Molecular cloning and expression of the 3-chlorobenzoate-degrading genes from Pseudomonas sp. strain B13.

The genes specifying the utilization of 3-chlorobenzoate by Pseudomonas sp. strain B13 WR1 have been cloned by using a broad-host-range cosmid cloning system. Analysis of the catabolic products of the enzymatic reactions encoded by two hybrid cosmids, pMW65 and pMW90, by thin-layer and high-performance liquid chromatography demonstrated that both encoded the genes for the complete catabolism of 3-chlorobenzoate. Physical analysis of one of the cosmid derivatives, pMW65, by restriction endonuclease mapping and subcloning demonstrated that the pathway genes are encoded on a fragment no larger than 11 kilobases.     

Activation of phospholipase C in platelets by platelet activating factor and thrombin causes hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate

Despite their physicochemical and mechanistic differences platelet activating factor (or acetylglycerylether phosphorylcholine; AGEPC) and thrombin, both platelet stimulatory agents, induce phosphoinositide turnover in platelets. We therefore investigated the stimulation of the phosphoinositide phosphodiesterase by these agents and questioned whether they evoked hydrolysis of the same or different pools of phosphoinositides. [3H]Inositol-labelled rabbit platelets were challenged with thrombin and/or AGEPC under a variety of protocols, and the phospholipase C mediated production of radioactive inositol monophosphate (IP); inositol bisphosphate (IP2) and inositol trisphosphate (IP3) was used as the parameter. AGEPC (1 X 10(-9) M) caused a transient maximum (5 to 6-fold) increase in [3H]IP3 at 5 s followed by a decrease. Thrombin (2 U/ml) elicited an increase in [3H]IP3 at a much slower rate than AGEPC; 2 fold at 5 s, 5 fold at 30 s and a maximum 6 to 8-fold at 2-5 min. Compared to AGEPC, thrombin stimulated generation of [3H]IP2 and [3H]IP were severalfold higher. When thrombin and AGEPC were added together to platelets there was no evidence for an additive increase in inositol polyphosphate levels except at earlier time points where increases were submaximal. When AGEPC was added at various time intervals after thrombin pretreatment, no additional increases in [3H]IP3 were observed over that maximally seen with thrombin or AGEPC alone. In another set of experiments, submaximal increases (about 1/4 and 1/2 of maximum) in [3H]IP3 were achieved by using selected concentrations of thrombin (0.1 U and 0.3 U, respectively) and then AGEPC (1 X 10(-9) M) was added for 5 s. Once again the increase in [3H]IP3 was close to the maximal level seen with thrombin or AGEPC individually. It is concluded that thrombin and AGEPC differentially activated phosphoinositide phosphodiesterase (phospholipase C) in rabbit platelets and that the stimulation of the phospholipase C by these two stimuli causes IP3 production via hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate.     

Induction of T4 DNA ligase in a recombinant strain of Escherichia coli

The kinetics of cell growth and foreign protein production, as well as factors affecting protein stability, were studied and optimized in batch and fed-batch fermentations of a recombinant strain of Escherichia coli. The pL promoter from bacteriophage lambda under the control of a temperature-sensitive cl represser, with the entire construct integrated into the E. coli chromosome through the use of a defective bacteriophage lambda lysogen, was used to direct the synthesis of T4 DNA ligase. The biphasic fermentations consisted of a primary growth phase at 30 degrees C followed by an induction phase which was initiated by shifting the temperature to 42 degrees C. In the fed-batch fermentations, additional nutrients were added at the time of initiating induction. Maintenance of sufficiently high concentrations of the organic substrates (glucose and casamino acids) during the induction phase was required for continued cell growth at 42 degrees C. Such growth was essential for T4 DNA ligase formation and in vivo stability. Hence, fed-batch fermentations produced the highest yield of the foreign protein Commensurate with providing lower total amounts of substrates. In such cases, high cell densities (6 g dry wt/L) with substantial intracellular levels of T4 DNA ligase (4.6% total cellular protein, or 2.7% of the dry biomass) were achieved.     

Angiotensin 'antipeptides': (-)messenger RNA complementary to human angiotensin II (+)messenger RNA encodes an angiotensin receptor antagonist

(-)mRNA complementary to human angiotensin II (+)mRNA encodes the 'antipeptide' Glu-Gly-Val-Tyr-Val-His-Pro-Val which is structurally related to angiotensin II. Angiotensin II 'antipeptide' (antiANG II) and the desglutamyl heptapeptide (antiANG III) are Type I antagonists which inhibit the contractile action of angiotensin at smooth muscle receptors by binding to a negative modulatory site on the angiotensin receptor which is distinct from the angiotensin binding site. These findings may illustrate that the inhibitory binding site on the angiotensin receptor exists to accomodate a naturally occurring inhibitor(s), which is encoded by the DNA strand complementary to that encoding angiotensin II.     

65Zinc uptake from blood into brain and other tissues in the rat

Zinc is essential for normal growth, development and brain function although little is known about brain zinc homeostasis. Therefore, in this investigation we have studied⁶⁵Zn uptake from blood into brain and other tissues and have measured the blood-brain barrier permeability to⁶⁵Zn in the anaesthetized rat in vivo. Adult male Wistar within the weight range 500–600 g were used.⁶⁵ZnCl₂ and [¹²⁵I]albumin, the latter serving as a vascular marker, were injected in a bolus of normal saline I.V. Sequential arterial blood samples were taken during experiments that lasted between 5 min and 5 hr. At termination, samples from the liver, spleen, pancreas, lung, heart, muscle, kidney, bone, testis, ileum, blood cells, csf, and whole brain were taken and analysed for radio-isotope activity. Data have been analysed by Graphical Analysis which suggests⁶⁵Zn uptake from blood by all tissues sampled was unidirectional during this experimental period except brain, where at circulation times<30 min,⁶⁵Zn fluxes were bidirectional. In addition to the blood space, the brain appears to contain a rapidly exchanging compartment(s) for⁶⁵Zn of about 4 ml/100g which is not csf.     

High hydrostatic pressure effects in vivo: changes in cell morphology, microtubule assembly, and actin organization

We present the first study of the changes in the assembly and organization of actin filaments and microtubules that occur in epithelial cells subjected to the hydrostatic pressures of the deep sea. Interphase BSC-1 epithelial cells were pressurized at physiological temperature and fixed while under pressure. Changes in cell morphology and cytoskeletal organization were followed over a range of pressures from 1 to 610 atm. At atmospheric pressure, cells were flat and well attached. Exposure of cells to pressures of 290 atm or greater caused cell rounding and retraction from the substrate. This response became more pronounced with increased pressure, but the degree of response varied within the cell population in the pressure range of 290-400 atm. Microtubule assembly was not noticeably affected by pressures up to 290 atm, but by 320 atm, few microtubules remained. Most actin stress fibers completely disappeared by 290 atm. High pressure did not simply induce the overall depolymerization of actin filaments for, concurrent with cell rounding, the number of visible microvilli present on the cell surface increased dramatically. These effects of high pressure were reversible. Cells re-established their typical morphology, microtubule arrays appeared normal, and stress fibers reformed after approximately 1 hour at atmospheric pressure. High pressure may disrupt the normal assembly of microtubules and actin filaments by affecting the cellular regulatory mechanisms that control cytological changes during the transition from interphase into mitosis.     

Confidence Intervals on Ratios of Variance Components for the Unbalanced Two Factor Nested Model

The model considered in this article is the two-factor nested unbalanced variance component model: for p = 1, 2, …, P; q = 1, 2, …, Q_p; and r = 1, 2, …, R_pq. The random variables Y_pqr are observable. The constant μ is an unknown parameter, and A_p, B_pq and C_pqr are (unobservable) normal and independently distributed random variables with zero means and finite variances σ²_A, σ²_B, and σ²_C, respectively. Approximate confidence intervals on ?_A and ?_B using unweighted means are derived, where The performance of these approximate confidence intervals are evaluated using computer simulation. The simulated results indicate that these proposed confidence intervals perform satisfactorily and can be used in applied problems.