Search

The objective was to evaluate treatments designed to increase passage rate on forage intake and digestion. Eight ruminally-cannulated steers were assigned to a replicated 4 × 4 Latin square design. All steers were fed an ad libitum diet of prairie grass hay (10.4% CP) with the following treatments designed to increase passage rate: no treatment (control; CON), pelleted (PEL), 7% Ca(OH)2 treated (CAOH), and six 2 kg weights inserted into the rumen (WTS). Molasses (3% of diet DM) was added to all treatments. Feed, orts, and total feces was collected on d 12–17 to determine total tract digestibility. On d 17–20, ruminal pH was recorded every 8 hr. Data were analyzed using the MIXED procedure of SAS 9.4 with treatment and period as fixed effects and steer as a random effect. A treatment effect (P < 0.01) was observed for ruminal pH; steers fed CAOH had the highest pH (6.97) with WTS and CON intermediate and PEL being the lowest (6.34). Organic matter intake (OMI) was affected (P < 0.01) by treatment with PEL steers consuming the most (7.86 kg). While steers consuming CON had greater OMI than CAOH (P = 0.04), WTS was not different from CON (P = 0.50). Organic matter digestibility (OMD) was also affected (P < 0.01) by treatment as WTS and CON were the greatest (62.8% and 61.7%, respectively) and CAOH (58.7%) tended (P = 0.08) to be greater than PEL (55.9%). Total digestible organic matter intake (TDOMI) was affected (P < 0.01) by treatment; PEL had the greatest TDOMI with CON being intermediate, and CAOH and WTS being the least. Treatments designed to increase passage rate did affect forage intake and digestion with the greatest effect resulting from pelleting grass hay due to the severe reduction in particle size.

Hepatitis C virus NS3 helicase can unwind double-stranded DNA and RNA and has been proposed to form oligomeric structures. Here we examine the DNA unwinding activity of monomeric NS3. Oligomerization was measured by preparing a fluorescently labeled form of NS3, which was titrated with unlabeled NS3, resulting in a hyperbolic increase in fluorescence anisotropy and providing an apparent equilibrium dissociation constant of 236 nm. To evaluate the DNA binding activity of individual subunits within NS3 oligomers, two oligonucleotides were labeled with fluorescent donor or acceptor molecules and then titrated with NS3. Upon the addition of increasing concentrations of NS3, fluorescence energy transfer was observed, which reached a plateau at a 1:1 ratio of NS3 to oligonucleotides, indicating that each subunit within the oligomeric form of NS3 binds to DNA. DNA unwinding was measured under multiple turnover conditions with increasing concentrations of NS3; however, no increase in specific activity was observed, even at enzyme concentrations greater than the apparent dissociation constant for oligomerization. An ATPase-deficient form of NS3, NS3(D290A), was prepared to explore the functional consequences of oligomerization. Under single turnover conditions in the presence of excess concentration of NS3 relative to DNA, NS3(D290A) exhibited a dominant negative effect. However, under multiple turnover conditions in which DNA concentration was in excess to enzyme concentration, NS3(D290A) did not exhibit a dominant negative effect. Taken together, these data support a model in which monomeric forms of NS3 are active. Oligomerization of NS3 occurs, but subunits can function independently or cooperatively, dependent upon the relative concentration of the DNA.

Hepatitis C virus (HCV) infects over 170 million persons worldwide. It is the leading cause of liver disease in the U.S. and is responsible for most liver transplants. Current treatments for this infectious disease are inadequate; therefore, new therapies must be developed. Several labs have obtained evidence for a protein complex that involves many of the nonstructural (NS) proteins encoded by the virus. NS3, NS4A, NS4B, NS5A, and NS5B appear to interact structurally and functionally. In this study, we investigated the interaction between the helicase, NS3, and the RNA polymerase, NS5B. Pull-down experiments and surface plasmon resonance data indicate a direct interaction between NS3 and NS5B that is primarily mediated through the protease domain of NS3. This interaction reduces the basal ATPase activity of NS3. However, NS5B stimulates product formation in RNA unwinding experiments under conditions of excess nucleic acid substrate. When the concentrations of NS3 and NS5B are in excess of nucleic acid substrate, NS5B reduces the rate of NS3-catalyzed unwinding. Under pre-steady-state conditions, in which NS3 and substrate concentrations are similar, product formation increased in the presence of NS5B. The increase was consistent with 1:1 complex formed between the two proteins. A fluorescently labeled form of NS3 was used to investigate this interaction through fluorescence polarization binding assays. Results from this assay support interactions that include a 1:1 complex formed between NS3 and NS5B. The modulation of NS3 by NS5B suggests that these proteins may function together during replication of the HCV genome.

Paleoindian unifacial stone tools frequently exhibit distinct, sharp projections, known as “spurs”. During the last two decades, a theoretically and empirically informed interpretation–based on individual artifact analysis, use-wear, tool-production techniques, and studies of resharpening–suggested that spurs were sometimes created intentionally via retouch, and other times created incidentally via resharpening or knapping accidents. However, more recently Weedman strongly criticized the inference that Paleoindian spurs were ever intentionally produced or served a functional purpose, and asserted that ethnographic research “demonstrates that the presence of so called ‘graver’ spurs does not have a functional significance.” While ethnographic data cannot serve as a direct test of the archaeological record, we used Weedman’s ethnographic observations to create two quantitative predictions of the Paleoindian archaeological record in order to directly examine the hypothesis that Paleoindian spurs were predominantly accidents occurring incidentally via resharpening and reshaping. The first prediction is that the frequency of spurs should increase as tool reduction proceeds. The second prediction is that the frequency of spurs should increase as tool breakage increases. An examination of 563 unbroken tools and 629 tool fragments from the Clovis archaeological record of the North American Lower Great Lakes region showed that neither prediction was consistent with the notion that spurs were predominately accidents. Instead, our results support the prevailing viewpoint that spurs were sometimes created intentionally via retouch, and other times, created incidentally via resharpening or knapping accidents. Behaviorally, this result is consistent with the notion that unifacial stone tools were multifunctional implements that enhanced the mobile lifestyle of Pleistocene hunter-gatherers.

HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNA unwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nm) is equimolar with the substrate, a small burst amplitude of approximately 8 nm is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only approximately 10% of NS3 helicase domain and approximately 20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of full-length NS3 is part of an oligomeric species in vitro.

A 7-year-old boy presented with an annular verrucous plaque on the chin of 5 weeks duration. The lesion occurred after a bicycle accident and was unresponsive to antibiotics. Fungal culture grew Sporothrix schenckii. Sporotrichosis should be considered and fungal culture obtained whenever a nodule or plaque fails to respond to initial treatment. Language: en

Mitogenic activity is present at a variety of sites in the central nervous system. A growth factor was purified from neonatal bovine spinal cord. It has a pI of 9.5-9.8 and a molecular weight of about 11,000 daltons. Spinal cord growth factor is a basic polypeptide that is inactivated by extremely acid or basic conditions. Its mobility on SDS polyacrylamide gels suggests that this factor is different from pituitary FGF and brain FGF-1.