Chronic inflammation decreases arcuate kisspeptin expression in male sheep.

Lipopolysaccharide (LPS) from Gram-negative bacteria induces an immune response and impairs reproduction through suppression of gonadotropin releasing hormone (GnRH), subsequently luteinizing hormone (LH) secretion. While there is evidence that acute inflammation inhibits kisspeptin, little is known about the impact of chronic inflammation on this key reproductive neuropeptide in livestock species. Thus, we sought to examine a central mechanism whereby LPS suppresses LH secretion in sheep. Twenty wethers were randomly assigned to one of five treatment groups: control (CON; n=4), single acute IV LPS dose (SAD; n=4), daily acute IV LPS dose (DAD; n=4), daily increasing IV LPS dose (DID; n=4), and chronic subcutaneous LPS dose (CSD; n=4). On Days 1 and 7, blood samples were collected every 12 minutes for 360 minutes using jugular venipuncture. Following blood collection on Day 7, all animals were euthanized, brain tissue was perfused with 4% paraformaldehyde, and hypothalamic blocks were removed and processed for immunohistochemistry. On Day 1, LH pulse frequency was significantly lower (p=0.02) in SAD (0.25 ± 0.1 pulses/hour), DAD (0.25 ± 0.1 pulses/hour), DID (0.35 ± 0.1 pulses/hour), and CSD (0.40 ± 0.1 pulses/hour) compared to CON (0.70 ±0.1 pulses/hour). On Day 7, only DID animals (0.35 ± 0.1 pulses/hour) had significantly lower (p=0.049) LH pulse frequency compared to controls (0.85 ± 0.1 pulse/hour). Furthermore, only DID animals (33.3 ± 10.9 cells/section/animal) had significantly fewer (p=0.001) kisspeptin-immunopositive cells compared to controls (82.6 ± 13.6 cells/section/animal). Taken together, we suggest that daily increasing doses of LPS is a powerful inhibitor of kisspeptin neurons in young male sheep and a physiologically relevant model to examine the impact of chronic inflammation on the reproductive axis in livestock.

Characterization of placentome vascular perfusion in relation to pregnancy associated glycoproteins throughout gestation in pregnant beef heifers.

During pregnancy, blood flow to the uterus changes to support fetal demand. Placentomes serve as vascular attachment sites on the placenta for exchange of gases, nutrients, and metabolic products. Non-invasive methods of ultrasonography and biomarkers have been described to assess placental health and fetal viability. Pregnancy associated glycoproteins (PAGs) are produced by the ruminant placenta and are detected in maternal circulation. In cattle, changes in circulating PAG concentrations are associated with embryonic and fetal outcomes. The objective of this study was to determine the association between placentome blood perfusion and circulating PAG concentrations as they relate to the health of the developing fetus. We hypothesized that placentome perfusion and PAG concentration will be positively correlated and associated with neonatal outcome. A prospective, observational study was designed using 26 pregnant, nulliparous, Angus heifers in which PAG concentration and placentome blood perfusion were assessed throughout gestation, with assessment of calving characteristics following parturition. Placentome blood perfusion was visualized at 30-day intervals via transrectal Doppler ultrasonography with power flow function. Ultrasound images were analyzed using ImageJ software to determine the percent area of perfusion and integrated pixel densities. Venous blood was collected and PAG concentrations were determined via serum PAG enzyme-linked immunoassay. Mean placentome blood perfusion increased as gestation advanced. PAG concentrations demonstrated the expected temporal trend, increasing with gestation length, and were positively linearly correlated with placentome perfusion (P < 0.0001). The relationship identified between circulating PAG concentration and placentome blood perfusion suggests the use of transrectal power flow Doppler ultrasonography as a noninvasive technique to determine placental blood flow morphometrics to assess conceptus wellbeing throughout pregnancy.

Evaluation of glucose response to 3 types of insulin using a continuous glucose monitoring system in healthy alpacas.

BACKGROUND: Persistent hyperglycemia is common in alpacas and typically requires insulin administration for resolution; however, little is known about alpacas' response to different insulin formulations. OBJECTIVES: To evaluate the effects of 3 insulin formulations on blood glucose concentrations and the use of a continuous glucose monitoring (CGM) system in alpacas. ANIMALS: Six healthy alpacas. METHODS: The CGM was installed in the left paralumbar fossa at the start of this crossover study and recorded data every 5 minutes. Regular insulin, NPH insulin, insulin glargine, and dextrose were administered to each alpaca over a 2-week period. Blood samples were collected for glucose testing at 0, 1, 2, 4, 6, 8, and 12 hours, and then every 6 hours after each administration of insulin or dextrose. Data were compared by using method comparison techniques, error grid plots, and ANOVA. RESULTS: Blood glucose concentrations decreased most rapidly after regular insulin administration when administered IV or SC as compared to the other formulations. The NPH insulin produced the longest suppression of blood glucose. The mean CGM interstitial compartment glucose concentrations were typically lower than the intravascular compartment glucose concentrations. The alpacas had no adverse reactions to the different insulin formulations. CONCLUSIONS AND CLINICAL IMPORTANCE: The NPH insulin might be more appropriate for long-term use in hyperglycemic alpacas because of its extended duration of action. A CGM is useful in monitoring glucose trends and reducing blood collection events, but it should not be the sole method for determining treatment protocols.

Subchronic JP-8 jet fuel exposure enhances vulnerability to noise-induced hearing loss in rats.

Both laboratory and epidemiological studies published over the past two decades have identified the risk of excess hearing loss when specific chemical contaminants are present along with noise. The objective of this study was to evaluate the potency of JP-8 jet fuel to enhance noise-induced hearing loss (NIHL) using inhalation exposure to fuel and simultaneous exposure to either continuous or intermittent noise exposure over a 4-wk exposure period using both male and female Fischer 344 rats. In the initial study, male (n = 5) and female (n = 5) rats received inhalation exposure to JP-8 fuel for 6 h/d, 5 d/wk for 4 wk at concentrations of 200, 750, or 1500 mg/m³. Parallel groups of rats also received nondamaging noise (constant octave band noise at 85 dB(lin)) in combination with the fuel, noise alone (75, 85, or 95 dB), or no exposure to fuel or noise. Significant concentration-related impairment of auditory function measured by distortion product otoacoustic emissions (DPOAE) and compound action potential (CAP) threshold was seen in rats exposed to combined JP-8 plus noise exposure when JP-8 levels of 1500 mg/m³ were presented with trends toward impairment seen with 750 mg/m³ JP-8 + noise. JP-8 alone exerted no significant effect on auditory function. In addition, noise was able to disrupt the DPOAE and increase auditory thresholds only when noise exposure was at 95 dB. In a subsequent study, male (n = 5 per group) and female (n = 5 per group) rats received 1000 mg/m³ JP-8 for 6 h/d, 5 d/wk for 4 wk with and without exposure to 102 dB octave band noise that was present for 15 min out of each hour (total noise duration 90 min). Comparisons were made to rats receiving only noise, and thosereceiving no experimental treatment. Significant impairment of auditory thresholds especially for high-frequency tones was identified in the male rats receiving combined treatment. This study provides a basis for estimating excessive hearing loss under conditions of subchronic JP-8 jet fuel exposure.

Development of a wireless system for auditory neuroscience.

In order to study how the auditory cortex extracts communication sounds in a realistic acoustic environment, a wireless system is being developed that will transmit acoustic as well as neural signals. The miniature transmitter will be capable of transmitting two acoustic signals with 37.5 KHz bandwidths (75 KHz sample rate) and 56 neural signals with bandwidths of 9.375 KHz (18.75 KHz sample rate). These signals will be time-division multiplexed into one high bandwidth signal with a 1.2 MHz sample rate. This high bandwidth signal will then be frequency modulated onto a 2.4 GHz carrier, which resides in the industrial, scientic, and medical (ISM) band that is designed for low-power short-range wireless applications. On the receiver side, the signal will be demodulated from the 2.4 GHz carrier and then digitized by an analog-to-digital (A/D) converter. The acoustic and neural signals will be digitally demultiplexed from the multiplexed signal into their respective channels. Oversampling (20 MHz) will allow the reconstruction of the multiplexing clock by a digital signal processor (DSP) that will perform frame and bit synchronization. A frame is a subset of the signal that contains all the channels and several channels tied high and low will signal the start of a frame. This technological development will bring two benefits to auditory neuroscience. It will allow simultaneous recording of many neurons that will permit studies of population codes. It will also allow neural functions to be determined in higher auditory areas by correlating neural and acoustic signals without apriori knowledge of the necessary stimuli.

Orf virus encodes a novel secreted protein inhibitor of granulocyte-macrophage colony-stimulating factor and interleukin-2.

The parapoxvirus orf virus encodes a novel soluble protein inhibitor of ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). The GM-CSF- and IL-2-inhibitory factor (GIF) gene was expressed as an intermediate-late viral gene in orf virus-infected cells. GIF formed homodimers and tetramers in solution, and it bound ovine GM-CSF with a K(d) of 369 pM and ovine IL-2 with a K(d) of 1.04 nM. GIF did not bind human GM-CSF or IL-2 in spite of the fact that orf virus is a human pathogen. GIF was detected in afferent lymph plasma draining the skin site of orf virus reinfection and was associated with reduced levels of lymph GM-CSF. GIF expression by orf virus indicates that GM-CSF and IL-2 are important in host antiviral immunity.

Cytokines and their inhibitors in orf virus infection.

The epitheliotropic parapoxvirus, orf virus, can repeatedly infect sheep skin. A specific immune response is generated as reinfections induce smaller lesions with quicker resolution times than primary lesions. Cyclosporin-A treatment abrogates this partial immunity. Cytokine mRNAs detected in lesion biopsies include the transcripts for IL-1 beta, IL-3 GM-CSF, TNF-alpha and, less reproducibly, IFN-gamma. CD4+ T-cells predominate in afferent lymph draining the site of infection, and are the major source of GM-CSF and IFN-gamma. IL-1 beta and IL-8 are also detected. The orf virus genome contains a homologue of mammalian vascular endothelial growth factor that may enhance virulence and a vaccinia virus E3L-like gene which may inhibit the anti-viral effect of the interferons. A GM-CSF inhibitory activity has also been discovered and has been 'chased' into a 10 kb DNA segment of the orf virus genome. These studies indicate that orf virus may temporarily avoid host immunity by a combination of acute, rapid infection and replication in the epidermis and by producing virulence factors that inhibit protective proteins of the host immune and inflammatory response.

Evidence that a kissing loop structure facilitates genomic RNA dimerisation in HIV-1.

Genomic RNA isolated from retroviral particles is a dimer composed of two identical strands. A region called the dimer linkage signal close to the 5' end of the RNA may be involved in forming the dimer. Several models for the formation of the HIV-1 RNA dimer have been proposed. In the kissing loop model, dimerisation results from base-pairing between homologous sequences in an RNA stem-loop. In the guanine tetrad model interstrand guanine contacts from the dimer. We have made mutations preventing the dimerisation of subgenomic RNAs in vitro by these mechanisms. To prevent the kissing loop dimer forming we changed the complementary loop sequence from 711GCGCGC716 to 711AAACGC716. To prevent the guanine tetrad dimer forming we changed G819 to U. These mutations were introduced into a clone of HIV-1NL4-3 separately and collectively. All three clones produced infectious virions. Dimeric RNA with similar thermal stabilities was isolated from viruses containing either the single or the double mutations. The results suggest that sequences involved in forming a guanine tetrad are not important for HIV-1 RNA dimerisation. In contrast sequences involved in forming a kissing loop complex are not absolutely required, but are important in forming a stable HIV-1 RNA dimer.

A study of the dimerization of Rous sarcoma virus RNA in vitro and in vivo.

The Rous sarcoma virus dimer linkage site (DLS) has been located by electron microscopy at position 511 +/- 28 nucleotides. We have studied the dimerization of RNAs encompassing the first 634 nucleotides of Rous sarcoma virus and conclude that there are at least two dimerization signals. One is located between nucleotides 531 and 634 and may involve Watson-Crick pairing of an imperfect inverted repeat. The other signal is located between nucleotides 496 and 530. A tetraguanine sequence at nucleotides 523-526 is required for dimerization of this domain. The guanines are not involved in an identifiable Watson-Crick interaction or in guanine tetrad formation. Either dimerization domain can initiate the dimerization of RNA 1-634. It is possible that these domains are two parts of a single dimerization signal. Interstrand RNA contacts within the virion are not limited to the DLS but occur along the length of the genome. Nascent virions contain monomeric RNA which slowly associates to form an RNA dimer. The limiting step in dimerization is not proteolytic cleavage of the gag precursor because only the mature capsid protein p27 can be detected in these nascent virions.

Transcripts from the Epstein-Barr virus BamHI A fragment are detectable in all three forms of virus latency.

An unexpected feature of the latency II form of Epstein-Barr virus (EBV) infection seen in the epithelial tumor nasopharyngeal carcinoma (NPC) is the presence of spliced polyadenylated RNAs encoded from the BamHI A fragment of the viral genome and running in the opposite orientation to several BamHI-A lytic cycle genes. The importance of these BamHI-A transcripts and the specificity of their association with NPC remain to be determined. In this study, we examined the extent to which such RNAs are present in other transcriptionally distinct forms of EBV latency seen in B cells. Two independent assays of BamHI-A transcription were employed: amplification across defined splice junctions in cDNAs, using the polymerase chain reaction, and in situ hybridization with a radiolabeled riboprobe specific for a putative open reading frame downstream of these splice junctions. Such methods, which easily detected BamHI-A RNAs in fresh NPC biopsies and transplantable NPC lines, also revealed consistent expression of these transcripts in all EBV-positive Burkitt's lymphoma cell lines displaying the highly restricted latency I form of infection (BamHI-F promoter usage) as well as in all EBV-transformed lymphoblastoid cell lines (LCLs) displaying the latency III form of infection (BamHI-C/W promoter usage). Expression in established LCLs, occurring irrespective of virus producer status, was not a consequence of continued in vitro passage; thus, appropriately spliced BamHI-A transcripts could be amplified from normal B cells within 1 day of their experimental infection in vitro, along with BamHI-C/W promoter-initiated but not BamHI-F promoter-initiated mRNAs. In situ hybridization both on Burkitt's lymphoma cell lines and on LCLs showed that essentially every cell contained BamHI-A transcripts, although at levels apparently lower than those observed in NPC. We conclude that expression of the BamHI-A RNAs is a consistent feature shared by all known forms of latent EBV infection.

The Epstein-Barr virus (EBV) nuclear antigen 1 BamHI F promoter is activated on entry of EBV-transformed B cells into the lytic cycle.

In Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cell lines exhibiting the latency I form of infection (i.e., EBV nuclear antigen 1 [EBNA1] positive in the absence of other latent proteins), the EBNA1 mRNA has a unique BamHI Q/U/K splice structure and is expressed from a novel promoter, Fp, located near the BamHI FQ boundary. This contrasts with the situation in EBV-transformed lymphoblastoid cell lines (LCLs) exhibiting the latency III form of infection (i.e., positive for all latent proteins), in which transcription from the upstream Cp or Wp promoters is the principal source of EBNA mRNAs. We carried out cDNA amplifications with oligonucleotide primer-probe combinations to determine whether Fp is ever active in an LCL environment. The results clearly showed that some LCLs express a Q/U/K-spliced EBNA1 mRNA in addition to the expected Cp/Wp-initiated transcripts; this seemed inconsistent with the concept of Cp/Wp and Fp as mutually exclusive promoters. Here we show that Fp is indeed silent in latency III cells but is activated at an early stage following the switch from latency III into the virus lytic cycle. Four pieces of evidence support this conclusion: (i) examples of coincident Cp/Wp and Fp usage in LCLs are restricted to those lines in which a small subpopulation of cells have spontaneously entered the lytic cycle; (ii) transcripts initiating from Fp can readily be demonstrated in spontaneously productive lines by S1 nuclease protection; (iii) the presence of Fp-initiated transcripts is not affected by acyclovir blockade of the late lytic cycle; and (iv) infection of latently infected LCLs with a recombinant vaccinia virus encoding the EBV immediate-early protein BZLF1, a transcriptional transactivator which normally initiates the lytic cycle, results in the appearance of the diagnostic Q/U/K-spliced transcripts.

Three transcriptionally distinct forms of Epstein-Barr virus latency in somatic cell hybrids: cell phenotype dependence of virus promoter usage.

Phenotypically distinct human B cell lines display two transcriptionally distinct forms of Epstein-Barr virus (EBV) latency. Latency I (Lat I) in group I Burkitt's lymphoma (BL) cell lines is characterized by selective expression of the virus-coded nuclear antigen EBNA 1 from a uniquely spliced mRNA driven by the Fp promoter. Latency III (Lat III) in group III BL and EBV-transformed lymphoblastoid cell lines (LCLs) is characterized by expression of EBNAs 1, 2, 3a, 3b, 3c, and -LP from mRNAs driven by the Cp or Wp promoter and of the latent membrane proteins (LMPs 1, 2A, and 2B) from mRNAs driven by the LMP promoters. Here we have altered the group I BL and LCL phenotypes by cell hybridization and screened for attendant changes in EBV latency by PCR analysis of viral mRNAs and immunoblotting of viral proteins. Fusion of group I BL cells with LCLs activated the BL virus genome from a Lat I to Lat III pattern of gene expression. Fusion of LCLs with nonlymphoid lines repressed virus gene expression from Lat III either to Lat I or to another form of latency (Lat II) hitherto not seen in vitro and characterized by selective expression of the Fp-driven EBNA 1 mRNA and of the LMP 1, 2A, and 2B transcripts. There are therefore three forms of EBV latency which can be interconverted by altering cellular phenotype and thereby virus promoter usage.

Three pathways of Epstein-Barr virus gene activation from EBNA1-positive latency in B lymphocytes.

Previous studies on Epstein-Barr virus (EBV)-positive B-cell lines have identified two distinct forms of virus latency. Lymphoblastoid cell lines generated by virus-induced transformation of normal B cells in vitro, express the full spectrum of six EBNAs and three latent membrane proteins (LMP1, LMP2A, and LMP2B); furthermore, these lines often contain a small fraction of cells spontaneously entering the lytic cycle. In contrast, Burkitt's lymphoma-derived cell lines retaining the tumor biopsy cell phenotype express only one of the latent proteins, the nuclear antigen EBNA1; such cells do not enter the lytic cycle spontaneously but may be induced to do so by treatment with such agents as tetradecanoyl phorbol acetate and anti-immunoglobulin. The present study set out to determine whether activation of full virus latent-gene expression was a necessary accompaniment to induction of the lytic cycle in Burkitt's lymphoma lines. Detailed analysis of Burkitt's lymphoma lines responding to anti-immunoglobulin treatment revealed three response pathways of EBV gene activation from EBNA1-positive latency. A first, rapid response pathway involves direct entry of cells into the lytic cycle without broadening of the pattern of latent gene expression; thereafter, the three "latent" LMPs are expressed as early lytic cycle antigens. A second, delayed response pathway in another cell subpopulation involves the activation of full latent gene expression and conversion to a lymphoblastoidlike cell phenotype. A third response pathway in yet another subpopulation involves the selective activation of LMPs, with no induction of the lytic cycle and with EBNA expression still restricted to EBNA1; this type of latent infection in B lymphocytes has hitherto not been described. Interestingly, the EBNA1+ LMP+ cells displayed some but not all of the phenotypic changes normally induced by LMP1 expression in a B-cell environment. These studies highlight the existence of four different types of EBV infection in B cells, including three distinct forms of latency, which we now term latency I, latency II, and latency III.

Hierarchy for 5' splice site preference determined in vivo.

The relationship between preferences among alternative 5' splice sites and their sequences has been investigated for 37 sequences by assessing their use in splicing relative to the 5' splice site of IVS-2 of rabbit beta-globin. There are strong correlations between the intrinsic strength of a 5' splice site and both the extent to which it resembles the consensus sequence and the calculated stability of its interactions with U1 small nuclear RNA. However, present methods of calculating either of the latter values do not allow predictions to be made of the relative preferences among a small number of sequences.

Identification of a talin binding site in the cytoskeletal protein vinculin.

Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746-867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.

Progress of O-acetylation and cross-linking of peptidoglycan in Neisseria gonorrhoeae grown in the presence of penicillin.

The synthesis, cross-linking and O-acetylation of gonococcal peptidoglycan in growing cells were studied by following incorporation of radioactive glucosamine and separation of the SDS-insoluble peptidoglycan into uncross-linked (monomer) and cross-linked (dimer and oligomer) fragments. Cultures to which penicillin or piperacillin at concentrations near the minimum growth inhibitory concentration (MIC) had been added 20 min before the radioactive label were compared with controls. The beta-lactams affected the early stage of cross-linking (up to 3 min) but had no effect thereafter. The deficit of cross-linking, however, did not recover. The O-acetylation, particularly of the monomer fraction, was decreased by beta-lactams even at concentrations that had no effect on culture optical density, viable counts or overall peptidoglycan synthesis. These effects on O-acetylation occurred mainly after the first 3 min of incorporation, rather than before. O-Acetylation of the oligomer fraction was also followed. Here penicillin led to increased levels of O-acetylation during the early stages of incorporation but the final value was never exceeded; indeed at higher drug concentrations the later stages of O-acetylation of oligomers were inhibited (e.g. almost completely at 2.5 X MIC).

O-acetylation of peptidoglycan in Neisseria gonorrhoeae. Investigation of lipid-linked intermediates and glycan chains newly incorporated into the cell wall.

Radioactive labelling of the amino sugars in gonococcal peptidoglycan was followed by treatment with Chalaropsis muramidase and TLC separation of the products. Even after very brief periods of labelling (0.5 min) the peptidoglycan was already cross-linked to some 80% of the final value and little change occurred within 2 min. The remaining cross-linking was achieved only over a period of about one generation time. Streptomycete endopeptidase was used to show the extent to which new chains were cross-linked to old. Even at the earliest times many cross-linked units contained new material in both moieties and by 3 min there was little distinction in relative labelling, indicating that in Neisseria gonorrhoeae most newly synthesized glycan chains are cross-linked to other new chains rather than to pre-existing peptidoglycan. A model is proposed in which newly polymerized monomer units are predestined either towards dimer formation with other new chains, which are then rapidly O-acetylated and not further cross-linked, or towards the formation of trimers and higher oligomers, the latter being a slower process. Although significant O-acetylation of peptidoglycan was detectable even at the earliest times, efforts to detect O-acetylated lipid intermediates were unsuccessful. The chief lipid intermediate found was apparently the disaccharide-peptide unit linked to undecaprenol.

Degrees of O-acetylation and cross-linking of the peptidoglycan of Neisseria gonorrhoeae during growth.

The progress of incorporation of radioactive glucosamine into the O-acetylated and non-O-acetylated sub-units of the insoluble peptidoglycan of growing Neisseria gonorrhoeae was examined. More than 80% of the final degree of cross-linking was achieved within 3 min; the remainder of the process took much longer. Rapid O-acetylation occupied up to 10 min, at which time only about 65% of the maximum value had been reached. There was thus evidence for maturation of peptidoglycan both in regard to cross-linking and to O-acetylation.