In order to assess the extent of regeneration, creatine kinase contents of injected gastrocnemius muscle were determined at four time intervals after inoculation. Simultaneously, the diameters of regenerating fibers were measured, the wet weight of the gastrocnemius was recorded and electrophoretic patterns of muscle proteins and histology of the tissue were analyzed. Injection of crude B. asper venom caused myonecrosis and hemorrhage; in these cases regeneration was very poor, as there was only a small increase in creatine kinase content by four weeks. Histologically, large areas of fibrosis were observed, intermixed with small areas of regenerating muscle. Furthermore, the diameter of regenerating muscle cells was abnormally small, even four weeks after envenomation. In order to test the role of hemorrhage in impairment of regeneration, we also studied the effects of B. asper venom that had been incubated with a volume of antivenom that completely neutralized hemorrhagic activity, but not myotoxic activity. At 24 hr mice showed a generalized picture of myonecrosis, without hemorrhage. Afterwards, regeneration proceeded and significant increases in creatine kinase content were demonstrated at 1, 2 and 4 weeks. Also, histological observations showed the presence of many areas of regenerating muscle, intermixed with small areas of fibrosis and adipose tissue. The diameter of regenerating cells increased with time, reaching values of 25.8 μm by four weeks. Electrophoretic analyses of muscle proteins showed that muscle injected with partially neutralized venom underwent a more complete regeneration than muscle injected with crude venom. Even by 28 days, however, regeneration was not complete in gastrocnemius inoculated with partially neutralized venom, suggesting that factors other than hemorrhagic toxins may be interfering with muscle regeneration.
This review covers previous data, together with new information from our laboratories, on the subject of the anthocerote chioroplast. Unlike all other archegoniates, most species of anthocerote have pyrenoids in their chloroplasts. The pyrenoid is the site of accumulation of the first enzyme in the C3 photosynthetic cycle, ribulose bisphosphate carboxylase/oxygenase. Unlike most algae, the hornwort pyrenoid is composed of distinct subunits, numbering up to several hundred. Pyrenoid morphology is quite variable among the genera in shape, fine structure, and distribution of inclusions. Another unique feature of the anthocerote chloroplast is the presence of thylakoids that connect adjacent granal stacks at right angles to the long axis of the granum (so-called channel thylakoids), resulting in a 'spongy' arrangement of the thylakoid system. The granal stacks of anthocerotes are like the 'pseudograna' of green algae because they lack the highly-curved end membranes typical of all other embryophytes. The channel thylakoids are enriched in photosystem (PS) I and the grana are enriched in PS II. The chloroplast envelope is a double membrane structure with regions of appression, much like that of other green plants. The apical cell of the gametophyte contains chloroplasts similar to the mature chloroplasts of the thallus, although certain gametophytic tissues may contain underdeveloped plastids. Chloroplasts in cells around Nostoc colonies and in cells invaded by mycorrhizal fungi have thylakoids mainly in pairs, and small or absent pyrenoids. A number of similarly reduced plastids are noted in the placental cells at the sporophybe/gametophyte junction and in developing spores. The greatest reduction is observed in spermatid cell plastids, which at maturity consist of only a small starch grain surrounded by the envelope. Chromoplast-like organelles are found in the cells of the antheridial jacket in some genera; these contain numerous osmiophilic globules that are probably pigment aggregations. Colourless bead-like plastids occur in the rhizoids; these seem to develop by fragmentation of the single chioroplast in the rhizoid initials, concomitant with the loss of chlorophyll. Chloroplast division is a tightly controlled process and, in uniplastidic species, always occurs just before nuclear division, with the participation of a unique system of chioroplast-associated microtubules. The number of chloroplasts per cell is quite variable in some genera, although most species have but a single chloroplast in each cell of the gametophyte. Chioroplast shape is also variable from ellipsoidal, dumbbell-shaped, to irregular. These data indicate that the anthocerote chloroplast is unique among the embrvophvtes and are in line with the notion of an isolated position in the plant kingdom. Certain features of chioroplast morphology appear to be typical of certain genera and might prove useful in taxonomic decisions at the generic level.
