Reversible inhibition of osteoclastic activity by bone-bound gallium (III).

Gallium(III) is a new therapeutic agent for hypercalcemia. Ga3+ reduces osteoclast action, but how it inhibits the cell's physiology is unknown. In vivo, 7-12 microM Ga(III) reduces calcium release from bone, but surprisingly, 10-100 microM Ga3+ added to isolated avian osteoclasts did not reduce their degradation of L-(5-3H)-proline bone. 3H-proline labels bone collagen specifically, and collagenolysis is an excellent indicator of bone dissolution because collagen is the least soluble component of bone. Ga(III) greater than 100 microM inhibited osteoclasts in vitro, but also killed the cells. To resolve this apparent conflict, we measured 67Ga distribution between bone, cells, and media. Gallium binds avidly but slowly to bone fragments. One hundred micrograms of bone clears 60% of 1 microM gallium from 500 microliters of tissue culture medium, with steady state at greater than 24 h. Osteoclasts on bone inhibited gallium binding capacity approximately 40%, indicating a difference in available binding area and suggesting that osteoclasts protect their substrate from Ga binding. Less gallium binds to bone in serum-containing medium than in phosphate-buffered saline; 30% reduction of the affinity constant suggests that the serum containing medium competes with bone binding. Consequently, the effect of [Ga] on bone degradation was studied using accurately controlled amounts of Ga(III) pre-bound to the bone. Under these conditions, gallium sensitivity of osteoclasts is striking. At 2 days, 100 micrograms of bone pre-incubated with 1 ml of 1 microM Ga3+, with 10 pmoles Ga3+/micrograms bone, was degraded at 50% the rate of control bone; over 50 pM Ga3+/micrograms bone, resorption was essentially zero. In contrast, pre-treatment of bone with [Ga3+] as high as 15 microM had no significant effect on bone resorption rate beyond 3 days, indicating that gallium below approximately 150 pg/micrograms bone acts for a limited time and does not permanently damage the cells. We conclude that bone-bound Ga(III) from medium concentrations less than 15 microM inhibits osteoclasts reversibly, while irreversible toxicity occurs at solution [Ga3+] greater than 50 microM.     

Differential expression of K+ channel mRNAs in the rat brain and down-regulation in the hippocampus following seizures.

K+ channels are major determinants of membrane excitability. Differences in neuronal excitability within the nervous system may arise from differential expression of K+ channel genes, regulated spatially in a cell type-specific manner, or temporally in response to neuronal activity. We have compared the distribution of mRNAs of three K+ channel genes, Kv1.1, Kv1.2, and Kv4.2 in rat brain, and examined activity-dependent changes following treatment with the convulsant drug pentylenetetrazole. Both regional and cell type-specific differences of K+ channel gene expression were found. In addition, seizure activity caused a reduction of Kv1.2 and Kv4.2 mRNAs in the dentate granule cells of the hippocampus, raising the possibility that K+ channel gene regulation may play a role in long-term neuronal plasticity.     

''Cross-talk'' between phospholipase C and adenylyl cyclase involves regulation of G-protein levels in GH3 rat pituitary cells.

We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the -subunits of the stimulatory and inhibitory G-proteins of AC (G_s and G_i−2) in cultured prolactin-producing rat pituitary adenoma cells (GH₃ cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6–48 h) with ionomycin (1 μM) or 1-oleoyl-2-acetylglycerol (OAG; 1μM) showed that ionomycin regulated G_s levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered G_s levels by more than 50% at all time-points. G_i−2 levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane G_s protein -subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of ‘cross-talk’ between the PLC- and AC-dependent signalling pathways.     

Viability measurements of hybridoma cells in suspension cultures

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.     

Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli

Summary The effects of 25-fold overproduction ofEscherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between signal sequence functions selected from theBacillus subtilis chromosome and the mature part of TEM-β-lactamase, were studied inE. coli. One precursor (pre[A2d]-β-lactamase) showed an enhanced processing rate, and consequently, a highly improved release of the mature enzyme into the periplasm. A minor fraction of a second hybrid precursor (pre[Al3i]-β-lactamase), which was not processed under standard conditions of SPase I synthesis, was shown to be processed under conditions of SPase I overproduction. However, this did not result in efficient release of the mature β-lactamase into the periplasm. In contrast, the processing rates of wild-type pre-β-lactamase and pre(A2)-β-lactamase, already high under standard conditions, were not detectably altered by SPase I overproduction. These results demonstrate that the availability of SPase I can be a limiting factor in protein export inE. coli, in particular with respect to (hybrid) precursor proteins showing low (SPase I) processing efficiencies.     

Correlation between cell wall extensibility and the content of diferulic and ferulic acids in cell walls of Oryza sativa coleoptiles grown under water and in air

Rice ( Oryza sativa L. cv. Sasanishiki) coleoptiles grown under water achieved greater length than those grown either in air or under water with constant air bubbling. The extensibility of cell walls in coleoptiles grown under water was larger than that in the other treatments. Per unit length of the coleoptile, the content of ferulic and diferulic acids ester-linked to hemicelluloses was higher in air and bubbling type coleoptiles than in water type ones. The extensibility of the coleoptile cell walls correlated with the content of diferulic acids per unit length and per hemicellulose, suggesting that the enhancement of the formation of diferulic acid bridges in hemicelluloses in air or under water with air bubbling makes the cell walls mechanically rigid; thereby inhibiting cell elongation in rice coleoptiles. In addition, the ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age, zone and growth conditions, suggesting that the feruloylation of hemicelluloses is rate-limiting in the formation of diferulic acid bridges in the cell walls of rice coleoptiles.     

Effects of the Introduction of Agrobacterium tumefaciens T-DNA ipt Gene in Nicotiana tabacum L. cv. Petit Havana SR1 Plant Cells

Transformation of tobacco leaf discs with the ‘cytokinin’ipt gene yielded several transgenic callus tissue lines, respectiveto the kind of ipt construction present in the A. tumefacienscointegrates. Those calli containing an active ipt gene wereable to grow hormone-autotrophically and showed an increasedendogenous cytokinin level in comparison with controls. Analysisof endogenous IAA level did not allow any quantitative correlationwith the cytokinin content. However, a minimal level of auxinseems to be necessary to obtain hormone-autotrophic growth.Exogenously supplied NAA significantly reduced the endogenouscytokinin content without modifying growth characteristics. The varying chlorophyll content in the different callus lineselicited the study of the ultrastructure of the plastids. Thecontrols contained small plastids, often filled with starchor accumulated vesicles that did not allow observation of theinternal membrane system. The ‘Pssu-ipt’ line, havinga higher cytokinin content, showed plastids with an internalmembrane system consisting of stroma and grana thylakoids, butthis structure was lost during subculture. Swollen thylakoidsappeared, the amount of starch was reduced and vesicles wereaccumulating. (Received November 15, 1990; Accepted March 4, 1991)     

Nodulation of lucerne (Medicago sativa L.) in an acid soil: Effects of inoculum size and lime-pelleting

The effects of inoculum level and lime-pelleting were studied in an acid soil with respect to the nodulation and growth of lucerne (Medicago sativa cv Resis) and the population dynamics of Rhizobium meliloti. In small root-boxes (rhizotrons), the in-situ survival of inoculated rhizobia was studied in the micro-environment around the seed for a period of 12 days after sowing. During the initial 24 hours, a strong increase in rhizobial numbers was measured, concomitantly with the development of roots. As a result of lime-pelleting, rhizobial numbers were higher only at 3 days after sowing (P<0.05). Later, this difference diminished steadily. Addition of lime did not increase the adhesion of the rhizobia to the seedling tap root. Plant responses to inoculation were studied in pots. To obtain optimal nodulation, the soil had to be neutralized around the seed with lime and at least 10⁵ cells of R. meliloti were required. With more than 10⁵ rhizobia per seed, lime-pelleting increased the number of crown-nodulated seedlings from 24% to 77%. Higher numbers of rhizobia could not compensate the effect of lime. A strong correlation was found between crown nodulation, nitrogen content and dry weight of the shoots.     

Role of magnesium in combination with liming in alleviating acid-soil stress with the aluminium-sensitive sorghum genotype CV323

An experiment to study the effects of Mg nutrition on root and shoot development of the Al-sensitive sorghum (Sorghum bicolor (L.) Moench) genotype CV323 grown in pots of sandy loam under different acid soil stress is reported. This experiment had a factorial design: four rates of liming were combined with four rates of Mg fertilization. When no Mg was added, the pH of the soil solutions (collected in ceramic cups) increased from 4.0 (unlimed) to 4.2, 4.7 and 5.9 at the increasing rates of liming. After 30 days of growth dry matter yields of the limed treatments were 40%, 115% and 199% higher than that of the unlimed treatment. Without liming and at the highest liming rate, adding Mg did not affect plant biomass significantly. At the two intermediate levels of liming, however, 11.3 mg extra Mg per kg soil increased dry matter yield to the same levels as found at the highest liming rate. Concentrations of Mg in the soil solution rose after Mg was added and fell when lime was added, but adding both Mg and lime increased Mg concentrations in the plant shoots. In plants of the limed treatments, dry matter yield was correlated closely with the Mg concentration in the shoot. This was not so in the unlimed treatment. Furthermore, in the unlimed treatments root development was inhibited, but reduced Mg uptake by the plants resulted mainly from the direct effect of Al- (or H-) ions in the soil solution rather than from impaired root development. It is concluded that Mg fertilization counteracted the interfering effects of Al- and H ions on Mg uptake.     

Overnight (1 mg) dexamethasone suppression testing reliably distinguishes non-cushingoid obesity from Cushing's syndrome.

To determine the sensitivity of the overnight 1-mg dexamethasone suppression test in diagnosing Cushing's syndrome, we evaluated the cortisol responses of 55 subjects (25 non-obese individuals with body mass index less than 25 kg/m2, 20 obese individuals with body mass index greater than 30 kg/m2, and 10 patients with surgically proven Cushing's syndrome) following ingestion of 1 mg dexamethasone at midnight. The basal 8 AM plasma cortisol levels among non-obese and obese individuals and patients with Cushing's syndrome were 310 +/- 85, 377 +/- 91, and 813 +/- 270 nmol/L, respectively. Following 1 mg of dexamethasone, Cushing's syndrome patients showed minimal suppression of cortisol to 609 +/- 180 nmol/L (P = 0.79). Non-obese and obese individuals suppressed to 18.7 +/- 6.0 nmol/L (P less than 0.001) and 22 +/- 7.1 nmol/L (P = 0.003), respectively. The results demonstrated similar cortisol responses to overnight dexamethasone suppression in obese and non-obese groups, and clearly distinguished these subjects from those with Cushing's syndrome. Obesity is not a confounding factor in the 1-mg dexamethasone suppression test.