Studies on a Morphologically Distinct Colchicine-Resistant Variant of Entamoeba sp.*

Colchicine has a temperature-dependent cytotoxic effect on Entamoeba sp. (Laredo isolate) that is most apparent when the drug is applied during the initiation of cultures at a concentration of 7.5 mM or higher. Continued transfer of cultures in medium containing progressively increasing concentrations of colchicine has resulted in a variant that grows prolifically in the presence of colchicine (7.5 mM) with a generation time comparable to that of the parent stock. Comparison of a number of parameters of the 2 variants revealed that colchicine resistance was accompanied by a change in cell shape, a reduced membrane permeability, which could partially be overcome by the addition of dimethyl sulfoxide (DMSO), and a reduced tolerance to osmotic stress. However, the parent strain and resistant variant were equally susceptible to cycloheximide and puromycin suggesting that the acquired colchicine resistance may not be explained on the basis of an entirely unspecific generalized reduced ability for drug uptake. Colchicine resistance and altered structure were found to be stable over a long period of time. The possible interdependence of these 2 parameters and their relation to cell motility in Entamoeba sp. are discussed.     

Freeze-Fracture Study of Malaria Sporozoites: Antibody-Induced Changes of the Pellicular Membrane*

Plasmodium cynomolgi, Plasmodium knowlesi, and Plasmodium berghei sporozoites, before and after incubation with immune serum, were studied after freeze-fracture by electron microscopy. There were evenly distributed numerous intramembranous particles (IMP) on the P face of the outer membrane. The E face of the plasma membrane had fewer IMP than its P face. The E face of the intermediate membrane had few IMP and also linear arrays of slightly raised ridges running the length of the parasite. The P face of the intermediate membrane had many IMP aligned along the long axis of the sporozoite. On the P face of the inner membrane. IMP were arranged in very distinct rows conforming to the long axis of the parasite; the E face of this membrane had a few randomly distributed IMP. A prominent change in the sporozoite incubated in immune serum was the appearance of a layer of aggregated particles around the parasite. The P face of the plasma membrane had several clear areas devoid of IMP and IMP aggregates. No changes were seen in the other fractured faces of the pellicle. These observations suggest that immune serum acts only on the P face of the plasma membrane.     

Morphogenesis in the Heterotrich Ciliate Climacostomum virens. I. Oral Development During Cell Division

The principal characteristics of stomatogenesis during division in Climacostomum virens are: (A) Kinetosomal proliferation on the left side of a variable number of kineties in the ventral somatic cortex forms an oral primordium consisting of several kinetosomal fields, which then fuse to form a single anarchic field. (B) A constant topographical relationship exists between the primordium and a well defined cortical pattern, the zone of discontinuity. (C) The anarchic field primordium divides into 2 unequal parts—to the left, the AZM primordium, and to the right, the paroral primordium, which differentiates into the apical membranelles, the peristomial field, and the buccal tube. (D) Preoral and oblique kineties of the somatic cortex form along the right side of the paroral primordium. (E) Parental oral structures are partially dedifferentiated. Stomatogenesis in C. virens and other heterotrichs is compared.     

Effect of Exposure Period and Algal Concentration on the Frequency of Infection of Aposymbiotic Ciliates by Symbiotic Algae from Paramecium bursaria

The effect of exposure period and concentration of algae on the frequency of infection of aposymbiotic ciliates by algae obtained from the same clone of Paramecium bursaria syngen 2, was studied. The frequency of infection was roughly proportional to the algal concentration and to the exposure time of ciliates to algae. The relationship of algal concentration to infection frequency closely fitted the Poisson distribution curve for N = 1, suggesting that the minimum number of algae required to infect a single ciliate is 1. However, the data also strongly suggested that the average number of algae required to initiate infection of an average ciliate was ? 1,000. Three possible resolutions of this situation are: (a) the selection by the ciliate of a rare infective variant from a heterogeneous population: (b) the rare escape of an alga from digestion by the ciliate; and (c) the requirement for a large number of algae-ciliate contacts to induce susceptibility in the ciliate. Splitting the exposure of ciliates to algae into 2 periods of 0.5 h, separated by 5 h in the absence of algae, produced a much higher frequency of infection than a single l-h exposure, supporting the suggestion that the large number of algae is required to induce susceptibility in the ciliate which can then be infected by as few as a single algal cell.     

Plasmodium falciparum in Culture: Improved Continuous Flow Method*

SYNOPSIS. A new design of flow vessel provides a method for continuous culture of P. falciparum in a settled layer of human erythrocytes with a slow flow of culture medium over them. The parasitemia is kept fluctuating from ? 1%, just after addition of fresh erythrocytes. to ? 10%, 2 or 3 days later. Each vessel provides each week 3 harvests, each containing ? 0.6–1 × 10⁹ parasites.     

The most prevalent genetic cause of ALS-FTD,C9orf72 synergizes the toxicity of ATXN2 intermediate polyglutamine repeats through the autophagy pathway

The most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) is repeat expansion of a hexanucleotide sequence (GGGGCC) within the C9orf72 genomic sequence. To elucidate the functional role of C9orf72 in disease pathogenesis, we identified certain molecular interactors of this factor. We determined that C9orf72 exists in a complex with SMCR8 and WDR41 and that this complex acts as a GDP/GTP exchange factor for RAB8 and RAB39, 2 RAB GTPases involved in macroautophagy/autophagy. Consequently, C9orf72 depletion in neuronal cultures leads to accumulation of unresolved aggregates of SQSTM1/p62 and phosphorylated TARDBP/TDP-43. However, C9orf72 reduction does not lead to major neuronal toxicity, suggesting that a second stress may be required to induce neuronal cell death. An intermediate size of polyglutamine repeats within ATXN2 is an important genetic modifier of ALS-FTD. We found that coexpression of intermediate polyglutamine repeats (30Q) of ATXN2 combined with C9orf72 depletion increases the aggregation of ATXN2 and neuronal toxicity. These results were confirmed in zebrafish embryos where partial C9orf72 knockdown along with intermediate (but not normal) repeat expansions in ATXN2 causes locomotion deficits and abnormal axonal projections from spinal motor neurons. These results demonstrate that C9orf72 plays an important role in the autophagy pathway while genetically interacting with another major genetic risk factor, ATXN2, to contribute to ALS-FTD pathogenesis.     

A biomarker for severity of Alzheimer’s disease: 1H-NMR resonances in cerebrospinal fluid correlate with performance in mini-mental-state-exam

Context: There is no valid biomarker for severity of Alzheimer’s disease (AD) available until now.

Objective: Therefore, we investigated ¹H-NMR spectroscopy for specific resonances as biomarkers for severity of AD.

Materials and methods: Cerebrospinal fluid (CSF) of patients with diagnosed AD and healthy control subjects was analysed by one-dimensional water-suppressed ¹H-NMR spectroscopy. The resonances were correlated with the cognitive performance of the patients and controls.

Results: Specific ¹H-NMR resonances showed higher intensities in AD patients than in control subjects. Mini-mental-state-exam scores correlated with ¹H-NMR resonances in AD patients.

Discussion and conclusion: ¹H-NMR resonances of CSF are obviously valid biomarker for severity of AD, despite the lack of knowledge of the underlying molecular structure. Successful isolation and identification of these substances will most likely provide details to the pathophysiology of AD.     

OCCURRENCE OF PHYIWATED CHLOROPHYLL c IN ISOCHRYSIS GALBANA AND ISOCHRYSIS SP. (CLONE T-ISO) (PRYMNESIOPHYCEAE)1

The pigment composition of two clones of Isochrysis galbana Parke (CCMP 1323 and CCAP 927/1), and Isochrysis sp. (clone T-ISO) was analyzed by high-performance liquid chromatography using a polymeric octadecylsilica column. Fluorescent peaks with retention times higher than chlorophyll a were detected for all three clones. The corresponding pigments were isolated and characterized in terms of their visible absorbance and fluorescence spectra. The pigments were similar to phytol-substituted chlorophyll c, previously isolated from Emiliania huxleyi (Lohm.) Hay and Mohler and other species containing chlmophyll c₃. The presence of phytol-substituted chlorophyll c in I. galbana which lacked chlorophyll c₃, increases the diversity of chlorophyll patterns for the Haptophyta, which can be grouped, at present, into six different pigment types. This is the jrst observation of a haptophyte containing the apolar phytylated chlorophyll c-like pigment but lacking chlorophyll c₃.