darkness greatly decreased the percentage of 1st longitudinal divisions occurring at the 3rd division, and increased the number of transverse divisions. Fifty percent reduction of induction of 1st longitudinal division was caused by ca. 50 hr exposure to red light between 1st and 2nd division and by ca. 20 hr between 2nd and 3rd division, and total loss was induced by an exposure of ca. 100 hr or longer to red light in the former and by ca. 40 hr longer in the latter. Thus, by using an appropriate intervening dark period or exposure to red light, the orientation and timing of cell division could be controlled in apical cell of the fern protonemata.     

Fate of ($ ) S-methyl-L-cysteine sulfoxide in Chinese cabbage, Brassica pekinensis RUPR.

($) S-methyl-L-cysteine sulfoxide (MCS) was scarcely found inseeds of Chinese cabbage, but was present in relatively largeamounts in all plant parts after germination. Changes in MCScontent paralleled those for soluble sulfur content of tissue. When Na₂³⁵SO₄ was fed to plants, the ³⁵S was predominantly incorporatedinto MCS in the free amino acid fraction in both sulfur-sufficientand deficient plants, but it was incorporated to a greater extentin the former. ³⁵S-MCS was metabolized more readily in deficientthan in sufficient plants, and its sulfur was found not onlyin various soluble compounds but in the insoluble fractionsfrom plants as well. These results indicate that MCS is a conspicuousconstituent in the free amino acid pool of Chinese cabbage andmay play an important role in sulfur metabolism by acting asa soluble pool for organic sulfur. (Received June 22, 1970; )     

Transmission and Scanning Electron Microscopic Studies of the Micronemata of Trypanosoma gambiense1

The structure of micronemata arising from the surface of the bloodstream form of Trypanosoma gambiense was studied by electron microscopy. In order to produce micronemata, trypanosomes were incubated in either 1) phosphate buffered saline supplemented with glucose (PBSG), 2) immune mouse serum or 3) PBSG after passage through a DEAE-cellulose column. Electron microscopic examination of the parasite revealed the presence of thread-like micronemata arising from the anterior end and from the flagellar pocket regardless of the incubation conditions. Negative staining revealed a distinct peripheral fringe layer with nodular protrusions covering the entire surface of the micronema. The distribution and number of intramembrane particles (IMP) on the P and E faces of the micronema were similar to those of the flagellum of T. gambiense, indicating a close relationship between the membrane structure of the micronema and the flagellum. Micronemata became fragmented and adhered to each other after incubation of the parasite in the media for 12 h. Since micronemata tend to have the characteristics of adhesiveness and fragmentation, fragments of these structures might adhere to various host organs. Dispersal of potential antigenic material might be responsible, in part, for the induction of the host immune response.     

Phenotypic and Genetic Diversity of Erwinia carotovora ssp. carotovora Strains from Asia

A collection of 87 strains of the soft rot pathogen Erwinia carotovora ssp. carotovora (Ecc) isolated from various host plants in Japan, Korea and Thailand was characterized by bacteriological, pathological and genetic properties. On the basis of pathogenicity on the potato, tomato, onion and cucumber, strains were divided into four groups. They were also characterized by PCR‐restriction fragment length polymorphisms (RFLP) of 16S ribosomal DNA (rDNA), 16S‐23S rDNA intergenic spacer regions (ISRs) and a pel gene encoding pectate lyase. By analysis of 16S rDNA RFLP generated by Hinf I, Ecc strains were differentiated into two groups where it was discovered that most strains from Korea and Japan belonged to the same group. In the analysis of ISRs RFLP with MboI, two patterns were found. All Thai strains showed the same pattern. In the analysis of the pel gene RFLP with Sau3AI, all strains were separated into two independent patterns except for one strain. The strain (MAFF 301937) isolated from the mulberry showed a unique RFLP pattern of the pel gene. In cluster analysis based on 26 phenotypic characters, Ecc strains were composed of two groups, A and B. Group A contained typical Ecc strains which provided negative reactions in testing the production of reducing substances from sucrose and acids from α‐methyl glucoside. All Thai strains and most of the Korean strains belonged to group A, whereas group B contained atypical Ecc strains, which were isolated in Japan and Korea; the properties of this group were similar to those of E. carotovora ssp. atroseptica. The research reported here was undertaken to provide information on the strains of E. carotovora ssp. carotovora in Asia.