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Antarctic ecosystems are under increasing anthropogenic pressure, but efforts to predict the responses of Antarctic biodiversity to environmental change are hindered by considerable data challenges. Here, we illustrate how novel data capture technologies provide exciting opportunities to sample Antarctic biodiversity at wider spatiotemporal scales. Data integration frameworks, such as point process and hierarchical models, can mitigate weaknesses in individual data sets, improving confidence in their predictions. Increasing process knowledge in models is imperative to achieving improved forecasts of Antarctic biodiversity, which can be attained for data-limited species using hybrid modelling frameworks. Leveraging these state-of-the-art tools will help to overcome many of the data scarcity challenges presented by the remoteness of Antarctica, enabling more robust forecasts both near- and long-term., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objectives: High-level β-lactam resistance in MRSA is mediated in the majority of strains by a mecA or mecC gene. In this study, we identified 10 mec gene-negative MRSA human isolates from Austria and 11 bovine isolates from the UK showing high levels of β-lactam resistance and sought to understand the molecular basis of the resistance observed., Methods: Different antimicrobial resistance testing methods (disc diffusion, Etest and VITEK® 2) were used to establish the β-lactam resistance profiles for the isolates and the isolates were further investigated by WGS., Results: A number of mutations (including novel ones) in PBPs, AcrB, YjbH and the pbp4 promoter were identified in the resistant isolates, but not in closely related susceptible isolates. Importantly, a truncation in the cyclic diadenosine monophosphate phosphodiesterase enzyme, GdpP, was identified in 7 of the 10 Austrian isolates and 10 of the 11 UK isolates. Complementation of four representative isolates with an intact copy of the gdpP gene restored susceptibility to penicillins and abolished the growth defects caused by the truncation., Conclusions: This study reports naturally occurring inactivation of GdpP protein in Staphylococcus aureus of both human origin and animal origin, and demonstrates clinical relevance to a previously reported association between this truncation and increased β-lactam resistance and impaired bacterial growth in laboratory-generated mutants. It also highlights possible limitations of genomic determination of antibiotic susceptibility based on single gene presence or absence when choosing the appropriate antimicrobial treatment for patients., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Agriculture is important to New Zealand's economy. Like other primary producers, New Zealand strives to increase agricultural output while maintaining environmental integrity. Utilising modelling to explore the economic, environmental and land use impacts of policy is critical to understand the likely effects on the sector. Key deficiencies within existing land use and land cover change models are the lack of heterogeneity in farmers and their behaviour, the role that social networks play in information transfer, and the abstraction of the global and regional economic aspects within local-scale approaches. To resolve these issues we developed the Agent-based Rural Land Use New Zealand model. The model utilises a partial equilibrium economic model and an agent-based decision-making framework to explore how the cumulative effects of individual farmer's decisions affect farm conversion and the resulting land use at a catchment scale. The model is intended to assist in the development of policy to shape agricultural land use intensification in New Zealand. We illustrate the model, by modelling the impact of a greenhouse gas price on farm-level land use, net revenue, and environmental indicators such as nutrient losses and soil erosion for key enterprises in the Hurunui and Waiau catchments of North Canterbury in New Zealand. Key results from the model show that farm net revenue is estimated to increase over time regardless of the greenhouse gas price. Net greenhouse gas emissions are estimated to decline over time, even under a no GHG price baseline, due to an expansion of forestry on low productivity land. Higher GHG prices provide a greater net reduction of emissions. While social and geographic network effects have minimal impact on net revenue and environmental outputs for the catchment, they do have an effect on the spatial arrangement of land use and in particular the clustering of enterprises.

Unlabelled: Methicillin-resistant Staphylococcus aureus (MRSA) is a global human health problem causing infections in both hospitals and the community. Companion animals, such as cats, dogs, and horses, are also frequently colonized by MRSA and can become infected. We sequenced the genomes of 46 multilocus sequence type (ST) 22 MRSA isolates from cats and dogs in the United Kingdom and compared these to an extensive population framework of human isolates from the same lineage. Phylogenomic analyses showed that all companion animal isolates were interspersed throughout the epidemic MRSA-15 (EMRSA-15) pandemic clade and clustered with human isolates from the United Kingdom, with human isolates basal to those from companion animals, suggesting a human source for isolates infecting companion animals. A number of isolates from the same veterinary hospital clustered together, suggesting that as in human hospitals, EMRSA-15 isolates are readily transmitted in the veterinary hospital setting. Genome-wide association analysis did not identify any host-specific single nucleotide polymorphisms (SNPs) or virulence factors. However, isolates from companion animals were significantly less likely to harbor a plasmid encoding erythromycin resistance. When this plasmid was present in animal-associated isolates, it was more likely to contain mutations mediating resistance to clindamycin. This finding is consistent with the low levels of erythromycin and high levels of clindamycin used in veterinary medicine in the United Kingdom. This study furthers the "one health" view of infectious diseases that the pathogen pool of human and animal populations are intrinsically linked and provides evidence that antibiotic usage in animal medicine is shaping the population of a major human pathogen., Importance: Methicillin-resistant Staphylococcus aureus (MRSA) is major problem in human medicine. Companion animals, such as cats, dogs, and horses, can also become colonized and infected by MRSA. Here, we demonstrate that a shared population of an important and globally disseminated lineage of MRSA can infect both humans and companion animals without undergoing host adaptation. This suggests that companion animals might act as a reservoir for human infections. We also show that the isolates from companion animals have differences in the presence of certain antibiotic resistance genes. This study furthers the "one health" view of infectious diseases by demonstrating that the pool of MRSA isolates in the human and animal populations are shared and highlights how different antibiotic usage patterns between human and veterinary medicine can shape the population of bacterial pathogens., (Copyright © 2014 Harrison et al.)

Objectives: There are limited data available on the epidemiology and prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the human population that encode the recently described mecA homologue, mecC. To address this knowledge gap we undertook a prospective prevalence study in England to determine the prevalence of mecC among MRSA isolates., Patients and Methods: Three hundred and thirty-five sequential MRSA isolates from individual patients were collected from each of six clinical microbiology laboratories in England during 2011-12. These were tested by PCR or genome sequencing to differentiate those encoding mecA and mecC. mecC-positive isolates were further characterized by multilocus sequence typing, spa typing, antimicrobial susceptibility profile and detection of PBP2a using commercially available kits., Results: Nine out of the 2010 MRSA isolates tested were mecC positive, indicating a prevalence among MRSA in England of 0.45% (95% CI 0.24%-0.85%). The remainder were mecA positive. Eight out of these nine mecC MRSA isolates belonged to clonal complex 130, the other being sequence type 425. Resistance to non-β-lactam antibiotics was rare among these mecC MRSA isolates and all were phenotypically identified as MRSA using oxacillin and cefoxitin according to BSAC disc diffusion methodology. However, all nine mecC isolates gave a negative result using three different commercial PBP2a detection assays., Conclusions: mecC MRSA are currently rare among MRSA isolated from humans in England and this study provides an important baseline prevalence rate to monitor future changes, which may be important given the increasing prevalence of mecC MRSA reported in Denmark.

Objectives: Methicillin resistance in Staphylococcus spp. results from the expression of an alternative penicillin-binding protein 2a (encoded by mecA) with a low affinity for β-lactam antibiotics. Recently, a novel variant of mecA known as mecC (formerly mecALGA251) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified two Staphylococcus sciuri subsp. carnaticus isolates from bovine infections that harbour three different mecA homologues: mecA, mecA1 and mecC., Methods: We subjected the two isolates to whole-genome sequencing to further understand the genetic context of the mec-containing region. We also used PCR and RT-PCR to investigate the excision and expression of the SCCmec element and mec genes, respectively., Results: Whole-genome sequencing revealed a novel hybrid SCCmec region at the orfX locus consisting of a class E mec complex (mecI-mecR1-mecC1-blaZ) located immediately downstream of a staphylococcal cassette chromosome mec (SCCmec) type VII element. A second SCCmec attL site (attL2), which was imperfect, was present downstream of the mecC region. PCR analysis of stationary-phase cultures showed that both the SCCmec type VII element and a hybrid SCCmec-mecC element were capable of excision from the genome and forming a circular intermediate. Transcriptional analysis showed that mecC and mecA, but not mecA1, were both expressed in liquid culture supplemented with oxacillin., Conclusions: Overall, this study further highlights that a range of staphylococcal species harbour the mecC gene and furthers the view that coagulase-negative staphylococci associated with animals may act as reservoirs of antibiotic resistance genes for more pathogenic staphylococcal species.

Objectives: mecC methicillin-resistant Staphylococcus aureus (MRSA) represent a newly recognized form of MRSA, distinguished by the possession of a divergent mecA homologue, mecC. The first isolate to be identified came from bovine milk, but there are few data on the prevalence of mecC MRSA among dairy cattle. The aim of this study was to conduct a prevalence study of mecC MRSA among dairy farms in Great Britain., Methods: Test farms were randomly selected by random order generation and bulk tank samples were tested for the presence of mecC MRSA by broth enrichment and plating onto chromogenic agar. All MRSA isolated were screened by PCR for mecA and mecC, and mecC MRSA were further characterized by multilocus sequence typing, spa typing and antimicrobial susceptibility testing., Results: mecC MRSA were detected on 10 of 465 dairy farms sampled in England and Wales (prevalence 2.15%, 95% CI 1.17%-3.91%), but not from 625 farms sampled in Scotland (95% CI of prevalence 0%-0.61%). Seven isolates belonged to sequence type (ST) 425, while the other three belonged to clonal complex 130. Resistance to non-β-lactam antibiotics was uncommon. All 10 isolates produced a negative result by slide agglutination for penicillin-binding protein 2a. mecA MRSA ST398 was detected on one farm in England., Conclusions: mecC MRSA is widely distributed among dairy farms in Great Britain, but this distribution is not uniform across the whole country. These results provide an important baseline dataset to monitor the epidemiology of this emerging form of MRSA.

Recently, a novel variant of mecA known as mecC (mecA(LGA251)) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified a Staphylococcus xylosus isolate that harbors a new allotype of the mecC gene, mecC1. Whole-genome sequencing revealed that mecC1 forms part of a class E mec complex (mecI-mecR1-mecC1-blaZ) located at the orfX locus as part of a likely staphylococcal cassette chromosome mec element (SCCmec) remnant, which also contains a number of other genes present on the type XI SCCmec.

Livestock-associated meticillin-resistant Staphylococcus aureus belonging to clonal complex 398 (LA-MRSA CC398) is an important cause of zoonotic infections in several countries, but there is only a single published report of this lineage from the United Kingdom (UK). Here, we describe the isolation of LA-MRSA CC398 from bulk tank milk from five geographically dispersed farms in the UK. Our findings suggest that LA-MRSA CC398 is established in livestock in the UK. Awareness of the potential occupational risks and surveillance in other food-producing animal species should be promoted.

Intracellular replication within specialized vacuoles and cell-to-cell spread in the tissue are essential for the virulence of Salmonella enterica. By observing infection dynamics at the single-cell level in vivo, we have discovered that the Salmonella pathogenicity island 2 (SPI-2) type 3 secretory system (T3SS) is dispensable for growth to high intracellular densities. This challenges the concept that intracellular replication absolutely requires proteins delivered by SPI-2 T3SS, which has been derived largely by inference from in vitro cell experiments and from unrefined measurement of net growth in mouse organs. Furthermore, we infer from our data that the SPI-2 T3SS mediates exit from infected cells, with consequent formation of new infection foci resulting in bacterial spread in the tissues. This suggests a new role for SPI-2 in vivo as a mediator of bacterial spread in the body. In addition, we demonstrate that very similar net growth rates of attenuated salmonellae in organs can be derived from very different underlying intracellular growth dynamics.

Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its importance to virulence. In this study we have explored the contribution of the bacterial cytoskeleton to the ability of Salmonella to express and assemble virulence factors and cause disease. The bacterial actin-like protein MreB polymerises into helical filaments and interacts with other cytoskeletal elements including MreC to control cell-shape. As mreB appears to be an essential gene, we have constructed a viable ΔmreC depletion mutant in Salmonella. Using a broad range of independent biochemical, fluorescence and phenotypic screens we provide evidence that the Salmonella pathogenicity island-1 type three secretion system (SPI1-T3SS) and flagella systems are down-regulated in the absence of MreC. In contrast the SPI-2 T3SS appears to remain functional. The phenotypes have been further validated using a chemical genetic approach to disrupt the functionality of MreB. Although the fitness of ΔmreC is reduced in vivo, we observed that this defect does not completely abrogate the ability of Salmonella to cause disease systemically. By forcing on expression of flagella and SPI-1 T3SS in trans with the master regulators FlhDC and HilA, it is clear that the cytoskeleton is dispensable for the assembly of these structures but essential for their expression. As two-component systems are involved in sensing and adapting to environmental and cell surface signals, we have constructed and screened a panel of such mutants and identified the sensor kinase RcsC as a key phenotypic regulator in ΔmreC. Further genetic analysis revealed the importance of the Rcs two-component system in modulating the expression of these virulence factors. Collectively, these results suggest that expression of virulence genes might be directly coordinated with cytoskeletal integrity, and this regulation is mediated by the two-component system sensor kinase RcsC.

Salmonella enterica serovar Typhi, the agent of typhoid fever in humans, expresses the surface Vi polysaccharide antigen that contributes to virulence. However, Vi expression can also be detrimental to some key steps of S. Typhi infectivity, for example, invasion, and Vi is the target of protective immune responses. We used a strain of S. Typhimurium carrying the whole Salmonella pathogenicity island 7 (SPI-7) to monitor in vivo Vi expression within phagocytic cells of mice at different times after systemic infection. We also tested whether it is possible to modulate Vi expression via the use of in vivo-inducible promoters and whether this would trigger anti-Vi antibodies through the use of Vi-expressing live bacteria. Our results show that Vi expression in the liver and spleen is downregulated with the progression of infection and that the Vi-negative population of bacteria becomes prevalent by day 4 postinfection. Furthermore, we showed that replacing the natural tviA promoter with the promoter of the SPI-2 gene ssaG resulted in sustained Vi expression in the tissues. Intravenous or oral infection of mice with a strain of S. Typhimurium expressing Vi under the control of the ssaG promoter triggered detectable levels of all IgG subclasses specific for Vi. Our work highlights that Vi is downregulated in vivo and provides proof of principle that it is possible to generate a live attenuated vaccine that induces Vi-specific antibodies after single oral administration.

The interaction between Salmonella enterica and the host immune system is complex. The outcome of an infection is the result of a balance between the in vivo environment where the bacteria survive and grow and the regulation of fitness genes at a level sufficient for the bacteria to retain their characteristic rate of growth in a given host. Using bacteriological counts from tissue homogenates and fluorescence microscopy to determine the spread, localization, and distribution of S. enterica in the tissues, we show that, during a systemic infection, S. enterica adapts to the in vivo environment. The adaptation becomes a measurable phenotype when bacteria that have resided in a donor animal are introduced into a recipient naïve animal. This adaptation does not confer increased resistance to early host killing mechanisms but can be detected as an enhancement in the bacterial net growth rate later in the infection. The enhanced growth rate is lost upon a single passage in vitro, and it is therefore transient and not due to selection of mutants. The adapted bacteria on average reach higher intracellular numbers in individual infected cells and therefore have patterns of organ spread different from those of nonadapted bacteria. These experiments help in developing an understanding of the influence of passage in a host on the fitness and virulence of S. enterica.

Human CMV (HCMV) encodes multiple genes that control NK cell activation and cytotoxicity. Some of these HCMV-encoded gene products modulate NK cell activity as ligands expressed at the cell surface that engage inhibitory NK cell receptors, whereas others prevent the infected cell from upregulating ligands that bind to activating NK cell receptors. A major activating NKR is the homodimeric NKG2D receptor, which has eight distinct natural ligands in humans. It was shown that HCMV is able to prevent the surface expression of five of these ligands (MIC A/B and ULBP1, 2, and 6). In this article, we show that the HCMV gene product UL142 can prevent cell surface expression of ULBP3 during infection. We further show that UL142 interacts with ULBP3 and mediates its intracellular retention in a compartment that colocalizes with markers of the cis-Golgi complex. In doing so, UL142 prevents ULBP3 trafficking to the surface and protects transfected cells from NK-mediated cytotoxicity. This is the first description of a viral gene able to mediate downregulation of ULBP3.

The three-dimensional structure of human chorionic gonadotropin shows that each of its two different subunits has a similar topology, with three disulphide bonds forming a cystine knot. This same folding motif is found in some protein growth factors. The heterodimer is stabilized by a segment of the beta-subunit which wraps around the alpha-subunit and is covalently linked like a seat belt by the disulphide Cys 26-Cys 110. This extraordinary feature appears to be essential not only for the association of these heterodimers but also for receptor binding by the glycoprotein hormones.

Conditions in which modest intracavity anisotropic losses generate highly polarized pulsed XeCI laser output are clarified. These conditions are generally applicable to other pulsed lasers.

Synthetic peptides meeting certain guidelines have been used as immunogens to generate antibodies with predefined specificity. We have raised and characterized using established methods a monoclonal antibody against a synthetic peptide corresponding to the 18-amino acid carboxyterminal sequence (A194-211) of the platelet-derived growth factor (PDGF) A chain expressed by the U343 human glioma cell line. This antibody was generated in order to carry out structure-function studies on this region of PDGF whose biological significance is not yet clear. Anti-PDGF-A194-211 was found to be a low titre, IgM kappa molecule, with a Kd of 2.8 x 10(-7) M. When antibody reactivity was tested with parent PDGF-AAL (A chain homodimer containing a carboxyterminal extension) significant binding was observed. Surprisingly, 125I-PDGF-AAS, consisting of truncated A chains but lacking the extension was also bound. Moreover, poly-L-lysine, beta-thromboglobulin, PDGF-A194-211, and myoglobin competed dose-dependently with 125I-PDGF-AAL for antibody. 125I-bovine serum albumin was also bound. Examination of the primary sequence of proteins and peptides bound by the antibody revealed only one shared structural motif: a lysyl-lysine moiety. Selected small synthetic peptides containing this and other sequences were used as potential competitors of 125I-PDGF-A194-211 in antibody binding. Lysyl-lysyl-glycyl-glutamic acid [corrected] and lysyl-lysine competed, whereas lysyl-leucine did not. These results suggest that as few as two amino acid residues constitute a functional antigenic determinant and contrast with most previous estimates of the minimum number of residues required. Furthermore, we show that guidelines governing the design of synthetic peptides for their use as antigens to produce monoclonal antibodies of predetermined specificity may be unreliable.

Eight human subjects were fed diets enriched in saturated fat (SF), or polyunsaturated fat (PUF) and after each dietary regimen the plasma heparinthrombin clotting time (HTCT) was determined. The HTCT of citrated plasma indicated reduced heparin-neutralizing activity (HNA) after PUF feeding compared with SF feeding. Platelet factor 4 (PF4) levels in the citrated plasma samples demonstrated an inverse correlation with the HTCT (r = 0.62). Experiments with purified PF4 indicated that the PF4 present in citrated plasma could only account for approximately 10% of the HNA. Plasma prepared in a manner which minimized in vitro release of platelet constituents contained significantly less PF4 after PUF feeding and indicated that most of the PF4 found in citrated plasma resulted from in vitro release. The factor Xa inhibitory activity of citrated plasma was not significantly altered by either of the dietary regimens.

Experiments are described whose aim is to understand the way in which the light from a pulsed dye laser becomes polarized when there is a simple tilted glass plate in the cavity. If the process consists of a competition between orthogonal linear polarizations, the way the degree of polarization depends on the anisotropy of the cavity loss can be understood approximately by using results of a two-mode rate equation analysis of the laser. It is necessary to suppose that there is a delay between onset of laser action and detection of the laser pulse, however; the delay required is much less than the pulse length. The detailed time dependences of the light in the two orthogonal modes do not follow this model.

Platelet release products and purified platelet-derived growth factor stimulated the phosphorylation of ribosomal protein S6 in cultured mouse Balb/c 3T3 fibroblasts. The post-nuclear fraction of the stimulated cells was enriched in S6 kinase activity specific for sites resembling those phosphorylated within intact cells in response to PDGF as determined by tryptic peptide mapping. 3T3-S6 sites closely resembled those phosphorylated in S6 of rat hepatocytes stimulated with insulin and included sites for both cAMP-dependent and independent kinases.

Androgen receptor assays have been performed on 1371 specimens of histologically confirmed primary and recurrent breast cancer. Forty-two patients who had received tamoxifen as treatment for advanced disease were assessed for objective response. Another 42 patients who had received chemotherapy were similarly studied. Patients with androgen receptor-negative tumors had a significantly poorer response rate to hormone therapy than those with receptor-positive tumors (P less than 0.05). This clinical correlation is supported by survival data of 1181 patients with primary breast cancer which showed that patients with androgen receptor-negative tumors had a highly significant trend toward shorter overall survival than those with receptor-positive tumors (P less than 0.001). Androgen receptor data added significantly to the information provided by estrogen receptor data both in terms of response to hormone treatment and survival.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.

Platelet-derived growth factor was isolated from the release products of washed, human platelets and from freeze-thawed outdated platelet concentrates. On the basis of sodium dodecyl sulphate polyacrylamide gel electrophoresis and amino acid sequence determination we conclude that platelet derived growth factor released from platelets by the agonist thrombin (EC 3.4.4.13) is structurally similar to that isolated from lysed platelets and from platelet concentrates stored for more than 72 hr at room temperature.

Over a five-year period, from 1976 to 1981, hormone-receptor assays were performed in breast cancer specimens from 1868 women and 10 men. Oestrogen, progesterone, and androgen receptor results were distributed from zero to high levels without any clear separation into identifiable groups, which suggests that the division of results into negative and positive is an arbitrary one. The number of positive results increased significantly during the course of the study, probably due to improved tissue handling and increasing experience with assays. Over-all results showed that 61% of primary breast cancers in women compared with only 49% of secondary cancers gave positive results for oestrogen receptor. The incidence of progesterone and androgen receptors was also higher in primary cancers than in secondary cancers. In women with primary cancers, 72% of cancers were women with primary cancers, 72% of cancers were oestrogen-receptor positive in women after menopause, whereas in those before menopause only 48% were positive. Oestrogen receptor was detected in nine out of 10 male breast cancers.

Survival data for 2006 women who had oestrogen receptor assay carried out on primary breast cancer tissue between 1976 and 1982 are presented. There was a significant trend to shorter survival in patients with low ER levels than in those with high ER levels (P less than 0.01). This trend was evident in both pre- and post-menopausal women. The point of maximum discrimination between prognostic groups occurred at 8 fm in premenopausal women and the four year survival rates of patients above and below this level were 84% and 48%, respectively. In post-menopausal women, maximum discrimination occurred at 90 fm, and the four year survival rates above and below this level were 82% and 64%, respectively.

The amino acid sequence of a type 1 copper protein, the 96-residue basic blue protein from cucumber seedlings, has been determined by Edman degradation of the intact molecule and of fragments produced by cleavage with cyanogen bromide and with trypsin. The cucumber basic blue protein shows a marked sequence homology with stellacyanin, and to a smaller degree with plastocyanin and azurin. The known copper ligands of plastocyanin and azurin (corresponding to histidine-37, cysteine-84, histidine-87, and methionine-92 in plastocyanin) are present in the cucumber basic blue protein. However, the latter also contains a half-cystine residue analogous to the suggested fourth ligand of stellacyanin, where methionine is absent.

The expression of the proopiomelanocortin (POMC) gene by normal lung and thyroid was examined by measurement of the content of ACTH, beta-lipotropin (beta LPH), and beta-endorphin (beta EP) in porcine lung and thyroid tissue. Acid extracts of normal porcine lung and thyroid tissue each contained appreciable amounts of immunoreactive (ir) ACTH, ir-beta LPH, and ir-beta EP. The content of ir-beta LPH in both tissues exceeded by severalfold, on a molar basis, the content of ir-ACTH and ir-beta EP, suggesting that the common precursor POMC was processed predominantly to peptides other than ir-ACTH and ir-beta EP. A porcine thyroid extract (Calcitare, porcine calcitonin, Armour) showed equivalent levels of beta EP-like immunoreactivity and bioactivity, measured by opiate radioreceptor assay; in contrast, ACTH-like bioactivity, measured by rat zona fasciculata steroidogenesis, was only 4% of ACTH-like immunoreactivity. On reversed phase high performance liquid chromatography, Calcitare showed multiple peaks of ACTH-like immunoreactivity, one of which coeluted with porcine ACTH-(1-39), and two much smaller peaks of beta EP-like immunoreactivity, of which the smaller coeluted with porcine beta EP. These data suggest that both lung and thyroid gland synthesize POMC, which in normal tissue is usually predominantly processed to species other than ACTH and beta EP. Ectopic secretion of ACTH and beta EP by lung and thyroid neoplasms may thus represent the loss of a system(s) normally responsible for processing the precursor beyond ACTH and beta EP.

The present studies were undertaken to characterize the immunoreactive-corticotropin-releasing factor (ir-CRF) in two areas of the ovine tuberoinfundibular system, hypophysial portal blood, and pituitary. With an antiserum raised against synthetic ovine (o)CRF(1-41) and 125I-Tyro-oCRF(1-41) as the tracer, concentrations of ir-CRF (pg/mg wet weight, n = 5) were: paraventricular hypothalamus (PVN), 11.7 +/- 2.5; median eminence (ME), 2276 +/- 296; anterior pituitary (AP), less than 0.5; posterior pituitary (PP), 10.0 +/- 2.2. Analysis of the ir-CRF in these areas on G-75 Sephadex chromatography revealed two main peaks--a 'major' peak which coeluted with synthetic oCRF(1-41) and a 'minor' peak which eluted eight fractions later. These two immunoreactive species of CRF were also found in hypophysial portal blood. When ME extract was analyzed by reverse-phase high performance liquid chromatography (HPLC), the 'minor' peak of ir-CRF eluted before that of CRF(1-41). Since CRF contains Arg35-Lys36 within its sequence, we tested the hypothesis that the 'minor' peak of ir-CRF represented a fragment, or fragments, of the molecule derived by proteolytic cleavage at this site. Tyro-oCRF(34-41) was digested with trypsin and the reaction products were identified by amino acid analysis. Two of these products were CRF(36-41) and CRF(37-41), and both migrated in the 'minor' peak area on G-75 Sephadex chromatography and HPLC. In the CRF(1-41) RIA, serial dilution of both fragments yielded nonparallel displacement curves. However, with 125I-Tyro-oCRF(34-41) as the radiolabeled ligand and Tyro-oCRF(34-41) as the standard, serial dilutions of CRF(1-41), CRF(36-41), and CRF(37-41) generated parallel displacement curves, and the molar cross-reactivities were 90%, 45% and 10% respectively. When the ir-CRF in HPLC fractions of ovine ME was measured in the Tyro-oCRF(34-41) RIA, the molar abundance of the hexapeptide and pentapeptide could be obtained. Calculations based on the premise that the 'minor' peak was solely composed of either the hexapeptide or pentapeptide indicated that CRF(36-41) could account for up to 37% of the total ir-CRF, or that CRF(37-41) could account for up to 73% of the total immunoreactivity. On more discriminating HPLC systems, immunoreactive (ir-) oCRF in the sheep median eminence (ME) could be resolved into five different molecular forms. On three distinct chromatographic systems, four of these immunoreactive species shared retention characteristics identical with synthetic oCRF(37-41), oCRF(36-41), oCRF(16-41) and oCRF(1-41), with the fifth immunoreactive peak as yet unidentified.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat liver ribosomes and 40 S ribosomal subunits were phosphorylated with the purified catalytic subunit of cAMP-dependent protein kinase. Phosphorylation of ribosomal protein S6 plateaued at around 2 mol of phosphate/mol of protein with both substrates. Peptide map analyses showed that the most prominent phosphorylation sites associated with 40 S substrates were the adjacent serines in the Arg-Leu-Ser-Ser-Leu-Arg segment of S6. The first serine residue appeared to be the preferred site as has been established previously for 80 S ribosomes (Wettenhall, R.E.H., and Cohen, P. (1982) FEBS Lett. 140, 263-269). Additional phosphorylation sites were apparent from the peptide maps. One of these was associated with the triphosphopeptide (termed T1a) having the sequence Arg-Leu-Ser-Ser-Leu-Arg-Ala-Ser-Thr-Ser-Lys. A larger fragment of S6 (termed Tlc) isolated from mild tryptic digests of whole ribosomes, consisted of the T1a sequence extended by the sequence Ser-Glu-Glu-Ser-Gln-(Lys) at the COOH terminus. A comparison of the size and chromatographic and isoelectric focusing properties of the T1a/T1c peptides and prominent tryptic peptides of S6 from insulin-stimulated hepatocytes indicated a relationship between these peptides. Thus, it appeared that some of the potential phosphorylation sites in the T1a/T1c region of S6 are phosphorylated by an insulin-regulated kinase in hepatocytes.

Platelet survival and plasma concentrations of beta thromboglobulin and platelet factor 4 were measured in 44 patients before and 6 months after coronary artery bypass grafting. Postoperatively, patients were randomized to receive sulfinpyrazone, 800 mg/day, or placebo. Preoperatively, platelet survival was significantly shorter than normal, and plasma concentrations of both platelet-specific proteins were significantly elevated. Postoperatively, all three indexes of platelet function tended to become normal, but these changes were statistically significant only in patients treated with sulfinpyrazone. Postoperative exercise testing correlated significantly with plasma concentrations of beta thromboglobulin and platelet factor 4 measured preoperative and postoperative. These results are consistent with reports of the effects of sulfinpyrazine on platelet involvement in other conditions, and suggest that the drug reduces platelet activation and inhibits actual destruction. The results also show a relationship between abnormalities of platelet function and an index of postoperative myocardial ischemia.

Monoclonal antibody PH7 has specificity for the phosphorylated form of the human liver phenylalanine hydroxylase and negligible reactivity towards the dephosphorylated form of the native enzyme by enzyme-linked immunoassay. PH7 binds specifically to the phosphorylated form of the liver enzyme after SDS/polyacrylamide-gel electrophoresis and transfer to nitrocellulose. Competitive blocking assays have been applied in conjunction with reversed-phase h.p.l.c. of purified tryptic fragments of human liver phenylalanine hydroxylase to localize the epitope. The major immunoreactive tryptic peptide cross-reacting with PH7 had an amino acid analysis corresponding to the first 41 amino acids of the human liver phenylalanine hydroxylase sequence and included the serine residue that is thought to be the phosphorylation site. The monoclonal antibody recognized the phosphorylated form of the synthetic decapeptide corresponding to the local phosphorylation-site sequence Gly-Leu-Gly-Arg-Lys-Leu-Ser(P)-Asp-Phe-Gly, but not the dephosphodecapeptide. Thermolysin digestion of the peptide demonstrated the monoclonal antibody bound to the pentapeptide Leu-Ser(P)-Asp-Phe-Gly. Monoclonal antibody PH7 recognized the phosphodecapeptide at concentrations 10(3)-fold higher than with phenylalanine hydroxylase, compared with 10(4)-10(7)-fold higher for other phosphopeptides and phosphoproteins. The results demonstrate that monoclonal antibody PH7 has specificity for the phosphorylated form of phenylalanine hydroxylase at the phosphorylation site.

Classical phenylketonuria, an inborn error in metabolism, is caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase. The identification of putative cDNA clones coding for rat liver phenylalanine hydroxylase by hybrid-selected translation has previously been reported [Robson, K. J., Chandra, T., MacGillivray, R. T. A., & Woo, S. L. C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4701-4705]. The authenticity of the clones, however, could not be definitively ascertained at the time because of a lack of amino acid sequence data of the enzyme in the literature. Purified rat liver phenylalanine hydroxylase was subjected to cyanogen bromide treatment, and the resulting fragments were used for N-terminal amino acid sequence analysis. The partial amino acid sequence was then compared to that deduced from an open reading frame in the nucleotide sequence of the cDNA clones. A perfect match of 17 amino acid residues was found between the two sequences following a unique methionine codon present in the nucleotide sequence, thereby providing unambiguous evidence for the identity of the rat liver phenylalanine hydroxylase cDNA clones.

Mice of strain A/J responded to repeated intraperitoneal injection of Limulus hemocyanin derivatized with arsanilic acid by producing large quantities (approximately 5 mg/mL of ascites fluid) of IgG antibodies specific for this hapten. The antibodies possessed a characteristic idiotypic determinant and exhibited restricted heterogeneity as demonstrated by isoelectric focusing and primary N-terminal amino acid sequence analysis of isolated light and heavy polypeptide chains. Both light- and heavy-chain sequences were comparable to those of myeloma proteins in lack of heterogeneity. The N terminus of the light chain exhibited V KI sequence and only one position in the first 30 residues showed more than one amino acid. No variability was observed in the first 10 N-terminal residues of the heavy chain. Rabbit antiserum to the idiotype blocked binding of hapten by the purified antibody. The presence of both light- and heavy-chain antigenic determinants was required for optimal formation of the idiotypic determinant.

A pulsed dye laser is polarized by insertion of a tilted glass plate in the cavity. The degree of polarization depends upon the cavity losses for light of the two polarizations, and we have measured the dependence for lasers in which the dye (Rh 6G) is dissolved in solvents of various viscosities. It is found that the polarization is less for higher viscosity solvents. A rate equation model is used to make numerical estimates of the effect, and these are compared with the experimental data.

This report describes the application of high performance liquid chromatography (HPLC) and associated techniques to the isolation of inhibin from ovine follicular fluid (oFF). Two modes of HPLC have been assessed. High speed gel permeation chromatography on a Waters I-125 support showed that at pH 7.0 inhibin bioactivity was associated with an approx. 80000 mol. wt protein fraction which co-eluted with the bulk of the protein in oFF and gave a yield of 30-44% of the applied activity. Reversed-phase HPLC (RP-HPLC) of a peptide-rich fraction extracted from oFF was also investigated. Using analytical liquid chromatography on octadecylsilylsilica (ODS-silica), two distinct inhibin bioactive fractions were obtained, with a resultant purification of 23-fold and 3-fold respectively and an overall recovery of 8.5%. These results demonstrate that RP-HPLC may be employed as a direct and rapid technique for the isolation of inhibin from oFF.

A rapid procedure for the isolation of ribosomal protein S6 from rat liver ribosomes has been developed in which proteins were separated by reversed-phase HPLC using wide-pore n-butyl-, n-octyl-, or diphenyl-bonded silica phases. Rapid processing of whole ribosomal material was achieved by the extraction of proteins in 6 M guanidinium hydrochloride and subsequent precipitation of RNA by acidification. Highly purified S6 was obtained in two chromatographic steps as shown by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, and automated microsequencing. The purification of S6 was monitored using 32P-labeled S6 as a marker which cochromatographed with unphosphorylated S6 under the low-pH elution conditions employed. Other ribosomal proteins were also purified using these reversed-phase supports, although in the case of more hydrophobic proteins such as S4 and S10 further optimization of the gradient conditions was required.