Immunogenicity in mice of human metapneumovirus with a truncated SH glycoprotein.

The SH glycoprotein of human metapneumovirus (HMPV) is twice the size of that of human respiratory syncytial virus and possesses a large, hydrophilic luminal domain. The glycoprotein is located on the surface of the virion and of virus infected cells and, if immunogenic, might be expected to play a role in anti-viral immunity. Initial attempts to study anti-SH antibody immunogenicity were thwarted by the instability of the SH gene on passage both in human bronchial epithelial cells and in mice. Repeated passage of virus isolates in human bronchial epithelial cells in culture resulted in the appearance and eventual predominance of HMPV mutants lacking all or most of the luminal domain of SH coincidental with the loss of productive infection in mouse lungs. Where infection was established in mice with an early cell culture passage, the virus recovered from mouse lung differed markedly from the inoculum, carrying 19 coding mutations in the SH luminal domain. Immunization of mice with a mutant virus variant expressing only 14 amino acids of the luminal domain of SH induced a cross-reactive antibody response to both the F glycoprotein and the SH glycoprotein but a largely sub-group specific response to the G glycoprotein. Similar patterns of response were achieved by immunization with individual HMPV glycoproteins expressed from recombinant vaccinia viruses. Recombinant truncated SH glycoprotein induced sub-group cross-reactive antibodies capable of neutralizing wild-type virus. Recombinant F glycoprotein also induced cross-reactive neutralizing antibodies whilst recombinant G glycoprotein induced largely strain-specific, non-neutralizing antibodies.

The rise and fall of XMRV.

Due to the relatively recent emergence of the human T-lymphotropic and the human immunodeficiency viruses, enthusiasm for the identification of novel viruses, especially retroviruses, with pathogenic potential in humans, remains high. Novel technologies are now available with the ability to search for unknown viruses, such as gene arrays and new generation sequencing of tissue and other samples. In 2006, chip technology identified a novel retrovirus in human prostate cancer (PCa) tissue samples. Due to close homology to a mouse retrovirus, the virus was named xenotropic murine leukaemia virus-related virus (XMRV). Ever since the initial disease association with PCa, XMRV has stirred a lot of attention and concern worldwide for the medical community, public health officials and in particular global transfusion services. Public response, in this new era of electronic communication and advocacy was rapid, wide and unprecedented. In this review, we outline the course of biomedical research efforts that were put forward internationally in the process of determining the risk to the human population, the response of the blood banking community and review the current state of knowledge of xenotropic murine retroviruses. Although XMRV is no longer regarded as an infection of humans, a lesson was learnt in modern virology that holds deeper implications for biomedical research, particularly stem cell generation and transplantation practices.

The effect of level of crude protein and available lysine on finishing pig performance, nitrogen balance and nutrient digestibility.

Two trials were conducted to investigate the effect of decreasing the crude protein (CP) content of diets for finishing pigs containing two levels of available lysine on nutrient digestibility, nitrogen (N) balance and production performance. Ten finishing diets containing five levels of CP (on average 144, 155, 168, 182 and 193 g/kg fresh basis) and two levels of available lysine (6.9 and 8.2 g/kg fresh basis) were formulated. The diets were offered to pigs on a performance trial (n = 800 Large White (LW)×Landrace (LR) pigs) from 10 wk of age until finish at 21 wks+5 d of age. Average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) were calculated. In addition, a digestibility/N balance trial was conducted using pigs (n = 80 LW×LR) housed in metabolism crates. Digestibility of dry matter (DM), CP, oil, fibre and energy was determined. N balance values were determined through analysis of N content of urine and faeces ('as determined'). N balance values were also calculated using ADG values and assuming that 16% of growth is protein deposition ("as calculated"). Pig performance was poor between 10 and 13 wk of age which indicated that the dietary treatments were nutritionally inadequate for pigs less than 40 kg. There was a significant (p<0.01) quadratic effect of increasing CP level on feed intake, ADG and FCR from 10 to 13 wk which indicated that the lower CP levels did not supply adequate levels of essential or non-essential amino acids. There was no effect of increasing available lysine level throughout the early period, which in conjunction with the response in older pigs, suggested that both 8.2 and 6.9 g/kg available lysine were insufficient to drive optimum growth. There was a positive response (p<0.05) to increasing available lysine level from 13 wk to finish which indicated that 6.9 g/kg available lysine was not adequate for finishing pigs. Energy digestibility decreased with decreasing CP level of diets containing 6.9 g/kg available lysine which may be attributed to the higher fibre content of the lower CP diets. Nitrogen excretion (g/d) was lowered when dietary CP was reduced regardless of whether the values were determined through balance or calculated using ADG. Calculated N excretion decreased linearly (p<0.001) and quadratically (p<0.001) with decreasing dietary CP content. When the N balance figures calculated in this study were compared with those quoted in the Northern Ireland and English Nitrates Directive Action Programmes, N excretion was less per pig (wean to finish) offered a 169 g/kg CP, 8.2 g/kg available lysine diet (2.39 kg vs 3.41 kg (Northern Ireland) and 2.93 kg (England)).

Mitogen-activated protein kinase pathways.

Nearly all cell surface receptors utilize one or more of the mitogen-activated protein kinase cascades in their repertoire of signal transduction mechanisms. Recent advances in the study of such cascades include the cloning of genes encoding novel members of the cascades, further definition of the roles of the cascades in responses to extracellular signals, and examination of cross-talk between different cascades.

Phosphorylation of TPL-2 on serine 400 is essential for lipopolysaccharide activation of extracellular signal-regulated kinase in macrophages.

Tumor progression locus 2 (TPL-2) kinase is essential for Toll-like receptor 4 activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) and for upregulation of the inflammatory cytokine tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-stimulated macrophages. LPS activation of ERK requires TPL-2 release from associated NF-kappaB1 p105, which blocks TPL-2 access to its substrate, the ERK kinase MEK. Here we demonstrate that TPL-2 activity is also regulated independently of p105, since LPS stimulation was still needed for TPL-2-dependent activation of ERK in Nfkb1(-/-) macrophages. In wild-type macrophages, LPS induced the rapid phosphorylation of serine (S) 400 in the TPL-2 C-terminal tail. Mutation of this conserved residue to alanine (A) blocked the ability of retrovirally expressed TPL-2 to induce the activation of ERK in LPS-stimulated Nfkb1(-/-) macrophages. TPL-2(S400A) expression also failed to reconstitute LPS activation of ERK and induction of TNF in Map3k8(-/-) macrophages, which lack endogenous TPL-2. Consistently, the S400A mutation was found to block LPS stimulation of TPL-2 MEK kinase activity. Thus, induction of TPL-2 MEK kinase activity by LPS stimulation of macrophages requires TPL-2 phosphorylation on S400, in addition to its release from NF-kappaB1 p105. Oncogenic C-terminal truncations of TPL-2 that remove S400 could promote its transforming potential by eliminating this critical control step.

Translating concepts of risk and loss in rodent models of gambling and the limitations for clinical applications.

Gambling involves placing something of value at risk in exchange for the opportunity to potentially gain something of greater value in return. A variety of gambling paradigms have been designed to study the maladaptive decision-making that underlies problematic gambling. Central to these gambling models are the definitions of "risk" and "loss", especially when translating the results from rodent studies to clinical applications. Risk and loss are not mutually exclusive but rather share some overlap. With careful interpretation and consideration of the limitations of these behavioral paradigms, results from rodent models may provide insights into the neurobiology of risky decision-making that leads to problematic gambling in humans.

A constitutively active and nuclear form of the MAP kinase ERK2 is sufficient for neurite outgrowth and cell transformation.

BACKGROUND: Mitogen-activated protein (MAP) kinases are ubiquitous components of many signal transduction pathways. Constitutively active variants have been isolated for every component of the extracellular-signal-regulated kinase 1 (ERK1) and ERK2 MAP kinase pathway except for the ERK itself. RESULTS: To create an activated ERK2 variant, we fused ERK2 to the low activity form of its upstream regulator, the MAP kinase kinase MEK1. The ERK2 in this fusion protein was active in the absence of extracellular signals. Expression of the fusion protein in mammalian cells did not activate endogenous ERK1 or ERK2. It was sufficient, however, to induce activation of the transcription factors Elk-1 and AP-1, neurite extension in PC12 cells in the absence of nerve growth factor, and foci of morphologically and growth-transformed NIH3T3 cells, if the fusion protein was localized to the nucleus. A cytoplasmic fusion protein was without effect. CONCLUSIONS: Activation of ERK2 is sufficient to cause several transcriptional and phenotypic responses in mammalian cells. Nuclear localization of activated ERK2 is required to induce these events.

Diagnosis of human metapneumovirus by immunofluorescence staining with monoclonal antibodies in the North-East of England.

BACKGROUND: Since its discovery in 2001 human metapneumovirus (hMPV) has been shown to be a significant cause of human respiratory disease, responsible for 5-8% of respiratory infections in hospitalised children. Diagnosis hitherto has been largely carried out by reverse tanscriptase polymerase chain reaction (RT-PCR) but immunofluorescence staining of cells from nasopharyngeal secretions (IF) offers advantages for some laboratories and may produce a more rapid result in urgent cases. We have recently demonstrated that IF with a rabbit antiserum gave sensitivity equal to that of RT-PCR. However, monoclonal antibodies offer a more plentiful, uniform IF reagent. OBJECTIVES: Here we have evaluated a pool of anti-hMPV monoclonal antibodies in the routine diagnosis of respiratory infections in hospitalised infants and children. STUDY DESIGN: Eight hundred and fifty-seven routine respiratory specimens were tested by IF with rabbit polyclonal antiserum and monoclonal antibody pool in parallel. A further 1003 specimens were tested with the monoclonal antibody pool alone. All specimens were also tested for a panel of other respiratory viruses by IF. RESULTS: Both rabbit polyclonal antiserum and monoclonal antibody pool gave positive results in 56 and negative results in 797 specimens. The rabbit polyclonal antibody detected virus in a further two specimens which were negative when tested with the monoclonal pool giving a concordance of 96.6% and a specificity of 100% for the monoclonal antibody pool. Overall hMPV was detected in 5% of specimens whilst 18.4% were positive for hRSV. CONCLUSIONS: The monoclonal antibody pool-based IF is a robust assay suitable for routine use with a sensitivity only slightly less than that of the other major diagnostic methodologies available.

Effects of anisomycin on consolidation and reconsolidation of a morphine-conditioned place preference.

Protein synthesis inhibitors block consolidation of memory and may also block the reconsolidation of a reactivated memory in paradigms of aversive learning, but the evidence for reconsolidation effects is conflicting in appetitive paradigms. We now report that intra-cerebroventricular (ICV) anisomycin (400microg) prevents consolidation of morphine-induced place preference (CPP), but does not impair its reconsolidation unless the reactivation procedure associates anisomycin with the morphine context. Rats were injected alternately with morphine (5mg/kg, IP) or vehicle, and confined to one of two distinctive compartments in a three compartment apparatus. On a subsequent day rats were allowed to choose the compartment they preferred in a 20min test session. In the first experiment, rats that were injected with vehicle or with anisomycin before or 3h after training sessions, developed a CPP. However, rats that received anisomycin ICV immediately after training sessions did not develop a CPP. In experiment 2, rats received no ICV injections during initial training. Once a CPP was established, they received four additional training sessions on which they received vehicle or anisomycin ICV. All groups continued to prefer the morphine-paired compartment after reactivation sessions with vehicle or anisomycin ICV. In experiment 3, ICV anisomycin was administered selectively on morphine-paired reactivation trials or saline-paired reactivation trials and the CPP was weakened or strengthened, respectively. This suggests that associations between aversive effects of the amnestic treatment and the morphine context might mimic disruption of reconsolidation.

Central but not peripheral beta-adrenergic antagonism blocks reconsolidation for a morphine place preference.

Blocking the process of memory reconsolidation by means of amnestic agents may prove to have therapeutic applications. Here we used a morphine-induced conditioned place preference as an index of drug seeking. After inducing in rats a preference for a distinctive compartment paired with morphine, the memory for drug experience was reactivated by a 20-min test session and saline, the beta-antagonist propranolol, or the peripherally acting beta-antagonist nadolol were administered. Animals which received saline or nadolol upon reactivation, or propanolol without memory reactivation, maintained their preference for the drug-paired compartment 24h and seven days later. However, animals that received propranolol upon reactivation no longer displayed a morphine preference on either test, although these animals once again expressed a preference when given a morphine-primed retest at 10 days. Our results suggest that beta-blockers may have potential for attenuating the impact of cue-induced craving which is a major cause of relapse in detoxified addicts.

Lipopolysaccharide activation of the TPL-2/MEK/extracellular signal-regulated kinase mitogen-activated protein kinase cascade is regulated by IkappaB kinase-induced proteolysis of NF-kappaB1 p105.

The MEK kinase TPL-2 (also known as Cot) is required for lipopolysaccharide (LPS) activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade in macrophages and consequent upregulation of genes involved in innate immune responses. In resting cells, TPL-2 forms a stoichiometric complex with NF-kappaB1 p105, which negatively regulates its MEK kinase activity. Here, it is shown that lipopolysaccharide (LPS) stimulation of primary macrophages causes the release of both long and short forms of TPL-2 from p105 and that TPL-2 MEK kinase activity is restricted to this p105-free pool. Activation of TPL-2, MEK, and ERK by LPS is also demonstrated to require proteasome-mediated proteolysis. p105 is known to be proteolysed by the proteasome following stimulus-induced phosphorylation of two serines in its PEST region by the IkappaB kinase (IKK) complex. Expression of a p105 point mutant, which is not susceptible to signal-induced proteolysis, in RAW264.7 macrophages impairs LPS-induced release of TPL-2 from p105 and its subsequent activation of MEK. Furthermore, expression of wild-type but not mutant p105 reconstitutes LPS stimulation of MEK and ERK phosphorylation in primary NF-kappaB1-deficient macrophages. Consistently, pharmacological blockade of IKK inhibits LPS-induced release of TPL-2 from p105 and TPL-2 activation. These data show that IKK-induced p105 proteolysis is essential for LPS activation of TPL-2, thus revealing a novel function of IKK in the regulation of the ERK MAP kinase cascade.

Roles of "Wanting" and "Liking" in Motivating Behavior: Gambling, Food, and Drug Addictions.

The motivation to seek out and consume rewards has evolutionarily been driven by the urge to fulfill physiological needs. However in a modern society dominated more by plenty than scarcity, we tend to think of motivation as fueled by the search for pleasure. Here, we argue that two separate but interconnected subcortical and unconscious processes direct motivation: "wanting" and "liking." These two psychological and neuronal processes and their related brain structures typically work together, but can become dissociated, particularly in cases of addiction. In drug addiction, for example, repeated consumption of addictive drugs sensitizes the mesolimbic dopamine system, the primary component of the "wanting" system, resulting in excessive "wanting" for drugs and their cues. This sensitizing process is long-lasting and occurs independently of the "liking" system, which typically remains unchanged or may develop a blunted pleasure response to the drug. The result is excessive drug-taking despite minimal pleasure and intense cue-triggered craving that may promote relapse long after detoxification. Here, we describe the roles of "liking" and "wanting" in general motivation and review recent evidence for a dissociation of "liking" and "wanting" in drug addiction, known as the incentive sensitization theory (Robinson and Berridge 1993). We also make the case that sensitization of the "wanting" system and the resulting dissociation of "liking" and "wanting" occurs in both gambling disorder and food addiction.

Kinetics of histone hyperacetylation and deacetylation in human diploid fibroblasts.

We have utilized sodium butyrate-treated normal human diploid fibroblasts to study core histone hyperacetylation kinetics. We report a small, distinct population of core histone characterized by a very rapid rate of hyperacetylation (t1/2 approximately equal to 10-15 min for monoacetylated histone H4) compared to the slower rate (t1/2 approximately equal to 140-200 min for monoacetylated H4) observed for bulk histone. Two rates of core histone deacetylation were also detected and we demonstrated that the rapidly hypermodified histone H4 population was also rapidly deacetylated. The kinetics of histone H4 hyperacetylation and deacetylation in these cells were not significantly altered, regardless of whether cultures were exponentially growing, confluent or arrested in an essentially non-mitotic state.

Contractile responses to endothelin in feline cortical vessels in situ.

In this study in chloralose-anaesthetised cats, the vasomotor responses of individual pial vessels on the cortical surface to perivascular subarachnoid microapplication of endothelin were examined. Endothelin (3 x 10(-10) -3 x 10(-6) M) induced marked vasoconstriction of pial arterioles (-33.5 +/- 3.8% at 3 x 10(-6) M) and pial veins (-35.1 +/- 2.7% at 3 x 10(-6) M). The concentration of endothelin inducing half-maximal response was in the nanomolar range, with pial veins being slightly more sensitive to the peptide than pial arterioles. Vasoconstrictions induced by endothelin were extremely prolonged, persisting for approximately 90 min after a single microapplication. Arterioles constricted by endothelin remained responsive to perivascular microapplication of K+ (10 mM) or alkalotic CSF (pH 7.48).

Endothelin-1-induced reductions in cerebral blood flow: dose dependency, time course, and neuropathological consequences.

The capacity of endothelin-1 to induce severe reductions in cerebral blood flow and ischaemic neuronal damage was assessed in anaesthetised rats. Endothelin-1 (25 microliters of 10(-7)-10(-4) M) was applied to the adventitial surface of an exposed middle cerebral artery and striatal blood flow assessed by the hydrogen clearance technique. Endothelin-1 induced severe dose-dependent reductions in cerebral blood flow (e.g., minimum CBF at 10(-5) M of 9 +/- 11 ml 100 g-1 min-1 compared to 104 +/- 22 ml 100 g-1 min-1 with vehicle, p < 0.05), which persisted for at least 60 min at each concentration of endothelin-1. Application of endothelin-1 to the middle cerebral artery produced dose-dependent ischaemic brain damage (e.g., volume of damage of 65 +/- 34 mm3 at 10(-5) M compared to 0.22 +/- 0.57 mm3 for vehicle, p < 0.01). These data demonstrate that endothelin-1 is capable of reducing blood flow to pathologically low levels and provide a new model of controlled focal ischaemia followed by reperfusion.

Characterisation of glucose transport in the hyperthermophilic Archaeon Sulfolobus solfataricus.

Sulfolobus solfiataricus is a hyperthermophilic Archaeon growing at 80 degrees C, pH 3. The glucose transport system of this organism has been characterised kinetically at this temperature and pH using 2-deoxy-D-glucose: the sugar analogue is transported into the cells with a Km = 1.8 +/- 0.3 microM and a Vmax = 3.6 +/- 0.1 nmol min(-1) (mg protein)(-1), with an intracellular accumulation of up to 200-fold over the extracellular concentration. Transport was significantly reduced at pH 5. Inhibition of 2-deoxy-D-glucose transport was investigated using a variety of sugars and sugar analogues; D-glucose, D-galactose and D-mannose showed the highest affinity for the transporter, with D-glucose possessing a Ki = 120 +/- 20 nM.

Histone acetylation and deacetylation in senescent human diploid fibroblasts.

We measured the kinetics of histone H4 hyperacetylation and kinetics of deacetylation for all core histones in young and senescent human diploid fibroblast-like cells. Both cell populations contained a fraction of histone H4 characterized by very rapid acetylation and deacetylation. The kinetics of these reactions were similar in young and senescent cells. The distribution of acetylated species of core histones was also similar in young and senescent human diploid fibroblast-like cells. These results indicated that previously-reported alterations in chromatin template activity in senescent cells were due to a mechanism other than histone acetylation.

The effects of histones, in vitro age, and culture state on the digestion of DNA by micrococcal nuclease and deoxyribonuclease I.

Nuclei from confluent and mitotically arrested populations of human diploid fibroblast-like cells were subjected to digestion by micrococcal nuclease and deoxyribonuclease (DNase I) following the removal of various histone components by salt extraction. There was no age or culture state variation in the susceptibility of DNA to micrococcal nuclease digestion. There was an age related inhibition of DNA digestion by DNase I in nuclei from older confluent cells before and after the removal of H1 histone but not after the removal of core particle histones. This inhibition was not detected in older arrested populations. These results indicate that an age-related masking by nucleosome core histones may limit the accessibility of DNA to enzymatic activities in older confluent cells. Since this inhibition was absent in older arrested populations, the importance of limited DNA accessibility as a primary cause of cellular senescence is questionable.

Pyloric gland adenoma of the main pancreatic duct.

A case of a pyloric gland type adenoma of the main pancreatic duct in a 69-year-old woman is reported. The tumor led to occlusion and cystic dilatation of the main duct in the pancreatic tail. The surgical resection specimen disclosed a polypoid, bilobed mass attached to the wall of the main pancreatic duct by a thin fibrous stalk. Light-microscopic examination revealed a well-demarcated nodule composed of closely packed tubular glands lined by columnar, mucin-secreting cells with abundant clear cytoplasm and basally oriented nuclei. Focal, mild cytologic atypia was seen. Pyloric metaplasia and focal papillary hyperplasia was present in the adjacent ductal epithelium. Periodic acid-Schiff reactions, with and without diastase predigestion, showed reactivity in the tubular glands, whereas alcian blue (pH 2.5) was negative. Immunohistochemical stains for chromogranin, serotonin, somatostatin, and gastrin failed to detect the respective antigens. Genetic analysis using polymerase chain reaction with mutant enrichment and allele specific oligonucleotide hybridization detected a single mutation at codon 12 of K-ras, which changed the wild-type glycine to arginine. This mutation is commonly found in invasive pancreatic ductal carcinomas. Although tumors with microscopic and immunohistochemical features consistent with pyloric gland adenoma have been described in the gallbladder, to our knowledge, this is the first reported case within the pancreatic ductal system. The finding of a K-ras, codon 12 mutation and the presence of focal dysplasia may denote neoplastic potential in association with this lesion.

Pulmonary granulomas secondary to embolic prosthetic valve material.

A patient is reported with unusually severe variance of a prosthetic Beall fabricated Teflon disc valve in the tricuspid position. Foreign material was found in the vessels, vessel walls, and parenchyma of the lung associated with a foreign body type of inflammatory reaction. This material was identified by scanning electron microscopy and high energy dispersive x-ray analysis as Teflon that was embolic from the eroded valve.