Adelphamoeba galeacystis n. g., n. sp., an Amoeboflagellate Isolated from Soil*

SYNOPSIS. An amoeboflagellate isolated from common soil is described. The amoeboid stage is typically limax and contains a well differentiated uroid region. The flagellate has 2 flagella, which emerge anteriorly and are equal in length. It has a ventral cytostome near the anterior border. The cyst is helmet-shaped and without opercula. Polar masses are present during nuclear division.     

Protein Kinase C and Melanocyte Transformation

Melanoma cells which have been isolated from metastatic melanoma tissue are able to survive and proliferate in serum supplemented media. In contrast, normal human melanocytes require the presence of growth stimulators if they are to survive in culture. A tumor promotor, 12–0-tetradecanoyl-phorbol-13-acetate (TPA) and substances that increase intracellular levels of cyclic-adenosine-monophosphate (cAMP), such as cholera toxin or isobutylmethyl xanthine, have been widely used for this purpose. The phorbol diester receptor was shown in 1982 to be the phospholipid- and calcium-dependent enzyme protein kinase C (PKC). We therefore directed our studies to the role of PKC regulation in the growth of normal human melanocytes and their transformation. Our studies show that while melanoma cells are inhibited by TPA, the growth of normal melanocytes is stimulated in a dose-dependent manner. The inhibitor, 1-(5-isoquinolinesulfonyl)-2-methyl-piperizine dihydrochloride (H7), which has been found to be the most specific for PKC, had no effect on the growth of normal melanocytes, but inhibited the growth of melanoma cells in a dose-dependent manner. PKC was isolated from the membrane and cytosol of normal melanocytes and melanoma cells. The basal (resting) levels of PKC activity in normal melanocytes was low compared to that measured in melanoma cells, and after short-term (1 hour) treatment with TPA the PKC activity was greatest at the membrane, with the activity decreasing the cytosol. Upon prolonged (48 hours) treatment with TPA, this redistribution of activity continued in normal melanocytes and the total activity increased. In melanoma cells, however, the total PKC activity decreased, particularly in the membrane fraction. A difference in activity and distribution of the enzyme was also seen after short-term (1 hour) treatment with H7. There was very little effect seen on PKC in normal melanocytes; however, the effect on melanoma cells was similar to that seen after 48 hours of exposure to TPA with a decrease in total activity, particularly in the membrane fraction. These results indicate that the regulation of PKC, in particular its activation by TPA, is altered during the transformation of normal human melanocytes     

Cytochemical Localization of Reserves during Seed Development in Arabidopsis thaliana under Spaceflight Conditions

Successful development of seeds under spaceflight conditionshas been an elusive goal of numerous long-duration experimentswith plants on orbital spacecraft. Because carbohydrate metabolismundergoes changes when plants are grown in microgravity, developingseed storage reserves might be detrimentally affected duringspaceflight. Seed development in Arabidopsis thaliana plantsthat flowered during 11 d in space on shuttle mission STS-68has been investigated in this study. Plants were grown to therosette stage (13 d) on a nutrient agar medium on the groundand loaded into the Plant Growth Unit flight hardware 18 h priorto lift-off. Plants were retrieved 3 h after landing and siliqueswere immediately removed from plants. Young seeds were fixedand processed for microscopic observation. Seeds in both theground control and flight plants are similar in their morphologyand size. The oldest seeds from these plants contain completelydeveloped embryos and seed coats. These embryos developed radicle,hypocotyl, meristematic apical tissue, and differentiated cotyledons.Protoderm, procambium, and primary ground tissue had differentiated.Reserves such as starch and protein were deposited in the embryosduring tissue differentiation. The aleurone layer contains alarge quantity of storage protein and starch grains. A seedcoat developed from integuments of the ovule with gradual changein cell composition and cell material deposition. Carbohydrateswere deposited in outer integument cells especially in the outsidecell walls. Starch grains decreased in number per cell in theintegument during seed coat development. All these characteristicsduring seed development represent normal features in the groundcontrol plants and show that the spaceflight environment doesnot prevent normal development of seeds in Arabidopsis. Arabidopsis ; spaceflight; embryo; endosperm; seed coat; storage reserves     

Molecular Characterization of a Cytoplasmic Dynein from Dictyostelium

Cytoplasmic dynein is a high molecular weight, microtubule-based mechanochemical ATPase that is believed to provide motive force for a number of intracellular motilities, including transport of membrane-bound organelles. Cytoplasmic dynein also localizes to the mitotic spindles of some organisms and to the kinetochore regions of some condensed chromosomes, where it may play an active role in spindle assembly, spindle position, and/or chromosome movement during cell division. Despite active research efforts from a number of laboratories, little detail is yet available about dynein-based cellular activities. This paper describes our efforts to characterize cytoplasmic dynein from Dictyostelium and to use this protist as a molecular genetic factory to probe structure-function relationships of this molecule.     

Micronuclear and Macronuclear Sequences of a Euplotes crassus Gene Encoding a Putative Nuclear Protein Kinase

The sequences of a 1.8-kbp macronuclear DNA molecule (V3), and the majority of its micronuclear counterpart, are reported. The macronuclear V3 DNA molecule contains an open reading frame that is interrupted by a single intron, while the micronuclear copy is interrupted by four internal eliminated sequences, one of which is located within the intron. The predicted protein product of the macronuclear V3 gene is a 471-amino acid polypeptide that is very similar to a group of protein-serine/threonine kinases from both plant and animal species, some of whose members appear to be involved in cell cycle or growth control.     

The effect of season and group size on survivorship and larval growth in Plagiodera versicolora

Abstract.

• 1 The larvae of the multivoltine imported willow leaf beetle, Plagiodera versicolora (Laicharting), are found in larval aggregations during the first half of the larval stage. An experimental study was conducted to test for seasonal (brood) and group size effects on larval fitness. For each of the three broods, egg group sizes of 1, 4, 8, 16 , 24 and 32 were established in the field and monitored for the first 6 days post-hatching.
• 2 There was no effect of group size on larval weight, but there was a significant effect of group size on survival. Individuals from group size 16 had the highest survival, which coincides with the mean natural cluster size of 15.3 eggs.
• 3 There were no significant interactions between brood and group size.
• 4 The effect of larval weight on adult fitness was determined in the laboratory and the field. Larval weight was correlated with egg production in the laboratory but not in the field.

     

Some Factors Influencing the in vitro Infectivity and Replication of Encephalitozoon cuniculi*

SYNOPSIS. Rabbit Encephalitozoon cuniculi were propagated in vitro using rabbit choroid plexus (RCP) cells. The organisms reached maximum titer and numbers by 15 days. The source and in vitro passage level of RCP cells moderately influenced the sensitivity of the cells to infection. Cells less than 1 week old were significantly less sensitive than older cells. A moderate increase in infectivity for RCP cells was demonstrated with increasing organism passage level in vitro. Rabbit E. cuniculi were not affected by penicillin-streptomycin or gentamicin in the culture medium. The organism survived more than 9 days in buffer at 37 C and least 24 days at 4 and 20 C. Storage at -70 C or in liquid nitrogen was successful for at least 6 months. Encephalitozoon cuniculi survived 60 but not 120 min at 56 C. They were killed after 10 min of autoclaving and by 2% (v/v) Lysol, 10% (v/v) formalin and 70% (v/v) ethyl alcohol. The organisms survived at least 24 h at pH 9 or pH 4 and were not affected by sonication, freezing and thawing, or distilled water but lost significant infectivity after 24 h in CsCl or 40% (w/v) sucrose.     

Heterogeneity of Intermediate Filament Expression in Vascular Smooth Muscle: A Gradient in Desmin Positive Cells from the Rat Aortic Arch to the Level of the Arteria Iliaca Communis

The display of the two distinct intermediate filament proteins, desmin and vimentin, in rat vascular smooth muscle tissue was studied by immunofluorescence microscopy on frozen sections of aorta and other blood vessels. Vascular smooth muscle cells present in these vessels always appeared rich in vimentin. However, staining of sections covering six distinct but contiguous parts of the aorta showed that the number of desmin containing cells was low distal to the truncus brachiocephalicus, but increases until in distal parts of the aorta and in the arteria iliaca communis almost all cells appear positive for desmin. Thus blood vessels show heterogeneity of intermediate filament expression not only in cross-section but can also display heterogeneity along their length. Muscular arteries such as the renal artery and the arteria femoralis, as well as arterioles and veins including the vena jugularis and the vena cava also contain desmin. Thus it may be that low numbers of desmin-positive cells are typical of elastic arteries, while muscular arteries and other blood vessels are characterized by large numbers of desmin-positive cells. We discuss whether desmin-positive and desmin-negative vascular smooth muscle cells may perform different functions and raise the possibility that desmin expression may coincide with the turn on of a specially regulated contractility program.