Non-invasive assessment of fecal glucocorticoid, progesterone, and androgen metabolites and microbiome in free-ranging southern white rhinoceros (Ceratotherium simum simum) in South Africa.

Increased poaching in northern South Africa has necessitated relocation of large numbers of southern white rhinoceros (Ceratotherium simum simum) to the Eastern Cape Province. The climate and grassland ecology of this province differ from that of northern South Africa which may impact the health of this species. This assessment of fecal steroid levels and microbiome in 10 free-ranging southern white rhinoceros in the Eastern Cape will provide insights into white rhinoceros physiology in this biome. Fecal steroid metabolites were analyzed using enzyme immunoassay (EIA) and ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). Fecal microbial composition was assessed via next generation sequencing. EIAs with antibodies raised against progesterone (P4; mouse monoclonal - CL425 clone), testosterone (T; rabbit polyclonal), corticosterone (B; sheep polyclonal) were utilized. Pregnant females had large quantities of fecal progesterone metabolites (FPMs) detected by CL425 EIA. Pregnant females also had native P4 and 11α-hydroxydihydroprogesterone (11αOHDHP4; 4-pregnen-11α-ol-3,20-dione) detected by UPC2-MS/MS but these concentrations were 1000-fold less than the concentrations of FPMs detected by the CL425 EIA. By contrast, non-pregnant females had FPM concentrations detected by CL425 EIA which were similar to native P4 and 11αOHDHP4 concentrations detected by UPC2-MS/MS. Mean fecal androgen metabolite (FAM) concentrations detected by the T EIA were similar between males and females. 11-ketoandrostenedione (11KA4) detected by UPC2-MS/MS was higher in females than males. However, there was no difference between males and females in the concentration of fecal glucocorticoid metabolites (FGMs) detected by the B EIA. Bacteroidia, followed by Clostridia, was the most abundant classes of fecal microbes. The unfiltered microbiome of females was more diverse than that of males. The core fecal microbiome of young rhinoceros had a higher observed species richness (Shannon diversity index, and Simpson diversity index) than that of old rhinoceros. In the alpha male, immobilization was associated with an increase in FGMs detected by 11-deoxycortisol (S) detected by UPC2-MS/MS coupled with decreased abundance of Spirochaetia. We detected substantially different FAM and FPM concentrations from those previously reported for both captive and wild white rhinoceros. Comparison of our UPC2-MS/MS and EIA results underscores the fact that most EIAs are highly cross reactive for many steroid metabolites. Our data also demonstrates a distinct effect of stress not only on FGMs but also on the fecal microbiome. This is the first non-invasive assessment of fecal steroid metabolites by UPC2-MS/MS and the fecal microbiome in wild white rhinoceros.

Renin mRNA is upregulated in testes and testicular cells in response to treatment with aflatoxin B1.

Aflatoxin B(1) (AFB(1)) has been shown to affect fertility in many species; however, the exact molecular mechanisms associated with the disruption are not known. Our objectives were to determine changes in testicular gene expression due to exposure to AFB(1) and to investigate which cell types were affected by treatment with AFB(1). Male mice 4 wk of age were administered a daily placebo (control; N = 9) or 50 µg/kg AFB(1) (AFB(1) treated; N = 10) daily for 45 days. Males were then mated to four females each for 8 days. Male mice were characterized as being "Tolerant" (N = 3) or "Intolerant" (N = 3) to the effects of AFB(1) based on positive terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in the testes and the number of pups sired. Tolerant males produced a similar average number of fetuses (mean ± SEM) (12.5 ± 1.2) per male as selected control males (13.4 ± 1.2), but more fetuses (P = 0.01) than Intolerant males (7.6 ± 1.2). The number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells in Intolerant males tended to be (P = 0.10) greater (136.5 ± 27.2) than in Tolerant (55.0 ± 22.2) and selected control (54.3 ± 22.2) males. Affymetrix microarray (Sunnyvale, CA, USA) analysis revealed differential expression (P < 0.05) of 193 extra cellular space and signaling genes, 49 signal transduction genes, 45 immune regulation genes, and 230 cell differentiation genes in the testis. Renin was commonly represented amongst many clusters and was chosen for further analyses. Upregulation (P < 0.001) of Renin in Tolerant mice (N = 3) compared with Intolerant mice (N = 3) was confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR) (P = 0.05). This upregulation (P = 0.01) was also observed in representative AFB(1) treated males (N = 8) compared with control males (N = 8) selected for real-time reverse transcription polymerase chain reaction analysis. Spermatogonia cultured in vitro and treated with 0, 5, 10, or 20 µg/mL AFB(1) (N = 6 per treatment) resulted in a 10-fold upregulation (P = 0.01) of Renin message at the 20 µg/mL level, whereas Leydig tumor cells showed similar differences (P = 0.03) in message for Renin in treated (10 and 20 µg/mL) versus control cell cultures. Based on these results, we inferred a role for Renin at the molecular level in the response to the adverse effects of AFB(1) in male mice.

Glycoproteins gM and gN of pseudorabies virus are dispensable for viral penetration and propagation in the nervous systems of adult mice.

Glycoproteins gM and gN are conserved throughout the herpesviruses but are dispensable for viral replication in cell cultures. To assay for a function of these proteins in infection of an animal, deletion mutants of pseudorabies virus lacking gM or gN and corresponding revertants were analyzed for the ability to penetrate and propagate in the nervous systems of adult mice after intranasal inoculation. We demonstrate that neither of the two glycoproteins is required for infection of the nervous systems of mice by pseudorabies virus.

Potency of an experimental DNA vaccine against Aujeszky's disease in pigs.

Intradermal vaccination with plasmid DNA encoding envelope glycoprotein C (gC) of pseudorabies virus (PrV) conferred protection of pigs against Aujeszky's disease when challenged with strain 75V19, but proved to be inadequate for protection against the highly virulent strain NIA-3. To improve the performance of the DNA vaccine, animals were vaccinated intradermally with a combination of plasmids expressing PrV glycoproteins gB, gC, gD, or gE under control of the major immediate-early promotor/enhancer of human cytomegalovirus. 12.5 microg per plasmid were used per immunization of 5-week old piglets which were injected three times at biweekly intervals. Five out of six animals survived a lethal challenge with strain NIA-3 without exhibiting central nervous signs, whereas all the control animals succumbed to the disease. This result shows the increased protection afforded by administration of the plasmid mixture over vaccination with a gC expressing plasmid alone. A comparative trial was performed using commercially available inactivated and modified-live vaccines and a mixture of plasmids expressing gB, gC, and gD. gE was omitted to conform with current eradication strategies based on gE-deleted vaccines. All six animals vaccinated with the live vaccine survived the lethal NIA-3 challenge without showing severe clinical signs. In contrast, five of six animals immunized with the inactivated vaccine died, as did two non-vaccinated controls. In this test, three of six animals vaccinated with the DNA vaccine survived without severe clinical signs, whereas three succumbed to the disease. Comparing weight reduction and virus excretion, the DNA vaccine also ranged between the inactivated and modified-live vaccines. Thus, administration of DNA constructs expressing different PrV glycoproteins was superior to an adjuvanted inactivated vaccine but less effective than an attenuated live vaccine in protection of pigs against PrV infection. Our data suggest a potential use of DNA vaccination in circumstances which do not allow administration of live attenuated vaccines.

Different point mutations within the conserved N-glycosylation motif of pseudorabies virus glycoprotein M result in expression of a nonglycosylated form of the protein.

Glycoprotein M (gM) constitutes one of the rare examples of a nonessential glycoprotein conserved throughout all herpesvirus subfamilies. Whereas gM in wild-type pseudorabies virus (PrV) strains carries an N-glycan, gM of the attenuated strain Bartha is not glycosylated due to a point mutation in the N-glycosylation motif. Since PrV Bartha lacks glycoproteins E and I and carries a mutated gC, we analysed glycosylation of gM in isogenic PrV glycoprotein deletion mutants. Whereas gM was glycosylated normally in most mutants, two independent gC deletion mutants and a gI mutant expressed a nonglycosylated form of gM. DNA sequence analyses revealed the presence of point mutations in the N-glycosylation consensus motif. Surprisingly, mutations in strain Bartha, the two gC-deletion mutants and the gI mutant proved to be different, although all affected the N-glycosylation motif. Thus, our data show that different, apparently independent point mutations cause expression of nonglycosylated gM.

Glycoproteins M and N of pseudorabies virus form a disulfide-linked complex.

Genes homologous to the herpes simplex virus UL49.5 open reading frame are conserved throughout the Herpesviridae. In the alphaherpesvirus pseudorabies virus (PrV), the UL49.5 product is an O-glycosylated structural protein of the viral envelope, glycoprotein N (gN) (A. Jöns, H. Granzow, R. Kuchling, and T. C. Mettenleiter, J. Virol. 70:1237-1241, 1996). For functional characterization of gN, a gN-negative PrV mutant, PrV-gNbeta, and the corresponding rescuant, PrV-gNbetaR, were constructed, gN-negative PrV was able to productively replicate on noncomplementing cells, and one-step growth in cell culture was only slightly reduced compared to that of wild-type PrV. However, penetration was significantly delayed. In indirect immunofluorescence assays with rabbit serum directed against baculovirus-expressed gN, specific staining of wild-type PrV-infected cells occurred only after permeabilization of cells, whereas live cells failed to react with the antiserum. This indicates the lack of surface accessibility of gN in the plasma membrane of a PrV-infected cell. Western blot analyses and radioimmunoprecipitation experiments under reducing and nonreducing conditions led to the discovery of a heteromeric complex composed of gM and gN. The complex was stable in the absence of 2-mercaptoethanol but dissociated after the addition of the reducing agent, indicating that the partners are linked by disulfide bonds. Finally, gN was absent from gM-negative PrV virions, whereas gM was readily detected in virions in the absence of gN. Thus, gM appears to be required for virion localization of gN.

Protection of pigs against Aujeszky's disease by DNA vaccination.

Vaccination with DNA constructs encoding viral antigens has been shown to induce antiviral immunity in various model hosts. However, relevant natural virus-host systems have so far been analysed to only a very limited extent. To test the efficacy of DNA vaccination in an economically important large animal, pigs were immunized against Aujeszky's disease, a serious virus infection caused by the alphaherpesvirus pseudorabies virus (PrV), which is characterized by severe central nervous and respiratory symptoms. After vaccination with plasmid vectors containing genes for immunogenic envelope glycoproteins C or D (gC or gD) of PrV under control of the major immediate early promotor of human cytomegalovirus, animals developed serum antibodies which recognized the respective antigen in immunoblot and exhibited neutralizing activity. Animals vaccinated with the gC expression plasmid were fully protected against a lethal challenge with PrV strain 75V19, and showed partial protection against the highly virulent NIA-3 strain. In contrast, protection was not observed after vaccination with the gD plasmid. Three intramuscular or intradermal immunizations with as little as 1 microgram of gC plasmid DNA resulted in sero-conversion and partial protection against lethal NIA-3 Infection. Specific antibodies were detected until at least 9 months after vaccination. In addition, a cellular immune response specific for gC could be demonstrated in proliferation assays of peripheral mononuclear lymphocytes. Our results thus demonstrate the potency of DNA vaccination for protection of large animals against a lethal virus infection.

Green fluorescent protein expressed by recombinant pseudorabies virus as an in vivo marker for viral replication.

We isolated and characterized a pseudorabies virus (PrV) mutant expressing an engineered green fluorescent protein (GFP) optimized for expression in human cells. The GFP DNA was inserted in the non-essential glycoprotein G (gG) gene of the attenuated PrV strain Bartha. The coding sequence was cloned in frame behind the first seven codons of the gG gene under control of the strong gG promotor. On excitation with blue light, live cells infected with the recombinant PrV B80eGFP exhibited bright fluorescence when examined microscopically using filters for FITC fluorescence. In fixed samples detection sensitivity was increased by immunofluorescence using an anti-GFP antibody. Specifically labelled PrV mutants have been used successfully as transsynaptic circuit tracers for definition of central command neurons in the brain (Jansen et al., 1995. Central command neurons of the sympathetic nervous system: basis of the fight-or-flight response. Science 270, 644-646). Availability of this recombinant allows the study of even more complex interactions using differentially labelled PrV mutants, and provides a means to monitor viral replication and spread without destruction of the cell.

Ultrastructural analysis of the replication cycle of pseudorabies virus in cell culture: a reassessment.

We reinvestigated major steps in the replicative cycle of pseudorabies virus (PrV) by electron microscopy of infected cultured cells. Virions attached to the cell surface were found in two distinct stages, with a distance of 12 to 14 nm or 6 to 8 nm between virion envelope and cell surface, respectively. After fusion of virion envelope and cell membrane, immunogold labeling using a monoclonal antibody against the envelope glycoprotein gE demonstrated a rapid drift of gE from the fusion site, indicating significant lateral movement of viral glycoproteins during or immediately after the fusion event. Naked nucleocapsids in the cytoplasm frequently appeared close to microtubules prior to transport to nuclear pores. At the nuclear pore, nucleocapsids invariably were oriented with one vertex pointing to the central granulum at a distance of about 40 nm and viral DNA appeared to be released via the vertex region into the nucleoplasm. Intranuclear maturation followed the typical herpesvirus nucleocapsid morphogenesis pathway. Regarding egress, our observations indicate that primary envelopment of nucleocapsids occurred at the inner leaflet of the nuclear membrane by budding into the perinuclear cisterna. This nuclear membrane-derived envelope exhibited a smooth surface which contrasts the envelope obtained by putative reenvelopment at tubular vesicles in the Golgi area which is characterized by distinct surface projections. Loss of the primary envelope and release of the nucleocapsid into the cytoplasm appeared to occur by fusion of envelope and outer leaflet of the nuclear membrane. Nucleocapsids were also found engulfed by both lamella of the nuclear membrane. This vesiculation process released nucleocapsids surrounded by two membranes into the cytoplasm. Our data also indicate that fusion between the two membranes then leads to release of naked nucleocapsids in the Golgi area. Egress of virions appeared to occur via transport vesicles containing one or more virus particles by fusion of vesicle and cell membrane. Our data thus support biochemical data and mutant virus studies of (i) two steps of attachment, (ii) the involvement of microtubules in the transport of nucleocapsids to the nuclear pore, and (iii) secondary envelopment in the trans-Golgi area in PrV infection.

The UL49.5 gene of pseudorabies virus codes for an O-glycosylated structural protein of the viral envelope.

Sequence analysis of BamHI fragment 1 of the pseudorabies virus (PrV) genome identified a novel PrV gene located upstream of the UL50 gene encoding PrV dUTPase. The deduced protein product displayed homology to the product of the herpes simplex virus type 1 UL49.5 protein. The predicted PrV UL49.5 protein consists of 98 amino acids with a calculated molecular mass of 10,155 Da. It contains putative signal peptide and transmembrane domains but lacks a consensus sequence for N glycosylation. PrV UL49.5 was expressed as a fusion protein with glutathione S-transferase in Escherichia coli, and a rabbit antiserum was generated. In Western blots (immunoblots) of purified virions, the antiserum detected a protein with an apparent molecular mass of 14 kDa. After fractionation of the virions, the 14-kDa protein was detected in the envelope fraction. Localization of the UL49.5 protein in the viral envelope was confirmed by immunoelectron microscopy. The treatment of purified virions with glycosidases led to a reduction of the apparent molecular mass in Western blots by approximately 2 kDa following digestion with neuraminidase and O-glycosidase. Our results demonstrate that the PrV UL49.5 protein is an O-glycosylated structural component of the viral envelope. It represents the 10th PrV glycoprotein described. According to the unified nomenclature for alphaherpesvirus glycoproteins, we propose to designate it glycoprotein N (gN).

Anion exchanger 1 in human kidney and oncocytoma differs from erythroid AE1 in its NH2 terminus.

Acid-secreting intercalated cells of the kidney collecting duct and tumor cells of renal oncocytoma express an anion exchanger that is immunologically related but not identical to the chloride-bicarbonate anion exchanger of erythrocytes (AE1). In this study, we have mapped the binding site of a monoclonal antibody against erythroid AE1 that does not react with either intercalated cells or oncocytoma. The epitope is located close to the NH2 terminus of AE1, indicating that AE1 in intercalated cells and oncocytoma differs in its NH2 terminus from erythroid AE1. This conclusion was supported by an antibody directed against residues 1-14 of erythroid AE1 that does not react with intercalated cells in oncocytoma. Polymerase chain reaction performed with mRNA from a human kidney revealed that the sequence containing the codons for Met-1 and Met-33 in erythroid mRNA is missing in the kidney transcript, whereas the sequence coding for Met-66 is present. DNA sequence data derived from cloning the 5' end of the human kidney AE1 mRNA clearly showed that the 5' untranslated region comprises part of intron 3, the complete exon 4 that is followed by exon 5 containing Met-66 as the site of translation initiation. Altogether, the results indicate that AE1 in the human kidney is an amino-terminally truncated form of erythroid AE1 that is restricted to the basolateral membrane domain of the acid-secreting intercalated cells of the collecting duct and is also expressed in oncocytoma.

Cytoskeleton and epithelial polarity.

The membrane surface of polarized epithelial cells can be divided in apical and basolateral domains that differ in molecular composition and function. Components of the cytoskeleton are involved in critical steps of both generation and maintenance of cell polarity. Generation of polarity is controlled by microtubules that serve as uniformly aligned and polarized cytoplasmic guiding structures for the vectorial and selective transport of Golgi-derived carrier vesicles to the apical cell surface. Targeting of membrane proteins to the basolateral cell surface does not depend on microtubules but follows the constitutive bulk flow of membranes. Once inserted into the lipid bilayer several membrane proteins such as the kidney anion exchanger 1 (AE1) and the sodium pump become immobilized at specialized microdomains of the lateral cell surface. Evidence is provided that both membrane proteins are linked via ankyrin to the spectrin-based membrane cytoskeleton that underlies the basolateral membrane domain. Linkage of these and other integral membrane proteins to the cytoskeleton may not only place them to specialized sites of the plasma membrane but may also prevent these transporters from clustering and endocytosis, thus helping them to stay at the cell surface. In search of sequence motifs involved in binding of integral membrane proteins to components of the cytoskeleton we found that the binding interface of AE1 to protein 4.1 (an actin and spectrin cross-linking protein) consists of a cluster of five amino acid residues, namely IRRRY in AE1 and LEEDY on protein 4.1. This motif may play a more general role in cytoskeleton membrane linkages.

Gas exchange of ears of cereals in response to carbon dioxide and light : I. Relative contributions of parts of the ears of wheat, oat, and barley to the gas exchange of the whole organ.

One cultivar each of spring wheat (Triticum aestivum L. cv. Arkas), oat (Avena sativa L. cv. Lorenz), and barley (Hordeum vulgare L. cv. Aramir) was chosen in order to study the relative contributions of individual bracts to the gas exchange of whole ears. The distribution and frequency of the stomata on the bracts were examined. Gas exchange was measured at normal atmospheric CO2 (330 µbar) and at high CO2 (2000 µbar) on intact ears and on ears from which glumes or lemmas and pleae (wheat and oat) or awns (barley) had been removed.The relative contribution to the gas exchange of the whole organ is highest for the awns of barley ears. In wheat, the contribution of the glumes is slightly higher than that of the inner bracts before anthesis. Two weeks after anthesis the inner bracts contribute more than the glumes. This tendency of increasing importance of the inner bracts is also found in oat ears, but the relative amount of CO2 uptake by the glumes is higher than in wheat. These changes during ontogeny result from the better supply of light to the inner bracts caused by opening of the ears' structures during grain filling, which in part compensates for the decreasing photosynthetic capacity.The ratio of the photosynthesis rate at high CO2 to that at normal CO2 is lower for the glumes of oat and for the awns of barley than for the other bracts.

Gas-exchange of ears of cereals in response to carbon dioxide and light : II. Occurrence of a C3-C 4 intermediate type of photosynthesis.

Data for the maximum carboxylation velocity of ribulose-1,5-biosphosphate carboxylase, Vm, and the maximum rate of whole-chain electron transport, Jm, were calculated according to a photosynthesis model from the CO2 response and the light response of CO2 uptake measured on ears of wheat (Triticum aestivum L. cv. Arkas), oat (Avena sativa L. cv. Lorenz), and barley (Hordeum vulgare L. cv. Aramir). The ratio Jm/Vm is lower in glumes of oat and awns of barley than it is in the bracts of wheat and in the lemmas and paleae of oat and barley. Light-microscopy studies revealed, in glumes and lemmas of wheat and in the lemmas of oat and barley, a second type of photosynthesizing cell which, in analogy to the Kranz anatomy of C4 plants, can be designated as a bundle-sheath cell. In wheat ears, the CO2-compensation point (in the absence of dissimilative respiration) is between those that are typical for C3 and C4 plants.A model of the CO2 uptake in C3-C4 intermediate plants proposed by Peisker (1986, Plant Cell Environ. 9, 627-635) is applied to recalculate the initial slopes of the A(p(c)) curves (net photosynthesis rate versus intercellular partial pressure of CO2) under the assumptions that the Jm/Vm ratio for all organs investigated equals the value found in glumes of oat and awns of barley, and that ribulose-1,5-bisphosphate carboxylase is redistributed from mesophyll to bundle-sheath cells. The results closely match the measured values. As a consequence, all bracts of wheat ears and the inner bracts of oat and barley ears are likely to represent a C3-C4 intermediate type, while glumes of oat and awns of barley represent the C3 type.