The formation of inositol 1,2-cyclic phosphate on agonist stimulation of phosphoinositide breakdown in mouse pancreatic minilobules. Evidence for direct phosphodiesteratic cleavage of phosphatidylinositol

It is generally thought that formation of inositol 1,2-cyclic phosphate (IcP) on agonist-stimulated "breakdown" of endogenous phosphatidylinositol in intact cells would provide strong evidence for the direct phosphodiesteratic cleavage of phosphatidylinositol. We report here that on ionophoresis of extracts of pancreatic minilobules incubated with the cholecystokinin/pancreozymin congener, caerulein, the usual inositol phosphates, i.e. inositol 1-phosphate (IP), inositol 4,5-bisphosphate (IP2), and inositol 1,4,5-trisphosphate (IP3) were seen. In addition, an [3H]inositol-labeled unknown was present with the correct electrophoretic mobility of IcP. There was only a trace of "IcP" in the unstimulated pancreatic minilobules. Several lines of evidence indicate that the unknown peak was IcP. 1) It ran on ionophoresis with standard [14C]IcP, and the ratio of 3H to 14C for each point on the peak was a constant within experimental error. 2) The putative IcP peak which had been eluted from the electropherogram also coincided with standard [14C]IcP on paper chromatography. 3) On mild acid hydrolysis in the presence of standard 14C-labeled IP, the putative [3H] IcP peak disappeared and appeared in the exact position of the standard [14C]IP peak, as to be predicted of IcP. The formation of IcP on agonist stimulation supports direct phosphodiesteratic cleavage of phosphatidylinositol on stimulation of phosphoinositide breakdown in pancreatic minilobules.     

The effects of sodium and magnesium-ion interactions on chromatin structure and solubility

The effects of sodium and magnesium-ion interactions on chromatin structure and solubility were examined in isolated mouse liver nuclei. To facilitate this study, a simple assay of chromatin structure was developed, based on the absorbances at 260 nm (A260) and 320 nm (A320) of nuclei in test solutions. By subtracting the A320 from the A260, a single "spectral index" was obtained which served as a useful, but not absolute, indicator of chromatin structure. Electron microscopy verified the validity of this approach. The results indicate that either 200 mM NaCl or 0.5 mM MgCl2 were capable of preserving the native 20 to 30 nm chromatin fiber structure. Below 200 mM NaCl, the native fiber progressively uncoiled to the 10 nm unit fiber. The presence of 0.5 mM MgCl2 inhibited this uncoiling. Only divalent cations stabilized condensed chromatin (heterochromatin) within the nucleus. Monovalent and divalent cations interacted with one another at critical concentrations and modified their individual effects on chromatin structure; e.g., 10 to 25 mM NaCl interfered with the action of 0.5 to 1.5 mM MgCl2, causing a complete loss of condensed chromatin. Maximum solubility of micrococcal nuclease-digested chromatin occurred at 10 mM NaCl, which treatment allowed the chromatin to unfold to the 10 nm fiber. However, ionic conditions that disrupted condensed chromatin but maintained the native chromatin fiber morphology still resulted in relatively high yields of soluble chromatin. Minimum solubility occurred under conditions which preserved the structure of condensed chromatin.     

Biochemical pathways in prokaryotes can be traced backward through evolutionary time

For the first time, a credible prokaryotic phylogenetic tree is being assembled by Woese and others using quantitative sequence analysis of oligonucleotides in the highly conservative rRNA. This provides an evolutionary scale against which the evolutionary steps that led to the arrangement and regulation of contemporary biochemical pathways can be measured. This paper presents an emerging evolutionary picture of aromatic amino acid biosynthesis within a large superfamily assemblage of prokaryotes that is sufficiently developed to illustrate a new perspective that will be applicable to many other biochemical pathways.      

Fatal myocarditis in mice fed rancid purified feed

An outbreak of disease characterized by diarrhea, severe myocarditis, and high mortality occurred in a group of 800 male B6C3F1/ Har mice fed powdered purified diets. A total of 292 animals died. No evidence of an infectious agent was found, and the disease was reproduced in healthy mice by feeding the purified diets, suggesting a nutritional deficiency or toxicity. Analysis of the feed revealed adequate levels of vitamin E, reduced levels of thiamine, and elevated levels of lipoperoxide. It was concluded that mortality was due to myocarditis associated with the ingestion of rancid feed.     

Secretogogue-stimulated phosphatidylinositol breakdown in the exocrine pancreas liberates arachidonic acid, stearic acid, and glycerol by sequential actions of phospholipase C and diglyceride lipase

When mouse pancreatic "minilobules" prelabeled with either [14C]arachidonic acid (AA), [14C]stearic acid (SA), or [3H]glycerol were stimulated with the secretogogue, caerulein, there was a 60-70% loss in radioactivity in phosphatidylinositol (PI) at 30 min. This loss was accompanied by the formation of [14C] phosphatidic acid (PA), [14C]diacylglycerol (DG), [14C] triacylglycerol (TG), and free [14C]AA, [14C]SA, and [3H]glycerol. The loss in radioactive PI was the same as the loss in chemically measured PI-phosphorus. Thirty to fifty per cent of the caerulein-induced loss of prelabeled PI could be accounted for as free [14C]AA, [14C]SA, or [3H]glycerol. Increased incorporation of fatty acid or glycerol residues into DG, PA, and TG accounted for the balance of the loss in PI. The specific DG-lipase inhibitor, RHC 80267, markedly inhibited the caerulein-stimulated release of [14C]AA, [14C]SA, and [3H]glycerol and roughly doubled the caerulein-induced increment in [14C]AA-, [14C]SA-, or [3H]glycerol-labeled DG, showing that the source of the caerulein-induced increment in fatty acids and glycerol was DG. When the PI was prelabeled with either [32P] orthophosphate, [3H]myoinositol, or [3H]glycerol, only 1% or less of the radioactivity in PI was in lysophosphatidylinositol (LPI), and there was no increase in radioactivity in LPI on stimulation with caerulein. These observations, taken together, argue strongly for a phospholipase C-catalyzed breakdown of PI followed by DG-lipase and argue against any significant involvement of phospholipase A2 in PI degradation in mouse pancreas. The formation of substantial amounts of free [14C]AA on stimulation supports the view that, among other things, the phosphoinositide effect in the exocrine pancreas serves to generate arachidonate (and its metabolites). The release of appreciable amounts of free fatty acids and glycerol shows that a significant portion of the DG formed as a result of caerulein-stimulated PI breakdown is not conserved in the phosphoinositide cycle.     

Cavitation Events in Thuja occidentalis L.? : Utrasonic Acoustic Emissions from the Sapwood Can Be Measured

Ultrasonic acoustic emissions (AE) in the frequency range of 0.1 to 1 megahertz appear to originate in the sapwood of Thuja occidentalis L. The AE are vibrations of an impulsive nature. The vibrations can be transduced to a voltage waveform and amplified. The vibrations of each AE event begin at a large amplitude which decays over 20 to 100 microseconds. Strong circumstantial evidence indicates that the ultrasonic AE result from cavitation events because: (a) they occur only when the xylem pressure potential Ψ_xp is more negative than a threshold level of about —1 megapascal; (b) the rate of AE events increases as Ψ_xp decreases and when the net rate of water loss increases; (c) the AE can be stopped by raising Ψ_xp above —1 megapascal. Ultrasonic AE have been measured in whole terminal shoots allowed to dry in the laboratory, in isolated pieces of sapwood as they dried in the laboratory, and in whole terminal shoots in a pressure bomb when Ψ_xp was decreased by lowering the gas pressure in the pressure bomb.