Effects of denervation on the composition and synthesis of sarcolemmal proteins

In vitro synthesis of proteins and changes in polypeptide composition of sarcolemma were studied in innervated and denervated extensor digitorum longus muscle of the rat. A technique of evacuating myoplasm from muscle slices was used as a preliminary step in the preparation of three membrane fractions, M, H and S, containing sarcolemma. On the basis of findings from the previous study and the present investigation, it was concluded that the M fraction was most enriched with extrajunctional sarcolemma.In vitro incorporation of [³H]leucine into membrane proteins of the M fraction showed an apparent linear increase in the rate of protein synthesis from 1–10 days after denervation. The relative increase at 10 days was 137% greater than that of innervated controls. Fractions H and S showed a smaller relative increase.Polypeptide composition of M, H and S fractions based on SDS gel electrophoresis of innervated and denervated muscle, showed qualitative and quantitative changes. The most striking difference was a nominal 29 000 component in M that constituted a disproportionately large peak. Following 10 days of denervation the M fraction underwent significant compositional changes in its electrophoretic profile, the most dramatic of which was a large reduction in the proportion of the 29 000 component. The denervation-induced compositional change is discussed in light of known alterations in the chloride conductance of the muscle plasmalemma.     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

Determination of 2,4-diamino-5-benzylpyrimidines in combination with sulphadiazine in biological fluids using high-performance liquid chromatography

High-performance liquid chromatographic methods for the simultaneous analysis of tetroxoprim (TXP)/sulphadiazine (SDZ) and metioprim (MTP)/SDZ in serum and prostatic secretion (PS) are described. The detection limits in serum and PS were 50 ng TXP per ml and 100 ng SDZ per ml, and 40 ng MTP per ml and 100 ng SDZ per ml, respectively. The intra-assay coefficients of variation were in the range of 2.7–2.19%. Some preliminary data from a pharmacokinetic study in geriatric patients and a distribution study in dogs are presented. These methods enable the investigator to process a large number of TXP/SDZ and MTP/SDZ samples in one working day.     

Regeneration of pleated filters used to concentrate enteroviruses from large volumes of tap water.

Pleated cartridge filters are capable of concentrating enteroviruses from large volumes (well over 2,000 liters) of tap water. These epoxy-fiberglass filters can be regenerated if they are treated with 0.1 N NaOH or autoclaved to inactivate any contaminating virus. The regenerated filters regained their ability to concentrate viruses from water at high flow rates.     

Ehlers Danlos syndrome type VIIB. Incomplete cleavage of abnormal type I procollagen by N-proteinase in vitro results in the formation of copolymers of collagen and partially cleaved pNcollagen that are near circular in cross-section.

We have shown that a child with Ehlers Danlos syndrome (EDS) type VII has a G to A transition at the first nucleotide of intron 6 in one of her COL1A2 alleles. Half of the cDNA clones prepared from the proband's pro alpha 2(I) mRNA lacked exon 6. The type I procollagen secreted by the proband's dermal fibroblasts in culture was purified, and collagen fibrils were generated in vitro by cleavage of the procollagen with the procollagen N- and C-proteinases. Incubation of the procollagen with N-proteinase resulted in a 1:1 mixture of pCcollagen and uncleaved procollagen. Incubation of this mixture with C-proteinase generated collagen and abnormal pNcollagen (pNcollagen-ex6) that readily copolymerized into fibrils. By electron microscopy these fibrils resembled the hieroglyphic fibrils seen in the N-proteinase-deficient skin of dermatosparactic animals and humans and were distinct from the near circular cross-section fibrils seen in the tissues of individuals with EDS type VII. Further incubation of the hieroglyphic fibrils with N-proteinase resulted in partial cleavage of the pNcollagen-ex6 in which the abnormal pN alpha 2(I) chains remained intact. These fibrils were not hieroglyphic but were near circular in cross-section. Fibrils formed from collagen and pNcollagen-ex6 that had been partially cleaved with elevated amounts of N-proteinase prior to fibril formation were also near circular in cross-section. The results are consistent with a model of collagen fibril formation in which the intact N-propeptides are located exclusively at the surface of the hieroglyphic fibrils. Partial cleavage of the pNcollagen-ex6 by N-proteinase allows the N-propeptides to be incorporated within the body of the fibrils. The model provides an explanation for the morphology and molecular composition of collagen fibrils in the tissues of patients with EDS type VII.     

Identification of CpG islands in a physical map encompassing the Friedreich's ataxia locus

The Friedreich's ataxia locus has been previously assigned to chromosome 9q 13-21.1 by the demonstration of tight linkage to two anonymous DNA markers. MCT112 (Z greater than 80, theta = 0) and DR47 (Z greater than 50, theta = 0). The absence of recombination between these three loci has prevented the resolution of gene/probe order in this region, impeding strategies for gene isolation. We report physical mapping over a 4-Mb genomic interval, linking the markers MCT112 and DR47 on a common 460-kb NotI fragment and identifying 11 CpG islands in the 1.7-Mb interval most likely to contain the Friedreich's ataxia locus. Four of these islands were detected only by analysis of three YAC clones spanning a 700-kb interval including the MCT112/DR47 cluster. Without clear evidence of the precise location of the disease locus from recombination events, each of these regions must be considered as specifying a potential "candidate" sequence for the mutated gene.     

Bacterial Streak Disease of Maize (Zea mays L.) in South Africa

Bacterial streak disease of maize is currently causing some concern among breeders in South Africa. The causal organism of this previously undescribed disease was successfully isolated and its pathogenicity established using KoCH's postulates. Standard physiological and biochemical tests used to identify phytopathogenic bacteria indicated that the bacterium is a Xanthomonas campestris pathovar. Comparisons between this organism and other recognized X. campestris pathovars of the Poaceae indicated that apart from some minor differences the maize streak pathogen is physiologically similar to X. campestris pv. holcicola. However, in repeated reciprocal inoculation experiments all attempts to induce disease symptoms in sorghum with the maize streak pathogen were unsuccessful. Conversely, X. campestris pv. holcicola did produce symptoms in maize leaves. In all the maize cultivars tested the symptoms produced by the maize streak pathogen were, however, always considerably more severe than those caused by X. campestris pv. holcicola. Notwithstanding its physiological similarity to X. campestris pv. holicola it would appear that on the grounds of host specificity the maize streak pathogen warrants new pathovar status. The name X. campestris pv. zeae is proposed.     

Ornithine decarboxylase induction and the cytoskeleton in normal and transformed cells

ODC induction by fresh medium added to stationary, medium-depleted, confluent cultures has been studied in transformed HeLa and CHO cells, and in normal human fibroblasts as an indicator of the resumption of cell multiplication. The transformed HeLa cell displays a more easily reversed G₁ block, a higher peak ODC level, and a shorter time period for achievement of the peak ODC value than does the normal fibroblast. Low concentrations of microtubule depolymerizing agents like colchicine suppress ODC induction almost completely in the normal fibroblast, but hardly at all in the HeLa or CHO cells. Both transformed cells occasionally reveal a superinduction of ODC at very low colchicine levels (10^?8-10^?7 M) and a more variable response to such agents than does the normal fibroblast. Higher concentrations of colchicine suppress ODC induction in all cells. Experiments with actinomycin D and cycloheximide indicate that the principal colchicine action involves inhibition at the level of protein or mRNA synthesis, rather than inactivation of the already synthesized enzyme. These experiments are provisionally interpreted as an indication that a microtubular system is needed to reinitiate certain steps associated with growth in G₁-blocked, normal cells, and that a second microtubular action terminating enzyme biosynthesis may exist. This microtubular control is defective in the transformed cells here studied. Specific microtubular actions necessary for initiation and termination of protein syntheses may occur throughout the cell reproductive cycle, and in the course of normal differentiation processes.     

A simple enzyme assay for dry matter digestibility and its value in studying food selection by generalist herbivores

Summary The dry matter digestibility of 94 species of leaf was assayed by a simple method involving sequential treatment with pepsin and fungal cellulase enzymes. It was demonstrated that for foliage from rainforest trees of a wide range of dicotyledonous plant families the assay showed high positive correlation with estimates of dry matter digestibility obtained using rumenliquor from a fistulated steer. Both assays were found to reflect negative correlates of digestibility, notably fibre and condensed tannin, rather than the nutritional value of an item. The higher dry matter digestibility of immature leaves relative to mature leaves appeared to be accounted for by their lower fibre content. It is suggested that the pepsin/cellulase assay offers a cheap, quick, routine method of gaining information on the effects of some types of plant secondary compounds (digestibility reducers) on the food potential of different kinds of foliage to herbivores. Its use in studies of herbivory in rainforest areas in relation to analyses for plant secondary compounds and food selection by herbivores is discussed.Publication 20-018 of the Wisconsin Regional Primate Research Center     

Influence of Estrogen and Progesterone on Glutamic Acid Decarboxylase Activity in Discrete Regions of Rat Brain

Abstract: Female Charles River rats were ovariectomized and treated for three days with 17/8-estradiol benzoate (E) (1.0 /μg/day), progesterone (P) (500 μg/day), vehicle, or a combined treatment (2 days E, one day P). Animals were killed on day 3 and the brains were dissected by the micropunch technique. Glutamic acid decarboxylase (GAD) activity was measured by collection of ¹⁴CO². Estradiol benzoate and progesterone were potent inhibitors of GAD activity in regions such as the arcuate nucleus, ventromedial hypothalamus and corticomedial amygdala. Estrogen reduced the V_max of GAD for glutamate as a substrate without changing the K_m. Estrogen also failed to change the K_m for pyridoxal phosphate. Combined treatment with estrogen and progesterone did not reduce GAD from ovariectomized levels except in the septum, indicating an interaction of the two hormones at the level of GAD. The suggestion is made that under conditions that inhibit LH secretion GAD activity is low, but when LH secretion is stimulated GAD activity may be comparatively high.