Apamin-sensitive Ca(2+)-activated K+ channels regulate pacemaker activity in nigral dopamine neurons

Mesencephalic dopamine-containing neurons exhibit a Ca(2+)-dependent oscillation in membrane potential believed to underlie the ability of these cells to maintain spontaneous activity in the absence of afferent synaptic drive. In the present series of experiments, sharp electrode intracellular recording techniques were used in conjunction with an in vitro brain slice preparation to explore the ionic mechanisms underlying rhythmogenesis in nigral dopamine neurons in the rat. Our results indicate that the K+ channel producing the prolonged post-spike afterhyperpolarization exhibited by these neurons is also principally responsible for generating the falling phase of the autogenous pacemaker oscillation. Alterations in the expression of this conductance are associated with marked changes in neuronal firing pattern, indicating that modulation of ligand-gated Ca(2+)-activated K+ channels may constitute a functional means of altering temporal coding among the major mesotelencephalic dopamine systems.     

Discovery of novel pyridinopolyamines with potent antimicrobial activity: deconvolution of mixtures synthesized by solution-phase combinatorial chemistry

A 1638-member pyridinopolyamine library, consisting of 13 sublibraries of 126 members prepared by a solution-phase approach, was completely deconvoluted from orthogonally protected intermediates by a combination of iterative and positional scanning procedures. Antibacterial assays against Streptococcus pyogenes and Escherichia coli imp- and a Candida albicans yeast specificity assay were employed to follow the activity of sublibraries. Screening of the 13 sublibraries, which were prepared by a synthetic method that places the differentiating functionality in a selected position A (secondary amine), at the end of the synthesis (fix last), provided several first-round activities. Subsequently, six single pyridinopolyamines (2-7) were prepared where the first-round winner, a hydrogen atom, is in the first deconvoluted position and the remaining three positions contained the same functionalities. The range of antibacterial and yeast activities of these single compounds suggested that a more active and selective compound may be discovered by completely deconvoluting the first-round active sublibraries. Pyridinopolyamine positions B (secondary benzylamine) and C (primary benzylamine) were then sequentially positionally scanned with a set of six meta-substituted benzyl functionalities to generate two sets of second/third-round sublibraries, containing 21 or 36 compounds in each sublibrary, respectively. High-throughput screening yielded sublibraries 15, 18, and 21 with MICs of 1-5 microM against S. pyogenes and E. coli imp-. Using rounds 1 and 2/3 screening data, two sets of single compounds (22-27) and (28-32) with the combination of m-(trifluoromethyl)-benzyl group at position C and m-(trifluoromethyl)benzyl or m-methylbenzyl group at position B with position D (primary benzylamine) fixed were synthesized in the fourth round deconvolution. Subsequently, broader screening of deconvoluted compounds against a tier II panel of wild-type bacteria identified eight compounds (5, 7, 27, and 29-32) with approximately 100-fold greater selectivity for Gram-positive than Gram-negative bacteria. Thus, S. pyogenes, S. pyogenes (wild-type), Streptomyces aureus, and Enterococcus faecalis were inhibited at MICs of 1-12 microM, whereas MICs for E. coli, Klebsiella pneumoniae, Proteus vulgaris, and Pseudomonas aeruginosa were > 100 microM. These eight compounds were not active (> 100 microM) against fungus C. albicans.     

Intravascular coagulation activation in a murine model of thrombomodulin deficiency: effects of lesion size, age, and hypoxia on fibrin deposition

We consecutively inactivated both alleles of the thrombomodulin (TM) gene in murine embryonic stem (ES) cells and generated TM-deficient (TM-/-) chimeric mice. Quantitation of an ES-cell marker and protein C cofactor activity indicates that up to 50% of pulmonary endothelial cells are ES-cell derived and therefore TM deficient. Infusions of 125I-fibrinogen into mice show a significant increase (fourfold, P <.005) in radiolabeled cross-linked fibrin in TM-/- chimeric mouse lung as compared with wild-type mice. However, only chimeric mice that exhibit at least a 30% reduction in protein C cofactor activity and are at least 15 months old display this phenotype. Immunocytochemical localization of TM in chimeras shows a mosaic pattern of expression in both large and small blood vessels. Colocalization of cross-linked fibrin and neo (used to replace TM) reveals that fibrin is deposited in TM-/- regions. However, the fibrin deposits were largely restricted to pulmonary vessels with a lumenal area greater than 100 micrometer2. The hypercoagulable phenotype can be induced in younger chimeric mice by exposure to hypoxia, which causes a fivefold increase in beta-fibrin levels in lung. Our findings show that TM chimerism results in spontaneous, intravascular fibrin deposition that is dependent on age and the magnitude of the TM deficiency.     

Iatrogenic superior gluteal mononeuropathy

To prevent barotitis during descent in aviation, the ears have to be cleared several times by performing the Valsalva's manoeuvre. The manoeuvre is difficult for children to perform, and they are therefore at high risk of developing barotitis. The treatment of barotitis is either inflation by a Politzer balloon or myringotomy. An alternative treatment is autoinflation using the Otovent. This prophylaxis/treatment can be performed by the child with assistance from its parents as soon as or preferably before the descent has started. The prevalence of barotitis amongst 45 children and 49 adults in transit was found to be highest in children, 28%, compared with adults, 10%. Only 6% of the children with negative middle ear pressure after flight managed a successful Valsalva manoeuvre, whereas 33% could normalise the middle ear pressure by inflating the Otovent. In conclusion we recommend autoinflation using the Otovent set by children and adults who have problems clearing their ears during flight.     

Renal and depressor activities of inhibitors of neutral endopeptidase and angiotensin converting enzyme in monkeys infused with angiotensin II

In a previous study, the depressor activity of combined selective inhibitors of neutral endopeptidase EC 3.4.24.11 (NEP) and angiotensin-converting enzyme (ACE) depended on the level of ACE inhibition, whereas the renal responses were determined by NEP inhibition. Our study confirmed that a mixed NEP/ACE inhibitor BMS-182657 ([S-(R*,R*)]-2,3,4,5-tetrahydro-3-[(2-mercapto-1-oxo-3- phenylpropyl)amino]-2-oxo-1H-benzazepine-1-acetic acid) reduced mean arterial pressure (MAP) when renin release was reduced by a sodium load, suggesting that the depressor response did not require suppression of endogenous angiotensin II generation. Furthermore, a pressor dose of 30 ng/min of angiotensin II was required to block the depressor response to BMS-182657 in the presence or absence of exogenous human atrial natriuretic peptide (hANP 99-126). Thirty ng/min of angiotensin II also significantly enhanced the natriuresis induced by hANP 99-126 after BMS-182657 administration. In contrast, a nonpressor dose of angiotensin II (3 ng/min) reduced basal sodium excretion and the natriuretic responses to exogenous hANP 99-126 in the presence or absence of BMS-182657. The potentiation of the urinary ANP and cyclic guanosine monophosphate (cGMP) responses to hANP 99-126 by BMS-182657 was similar for all doses of angiotensin II; therefore angiotensin did not alter the effects of BMS-182657 on ANP metabolism or cGMP accumulation in the kidney. In summary, the renal responses to mixed metalloprotease inhibitors were apparently mediated by ANP potentiation and were modulated by angiotensin II. The depressor activity depended on ACE inhibition but was not mediated solely by reductions in endogenous angiotensin II levels.     

Superior outcomes in pediatric renal transplantation

BACKGROUND: Nationally, results of renal transplantation in children, particularly in small children, are inferior to those obtained in adults. OBJECTIVE: To determine factors important for success in renal transplantation in children. DESIGN: Results of 108 consecutive renal transplantations performed in patients aged 7 months to 18 years were reviewed and compared with those reported by the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS), the national registry. RESULTS: One-, 2-, and 3-year graft survival rates (+/-SE) were 99% +/- 1%, 95% +/- 3%, and 93% +/- 4%, respectively, for living donor grafts and 97% +/- 3%, 92% +/- 6%, and 92% +/- 6%, respectively, for cadaver grafts. Incidence of acute rejection was half that reported by NAPRTCS. There were no graft losses for technical reasons (19% in NAPRTCS). Twelve percent of patients were younger than 2 years (6% in NAPRTCS); 17% were 2 to 5 years old (16% in NAPRTCS). Most small children received an adult-sized kidney. Ninety-three percent of recipients weighing 15 kg or less received postoperative mechanical ventilation assistance to optimize fluid resuscitation and perfusion of adult-sized kidneys. Structural abnormalities of the urinary tract were present in 53.7% of the patients (48.5% in NAPRTCS; adults, 5.3%). Nephroureterectomy was required in 38 children; in 27 (71%) of them, it was performed at the time of transplant surgery. CONCLUSIONS: Excellent results can be obtained in pediatric renal transplantation by strict adherence to surgical detail, tight immunosuppressive management, aggressive fluid management in the small child, and careful integration of urologic and transplant surgery.     

Carbonyl histochemistry in rat reperfusion nerve injury

Free radical mediated, site-specific lipid and protein oxidation has been implicated in the pathophysiology of an ischaemic/reperfusion injury. The aim of the present study was to determine whether carbonyl formation could be detected histochemically in reperfused rat sciatic nerves. We also examined the effects of preischaemic alpha-tocopherol supplementation on carbonyl formation in reperfused nerves. Seven hours of near-complete ischaemia was induced in rat right hindlimb by occlusion of major arteries using microvascular clips. Histochemical detection of carbonyl compounds, applying naphthoic acid hydrazide (NAH) and Fast Blue B (FBB), was undertaken at thigh, knee and calf levels of sciatic, tibial and peroneal nerves. NAH-FBB reactivity was confined to vessels in reperfused nerves. Positively stained epi-, peri- and endoneurial vessels were invariably observed after 2 h of reperfusion at all levels examined. After 24 and 48 h and 7 days of reperfusion, NAH-FBB-positive vessels were more frequently found at knee and calf levels than at the thigh level. Following preischaemic alpha-tocopherol supplementation, no vessels were stained positively with NAH-FBB, except for some epineurial vessels at knee and calf levels after 2 h of reperfusion. Morphometry in endoneurial vessels at the knee level revealed that endothelial cell area in alpha-tocopherol-treated reperfused nerves was significantly less when compared with those in reperfused nerves without alpha-tocopherol. In conclusion, we have demonstrated histochemical evidence of carbonyl formation in vessels, but not with nerve fibres, in ischaemic/reperfused rat sciatic nerves. These abnormalities were prevented with preischaemic supplementation of alpha-tocopherol.     

Conjugate imagery in the automated reproduction of three dimensional coordinates from two dimensional coordinate data

The three-dimensional video analysis of human motion commonly utilises automated image processing and digitisation processes to produce real-time unidentified two-dimensional coordinate data of segmental markers. In what can be a time-consuming process the two-dimensional data are then identified and tracked to produce three-dimensional coordinates. This paper presents an approach to the automated reproduction of three-dimensional coordinates from two-dimensional coordinates data. Conjugate imaging techniques were utilised in the development of four criterion measures for determining the validity of conjugate (corresponding) image points. An algorithm based on the criterion measures was then developed for the automated reproduction of three dimensional coordinates from camera image coordinate data. The algorithm was tested with a 55 point marker system viewed in four video cameras (digitisation error approx. 0.2%, lab point separation > or = 6 cm). The success of the algorithm was dependent on the closeness of markers, the accuracy of the photogrammetric system, and the number of markers visible in two camera images. The present research has developed techniques based on conjugate imagery for the automated reproduction of three-dimensional coordinates from two-dimensional data, and provided a bases for further development of automated three-dimensional tracking.     

Constitutive intra-articular expression of human IL-1 beta following gene transfer to rabbit synovium produces all major pathologies of human rheumatoid arthritis

To investigate the pathophysiologic effects of chronically elevated intra-articular levels of IL-1 beta, we used an ex vivo gene transfer method to deliver and express human IL-1 beta (hIL-1 beta) in the knee joints of rabbits. Expression of hIL-1 beta resulted in a severe, highly aggressive form of arthritis analogous to chronic rheumatoid arthritis in humans. Intra-articular manifestations included intense inflammation, leukocytosis, synovial hypertrophy and hyperplasia, and highly aggressive pannus formation with erosion of the articular cartilage and periarticular bone. Systemic effects were also observed, including diarrhea, fever, weight loss, and an increased erythrocyte sedimentation rate. In addition, the hIL-1 beta was found to induce elevated levels of both rabbit IL-1 beta and TNF-alpha in synovial fluid. Following the loss of hIL-1 beta transgene expression between 14 and 28 days post-transplantation, many of these changes began to normalize. These results suggest that chronically elevated intra-articular levels of IL-1 beta alone are sufficient to produce virtually all the pathologies found in rheumatoid arthritis, and furthermore, demonstrate that gene transfer can be used to investigate the roles of specific gene products in the pathogenesis of arthritis.     

Selective disruption of lymphotoxin ligands reveals a novel set of mucosal lymph nodes and unique effects on lymph node cellular organization

Lymphotoxin (LT) provides a critical signal for the genesis of lymph nodes (LN) in mice. Here we show that mice treated in utero with LT beta-R-Ig, which binds to the membrane LT alpha 1 beta 2 heterotrimer, lacked most LN, yet retained a set of mucosal surface draining LN. Since mice genetically deficient in LT alpha lack all LN, including the mucosal set, we hypothesize that a novel LT alpha-dependent pathway controls their genesis. This novel set of mucosal LN cannot be discriminated on the basis of addressin expression. The discovery of LN in mice treated with LT beta-R-Ig fusion protein in utero allowed us to compare the roles of membrane LT alpha beta or soluble LT alpha/tumor necrosis factor (TNF) in the development of cellular organization in LN and spleen. Our results indicate that both membrane LT alpha beta and soluble LT alpha/TNF mediate T-B cell segregation and the organization of B cell follicles in spleen and LN. Interestingly, while antagonism of membrane LT alpha beta or soluble LT alpha/TNF prevented germinal center (GC) formation in spleen, antagonism of soluble LT alpha/TNF had no effect on LN formation. The data suggest that multiple LT/TNF ligands control B cell follicle organization in the spleen and LN of adult mice, and that the requirements for LT/TNF ligands in GC formation are distinct in the different lymphoid organs.