Autoradiographic localization of putrescine uptake to type II pneumocytes of rabbit lung slices

At low concentrations (0.4-5.0 microM), the polyamine, putrescine, is accumulated predominantly by a saturable process in rabbit lung slices. The question of why the lung should possess polyamine transport capabilities and the known cellular compartmentalization of various other pulmonary functions prompted us to investigate whether putrescine uptake was localized to a specific cell type(s) within rabbit lung. Localization of putrescine uptake in rabbit lung was investigated by frozen and plastic section autoradiography, combined with electron microscopy. Rabbit lung slices were incubated with [3H]putrescine at a concentration (0.4 microM) at which the saturable component of uptake predominates. Both frozen and plastic section autoradiograms revealed relatively minor amounts of radiolabel associated with either vascular or bronchiolar tissues, whereas a greater density of radiolabel was present over the alveolar epithelium. Highly localized areas of radiolabel were found at the junctions of adjacent alveoli and these labeled cells were identified by electron microscopy as type II pneumocytes. We conclude that the type II pneumocyte is a major site of putrescine uptake in rabbit lung slices. Our study also indicates that different rabbit lung cell types possess differing abilities to accumulate putrescine.     

A mathematical model of optimized radioiodine-131 therapy of Graves' hyperthyroidism

Background

The current status of radioiodine-131 (RaI) dosimetry for Graves' hyperthyroidism is not clear. Recurrent hyperthyroidism and iatrogenic hypothyroidism are two problems which interact such that trying to solve one leads to exacerbation of the other. Optimized RaI therapy has therefore begun to be defined just in terms of early hypothyroidism (ablative therapy) as physicians have given up on reducing hypothyroidism.

Methods

Optimized therapy is evaluated both in terms of the greatest separation of cure rate from hypothyroidism rate (non-ablative therapy) or in terms of early hypothyroidism (ablative therapy) by mathematical modeling of outcome after radioiodine and critically discussing the three common methods of RaI dosing for Graves' disease.

Results

Cure follows a logarithmic relationship to activity administered or absorbed dose, while hypothyroidism follows a linear relationship. The effect of including or omitting factors in the calculation of the administered I–131 activity such as the measured thyroid uptake and effective half-life of RaI or giving extra compensation for gland size is discussed.

Conclusions

Very little benefit can be gained by employing complicated methods of RaI dose selection for non-ablative therapy since the standard activity model shows the best potential for cure and prolonged euthyroidism. For ablative therapy, a standard MBq/g dosing provides the best outcome in terms of cure and early hypothyroidism.     

Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55 degrees C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n = 72), C. dubliniensis (n = 58), C. glabrata (n = 5), C. krusei (n = 2), C. parapsilosis (n = 4), and C. tropicalis (n = 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management.     

Identification and characterization of aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus with partial vasopressin response

Congenital nephrogenic diabetes insipidus (NDI) is a rare disease caused most often by mutations in the vasopressin V2 receptor (AVPR2). We studied a family which included a female patient with NDI with symptoms dating from infancy. The patient responded to large doses of desmopressin (dDAVP) which decreased urine volume from 10 to 4 I/day. Neither the parents nor the three sisters were polyuric. The patient was found to be a compound heterozygote for two novel recessive point mutations in the aquaporin-2 (AQP2) gene: L22V in exon 1 and C181W in exon 3. Residue Cys181 in AQP2 is the site for inhibition of water permeation by mercurial compounds and is located near to the NPA motif conserved in all aquaporins. Osmotic water permeability (Pf) in Xenopus oocytes injected with cRNA encoding C181W-AQP2 was not increased over water control, while expression of L22V cRNA increased the Pf to approximately 60% of that for wild-type AQP2. Co-injection of the mutant cRNAs with the wild-type cRNA did not affect the function of the wild-type AQP2. Immunolocalization of AQP2-transfected CHO cells showed that the C181W mutant had an endoplasmic reticulum-like intracellular distribution, whereas L22V and wild-type AQP2 showed endosome and plasma membrane staining. Water permeability assays showed a high Pf in cells expressing wild-type and L22V AQP2. This study indicates that AQP2 mutations can confer partially responsive NDI.      

Antiarthritic profile of BF-389 — A novel anti-inflammatory agent with low ulcerogenic liability

BF-389, dihydro-4-(3,5-di-tert-butyl-4-hydroxybenzylidene)-2methyl-2H-1,2-oxazin-3(4H)-one, is a potent, orally active, antiarthritic and analesis agent with low ulcerogenic potential. A comparison of the activity profiles of BF-389 and naproxen showed similarities in: (1) suppression of developing and chronic adjuvant arthritis (AA); (2) maximal inhibitory response, as shown by theE(max0 values in the developing and established AA models; (3) inhibition of bone degenerative changes associated with chronic adjuvant arthritis; and (4) analgesic activity in the acetic acid and phenylquinone writhing assays. Though BF-389 has been shown to be a potent inhibitor of cyclooxygenase, IC₅₀=0.84±0.25 M against the production of PGE₂ in vitro, there is a great difference from most cyclooxygenase inhibitors; it also inhibits the 5-lipoxygenase enzyme. For BF-389, the IC₅₀ for invitro LTB₄ formation was found to be 3.65±1.19 M. The ulcerogenic potential of BF-389 was compared to that of naproxen using a five-dayin vivo ulcerogenic rat assay. The UD₅₀ for naproxen was found to be approximately 30 mg/kg/day, p.o. Based upon efficacy in the DEV AA and EST AA models, UD₅₀/ED₅₀ values for naproxen were estimated to be 0.7 and 1.9, respectively. For BF-389 the UD₅₀ was shown to be 520 (389–695) mg/kg/day, p.o., and the corresponding UD₅₀/ED₅₀ values were calculated to be 84 and 28, respectively, thus demonstrating the wide margin of safety between efficacy and ulcerogenicity in rats.     

Reciprocal effect of Waardenburg syndrome mutations on DNA binding by the Pax-3 paired domain and homeodomain

The Pax-3 protein contains two DNA-binding domains, a paired domain and a homeodomain. Mutations in Pax-3 cause Waardenburg syndrome (WS) in humans and the mouse Splotch (Sp) phenotype. In the Sp-delayed mouse, a mutation in the Pax-3 paired domain (G9R) abrogates the DNA-binding activity of both the paired domain and the homeodomain, suggesting that they may functionally interact. To investigate this possibility further, we have analyzed the DNA-binding properties of additional point mutants in the Pax-3 paired domain and homeodomain that occur in WS patients (F12L, N14H, G15S, P17L, R23L, G48A, S51F and G66D in the paired domain, V47F and R53G in the homeodomain), the Pax-1 un mutation (G15A) and a substitution associated with Peters' anomaly in the PAX-6 gene (R23G). Within the paired domain, seven of 10 mutations were found to abrogate DNA-binding by the paired domain. Remarkably, these seven mutations also affected DNA binding by the homeodomain, causing either a complete loss (P17L and G66D), a reduction (R23G, R23L, G15S and G15A) or an increase in DNA-binding activity (N14H). In addition, the effect of paired domain mutations occurred at the level of monomer formation by the homeodomain, while the dimerization potential of this domain seemed unaffected in mutants where it could be analyzed. Furthermore, while both homeodomain mutations were found to abolish DNA binding by this domain, the R53G mutation also abrogated DNA binding by the paired domain. The important observation that independent mutations in either domain can affect DNA binding by the other in the intact Pax- 3 protein strongly suggests that the two domains are not functionally independent but bind DNA through cooperative interactions. Modeling the deleterlous mutations on the three-dimensional structure of the paired domain of Drosophila Prd shows that these mutations cluster at the DNA interface, thus suggesting that a series of DNA contacts are essential for DNA binding by both the paired domain and the homeodomain of Pax-3.      

The spectrum of atrioventricular discordance. A clinical study.

The clinical, investigative, and surgical findings were reviewed in 47 patients with atrioventricular discordance who presented to the Brompton Hospital between January 1962 and June 1981. Although the unifying feature was the atria connecting to morphologically inappropriate ventricles, the hearts differed widely in other respects. In most cases there was the usual visceral and atrial arrangement, but six had a mirror image arrangement of the atria and viscera. Among those patients with usual atrial arrangement (solitus) the aorta was not always anterior and left sided, and ventricular "inversion" was not invariable. Only 35 of the 47 patients also had a discordant ventriculoarterial connexion, the majority of the remainder having a double outlet right ventricle. The specific diagnosis of atrioventricular discordance depended on echocardiographic and angiographic examination. The other investigations did not distinguish between different ventriculoarterial connexions and were not specific even for the presence of atrioventricular discordance. For those patients with the usual atrial arrangement the anticipated right heart border was present in only just over one third, and the reversal of Q wave progression in the precordial leads of the standard electrocardiogram was found in under a half. Many patients with atrioventricular discordance progressed normally to adult life. In 20 cases no surgery was performed. The results of total correction showed a mortality of 25% (three cases), including two deaths after a modified Fontan procedure for exceedingly complex associated lesions. The results of surgery in the survivors were excellent, and awareness of the disposition of the atrioventricular conduction tissue made it possible to avoid atrioventricular dissociation.     

Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn.

The present study investigated the regional distribution of the N-methyl-D-aspartate (NMDA) receptor containing the NR2B subunit protein in rat lumbar spinal cord and examined whether selective NR2B antagonists would exhibit antinociception with reduced side-effect liability than subtype non-selective NMDA antagonists and anticonvulsants. Immunocytochemical studies showed the NR2B subunit had a restricted distribution, with moderate labelling of fibres in laminas I and II of the dorsal horn suggesting a presynaptic location on primary afferent fibers and possible involvement in pain transmission. In the in vivo studies, the NMDA/glycine antagonists (MK-801, 0.02-1 mg/kg i.p., L-687,414 10-300 mg/kg i.p., and L-701,324 1-10 mg/kg i.p.) and the anticonvulsant, gabapentin (10-500 mg/kg p.o.), induced rotarod deficits at antinociceptive doses. In contrast, the selective NR2B antagonists, (+/-)-CP-101,606 (1-100 mg/kg p.o.) and (+/-)-Ro 25-6981 (3-100 mg/kg i.p.) showed a significant dose window. (+/-)-CP-101,606 caused no motor impairment or stimulation in rats at doses up to 100 mg/kg p.o., which is far in excess of those inhibiting allodynia in neuropathic rats (ID50 4.1 mg/kg, p.o.). (+/-)-Ro 25-6981 also showed a significant separation (ID50 allodynia 3.8 mg/kg, i.p.), however, some disruption of rotarod performance was observed at 100 mg/kg. The anticonvulsant lamotrigine (3-500 mg/kg p.o.) also showed a good dose window. These findings demonstrate that NR2B antagonists may have clinical utility for the treatment of neuropathic and other pain conditions in man with a reduced side-effect profile than existing NMDA antagonists.