Diabetes mellitus is a chronic disease associated with vascular complications. Vascular endothelial dysfunction caused by increased endothelial cell apoptosis contributes to diabetic cardiovascular complications. The glucose metabolic by-product, D-lactate, is elevated in diabetics and it is unknown whether it contributes to endothelial cell apoptosis. We hypothesized that diabetic D-lactate levels induce apoptosis in human vascular endothelial cells (HUV-EC-C). HUV-EC-C were incubated with 0.2 mM D-lactate (DLA) and mRNA expression of PI3K/AKT pathway members (AKT1, Bcl-2, BAD, eNOS, PI3K) were measured using Quantitative RT-PCR. DLA downregulated all genes at 6 and 24 hours, followed by increase in expression after 48 hours except PI3K, which remained below control. To further investigate apoptosis, the Human Apoptosis PCR Array was used and expression of all proapoptotic genes (TNF family members) and antiapoptotic genes (IAP family members) were decreased and increased, respectively, at 24 hours followed by an increase and decrease, respectively, at 48 hours. Caspase activity, measured using the Caspase-Glo® 3/7 Assay after HUV-EC-C exposure to 0.2 mM DLA alone or in combination with 20 mM glucose (GLU) or 5 µM methylglyoxal (MG), was increased after 1, 72, and 96 hours. Furthermore, to know whether DLA (0.2 mM) and DLA (0.2 mM), GLU (20 mM) and MG (5 µM) combined cause changes in cellular energy metabolism, creatine (Cr) and high-energy phosphate substrates (CrP, ATP, ADP, AMP) were quantified using HPLC and no changes were observed. We further measured ROS production in HUV-EC-C treated with 0.06-2 mM DLA alone or 0.2 mM DLA with 5-30 mM GLU or 5-160 μM MG. All DLA concentrations increased ROS production by 160% to 216%. DLA with GLU or MG significantly increased ROS production compared to GLU or MG alone. Lastly, D-lactate dehydrogenase (D-LDH) expression was determined using Quantitative RT-PCR and D-LDH was not detected in HUV-EC-C. In conclusion, DLA altered expression of different pro- and anti-apoptotic genes in HUV-EC-C. Furthermore, exposure of HUV-EC-C to DLA levels typically present in diabetics resulted in time-dependent changes in caspase activity, possibly due to excessive ROS production. Whether these changes eventually lead to endothelial dysfunction in diabetes needs further investigation.

The glyoxalase pathway is widespread in both prokaryotic and eukaryotic organisms. This system utilizes two enzymes (glyoxalase I (GlxI) and glyoxalase II (GlxII)) to catalyze the formation of D-lactate from the substrates glutathione (GSH) and methylglyoxal (MG). The latter chemical is a harmful byproduct of glycolysis. This thesis gives detailed studies of the behavior of the GlxI enzyme as it pertains to its thiol co-substrate specificity, its structural similarity among its superfamily members (most particularly with the fosfomycin resistance protein (FosA)) and residue identification that would alter its metal selectivity. The thiol co-substrate GSH was thought to be the only thiol utilizied by the glyoxalase system. However, reports identified organisms that utilized the thiols trypanothione (T(SH)2) and glutahionylspermidine (GspdSH) as co-substrates. These organisms, known as the trypanosomes, are very well known in tropical environments to cause diseases. E. coli does not contain T(SH)2 but does contain GspdSH and manufactures the latter in increasing amounts under conditions of cell duress. Substrate specificity studies were conducted replacing GSH with GspdSH and T(SH)2. In addition to this, to ensure the thiols reacted in a true glyoxalase system, substrate specificity studies were also conducted on the second enzyme GlxII and verification of the product D-lactate was performed. To continue, structurally, the enzyme GlxI belongs to the βαβββ superfamily of proteins that are known to have very similar structure but to catalyze very different reactions. Comparing the active site of E. coli GlxI and FosA, there is one significant difference at one residue. Therefore an E56A mutation was performed on GlxI and the mutant bacterium were subjected to growth analysis in the presence of fosfomycin and MG. The mutant enzyme was also tested for its performance in the presence of MG and various divalent metals. Further, the Glx I enzyme from E. coli is known to be active in the presence of non-zinc bivalent metals, while the human counterpart is active in the presence of Zn2+. When one compares GlxI from E. coli with the human GlxI, there are many differences in the primary structure that could be viable areas that determine the metal specificity of the enzyme. Mutation analysis was performed on these areas to determine catalytic performance as well as metal specificity. These studies display how versatile the glyoxalase system is with regard to the use of its thiol co-substrates. These thiols participate in the detoxification pathway for MG in the cell especially under late log phase conditions. Structural studies can give some knowledge concerning the possible evolution of the enzyme among its family members, and is of monumental significance to the scientific community as it relates to enzyme metal selectivity and the development of enzymes over time.

The glyoxalase pathway is widespread in both prokaryotic and eukaryotic organisms. This system utilizes two enzymes (glyoxalase I (GlxI) and glyoxalase II (GlxII)) to catalyze the formation of D-lactate from the substrates glutathione (GSH) and methylglyoxal (MG). The latter chemical is a harmful byproduct of glycolysis. This thesis gives detailed studies of the behavior of the GlxI enzyme as it pertains to its thiol co-substrate specificity, its structural similarity among its superfamily members (most particularly with the fosfomycin resistance protein (FosA)) and residue identification that would alter its metal selectivity. The thiol co-substrate GSH was thought to be the only thiol utilizied by the glyoxalase system. However, reports identified organisms that utilized the thiols trypanothione (T(SH)2) and glutahionylspermidine (GspdSH) as co-substrates. These organisms, known as the trypanosomes, are very well known in tropical environments to cause diseases. E. coli does not contain T(SH)2 but does contain GspdSH and manufactures the latter in increasing amounts under conditions of cell duress. Substrate specificity studies were conducted replacing GSH with GspdSH and T(SH)2. In addition to this, to ensure the thiols reacted in a true glyoxalase system, substrate specificity studies were also conducted on the second enzyme GlxII and verification of the product D-lactate was performed. To continue, structurally, the enzyme GlxI belongs to the βαβββ superfamily of proteins that are known to have very similar structure but to catalyze very different reactions. Comparing the active site of E. coli GlxI and FosA, there is one significant difference at one residue. Therefore an E56A mutation was performed on GlxI and the mutant bacterium were subjected to growth analysis in the presence of fosfomycin and MG. The mutant enzyme was also tested for its performance in the presence of MG and various divalent metals. Further, the Glx I enzyme from E. coli is known to be active in the presence of non-zinc bivalent metals, while the human counterpart is active in the presence of Zn2+. When one compares GlxI from E. coli with the human GlxI, there are many differences in the primary structure that could be viable areas that determine the metal specificity of the enzyme. Mutation analysis was performed on these areas to determine catalytic performance as well as metal specificity. These studies display how versatile the glyoxalase system is with regard to the use of its thiol co-substrates. These thiols participate in the detoxification pathway for MG in the cell especially under late log phase conditions. Structural studies can give some knowledge concerning the possible evolution of the enzyme among its family members, and is of monumental significance to the scientific community as it relates to enzyme metal selectivity and the development of enzymes over time.

No description available.

The objective of this study was to investigate whether it is possible to create acute laminitis in young ruminating calves by feeding high carbohydrate diets. Three experiments were performed. In the first, 16 calves, 17 wk of age, were fed one of four diets (4 replications) that contained either 71 or 81% of TDN and either 15 or 20% CP. Jugular blood and rumen fluid were sampled and hoof temperature measured at frequent intervals over a subsequent 2-d period. Calves responded acutely to the 81% TDN diets by anorexia, stiffness and diarrhea. Ruminal pH was lower and L- and D-lactates greater in the rumen of 81% TDN treatments. Total ruminal VFA decreased as pH declined. Proportion of acetate increased while propionate decreased in 81% TDN treatments. Butyrate differed but was not dietary related. Whole blood L-lactate did not differ by treatments. Blood D-lactate increased significantly in the calves fed 81% TDN, peaking at 32 h (65 mg/dL). Hoof temperature was significantly lower in 81% TDN treatments at 28-32 h. In the second experiment dietary sodium bicarbonate (.9% of DM) attenuated lactic acidosis in animals which consumed high quantities of concentrate. Although some animals in the buffer group suffered from acidosis, sickness was potentiated in nonbuffer group. Buffer inhibited the decline of ruminal fluid pH, and the increase of lactate in the rumen. Total VFA in the rumen declined with pH. Proportions of major VFA remained unchanged. Blood L-lactate increased at 28 h in animals which did not receive buffer. Blood D-lactate increased in both treatments and was greater in nonbuffer treatment. In the third experiment, 24 calves, 17 wk of age, were fed diets containing either 68 or 80% TDN. The latter diet was supplemented with either ionophore, buffer or contained no supplementation. Sudden introduction of the diets resulted in transient lactic acidosis. Buffer was more efficient in the prevention of acidosis than ionophore. The acetate : propionate (Ac:Pr) ratio tended to be lower in ionophore treatment. Growth performance did not differ between the treatments. Acute laminitis was not detected but the reduction of ruminal pH and a many fold increase in blood D-lactate observed in this study, may contribute to occurrence of laminitis. / Ph. D.