Differential expression of the amyloid SAA 3 gene in liver and peritoneal macrophages of mice undergoing dissimilar inflammatory episodes

The three active serum amyloid A (SAA) genes of mice, SAA 1, SAA 2, and SAA 3, are coordinately expressed in liver during acute and chronic inflammatory stimulation and experimental amyloidosis. The genes, primarily SAA 3, are also expressed extrahepatically. The apoprotein SAA 2 is the precursor of the amyloid A (AA) fibril protein that is deposited as insoluble fibrils extracellularly in spleen and other organs when amyloidosis occurs secondarily to inflammation. The exact cause of AA fibril formation is unknown. Amyloid enhancing factor is a high m.w. glycoprotein extracted from amyloidotic organs. Administration of amyloid enhancing factor alters experimental inflammation to bring about accelerated deposition of amyloid A fibrils first in spleen and later in other organs. In this study, hepatic and extrahepatic expression of the SAA genes were compared during accelerated amyloidosis relative to inflammation uncomplicated by amyloidosis. Differences in kinetics and pattern of SAA gene expression by resident peritoneal macrophages and liver were detected during four dissimilar inflammatory episodes. Macrophages expressed the SAA 3 gene solely, and to a greater extent in chronic than in acute inflammation. In accelerated amyloid induction, macrophage SAA 3 expression increased as SAA 1 and SAA 2 expression in liver decreased. However, alpha-1-acid glycoprotein expression remained elevated throughout the course of amyloid induction. The greatly increased expression of the SAA 3 gene by macrophages and decreased expression of the SAA 1 and SAA 2 genes in liver during amyloidosis, suggests that altered SAA gene expression may play a pathogenetic role in experimental amyloid deposition.     

bcd: A mutation affecting the width of organelle domains in the cortex of Tetrahymena thermophila

Summary A single-gene recessive mutation, bcd (broadened cortical domains), of Tetrahymena thermophila is characterized by a variable broadening of the spatial domains within which cortical organelles, including both the contractile vacuole pores (CVP) and oral apparatus (OA), are formed. The phenotype is not temperature-sensitive. During the development of the organelles of the mutant prior to cell division, extra CVPs and extra oral primordia (OP) appear near ciliary rows adjacent to the rows at which these structures normally form. In the later stages of development, some, but not all, of these extra structures are resorbed, or in the case of the oral domain, multiple adjacent OPs may be completely or partially integrated into a single enlarged OA. When multiple OAs persist, one or more of these may display a reversed orientation reminiscent of those encountered in janus mutants. However, unlike janus, bcd cells do not express any sign of a mirror-image global organization.Our results can best be accounted for by postulating that the bcd mutation affects some common determinant of the widths of both CVP and OA domains. Studies are in progress which explore the relationship between this width-determining mechanism(s) and the mechanism(s) determining the location of cortical organelles around the cell circumference.     

Cytochalasin D and cationized ferritin as probes for the morphological investigation of blebbing in two human cell lines

Summary The potent fungal metabolite cytochalasin D (CD) and cationized ferritin (CF) are used in combination to test for negative charge distribution on blebs (knobs). Two established human epithelial cell lines, WISH and HeLa, that display blebs in various phases of the cell cycle or under certain culture conditions (37,46) are investigated. CD alone, applied at a low concentration (1.0 μg/ml) and for a short time period (3 min), causes blebs to appear as the prevalent surface feature. These are filled mainly with free ribosomes. Additionally, feltlike mats, presumed to be disorganized, compacted microfilaments, are formed directly beneath the cell membrane. These are especially evident in the cortical cytoplasm below the blebs or bleb clusters. CF (0.345 mg/ml), applied for a 5-min period after CD administration (1.0 μg/ml) for 3 min, appears along the surface of microvilli, at the base of blebs, and in vesicles beneath the bleb clusters. In some cases, microfilaments (6 nm in diameter) are closely related to the vesicles. CF does not preferentially bind to the apical cell membrane of blebs. Above areas of the subplasmalemmal microfilaments, CF membrane binding is apparent, even under circumstances where the filaments are disorganized by cytochalasin treatment. These results seem to show the following: (a) bleb membranes are different from the remainder of the cell and do exhibit a loss of negative charge and (b) surface charge may be dependent on the presence or structural integrity of membrane-related 6-nm microfilaments. The support of this research by a grant from the Baylor College of Dentistry and The Oklahoma College of Osteopathic Medicine and Surgery is gratefully acknowledged. The assistance of Dr. J. H. Martin, Department of Pathology, Baylor University Medical Center, is also greatly appreciated.     

γ-Aminobutyric Acid Turnover in Rat Striatum: Effects of Glutamate and Kainic Acid

The turnover rate of gamma-aminobutyric acid (GABA) in the rat striatum was estimated by measuring its accumulation after inhibition of GABA-transaminase (GABA-T) with gabaculine. Intrastriatal injections of 100 micrograms gabaculine induced a rapid and complete inhibition of GABA-T. GABA accumulation was linear with time for at least 60 min (estimated turnover rate = 25 nmol/mg protein/h). The accumulation of GABA after gabaculine administration in animals that had been treated with kainic acid (5 nmol intrastriatally, 7 days) was only 40% of the control value, indicating that a major fraction of the net increase in GABA content induced by gabaculine originates in kainic acid-sensitive neurons. Intrastriatal injection of a mixture of kainic acid (5 nmol) and gabaculine caused a net increase in striatal GABA content significantly greater than that observed in controls, suggesting that neuronal death induced by kainic acid is preceded by a period of increased neuronal activity. Glutamic acid, the putative neurotransmitter for the excitatory corticostriatal pathway, also produced a significant increase in striatal GABA accumulation when injected together with gabaculine. This effect was blocked by the administration of the glutamate receptor antagonist glutamic acid diethyl ester. The interactions between GABAergic neurons and other neurotransmitters present in the striatum were also analyzed.     

Acetylcholine and Choline in Neuronal Tissue Measured by HPLC with Electrochemical Detection

Abstract: A simple, rapid method is presented for the determination of acetylcholine (ACh) and choline (Ch) in neuronal tissue using HPLC with electrochemical detection. The method is based on the separation of ACh and Ch by reverse-phase HPLC and mixing the effluent as it emerges from the column with acetylcholinesterase and Ch oxidase, which converts endogenous Ch and Ch produced by the hydrolysis of ACh to betaine and hydrogen peroxide. Production of hydrogen peroxide is continuously monitored electrochemically. The sensitivity of the procedure is 1 pmol for Ch and 2 pmol for ACh. Specificity of the method is based on HPLC, two specific enzymatic reactions, and the detection of hydrogen peroxide.     

MICROTUBULATION OF THE INNER MEMBRANE OF THE NUCLEAR ENVELOPE

In the course of a light and electron microscopy study of spermatogenesis in the European crayfish, Astacus fluviatilis, spermatocytes of abnormal appearance were observed in two instances in individuals that had passed the mating period. The electron microscope showed that the inner membrane of the nuclear envelope of these cells was erupting into a mass of microtubules, 15 to 18 mµ in diameter and 0.5 µ or more in length, while the outer membrane transformed into cytoplasmic vesicles. Stages in the formation of these novel processes were followed. The plasma membrane of the affected cells was seen in some cases to erupt into similar although shorter microtubules. It is concluded that the phenomenon is part of a degenerative process in which the spermatocytes are being absorbed by sustentacular cells. It is suggested that the observations provide further evidence for a fundamental functional as well as a morphological similarity between the membranes bounding the nucleus and the plasma membrane.