We have presented direct evidence that at least one of the traits associated with killing of paramecia by kappa particles is determined by an extrachromosomal genetic element. Plasmid DNA was isolated from Caedibacter taeniospiralis 47 (commonly known as 47 kappa), which is an obligate cytoplasmic endosymbiont of Paramecium tetraurelia. Fragments of pKAP47 DNA generated by Pst I digestion were inserted into pBR328 and then introduced into Escherichia coli 294 by transformation. Clones carrying recombinant plasmids were screened for toxicity toward sensitive strains of paramecia or for the ability to produce R bodies. None of the clones appeared to be toxic. However, three clones were found to have the ability to produce R bodies, which are proteinaceous ribbons (10-20 ,um long, 0.5 jam wide, and 13 nm thick) rolled up inside the cell to form a hollow cylinder about 0.5 ,um in diameter and 0.5 jzm long. Each of these clones carry plasmids that contain the Pst I B fragment from pKAP47. Subclones of one of the recombinant plasmids, pBQ51, were constructed to determine the approximate location of DNA sequences necessary for R-body synthesis. The left-hand boundary of the required sequences was found to occur within a 600-base-pair region, and the location ofthe right-hand boundary was determined to occur within a 700-base-pair region. The minimum and maximum sizes ofsequences required for R-body synthesis are between 1,300 and 2,600 base pairs.Killer traits in paramecium are genetically determined by a variety of bacterial endosymbionts that live in the cytoplasm of killer paramecia (1, 2). The most notable group ofthese bacteria are kappa particles, which comprise the genus Caedibacter. Killer paramecia release these endosymbionts, some of which carry toxin, at a slow rate into the environment. These endosymbiont-bearing paramecia are killers because uninfected paramecia that are sensitive to the toxin may suffer lethal consequences when toxic forms of endosymbionts are ingested.All members of the genus Caedibacter, in addition to being cytoplasmic endosymbionts that can confer a killer trait on their host paramecia, have two distinct morphological forms (3). The predominant form (about 95% of most populations) does not exhibit any unusual morphological features. The second form, which accounts for about 5% of the individuals in most populations, contains a large inclusion body (Fig. 1A) known as an R body. Inside the cell, R bodies appear as hollow cylinders approximately 0.5 ,um long and 0.5 Am in diameter. An R body (Fig. 1B) is actually a long (8-20 ,um) proteinaceous ribbon, about 0.5 ,um wide and 13 nm thick, that is rolled up inside the bacterial cell to form a hollow cylinder (4). Several investigators have shown that it is the R-body-containing forms of the endosymbionts that are toxic and that they are the progeny ofcells that do not contain R bodies (5-8).Indirect evidence indicates that the genetic determinants for R-body and toxin synthesis are extrachromosomal (2, 9, 10). In three of the fo...

This study was designed to determine the source of tumor necrosis factor (TNF) alpha within the porcine corpus luteum (CL). 1) Sections of frozen or paraffin-embedded CL from various stages of the estrous cycle were incubated with the following primary antibodies: anti-human recombinant TNFalpha, anti-porcine macrophage-specific antigen, or anti-alpha-actin (marker of pericyte and smooth muscle cells). Dolichos biflorus lectin-peroxidase was used as an endothelial cell label. Positive immunostaining for TNFalpha was apparent in porcine CL throughout the estrous cycle. TNFalpha immunoreactivity was primarily localized in cells along septal/vascular tracts, and exhibited spatial and temporal distribution similar to that of cells labeled with anti-macrophage antibodies. Large luteal cells exhibited weak staining for TNFalpha in paraffin sections, whereas microvascular endothelial cells were consistently negative in both frozen and paraffin sections. 2) Enriched subpopulations of macrophages, endothelial cells, and large and small luteal cells were isolated by density gradient and immunomagnetic bead separation techniques. TNFalpha secretion by each subpopulation was determined by measuring bioactive TNFalpha in incubation media using a specific in vitro bioassay. Macrophage subpopulations secreted up to 100-fold greater quantities of bioactive TNFalpha (up to 400 pg/10(6) cells) than did other subpopulations. In contrast, endothelial cell and small luteal cell subpopulations released very small amounts (< 8 pg/10(6) cells) of bioactive TNFalpha. Large luteal cells secreted slightly greater amounts of TNFalpha (10-15 pg/10(6) cells). Local macrophages appear to be the primary source of TNFalpha in the porcine CL.

Differences in gross and microscopic morphology, fiber-size distribution, and fiber-type composition were present in the diaphragm of 35-, 130-, and 180-day-old dystrophic (Bio 14.6) compared with age-matched control (Bio F1B) hamsters. The dystrophic diaphragm was significantly thicker than the control at 130 and 180 days. Increases in wet-to-dry weight ratios, connective tissue per unit area, and muscle fiber number suggest that increased tissue hydration, fibrosis, and fiber hyperplasia contribute to diaphragm hypertrophy. Marked variations of fiber areas and diameters were evident in each fiber type at each age, but generalized atrophy predominated over hypertrophy, resulting in significant decreases in cross-sectional areas of each fiber type. Significant differences in fiber-type composition were noted in the dystrophic vs. control diaphragm at each age: at 35 days the percentage of slow-oxidative fibers was lower and in fast-oxidative fibers was higher; at 130 days the percentage of fast-oxidative fibers remained elevated; at 180 days the percentage of fast-glycolytic fibers was reduced. In vitro contractility studies of 130-day-old animals showed that twitch and peak tension development were significantly lower in the dystrophic compared with the control diaphragm, whereas optimal length, contraction time, and half-relaxation time were within control limits. Microscopic and physiological abnormalities were also present in the soleus of 130-day-old dystrophic animals. As in the diaphragm, fiber areas were reduced, connective tissue area increased, and peak and twitch tension decreased significantly compared with the control soleus. The histopathological and pathophysiological changes in the diaphragm correlated well with each other and are consistent with the slowly evolving inability of the dystrophic hamster to increase tidal volume and minute ventilation in response to a hypercapnic challenge.

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Male dystrophic hamsters (DH) were treated with pellets containing thyroxine (T hamsters) or placebo (P hamsters) for 8 wk. O2 consumption, ventilation, and ventilation in response to 8% CO2 in O2 and 10% O2 in N2 were evaluated 1 and 8 wk after treatment began. O2 consumption was elevated in T hamsters at 1 and 8 wk, whereas ventilation was similar in the two groups on the first week. By 8 wk, ventilation and ventilatory responses to hypoxic and hypercapnic challenges were 100% greater in T than in P hamsters (P < 0.05). Morphometric evaluations at the end of the treatment period indicated that air space surface density, tissue volume density, and surface density-to-air space volume ratio of the lung parenchyma were greater (P < 0.05) in T than in P hamsters. In contrast, chord length within the lung parenchyma was shorter and necrosis in the diaphragm and tongue, but not in the heart, was lower (P < 0.05) in T than in P hamsters. Taken together, these results suggest that T treatment of DH for 8 wk affects O2 consumption, ventilation, lung architecture, and skeletal muscle without increasing triiodothyronine levels.