Genome segment RNA-1 of a flat apple isolate of <Emphasis Type="Italic">Cherry rasp leaf virus</Emphasis>: nucleotide sequence analysis and RT-PCR detection

Summary. The sequence of the RNA-1 of a flat apple isolate of Cherry rasp leaf virus (CRLV-FA) was determined using overlapping cDNA fragments. CRLV-FA RNA-1 consists of 6992 nucleotides (nt), excluding a 3′ poly (A) tail. A single open reading frame (ORF) consisting of 6705 nt was identified. This ORF encodes a putative polyprotein consisting of 2235 amino acid (aa) residues, approximately 249.6 kDa. When compared to CRLV-pot (potato isolate) RNA-1 ORF, 2 deletions of 5 aa and 10 aa (total 15 aa) were observed at the variable N-terminus of the protease cofactor of CRLV-FA. Non-coding regions were identified at the 5′-(142 nt) and 3′-end (145 nt). CRLV-FA and CRLV-pot are isolates of the same virus with identity levels for the RNA-1 associated nt and deduced aa of 94% and 95%, respectively. RT-PCR targeting CRLV-FA RNA-1 appear to be of similar sensitivity and just as reliable as RT-PCR targeting RNA-2.     

Bb2Bb3 regulation of murine Lyme arthritis is distinct from Ncf1 and independent of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase

Several quantitative trait loci regulating murine Lyme arthritis severity have been mapped, including a highly significant linkage found on chromosome 5, termed Bb2Bb3. Within this region, the Ncf1 gene of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been identified as a major regulator of arthritis severity in rodent models of rheumatoid arthritis, an effect attributed to protective properties of reactive oxygen species. To assess the role of Ncf1 in Lyme arthritis, we introgressed Bb2Bb3 from severely arthritic C3H/He mice onto mildly arthritic C57BL/6 mice. This increased Lyme arthritis severity, whereas the reciprocal transfer conferred protection from disease. A single nucleotide polymorphism was identified in the Ncf1 gene that did not influence the protein sequence or expression of Ncf1. Although polymorphonuclear leukocytes from C57BL/6 mice generated a greater oxidative burst than polymorphonuclear leukocytes from C3H/He mice, studies with the Bb2Bb3 congenic mice demonstrated this difference was not linked to Ncf1 alleles. Furthermore, Lyme arthritis severity was not altered in mice lacking either the Ncf1 or Gp91phox subunits of the NADPH oxidase complex. Together, these results argue that Ncf1 is not a candidate gene for regulation of Lyme arthritis and reveal Lyme arthritis to be independent of NADPH oxidase activity, distinguishing it from other models of rheumatoid arthritis.     

The effects of head and trunk position on torsional vestibular and optokinetic eye movements in humans

We measured torsional vestibular and optokinetic eye movements in human subjects with the head and trunk erect, with the head supine and the trunk erect, and with the head and trunk supine, in order to quantify the effects of otolithic and proprioceptive modulation. During active head movements, the torsional vestibulo-ocular reflex (VOR) had significantly higher gain with the head upright than with the head supine, indicating that dynamic otolithic inputs can supplement the semicircular canal-ocular reflex. During passive earth-vertical axis rotation, torsional VOR gain was similar with the head and trunk supine and with the head supine and the trunk erect. This finding implies that static proprioceptive information from the neck and trunk has little effect upon the torsional VOR. VOR gain with the head supine was not increased by active, self-generated head movement compared with passive, whole body rotation, indicating that the torsional VOR is not augmented by dynamic proprioceptive inputs or by an efference copy of a command for head movement. Viewing earth-fixed surroundings enhanced the torsional VOR, while fixating a chair-fixed target suppressed the VOR, especially at low frequencies. Torsional optokinetic nystagmus (OKN) evoked by a full-field stimulus had a mean slow-phase gain of 0.22 for 10°/s drum rotation, but gain fell to 0.06 for 80°/s stimuli. Despite this fall in gain, mean OKN slow-phase velocities increased with drum speed, reaching maxima of 2.5°/s–8.0°/s in our subjects. Optokinetic afternystagmus (OKAN) was typically absent. Torsional OKN and OKAN were not modified by otolithic or proprioceptive changes caused by altering head and trunk position with respect to gravity. Torsional velocity storage is negligible in humans, regardless of head orientation.Presented in part at the Society for Neuroscience Annual Meeting, October 31, 1989, Phoenix, AZ     

Distribution of collagens and fibronectin in the subepicardium during avian cardiac development

Summary The development of the layer of connective tissue between ventricular epicardium and myocardium was studied during chick morphogenesis using electron microscopy, light microscopy and immunohistochemical techniques. This layer, called the subepicardium, increases rapidly in volume from embryonic day 6 to 11 (E6–E11) during mesenchymal cell invasion. Fibrous, matrix components are initially apparent at E11 to E16, and as fibrous connective tissue structures accumulate, subepicardial volume decreases. Antibody labeling shows that fibronectin is an early, prominent constituent of the subepicardium, and by E8, the subepicardium is the cardiac site most enriched in fibronectin. Collagen type III is present in circumferentially-oriented fibers at E8. During subsequent cardiac growth, collagen type III fibers become broadly distributed in the subepicardium, with some fibers appearing to attach myocardium to epicardium. Collagen type I fibers are not apparent until E10. At E12 collagen type I fibers are distributed circumferentially around the heart in bundles crimped into waves of low amplitude. Other collagen type I fibers are oriented radially in the subepicardium. During late cardiac morphogenesis and in fully-differentiated hearts, fibronectin and collagen types I and III are more concentrated in the subepicardium than within the myocardium. These observations suggest that the composition and organization of the subepicardial connective tissue may make important contributions to cardiac mechanics from the latter half of embryonic development through adulthood.     

A UV/visible spectroscopic study of water-soluble conjugated polyenes prepared via the ring-opening metathesis polymerization reaction

A series of water-soluble conjugated polyenes, based on the poly(furanylvinylene) structure, have been prepared from readily available starting materials using ring-opening metathesis polymerization followed by dehydrogenation of the resulting polymer. ¹³C NMR analysis of the precursor polymers shows that the stereochemistry of the main-chain double bonds varies from 93% trans to 10% trans and is retained in the water-soluble derivatives, allowing different degrees of extended conjugation. This is reflected in the UV/vis spectra of the materials. The polymers may be cast into films from aqueous solution, and the UV/vis spectra exhibit vibrational fine structure indicative of conformational rigidity; significantly this fine structure is retained on redissolving the high-trans but not the high-cis films. The absorption maximum of the visible spectrum shows a stronger pH dependence in the high-cis polymers than in the high-trans, and this is interpreted in terms of the different modes of hydrogen bonding which cis and trans units can adopt.     

Muscarinic action of acetylcholine in the rat ventromedial thalamic nucleus

Summary Several lines of evidence suggest a role for ACh in the mediation of cerebello-thalamic transmission. The physiological, pharmacological and biochemical experiments described were designed to test this hypothesis for the rat cerebello-thalamic pathway. Unilateral electrolytic lesions of the superior cerebellar peduncle resulted in modest falls of CAT from both ventromedial thalamic nuclei (contralateral 35%, ipsilateral 15%). Iontophoretic application of ACh to relay cells evokes three types of response (i) excitation (ii) inhibition (iii) polyphasic combinations of (i) and (ii). The type of response evoked was directly related to the firing pattern of the cell. Thus, for example, excitatory responses were never recorded during high-frequency bursting but were easily evoked following a switch to tonic, single-spike activity. All responses to ACh and synaptic responses to cerebellar stimulation were sensitive to muscarinic but not to nicotinic cholinergic antagonists. The nicotinic antagonist mecamylamine was a potent blocker of excitant amino acid responses but had no effect on cerebellarevoked synaptic responses. Cholinergic and anticholinergic agents had a profound action on relay cell firing pattern. ACh promoted single-spike activity whereas atropine promoted high-frequency bursting. The actions of ACh are discussed with reference to recently discovered voltage-sensitive ionic conductances. Because of the modulatory action of ACh on relay cell firing pattern and excitability no firm conclusion can be reached concerning the hypothesis under test here. We tentatively suggest a dual role for ACh as both neurotransmitter and neuromodulator.     

Analysis of the MTHFR 1298A-->C and 677C-->T polymorphisms as risk factors for neural tube defects

The thermolabile variant (677TT) of methylenetetrahydrofolate reductase (MTHFR) is a known risk factor for neural tube defects (NTDs). The relationship between a second MTHFR polymorphism (1298A→C) and NTD risk has been inconsistent between studies. We genotyped 276 complete NTD triads (mother, father and child affected with an NTD) and 256 controls for MTHFR 1298A→C. Our findings do not support a role for the 1298A→C polymorphism in NTDs (OR 0.85 (95% CI 0.49–1.47), p= 0.55), nor do we observe a combined effect with the 677C→T polymorphism. Electronic Publication     

P2X receptors in trigeminal subnucleus caudalis modulate central sensitization in trigeminal subnucleus oralis

This study investigated the role of trigeminal subnucleus caudalis (Vc) P2X receptors in the mediation of central sensitization induced in nociceptive neurons in subnucleus oralis (Vo) by mustard oil (MO) application to the tooth pulp in anesthetized rats. MO application produced a long-lasting central sensitization reflected in neuroplastic changes (i.e., increases in neuronal mechanoreceptive field size and responses to innocuous and noxious mechanical stimuli) in Vo nociceptive neurons. Twenty minutes after MO application, the intrathecal (i.t.) administration to the rostral Vc of the selective P2X(1), P2X(3), and P2X(2/3) receptor antagonist, 2'-(or 3'-)O-trinitrophenyl-ATP (TNP-ATP), significantly and reversibly attenuated the MO-induced central sensitization for more than 15 min; saline administration had no effect. Administration to the rostral Vc of the selective P2X(1), P2X(3), and P2X(2/3) receptor agonist, alpha,beta-methylene ATP (alpha,beta-meATP, i.t.) produced abrupt and significant neuroplastic changes in Vo nociceptive neurons, followed by neuronal desensitization as evidenced by the ineffectiveness of a second i.t. application of alpha,beta-meATP and subsequent MO application to the pulp. Administration to the rostral Vc of the selective P2X(1) receptor agonist beta,gamma-methylene ATP (beta,gamma-meATP, i.t.) produced no significant neuroplastic changes per se and did not affect the subsequent MO-induced neuroplastic changes in Vo nociceptive neurons. These results suggest that P2X(3) and possibly also the P2X(2/3) receptor subtypes in Vc may play a role in the initiation and maintenance of central sensitization in Vo nociceptive neurons induced by MO application to the pulp.     

A course on the transition to adult care of patients with childhood-onset chronic illnesses.

Children with special health care needs born today have a 90% chance of surviving into adulthood, making their transition to adult systems of care an issue that will affect almost all physicians. However, many adult generalists and specialists are not familiar with the management of chronic diseases that begin in childhood. While the public health system has made transition to appropriate adult care a priority, and many specialty organizations have endorsed this concept, there are no published studies addressing how the concept of transition can be taught to medical students or residents. The authors describe a one-week course for medical students, begun in 2001 at their institution, that addresses the transition for youth with special health care needs, emphasizing patient and family-centered care, cultural competence, and decision making in end-of-life issues. Cystic fibrosis, a common genetic disease with increasing life expectancy, is used as the model for the course. Involvement of interdisciplinary faculty, interviews with youth with special health care needs and family caregivers, readings from academic and nonacademic literature, and group discussions are presented as teaching methods. Key insights based on experience with the course are the need to include the voices of patients and families, the use of faculty from various professions and specialties to model interdisciplinary care, and the insight that problems specific to transition offer into contemporary health care financing. Future studies should measure the impact of such courses on students' knowledge of transition issues, and determine essential information required for physicians in practice.     

Chimaeric anti-CD4 monoclonal antibody cross-linked by monocyte Fcγ receptor mediates apoptosis of human CD4 lymphocytes

Previous studies have shown that murine anti-CD4 monoclonal antibody, cross-linked by rabbit anti-mouse immunoglobulin, could mediate apoptosis of murine CD4⁺ lymphocytes when they were stimulated by T cell receptor antibody. In this study, we have shown that the murine anti-CD4 monoclonal antibody, OKT4, can induce apoptosis in human CD4⁺ T cells stimulated by the recall antigen tuberculin purified protein derivative (PPD) only when cross-linked by rabbit anti-mouse immunoglobulin. The chimeric anti-CD4 monoclonal antibody, cM-T412 whose Fc fragment is human, was able to cause apoptosis without cross-linking by a second antibody. Similarly, abolition of PPD-induced proliferation of peripheral blood mononuclear cells by cM-T412 did not require cross-linking with rabbit anti-human immunoglobulin. Inhibition of proliferation by cM-T412 could be reduced by pre-treating monocytes with heat-aggregated human IgG. This suggested that monocyte Fcγ receptors might be cross-linking the human Fc of cM-T412. Propidium iodide staining together with immunofluorescence showed that the apoptotic cells were indeed CD4⁺ lymphocytes. It is proposed that during treatment with cM-T412 in autoimmune disease such as rheumatoid arthritis, cM-T412-coated CD4 T cells, when they are subsequently stimulated by the unknown arthritogenic antigen, may undergo apoptotic cell death through cross-linking of cM-T412 on Fey receptor-positive cells within the joint.