Amyloid protein AA is the presumptive fragment of an acute phase serum apolipoprotein, apoSAA. Two major murine apoSAA isotypes (apoSAA1 and apoSAA2) have been identified. The NH2-terminal amino acid sequences of purified murine apoSAA1 and apoSAA2 have been examined and compared with that of murine amyloid protein AA. Our results indicate that apoSAA1 and apoSAA2 are separate gene products and that amyloid protein AA has NH2-terminal amino acid sequence identity with only one of these isotypes, namely apoSAA2.

Induction of metallothionein-I (MT-I) mRNA by bacterial endotoxin (LPS) was examined. A single injection of LPS induced MT-I mRNA accumulation in both liver and kidney comparable to that induced by heavy metals. Maximal message levels were achieved 6 hr after LPS administration, prior to any increase in either hepatic or renal Zn or Cu. Experiments in which LPS was administered to transgenic mice harboring recombinant genes made by fusing the MT-I gene promoter to the herpes simplex virus thymidine kinase structural gene revealed that the response to LPS is independent of glucocorticoid hormones. These experiments begin to define the region of the MT-I gene promoter required for the LPS response.It has been well documented that the synthesis of metallothionein (MT) is regulated by both heavy metals, such as Cd and Zn, and glucocorticoid hormones (1-4). We have shown both in vivo and in cultured cells that the regulation of the mouse MT-I gene occurs primarily at the level of gene transcription for both classes of inducers (5-7). Definition of the region(s) of the MT-I gene promoter necessary for regulation by heavy metals has been approached by fusing deleted variants of the MT-I gene promoter to easily assayable genes, such as the herpes simplex virus (HSV) thymidine kinase (TK) gene, and analyzing metal regulation after the fused genes were introduced into either tissue culture cells or mouse eggs (8,9). These studies indicate that the region of the mouse MT-I promoter necessary for metal regulation maps within 90 nucleotides 5' of the normal MT-I transcription initiation site. Similar analysis of the region important for glucocorticoid regulation has not been possible because the mouse MT-I promoter is not responsive to glucocorticoids when transferred to other cells (9-11).In Like MT, some acute-phase proteins are regulated by elevated glucocorticoid levels resulting from stresses such as surgical trauma, pain, apprehension, and exposure to heat and cold (14-17). However, for MTs the response to inflammatory agents does not appear to be glucocorticoid-mediated because it has been recently shown that the injection of turpentine induced comparable amounts of hepatic MT in adrenalectomized and control rats (18). This suggests that there may be several mechanisms regulating MT synthesis that are operative during different types of stress and implies that perhaps a third class of regulatory molecule can affect MT-I gene transcription. Here we present several lines of evidence that rule out metal or glucocorticoid involvement in the response to the potent inflammatory agent LPS and thus support the notion that a third class of molecules may mediate MT-I gene expression during stress. MATERIALS AND METHODSMT-I mRNA Accumulation Time Course. Male SwissWebster mice, 14-17 g, were injected subcutaneously with ZnSO4 (5 mg/kg) or intraperitoneally with LPS (3 mg/kg; Difco; lipopolysaccharide W, Salmonella typhosa 0901). Tissues were removed and MT-I mRNA levels were measured in total nucleic acid samples as desc...

There is compelling evidence that the eukaryotic cell cycle is controlled by a family of proteins called cyclins, which complex with cyclin-dependent kinases (CDK) to modulate key events during cell division. We have examined the regulation of these genes in models of experimental liver regeneration and their expression in human liver diseases. Seventy percent partial hepatectomy (PH) was performed on rats and normal BALB/c and athymic nude mice to determine patterns of cyclin and CDK1 mRNA expression. It has been previously shown by [3H]thymidine incorporation that athymic nude mice manifest impaired regeneration after PH. Our results demonstrate a sequential pattern of cyclin and CDK1 transcript expression in each of the models. Cyclin D1 was the most abundant mRNA steady-state transcript in the regenerating livers. CDK1 and cyclins associated with later stages of the cell cycle showed delayed and diminished expression in nude mice compared with normals. Nuclear run-off assays performed at key time points post-PH revealed little change in transcription rates, suggesting that steady-state mRNA expression of the cyclin genes is regulated primarily by posttranscriptional events. Human liver tissue from various acute and chronic hepatic diseases showed increased expression of cyclins A and D1. We conclude that the regenerating liver post-PH offers an excellent in vivo model for studying cyclin and CDK gene expression. Impaired regeneration in the nude mouse is associated with altered cyclin and CDK1 mRNA transcript expression. Furthermore, cyclins may eventually provide clinically relevant molecular markers of regenerative activity in human liver diseases.

The deposits of fibrils found in amyloidosis of the A type are derived from only one of the three serum amyloid A (SAA) gene products, namely SAA2. In order to explore the mechanism of SAA isotype-specific amyloid protein AA deposition, the molecular kinetics of the serum amyloid proteins were examined in CBA mice during casein induction of amyloidosis. The presence of SAA mRNA in spleen was searched for; hepatic SAA1 and SAA2 mRNA levels, rates of specific protein synthesis and secretion by hepatocytes, and serum levels were measured during a 20-d period of amyloid induction. We observed the following: small amounts of amyloid substance appeared in the spleen by day 5 and increased steadily over the ensuing 15 d to occupy nearly 30% of splenic volume by day 20. No SAA mRNA was detected in spleen at any time during induction of amyloid formation. Total serum SAA levels peaked 1 d after we began casein treatment, and thereafter declined. This decline was accounted for entirely by a dramatic fall in SAA2, while SAA1 levels remained nearly constant throughout. The ratios of hepatic SAA2:SAA1 mRNA, as determined by in vitro translation, remained constant during the 20-d period, as did amounts of SAA1 and SAA2 synthesized and secreted by freshly isolated hepatocytes. These data indicate that the deposition of amyloid A protein derived from SAA2 is not due to local SAA production in spleen, nor excessive SAA2 production compared with SAA1, but involves the selective and accelerated removal of SAA2 from the circulating pool of both SAA1 and SAA2.

A B S T R A C T The plasma clearance kinetics of the amyloid-related high density lipoprotein (HDL) apoprotein serum amyloid protein (apoSAA) was examined in BALB/c mice by two different methods, using labeled '25I-apoSAA-rich HDL and unlabeled plasma apoSAA (clearance monitored by radioimmunoassay). The plasma half-life of apoSAA, estimated by both methods, was on the order of 75-80 min, as compared with a value of -11 h for mouse apoA-I. In tracelabeling studies, the rapid plasma clearance of both major '25I-labeled apoSAA isotypes was observed; this metabolic behavior was unique to these polypeptides among HDL apoproteins. The property of rapid plasma clearance was lost upon purification and reconstitution of '251I-apoSAA with HDL, indicating that this property is labile to denaturing conditions. Studies aimed at determining the metabolic fate of 1251_ apoSAA gave no evidence for either the selective excretion of '251-apoSAA or clearance to unique tissue sites as compared with other 251I-HDL apoproteins.