Axon growth from limb motorneurons in the locust embryo: the effect of target limb removal on the pattern of axon branching in the periphery

Metathoracic limb buds have been unilaterally ablated from locust embryos at 25 to 30% of embryonic development and the effect of this operation on the axon morphology of the motorneuron fast extensor tibiae (FETi) observed at later embryonic stages. In control embryos this neuron sends a single axon out the main leg nerve, nerve 5, to the extensor tibiae muscle in the femur. In limb ablated embryos the axon of FETi is found in a wide variety of aberrant peripheral nerve pathways and projects to a wide range of foreign muscles. There is a degree of apparent selectivity, but no rigid hierarchy, in the choice of pathway and muscle made by FETi. A high degree of variability is found between one embryo and another in the extent and pattern of axon branching. The axon of FETi is generally found in pathways that correspond to nerves in control embryos but on occasion grows along novel routes. An anteriorly directed dendritic branch, seldom seen in control FETi neurons, is frequently seen in experimental FETis. These findings are discussed in terms of the rules for specific axon growth in normal development.     

Cryopreservation of Plasmodium chabaudi. II. Cooling and warming rates

Various cooling and warming rates were investigated to determine the optimum conditions for cryopreserving the intraerythrocytic stages of Plasmodium chabaudi. Infected blood, equilibrated in 10% v/v glycerol at 37 degrees C or in 15% v/v Me2SO at 0 degree C for 10 min, was cryopreserved using cooling rates between 1 and 5100 degrees C min-1. After overnight storage in liquid nitrogen the samples were warmed at 12,000 degrees C min-1. Warming rates between 1 and 12,000 degrees C min-1 were investigated using samples previously cooled at 3600 degrees C min-1. After thawing, the glycerol and Me2SO were removed by dilution in 15% v/v glucose-supplemented phosphate-buffered saline. Survival was assayed by inoculation of groups of five mice each with 10(6) infected cells and the time taken to reach a level of 2% parasitemia estimated. The optimum cooling rate was 3600 degrees C min-1 for parasites frozen using either 10% glycerol or 15% Me2SO; the pre-2% patent periods were 0.90 and 1.01 days above control values (representing survival levels of 21 and 17.5%, respectively). The optimum warming rate was 12,000 degrees C min-1; the pre-2% patent periods were 1.01 and 1.32 days above control values, respectively (18 and 10% survival), for glycerol and Me2SO. With ethanediol (5% v/v) and sucrose (15% w/v) as cryoprotectants the optimum warming rates were also 12,000 degrees C min-1 while the optimum cooling rates were 330 and 3600 degrees C min-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversible inhibition of aggregation-related cohesivity in Dictyostelium discoideum by diffusible metabolites

Evidence presented elsewhere (G.B. Williams, E.M. Elder, and M. Sussman 1984, Dev. Biol. 105, 377-388) indicates that NH3 and certain carboxylic acids including propionate, succinate, and acetate modulate the cAMP relay in Dictyostelium discoideum. The former appears to act as a cAMP accumulation inhibitor, the latter as cAMP release inhibitors. The cohesive properties of aggregation competent cells have been assayed quantitatively in the presence of these modulators. The following results were obtained: (1) At pH 7.5, EDTA-resistant cohesivity was greatly inhibited by NH4C within the concentration range tested (30-3.8 mM). Even at the higher concentrations the effect was not immediate but required ca. 10 min for full expression. At the lower concentrations, the inhibitory level was only slightly reduced but the time for full expression progressively increased. At pH 6.5, the level of inhibition was marginal, indicating that NH3 is the active molecular species. By themselves, neither ambient pH nor ionic strength appeared to affect cohesive performance within the ranges employed. The inhibition was immediately and completely reversed upon removal of NH4Cl or a shift of ambient pH from 7.5 to 6.5. The presence of cycloheximide did not affect the recovery of cohesivity after NH4Cl removal. (2) The presence of 15 mM succinate, propionate, or acetate also reduced cell cohesivity. The timing and extent of the inhibition were identical at pH 7.5 and 6.5. The inhibition was expressed immediately and was reversible. Each of the acids acted synergistically with NH4Cl. The relative potencies of these metabolites acting singly or in combination as inhibitors of cohesivity corresponded roughly to their potencies as modulators of the cAMP relay (Williams et al., 1984). (3) The sensitivity to the metabolites was stage specific, being maximal during and shortly after aggregation and disappearing abruptly at 11-12 hr. This corresponds to the time at which this cohesive system, responsible for the end-to-end cell associations evident during aggregation (H. Beug, G. Gerisch, S. Kempff, V. Riedel, and G. Cremer, 1970, Exp. Cell. Res. 63, 147-158) is supplanted by a newly arisen, serologically and genetically distinct system which thereafter maintains the integrity of the aggregate (C. Steinemann and R.W. Parish, 1980, Nature (London) 286, 721-724; D.K. Wilcox and M. Sussman, 1981, Dev. Biol. 82, 102-112, and Proc. Natl. Acad. Sci. USA 78, 358-362; C.L. Saxe III and M. Sussman, 1982, Cell 29, 755-759). The activities of the metabolites, detailed above, are discussed in relation to their previously demonstrated activities as morphogens.     

Optimizing conditions for cryopreservation of an insect cell line

A cell line (UM-BGE-2) derived from embryos of the cockroach Blattella germanica was frozen to ?196 °C under a variety of conditions and cell viability was assayed after warming. It was found that cell viability was affected by the cooling rate, the warming rate, the controlled cooling endpoint temperature, and the type and concentration of cryoprotectant. The best survival for cells suspended in Grace's tissue culture medium containing 1 M Me₂SO was obtained when cells were cooled at 1 °C/ min to at least ?90 °C before being placed in liquid nitrogen and warmed at more than 900 °C/min. Cultures initiated from these frozen cells produce typical growth curves and appear normal after several passages.     

Heterogeneity of heparan sulfate proteoglycans synthesized by PYS-2 cells

Antibodies to the basement membrane proteoglycan produced by the EHS tumor were used to immunoprecipitate [35S]sulfate-labeled protoglycans produced by PYS-2 cells. The immunoprecipitated proteoglycans were subsequently fractionated by CsCl density gradient centrifugation and Sepharose CL-4B chromatography. The culture medium contained a low-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.18, containing heparan sulfate side chains of Mr = 35-40,000. The medium also contained a high-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.23, containing heparan sulfate side chains of Mr = 30,000. The corresponding proteoglycans of the cell layer were all smaller than those in the medium. Since the antibodies used to precipitate those proteoglycans were directed against the protein core, this suggests that these proteoglycans share common antigenic features, and may be derived from a common precursor which undergoes modification by the removal of protein segments and a portion of each heparan sulfate chain.     

Biochemical and morphological characterization of a plasma membrane-enriched fraction from bovine parathyroid cells

A fraction of enriched plasma membranes from bovine parathyroid cells has been prepared by differential centrifugation. Biochemical characterization shows that this fraction has a specific activity enrichment of 7.2-fold in ouabain-sensitive Na+-K+ ATPase, and 3.5-fold in 5'-nucleotidase. Less than 4% of the total mitochondria and lysosomes are present within the plasma membranes, while microsomal contamination accounts for 14% of total specific activity. Parathyroid hormone radioimmunoassay also reveals the presence of some secretory granules within the plasma membrane fraction. The characteristic morphological aspect of the unusual surface membrane is shown by freeze-fracture electron microscopy. In the enriched pellets, vesicles identified as having a plasma membrane origin have variable sizes, and 50% show an inside-out conformation. Even though the plasma membrane fraction described herein is not absolutely free from contamination by other subcellular components, this protocol represents the first attempt to purify surface membrane from parathyroid tissue and provide the starting material for understanding, at a molecular level, the properties of extracellular Ca2+ regulation and its coupling with secretion of parathyroid hormone.     

N-demethylation of p-chloro-N-methylaniline catalyzed by subcellular fractions from the avocado pear (Persea americana)

Subcellular fractions from the avocado pear ( Persea americana) catalyzed formation of p-chloroaniline from p-chloro-N-methylaniline. Fractions prepared by centrifugation of avocado homogenates at 20, 000g for 20 min formed p-chloroaniline (2900 +/- 500 pmol min-1 mg protein-1) with an NADPH-generating system. p-Chloroaniline formation required reduced pyridine nucleotide (NADPH was 6-7 times more effective than NADH) and O2. N-Demethylation was inhibited by CO (55% inhibition at CO:O2 = 1) and was not inhibited by CN. Cytochrome P-450 was detected in the 20, 000g pellet at levels of 300-380 pmol/mg protein. This particulate preparation was also active in catalyzing the NADPH-dependent epoxidation of the chlorinated cyclodiene aldrin. Improvements to a colorimetric procedure for measuring p-chloroaniline increased the sensitivity of the procedure fourfold, and allowed use of samples containing high amounts of lipid. Avocado pear is suitable tissue for further studies on the oxidation of foreign compounds by higher plants.     

Turnover of proteoglycans in cultures of bovine articular cartilage

Proteoglycans in cultures of adult bovine articular cartilage labeled with [35S]sulfate after 5 days in culture and maintained in medium containing 20% fetal calf X serum had longer half-lives (average 11 days) compared with those of the same tissue maintained in medium alone (average 6 days). The half-lives of proteoglycans in cultures of calf cartilage labeled after 5 days in culture and maintained in medium with serum were considerably longer (average 21 days) compared to adult cartilage. If 0.5 mM cycloheximide was added to the medium of cultures of adult cartilage, or the tissue was maintained at 4 degrees C after labeling, the half-lives of the proteoglycans were greater, 24 and greater than 300 days, respectively. Analyses of the radiolabeled proteoglycans remaining in the matrix of the tissue immediately after labeling the tissue and at various times in culture revealed two main populations of proteoglycans; a large species eluting with Kav of 0.21-0.24 on Sepharose CL-2B, of high bouyant density and able to form aggregates with hyaluronate, and a small species eluting with a Kav of 0.63-0.70 on Sepharose CL-2B, of low buoyant density, containing only chondroitin sulfate chains, and unable to form aggregates with hyaluronate. The larger proteoglycan had shorter half-lives than the smaller proteoglycan; in cartilage maintained with serum, the half-lives were 9.8 and 14.5 days, respectively. Labeling cartilage with both [3H]leucine and [35S]sulfate showed the small proteoglycan to be a separate synthetic product. The size distribution of 35S-labeled proteoglycans lost into the medium was shown to be polydisperse on Sepharose CL-2B, the majority eluting with a Kav of 0.27 to 0.35, of high buoyant density, and unable to aggregate with hyaluronate. The size distribution of glycosaminoglycans from 35S-labeled proteoglycans appearing in the medium did not differ from that associated with labeled proteoglycans remaining in the matrix.     

Modification of rat liver iodothyronine 5'-deiodinase activity with diethylpyrocarbonate and rose bengal; evidence for an active site histidine residue

Iodothyronine 5'-deiodinase activity of rat liver microsomes was rapidly and completely lost by treatment with diethylpyrocarbonate (DEP) and by photo-oxidation with Rose Bengal (RB). In both cases inactivation followed pseudo first order reaction kinetics. Inactivation by DEP was diminished in the presence of substrate or competitive inhibitors, and was reversed by hydroxylamine treatment. In addition to photo-oxidation, deiodinase activity was also inhibited by RB in the dark. This inhibition was reversible and competitive with substrate (Ki 60 nM). These results suggest the location of an essential histidine residue at or near the active site of rat liver iodothyronine deiodinase.     

Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities

The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated. The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease. The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined. The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted. Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates. Magnesium was found to reinforce the binding of the enzyme to these affinity supports. DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns. These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases.