External Ca2+ acts upstream of adenylyl cyclase SACY in the bicarbonate signaled activation of sperm motility

The HCO₃⁻ anion activates sperm motility, an important early step in capacitation, by increasing flagellar beat frequency through a pathway that requires the atypical adenylyl cyclase SACY and the sperm-specific Cα₂ catalytic subunit of PKA. Here we show that the accelerating action of HCO₃⁻ also requires the continued presence of external Ca²⁺ (EC₅₀ ∼ 0.5 mM), and find that Ca²⁺ can be replaced by Sr²⁺ but not by Mn²⁺. Ca²⁺ is required for HCO₃⁻ to elevate cAMP, but not for cAMP-AM to increase beat frequency, indicating that external Ca²⁺ acts before rather than after stimulation of SACY by HCO₃⁻. With external Ca²⁺ present, HCO₃⁻ does not alter cytosolic or near-membrane [Ca²⁺]. Removal of external Ca²⁺ initiates a slow decline in intracellular [Ca²⁺] and rapid block of the HCO₃⁻-evoked acceleration that is not relieved upon increasing internal [Ca²⁺] by rapid photolysis of caged Ca²⁺. We also find that the rapid (t_1/2 ∼ 10 s) accelerating action of HCO₃⁻ is slowed more than three-fold by the carbonic anhydrase inhibitor acetazolamide. It is unaltered by the broad spectrum anion transport inhibitor SITS, and is not accompanied by detectable changes in intracellular pH. We propose that external Ca²⁺ binds an unidentified extracellular protein that is required for HCO₃⁻ to engage cAMP-mediated activation of motility.     

A Neuroprotective Function of NSF1 Sustains Autophagy and Lysosomal Trafficking in Drosophila

A common feature of many neurodegenerative diseases is the accumulation of toxic proteins that disrupt vital cellular functions. Degradative pathways such as autophagy play an important protective role in breaking down misfolded and long-lived proteins. Neurons are particularly vulnerable to defects in these pathways, but many of the details regarding the link between autophagy and neurodegeneration remain unclear. We previously found that temperature-sensitive paralytic mutants in Drosophila are enriched for those exhibiting age-dependent neurodegeneration. Here we show that one of these mutants, comatose (comt), in addition to locomotor defects, displays shortened lifespan and progressive neurodegeneration, including loss of dopaminerigic (DA) neurons. comt encodes N-ethyl-maleimide sensitive fusion protein (NSF1), which has a well-documented role in synaptic transmission. However, the neurodegenerative phenotypes we observe in comt mutants do not appear to depend on defects in synaptic transmission, but rather from their inability to sustain autophagy under stress, due at least in part to a defect in trafficking of lysosomal proteases such as cathepsin-L. Conversely, overexpression of NSF1 rescues α-synuclein-induced toxicity of DA neurons in a model of Parkinson’s disease. Our results demonstrate a neuroprotective role for NSF1 that involves mediation of fusion events crucial for degradative pathways such as autophagy, providing greater understanding of cellular dysfunctions common to several neurodegenerative diseases.     

Analytical procedures for the quantification of isotopic amino acid incorporation into photosynthetic proteins of Synechocystis PCC 6803

The mechanism of oxygen evolution has been an enigma for nearly two centuries. Pioneering work by Bessel Kok, Pierre Joliot, and many others during the last quarter century has provided valuable insight into this most unique and important chemical reaction. The late 1970s and early 1980s saw the introduction of biochemical techniques for the purification of photosynthetic complexes that have, in turn, stimulated the biophysical chemists and spectroscopists to apply high resolution techniques in order to resolve the structure/function relationships in these protein complexes. Valuable information about events at the atomic level can be gained through isotopic substitution of particular amino acids thought to be important in the catalytic process. The ability to generate functional auxotrophs in the photosynthetic cyanobacterium Synechocystis 6803 has been used successfully to identify the redox active components Z and D as tyrosine residues in the reaction center of Photosystem II. In this report, we present results of the application of specific isotopic labeling for high resolution spectroscopy of purified PS II particles. We have developed analytical procedures for monitoring the incorporation of both ²H and ¹⁷O labeled amino acids by gas chromatography-mass spectroscopic analysis. We also show that the growth curve of cells subjected to obligate auxotrophy displays two distinct stationary phases; one that corresponds to depletion of exogenous amino acids, and a second that corresponds to the normal cell density at stationary phase. Cells harvested at the second stationary phase show little or no retention of the labeled amino acid.Abbreviations D1 D2 reaction center core proteins of Photosystem II encoded by the psbA and psbD genes, respectively - FTIR Fourier transform infrared - ENDOR electron-nuclear double resonance - ESEEM electron spin-echo envelope modulation - EXAFS extended X-ray absorption fine structure - iBuCF isobutylchloroformate - OD optical density - XANES X-ray absorption near-edge structure - Y_D Y_Z redox active tyrosines of PS II     

Pharmacokinetic Properties of 2nd-Generation Fibroblast Growth Factor-1 Mutants for Therapeutic Application

Fibroblast growth factor-1 (FGF-1) is an angiogenic factor with therapeutic potential for the treatment of ischemic disease. FGF-1 has low intrinsic thermostability and is characteristically formulated with heparin as a stabilizing agent. Heparin, however, adds a number of undesirable properties that negatively impact safety and cost. Mutations that increase the thermostability of FGF-1 may obviate the need for heparin in formulation and may prove to be useful “2nd-generation” forms for therapeutic use. We report a pharmacokinetic (PK) study in rabbits of human FGF-1 in the presence and absence of heparin, as well as three mutant forms having differential effects upon thermostability, buried reactive thiols, and heparin affinity. The results support the hypothesis that heparan sulfate proteoglycan (HSPG) in the vasculature of liver, kidney and spleen serves as the principle peripheral compartment in the distribution kinetics. The addition of heparin to FGF-1 is shown to increase endocrine-like properties of distribution. Mutant forms of FGF-1 that enhance thermostability or eliminate buried reactive thiols demonstrate a shorter distribution half-life, a longer elimination half-life, and a longer mean residence time (MRT) in comparison to wild-type FGF-1. The results show how such mutations can produce useful 2nd-generation forms with tailored PK profiles for specific therapeutic application.     

Orchestration of the S-phase and DNA damage checkpoint pathways by replication forks from early origins

The S-phase checkpoint activated at replication forks coordinates DNA replication when forks stall because of DNA damage or low deoxyribonucleotide triphosphate pools. We explore the involvement of replication forks in coordinating the S-phase checkpoint using dun1Delta cells that have a defect in the number of stalled forks formed from early origins and are dependent on the DNA damage Chk1p pathway for survival when replication is stalled. We show that providing additional origins activated in early S phase and establishing a paused fork at a replication fork pause site restores S-phase checkpoint signaling to chk1Delta dun1Delta cells and relieves the reliance on the DNA damage checkpoint pathway. Origin licensing and activation are controlled by the cyclin-Cdk complexes. Thus, oncogene-mediated deregulation of cyclins in the early stages of cancer development could contribute to genomic instability through a deficiency in the forks required to establish the S-phase checkpoint.     

Breaking the diffraction barrier: super-resolution imaging of cells

Anyone who has used a light microscope has wished that its resolution could be a little better. Now,?after centuries of gradual improvements, fluorescence microscopy has made a quantum leap in its resolving power due, in large part, to advancements over the past several years in a new area of research called super-resolution fluorescence microscopy. In this Primer, we explain the principles of various super-resolution approaches, such as STED, (S)SIM, and STORM/(F)PALM. Then, we describe recent applications of super-resolution microscopy in cells, which demonstrate how these approaches are beginning to provide new insights into cell biology, microbiology, and neurobiology.     

Appearance of the v(FeIV = O) vibration from a ferryl-oxo intermediate in the cytochrome oxidase/dioxygen reaction

Time-resolved resonance Raman spectra have been recorded during the reaction of fully reduced (a2+a3(2+)) cytochrome oxidase with dioxygen at room temperature. In the spectrum recorded at 800 microseconds subsequent to carbon monoxide photolysis, a mode is observed at 790 cm-1 that shifts to 755 cm-1 when the experiment is repeated with 18O2. The frequency of this vibration and the magnitude of the 18O2 isotopic frequency shift lead us to assign the 790-cm-1 mode to the FeIV = O stretching vibration of a ferryl-oxo cytochrome a3 intermediate that occurs in the reaction of fully reduced cytochrome oxidase with dioxygen. The appearance and vibrational frequency of this mode were not affected when D2O was used as a solvent. This result suggests that the ferryl-oxo intermediate is not hydrogen bonded. We have also recorded Raman spectra in the high-frequency (1000-1700 cm-1) region during the oxidase/O2 reaction that show that the oxidation of cytochrome a2+ is biphasic. The faster phase is complete within 100 microseconds and is followed by a plateau region in which no further oxidation of cytochrome a occurs. The plateau persists to approximately 500 microseconds and is followed by the second phase of oxidation. These results on the kinetics of the redox activity of cytochrome a are consistent with the branched pathway discussed by Hill et al. [Hill, B., Greenwood, C., & Nichols, P. (1986) Biochim. Biophys. Acta 853, 91-113] for the oxidation of reduced cytochrome oxidase by O2 at room temperature.     

Forensic Palynology as Classroom Inquiry

This activity introduces the science of forensic palynology: the use of microscopic pollen and spores (also called palynomorphs) to solve criminal cases. Plants produce large amounts of pollen or spores during reproductive cycles. Because of their chemical resistance, small size, and morphology, pollen and spores can be used to link individuals or objects to specific locations where the parent plants grow. Students will use a digital pollen database and Google Earth to link pollen trace evidence to a specific crime scene. The methods presented are based on those used in criminal cases and palynological techniques used by forensic scientists. Step-by-step instructions for a hands-on investigation and a case simulation are presented.