Pharmacology and subcellular distribution of [3H]rilmenidine binding sites in rat brain

We have previously reported that in rat brain membranes, [3H]rilmenidine, in addition to labelling alpha2-adrenoceptors and the I2B-subtype of imidazoline receptor binding site (I2B-RBS), may label an additional I-RBS population, distinct from previously classified I1-RBS and I2-RBS. In this study, using crude or fractionated rat brain membranes we examined the possible association of [3H]rilmenidine-labelled I-RBS with the A- and B-isoforms of monoamine oxidase (MAO) by studying the inhibition of [3H]rilmenidine binding by a number of MAO inhibitors; and comparing the maximal binding density (Bmax) and subcellular distribution of [3H]rilmenidine binding sites with that of MAO-A and MAO-B catalytic sites labelled by [3H]RO41-1049 and [3H]RO19-6327 and 12-RBS labelled by [3H]2-BFI. Inhibition of [3H]rilmenidine binding by all MAO inhibitors tested produced very shallow curves (slope 0.29-0.56). Clorgyline and moclobemide (selective MAO-A inhibitors) displayed moderate affinities (60-140 nM), while pargyline (non-selective MAO-inhibitor), RO41-1049 (selective MAO-A inhibitor) and RO19-6327 (selective MAO-B inhibitor) exhibited very low affinities (> 2 microM) for 50-75% of [3H]rilmenidine-labelled I-RBS in crude brain membranes and even lower affinity for the remaining binding. Under identical buffer conditions, the Bmax of [3H]rilmenidine-labelled I-RBS (1.45+/-0.14 pmol/mg protein) was considerably lower than those of MAO-A (13.10+/-0.15 pmol/mg) and MAO-B (10.35+/-0.50 pmol/mg) sites. These results suggest that [3H]rilmenidine does not interact directly with the active catalytic site of either MAO enzyme and could at best only associate with a subpopulation of MAO molecules. Binding studies on five fractions of rat cortex homogenates-nuclear (N), heavy (M) and light (L) mitochondrial, microsomal non-mitochondrial (P), and soluble cytosolic (S) fractions-revealed that 45% of total [3H]rilmenidine binding was present in the P fraction cf. 20 and 23% in the M and L fractions, in contrast to [3H]RO19-6327 and [3H]2-BFI which bound 11-13% in the P fraction and 36-38% and 35-44% in the M and L fractions, respectively. Binding of all ligands in the N fraction was 6-15% of total. These studies reveal that [3H]rilmenidine-labelled I-RBS, unlike the I2-RBS, are not predominantly associated with mitochondrial fractions containing the MAO enzymes (and cytochrome oxidase activity), but appear to be distributed in both the mitochondrial and plasma membrane fractions in rat cerebral cortex.     

Mast cell activation and migration to lymph nodes during induction of an immune response in mice

The mast cell response in skin and lymph nodes was examined during the sensitization phase of dinitrofluorobenzene (DNFB)-induced contact hypersensitivity in mice. Degranulation of 62% of mast cells in DNFB-exposed skin was evident within 30 min of a dual application of DNFB, reaching a peak of 77% at 24 h, and persisting in 42% after 5 d. Abundant expression of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta mRNAs and proteins was observed in keratinocytes, and mast cell degranulation was significantly inhibited after administration of neutralizing antibodies to MIP-1alpha, but not MIP-1beta. During DNFB sensitization, the mast cell density in the skin decreased by half, concurrent with a fivefold expansion of mast cell numbers in draining lymph nodes. Fluorescent-labeled mast cells injected into the skin appeared in draining lymph nodes after application of DNFB, followed by subsequent migration to the spleen. In lymph nodes, mast cells were an abundant and predominant source of MIP-1beta, neutralization of which partially inhibited T lymphocyte recruitment. These results indicate that mast cells contribute to the induction of this primary immune response by activation at and migration from the site of antigen encounter to draining lymph nodes, wherein they mediate T lymphocyte recruitment by production of MIP-1beta.     

Relapsing cutaneous polyarteritis nodosa associated with streptococcal infections

BACKGROUND: Polyarteritis nodosa is an aggressive, often fatal form of vasculitis associated with multi-organ involvement. Cutaneous polyarteritis nodosa is purported to be a more benign form of this disorder with involvement limited to the skin. METHODS: The identification of a female patient from childhood to adulthood documenting repeated episodes of cutaneous polyarteritis nodosa following bouts of recurrent streptococcal pharyngitis. RESULTS: Repeated bouts of streptococcal pharyngitis at ages 11, 28, and 33 years were followed by episodes of cutaneous polyarteritis nodosa, documented by histopathologic skin changes and clinical presentation, and confirmed by therapeutic management. CONCLUSIONS: Various infectious and non-infectious conditions have been linked both to the initiation and relapse of this disease. We describe a patient with recurrent episodes of cutaneous polyarteritis nodosa spanning a period of over 20 years with each episode appearing to be linked to a prior streptococcal infection.     

Clonidine reduces dopamine and increases GABA in the nucleus accumbens: an in vivo microdialysis study

The spectrum of infectious disease (ID) emergencies in hospitalized patients was assessed in a prospective study of 3,626 inpatient ID consultations in a 1,350-bed teaching hospital. ID emergencies, defined by a need or anticipated need for advanced life support or by irreversible organ damage leading to permanent functional loss, were encountered in 175 patients. Infections of the central nervous system (26.3%), cardiovascular system (14.9%), alimentary system (13.1%), and lower respiratory tract (7.4%) and adverse reactions to antimicrobial agents (7.4%) were most common. In 18.9% of the cases, the referring clinicians were unaware of the emergency at the time of referral. Drug reactions (46.1%), severe alimentary and peritoneal infections (32.0%), upper respiratory tract infections (28.6%), and skin and soft-tissue infections (27.3%) were most frequently missed. The emergency ID conditions were not recognized because they had an atypical presentation (51.5%), were not commonly seen in the referring specialty (24.2%), were due to rare organisms (15.2%), or had unusual anatomical sites of involvement (9.1%). A close liaison between clinicians and the ID team is crucial for recognition of ID emergencies at their early stages so that appropriate investigations and management can be instituted expediently, before the occurrence of irreversible damage.     

Effect of plasma TNF-alpha on filgrastim-stimulated hematopoiesis in mice and humans

Marshall syndrome is a rare, autosomal dominant skeletal dysplasia that is phenotypically similar to the more common disorder Stickler syndrome. For a large kindred with Marshall syndrome, we demonstrate a splice-donor-site mutation in the COL11A1 gene that cosegregates with the phenotype. The G+1-->A transition causes in-frame skipping of a 54-bp exon and deletes amino acids 726-743 from the major triple-helical domain of the alpha1(XI) collagen polypeptide. The data support the hypothesis that the alpha1(XI) collagen polypeptide has an important role in skeletal morphogenesis that extends beyond its contribution to structural integrity of the cartilage extracellular matrix. Our results also demonstrate allelism of Marshall syndrome with the subset of Stickler syndrome families associated with COL11A1 mutations.     

Biocatalytic sulfur removal from fuels: applicability for producing low sulfur gasoline

Environmental regulations are driving R&D efforts to produce low sulfur fuels, including diesel fuel and gasoline for motor vehicles. Biocatalytic sulfur removal from fuels has potential applicability for producing low sulfur gasoline. Microbial biocatalysts have been identified that can biotransform sulfur compounds found in fuels, including ones that selectively remove sulfur from dibenzothiophene heterocyclic compounds. Most attention is give to the 4S pathway of Rhodococcus, which can remove sulfur from substituted and unsubstituted dibenzothiophenes, including sulfur compounds that hinder chemical catalysis and that resist removal by mild hydrotreatment. Various bioreactor and bioprocess designs are being tested for use with biocatalysts, including recombinant biocatalysts, for use in removing sulfur from fuels and feedstocks within the petroleum refinery stream. With bioprocess improvements that enhance biocatalyst stability, achieve faster kinetics, improve mass transfer limitations, temperature and solvent tolerance, as well as broaden substrate specificity to attack a greater range of heterocyclic compounds, biocatalysis may be a cost-effective approach to achieve the production of low sulfur gasoline. The challenge will be to accomplish these improvements by the time the regulations for low sulfur gasoline and other vehicle fuels go into effect in order to be competitive with emerging nonbiological desulfurization technologies.     

Definition of the specific roles of lysolecithin and palmitic acid in altering the susceptibility of dipalmitoylphosphatidylcholine bilayers to phospholipase A2

Bilayers composed of phosphatidylcholine initially resist catalysis by phospholipase A2. However, after a latency period, they become susceptible when sufficient reaction products (lysolecithin and fatty acid) accumulate in the membrane. Temperature near the main bilayer phase transition and calcium concentration modulate the effectiveness of the reaction products. The purpose of this study was to examine the individual contributions of lysolecithin and palmitic acid to the susceptibility of dipalmitoylphosphatidylcholine vesicles and to rationalize the effects of temperature and calcium. Various fluorescent probes (Prodan, Laurdan, pyrene-labeled fatty acid, and dansyl-labeled phospholipid) were used to assess changes in the ability of the reaction products to perturb the bilayer and to affect the interactions with the enzyme. Un-ionized palmitic acid decreased bilayer polarity and perturbed the membrane surface exposing some of the Prodan to bulk water. Lysolecithin increased bilayer polarity and the rate of dipolar relaxation in response to the excited states of Laurdan and Prodan. A combination of the individual contributions of each product was observed when palmitic acid and lysolecithin were present together at low calcium, and the effects of lysolecithin dominated at high calcium. Palmitic acid, but not lysolecithin, promoted the binding of phospholipase A2 to the bilayer surface in the absence of calcium. Lysolecithin reduced the ability of fatty acid to enhance binding apparently by altering the structure of fatty acid domains in the membrane. Furthermore, increased temperature and ionization of the fatty acid tended to cause segregation of bound phospholipase A2 into domains poor in phospholipid content which presumably impeded bilayer hydrolysis. In contrast, un-ionized palmitic acid and lysolecithin promoted hydrolysis by augmenting a step distal to the adsorption of enzyme to the bilayer. This kinetic response to lysolecithin was calcium-dependent. A model accounting for these varied influences of the reaction products is presented.     

Botulinum toxin injections in the internal anal sphincter for the treatment of chronic anal fissure: long-term results after two different dosage regimens

OBJECTIVE: To determine whether the recently published guidelines on neuroimaging in patients with new-onset seizures are applicable to children. METHODS: We carried out a retrospective analysis of 107 neurologically normal children (excluding children with simple febrile seizures) who had undergone neuroimaging when they presented to the emergency department with a possible "first seizure." RESULTS: Eight of the 107 children had nonepileptic events (gastroesophageal reflux, syncopal event, rigor). Of the remaining 99 children, 49 had provoked seizures (complicated febrile seizure, meningo-encephalitis, toxic or metabolic abnormalities), and 50 had unprovoked seizures. A total of 19 children had brain abnormalities identified on computed tomography (CT) scan; 7 received further investigation or intervention as a result of CT scan findings (2 with tumors, 3 with vascular anomalies, 1 with cysticercosis, and 1 with obstructive hydrocephalus). CT scan abnormalities requiring treatment or monitoring were more frequently seen in children with their first unprovoked seizure (P < .01) and in those children whose seizure onset had been focal or who had focal abnormalities identified on postictal neurologic examination (P < .04). CONCLUSION: In a child, a seizure in the setting of a fever rarely indicates the presence of an unexpected CT scan lesion requiring intervention.     

Clonal analysis of intrahepatic B cells from HCV-infected patients with and without mixed cryoglobulinemia

Clonal rearrangements of Ig heavy chain (IgH) genes and hepatitis C virus (HCV) genomic sequences were assayed on intrahepatic B lymphocytes isolated from HCV chronically infected patients with and without type II mixed cryoglobulinemia (MC). Liver tissue samples from eight patients with and nine without MC were subjected to routine histologic studies, immunophenotyping, and genotypic analysis including IgH V-D-J region gene rearrangements by PCR. RT-PCR, signal amplification by branched DNA assay, and in situ hybridization technique were used to detect and quantitate HCV RNA genomic sequences in selected B cells purified from each tissue sample. Although HCV infection of intrahepatic B cells was shown in all patients both with and without MC, frank B cell monoclonal and oligoclonal patterns were found in only three and four patients with MC, respectively. No monoclonal profile was seen in the noncryoglobulinemic patients, whereas an oligoclonal profile was demonstrated in four of them. No clonalities were shown in HCV-unrelated patients matched for age and severity of liver disease. No obvious difference in HCV genotype distribution was found in relation to the clonal expansion profile. Noncryoglobulinemic patients showing clonal expansion in liver tissue had higher titers of serum rheumatoid factor (RF). Spontaneous production of RF was shown in cell cultures of intrahepatic B cells, suggesting their persistent stimulation in vivo. These data indicate that HCV infection of B cells and B cell clonal expansions occur in the liver microenvironment and preferentially involve RF-producing cells.