Fluorometric analysis of transferable membrane pores

When pore-forming factors insert into the hyperpolarized membranes of lipid vesicles, ion gradients are rapidly equilibrated, effecting complete depolarization. This process can be conveniently followed with a potentiometric cyanine dye. The generality of the method is demonstrated by applications to three diverse materials. The well-studied gramicidin channel is used to demonstrate that the method is sensitive down to concentrations of 10(-12)M. An extract from the shark repellent skin secretion of the Red Sea flatfish displays activity in the assay and is used to demonstrate the potential of the method to elucidate some of the characteristics of the pore, including its molecularity. That membrane-active factors can be detected and assayed in crude preparations is demonstrated with an impure extract of "amoebapore" from Entamoeba histolytica. In addition, variation of the buffer composition surrounding the vesicles can provide information about the ion selectivity of the pore under investigation.     

Effectors of lipoprotein lipase secretion from isolated cardiac muscle cells incubated in vitro

Cycloheximide, colchicine, tunicamycin, glucagon, dibutyryl-3′–5′-cyclic AMP, dexamethasone and hydrocortisone had no effect on the lipoprotein lipase activity associated with rat cardiac muscle cells incubated $\text{in vitro}$. However, the steroid hormones and inhibitors affected profoundly the appearance of extracellular enzyme during the incubations. The pattern of effects, was consistent with lipoprotein lipase being a normal secretory product of heart muscle cells.     

Estimating dataset size requirements for classifying DNA microarray data.

A statistical methodology for estimating dataset size requirements for classifying microarray data using learning curves is introduced. The goal is to use existing classification results to estimate dataset size requirements for future classification experiments and to evaluate the gain in accuracy and significance of classifiers built with additional data. The method is based on fitting inverse power-law models to construct empirical learning curves. It also includes a permutation test procedure to assess the statistical significance of classification performance for a given dataset size. This procedure is applied to several molecular classification problems representing a broad spectrum of levels of complexity.     

Pathology Associated with AAV Mediated Expression of Beta Amyloid or C100 in Adult Mouse Hippocampus and Cerebellum

Accumulation of beta amyloid (Aβ) in the brain is a primary feature of Alzheimer’s disease (AD) but the exact molecular mechanisms by which Aβ exerts its toxic actions are not yet entirely clear. We documented pathological changes 3 and 6 months after localised injection of recombinant, bi-cistronic adeno-associated viral vectors (rAAV2) expressing human Aβ40-GFP, Aβ42-GFP, C100-GFP or C100^V717F-GFP into the hippocampus and cerebellum of 8 week old male mice. Injection of all rAAV2 vectors resulted in wide-spread transduction within the hippocampus and cerebellum, as shown by expression of transgene mRNA and GFP protein. Despite the lack of accumulation of Aβ protein after injection with AAV vectors, injection of rAAV2-Aβ42-GFP and rAAV2- C100^V717F-GFP into the hippocampus resulted in significantly increased microgliosis and altered permeability of the blood brain barrier, the latter revealed by high levels of immunoglobulin G (IgG) around the injection site and the presence of IgG positive cells. In comparison, injection of rAAV2-Aβ40-GFP and rAAV2-C100-GFP into the hippocampus resulted in substantially less neuropathology. Injection of rAAV2 vectors into the cerebellum resulted in similar types of pathological changes, but to a lesser degree. The use of viral vectors to express different types of Aβ and C100 is a powerful technique with which to examine the direct in vivo consequences of Aβ expression in different regions of the mature nervous system and will allow experimentation and analysis of pathological AD-like changes in a broader range of species other than mouse.     

Growth characteristics of microorganisms on commercially available animal-free alternatives to tryptic soy medium

The growth characteristics of a range of bacteria and fungi that are routinely used in quality control practices were compared for two representative vegetable-based tryptic soy formulations. All of the representative microorganisms grew well on the vegetable-based media and the media provided suitable recoveries of the organisms following simulated storage. Subtle phenotypic changes were observed between cultures grown on different media, but these did not significantly change the strain identification.     

STIM1 and the noncapacitative ARC channels

Our understanding of the nature and regulation of receptor-activated Ca(2+) entry in nonexcitable cells has recently undergone a radical change that began with the identification of the stromal interacting molecule proteins (e.g., STIM1) as playing a critical role in the regulation of the capacitative, or store-operated, Ca(2+) entry. As such, current models emphasize the role of STIM1 located in the endoplasmic reticulum membrane, where it senses the status of the intracellular Ca(2+) stores via a luminal N-terminal Ca(2+)-binding EF-hand domain. Dissociation of Ca(2+) from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca(2+) channels (e.g., CRAC channels). Thus, the specific dependence on store-depletion, and the role of the Ca(2+)-binding EF-hand domain in this process, are critical to all current models of the action of STIM1 on Ca(2+) entry. However, until recently, the effects of STIM1 on other modes of receptor-activated Ca(2+) entry have not been examined. Surprisingly, we found that STIM1 exerts similar, although not identical, actions on the arachidonic acid-regulated Ca(2+)-selective (ARC) channels-a widely expressed mode of agonist-activated Ca(2+) entry whose activation is completely independent of Ca(2+) store depletion. Regulation of the ARC channels by STIM1 is not only independent of store depletion, but also of the Ca(2+)-binding function of the EF-hand, and translocation of STIM1 to the plasma membrane. Instead, it is the pool of STIM1 that constitutively resides in the plasma membrane that is critical for the regulation of the ARC channels. Thus, ARC channel activity is selectively inhibited by exposure of intact cells to an antibody targeting the extracellular N-terminal domain of STIM1. Similarly, introducing mutations in STIM1 that prevent the N-linked glycosylation-dependent constitutive expression of the protein in the plasma membrane specifically inhibits the activity of the ARC channels without affecting the CRAC channels. These studies demonstrate that STIM1 is a far more universal regulator of Ca(2+) entry pathways than previously assumed, and has multiple, and entirely distinct, modes of action. Precisely how this same protein can act in such separate and specific ways on these different pathways of agonist-activated Ca(2+)entry remains an intriguing, yet currently unresolved, question.     

The N-terminal domains of myosin binding protein C can bind polymorphically to F-actin

The regulation of vertebrate striated muscle contraction involves a number of different molecules, including the thin-filament accessory proteins tropomyosin and troponin that provide Ca²⁺-dependent regulation by controlling access to myosin binding sites on actin. Cardiac myosin binding protein C (cMyBP-C) appears to modulate this Ca²⁺-dependent regulation and has attracted increasing interest due to links with inherited cardiac diseases. A number of single amino acid mutations linked to clinical diseases occur in the N-terminal region of cMyBP-C, including domains C0 and C1, which previously have been shown to bind to F-actin. This N-terminal region also has been shown to both inhibit and activate actomyosin interactions in vitro. Using electron microscopy and three-dimensional reconstruction, we show that C0 and C1 can each bind to the same two distinctly different positions on F-actin. One position aligns well with the previously reported binding site that clashes with the binding of myosin to actin, but would force tropomyosin into an “on” position that exposes myosin binding sites along the filament. The second position identified here would not interfere with either myosin binding or tropomyosin positioning. It thus appears that the ability to bind to at least two distinctly different positions on F-actin, as observed for tropomyosin, may be more common than previously considered for other actin binding proteins. These observations help to explain many of the seemingly contradictory results obtained with cMyBP-C and show how cMyBP-C can provide an additional layer of regulation to actin-myosin interactions. They also suggest a redundancy of C0 and C1 that may explain the absence of C0 in skeletal muscle.     

Savanna chimpanzees, Pan troglodytes verus, hunt with tools

Although tool use is known to occur in species ranging from naked mole rats [1] to owls [2], chimpanzees are the most accomplished tool users [3-5]. The modification and use of tools during hunting, however, is still considered to be a uniquely human trait among primates. Here, we report the first account of habitual tool use during vertebrate hunting by nonhumans. At the Fongoli site in Senegal, we observed ten different chimpanzees use tools to hunt prosimian prey in 22 bouts. This includes immature chimpanzees and females, members of age-sex classes not normally characterized by extensive hunting behavior. Chimpanzees made 26 different tools, and we were able to recover and analyze 12 of these. Tool construction entailed up to five steps, including trimming the tool tip to a point. Tools were used in the manner of a spear, rather than a probe or rousing tool. This new information on chimpanzee tool use has important implications for the evolution of tool use and construction for hunting in the earliest hominids, especially given our observations that females and immature chimpanzees exhibited this behavior more frequently than adult males.