Myxoma Virus M083 Is a Virulence Factor Which Mediates Systemic Dissemination.

Poxviruses are large, DNA viruses whose protein capsid is surrounded by one or more lipid envelopes. Embedded into these lipid envelopes are three conserved viral proteins which are thought to mediate binding of virions to target cells. While the function of these proteins has been studied in vitro, their specific roles during the pathogenesis of poxviral disease remain largely unclear. Here we present data demonstrating that the putative chondroitin binding protein M083 from the leporipoxvirus myxoma virus is a significant virulence factor during infection of susceptible Oryctolagus rabbits. Removal of M083 results in a reduced capacity of virus to spread beyond the regional lymph nodes and completely eliminates infection-mediated mortality. In vitro, removal of M083 results in only minor intracellular replication defects but causes a significant reduction in the ability of myxoma virus to spread from infected epithelial cells onto primary lymphocytes. We hypothesize that the physiological role of M083 is therefore to mediate the spread of myxoma virus onto rabbit lymphocytes, allowing these cells to disseminate virus throughout infected rabbits.IMPORTANCE Poxviruses represent both a class of human pathogens and potential therapeutic agents for the treatment of human malignancy. Understanding the basic biology of these agents is therefore significant to human health in a variety of ways. While the mechanisms mediating poxviral binding have been well studied in vitro, how these mechanisms impact poxviral pathogenesis in vivo remains unclear. The current study advances our understanding of how poxviral binding impacts viral pathogenesis by demonstrating that the putative chondroitin binding protein M083 plays a critical role during the pathogenesis of myxoma virus in susceptible Oryctolagus rabbits by impacting viral dissemination through changes in the transfer of virions onto primary splenocytes.

Tendinous muscle insertions (scleromuscular junctions of the recti muscles) in patients with ocular alignment problems.

BACKGROUND: The purpose of this study was to prove the hypothesis whether the scleromuscular junction of extraocular recti muscle is tendinous. PATIENTS AND METHODS: Muscle samples of the 41 extraocular recti muscles of 33 patients and 4 muscle-/eye-matched samples from 2 postmortem eyes, were processed for light/electron microscopy and immunohistochemistry with antibodies against desmin, smooth-muscle actin and muscle regulating proteins like myf3 and myf4 (myogenin), tenascin C and for 8 samples against collagens I to IV. RESULTS: Histological examination of the muscle samples confirmed a thick collagen-structured tissue, specific for muscle tendon; without appearance of muscle tissue. This was confirmed by immunohistochemistry with antibodies against desmin, smooth-muscle actin, myf3 and myf4 (myogenin) and for eight samples with collagens I to IV. Anti-tenascin C marker was only strongly positive in the connective tissue of the blood vessel walls. Electron microscopy demonstrated collagen bundles composed of parallel oriented fibrils with a moderate amount of ground substance. CONCLUSIONS: The absence of contractile fibers at the sclerotendinous junction is an entirely normal finding in humans and cannot be related to ocular alignment pathogenesis.

The 2-global flash mfERG in glaucoma: attempting to increase sensitivity by reducing the focal flash luminance and changing filter settings.

PURPOSE: To test a new 2-flash multifocal electroretinogram (mfERG) paradigm in glaucoma using a reduced light intensity of the m-frame flash as opposed to the global flash, as it has been suggested that this may increase the responses induced by the global flash, which has been the part of the mfERG response where most changes have been noted in glaucoma. METHODS: A mfERG was recorded from one eye of 22 primary open angle glaucoma (POAG) patients [16 normal tension glaucoma (NTG), 6 high tension glaucoma (HTG)] and 20 control subjects. A binary m-sequence (2^13-1, Lmax 100 cd/m2, Lmin<1 cd/m2), followed by two global flashes (Lmax 200 cd/m2) at an interval of 26 ms (VERIS 6.0™, FMSIII), was used. The stimulus array consisted of 103 hexagons. Retinal signals were amplified (gain=50 K) and bandpass filtered at 1-300 Hz. For each focal response, the root mean square was calculated. We analyzed 5 larger response averages (central 15° and 4 adjoining quadrants) as well as 8 smaller response averages (central 10° and 7 surrounding response averages of approximately 7° radius each). Three epochs were analyzed: the direct component at 15-45 ms (DC) and the following two components induced by the effects of the preceding focal flash on the response to the global flashes at 45-75 ms (IC-1) and at 75-105 ms (IC-2). Statistical analysis was performed using linear mixed effects models adjusted for age. RESULTS: Responses differed significantly between POAG patients and controls in all central response averages. This difference was larger for the central 10° than for the response average of the central 15°. While these observations held true for all response epochs analyzed, the DC differed least and the IC-1 most when POAG was compared to control. For POAG, the most sensitive differential measure was IC-1 of the central 10° with an area under the ROC curve of 0.78. With a cutoff value of 12.52 nV/deg2, 80% of the POAG patients (100% HTG, 69% NTG) were correctly classified as abnormal, while 77% of the control subjects were correctly classified as normal. When the results of the mfERG were compared to the visual fields, there was a tendency for the mfERG to decrease as the mean defect increased. However, this correlation was only significant in the superior nasal quadrant when the IC-1 of the mfERG was compared to the corresponding area of the visual field. CONCLUSION: When compared to findings from previous studies, reducing the luminance of the m-frame flash in the 2-global flash paradigm did not increase the sensitivity and specificity of the mfERG to detect glaucoma further.

Neuropeptide Y stimulates luteinizing hormone-releasing hormone release from superfused hypothalamic GT1-7 cells.

The effects of neuropeptide Y (NPY) on LHRH release from an immortalized cell line were investigated using a flow-through cell culture superfusion system. Immortalized hypothalamic GT1-7 cells were cultured for 72 h and superfused for a total of 180 min. In initial experiments, discrete 5-min pulses of NPY (10(-12)-10(-5) M) were administered to the cells. A clear dose-dependent stimulatory effect on NPY on LHRH release from the cells was observed with a calculated 50% effectiveness concentration of 33 nM. The stimulatory effects of brief NPY exposure were rapid and robust, e.g. reaching and maintaining levels of 173% over baseline for 20 min at the 10(-7) dose. The lowest dose of NPY that showed a significant effect was 10(-10) M; maximal responses were observed at 10(-6) M and reached a plateau thereafter. Control pulses of Dulbecco's modified Eagle's medium (DMEM) and 10(-6) M substance P or arg-vasopressin were also presented to the cells to serve as controls for our pulse protocol, and these challenges produced no significant LHRH responses. The NPY receptor antagonists, PYX1 and PYX2, at 10(-8) M, completely blocked the observed NPY responses in these cells. To assess the NPY receptor subtypes that mediate the NPY effects pharmacologically, GT1-7 cells were challenged with a Y1 receptor agonist, (Leu31Pro34)NPY, a Y2 receptor agonist, NPY(13-36), or peptide YY, at doses 10(-12)-10(-5) M. All four peptides stimulated LHRH release from GT1-7 cells with a rank-ordered potency of NPY = peptide YY > Y1 agonist = Y2 agonist. To examine possible signal transduction mechanism(s) involved in mediating this effect, pertussis toxin, RpcAMPs (cyclic adenosine-3'5'-monophosphothioate Rp diastereomer), Ca(2+)-free DMEM and TMB-8 (3, 4, 5-trimethoxybenzoic acid 8-(diethylamino) octylester) were used to treat the cells before and during superfusion with NPY. Treatment with pertussis toxin, RpcAMPs, and Ca(2+)-free DMEM did not significantly alter NPY-stimulated LHRH release responses to 10(-7) M NPY. However, the addition of 100 microM and 250 microM TMB-8 to Ca(2+)-free DMEM almost completely blocked this NPY effect, as did 10 microM ryanodine. Finally, the locus of action for this NPY effect was examined using tetrodotoxin to reduce action potential propagation in the GT1-7 cells. Tetrodotoxin treatment blocked the LHRH response to NPY by more than 50%.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein-sparing effect of intravenously administered branched chain amino acids in adult rats.

Branched chain amino acids (BCAA) serve as intermediate energy substrates for peripheral tissues. Experiments were conducted to evaluate protein-sparing properties of BCAA at different doses without dextrose. Forty healthy male adult Wistar rats, weighing 437 +/- 34 gm, were randomly assigned to one of five groups. Four groups of eight rats were cannulated at the jugular vein and infused with 3% amino acid solutions containing 13, 23, 50 or 100% BCAA; an additional eight fasted rats served as controls. Results show that weight loss was not significantly different between groups, but was lower than in the fasted rats. Nitrogen balance (NB) was least negative in the 23% BCAA group, followed by 13, 50 and 100% BCAA. Protein-sparing index (PSI), defined as that portion of the infused nitrogen that is used for sparing body proteins, agreed with the calculated fat/protein ratios. Higher PSI and fat/protein ratios resulted from solutions containing balanced amino acid patterns (13 and 23%). No obvious imbalances in plasma amino acid patterns were observed. It is concluded that more protein conservation can be achieved with amino acid solutions containing a balanced amino acid pattern and that the effect of additional BCAA on protein conservation is limited in adult rats.

[Stress-related vertical double vision and ptosis]. / Belastungsabhängige vertikale Doppelbilder und Ptosis. Diagnose: Okuläre Pseudomyasthenie bei Keilbeinmeningeom am Sinus cavernosus links.

A 43-year-old patient presented to our department because her left eyelid had exhibited drooping to varying extents for 2-3 months. Furthermore, in extreme positions or after rapid eye movement, she perceived diplopic images and was sensitive to light. The diagnosis of sphenoid meningioma was reached. Subsequently two rounds of radiopeptide therapy with (90)Y-DOTATOC (somatostatin analog) were administered within 3 months. This treatment approach led to a reduction of tumor volume in our patient as well as clinical improvement followed by stabilization.

Can the lumpy distribution of galaxies be reconciled with the smooth X-ray background?

The fluctuations in X-ray background intensity that would be generated by clusters or superclusters of galaxies exceed those observed. It may be that the X-ray background arises from low-luminosity objects which are distributed much more smoothly and are more numerous than bright optical galaxies. Hierarchical universe models in which the distribution of X-ray sources follows the cosmological mass distribution are ruled out.

Blockade of singular follicle-stimulating hormone secretion and testicular development in photostimulated Djungarian hamsters (Phodopus sungorus) by a gonadotropin-releasing hormone antagonist.

Photostimulated male Djungarian hamsters following placement in a long-day photoperiod exhibit a characteristic rise in serum FSH levels that occurs in the absence of a simultaneous rise in LH levels. It is not known whether this singular FSH secretion is dependent upon a differential responsiveness of the gonadotrophs to the pattern of pulsatile GnRH release or is instead driven by a GnRH-independent mechanism. We have assessed the GnRH dependence of this singular FSH secretion by testing the ability of a potent GnRH antagonist (GnRHa: WY-45760) to block FSH and testicular responses to photostimulation. Photoinhibited hamsters were transferred from a short-day (6L:18D) to a long-day photoperiod (16L:8D). Hamsters received two daily injections of a GnRH antagonist or vehicle (VEH). After 0 (short day), 3, 5, 10, 30, or 40 days the hamsters were killed; plasma was assayed for FSH, LH, and testosterone (T), and testes weights were recorded. Testes were sectioned and analyzed for tubular development. In VEH-treated animals, testicular weights increased after photostimulation, reaching mean values of 514 mg by 30 days. Treatment with GnRHa resulted in a significant (p < 0.01) attenuation of testicular growth after 30 days of photostimulation (mean testes weight = 110.1 mg). In VEH-treated hamsters there was a rapid increase in FSH levels after photostimulation that became significant by 5 days and peaked at 10 days. In the GnRHa-treated group, however, these FSH increments were completely blocked at 5 days and significantly reduced at 10 days compared to the values in the corresponding VEH-treated groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-specific expression of the human gonadotropin-releasing hormone gene in transgenic animals.

We have previously demonstrated that 1131 base pairs (bp) of the human gonadotropin-releasing hormone (hGnRH) gene promoter can target simian virus 40 T antigen expression to GnRH neurons in transgenic mice. In these animals, GnRH neurons were transformed before they migrated to their final location in the rostral hypothalamus, complicating an analysis of cell-specific expression. To localize regions of the hGnRH promoter that are important for cell-specific expression, we created transgenic mice with various 5'-flanking regions of the hGnRH gene fused to the luciferase reporter gene. When 3828 or 1131 bp of the hGnRH promoter 5'-flanking DNA were used (-3828/+5LUC and -1131/+5LUC, respectively), luciferase expression in adult transgenic mice was observed in the rostral hypothalamus and olfactory tissues, regions which have been shown to be loci of GnRH-expressing neurons. Luciferase expression was not observed in other brain or peripheral tissues. Double-labeled in situ hybridization further demonstrated that luciferase expression was invariably colocalized with GnRH expression. When transgenic animals were created with a construct consisting of 484 bp of the hGnRH 5'-flanking DNA fused to the luciferase gene (-484/+5LUC), luciferase expression was not observed in the hypothalamus or in olfactory tissues. This is the first report localizing DNA sequences responsible for cell-specific expression of the GnRH gene in vivo.

Determination of intravenous non-protein energy and nitrogen requirements in growing rats.

Two experiments were conducted to estimate total non-protein energy and nitrogen requirements in growing healthy male Wistar rats nourished by parenteral nutrition. In experiment 1, non-protein energy varying from 30 to 70 kcal/day/rat were administered to animals receiving a constant dose of 80 mg nitrogen, plus vitamins and minerals. In experiment 2, nitrogen dosages varying from 0 to 280 mg N/day/rat with a constant dose of 60 kcal non-protein energy were studied. The formulation of the amino acid solution used in both experiments was based upon recommended oral amino acid requirements for growing rats. Dextrose served as the source of non-protein energy. Weight gain and nitrogen balance during a 6-day experimental period were used to determine requirements. Plasma free amino acids were also analyzed to evaluate the amino acid solution. Results indicate that under total parenteral nutrition conditions 578 to 621 mg/kg body weight3/4 nitrogen and 171 to 182 kcal/kg body weight3/4 non-protein energy are required to achieve growth of approximately 3 g/day. Inconsistent responses of plasma amino acid concentrations to the amounts infused were observed. It is suggested that the determined requirements can be applied as guidelines to research using the rat as an animal model in total parenteral nutrition.

Amplitude and frequency modulation of pulsatile luteinizing hormone-releasing hormone release.

1. A variety of neuroendocrine approaches has been used to characterize cellular mechanisms governing luteinizing hormone-releasing hormone (LHRH) pulse generation. We review recent in vivo microdialysis, in vitro superfusion, and in situ hybridization experiments in which we tested the hypothesis that the amplitude and frequency of LHRH pulses are subject to independent regulation via distinct and identifiable cellular pathways. 2. Augmentation of LHRH pulse amplitude is proposed as a central feature of preovulatory LHRH surges. Three mechanisms are described which may contribute to this increase in LHRH pulse amplitude: (a) increased LHRH gene expression, (b) augmentation of facilitatory neurotransmission, and (c) increased responsiveness of LHRH neurons to afferent synaptic signals. Neuropeptide Y (NPY) is examined as a prototypical afferent transmitter regulating the generation of LHRH surges through the latter two mechanisms. 3. Retardation of LHRH pulse generator frequency is postulated to mediate negative feedback actions of gonadal hormones. Evidence supporting this hypothesis is reviewed, including results of in vivo monitoring experiments in which LHRH pulse frequency, but not amplitude, is shown to be increased following castration. A role for noradrenergic neurons as intervening targets of gonadal hormone negative feedback actions is discussed. 4. Future directions for study of the LHRH pulse generator are suggested.

Further evidence for cosmological evolution of the fine structure constant.

We describe the results of a search for time variability of the fine structure constant alpha using absorption systems in the spectra of distant quasars. Three large optical data sets and two 21 cm and mm absorption systems provide four independent samples, spanning approximately 23% to 87% of the age of the universe. Each sample yields a smaller alpha in the past and the optical sample shows a 4 sigma deviation: Delta alpha/alpha = -0.72+/-0.18 x 10(-5) over the redshift range 0.5

Neuroendocrine regulation of the luteinizing hormone-releasing hormone pulse generator in the rat.

We have analyzed the mechanisms by which several known regulators of the LHRH release process may exert their effects. For each, we have attempted to determine how and where the regulatory input is manifest and, according to our working premise, we have attempted to identify factors which specifically regulate the LHRH pulse generator. Of the five regulatory factors examined, we have identified two inputs whose primary locus of action is on the pulse-generating mechanism--one endocrine (gonadal negative feedback), and one synaptic (alpha 1-adrenergic inputs) (see Fig. 29). Other factors which regulate LHRH and LH release appear to do so in different ways. The endogenous opioid peptides, for example, primarily regulate LHRH pulse amplitude (Karahalios and Levine, 1988), a finding that is consistent with the idea that these peptides exert direct postsynaptic or presynaptic inhibition (Drouva et al., 1981). Gonadal steroids exert positive feedback actions which also result in an increase in the amplitude of LHRH release, and this action may be exerted through a combination of cellular mechanisms which culminate in the production of a unique, punctuated set of synaptic signals. Gonadal hormones and neurohormones such as NPY also exert complementary actions at the level of the pituitary gland, by modifying the responsiveness of the pituitary to the stimulatory actions of LHRH. The LHRH neurosecretory system thus appears to be regulated at many levels, and by a variety of neural and endocrine factors. We have found examples of (1) neural regulation of the pulse generator, (2) hormonal regulation of the pulse generator, (3) hormonal regulation of a neural circuit which produces a unique, punctuated synaptic signal, (4) hormonal regulation of pituitary responsiveness to LHRH, and (5) neuropeptidergic regulation of pituitary responsiveness to LHRH. While an attempt has been made to place some of these regulatory inputs into a physiological context, it is certainly recognized that the physiological significance of these mechanisms remains to be clarified. We also stress that these represent only a small subset of the neural and endocrine factors which regulate the secretion or actions of LHRH. A more comprehensive list would also include CRF, GABA, serotonin, and a variety of other important regulators. Through a combination of design and chance, however, we have been able to identify at least one major example of each type of regulatory mechanism.