Novel Amiloride Derivatives That Inhibit Bacterial Motility across Multiple Strains and Stator Types.

The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H+) and sodium ions (Na+). The sodium-powered stators, like those in the PomA/PomB stator complex of Vibrio spp., can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium motility inhibitors have been described since the discovery of phenamil. In this study, we characterized two possible motility inhibitors, HM2-16F and BB2-50F, from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free-swimming bacteria, and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na+ motors comparable to phenamil at matching concentrations and could also stop rotation of tethered cells driven by H+ motors. Bead measurements in the presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na+ and H+ stator types and in pathogenic and nonpathogenic strains. IMPORTANCE Here, we characterized two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. These compounds were shown to inhibit flagellar motility at 10 µM across multiple strains: from nonpathogenic Escherichia coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (enterohemorrhagic E. coli or uropathogenic E. coli [EHEC or UPEC, respectively]), and finally, sodium-powered Vibrio alginolyticus. Broad antimotility compounds such as these are important tools in our efforts to control virulence of pathogens in health and agricultural settings.

Nuclear heterogeneity is prevalent in high-quality fractionated human sperm cells typically used for assisted conception.

STUDY QUESTION: What is the nuclear heterogeneity of high-density purified human spermatozoa typically used for IVF purposes. SUMMARY ANSWER: The data show that while density gradient separation has improved the overall sperm population, there is still a large degree of nuclear heterogeneity within these cells. WHAT IS KNOWN ALREADY: Chromomycin A3 (CMA3) is an important DNA binding fluorochrome for the assessment of male-factor fertility. It is typically used to predict IVF outcomes on entire sperm ejaculates with very high receiver operating characteristic. Here we used CMA3 to characterise typical populations of human spermatozoa that would be used for IVF purposes after density gradient separation. STUDY DESIGN, SIZE, DURATION: We compared the intensity of CMA3 binding within high-dense sperm populations obtained from men. Binding heterogeneity was confirmed through fluorescence microscopy and FACS analysis independently. We also looked at CMA3 staining directly with head morphology in this sperm population. Finally, we looked at electron micrographs of nuclear heterogeneity (vacuoles, chromatin compaction) of spermatozoa following density gradient sorting of CMA3-stained cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used sperm donors who had fathered one or more children. Semen was collected after 2 days abstinence and purified over Percoll gradients. Only the high-quality spermatozoa, the same used for assisted conception, were then used. Cells were stained with CMA3 and sorted using FACS. Following this, electron micrographs were used to assess nuclear heterogeneity of CMA3-dependent sorted spermatozoa. MAIN RESULTS AND THE ROLE OF CHANCE: CMA3 staining occurs within morphologically normal as well as abnormal spermatozoa. High-intensity CMA3-stained sperm possessed large vacuoles that were not seen in the low-CMA3 population. In addition, the high-CMA3 stained cells possess higher amounts of nuclear granulation. LIMITATIONS, REASONS FOR CAUTION: The present study only describes the issues within the chromatin of these cells and does not suggest an alternate selection technique. WIDER IMPLICATIONS OF THE FINDINGS: CMA3 is one of the better reported prognostic assays in predicting pregnancy outcomes, especially in cases where the male is at fault. However, it is clear that even in fractionated populations of human spermatozoa, there are sperm cells that are morphologically normal yet possess high levels of CMA3 staining and chromatin granulation. The implication of this is that the embryologist, whom selects on the basis of sperm morphology, may choose a cell with poor chromatin, which may lead to poor embryo outcomes. STUDY FUNDING/COMPETING INTEREST(S): The project was funded by the National Health and Medical Research council, APP1118943. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Pushing the super-resolution limit: recent improvements in microscopy below the diffraction limit.

Super-resolution microscopy has revolutionised the way we observe biological systems. These methods are now a staple of fluorescence microscopy. Researchers have used super-resolution methods in myriad systems to extract nanoscale spatial information on multiple interacting parts. These methods are continually being extended and reimagined to further push their resolving power and achieve truly single protein resolution. Here, we explore the most recent advances at the frontier of the 'super-resolution' limit and what opportunities remain for further improvements in the near future.

Please be upstanding - A narrative review of evidence comparing upright to supine lumbar spine MRI.

OBJECTIVES: The objective of the review was to examine the evidence comparing upright to supine MRI of the lumbar spine. KEY FINDINGS: A literature search identified 14 articles comparing data where subjects had been scanned in both supine and upright positions on the same scanner. Lumbar spine anatomy is dynamic and therefore subject to morphological changes when transitioning from the supine to the upright position. There is strong evidence to suggest structural changes in spinal morphology due to radiographic positioning, and that upright positioning is better for evaluating spondylolisthesis. CONCLUSION: It has been demonstrated that the scanning position is important in the outcome of the MRI examination of the lumbar spine. With this in mind, it would be beneficial for guidance to be written and adopted to improve the consistency and quality of scanning. IMPLICATIONS FOR PRACTICE: As upright MRI occupies a niche in the scanning sector, many professionals are unaware of its capabilities. This article aims to increase awareness of the use of upright MRI in evaluating the lumbar spine.

Signal transducer and activator of transcription (STAT) 1 and STAT3 are expressed in the human ovary and have Janus kinase 1-independent functions in the COV434 human granulosa cell line.

Ovarian granulosa cells are fundamental for oocyte maintenance and maturation. Recent studies have demonstrated the importance of members of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway in the granulosa cell population of mouse and horse ovaries, with perturbation of JAK1 signalling in the mouse shown to impair oocyte maintenance and accelerate primordial follicle activation. The presence and role of the JAK/STAT pathway in human granulosa cells has yet to be elucidated. In this study, expression of JAK1, STAT1 and STAT3 was detected in oocytes and granulosa cells of human ovarian sections from fetal (40 weeks gestation) and premenopausal ovaries (34-41 years of age; n=3). To determine the effects of JAK1 signalling in granulosa cells, the human granulosa-like cell line COV434 was used, with JAK1 inhibition using ruxolitinib. Chemical inhibition of JAK1 in COV434 cells with 100nM ruxolitinib for 72h resulted in significant increases in STAT3 mRNA (P=0.034) and p-Y701-STAT1 protein (P=0.0117), demonstrating a role for JAK1 in modulating STAT in granulosa cells. This study implicates a conserved role for JAK/STAT signalling in human ovary development, warranting further investigation of this pathway in human granulosa cell function.

Automated detection of outbreaks of antimicrobial-resistant bacteria in Japan.

BACKGROUND: Hospital outbreaks of antimicrobial-resistant (AMR) bacteria should be detected and controlled as early as possible. AIM: To develop a framework for automatic detection of AMR outbreaks in hospitals. METHODS: Japan Nosocomial Infections Surveillance (JANIS) is one of the largest national AMR surveillance systems in the world. For this study, all bacterial data in the JANIS database were extracted between 2011 and 2016. WHONET, a free software for the management of microbiology data, and SaTScan, a free cluster detection tool embedded in WHONET, were used to analyse 2015-2016 data of eligible hospitals. Manual evaluation and validation of 10 representative hospitals around Japan were then performed using 2011-2016 data. FINDINGS: Data from 1031 hospitals were studied; mid-sized (200-499 beds) hospitals accounted for 60%, followed by large hospitals (≥500 beds; 24%) and small hospitals (<200 beds; 16%). More clusters were detected in large hospitals. Most of the clusters included five or fewer patients. From the in-depth analysis of 10 hospitals, ∼80% of the detected clusters were unrecognized by infection control staff because the bacterial species involved were not included in the priority pathogen list for routine surveillance. In two hospitals, clusters of more susceptible isolates were detected before outbreaks of more resistant pathogens. CONCLUSION: WHONET-SaTScan can automatically detect clusters of epidemiologically related patients based on isolate resistance profiles beyond lists of high-priority AMR pathogens. If clusters of more susceptible isolates can be detected, it may allow early intervention in infection control practices before outbreaks of more resistant pathogens occur.

Involvement of homocysteine, homocysteine thiolactone, and paraoxonase type 1 (PON-1) in the etiology of defective human sperm function.

This study reports, for the first time, the significant (p ≤ 0.01) accumulation of homocysteine residues in low density, defective sperm suspensions isolated from patients attending an infertility clinic. This overabundance of homocysteine was not related to a deficiency in folate availability but may have been a reflection of the oxidative stress that characterizes such defective sperm populations. Direct addition of the homocysteine cyclic congener, homocysteine thiolactone, to human spermatozoa resulted in the rapid induction of mitochondrial reactive oxygen species (ROS) generation (p < 0.001), the stimulation of lipid peroxidation (p < 0.01), the promotion of tyrosine phosphorylation (p < 0.001), and the suppression of sperm motility (p < 0.001) in the absence of any significant impact on DNA integrity. The parent homocysteine molecule was less active and took 24 h to stimulate mitochondrial ROS production possibly because of the need to convert this compound to the corresponding thiolactone before it could exert a measureable biological effect. Thiolactone was also effective in suppressing the carboxymethylation of key proteins in the sperm tail, which are thought to be involved in the regulation of sperm movement. The major enzyme responsible for removing thiolactone from proteins, paraoxonase (PON-1), was shown to be a major target for alkylation by lipid aldehydes, such as 4-hydroxynonenal, generated as a consequence of oxidative stress. Exposure of human spermatozoa to such aldehydes resulted in a dose-dependent accumulation of homocysteine in spermatozoa (p < 0.03). These results suggest that one of the consequences of oxidative stress in mammalian spermatozoa is the inhibition of PON-1, which then enhances the availability of homocysteine thiolactone to interact with the epsilon-amino group of lysine residues on sperm proteins, triggering a raft of significant biological changes in these cells that ultimately compromise sperm function.

Imaging the lipid-phase-dependent pore formation of equinatoxin II in droplet interface bilayers.

Using phase-separated droplet interface bilayers, we observe membrane binding and pore formation of a eukaryotic cytolysin, Equinatoxin II (EqtII). EqtII activity is known to depend on the presence of sphingomyelin in the target membrane and is enhanced by lipid phase separation. By imaging the ionic flux through individual pores in vitro, we observe that EqtII pores form predominantly within the liquid-disordered phase. We observe preferential binding of labeled EqtII at liquid-ordered/liquid-disordered domain boundaries before it accumulates in the liquid-disordered phase.

Molecular characterization of the receptor binding structure-activity relationships of influenza B virus hemagglutinin.

Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA.

Functional deletion of Txndc2 and Txndc3 increases the susceptibility of spermatozoa to age-related oxidative stress.

Oxidative stress in the male germ line is known to be a key factor in both the etiology of male infertility and the high levels of DNA damage encountered in human spermatozoa. Because the latter has been associated with a variety of adverse clinical outcomes, including miscarriage and developmental abnormalities in the offspring, the mechanisms that spermatozoa use to defend themselves against oxidative stress are of great interest. In this context, the male germ line expresses three unique forms of thioredoxin, known as thioredoxin domain-containing proteins (Txndc2, Txndc3, and Txndc8). Two of these proteins, Txndc2 and Txndc3, retain association with the spermatozoa after spermiation and potentially play an important role in regulating the redox status of the mature gamete. To address this area, we have functionally deleted the sperm-specific thioredoxins from the male germ line of mice by either exon deletion (Txndc2) or mutation of the bioactive cysteines (Txndc3). The combined inactivation of these Txndc isoforms did not have an overall impact on spermatogenesis, epididymal sperm maturation, or fertility. However, Txndc deficiency in spermatozoa did lead to age-dependent changes in these cells as reflected by accelerated motility loss, high rates of DNA damage, increases in reactive oxygen species generation, enhanced formation of lipid aldehyde-protein adducts, and impaired protamination of the sperm chromatin. These results suggest that although there is considerable redundancy in the systems employed by spermatozoa to defend themselves against oxidative stress, the sperm-specific thioredoxins, Txndc2 and Txndc3, are critically important in protecting these cells against the increases in oxidative stress associated with paternal age.

On methods for the detection of reactive oxygen species generation by human spermatozoa: analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid.

Oxidative stress is known to have a major impact on human sperm function and, as a result, there is a need to develop sensitive methods for measuring reactive oxygen species (ROS) generation by these cells. A variety of techniques have been developed for this purpose including chemiluminescence (luminol and lucigenin), flow cytometry (MitoSOX Red, dihydroethidium, 4,5-diaminofluorescein diacetate and 2',7'-dichlorodihydrofluorescein diacetate) and spectrophotometry (nitroblue tetrazolium). The relative sensitivity of these assays and their comparative ability to detect ROS generated in different subcellular compartments of human spermatozoa, have not previously been investigated. To address this issue, we have compared the performance of these assays when ROS generation was triggered with a variety of reagents including 2-hydroxyestradiol, menadione, 4-hydroxynonenal and arachidonic acid. The results revealed that menadione predominantly induced release of ROS into the extracellular space where these metabolites could be readily detected by luminol-peroxidase and, to a lesser extent, 2',7'-dichlorodihydrofluorescein. However, such sensitivity to extracellular ROS meant that these assays were particularly vulnerable to interference by leucocytes. The remaining reagents predominantly elicited ROS generation by the sperm mitochondria and could be optimally detected by MitoSOX Red and DHE. Examination of spontaneous ROS generation by defective human spermatozoa revealed that MitoSOX Red was the most effective indicator of oxidative stress, thereby emphasizing the general importance of mitochondrial dysregulation in the aetiology of defective sperm function.

The Simmet lecture: new horizons on an old landscape--oxidative stress, DNA damage and apoptosis in the male germ line.

Our ability to diagnose and treat male infertility is gradually improving in concert with advances in our understanding of the molecular mechanisms underpinning defective sperm function. In this context, one of the factors to emerge as a major causative agent in male infertility is oxidative stress. Spermatozoa are particularly susceptible to such stress because they are exceptionally rich in vulnerable substrates such as polyunsaturated fatty acids, proteins and DNA. The lack of sperm cytoplasm also provides these cells with little capacity to protect themselves from oxidative attack or to effect any repair, should damage occur. Similarly, sperm chromatin is in a quasi-crystalline state and has very little capacity to respond to any DNA damage induced by oxidative attack. When the latter does occur, it appears to be initiated by reactive oxygen species (ROS) generated by the sperm mitochondria. These free radicals attack the lipids present in the sperm mitochondria generating electrophilic aldehydes, which bind to components of the mitochondrial electron transport chain stimulating yet more ROS production. The oxidative stress created via this self-propagating mechanism initiates an apoptotic cascade as a result of which the spermatozoa loose their capacity for fertilization and suffer damage to their DNA. Phosphatidylserine externalization is a late event in sperm apoptosis and may facilitate the silent phagocytosis of moribund cells in the female reproductive tract, that is, the phagocytosis of senescent spermatozoa without the accompanying generation of an inflammatory response. Encouragingly, the involvement of oxidative stress in the aetiology of male infertility has opened up new opportunities for therapeutic interventions involving the judicious administration of nucleophiles and other forms of antioxidants.

The looming epidemic of diabetes-associated tuberculosis: learning lessons from HIV-associated tuberculosis.

The prevalence of diabetes mellitus is increasing at a dramatic rate, and countries in Asia, particularly India and China, will bear the brunt of this epidemic. Persons with diabetes have a significantly increased risk of active tuberculosis (TB), which is two to three times higher than in persons without diabetes. In this article, we argue that the epidemiological interactions and the effects on clinical presentation and treatment resulting from the interaction between diabetes and TB are similar to those observed for human immunodeficiency virus (HIV) and TB. The lessons learned from approaches to reduce the dual burden of HIV and TB, and especially the modes of screening for the two diseases, can be adapted and applied to the screening, diagnosis, treatment and prevention of diabetes and TB. The new World Health Organization (WHO) and The Union Collaborative Framework for care and control of TB and diabetes has many similarities to the WHO Policy on Collaborative Activities to reduce the dual burden of TB and HIV, and aims to guide policy makers and implementers on how to move forward and combat this looming dual epidemic. The response to the growing HIV-associated TB epidemic in the 1980s and 1990s was slow and uncoordinated, despite clearly articulated warnings about the scale of the forthcoming problem. We must not make the same mistake with diabetes and TB. The Framework provides a template for action, and it is now up to donors, policy makers and implementers to apply the recommendations in the field and to 'learn by doing'.

Consultation meeting on tuberculosis and diabetes mellitus: meeting summary and recommendations.

The steadily growing epidemic of diabetes mellitus (DM) poses a threat for global tuberculosis (TB) control. Previous studies have identified an important association between DM and TB. However, these studies have limitations: very few were carried out in low-income countries, and none in Africa, raising uncertainty about the strength of the DM-TB association in these settings, and many critical questions remain unanswered. As a result of these questions and uncertainties, the International Union Against Tuberculosis and Lung Disease (The Union), the World Diabetes Foundation and the World Health Organization Stop TB Department undertook a series of consultations as of January 2009. A systematic review and meta-analysis was undertaken by the Department of Epidemiology, Harvard School of Public Health between May and August 2009, and a consultation meeting involving the experts who reviewed the report took place at The Union Headquarters in Paris on 6 and 7 November 2009. This paper constitutes a summary report of the findings, the research gaps and prioritised areas of research, and the recommendations from that meeting.

Growth and characterization of ceria thin films and Ce-doped γ-Al2O3 nanowires using sol-gel techniques.

γ-Al(2)O(3) is a well known catalyst support. The addition of Ce to γ-Al(2)O(3) is known to beneficially retard the phase transformation of γ-Al(2)O(3) to α-Al(2)O(3) and stabilize the γ-pore structure. In this work, Ce-doped γ-Al(2)O(3) nanowires have been prepared by a novel method employing an anodic aluminium oxide (AAO) template in a 0.01 M cerium nitrate solution, assisted by urea hydrolysis. Calcination at 500 °C for 6 h resulted in the crystallization of the Ce-doped AlOOH gel to form Ce-doped γ-Al(2)O(3) nanowires. Ce(3+) ions within the nanowires were present at a concentration of < 1 at.%. On the template surface, a nanocrystalline CeO(2) thin film was deposited with a cubic fluorite structure and a crystallite size of 6-7 nm. Characterization of the nanowires and thin films was performed using scanning electron microscopy, transmission electron microscopy, electron energy loss spectroscopy, x-ray photoelectron spectroscopy and x-ray diffraction. The nanowire formation mechanism and urea hydrolysis kinetics are discussed in terms of the pH evolution during the reaction. The Ce-doped γ-Al(2)O(3) nanowires are likely to find useful applications in catalysis and this novel method can be exploited further for doping alumina nanowires with other rare earth elements.

Nanocomposite hydroxyapatite formation on a Ti-13Nb-13Zr alloy exposed in a MEM cell culture medium and the effect of H2O2 addition.

Titanium alloys are known to nucleate an apatite layer when in contact with simulated body fluid. This improves the bioactivity of titanium implants and accelerates osseointegration. Promoting the formation of hydroxyapatite on biocompatible metals is, therefore, a very important topic of biomaterials research. In this paper, the formation of hydroxyapatite (HA) on the near-beta Ti-13Nb-13Zr alloy by immersion in minimal essential medium (MEM), with and without H(2)O(2) addition, has been studied using electrochemicals methods, scanning electron microscopy and X-ray photoelectron spectroscopy. The in vitro biocompatibility of this alloy was evaluated by cytotoxicity tests. The Ti-13Nb-13Zr alloy exhibits passive behaviour over a wide potential range in MEM and the passive film is composed of an inner barrier layer and an outer porous layer. The addition of H(2)O(2) leads to a thickening of the outer porous layer and strongly reduced current density. With regard to the surface composition, immersion in MEM solution results in the formation of an island-like distribution of HA+amino acids. Addition of H(2)O(2) to the MEM solution strongly promotes the formation of a thicker, continuous but porous nanocomposite layer of HA+amino acids. The Ti-13Nb-13Zr alloy is non-toxic and the nanocomposite HA+amino acid layer formed in the MEM solution favours the growth of osteoblast cells. For Ti alloys, the release of H(2)O(2) in the anti-inflammatory response appears to be an important beneficial process as it accelerates osseointegration.

Factors associated with the catastrophic decline of a cloudforest frog fauna in Guatemala

Comparison of recent and historical surveys of frog populations in cloudforest habitat in Sierra de las Minas,Guatemala,indicated population declines and local extirpation of several species.Pathological exams of diseased tadpoles indicated infection by amphibian chytridiomycosis. The local habitat has been severely altered by recent establishment of large-scale leatherleaf fern production.Analysis of water chemistry at our study site suggested increased nitrogenation associated with the leatherleaf industry

Una comparación entre un inventario anterior y otro reciente de poblaciones de ranas de bosque nublado en la Sierra de Las Minas de Guatemala demostró disminuciones poblacionales y ausencia localizada de varias especies.El examen patológico de un renacuajo muerto indicó infección por un hongo quítrido propio de los anfibios.El hábitat local ha sido gravemente alterado por el establecimiento reciente de producción a gran escala de helechos ornamentales.El análisis químico del agua en el área de estudio señaló un aumento en nitrogenación asociado al cultivo de helechos

A flow cytometric assay for global estimation of tyrosine phosphorylation associated with capacitation of spermatozoa from two marsupial species, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula).

The phosphorylation of tyrosine residues in cellular proteins is a major signal transduction event during sperm capacitation. In this study protein phosphorylation was monitored using a fluorescein isothiocyanate (FITC)-labeled antiphosphotyrosine monoclonal antibody and a flow cytometric procedure optimized for sperm. Using this technique, the correlation between tyrosine phosphorylation and sperm capacitation was examined in two marsupial species, the brushtail possum and the tammar wallaby and compared with that of ram spermatozoa. The levels of tyrosine phosphorylation in sperm from all three species were increased by the addition of cyclic AMP (cAMP) and vandate, a phosphotyrosine phosphatase inhibitor and were decreased by the addition of the phosphotyrosine kinase inhibitor, staurosporine. Oviductal conditioned media (CM) induced a progressive increase in tyrosine phosphorylation in both marsupial species and also induced morphological transition from a streamlined to a 'T'-shape configuration in brushtail possum spermatozoa but not in tammar wallaby spermatozoa. Transition to the 'T'-shape orientation associated with capacitation in marsupial spermatozoa was observed by 2 h of incubation in both species when tyrosine phosphorylation was increased by higher levels of cAMP i.e. 5 mM dibutyryl cAMP plus 3 mM pentoxyphylline. Thus the tyrosine phosphorylation trigger with CM may differ in these two marsupial species. Ram sperm tyrosine phosphorylation could be increased by addition of lower levels of cAMP (1 mM). These results support the finding that tyrosine phosphorylation is associated with sperm capacitation in marsupials. Similar results were obtained by using SDS PAGE/Western blot analysis of tyrosine phosphorylation in the brushtail possum spermatozoa. The specificity, efficiency and sensitivity of the procedure described here make it applicable for routine assessment of capacitation in large numbers of samples and in other species.

Factors associated with the catastrophic decline of a cloudforest frog fauna in Guatemala.

Comparison of recent and historical surveys of frog populations in cloudforest habitat in Sierra de las Minas, Guatemala, indicated population declines and local extirpation of several species. Pathological exams of diseased tadpoles indicated infection by amphibian chytridiomycosis. The local habitat has been severely altered by recent establishment of large-scale leatherleaf fern production. Analysis of water chemistry at our study site suggested increased nitrogenation associated with the leatherleaf industry.