Differential regulation of basic protein phosphorylation by calcium phospholipid and cyclic-AMP-dependent protein kinases

Myelin basic protein, an 80-kilodalton (kDa) protein in rat oligodendrocytes, and an 80-kDa basic protein in neuroblastoma x neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl-acetylglycerol). TPA-stimulated phosphorylation was inhibited by pre-incubation with 50 microM psychosine (galactosyl-sphingosine), confirming that it is mediated through the phospholipid-dependent protein kinase C (PK-C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic-AMP-dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte-specific 2',3'-cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.: Proceedings of the National Academy of Sciences of the United States of America 85:939, 1988). The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol for PK-C activation is discussed.     

Stimulation of tissue plasminogen activator production from epithelial cell lines

The aim of this study was to investigate the possibility of enhancing the yield of tissue plasminogen activator (tPA) from two epithelial cell lines of normal (non-malignant) derivation grown in tissue culture. The three agents used in this investigation were chosen because of their proven enhancing effect on analogous cells or products. The anabolic hormone stanozolol was found to have no significant stimulatory effect on these cell lines. A phorbol acetate (12-O-tetradecanoylphorbol 13-acetate) caused a twofold enhancement in tPA yield but the most significant results were obtained with 5-azacytidine. This agent increased the yield by up to fourfold in small stationary cultures and threefold in large-scale microcarrier cultures. A combination of azacytidine and phorbol acetate did not have an additive effect on total yield but did alter the kinetics of tPA expression with time. Indications were that the maximum yield with these types of potentiating agents was achieved as it could not be increased by using a combination of two different agents.     

A bivariate radioimmunoassay for arginine vasopressin and the synthetic antidiuretic agent 1-deamino-8-D-arginine vasopressin (desmopressin)

Pairs of radioimmunoassays, each of which include a two-dimensional matrix of standards, have been previously employed to resolve specificity problems in steroid immunoassay. In this study the bivariate radioimmunoassay principle has been applied to simultaneous measurement of plasma antidiuretic hormone, arginine vasopressin, and the synthetic antidiuretic agent 1-deamino-8-D-arginine vasopressin (desmopressin), by utilizing two arginine vasopressin antisera which show significantly different cross-reactivities with the synthetic analog. Data processing consists of mathematical representation of two curved dose-response surfaces followed by solution of this pair of nonlinear simultaneous equations for the unknown arginine vasopressin and desmopressin concentrations. Details of numerical procedures are given in the Appendix. The assay appears entirely adequate in terms of sensitivity, accuracy, and precision for measurement of these antidiuretic agents in clinical samples. No evidence of significant covariance in estimated concentrations could be detected but precision of estimation is (not unexpectedly) a function of the concentration of both agents. The plasma disappearance half-time of desmopressin (probably the second of a biphasic disappearance) was estimated as 37 min in one normal subject, which is in good agreement with a previously reported value of 30 min.     

Advanced weathering cracks as an improvement on split-line preparations for analysis of structural orientation in compact bone.

Weathering cracks have been demonstrated to be equivalent to split-lines in compact bone. The amount of cracking becomes more extensive with time. Advanced weathering cracks have the potential for giving more complete information on orientation patterns than split-lines. They can also be superior in accuracy and objectivity of results obtained, in preserving the original condition of the bone, and in elimination of much of the work involved in making split-line preparations.     

A Thionin Stain for Visualizing Bone Cells, Mineralizing Fronts and Cement Lines in Undecalcified Bone Sections

A staining method is described using thionin, for undecalcified deacrylated bone sections. RNA is stained purplish violet, allowing still active osteoblasts to be distinguished from lining cells. Staining intensity of mineralized bone is related to the degree of mineralization. Mineralizing fronts and cement lines are visualized clearly. Lamellae show an alternate pattern. Histomorphometric parameters such as osteon thickness and interstitial bone thickness can be measured without using polarized light. The mineralizing front can be assessed and expressed as a percentage of the osteoblast-covered interface between osteoid and mineralized bone. The stain is also useful for qualitative assessment of metabolic bone disease. Thionin stained sections can be kept for at least one year when stored hi the dark at 7 C.     

Bis(4-hydroxy-2H-chromen-2-one): Synthesis and effects on leukemic cell lines proliferation and NF-κB regulation

Synthesis of the bis-4-hydroxycoumarin-type compound, 3,3′-[3-(2-hydroxyphenyl)-3-oxopropane-1,1-diyl]bis(4-hydroxy-2H-chromen-2-one), was performed by two alternative pathways, either involving a basic organocatalyzed 1,4-conjugate addition tandem reaction of 4-hydroxycoumarin on chromone-3-carboxylic acid, or a double condensation of 4-hydroxycoumarin on ω-formyl-2′-hydroxyacetophenone. The anti-proliferative effects of the bis-4-hydroxycoumarin-type compound on human K-562 (chronic myeloid leukaemia) and JURKAT (acute T-cell leukaemia) cell lines using trypan blue staining, as well as its involvement in nuclear factor-kappa B (NF-κB) regulation analyzed by luciferase reporter gene assay, gene expression analysis and western blots were analysed. This compound inhibited TNFα-induced NF-κB activation in K-562 (IC₅₀ 17.5 μM) and JURKAT (IC₅₀ 19.0 μM) cell lines, after 8 h of incubation. Interestingly, it exerted mainly cytostatic effects at low doses on both cell lines tested, whereas it decreased JURKAT cell viability starting at 50 μM from 24 h of treatment. Importantly, it did not affect the viability of peripheral blood mononuclear cells (PBMCs) from healthy donors, even at concentrations above 100 μM.     

Antiproliferative activity of L-asparaginase of Tetrahymena pyriformis on human breast cancer cell lines

Purified L-asparaginase of Tetrahymena pyriformis is a multi-subunit enzyme exhibiting protein kinase activity as well. The enzyme's L-asparaginase activity is affected by its phosphorylation state. Both native and dephosphorylated L-asparaginase show antiproliferative activity on three breast cancer cell lines (T47D, BT20 and MCF-7) and on Walker 256 cells. These cells do not possess measurable L-asparaginase or L-asparagine synthetase activity. When T47D cells are treated for different times with L-asparaginase and then placed in fresh medium, the growth of cells treated for 1, 3, or 6 hours is initiated and parallels control curve, while the growth of cells treated for 24 or 48 hours with L-asparaginase stays at the same inhibitory level (24 h treatment) or continues to drop (48 h treatment). Addition of D-asparagine, a competitive inhibitor of T. pyriformis L-asparaginase, counteracts the antiproliferative activity of L-asparaginase, indicating that L-asparaginase and not the kinase activity is responsible for that effect.     

The Relativistic Quantum Defect Orbital Method and some of its Applications

Justification of a simple and reliable relativistic procedure for the prediction of a large body of transition probability data is made. The main features of the Relativistic Quantum Defect Orbital (RQDO) formalism are described, and several examples of the data yielded by this method are presented in tabular and graph forms.     

Peripheral and central nervous responses evoked by small water movements in a cephalopod

Potentials were recorded from the epidermal head lines and from the CNS of young cuttlefish, Sepia officinalis, in response to weak water movements. 1. Within the test range 0.5-400 Hz a sinusoidal water movement elicits up to 4 components of response if the electrode is placed on a headline: (i) a positive phasic ON response; (ii) a tonic frequency-following microphonic response; (iii) a slow negative OFF response; and (iv) compound nerve impulses. 2. The amplitude of both the ON wave and the microphonic potential depends on stimulus frequency, stimulus amplitude and stimulus rise time. Frequencies around 100 Hz and short rise times are most effective in eliciting strong potentials. The minimal threshold was 0.06 microns peak-to-peak water displacement at 100 Hz (18.8 microns/s as velocity). 3. Change of direction of tangential sphere movement (parallel vs. across the head lines) has only a small effect on the microphonic and the summed nerve potentials. 4. Frequency and/or amplitude modulations of a carrier stimulus elicit responses at the onset and offset of the modulation and marked changes in the tonic microphonic response. 5. Evoked potentials can be recorded from the brain while stimulating the epidermal lines with weak water movements. The brain potentials differ in several aspects from the potentials of the head lines and show little or no onset or offset wave at the transitions of a frequency and amplitude modulation.