Characterization of specific V1a vasopressin-binding sites on a rat mammary-tumour-cell line.

WRK 1, a cloned cell line derived from a rat mammary tumour, carries specific vasopressin-binding sites. Specific binding of 2-tyrosine-3H-labelled [8-lysine]vasopressin ([3H]vasopressin) was time-dependent, saturable and reversible. Scatchard-plot analysis of hormone binding indicated the presence of a single class of receptors with an equilibrium dissociation constant of 12.7 +/- 0.2 nM. The maximal binding capacity was 75 +/- 6 fmol/10(6) cells, which corresponds to approx. 45,000 sites per cell. Oxytocin and a highly potent oxytocin analogue were able to inhibit completely [3H]vasopressin binding, but, in this respect, they were far less potent than vasopressin. This clearly demonstrates the vasopressinergic nature of this receptor. Pharmacological studies using a series of 14 vasopressin or oxytocin analogues indicated that the ligand selectivity of the vasopressin receptor found on WRK 1 cells resembles that of the rat hepatocyte. This signifies that this vasopressin receptor is of the V1a subtype. This conclusion was confirmed by the observation that vasopressin did not influence the production of intracellular cyclic AMP in WRK 1 cells.     

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen

Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in well-preserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. The data are available via ProteomeXchange with identifier PXD001827.During the last decade, paleontology and taphonomy (the study of decaying organisms over time and the fossilization processes) have begun to overlap with the field of proteomics to shed new light on preserved organic matter in fossilized bones (1–4). These bones represent a time capsule of ancient biomolecules, owing to their natural resistance to post mortem decay arising from a unique combination of mechanical, structural, and chemical properties (4–7).Although bones can be cursorily described as a composite of collagen (protein) and hydroxyapatite (mineral), fossilized bones undergo three distinct diagenesis pathways: (i) chemical deterioration of the organic phase; (ii) chemical deterioration of the mineral phase; and (iii) (micro)biological attack of the composite (6). In addition, the rate of these degradation pathways are affected by temperature, as higher burial temperatures have been shown to accelerate these processes (6, 8). Though relatively unusual, the first of these three pathways results in a slower deterioration process, which is more generally mitigated under (6) specific environmental constraints, such as geochemical stability (stable temperature and acidity) that promote bone mineral preservation. Importantly, slower deterioration results in more preserved biological materials that are more amenable to downstream analytical assays. One example of this is the controversial case of bone and soft-tissue preservation from the Cretaceous/Tertiary boundary (9–22). In light of these and other studies of ancient biomolecules, paleontological models have proposed that organic biomolecules in ancient samples, such as collagen sequences from the 80 million-year-(my)-old Campanian hadrosaur, Brachylophosaurus canadensis (16) or 68-my-old Tyrannosaurus rex, might be protected by the microenvironment within bones. Such spaces are believed to form a protective shelter that is able to reduce the effects of diagenetic events. In addition to collagen, preserved biomolecules include blood proteins, cellular lipids, and DNA (4, 5). While the maximum estimated lifespan of DNA in bones is ∼20,000 years (ky) at 10 °C, bone proteins have an even longer lifespan, making them an exceptional target for analysis to gain relevant insights into fossilized samples (6). Indeed, the survival of collagen, which is considered to be the most abundant bone protein, is estimated to be in the range 340 ky at 20 °C. Similarly, osteocalcin, the second-most abundant bone protein, can persist for ≈45 ky at 20 °C, thus opening an unprecedented analytical window to study extremely old samples (2, 4, 23).Although ancient DNA amplification and sequencing can yield interesting clues and potential artifacts from contaminating agents (7, 24–28), the improved preservation of ancient proteins provides access to a reservoir of otherwise unavailable genetic information for phylogenetic inference (25, 29, 30). In particular, mass spectrometry (MS)-based screening of species-specific collagen peptides has recently been used as a low-cost, rapid alternative to DNA sequencing for taxonomic attribution of morphologically unidentifiable small bone fragments and teeth stemming from diverse archeological contexts (25, 31–33).For over five decades, researchers have presented biochemical evidence for the existence of preserved protein material from ancient bone samples (34–36). One of the first direct measurements was by amino acid analysis, which showed that the compositional profile of ancient samples was consistent with collagens in modern bone samples (37–39). Preservation of organic biomolecules, either from bone, dentin, antlers, or ivory, has been investigated by radiolabeled ¹⁴C fossil dating (40) to provide an avenue of delineating evolutionary divergence from extant species (3, 41, 42). It is also important to note that these parameters primarily depend on ancient bone collagen as the levels remain largely unchanged (a high percentage of collagen is retained, as gleaned by laboratory experiments on bone taphonomy (6)). Additionally, antibody-based immunostaining methods have given indirect evidence of intact peptide amide bonds (43–45) to aid some of the first evidence of protein other than collagen and osteocalcin in ancient mammoth (43) and human specimens (46).In the past, mass spectrometry has been used to obtain MS signals consistent with modern osteocalcin samples (2, 47), and eventually postsource decay peptide fragmentation was used to confirm the identification of osteocalcin in fossil hominids dating back ∼75 ky (48). More recently, modern “bottom-up” proteomic methods were applied to mastodon and T. rex samples (10), complementing immunohistochemistry evidence (13, 17). The results hinted at the potential of identifying peptides from proteolytic digest of well-preserved bone samples. This work also highlighted the importance of minimizing sources of protein contamination and adhering to data publication guidelines (20, 21). In the past few years, a very well-preserved juvenile mammoth referred to as Lyuba was discovered in the Siberian permafrost and analyzed using high-resolution tandem mass spectrometry (29). This study was followed with a report by Wadsworth and Buckley (30) describing the analysis of proteins from 19 bovine bone samples spanning 4 ky to 1.5 my. Both of these groups reported the identification of additional collagen and noncollagen proteins.Recently, a series of large extinct mammal bones were unearthed at a reservoir near Snowmass Village, Colorado, USA (49, 50). The finding was made during a construction project at the Ziegler Reservoir, a fossil site that was originally a lake formed at an elevation of ∼2,705 m during the Bull Lake glaciations ∼140 ky ago (49, 51). The original lake area was ∼5 hectares in size with a total catchment of ∼14 hectares and lacked a direct water flow inlet or outlet. This closed drainage basin established a relatively unique environment that resulted in the exceptional preservation of plant material, insects (52), and vertebrate bones (49). In particular, a cranial specimen from extinct Bison latifrons was unearthed from the Biostratigraphic Zone/Marine Oxygen Isotope Stage (MIS) 5d, which dates back to ∼120 ky (53, 54).Here, we describe the use of paleoproteomics, for the identification of protein remnants with a focus on a particularly unique B. latifrons cranial specimen found at the Ziegler site. We developed a simplified sample processing approach that allows for analysis of low milligram quantities of ancient samples for peptide identification. Our method avoids the extensive demineralization steps of traditional protocols and utilizes an acid labile detergent to allow for efficient extraction and digestion without the need for additional sample cleanup steps. This approach was applied to a specimen from B. latifrons that displayed visual and mechanical properties consistent with the meninges, a fibrous tissue that lines the cranial cavity. Bioinformatics analysis revealed the presence of a recurring glycosylation signature in well-preserved collagens. In particular, the presence of glycosylated hydroxylysine residues was identified as a unique feature of bone fossil collagen, as gleaned through meta-analyses of raw data from previous reports on woolly mammoth (Mammuthus primigenius) and bovine samples (29, 30). The results from these meta-analyses indicate a common, unique feature of collagen that coincides with, and possibly contributes to its preservation.     

Oxidation of polycyclic aromatic hydrocarbons and dibenzo[p]-dioxins by Phanerochaete chrysosporium ligninase

The lignin peroxidase (ligninase) of Phanerochaete chrysosporium catalyzes the oxidation of a variety of lignin-related compounds. Here we report that this enzyme also catalyzes the oxidation of certain aromatic pollutants and compounds related to them, including polycyclic aromatic hydrocarbons with ionization potentials less than or equal to approximately 7.55 eV. This result demonstrates that the H2O2-oxidized states of lignin peroxidase are more oxidizing than the analogous states of classical peroxidases. Experiments with pyrene as the substrate showed that pyrene-1,6-dione and pyrene-1,8-dione are the major oxidation products (84% of total as determined by high performance liquid chromatography), and gas chromatography/mass spectrometry analysis of ligninase-catalyzed pyrene oxidations done in the presence of H2(18)O showed that the quinone oxygens come from water. We found that whole cultures of P. chrysosporium also transiently oxidize pyrene to these quinones. Experiments with dibenzo[p]dioxin and 2-chlorodibenzo[p]dioxin showed that they are also substrates for ligninase. The immediate product of dibenzo[p]dioxin oxidation is the dibenzo[p]dioxin cation radical, which was observed in enzymatic reactions by its electron spin resonance and visible absorption spectra. The cation radical mechanism of ligninase thus applies not only to lignin, but also to other environmentally significant aromatics.     

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.     

Western equine encephalitis in avian populations in North Dakota, 1975

The involvement of wild birds in western equine encephalitis (WEE) and St. Louis encephalitis (SLE) virus activity in the Red River valley area of North Dakota (USA) during a WEE epidemic was investigated in August 1975. Free-ranging birds were captured with mist nets and nestlings by hand. Virologic and serologic results indicated that a similar rate of WEE virus activity occurred throughout Richland County and between permanent and summer resident birds. The rate of SLE virus activity in the birds of Richland County was lower than for WEE virus, but the SLE antibody prevalence was greater in rural areas than within urban locations. Seven of the nine WEE virus isolations were from nestling birds of four different species; the remaining two from adults of two different species. Overall prevalence of neutralizing (N) antibody against WEE virus was 5% in nestling and 14% in adult birds but was the opposite for N antibody against SLE virus, 17% in nestling and 5% in adult birds. Differences between the two viruses in the presence and persistence of maternal N antibody or differential mortality in nestling birds may have caused the disparity in antibody prevalences.     

Comparison of in vitro laboratory analyses with the fertility of cryopreserved stallion spermatozoa

Assessing the fertilizing potential of a semen sample is important for effective stallion management and for rapid progress in evaluating new cryopreservation technologies. Unfortunately, sperm motility does not estimate fertility well. These experiments established assays to measure cell viability, acrosomal integrity and mitochondrial function for cryopreserved stallion spermatozoa, using flow cytometry, and determined the variability associated with these assays. Correlations between results for these laboratory assays and stallion fertility were also determined. The inter-assay variability for visual motility, computer assisted motility, and sperm velocity, sperm viability, percent viable-acrosome intact cells and mitochondrial function of cells were all similar, however, intra-assay variability was lower for flow cytometric assays than for motility assays. The reliability of all assays were >0.72, except for sperm velocity (0.32). Although visual motility and the straightness of sperm motility conducted 90 min after thawing were correlated with seasonal fertility (0.56 and 0.55, respectively), data from no single assay were correlated with first-cycle fertility rates (P > 0.05). Best models using data from multiple assays explained 66 to 73, 76 to 89 and 79 to 94% of the variability in fertilizing potential, when two, three and four variables were included, respectively. Caution is required in interpreting these data, as only a few stallions were evaluated and relatively few mares were bred to each stallion, however, they do indicate that using a few rapid and inexpensive sperm assays, we can begin to understand factors important in stallion sperm fertilizing capacity, and we can use these assays to more effectively evaluate new methods for cryopreserving stallion spermatozoa.     

Jaw-muscle electromyography during chewing in Belanger's treeshrews (Tupaia belangeri)

We examined masseter and temporalis recruitment and firing patterns during chewing in five male Belanger's treeshrews (Tupaia belangeri), using electromyography (EMG). During chewing, the working-side masseters tend to show almost three times more scaled EMG activity than the balancing-side masseters. Similarly, the working-side temporalis muscles have more than twice the scaled EMG activity of the balancing-side temporalis. The relatively higher activity in the working-side muscles suggests that treeshrews recruit less force from their balancing-side muscles during chewing. Most of the jaw-closing muscles in treeshrews can be sorted into an early-firing or late-firing group, based on occurrence of peak activity during the chewing cycle. Specifically, the first group of jaw-closing muscles to reach peak activity consists of the working-side anterior and posterior temporalis and the balancing-side superficial masseter. The balancing-side anterior and posterior temporalis and the working-side superficial masseter peak later in the power stroke. The working-side deep masseter peaks, on average, slightly before the working-side superficial masseter. The balancing-side deep masseter typically peaks early, at about the same time as the balancing-side superficial masseter. Thus, treeshrews are unlike nonhuman anthropoids that peak their working-side deep masseters early and their balancing-side deep masseters late in the power stroke. Because in anthropoids the late firing of the balancing-side deep masseter contributes to wishboning of the symphysis, the treeshrew EMG data suggest that treeshrews do not routinely wishbone their symphyses during chewing. Based on the treeshrew EMG data, we speculate that during chewing, primitive euprimates 1) recruited more force from the working-side jaw-closing muscles as compared to the balancing-side muscles, 2) fired an early group of jaw-closing muscles followed by a second group of muscles that peaked later in the power stroke, 3) did not fire their working-side deep masseter significantly earlier than their working-side superficial masseter, and 4) did not routinely fire their balancing-side deep masseter after the working-side superficial masseter.     

Selective and quickly reversible inactivation of mammalian neurons in vivo using the Drosophila allatostatin receptor

Genetic strategies for perturbing activity of selected neurons hold great promise for understanding circuitry and behavior. Several such strategies exist, but there has been no direct demonstration of reversible inactivation of mammalian neurons in vivo. We previously reported quickly reversible inactivation of neurons in vitro using expression of the Drosophila allatostatin receptor (AlstR). Here, adeno-associated viral vectors are used to express AlstR in vivo in cortical and thalamic neurons of rats, ferrets, and monkeys. Application of the receptor's ligand, allatostatin (AL), leads to a dramatic reduction in neural activity, including responses of visual neurons to optimized visual stimuli. Additionally, AL eliminates activity in spinal cords of transgenic mice conditionally expressing AlstR. This reduction occurs selectively in AlstR-expressing neurons. Inactivation can be reversed within minutes upon washout of the ligand and is repeatable, demonstrating that the AlstR/AL system is effective for selective, quick, and reversible silencing of mammalian neurons in vivo.     

Holliday junctions in the eukaryotic nucleus: resolution in sight?

The Holliday junction is a key recombination intermediate whose resolution generates crossovers. Interplay between recombination, repair and replication has moved the Holliday junction to the center stage of nuclear DNA metabolism. Holliday junction resolvases in the eukaryotic nucleus have long eluded identification. The endonucleases Mus81/Mms4-Eme1 and XPF-MEI-9/MUS312 are structurally related to the archaeal resolvase Hjc and were found to be involved in crossover formation in budding yeast and flies, respectively. Although these endonucleases might represent one class of eukaryotic resolvases, their substrate preference opens up the possibility that junctions other than classical Holliday junctions might contribute to crossovers. Holliday junction resolution to non-crossover products can also be achieved topologically, for example, by the action of RecQ-like DNA helicases combined with topoisomerase III.