The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells.

MS-275 is a histone deacetylase (HDAC) inhibitor that has been reported to mediate its cytotoxic effect through generation of reactive oxygen species (ROS) in proliferating hematopoietic cell lines. We examined efficacy of MS-275 in nonproliferating chronic lymphocytic leukemia (CLL) cells from patients. In these cells, MS-275 demonstrated an in vitro LC(50) that was one log lower than for normal mononuclear cells. Following MS-275 treatment, histones H3 and H4 showed increased acetylation and HDAC enzymatic activity was reduced. Caspase-8, -9, and -3 were activated, and caspase substrates PARP and BID were cleaved. Additionally, FLICE-inhibitory protein (FLIP) was downmodulated following MS-275 incubation. MS-275 treatment caused detectable ROS generation after 15 h of incubation, which was blocked by the caspase inhibitor Z-VAD-fmk. Overexpression of Bcl-2 protein protected against MS-275-induced apoptosis. These data demonstrate that MS-275 is a promising therapy for the treatment of CLL, but that in contrast to previous reports, ROS generation does not precede commitment to apoptosis. Similar to many other therapeutic targets, MS-275-mediated apoptosis is reduced by overexpression of Bcl-2, justifying strategies to combine HDAC inhibitors with Bcl-2 antagonists.

Collagenase degradation decreases collagen fibril diameters--an in vitro study of the rabbit medial collateral ligament.

Based on the similarity of fibril diameters in healing and grafted ligaments, it has been speculated that all small fibrils represent newly synthesized collagen. Alternatively, small fibrils in grafts could be due to enzymatic degradation of endogenous large fibrils. This study examined the effect of collagenase on collagen fibril diameters in normal NZW rabbit MCLs. Midsubstance MCL slivers were incubated in buffer for 72 or 144 h for comparison with slivers incubated in buffer containing 4 units/ml bacterial collagenase. The samples were examined under TEM for fibril diameter analysis. Mean fibril diameters of 3-day and 6-day collagenase-treated MCLs were significantly reduced, resembling 40-week scar values. These results suggest that collagenase treatment can alter collagen fibril diameter and shape in normal rabbit MCL, thus it is possible that despite their similarity to ligament scars, that at least some small fibrils in ligament grafts may be enzymatically reduced endogenous fibrils.

Localization and characterization of porcine patellar tendon xenograft antigens in a rabbit model of medial collateral ligament replacement.

BACKGROUND: Ligament injuries of the knee are common and, if severe, can predispose to joint pain, instability, reinjury, and, ultimately, osteoarthritis. Xenograft replacement of ligaments could have potential; however, a limited understanding of the immunology of ligament xenograft rejection has inhibited their use. The purpose of this study was to characterize the antigenic elements of a fresh porcine tendon xenograft in a rabbit model and to provide a better understanding of what would need to be done to either block or extract these antigenic elements. METHODS: Three experimental situations were evaluated in a pig to rabbit ligament transplantation model: subcutaneous implantation of fresh porcine patellar tendon (PPT), implantation of fresh PPT into a medial collateral ligament midsubstance gap, and replacement of the entire medial collateral ligament complex with either fresh or guanidinium hydrochloride-extracted PPT. Preimmune and immune sera were collected from rabbits and used to localize antigenic targets in PPT, meniscus, and cartilage with indirect immunofluorescence techniques. The reactivities of the same rabbit sera towards tissue extracts of PPT, meniscus, and cartilage by Western immunoblot analyses were used to characterize the antigenic components. RESULTS: Indirect immunofluorescence with preimmune rabbit sera on PPT showed staining of tendon fibroblasts. Immune sera from rabbits transplanted with xenografts stained regions of the extracellular matrix of PPT. Fresh PPT induced antibodies that consistently recognized six extracellular matrix components with molecular masses of >200 kDa, 180 kDa, 135 kDa, 108 kDa, 63 kDa, and 59 kDa. CONCLUSIONS: Our results suggest that naturally occurring rabbit anti-pig antibodies of the IgG isotype recognize immunogenic components on tendon fibroblasts, whereas induced rabbit anti-pig antibodies recognize a specific subset of six extracellular matrix components of PPT. PPT xenografts appeared to induce a similar humoral immune response irrespective of graft location. Finally, our results indicate that selective extraction of PPT xenograft components before implantation altered the induced rabbit anti-pig antibody response; however, such extraction did not change the ultimate fate of the transplant tissue.

Influence of source and amount of dietary protein on milk yield by cows in early lactation.

The purpose of this research was to examine the effects of various amounts of CP and RUP on AA flow to the small intestine and milk yield of lactating dairy cows. The first trial was a 5 x 5 Latin square design using five ruminally and duodenally cannulated multiparous cows. Diets contained chopped alfalfa hay, corn silage, high moisture corn, solvent-extracted soybean meal, and specially processed soybean meal (60.2% RUP). Soybean meal replaced high moisture corn to increase dietary CP from 14.5 to 16.5 or 18.5%, and specially processed soybean meal replaced solvent-extracted soybean meal in diets containing 16.5 or 18.5% CP to provide 6.2, 7.3, 6.7, and 8.3% RUP. Increasing dietary CP increased the flows of all AA to the duodenum. Increasing dietary RUP increased flows of Arg, His, Lys, Phe, Asp, and Glu to the duodenum. In a second trial, 36 cows were fed diets similar to those used in trial 1. Increased amounts of RUP in diets tended to increase milk yield because of improved protein status, improved intake of metabolizable energy, or both.

Flows of nitrogen and amino acids in dairy cows fed diets containing supplemental feather meal and blood meal.

Four Holstein cows, fitted with ruminal and duodenal cannulas, were used to determine the effects of supplemental feather meal and blood meal on ruminal fermentation and flows of N and AA to the duodenum. The basal diet contained (DM basis) 9.9% chopped alfalfa hay, 39.7% corn silage, 34.7% cracked corn, 8.2% corn starch, 2.1% vitamins and minerals, 4.4% casein, and 1% urea. A combination of feather meal and blood meal (3:1 on an N basis) was used to replace 0, 33, 67, and 100% of the casein and urea in the basal diet in a Latin square design. Intakes of DM, OM, and N were similar for all diets. Flows of total N, NAN, individual AA, total AA, and total essential AA were unaltered as supplemental feather meal plus blood meal increased. Flows of microbial N tended to decrease, but flow of non-ammonia, nonmicrobial N increased as supplemental feather meal plus blood meal increased. Increased proportions of dietary feather plus blood meal decreased the proportions of Ile, Lys, Met, and Thr in duodenal digesta. Molar percentages of ruminal pH and VFA were unchanged, but concentrations of ruminal NH3 N decreased linearly as supplemental feather meal plus blood meal increased. For the diets fed in this study, inclusion of one-third of the supplemental protein from feather plus blood meal resulted in maximum flows of NAN and microbial N to the small intestine.

Addition of ruminally degradable crude protein and branched-chain volatile fatty acids to diets containing hydrolyzed feather meal and blood meal for lactating cows.

This study investigated the effects of amounts of RDP and branched-chain VFA on milk production and DMI by 32 early lactation Holstein cows fed diets based on corn silage and corn. All supplemental dietary protein was supplied by animal protein by-products and urea. Hydrolyzed feather meal and ring-dried blood meal served as sources of supplemental protein and were fed in a 3:1 ratio on a N basis. The experimental design was a completely randomized design with a 2 x 2 factorial arrangement of treatments. Main factors were percentage of RDP (8.0 vs. 9.5% of dietary DM) and amount of branched-chain VFA in the diet (0 vs. 90 g/d per cow). Urea was used to adjust the amount of degradable CP. Individual DMI, milk production, and milk composition were monitored during wk 5 to 19 of lactation. Ruminal fluid and blood were collected to examine the treatment effects on ruminal VFA patterns and plasma urea N concentrations. The DMI, total milk production, and milk component yield were unaffected by treatments. The molar percentages of isobutryate, isovalerate, and n-valerate increased when branched-chain VFA were fed, and concentrations of urea N in plasma increased with higher percentages of RDP. A combination of feather meal and blood meal can be used as supplemental protein to support high milk production (> 37 kg/d) in early lactation. No production benefits were observed by increased dietary RDP or branched-chain VFA.

Improved techniques for dissociating particle-associated mixed ruminal microorganisms from ruminal digesta solids.

Combinations of physical and chemical methods were evaluated for their ability to remove particle-associated microorganisms (PAM) from saline-washed ruminal digesta solids (SWRDS). Physical methods included chilling and storage, homogenization, multiple extraction, and agitation with marbles. Chemical methods included use of low pH, Tween 80, formaldehyde, methanol, tertiary butanol, and methylcellulose. Microbial removal from SWRDS was determined directly by using epifluorescence microscopy and indirectly by measuring removal of diaminopimelic acid and total purines. Different combinations of methods resulted in removals of 46 to 82% for particle-associated bacteria (PAB), 52 to 98% for particle-associated protozoa (PAP), and 60 to 83% for PAB plus PAP. Two methods were considered most effective, based on microscopy; both removed similar amounts of PAB (79 to 82%) and PAB plus PAP (80 to 83%). In one method, SWRDS were stored for 24 h at 4 degrees C in a solution of pH 2 saline, .1% Tween 80, 1.0% methanol, and 1.0% tertiary butanol. In the other method, SWRDS were incubated for 30 min in .1% methylcellulose before storage for 24 h at 4 degrees C in pH 2 saline, .1% Tween 80, and 1.0% methanol. Common to both treatments was subsequent homogenization of the suspensions for 15 s followed by washing the digesta solids seven times with the treatment solutions. Both methods resulted in values that exceeded those reported previously for removal of PAM from ruminal digesta solids.

Nutrient digestion, nitrogen, and amino acid flows in lactating cows fed soybean hulls in place of forage or concentrate.

Five Holstein cows in early lactation were used in a 5 x 5 Latin square design to study substitution of soybean hulls for portions of forage or concentrate in diets. The control diet consisted of (DM basis) 10% alfalfa hay, 40% corn silage, 25% high moisture corn, 23% protein supplement, and 2.3% vitamins and minerals. Soybean hulls were used to replace approximately 25 and 50% of the forage or concentrate DM in the control ration. The DMI decreased linearly as soybean hulls replaced forage. The DM and OM flow to the duodenum and apparent and true digestion of OM in the stomach were similar among treatments. Ruminal pH was similar among treatments, but concentrations of NH3 N decreased linearly when soybean hulls replaced forage. Total VFA concentrations were similar when soybean hulls replaced forage but showed a positive quadratic response when soybean hulls replaced concentrate. Microbial N averaged 59% of NAN flow and showed a negative quadratic response when soybean hulls replaced concentrate. Flows of total AA and total essential AA to the duodenum were not altered when soybean hulls were fed. Yields of milk and milk components were similar among treatments; hulls can be used effectively to replace forage or concentrate in lactation diets.