Immunolocalization of multiple Galpha subunits in mammalian spermatozoa and additional evidence for Galphas

Like somatic cells, mammalian spermatozoa appear to contain several different heterotrimeric G protein alpha-subunits that could mediate specialized cell responses. However, the precise Galpha subunits present, their subcellular location and their possible roles are still incompletely defined. In this study, using commercially available specific antibodies, we have shown by immunoblotting that Galpha(s) is present in human and mouse sperm lysates. Immunolocalization using intact spermatozoa from both species revealed this protein to be in the acrosomal cap region and the flagellum, particularly the principal piece. Treatment of permeabilized mouse spermatozoa with cholera toxin led to enhanced ADP-ribosylation of a protein the same size as Galpha(s), as well as an increase in cAMP, providing further proof for Galpha(s). Evidence for the presence and distinct localizations of Galpha(i2), Galpha(i3), Galpha(o), Galpha(q/11), and Galpha(olf) was also obtained. Of particular interest was Galpha(i2) which, like Galpha(s), was present in the acrosomal cap region and flagellum, the same regions where stimulatory and inhibitory adenosine receptors are localized. These observations are consistent with our hypothesis that G proteins mediate adenosine receptor modulation of adenylyl cyclase, with consequent alterations in cAMP production, apparently crucial for the spermatozoon's acquisition and maintenance of fertilizing ability.     

The structure, catalytic mechanism and regulation of adenylyl cyclase

The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity.     

Intraspecific brood mixing and reduced polyandry in a maternal mouth-brooding cichlid

Microsatellite loci were used to evaluate the level of polyandryand intraspecific brood mixing in Protomelas c.f. spilopterus,a paedophagous, maternal mouth-brooding cichlid from Lake Makairc.gif" ALT="w" BORDER="0">iAfrica. We found that broods were fertilized by one to threemales, which was a reduced level of multiple paternity comparedto other mouth-brooding cichlids. Low density of breeding malesand the risk of intraspecific predation are likely explanationsfor reduced polyandry. Intraspecific brood-mixing was foundin four out of the six broods examined, with the proportionsof foreign fry ranging from 6% to 65%. The potential originsof brood mixing are discussed, although no firm conclusionscan be drawn given the limited behavioral observations for thisspecies.     

Cofilin-1 is involved in regulation of actin reorganization during influenza A virus assembly and budding

Influenza A virus (IAV) assembly and budding on host cell surface plasma membrane requires actin cytoskeleton reorganization. The underlying molecular mechanism involving actin reorganization remains unclarified. In this study, we found that the natural antiviral compound petagalloyl glucose (PGG) inhibits F-actin reorganization in the host cell membrane during the late stage of IAV infection, which are associated with the suppression of total cofilin-1 level and its phosphorylation. Knock-down of cofilin-1 reduces viral yields. These findings provide the first evidence that cofilin-1 plays an important role in regulating actin reorganization during IAV assembly and budding.     

Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract

Respiratory syncytial virus (RSV) is the leading cause of viral bronchiolitis and pneumonia in infants and children. Currently, palivizumab is the only approved monoclonal antibody (mAb) for prophylaxis of RSV. However, a small percentage of patients are not protected by palivizumab; in addition, palivizumab does not inhibit RSV replication effectively in the upper respiratory tract. We report here the development and characterization of motavizumab, an ultra-potent, affinity-matured, humanized mAb derived from palivizumab. Several palivizumab variants that enhanced the neutralization of RSV in vitro by up to 44-fold were generated; however, in vivo prophylaxis of cotton rats with these antibodies conferred only about a twofold improvement in potency over palivizumab. This unexpected small increase of in vivo potency was caused by poor serum pharmacokinetics and lung bio-availability that resulted from unexpectedly broad tissue binding. Subsequent analyses revealed that changes at three amino acids arising from the affinity maturation markedly increased the non-specific binding to various tissues. Our results suggested that k(on)-driven mutations are more likely to initiate non-specific binding events than k(off)-driven mutations. Reversion of these three residues to the original sequences greatly diminished the tissue binding. The resulting mAb, motavizumab, binds to RSV F protein 70-fold better than palivizumab, and exhibits about a 20-fold improvement in neutralization of RSV in vitro. In cotton rats, at equivalent concentrations, motavizumab reduced pulmonary RSV titers to up to 100-fold lower levels than did palivizumab and, unlike palivizumab, motavizumab very potently inhibited viral replication in the upper respiratory tract. This affinity-enhanced mAb is being investigated in pivotal clinical trials. Importantly, our engineering process offers precious insights into the improvement of other therapeutic mAbs.     

Invasive aphids attack native Hawaiian plants

Invasive species have had devastating impacts on the fauna and flora of the Hawaiian Islands. While the negative effects of some invasive species are obvious, other species are less visible, though no less important. Aphids (Homoptera: Aphididae) are not native to Hawai’i but have thoroughly invaded the Island chain, largely as a result of anthropogenic influences. As aphids cause both direct plant feeding damage and transmit numerous pathogenic viruses, it is important to document aphid distributions and ranges throughout the archipelago. On the basis of an extensive survey of aphid diversity on the five largest Hawaiian Islands (Hawai’i, Kaua’i, O’ahu, Maui, and Moloka’i), we provide the first evidence that invasive aphids feed not just on agricultural crops, but also on native Hawaiian plants. To date, aphids have been observed feeding and reproducing on 64 native Hawaiian plants (16 indigenous species and 48 endemic species) in 32 families. As the majority of these plants are endangered, invasive aphids may have profound impacts on the island flora. To help protect unique island ecosystems, we propose that border vigilance be enhanced to prevent the incursion of new aphids, and that biological control efforts be renewed to mitigate the impact of existing species.     

Sternal gland structures in males of bean flower thrips,Megalurothrips sjostedti,and Poinsettia thrips,Echinothrips americanus,in comparison with those of western flower thrips,Frankliniella occidentalis (Thysanoptera: Thripidae)

Sternal pores are important features for identification of male thrips, especially within the subfamily Thripinae. They vary in shape, size and distribution even between species of one genus. Their functional role is speculated to be that of sex- and/or aggregation pheromone production. Yet, sexual aggregations are not reported in Echinothrips americanus, known to have sternal pores, while we observed aggregations in Megalurothrips sjostedti, previously reported to lack them.We examined the sternal glands and pores of the thripine species E. americanus and M. sjostedti males, in comparison with those of Frankliniella occidentalis using light microscopy, as well as scanning and transmission electron microscopy. Pore plates of F. occidentalis were ellipsoid and medial on sternites III–VII, while in E. americanus they were distributed as multiple micro pore plates on sternites III–VIII. In M. sjostedti they appeared as an extremely small pore in front of the posterior margin of each of sternites IV–VII. Pore plate and pore plate area were distributed similarly on sternites III–VII in F. occidentalis. However, in E. americanus the total pore plate area increased significantly from sternites III to VIII. Ultrastructure of cells associated with sternal glands showed typical characteristics of gland cells that differ in size, shape and number. The function of sternal glands is further discussed on the basis of morphological comparisons with other thrips species.     

Fish models in prion biology: Underwater issues

Transmissible spongiform encephalopathies (TSEs), otherwise known as prion disorders, are fatal diseases causing neurodegeneration in a wide range of mammalian hosts, including humans. The causative agents - prions - are thought to be composed of a rogue isoform of the endogenous prion protein (PrP). Beyond these and other basic concepts, fundamental questions in prion biology remain unanswered, such as the physiological function of PrP, the molecular mechanisms underlying prion pathogenesis, and the origin of prions. To date, the occurrence of TSEs in lower vertebrates like fish and birds has received only limited attention, despite the fact that these animals possess bona fide PrPs. Recent findings, however, have brought fish before the footlights of prion research. Fish models are beginning to provide useful insights into the roles of PrP in health and disease, as well as the potential risk of prion transmission between fish and mammals. Although still in its infancy, the use of fish models in TSE research could significantly improve our basic understanding of prion diseases, and also help anticipate risks to public health. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

GSTM1 modulation of IL-8 expression in human bronchial epithelial cells exposed to ozone

Exposure to the major air pollutant ozone can aggravate asthma and other lung diseases. Our recent study in human volunteers has shown that the glutathione S-transferase Mu 1 (GSTM1)-null genotype is associated with increased airway neutrophilic inflammation induced by inhaled ozone. The aim of this study was to examine the effect of GSTM1 modulation on interleukin 8 (IL-8) production in ozone-exposed human bronchial epithelial cells (BEAS-2B) and the underlying mechanisms. Exposure of BEAS-2B cells to 0.4 ppm ozone for 4 h significantly increased IL-8 release, with a modest reduction in intracellular reduced glutathione (GSH). Ozone exposure induced reactive oxygen species (ROS) production and NF-κB activation. Pharmacological inhibition of NF-κB activation or mutation of the IL-8 promoter at the κB-binding site significantly blocked ozone-induced IL-8 production or IL-8 transcriptional activity, respectively. Knockdown of GSTM1 in BEAS-2B cells enhanced ozone-induced NF-κB activation and IL-8 production. Consistently, an ozone-induced overt increase in IL-8 production was detected in GSTM1-null primary human bronchial epithelial cells. In addition, supplementation with reduced GSH inhibited ozone-induced ROS production, NF-κB activation, and IL-8 production. Taken together, GSTM1 deficiency enhances ozone-induced IL-8 production, which is mediated by generated ROS and subsequent NF-κB activation in human bronchial epithelial cells.     

Characterization of plasma triiodophenol binding proteins in vertebrates and tissue distribution of triiodophenol in Rana catesbeiana tadpoles

We investigated the interaction of 2,4,6-triiodophenol (TIP), a potent thyroid hormone disrupting chemical, with serum proteins from rainbow trout (Onchorhynchus mykiss), bullfrog (Rana catesbeiana), chicken (Gallus gallus), pig (Sus scrofa domesticus), and rat (Rattus norvegicus) using a [(125)I]TIP binding assay, gel filtration chromatography, and native polyacrylamide gel electrophoresis. [(125)I]TIP bound non-specifically to proteins in trout serum, specifically but weakly to proteins in bullfrog serum, and specifically and strongly to proteins in chicken, pig, and rat serum samples. Candidate TIP-binding proteins included lipoproteins (220-320kDa) in trout, albumin in bullfrog, albumin and transthyretin (TTR) in chicken and pig, and TTR in rat. TTR in the chicken, pig, and rat serum samples was responsible for the high-affinity, low-capacity binding sites for TIP (dissociation constant 2.2-3.5×10(-10)M). In contrast, a weak interaction of [(125)I]TIP with tadpole serum proteins accelerated [(125)I]TIP cellular uptake in vitro. Intraperitoneal injection of [(125)I]TIP in tadpoles revealed that the radioactivity was predominantly accumulated in the gallbladder and the kidney. The differences in the molecular and binding properties of TIP binding proteins among vertebrates would affect in part the cellular availability, tissue distribution and clearance of TIP.