Fusimotor innervation in the cat

The motor innervation of cat spindles was examined in hindlimb muscles using a variety of techniques employed in light and electron microscopy. Observations were made on teased, silver preparations of 267 spindles sampled from the peroneal, flexor hallucis longus, and soleus muscles, hereafter referred to as the PER/FHL/SOL series. The γ innervation. Trail endings are almost invariably present, and innervate both bag and chain muscle fibres. Trail fibres accounted for 64.6 to 74.8% of the total fusimotor supply to samples of spindle poles in the PER/FHL/SOL series, the mean number of fibres per pole varying from 2.7 to 5.0 in the different muscles, and the mean number of ramifications (areas of synaptic contact) per fibre being 3.7. By contrast, the p?innervation of a spindle pole generally consists of a single fibre supplying only one plate. In the above samples p(2) fibres accounted for 4.1 to 28.0% of the total fusimotor supply, and the mean number of fibres per pole varied from 0.3 to 1.2 in the different muscles. Ninety per cent of p(2) plates innervate bag fibres. The α innervation. The structure of p?plates as seen in both light and electron microscopy compares very closely with that of extrafusal plates. After nerve section p?plates degenerate at the same time as extrafusal plates, being the first of the three types of fusimotor ending to disappear. The frequency of the p?innervation is similar to that of the p?innervation. In the same samples of PER/FHL/SOL spindle poles as above p? fibres accounted for 6.0 to 28.8% of the total fusimotor supply, the mean number of fibres per pole varying from 0.25 to 2.1 in the different muscles. The majority of p? fibres enter a pole to terminate in one plate only. Seventy-five per cent of the plates innervate bag fibres. The three types of fusimotor ending are thus not selectively distributed to the two types of intrafusal muscle fibre. All three types of fusimotor fibre may branch within the spindle so as to innervate both bag and chain fibres. Bag fibres receive both types of plate ending as well as trail endings. Most chain fibres receive trail endings only; the rest receive either a p?or a p?plate innervation in addition, 25% of the p?and 10% of the p?innervation being distributed to chain fibres. The significance of this nonselective innervation is interpreted as indicating that the type of contraction elicited by stimulating a fusimotor fibre depends upon the type of ending initiating it rather than upon the type of muscle fibre executing it. Reasons are given for concluding that the dynamic response is controlled via the p?and p?plates, and that the static response is controlled by the trail endings. The participation of the α fibres in mammalian fusimotor innervation, previously regarded as a vestigial feature, proved to be widespread in the muscles studied and more prevalent in fast muscles (FHL, peroneus digiti quinti) than slow (soleus). A low frequency of p?innervation is offset by a high frequency of p?(as in peroneus longus), and vice versa (as in FHL). It is unlikely that collaterals from slow α fibres innervating type B muscle fibres are wholly responsible for the high frequency of the p?innervation in FHL, and it is suggested that collaterals may also be derived from fast α fibres innervating type C muscle fibres. The possibility of there being some motor fibres of α conduction velocity and with an exclusively fusimotor distribution is also taken into account.     

Specificity and Sensitivity of Insulin Staining by Aldehyde Fuchsin, Pseudoisocyanin and Toluidine Blue

Aldehyde fuchsin, pseudoisocyanin and toluidine blue, histochemical dyes reported to be specific for insulin-containing granules of the pancreatic beta cell, were applied to insulin fixed in polyacrylamide gel by disc electrophoresis. Two major and four minor bands were resolved as demonstrated by staining with amidoschwarz; only the two major bands, were stained by aldehyde fuchsin. The addition of serum did not affect this reaction. Serum or insulin components gave no metachromatic reactions to the other stains. Under the conditions applied, aldehyde fuchsin is the only one of these dyes specific for insulin in this, system, but this stain is not sufficiently sensitive to detect normal serum levels of the hormone.     

Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells

Rat calvaria osteoblasts derived from 21-day-old fetal rat pups undergo a temporal expression of markers of the osteoblast phenotype during a 5 week culture period. Alkaline phosphatase and osteocalcin are sequentially expressed in relation to collagen accumulation and mineralization. This pattern of expression of these osteoblast parameters in cultured rat osteoblasts (ROB) is analogous to that seen in vivo in developing fetal rat calvaria tissue (Yoon et. al: Biochem. Biophis. Res. Commun. 148:1129, 1987) and is similar to that observed in cultures of subcultivated 16-day-old embryonic chick calvaria-derived osteoblasts (COB) (Gerstenfeld, et.al: Dev. Biol. 122:46, 1987). While the cellular organization of subcultivated COB and primary ROB cultures are somewhat different, the temporal expression of the parameters remains. Both the rat and chick culture systems support formation of matrix mineralization even in the absence of beta-glycerol-phosphate. A systematic examination of factors which constitute conditions supporting complete expression of the osteoblast phenotype in ROB cultures indicate requirements for specific serum lots, ascorbic acid and the ordered deposition of mineral in the extracellular matrix. The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype. In ROB cultures, expression of osteocalcin synthesis occurs subsequent to initiation of alkaline phosphatase activity and accompanies the formation of mineralized nodules. Thus, extracellular matrix mineralization (deposition of hydroxyapatite) is required for complete development of the osteoblast phenotype, as reflected by a 200-fold increase in osteocalcin synthesis. These data show the temporal expression of the various osteoblast parameters during the formation and mineralization of an extracellular matrix can provide markers reflective of various stages of osteoblast differentiation/maturation in vitro.     

Characterization of envelope proteins from Pasteurella haemolytica and Pasteurella multocida

A method was devised for the reproducible isolation of envelopes from Pasteurella haemolytica serotype A2. It was also possible to prepare envelopes from other serotypes of P. haemolytica and Pasteurella multocida using this methodology. Examination of these preparations by SDS-PAGE showed major differences between strains of P. haemolytica and strains of P. multocida which allowed the clear distinction of isolates of these species. Amongst the P. haemolytica serotypes it was possible to distinguish envelope preparations made from A biotype and T biotype organisms easily, but it was not possible to identify individual serotypes from each other. Envelope profiles were sufficiently different between the individual P. multocida serotypes examined to allow each to be identified by its polypeptide profile. Experiments using radiolabelling, antibody absorption, and susceptibility to protease digestion, together with heat modifiability and detergent solubility characteristics indicated that 13 of the envelope proteins were probably surface-located. A high molecular mass immunogenic envelope protein was shown, by immunoblotting, to be present in all strains of P. haemolytica and P. multocida examined.     

Binding of apoE-rich high density lipoprotein particles by saturable sites on human blood platelets inhibits agonist-induced platelet aggregation

High density lipoproteins (HDL, d 1.063-1.21 g/ml) are reported to stimulate, to have no effect on, or to inhibit agonist-induced platelet aggregation. We have hypothesized that these conflicting reports might be explained by opposing effects of individual HDL subclasses on platelet aggregability. Physiologic concentrations of HDL3 had little effect on ADP-induced aggregation of washed platelet suspensions, although higher levels were stimulatory. Normal concentrations of HDL2 (0.2-0.4 mg of protein/ml) inhibited aggregation; further fractionation by heparin-Sepharose chromatography identified the particles rich in apolipoprotein E, termed HDL-E, as the major anti-aggregatory subclass. Washed platelets bound radioiodinated HDL-E to a uniform class of saturable sites; they numbered 4,200 per platelet and the KD was 7.9 x 10(-7) M. Binding of HDL-E by platelets, and its anti-aggregatory action, showed a similar rapidity and both occurred within the physiologic concentration range. Moreover, the two processes were independent of the presence of divalent ions and were impaired by chemical modification of the apolipoprotein constituents of HDL-E. We conclude that occupation of cell-surface receptors by HDL-E particles impairs platelet responsiveness to exogenous agonists and that platelet aggregability in the presence of whole HDL may reflect the relative concentrations of the individual subclasses in the particular sample.