[14C]-Glucose Metabolism during Shoot Bud Development in Cultured Cotyledon Explants of Pinus radiata

Excised cotyledons of Pinus radiata D. Don cultured under shoot-forming(plus benzyladenine) and non shoot-forming (minus benzyladenine)conditions for 10 and 21 days were fed U-[¹⁴C]-glucose for 3h in the light followed by a 3 h chase period. The labellingof individual metabolites as well as ¹⁴C incorporation intoprotein was assessed. It was found that the general metabolicpatterns were qualitatively the same in shoot-forming and nonshoot-forming conditions, however, metabolism leading to respirationas well as to the synthesis of some amino acids and proteinsynthesis was enhanced in the shoot-forming cultures. (Received February 16, 1987; Accepted July 8, 1987)     

Safety of Intracoronary Infusion of 20 Million C-Kit Positive Human Cardiac Stem Cells in Pigs

Background

There is mounting interest in using c-kit positive human cardiac stem cells (c-kit^pos hCSCs) to repair infarcted myocardium in patients with ischemic cardiomyopathy. A recent phase I clinical trial (SCIPIO) has shown that intracoronary infusion of 1 million hCSCs is safe. Higher doses of CSCs may provide superior reparative ability; however, it is unknown if doses >1 million cells are safe. To address this issue, we examined the effects of 20 million hCSCs in pigs.

Methods

Right atrial appendage samples were obtained from patients undergoing cardiac surgery. The tissue was processed by an established protocol with eventual immunomagnetic sorting to obtain in vitro expanded hCSCs. A cumulative dose of 20 million cells was given intracoronarily to pigs without stop flow. Safety was assessed by measurement of serial biomarkers (cardiac: troponin I and CK-MB, renal: creatinine and BUN, and hepatic: AST, ALT, and alkaline phosphatase) and echocardiography pre- and post-infusion. hCSC retention 30 days after infusion was quantified by PCR for human genomic DNA. All personnel were blinded as to group assignment.

Results

Compared with vehicle-treated controls (n=5), pigs that received 20 million hCSCs (n=9) showed no significant change in cardiac function or end organ damage (assessed by organ specific biomarkers) that could be attributed to hCSCs (P>0.05 in all cases). No hCSCs could be detected in left ventricular samples 30 days after infusion.

Conclusions

Intracoronary infusion of 20 million c-kit positive hCSCs in pigs (equivalent to ~40 million hCSCs in humans) does not cause acute cardiac injury, impairment of cardiac function, or liver and renal injury. These results have immediate translational value and lay the groundwork for using doses of CSCs >1 million in future clinical trials. Further studies are needed to ascertain whether administration of >1 million hCSCs is associated with greater efficacy in patients with ischemic cardiomyopathy.     

Sar1 GTPase Activity Is Regulated by Membrane Curvature

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5′-[(β,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.     

Shaking a leg and hot to trot: the effects of body size and temperature on running speed in ants

Abstract.  1. Data were compiled from the literature and our own studies on 24 ant species to characterise the effects of body size and temperature on forager running speed.
2. Running speed increases with temperature in a manner consistent with the effects of temperature on metabolic rate and the kinetic properties of muscles.
3. The exponent of the body mass-running speed allometry ranged from 0.14 to 0.34 with a central tendency of approximately 0.25. This body mass scaling is consistent with both the model of elastic similarity, and a model combining dynamic similarity with available metabolic power.
4. Even after controlling for body size or temperature, a substantial amount of inter-specific variation in running speed remains. Species with certain lifestyles [e.g. nomadic group predators, species which forage at extreme (>60 °C) temperatures] may have been selected for faster running speeds.
5. Although ants have a similar scaling exponent to mammals for the running speed allometry, they run slower than predicted compared with a hypothetical mammal of similar size. This may in part reflect physiological differences between invertebrates and vertebrates.     

The scaling of reproductive variability in trees

Seed output in perennial plant populations is temporally variable and often synchronous over large regions. The similarly complex spatiotemporal dynamics of animal populations have been characterized by the power‐law scaling of the variance in population numbers with mean abundance. Here we show that a large compilation of published reproductive time series exhibits largely invariant mean–variance scaling properties across both angiosperm and conifer tree species. A simple model of seed production in tree stands shows that observed values of the scaling exponent reflect very general aspects of plant ecology and life history as well as the temporal dynamics of seed production. Together, these results suggest that the continuum of reproductive variability and synchrony observed in trees may reflect the influence of a common set of ecological processes.     

Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies

In this report we investigated, within a group of closely related single domain camelid antibodies (V_HHs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast‐acting toxin and biothreat agent. The V1C7‐like V_HHs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin‐neutralizing activities. Using the X‐ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta‐based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7's binding affinity for ricin, whereas the reverse (ie, Gly for Arg at position 29) diminished V5C1's binding affinity by >10 fold. As expected, the V5C1_R29G mutant was largely devoid of toxin‐neutralizing activity (TNA). However, the TNA of the V1C7_G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen‐deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function.