Plasmid stability in immobilized and free recombinant Escherichia coli JM105(pKK223-200): importance of oxygen diffusion, growth rate, and plasmid copy number

Stability of the plasmid pKK223-200 in Escherichia coli JM105 was studied for both free and immobilized cells during continuous culture. The relationship between plasmid copy number, xylanase activity, which was coded for by the plasmid, and growth rate and culture conditions involved complex interactions which determined the plasmid stability. Generally, the plasmid stability was enhanced in cultured immobilized cells compared with free-cell cultures. This stability was associated with modified plasmid copy number, depending on the media used. Hypotheses are presented concerning the different plasmid instability kinetics observed in free-cell cultures which involve the antagonistic effects of plasmid copy number and plasmid presence on the plasmid-bearing/plasmid-free cell growth rate ratio. Both diffusional limitation in carrageenan gel beads, which is described in Theoretical Analysis of Immobilized-Cell Growth, and compartmentalized growth of immobilized cells are proposed to explain plasmid stability in immobilized cells.     

Further evidence from sectioned material in support of the existence of a linear terminal complex in cellulose synthesis

Summary Transmembrane linear terminal complexes considered to be involved in the synthesis of cellulose microfibrils have been described in the plasma membrane ofBoergesenia forbesii. Evidence for the existence of these structures has been obtained almost exlusively using the freeze etching technique. In the present study an attempt has been made to complete these studies using conventional fixation, staining, and sectioning procedures. In developing cells ofBoergesenia forbesii, strongly stained structures traversing the plasma membrane and averaging 598.9 nm ± 171.3 nm in length, 28.7 nm ± 4.2 nm in width, and 35.2 nm ± 6.6 nm in depth have been demonstrated. These structures are considered to be linear terminal complexes. At their distal (cell wall) surface, they appear to be closely associated with cellulose microfibrils. At the proximal (cytoplasmic) surface, they are associated with microtubules and polysomes. A model of the possible interrelation of the terminal complexes and microtubules leading to the generation of cell wall microfibrils is proposed.     

Effects of tin and lead on organ levels of essential minerals in rabbits

The effect of tin and lead on levels of essential metals (Zn, Cu, Ca, Fe) in rabbit tissues was compared in relation to the route of administration. Animals received intraperitoneally, or per os, SnCl2 (2 mg Sn/kg) or Pb(CH3COO)2 (3.5 mg Pb/kg) every day for 5 d or for 1 mo. Copper, zinc, iron, and calcium were determined by AAS in the liver, kidneys, spleen, brain, bone marrow, and blood; lead and tin concentration were measured in the blood of animals. Tin and lead administered per os caused either no changes or the decreased concentration of endogenous metals in several tissues. The other route of administration (ip) of both metals generally contributed to the increased storage of essential elements. Blood tin levels of tin treated animals were only about less than or equal to 1/10 of blood lead concentrations of rabbits exposed to lead.     

Identification of dexamethasone-binding sites on male-rat liver plasma membranes by affinity labelling.

Binding studies with [3H]dexamethasone identified two binding sites on plasma membranes prepared from the male rat liver, a low-capacity site with a KD of 7.0 nM and a higher-capacity site with a KD of 90.1 nM. Both sites exhibited glucocorticoid responsiveness and specificity for glucocorticoids and progestins. Triamcinolone acetonide, which competes well for the binding of dexamethasone to the cytosolic glucocorticoid receptor, did not compete well for the binding of [3H]dexamethasone to the plasma-membrane binding sites. The binding sites were sensitive to protease and neuraminidase treatment, and resistant to extraction with NaCl, but were extracted with the detergent Triton X-100. As these experiments indicated the presence of plasma-membrane protein components which bind glucocorticoids at physiological concentrations, affinity-labelling experiments with dexamethasone mesylate were conducted. Two peptides were specifically labelled, one at approx. Mr 66,000 and one at Mr 45,000. The Mr-66,000 peptide was not sensitive to glucocorticoids, and was extracted by NaCl, and so did not correspond to either of the sites identified in the dexamethasone-binding studies. The Mr-45,000 entity, on the other hand, resembled the dexamethasone-binding sites in its response to glucocorticoid manipulation of the animal and in its resistance to salt extraction. This peptide was not present in rat serum. Thus we have identified a plasma-membrane peptide which binds dexamethasone. Whether this peptide is involved in transport of the glucocorticoid across the plasma membrane remains to be determined.     

Temperature-induced modifications in size and pattern of microtubular organelles in a ciliate,Dileptus II. Formation and spatial arrangement of the microtubular skeleton of the oral parts

Summary Microtubular organelles formed during continuous exposure to high or low temperature were studied. Neither heat nor cold prevents formation of the microtubular skeleton in the oral parts of the ciliateDileptus. Elevated temperature causes the formation of short microtubular fibres, while the size of the oral structure is not diminished. This leads to failure in sculpturing of the cytostome. Heat-treatment may also alter the localization of the anchor site of fibres around the circumference of the basal bodies, and the orientation of the fibres. Cold-treatment evokes the formation of small mouthparts containing a lower number of organelles, although these are properly shaped and there are no deviations in the position or orientation of fibres. It seems that low temperature may suppress the rate of formation of microtubular organelles, while elevated temperature affects their patterning.Abbreviations MTOC microtubule organizing centre - T transverse fibres - B basal body - Cy cytostome - K kinetodesma - P postciliary fibre - C compound fibre - L lamina - F filamentous bundle - M monokinetid     

Electric organ polyamines and their effects on the acetylcholine receptor

1. The electric organ of Torpedo nobiliana contained putrescine (PUT), spermidine (SPD), spermine (SPM), and cadaverine (CAD). Traces of acetylated SPD and SPM were occasionaly seen. 2. Upon fractionation of the tissue by differential centrifugation, the polyamines (PA) were found predominantly in the soluble fraction. The postsynaptic membrane fraction, containing a high concentration of acetylcholine receptor (AChR), was proportionally enriched in SPM. The molar ratio of SPM to AChR was approximately two in these membranes. 3. The effect of exogeneous PA on AChR function was studied by two methods: carbamoylcholine (CCh)-dependent 86Rb+ influx into receptor-rich membrane vesicles and [alpha-125I]bungarotoxin (Bgt) binding to the AChR. 4. SPM inhibited both ion influx and the rate of Bgt binding at concentrations above 1 mM, and therefore it appears to act as a competitive antagonist of the AChR. 5. At submicromolar concentrations, and only after preincubation with the receptor-rich membrane, SPM and PUT increased the ion influx by about 20% over control values. 6. Preincubation with 100 nM SPM did not affect the equilibrium binding of iodinated toxin or the rate of toxin binding, and therefore SPM was not uncovering new receptors. 7. By measuring the initial rate of toxin binding after different periods of preincubation with 1 microM CCh, the rate of the slow phase of receptor desensitization was determined. This rate was not changed by 100 nM SPM. 8. Although these results suggest that at low concentrations SPM is a positive modulator of the AChR, the precise mechanism of action is not determined yet.     

The influence of proteases on the activity of glucoamylase from Aspergillus niger C

Summary Two proteases from Aspergillus niger C post-culture medium were isolated by fractionation on a DEAE-sepharose column and ultrafiltration. The four fractions of glucoamylase activity (GA1, GA2, GA3 and GA4) present in the medium showed different susceptibility to the influence of proteases. The effects of proteases on the different glucoamylase fractions during the growth of the fungus are demonstrated. The activity was found to decrease at the beginning of the culture, but by its end there was a stimulation of GA4 glucoamylase. After treating GA2 and GA3 with protease II, a new additional fraction of glucoamylase was detected.