Enhanced permeability of the antimicrobial agent 2,5-bis(4-amidinophenyl)furan across Caco-2 cell monolayers via its methylamidoidme prodrug

Purpose. DB75 [2,5-bis(4-amidinophenyl)furan] is a promising antimicrobial agent although it has poor oral potency. In contrast, its novel prodrug, 2,5-bis(4-amidinophenyl)furan-bis-O-methyl- amidoxime (DB289), has excellent oral potency. The mechanisms of transport of DB289 and DB75 across intestinal epithelium have been investigated in these studies to understand differences in their oral potency. Methods. Caco-2 cell monolayers were used as an in vitro model to examine the mechanisms of transport of DB289 and DB75. Samples collected from the transport studies were quantified using high-performance liquid chromatography with ultraviolet and fluorescence detection. Results. A low permeability coefficient (3.8 × 10^–7 cm/s for transport in apical [AP] to basolateral [BL] direction) and high sensitivity to extracellular Ca²⁺ suggest that AP to BL transport of DB75 across Caco-2 cell monolayers occurs predominantly via a paracellular route. DB289 has an 85-fold higher transport rate (322.0 × 10^–7 cm/s for transport in the AP to BL direction) across Caco-2 monolayers than that of DB75. This, with its insensitivity to extracellular Ca²⁺ indicates that AP to BL transport of DB289 across Caco-2 cell monolayers occurs predominantly via a transcellular route. Conclusions. DB75 is transported across Caco-2 cell monolayers predominantly via paracellular pathways, whereas the prodrug DB289 is transported via transcellular pathways. This could account for the much higher oral activity of DB289 over DB75.     

Structure-activity relationship for enhancement of paracellular permeability across Caco-2 cell monolayers by 3-alkylamido-2-alkoxypropylphosphocholines

Paracellular permeability enhancers have been used to improve the oral bioavailability of hydrophilic drugs; however, the mechanism of action of many enhancers is poorly understood. In this study, highly potent enhancers of paracellular permeability were identified in the 3-alkylamido-2-alkoxypropylphosphocholine series, and a structure-activity relationship was developed for enhancement of paracellular permeability across Caco-2 cell monolayers. Compounds with short (<5 carbons) hydrocarbon chains at both C-2 and C-3 were generally inactive. The potency exhibited a parabolic relationship with respect to the chain length at either C-2 or C-3. Linear molecules (i.e., compounds with a short hydrocarbon chain at C-2 or C-3 and a long hydrocarbon chain on C-3 or C-2, respectively) were more potent than the corresponding branched molecules with the same carbon load. The efficacy of 3-alkylamido-2-alkoxypropylphosphocholines as enhancers of paracellular permeability was not dependent on their existence in micellar form or their ability to alter the fluidity of cell membrane. Previously, a correlation between the potency of alkylphosphocholines as enhancers of paracellular permeability and the inhibitors of phospholipase C (PLC) was established in Madine Darby canine kidney (MDCK) cell monolayers. The potencies of selected 3-alkylamido-2-alkoxypropylphosphocholines as inhibitors of PLC and enhancers of paracellular permeability fit well into this correlation. Therefore, phosphocholines are likely to increase paracellular permeability by modulating the signal transduction pathway initiated by a PLC-catalyzed reaction rather than by physically altering the cell membrane.     

Somatic mutations in MEN type 1 tumors, consistent with the Knudson "two-hit" hypothesis

MEN type 1 is an autosomal dominant disorder characterized by the combined occurrence of tumors of the parathyroids, anterior pituitary, and pancreatic islet cells. The MEN1 gene, which is located on chromosome 11q13, consists of 10 exons and encodes a 610-amino acid protein named MENIN. The observation of LOH involving 11q13 in MEN type 1 tumors and the inactivating germline mutations found in patients suggest that the MEN1 gene acts as a tumor suppressor, in keeping with the "two-hit" model of hereditary cancer. The second hit in MEN type 1 tumors typically involves large chromosomal deletions that include 11q13. However, this only represents one mechanism by which the second hit may occur, and the other mechanisms, such as intragenic deletions or point mutations that inactivate the gene, have not been reported in MEN type 1 tumors. We have therefore undertaken studies to search for such mutations in six MEN type 1 tumors (four parathyroid tumors, one insulinoma, and one lipoma) that did not have LOH at 11q13 as assessed using the flanking markers D11S480, D11S1883 and PYGM centromerically and D11S449 and D11S913 telomerically. This revealed four somatic mutations, which consisted of two missense mutations and two frameshift mutations in two parathyroid tumors, one insulinoma, and one lipoma. Thus, our results, which represent the first small intragenic somatic mutations reported in MEN type 1 tumors, provide further evidence that the role of the MEN1 gene is consistent with that of a tumor suppressor gene, as postulated by Knudson's "two-hit" hypothesis.     

Modeling study of human renal chloride channel (hCLC-5) mutations suggests a structural-functional relationship

BACKGROUND: Dent's disease, a renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, and nephrolithiasis, is due to inactivating mutations in the X-linked renal-specific chloride channel, hCLC-5. The x-ray crystal structures of two bacterial chloride channels (CLCs) have recently been established, thereby allowing us to construct a model for hCLC-5 and further examine the role of its mutations. METHODS: The data regarding 49 hCLC-5 mutations were reviewed. Thirty-four mutations that predicted absent or truncated channels were excluded. The remaining 15 mutations (one in-frame insertion and 14 missense mutations), 12 of which have been studied electrophysiologically, were assessed. The hCLC-5 sequence was aligned with the Salmonella typhimurium and Escherichia coli sequences and used to map the hCLC-5 mutations onto a three-dimensional model. RESULTS: hCLC-5 is a homodimeric protein, with each subunit consisting of 18 helices. None of the missense mutations involved the chloride (Cl-) selectivity filter, but 12 of the 15 mutations were found to be clustered at the interface of the two subunits. Six of these mutations occurred in two of the helices that either form part of the interface or lie in close proximity to the interface, and three other mutations that did not lead to complete loss of Cl- conductance were at the edge of the interface. CONCLUSION: These results demonstrate a crucial role for the interaction between the two subunits at the interface of the homodimeric hCLC-5.     

Multifocal nodular episcleritis and scleritis with undiagnosed Hodgkin's lymphoma

PURPOSE: To report the case of a patient with undiagnosed Hodgkin's lymphoma who presented with coexistent unilateral nodular episcleritis and scleritis. DESIGN: Interventional case report and literature review METHODS: Review of clinical history, laboratory findings, histology of episcleral and cervical lymph node biopsies, and follow-up. RESULTS: A 20-year-old female presented with a 5-month history of redness and pain in her left eye, with associated symptoms of dyspnea, malaise, and fever. The patient was found to have multifocal nodular episcleritis and scleritis that was not responsive to topical steroids or systemic nonsteroidal anti-inflammatory treatment. Laboratory tests subsequently revealed evidence of systemic inflammation, and radiologic studies showed extensive mediastinal and cervical adenopathy. A cervical lymph node biopsy showed Reed-Sternberg cells and a chronic lymphocytic infiltrate consistent with nodular sclerosing Hodgkin's lymphoma. Histopathologic analysis of an episcleral nodule revealed a necrotizing granuloma with vasculitis. Systemic chemotherapy was instituted for the Hodgkin's disease; this therapy abolished the nodular scleritis. CONCLUSIONS: This case raises the possibility of concurrent undiagnosed systemic vasculitis with only an ocular manifestation with Hodgkin's lymphoma, either as a coincidence or as a paraneoplastic syndrome. Moreover, it emphasizes the important role of tissue biopsy in establishing diagnosis and directing treatment.     

Staphylococcal species in the oral cavity from patients in a regional burns unit

The aim of this study was to perform a quantitative and qualitative analysis of oral carriage of staphylococci in a range of oral specimens from patients admitted to a regional burns unit. The study recruited 28 patients and reasons for admittance were: burns (46%), skin grafting (39%), lacerations (7%), scalding (4%) and necrotizing fasciitis (4%). No patient had smoke inhalation injuries or trauma to the oro-pharynx. There were five patients from whom methicillin-sensitive S. aureus (MRSA) could be detected in oral specimens. For three patients only the wound and oral specimens were positive for MRSA. In one patient only the oral specimens were positive for MRSA. There were five patients from whom methicillin-sensitive S. aureus (MRSA) could be detected in the oral specimens. In one patient only the oral specimens were positive for MSSA. Staphylococci could be recovered from the dental plaque, denture and toothbrush specimens with a mean count of 1.1 x 10(4)cfu/mL (range 20-5.3 x 10(4)), 5.4 x 10(3) (range 40-2.1 x 10(4)) and 264 cfu/mL (range 20-500), respectively. Both MSSA and MRSA could be recovered from these specimen types. In one patient only the toothbrush was positive for MRSA and all other oral specimens were negative. This study suggests that staphylococci are not infrequent colonizers of the oral cavity, and that this site may serve as a potential reservoir for transmission to other body sites.     

Molecular and functional characterization of the extracellular calcium-sensing receptor in human colon cancer cells

Presence of a functional extracellular calcium-sensing receptor (CaR) is of particular relevance for the growth-inhibitory action of Ca2+ on human colon carcinoma cells. In order to detect CaR gene alterations that may have occurred during the tumorigenic process, we applied Southern blot, DNA sequence, and RT-PCR analysis to DNA from normal human colon mucosa and from cancerous lesions of different grading, as well as from primary cultured and established colonic carcinoma cell lines (e.g., Caco-2). No evidence was obtained for mutations or other sequence alterations in the CaR gene in any of the colon carcinoma cells analyzed. Only a differential expression of two splice variants of the CaR gene, which are generated by usage of different promoters in the 5'-untranslated region, was detected in colon carcinomas of different grade. From Western blot analysis a tendency towards lower CaR protein levels in carcinoma cells in parallel with tumor progression became apparent. Activation of the CaR by extracellular Ca2+ or by specific receptor agonists resulted in substantial growth inhibition in Caco-2 cells. Activation of the CaR was transduced into inhibition of phospholipase A2-mediated arachidonic acid formation, but also into increased production of cAMP and IP3. This provides evidence for a cell type-specific function of the CaR in human colonocytes. We conclude that neoplastic colon epithelial cells can respond to antimitogenic signals generated by activation of the CaR as long as they express sufficient amounts of the CaR protein. This provides a rationale for the use of calcium in chemoprevention of colon tumor development.     

Quantitative trait loci for hypercalciuria in a rat model of kidney stone disease

Hypercalciuria is the most common risk factor for kidney stones and has a recognized familial component. The genetic hypercalciuric stone-forming (GHS) rat is an animal model that closely resembles human idiopathic hypercalciuria, with excessive intestinal calcium absorption, increased bone resorption, and impaired renal calcium reabsorption; overexpression of the vitamin D receptor (VDR) in target tissues; and calcium nephrolithiasis. For identifying genetic loci that contribute to hypercalciuria in the GHS rat, an F2 generation of 156 rats bred from GHS female rats and normocalciuric WKY male rats was studied. The calcium excretion was six- to eightfold higher in the GHS female than in the WKY male progenitors. Selective genotyping of those F2 rats with the highest 30% and lowest 30% rates of calcium excretion was performed, scoring 98 markers with a mean interval of 23 cM across all 20 autosomes and the X chromosome. With the use of strict criteria for significance, significant linkage was found between hypercalciuria and a region of chromosome 1 at D1Rat169 (LOD, 2.91). Suggestive linkage to regions of chromosomes 4, 7, 10, and 14 was found. The proportion of phenotypic variance contributed by the region on chromosome 1, with appropriate adjustments, was estimated to be 7%. Candidate genes encoding the VDR and the calcium-sensing receptor were localized to regions on rat chromosomes 7 and 11, respectively, but the suggestive quantitative trait locus on chromosome 7 was not in the region of the VDR gene locus. Identification of genes that contribute to hypercalciuria in this animal model should prove valuable in understanding idiopathic hypercalciuria and kidney stone disease in humans.