The limited proteolysis of tumor necrosis factor-α

The limited proteolysis of human recombinant TNF- by trypsin yields two stable products resulting from cleavage after Arg6 and Arg44. In solution these two products remain associated together in a trimer with a Stokes' radius slightly greater than the radius of intact TNF- and, therefore, could not be separated from each other under nondenaturing conditions. This limited digest retains at least 20% of the activity of the original TNF- sample, and has a tertiary structure that is similar to that of the native protein by circular dichroism. On the other hand, incorrectly folded, inactive TNF- undergoes extensive digestion following similar treatment with trypsin. These results indicate that the active form of TNF- has a tight core structure which is maintained afterN-terminal cleavage and removal.     

Nucleotide sequence of the coat protein gene of canine parvovirus.

The nucleotide sequence of the canine parvovirus (CPV2) from map units 33 to 95 has been determined. This includes the entire coat protein gene and noncoding sequences at the 3' end of the gene, exclusive of the terminal inverted repeat. The predicted capsid protein structures are discussed and compared with those of the rodent parvoviruses H-1 and MVM.     

Both excision and replication of cloned autonomous parvovirus DNA require the NS1 (rep) protein.

When a bacterial plasmid containing the entire genome of LuIII virus except for the terminal 18 nucleotides from the right end is transfected into HeLa cells, the viral DNA is rescued and replicated, with production of infectious virus. This experimental system was used to examine the viral proteins and cis elements required for the excision and replication of viral DNA. The deletion of the entire NS1 gene provided a viral genome that was excised from the plasmid and replicated only when an NS1 gene was provided in trans. A frameshift mutation in the NS2 intron that truncates NS1 prevented excision and replication. Deletion of the left-end terminal inverted repeat or the right-end inverted repeat prevented excision of viral DNA from that end but not from the wild-type terminus. The viral terminus excised from the plasmid was protected from a processive degradation process, which began on the vector portion of the plasmid. The inhibitor of DNA polymerases alpha and delta, aphidicolin, blocked the excision reaction.     

Microbial retention of mercury from waste streams in a laboratory column containing merA gene bacteria

Abstract: The microorganisms used for the mercury retention experiments were natural isolates and genetically engineered bacteria. All mercury-resistant strains contained the merA gene. Column experiments with these strains were carried out by immobilizing them on different support materials. To obtain kinetic data of the reductase activity for whole cells and the crude extract, batch experiments were carried out under different conditions.     

Therapy of bovine ocular squamous-cell carcinoma with local doses of interleukin-2: 67% complete regressions after 20 months of follow-up

We have tested the therapeutic potency of peritumorally injected low doses of interleukin-2(IL-2). Seventy tumours of the bovine ocular squamous-cell carcinoma (BOSCC), 1–3 cm in diameter, were treated with 5000, 20 000 or 200 000 U IL-2 from Eurocetus (Chiron) to find the optimal dose for treatment. Injections were given peritumorally on Monday to Friday on 2 consecutive weeks. The size of the tumours was measured before treatment and 1, 3, 4, 9 and 20 months after treatment. After 9 months complete regression was observed in 89% of the tumours treated with 5000 U IL-2, 80% treated with 20 000 U and 67% treated with 200 000 U. After 20 months, there was complete regression of 35%, 31% and 67% of the tumours respectively. The 9-and 20-month results of the 200 000-U treatment are significantly better than those of the 5000-U and 20 000-U treatments taken together. This protocol may be useful to treat advanced inoperable tumours (e.g. of the nasopharynx or skin) of human patients.     

Replication process of the parvovirus H-1. VII. Electron microscopy of replicative-form DNA synthesis.

The geometry of replicative form (RF) DNA synthesis of the H-1 parvovirus was studied with the electron microscope using formamide or aqueous variations of the Kleinschmidt spreading procedure. H-1 DNA was isolated from human or hamster cells infected with a temperature-sensitive mutant, ts1, which is deficient in progeny single-stranded DNA synthesis at the restrictive temperature (S.L. Rhode, 1976), thus minimizing possible confusion between RF and progeny DNA replicative intermediates (RIs). The purity of the isolated H-1 DNA, as determined by gel electrophoresis, ethidium bromide staining, autoadiography, and digestion with endo R-EcoRI, was high. H-1 RF DNA'S WERE LINEAR DOUBLE-STRANDED MOLECULES, 1.53 MUM IN LENGTH. H-1 RIs of RF DNA replication were double-stranded, Y-shaped molecules, with the same length as RF DNAs. The replication origin was localized no more than 0.15 genome lengths from one end of the RF DNA, with replication proceeding toward the other end at a uniform rate. Similar RF and RI molecules of dimer size were also observed. The length of H-1 single-stranded DNA extracted from purified virions was measured relative to that of phiX174 and it had a very similar contour length, so that the molecular weight of H-1 single-stranded DNA would be at least 1.48 X 10(6) to 1.59 X 10(6) (Berkowitz and Day, 1974).     

Complementation for replicative form DNA replication of a deletion mutant of H-1 by various parvoviruses.

We established a persistent infection in L 929 cells with the DA strain of Theiler's murine encephalomyelitis virus. Our studies showed that only a small number of cells in the cultures contained infectious virus or viral antigen. A role for interferon in the maintenance of persistence was suggested. Viral isolates from the cultures were not temperature sensitive, nor did they contain viral capsid polypeptide mutations or defective interfering particles. T1 oligonucleotide maps showed evidence of mutation in two of three isolates.     

Dissecting the Inter-Substrate Navigation of Migrating Glioblastoma Cells with the Stripe Assay Reveals a Causative Role of ROCK

A hallmark of gliomas is the growth and migration of cells over long distances within the brain and proliferation within selected niches, indicating that the migrating cells navigate between complex substrates. We demonstrate in the present study a differential preference for migration that depends on Rho-associated coil kinase (ROCK) signaling, using the alternating Bonhoeffer stripe assay. Membrane fractions from nonmyelinated and myelinated brain areas from female rats, purified myelin also from female rats, and commercial extracellular matrix were used as substrates, with each substrate being tested against the others. The human tumor cell lines exhibited a clear preference for extracellular matrix over all other substrates and for myelinated over nonmyelinated tissue. ROCK signaling was different when cells were cultured on either substrate. The ROCK inhibitor Y27632 significantly attenuated and neutralized the preference for extracellular matrix and myelin, indicating that ROCK controls the substrate selectivity. The findings of this study pave the way for navigation-targeted therapeutics.