cappuccino (capu) is a Drosophila melanogaster gene required for establishing the dorsal-ventral and anterior-posterior axes of the developing egg and embryo. Egg chambers mutant for capu exhibit cytoplasmic streaming during mid-oogenesis that does not normally occur until late oogenesis. All known capu alleles stream at the same speed. Since capu alleles do differ in their effects on the dorsal-ventral axis, it is unlikely that premature streaming is a cause of the dorsal-ventral defects. Premature streaming may, however, cause the posterior defects. Streaming occurs at the same speed if isolated egg chambers are treated with the actin depolymerizing drug cytochalasin D, which suggests that CAPU may act in the actin cytoskeleton. A screen for proteins which physically interact with CAPU identified profilin, a regulator of the actin cytoskeleton as a probable partner of CAPU. This again suggests that CAPU acts in the actin cytoskeleton. CAPU is a member of the formin homology (FH) family of proteins. Sequence analysis of this family makes it possible to multiply align all family members throughout their carboxy-terminal halves. This makes possible better predictions of secondary structure, phylogenetic analysis, and identification of novel regions of conserved sequence. Analysis of the amino-terminal halves suggests that significant alignment is also possible in these more highly divergent regions. Members of the rho family of small GTPases have been implicated in the regulation of the actin cytoskeleton. They physically associate with members of the FH family, including CAPU. All four rho-like proteins tested associate with CAPU in the Interaction Trap system. There are also indications of genetic interactions between capu and the rho-like genes dcdc42 and drac1.

Forty-five obese postmenopausal Caucasian women were treated with phentermine hydrochloride (Fastin®) and a 1200 kcal diet for weight reduction. Total and regional body composition changes were measured by dual-energy X-ray absorptiometry (DXA) and by anthropometry at 3-mo intervals over 9 mo. Plasma lipids, and serum insulin and leptin were measured. After 3 mo, Fastin® therapy produced a 6.8 kg weight loss (P < 0.01), and DXA-assessed fat and lean mass losses (P < 0.01) of 11.9 and 3.0%. respectively. DXA-estimated regional composition revealed respective fat mass losses of 14.1 and 11.9%. From abdomen and thighs, suggesting primarily subcutaneous and central fat depot losses. DXA reliability was assessed by same-day duplicate measurements (n = 10) calculated using old (version 8.1a) and new (version 8.21) analysis software. A 1.5% between-duplicate difference in lean mass was obtained with the old software; the new software yielded 1.1, 1.4 and 1.6% differences between duplicates for fat, lean and leg lean masses, respectively. CVs ranged from 1.7% for bone mineral content to 12.0% for arm fat mass for both versions. The new software produced higher values for all variables, except arm fat and lean masses than the old software. Except for a 1.9% trunk fat loss detected with the old software, magnitude of body composition changes over 3 mo in 21 weight-stable subjects was the same. The new software estimated total weight more accurately and with less variability than the old software. Fastin®-treated women lost (P < 0.01) 10% of their baseline body weight over 9 mo which correlated with a 20% reduction (P < 0.01) in serum leptin concentration. Plasma HDL-cholesterol concentration increased (P < 0.01) by 15% over 9 mo while total- and LDL-cholesterol and triglycerides decreased 14.2 (P < 0.01), 25.4 (P < 0.01) and 12.2% (P < 0.05), respectively. Serum insulin was unaffected by weight reduction. These data suggest that FastinRTM therapy was effective in reducing weight, and in producing a healthier body fat distribution and plasma lipid profile, thereby lowering cardiovascular disease risk in obese postmenopausal women. The DXA instrument (Holologic QDR-4500A. Hologic Inc. Waltham, MA) gave reproducible estimates of composition change in this population regardless of the software version used.

The olfactory system of the hawkmoth Manduca sexta is able to discriminate among odors and to mediate a behavioral response, such as feeding or oviposition in females. This dissertation research describes attempts to elucidate neural coding involved in the processing of odors within the first-order olfactory center of the brain, the antennal lobe (AL), in female moths. This study focused on a small and identifiable subset of projection neurons within the AL, which have their cell bodies grouped on the anterior surface of the AL (AC neurons). The subpopulation of about 14-20 neurons was characterized physiologically and morphologically. The anatomical features of AC neurons and their physiological tuning specificities to plant-associated odors were investigated by means of electrophysiological recording and staining techniques. All AC neurons identified were output neurons, each having a distinct uniglomerular dendritic branching pattern. The entire population of AC neurons, which sometimes exhibited a separation of its cell body group into two clusters (primary and satellite), projected into two small groups of glomeruli. One group was located dorsally and medially in the posterior part of the AL and probably consists of five glomeruli. The other group was situated dorsally and anteriorly and possibly consists of one uniquely identifiable glomerulus. Furthermore, the results indicate response specificity and a narrow receptive range of certain AC neurons in response to flowers of Solanaceous hostplants. Among the odors tested were nitrogen (N)-bearing compounds, monoterpenes, aromatics and green-leaf volatiles. The response profiles were divided into different response categories: "excited" (multiphasic), "inhibited", "mixed" and "no response to odors tested". The majority of AC neurons were spontaneously active and exhibited rhythmic and periodic bursting (burst frequency 1-3 Hz). Also, AC neurons showed low-amplitude, membrane oscillations. These oscillations, as well as the bursts, are not odor-induced. In summary, the data show that the organization of a subset of unique, ordinary AC glomeruli is spatially distinct and recognizable, and functionally significant for hostplant odor processing in the AL. The mechanisms underlying processing seem to include a rate code (encoding concentration) and possibly a complex spatial code (encoding quality).

Growing beef steers were used in completely randomized block designs to evaluate effect of processing method, dry-rolled (DR) versus steam-flaked (SF) sorghum, and degree of processing (flaking density) of corn and sorghum grain on nitrogen (N) digestion and post-absorptive N metabolism. In experiments (Exp.) 1 and 2, seven duodenally and ileally cannulated steers were used to investigate N digestibilities. Experimental diets contained 77% grain: Exp. 1, DR or SF sorghum at densities of 437, 360 and 283 g/L (SF34, SF28, SF22); Exp. 2, corn at two flake densities (SF34 and SF22). Nitrogen intakes by steers fed sorghum (142 g/d) and corn (149 g/d) diets were similar among treatments. Steers fed SF compared to DR, increased ruminal (P =.04) DM digestibility. Microbial protein flow to the duodenum averaged 10% greater for SF vs DR. Reducing flaking density of sorghum grain linearly increased (P .05) ruminal DM and tended to increase linearly ruminal feed N (P =.11), total tract DM and N digestibilities and microbial efficiency (P .08). Unexpectedly, flake density did not affect DM or N digestibilities of SF corn diets by steers. In Exp. 3, eight steers implanted with indwelling catheters were used to measure blood flow, net absorption and uptake of ammonia-N, urea-N (UN) and alpha-amino N (AAN) across portal-drained viscera (PDV), hepatic and total splanchnic tissues. Experimental diets were the same as those in Exp. 1. Six arterial, portal and hepatic blood samples were collected per day at 2 h intervals for each diet and steer. Daily DM and N intakes averaged 7.0 kg and 142 g, respectively. Decreasing flake density of SF sorghum linearly increased net absorption of AAN (P =.04) and UN recycling to the gut (P =.02). Net UN recycling to the gut averaged 38% of N intake across treatments. Steers fed SF compared to DR decreased (P =.03) net splanchnic UN output (33 vs 50 g/d). The improved N retention and lower splanchnic UN output, contingent with greater ruminal microbial protein synthesis and flow to intestines may explain in part the observed higher performance of cattle fed SF compared to DR sorghum. Based on improved total tract N digestibilities and greater net absorption of AAN and UN recycling to the gut, optimum flake density for SF sorghum grain was 283 g/L (SF22).

Lactating Holstein dairy cows, fitted with appropriate blood vessel catheters, were used in two cross-over experiments to investigate effects of feeding diets with 40% steam-flaked (28 lb/bu; SF) vs dry-rolled (DR) sorghum (experiment 1; 8 cows), or 40% steam-flaked corn grain (experiment 2; 6 cows) of different flake densities, 38 lb/bu (SF38) vs 28 lb/bu (SF28), on postabsorptive nutrient fluxes. Net uptake or output of glucose, L-lactate, volatile fatty acids (VFA) and β-hydroxybutyrate (BHBA) was measured across portal-drained viscera (PDV), hepatic, splanchnic and mammary tissues. Compared to DR, feeding cows SF sorghum increased net PDV absorption of propionate by 25% (P =.08), tended to increase splanchnic output of glucose (+16%) and L-lactate (+80%; P ≤ .20), but did not alter mammary glucose or L-lactate uptake. Sorghum processing did not affect net splanchnic or mammary metabolism of acetate and butyrate. Splanchnic output of BHBA was greater (P =.08), but mammary uptake was lower (P =.09) for cows fed DR vs SF diets. For cows fed corn diets, hepatic synthesis of glucose was higher (3.0 vs 2.8 kg/d; P =.04) for SF28 vs SF38. Flake density did not affect net splanchnic or mammary metabolism of acetate. For cows fed SF38, higher (P =.03) net PDV absorption of butyrate possibly inhibited hepatic gluconeogenesis from propionate, which might explain a higher (P =.10) hepatic uptake of L-lactate. Processing of corn or sorghum did not alter estimated total net PDV absorption or total splanchnic output of energy. Net total PDV absorption of VFA tended to be greater for cows fed SF than DR (+17%; P =.17), and for SF38 than SF28 (+10%; P =.15). Total mammary uptake of energy was not different for DR vs SF, but was greater for cows fed SF28 vs SF38, due mainly to increased uptake of glucose (+25%; P < .01). In both experiments, hepatic glucose synthesis was more than adequate for milk lactose production. These changes in partitioning of nutrients and estimated energy across portal-drained viscera, liver, total splanchnic, and mammary tissues may partially explain increased milk protein synthesis (in longer term lactation studies) by dairy cows fed more extensively processed corn (SF28) and sorghum (SF) grain.

Diabetics suffer from a pro-thrombotic condition. It is established that platelets are activated in type 2 diabetes. However the mechanisms of platelet activation in this disease are not yet known. The purpose of these studies was to elucidate the mechanisms of platelet activation and the effect these mechanisms have on type 2 diabetic platelets. Apoptosis of cells is regulated by caspases, a group of cysteine proteases. When platelets become activated, they express phosphatidylserine (PS) on the outer leaflet of the plasma membrane as well as form platelet microparticles (PMPs). In addition, platelets aggregate when activated. We found that platelets from diabetic subjects contain activated caspases. These platelets also formed increased numbers of PMPs compared to platelets from non-diabetic subjects. We observed a 30-fold increase in thrombin activity in the plasma from diabetics. To determine if caspases were involved in platelet activation, we determined if caspase inhibition (using the pan-caspase inhibitor zVAD-fmk); (1) decreased PS expression and (2) decreased platelet aggregation following activation. We found that platelets treated with zVAD-fmk significantly decreased A23187-induced PS exposure as well as aggregation. There is limited information on the role of caspases on platelet adhesion proteins such as P-selectin or GPIIb/IIIa during platelet activation. Therefore, we tested if caspase inhibition attenuated P-selectin and GPIIb/IIIa expression. Using blood from non-diabetic volunteers, we found that treatment with zVAD-fmk caused a significant attenuation of P-selectin expression in stimulated platelets. Together these data suggest that caspases play a novel role in platelet activation. We also wanted to determine if treating type 2 diabetic rats (Zucker Diabetic Fatty rats, ZDF) with the caspase inhibitor zVAD-fmk in vivo ; (1) attenuated PS expression, (2) attenuated platelet microparticle (PMP) formation and (3) attenuated platelet aggregation. ZDF rats were treated in vivo with 40 μg zVAD-fmk for 4 days. We found attenuated PS exposure, PMP formation as well as decreased aggregation in ZDF rats treated with zVAD-fmk. Our overall results demonstrate a novel role of caspases in platelet activation. Together, these studies may lead to development of novel treatments for pathophysiologic states associated with platelet activation such as diabetes, myocardial infarction, and stroke.

Type 2 diabetes is a major risk factor in cardiovascular disease especially ischemic heart disease. The American Diabetes Association advocates that all diabetics are at risk for coronary myocardial infarctions (MI). Restoration of blood flow to the area of the myocardium that has undergone ischemia is the most important therapeutic goal in treating MI. However, restoration of blood to the myocardium contributes to further damage to the ischemic area. This phenomenon is known as ischemia-reperfusion (I-R) injury. Previous studies have found that experimentally induced diabetic animals are more susceptible to I-R injury compared to non-diabetic animals. One reason why diabetic hearts may be more susceptible to I-R injury may be due to the increased oxidative stress in diabetics. Diabetics also suffer from increased blood coagulation possibly due to increased thrombin activity. Thrombin, independent of its role in coagulation, may be contributing to additional myocardial damage following ischemia. This dissertation describes three experiments: First, determine if a non-thrombogenic dose of thrombin impairs cardiac recovery and compliance in isolated non-diabetic rat hearts using a red cell perfusate following 30 minutes of global ischemia. Second, determine if type 2 diabetic rats exhibit increased platelet microparticle formation (PMP), increased thrombin activity and increased coagulability. Third, determine if MI's were larger in diabetic rat hearts compared to non-diabetics and if vitamin E reduced MI size and improve cardiac function following coronary artery occlusion. We found that a non-thrombogenic thrombin dose significantly reduced recovery from global I-R and reduced myocardial compliance. Thrombin may act as a contributing factor in cardiovascular pathology independent of its role in coagulation. We found that thrombin production is increased 30 fold in type 2 diabetic rats compared to non-diabetic rats and PMP production is significantly increased in diabetics possibly resulting in a hypercoagulable state. Finally, we show that MI's are significantly larger in type 2 diabetic hearts compared to non-diabetics. Treatment with Vitamin E significantly reduced MI size and improved left ventricular function in treated diabetic rats compared to non-treated rats. These results help to explain the increased cardiovascular injury seen in the diabetic heart. Vitamin E may be a useful adjuvant to help diabetics reduce oxidative stress, minimizing cardiac damage done by oxidation compounds and activated blood coagulation components in the diabetic heart.

Diabetics have a much greater morbidity and mortality due to coronary heart disease (CHD) than non-diabetics. Furthermore, diabetic women have a 3.8 fold greater risk for CHD compared to diabetic men. Inflammation is now considered a risk factor for cardiovascular disease and also plays a role in diabetes. It is possible that diabetic women with cardiovascular disease (CVD) have a greater inflammatory response and increased interaction between white cells and platelets than diabetic men with CVD or non-diabetic women with CVD. This study tested the hypothesis that platelet-neutrophil conjugates, platelet activation, neutrophil activation, and cytokine production (interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and interleukin-1 (IL-1β) are increased in diabetic women with CVD compared to diabetic men with CVD and non-diabetic women with CVD. Neutrophil activation was assessed by measuring the expression of neutrophil CD11b and the production of Reactive Oxygen Species (ROS). We found that the baseline expression of CD11b and ROS was not statistically different among any of the groups. Platelet activation was quantified by the expression of GPIIb/IIIa and P-selectin. We found that the baseline expression of GPIIb/IIIa was not significantly different among any of the groups. Diabetic women with CVD had a 2 fold greater expression of platelet P-selectin compared to diabetic women without CVD. We also found the platelet-neutrophil conjugate reactivity to platelet activating factor (PAF) was significantly increased by 60% in diabetic and non-diabetic women with CVD in comparison to diabetic men with CVD. Finally, we found that IL-6 was increased over fourfold in diabetic women with CVD compared to non-diabetic women. These results indicate that platelets are chronically activated and IL-6 is chronically elevated in diabetic women with CVD compared to diabetic women without CVD and may contribute to thrombosis and the greater severity of coronary heart disease observed in diabetic women. The platelet-neutrophil conjugates may contribute to thrombosis/inflammation and the greater severity of coronary heart disease observed in diabetic women as compared to diabetic men. These aspects of inflammation may indicate one of the processes that exacerbate cardiovascular disease in diabetic women.

Multiple studies were conducted to evaluate management options for cows in a thermal stress environment. Those options included cooling to enhance cow comfort, recombinant bovine somatotropin (rbST; Posilac®) to potentially increase milk production, and reproductive hormone scheduling to theoretically coordinate follicular growth leading to ovulation. In the first series of trials measuring the effects of cooling and rbST on milk yield, reproductive performance, and health in Jersey cows during two summer of thermal stress, cows were divided into on of two pens. Both Year 1 and Year 2 control cows (n = 143 and n = 183, respectively) were housed in a pen with no cooling other than shade. The cooled treatment cows each year (n = 142, n = 180) were housed in a pen utilizing the Universal Fog cooling system. One half of the cows in each group was assigned to receive rbST on d 63 postpartum (pp). Cows were assigned to the trial at various days pp, but no cow was assigned prior to d 63, coincident with commencement of rbST injections. The main effect for cooling in combination with rbST increased milk yield compared to no cooling and no rbST for 1999 and 2000 (25.5 vs 21.8 kg/d, and 23.7 vs 20.5, respectively; P < 0.05). Cooling and rbST effects on milk yield were additive the first year, but had a synergistic interaction the second year. Incidences of mastitis (8 vs 17; P < 0.05) for both years and laminitis (2 vs 7; P < 0.05) for Year 1 were both reduced. Reproductive performance was improved in cows given access to cooling (126 pregnant and 6 abortions) vs shade only (112 pregnant vs 13 abortions) in Year 1 (P < 0.05). Additional income over cooling cost was 67¢/cow per day for Year 1 and 52¢/cow per day for Year 2. In the second series of experiments, Holstein and Brown Swiss cows at 56 ± 3.5 d pp were used to evaluate ovulation rates over three seasons, including two summers when half the cows were cooled. All cows received rbST beginning d 63 ± 3.5 pp regardless of treatment. For Exp.1, 58 cows were assigned at calving, beginning June 1, 1999, to either a cooled (Korral Kool™) or non-cooled (shade only, control) pen. At d 56 ± 3.5 pp, all cows commenced a hormonal program coined Select Synch, comprised of an injection of GnRH (100 μg) agonist (Factrel®) followed 7 d later with an injection of PGF₂α (25 mg In-Synch™), at which time ultrasonography was initiated and continued until ovulation or follicular turnover. Experiment 2 was conducted the same as Exp. 1, with the assignment of cows starting Nov. 1, 1999. In Exp.3, all cows were assigned the same as Exp. 1 and 2 beginning June 1, 2000. At d 56 ± 3.5 pp, cows were scheduled to commence Ovsynch, which was identical to Select Synch, except a second Factrel® injection was administered 33 h after 35 mg Lutalyse®. Ultrasonography was the same as described above. Ovsynch led to a higher frequency of ovulations for the non-cooled and cooled cows (77.3 and 69.6%, respectively), than Select Synch for the non-cooled, cooled, and winter treatments (27.6, 24.1 and 29.4%, respectively). Ovulation frequencies were related more to hormonal programming rather than the season.

The occupational chemicals 4-vinylcyclohexene (VCH) and butadiene diepoxide (BDE) have shown an ovarian effect in small pre-antral follicles in mice and rats (Flaws et al., 1994b; Doerr et al. , 1995). VCH exists as two enantiomers, S-VCH and R-VCH. Daily dosing with the enantiomers affected small primary follicles and wet tissue weights in B6C3F₁ mice. The S-enantiomer decreased small primary follicle numbers (P < 0.05) and both enantiomers reduced the weights of the ovary and uterus (p < 0.05). The results suggest the S-VCH enantiomer may have a greater ovotoxic effect as compared to the R-VCH enantiomer. In F344 rats, daily dosing with BDE reduced overall body weight (p < 0.05). There was no affect on ovarian follicle numbers. However, other ovarian effects were seen: decreased ovarian weights, delayed vaginal opening, decreased CL numbers and decreased caspase-3 activity in large antral follicles. Overall, BDE had a general toxic effect with a more specific effect in the ovary.