Synchronization of estrus in beef cows with Norgestomet-Alfaprostol or Syncro-Mate B

In the spring of 1986, 506 beef cows were used to evaluate the effectiveness of two estrus synchronization systems. Cows were synchronized with either a 6-mg Norgestomet implant placed in the ear for 14 d followed by a 6-mg Alfaprostol injection given 16 d after implant removal (Norgestomet-Alfaprostol) or with Syncro-Mate B (6-mg Norgestomet implant for 9 d with an injection containing 5 mg estradiol valerate and 3 mg Norgestomet at the time of implantation). The Alfaprostol injection in the Norgestomet-Alfaprostol group was given the same day as implant removal in the Syncro-Mate B group. These treatment groups were compared to a group of untreated controls. Cows were allotted to treatments by days postpartum, age and breed. Syncro-Mate B cows had a higher estrous response within 5 d after treatment (78.6 vs 64.0%) and a shorter interval to estrus (39.2 vs 66.7 h) than did Norgestomet-Alfaprostol cows (P < 0.05). Controls had a significantly lower estrous response compared to either of the synchronized groups (27.1%). The degree of estrus synchrony was identical in both synchronization systems (72.7%). Synchronized conception rate tended to be higher (P = 0.06) in the Norgestomet-Alfaprostol cows than in the Syncro-Mate B cows (74.5 vs 62.5%). Synchronized, 21-d, 25-d and breeding season pregnancy rates were 51.2, 70.8, 76.8 and 92.9% for Norgestomet-Alfaprostol cows; 48.5, 63.0, 73.2 and 87.8% for Syncro-Mate B cows; and 15.6, 56.3, 61.3 and 86.9% for control cows. The four pregnancy rates were not different between the two synchronization treatments (P > 0.10). Controls had lower synchronized and 25-d pregnancy rates when compared to either of the synchronized groups (P < 0.05). Days postpartum had no effect on the reproductive performance of cows synchronized with Norgestomet-Alfaprostol. Our results indicate that the Norgestomet-Alfaprostol system is as effective as Syncro-Mate B in synchronizing estrus in beef cows.     

Stochastic dynamics of the nerve growth cone and its microtubules during neurite outgrowth

The controlled extension of neurites is essential not only for nervous system development, but also for effective nerve regeneration after injury. This process is critically dependent on microtubule assembly since axons fail to elongate in the presence of drugs which disrupt normal assembly dynamics. For this reason, neurite outgrowth is potentially controllable by manipulation of the assembly state of the intracellular array of microtubules. Therefore, understanding how microtubule assembly dynamics and neurite outgrowth are coupled, in the absence of drugs, can lend valuable insight into the control and guidance of the outgrowth process. In the present study we characterized the stochastic dynamics of neurite outgrowth and its corresponding microtubule array, which advances concomitantly with the advance of the nerve growth cone, the highly motile structure at the terminus of the growing neurite, using reported fluorescent microscopic image sequences (Tanaka and Kirschner, 1991, J. Cell Biol. 115:345-363). Although previously modeled as an uncorrelated random walk, the stochastic advance of the growth cone was found to be anticorrelated over a time scale of approximately 4 min, meaning that growth cone advances tended to be followed by growth cone retractions approximately 4 min later. The observed anticorrelation most likely reflects the periodic stops and starts of neurite outgrowth that have been reported anecdotally. A strikingly similar pattern of anticorrelation was also identified in the advance of the growth cone's microtubule array. Cross-correlation analysis showed that growth cone dynamics tended to precede microtubule dynamics on a time scale of approximately 0-2 min, while microtubules tended to precede growth cone dynamics on a approximately 0-20-s time scale, indicating a close temporal coupling between microtubule and growth cone dynamics. Finally, the scaling of the mean-squared displacements with time for both the growth cone and microtubules suggested a fractional Brownian motion model which accounts for the observed anticorrelation of growth cone and microtubule advance. (c) 1996 John Wiley & Sons, Inc.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Synchronization of estrus in suckled postpartum beef cows with melengestrol acetate, 48-hour calf removal and PGF2alpha

One hundred and sixty-five suckled postpartum beef cows were utilized to evaluate the effectiveness of 2 estrus synchronization systems for the initiation and synchronization of estrus. The treatment groups consisted of 1) melengestrol acetate (MGA)-PGF2alpha (cows were given 0.5 mg MGA/head/day for 14 d with 25 mg PGF2alpha injected 17 d after the last day of MGA administration); 2) MGA-48-h calf removal (CR)-PGF2alpha (cows were given 0.5 mg MGA/head/day for 14 d with 48-h calf removal starting on the second day after completion of the MGA regimen plus 25 mg PGF2alpha administered 17 d after the last day of MGA); and 3) unsynchronized controls. Cows were assigned to treatments by the numbers of days post partum, body condition, age, and breed of sire. The cows were observed for estrus at 12-h intervals for 5 d after PGF2alpha administration and were artificially inseminated 12 to 18 h after the observed estrus. Both the MGA-PGF2alpha and MGA-CR-PGF2alpha treatments (64.8 and 61.8%) had greater (P < 0.05) 5-d estrus rates than the control treatments (34.5%). The synchronized pregnancy rate was greater (P < 0.05) for the MGA-CR-PGF2alpha than the control treatment.(52.7 vs 30.9%, respectively). The MGA-CR-PGF2alpha cows had a higher 25-d pregnancy rate than either the MGA-PGF2alpha (P < 0.05) or control cows (P < 0.08). Of the anestrous cows at the beginning of treatment, more MGA-CR-PGF2alpha (P = 0.1) and MGA-PGF2alpha cows were cyclic posttreatment than control cows (58.7 and 55.1 vs 44.7%, respectively), suggesting that treatment initiated estrous cycles in only a small number of the anestrous cows. Both MGA-PGF2alpha and MGA-CR-PGF2alpha treatments appear to be effective methods of synchronizing estrus in suckled postpartum beef cows. However, MGA-CR-PGF2alpha was more effective in establishing pregnancy earlier in the breeding season than MGA-PGF2alpha.     

Brownian Dynamics of Subunit Addition-Loss Kinetics and Thermodynamics in Linear Polymer Self-Assembly

The structure and free energy of multistranded linear polymer ends evolves as individual subunits are added and lost. Thus, the energetic state of the polymer end is not constant, as assembly theory has assumed. Here we utilize a Brownian dynamics approach to simulate the addition and loss of individual subunits at the polymer tip. Using the microtubule as a primary example, we examined how the structure of the polymer tip dictates the rate at which units are added to and lost from individual protofilaments. We find that freely diffusing subunits arrive less frequently to lagging protofilaments but bind more efficiently, such that there is no kinetic difference between leading and lagging protofilaments within a tapered tip. However, local structure at the nanoscale has up to an order-of-magnitude effect on the rate of addition. Thus, the kinetic on-rate constant, integrated across the microtubule tip (k_on,MT), is an ensemble average of the varying individual protofilament on-rate constants (k_on,PF). Our findings have implications for both catastrophe and rescue of the dynamic microtubule end, and provide a subnanoscale framework for understanding the mechanism of action of microtubule-associated proteins and microtubule-directed drugs. Although we utilize the specific example of the microtubule here, the findings are applicable to multistranded polymers generally.     

Determinants of Maximal Force Transmission in a Motor-Clutch Model of Cell Traction in a Compliant Microenvironment

The mechanical stiffness of a cell’s environment exerts a strong, but variable, influence on cell behavior and fate. For example, different cell types cultured on compliant substrates have opposite trends of cell migration and traction as a function of substrate stiffness. Here, we describe how a motor-clutch model of cell traction, which exhibits a maximum in traction force with respect to substrate stiffness, may provide a mechanistic basis for understanding how cells are tuned to sense the stiffness of specific microenvironments. We find that the optimal stiffness is generally more sensitive to clutch parameters than to motor parameters, but that single parameter changes are generally only effective over a small range of values. By contrast, dual parameter changes, such as coordinately increasing the numbers of both motors and clutches offer a larger dynamic range for tuning the optimum. The model exhibits distinct regimes: at high substrate stiffness, clutches quickly build force and fail (so-called frictional slippage), whereas at low substrate stiffness, clutches fail spontaneously before the motors can load the substrate appreciably (a second regime of frictional slippage). Between the two extremes, we find the maximum traction force, which occurs when the substrate load-and-fail cycle time equals the expected time for all clutches to bind. At this stiffness, clutches are used to their fullest extent, and motors are therefore resisted to their fullest extent. The analysis suggests that coordinate parameter shifts, such as increasing the numbers of motors and clutches, could underlie tumor progression and collective cell migration.     

Stable kinetochore-microtubule attachment constrains centromere positioning in metaphase

With a single microtubule attachment, budding-yeast kinetochores provide an excellent system for understanding the coordinated linkage to dynamic microtubule plus ends for chromosome oscillation and positioning. Fluorescent tagging of kinetochore proteins indicates that, on average, all centromeres are clustered, distinctly separated from their sisters, and positioned equidistant from their respective spindle poles during metaphase. However, individual fluorescent chromosome markers near the centromere transiently reassociate with their sisters and oscillate from one spindle half to the other. To reconcile the apparent disparity between the average centromere position and individual centromere proximal markers, we utilized fluorescence recovery after photobleaching to measure stability of the histone-H3 variant Cse4p/CENP-A. Newly synthesized Cse4p replaces old protein during DNA replication. Once assembled, Cse4-GFP is a physically stable component of centromeres during mitosis. This allowed us to follow centromere dynamics within each spindle half. Kinetochores remain stably attached to dynamic microtubules and exhibit a low incidence of switching orientation or position between the spindle halves. Switching of sister chromatid attachment may be contemporaneous with Cse4p exchange and early kinetochore assembly during S phase; this would promote mixing of chromosome attachment to each spindle pole. Once biorientation is attained, centromeres rarely make excursions beyond their proximal half spindle.     

The importance of lattice defects in katanin-mediated microtubule severing in vitro

The microtubule-severing enzyme katanin uses ATP hydrolysis to disrupt noncovalent bonds between tubulin dimers within the microtubule lattice. Although its microtubule severing activity is likely important for fundamental processes including mitosis and axonal outgrowth, its mechanism of action is poorly understood. To better understand this activity, an in vitro assay was developed to enable the real-time observation of katanin-mediated severing of individual, mechanically unconstrained microtubules. To interpret the experimental observations, a number of theoretical models were developed and compared quantitatively to the experimental data via Monte Carlo simulation. Models that assumed that katanin acts on a uniform microtubule lattice were incompatible with the in vitro data, whereas a model that assumed that katanin acts preferentially on spatially infrequent microtubule lattice defects was found to correctly predict the experimentally observed breaking rates, number and spatial frequency of severing events, final levels of severing, and sensitivity to katanin concentration over the range 6-300 nM. As a result of our analysis, we propose that defects in the microtubule lattice, which are known to exist but previously not known to have any biological function, serve as sites for katanin activity.     

Serosurvey of Brucella spp. infection in the Kafue lechwe (Kobus leche kafuensis) of the Kafue flats in Zambia

One of the diseases of veterinary and public health importance affecting the Kafue lechwe (Kobus leche kafuensis) on the Kafue flats is brucellosis, for which only scant information is available. During the 2003 (October), 2004 (December), and 2008 (July-December) hunting seasons in the Kafue flats, we conducted a study to determine the seroprevalence of Brucella spp. in the Kafue lechwe and to evaluate serologic tests for detection of Brucella spp. antibodies in lechwe. The Rose Bengal Test (RBT), competitive enzyme-linked immunosorbent assay (cELISA), and fluorescence polarization assay (FPA) were used. A total of 121 Kafue lechwe were hunted for disease investigations in 2003, 2004, and 2008 in the Kafue Flat Game Management Area. Of these, 21.6%, (95% confidence interval [CI]: 14.2-29.1%) had detectable antibodies to Brucella spp. The Kafue lechwe in Lochnivar National Park had higher antibody results than those in Blue Lagoon National Park (odds ratio=3.0; 95% CI: 0.94-9.4). Infection levels were similar in females (21.6%) and males (21.7%). Results were similar among RBT, FPA, cELISA tests, suggesting that these could effectively be used in diagnosing brucellosis in the Kafue lechwe. Our study demonstrates the presence of Brucella infections in the Kafue lechwe in two national parks located in the Kafue flats and further highlights the suitability of serologic assays for testing the Kafue lechwe. Because the Kafue lechwe is the most hunted wildlife species in Zambia, hunters need to be informed of the public health risk of Brucella spp. infection.     

Design of HIV-1 integrase inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75: a scaffold hopping approach using salicylate and catechol groups

HIV-1 integrase (IN) is a validated therapeutic target for antiviral drug design. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands the discovery of novel inhibitors that are structurally as well mechanistically different. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC(50)=5 μM) with more than 40-fold selectivity for the strand transfer over the 3'-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC(50) value of 8 μM. Using molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. Furthermore, the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site on IN. This work provides a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors.