The Ras/Rac1/Cdc42/SEK/JNK/c-Jun Cascade Is a Key Pathway by Which Agonists Stimulate DNA Synthesis in Primary Cultures of Rat Hepatocytes

The ability of signaling via the JNK (c-Jun NH2-terminal kinase)/stress-activated protein kinase cascade to stimulate or inhibit DNA synthesis in primary cultures of adult rat hepatocytes was examined. Treatment of hepatocytes with media containing hyperosmotic glucose (75 mM final), tumor necrosis factor α (TNFα, 1 ng/ml final), and hepatocyte growth factor (HGF, 1 ng/ml final) caused activation of JNK1. Glucose, TNFα, or HGF treatments increased phosphorylation of c-Jun at serine 63 in the transactivation domain and stimulated hepatocyte DNA synthesis. Infection of hepatocytes with poly-l-lysine–coated adenoviruses coupled to constructs to express either dominant negatives Ras N17, Rac1 N17, Cdc42 N17, SEK1−, or JNK1− blunted the abilities of glucose, TNFα, or HGF to increase JNK1 activity, to increase phosphorylation of c-Jun at serine 63, and to stimulate DNA synthesis. Furthermore, infection of hepatocytes by a recombinant adenovirus expressing a dominant-negative c-Jun mutant (TAM67) also blunted the abilities of glucose, TNFα, and HGF to stimulate DNA synthesis. These data demonstrate that multiple agonists stimulate DNA synthesis in primary cultures of hepatocytes via a Ras/Rac1/Cdc42/SEK/JNK/c-Jun pathway. Glucose and HGF treatments reduced glycogen synthase kinase 3 (GSK3) activity and increased c-Jun DNA binding. Co-infection of hepatocytes with recombinant adenoviruses to express dominant- negative forms of PI3 kinase (p110α/p110γ) increased basal GSK3 activity, blocked the abilities of glucose and HGF treatments to inhibit GSK3 activity, and reduced basal c-Jun DNA binding. However, expression of dominant-negative PI3 kinase (p110α/p110γ) neither significantly blunted the abilities of glucose and HGF treatments to increase c-Jun DNA binding, nor inhibited the ability of these agonists to stimulate DNA synthesis. These data suggest that signaling by the JNK/stress-activated protein kinase cascade, rather than by the PI3 kinase cascade, plays the pivotal role in the ability of agonists to stimulate DNA synthesis in primary cultures of rat hepatocytes.

Sequence-specific antitumor activity of a phosphorothioate oligodeoxyribonucleotide targeted to human C-raf kinase supports an antisense mechanism of action in vivo

To determine the mechanism of action responsible for the in vivo antitumor activity of a phosphorothioate antisense inhibitor targeted against human C-raf kinase (ISIS 5132, also known as CGP69846A), a series of mismatched phosphorothioate analogs of ISIS 5132 or CGP69846A were synthesized and characterized with respect to hybridization affinity, inhibitory effects on C-raf gene expression in vitro, and antitumor activity in vivo. Incorporation of a single mismatch into the sequence of ISIS 5132 or CGP69846A resulted in reduced hybridization affinity toward C-raf RNA sequences and reduced inhibitory activity against C-raf expression in vitro and tumor growth in vivo. Moreover, incorporation of additional mismatches resulted in further loss of in vitro and in vivo activity in a manner that correlated well with a hybridization-based (i.e., antisense) mechanism of action. These results provide important experimental evidence supporting an antisense mechanism of action underlying the in vivo antitumor activity displayed by ISIS 5132 or CGP69846A.

Vitamin B12 and hepatitis C: Molecular biology and human pathology

Cobalamins are stored in high concentrations in the human liver and thus are available to participate in the regulation of hepatotropic virus functions. We show that cyanocobalamin (vitamin B12) inhibited the HCV internal ribosome entry site (IRES)-dependent translation of a reporter gene in vitro in a dose-dependent manner without significantly affecting the cap-dependent mechanism. Vitamin B12 failed to inhibit translation by IRES elements from encephalomyocarditis virus (EMCV) or classical swine fever virus (CSFV). We also demonstrate a relationship between the total cobalamin concentration in human sera and HCV viral load (a measure of viral replication in the host). The mean viral load was two orders of magnitude greater when the serum cobalamin concentration was above 200 pM (P < 0.003), suggesting that the total cobalamin concentration in an HCV-infected liver is biologically significant in HCV replication.

Collection of Harvard College admission form letter and bills sent to Charles Walker and his son Charles Walker, Jr., 1785-1816

This collection consists primarily of quarter bills and butler's bills from Charles Walker and Charles Walker, Jr.'s years as students at Harvard College, from 1785 to 1789 and from 1815-1816. It includes the following materials from Charles Walker: a form of admission (a printed form letter with manuscript annotations and signatures) from August 1785, quarter bills and butler's bills from 1785 to 1789, and occasional receipts of payment. The documents from Charles Walker, Jr. are less numerous, consisting solely of quarter bills from 1815 and 1816. The bills for father and son include annotations explaining the basis of additional or unusual charges, including fines for absence from lectures and prayers. The form used for the son's quarter bills, issued in 1815 and 1816, separate the amounts owed into the following categories: Steward and Commons, Sizings, Study and Cellar Rent, Instruction, Librarian, Natural History, Episcopal Church, Books, Catalogue and Commencement Dinner, Repairs, Sweepers, Assessments for delinquency in payment of Quarter Bills, Wood, and Fines. All of the bills are printed forms which were then filled out by hand, by either the steward or the butler, and issued to the students. Caleb Gannett was the College steward during both father and son's era. Joshua Paine, William Harris, and Thomas Adams served, successively, as butler during the father's era. Some of the butler's bills are signed by Roger Vose, a student who appears to have been employed by the butler in 1786 and 1787.

Acquisition reform : DOD's efforts to streamline its acquisition system and reduce personnel : report to the Honorable William Roth, Jr., U.S. Senate /

"B-221205"--p. 1.

Acquisition reform : defense management report savings initiatives : report to the Honorable William V. Roth, Jr., U.S. Senate /

"B-241756"--p.1

Report of the Honorable Herbert Brownell, Jr., Attorney General of the United States, to the Judicial Conference of the United States.

"September 24, 1953."

Trade secrets : a collection of practical receipts for the use of sculptors, modellers, stone masons, builders, marble masons, polishers, &c. ; to which is added a description of the various stones in the United Kingdom suitable for monumental purposes, &c. /

A list of works by the author included at end of volume.