Increased visualization of microtubules by an improved fixation procedure.

We have found that when a buffer utilized for in vitro polymerization of microtubules, i.e., 1 mM guanosine triphosphate, 1 mM MgSO4, 2 mM ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid 100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.9 polymerization mix, was used in the glutaraldehyde prefixation regimen instead of classical fixative buffers, i.e., isotonic cacodylate or phosphate buffer, the following features were observed in thin-sections of the cytoplasm of interphase HeLa cells: (a) a greater than 2-fold increase in total microtubule contour length, (b) a 2-fold increase in a number of microtubules greater than or equal to 1 mu long, (c) an enhanced association of microtubules with cytoplasmic organelles, and (d) an increased clustering of 100 A filaments located in a perinuclear region of the cell. Furthermore, we found that after we incubated purified chick brain microtubules on a Sephadex G-25 column pre-equilibrated with polymerization mix, cacodylate or phosphate buffer at 37 degrees C, and then eluted the microtubules at 37 degrees C, the exposure to cacodylate or phosphate buffer caused extensive depolymerization, but exposure to polymerization mix buffer allowed reisolation of highly polymerized microtubules. Our results imply that prefixation with cacodylate or phosphate buffered glutaraldenyde destabilizes microtubules leading to the decreased visualization of microtubules.     

A novel terpene synthase controls differences in anti-aphrodisiac pheromone production between closely related Heliconius butterflies

Plants and insects often use the same compounds for chemical communication, but not much is known about the genetics of convergent evolution of chemical signals. The terpene (E)-β-ocimene is a common component of floral scent and is also used by the butterfly Heliconius melpomene as an anti-aphrodisiac pheromone. While the biosynthesis of terpenes has been described in plants and microorganisms, few terpene synthases (TPSs) have been identified in insects. Here, we study the recent divergence of 2 species, H. melpomene and Heliconius cydno, which differ in the presence of (E)-β-ocimene; combining linkage mapping, gene expression, and functional analyses, we identify 2 novel TPSs. Furthermore, we demonstrate that one, HmelOS, is able to synthesise (E)-β-ocimene in vitro. We find no evidence for TPS activity in HcydOS (HmelOS ortholog of H. cydno), suggesting that the loss of (E)-β-ocimene in this species is the result of coding, not regulatory, differences. The TPS enzymes we discovered are unrelated to previously described plant and insect TPSs, demonstrating that chemical convergence has independent evolutionary origins.

Plants and insects often use the same compounds for chemical communication, but little is known about the convergent evolution of such chemical signals. This study identifies a novel terpene synthase involved in production of an anti-aphrodisiac pheromone by the butterfly Heliconius melpomene. This enzyme is unrelated to other insect terpene synthases, providing evidence that the ability to synthesise terpenes has arisen multiple times independently within the insects.     

Control of DNA synthesis in polyploid mammalian cells

To test the hypothesis that the duration of DNA synthesis is an inverse function of nuclear size or DNA content, the S phase was calculated from PLM analysis for pseudodiploid, tetraploid, and octaploid lines of Chinese hamster cells growing as a monolayer or in suspension. S phase times were found not to be significantly different between polyploid lines and the diploid lines from which they were derived, regardless of the conformation of the nucleus. There is no evidence, therefore, that would implicate the nuclear membrane, or nuclear surface area/volume relationships, in the control of DNA synthesis.     

Nature and cellular origin of the adhesive coats of the lamprey egg (Petromyzon marinus)

Cell surface coats are important in adhesion and other cellular activities. The lamprey egg possesses a surface coat that has been divided into two morphologically and functionally distinct regions. The amorphous apical tuft forms a cap over the animal pole, while the elaborately-textured adhesive coat covers the ventral two-thirds of the egg. This latter area is composed of saccules that form rosettes over the egg surface and is derived from the remains of specialized follicular cells which break down during ovulation. The adhesive qualities of these coats may be inhibited or abolished by various proteins and sulphydryl-blocking agents, thereby implicating, as a possible source of this adhesion, classes of acid and sulphated glycoproteins and glycosaminoglycans which occur on the egg surface.     

Improvement of the Butyl Methacrylate-Paraffin Embedment

The excellent butyl methacrylate-paraffin method as an embedment for light microscopy has been technically improved. More uniform and reproducible polymerization has been obtained by using a vacuum oven to degas the polymerizing mixture and to replace the air with nitrogen at 650 Torr. The amount of benzoyl peroxide required must be determined for each batch of butyl methacrylate. A teflon lined, reusable metal mold and a method of one-step blocking of tissues in preparation for sectioning are also described.     

Before the storm: antecedent conditions as regulators of hydrologic and biogeochemical response to extreme climate events

While the influence of antecedent conditions on watershed function is widely recognized under typical hydrologic regimes, gaps remain in the context of extreme climate events (ECEs). ECEs are those events that far exceed seasonal norms of intensity, duration, or impact upon the physical environment or ecosystem. In this synthesis, we discuss the role of source availability and hydrologic connectivity on antecedent conditions and propose a conceptual framework to characterize system response to ECEs at the watershed scale. We present four case studies in detail that span a range of types of antecedent conditions and type of ECE to highlight important controls and feedbacks. Because ECEs have the potential to export large amounts of water and materials, their occurrence in sequence can disproportionately amplify the response. In fact, multiple events may not be considered extreme in isolation, but when they occur in close sequence they may lead to extreme responses in terms of both supply and transport capacity. Therefore, to advance our understanding of these complexities, we need continued development of a mechanistic understanding of how antecedent conditions set the stage for ECE response across multiple regions and climates, particularly since monitoring of these rare events is costly and difficult to obtain. Through focused monitoring of critical ecosystems during rare events we will also be able to extend and validate modeling studies. Cross-regional comparisons are also needed to define characteristics of resilient systems. These monitoring, modeling, and synthesis efforts are more critical than ever in light of changing climate regimes, intensification of human modifications of the landscape, and the disproportionate impact of ECEs in highly populated regions.