Pharmacological strategies for protection of extrahepatic islet transplantation.

The safety and effectiveness of islet transplantation has been proven through world-wide trials. However, acute and chronic islet loss has hindered the ultimate objective of becoming a widely used treatment option for type 1 diabetes. A large islet loss is attributed, in part, to the liver being a less-than-optimal site for transplantation. Over half of the transplanted islets are destroyed shortly after transplantation due to direct exposure to blood and non-specific inflammation. Successfully engrafted islets are continuously exposed to the liver micro-environment, a unique immune system, low oxygen tension, toxins and high glucose, which is toxic to islets, leading to premature islet dysfunction/death. Investigations have continued to search for alternate sites to transplant islets that provide a better environment for prolonged function and survival. This article gathers courses and conditions that lead to islet loss, from organ procurement through islet transplantation, with special emphasis on hypoxia, oxidative stress, and antigen non-specific inflammation, and reviews strategies using pharmacological agents that have shown effectiveness in protecting islets, including a new treatment approach utilizing siRNA. Pharmacological agents that support islet survival and promote ß-cell proliferation are also included. Treatment of donor pancreata and/or islets with these agents should increase the effectiveness of islets transplanted into extrahepatic sites. Furthermore, the development of methods designed to release these agents over an extended period, will further increase their efficacy. This requires the combined efforts of both islet transplant biologists and bioengineers.

Testing combinations of protease inhibitor and preservation solution to improve islet quality and yield.

UNLABELLED: Pancreas preservation using an oxygenated two-layer method (TLM) has been reported to improve islet yields, as has supplementation of Liberase with Pefabloc. We hypothesized that using both TLM and Pefabloc could enhance islet yield as compared with preservation in University of Wisconsin (UW) or Histidine-Tryptophan Ketoglutarate (HTK) solution. METHODS: Ninety-eight pancreata with no significant differences of age, body mass index, or cold ischemia time preserved randomly with UW (n = 40), TLM (n = 48), or HTK (n = 10) were processed with (n = 36) or without (n = 66) Pefabloc. RESULTS: The total islet equivalent (IEQ) from TLM-preserved pancreata processed with Pefabloc (n = 12) showed lower yields versus those processed without Pefabloc (n = 36): 216,120 +/- 27,906 vs. 301,427 +/- 21,447 IEQ (P < .05). Islets from 1 of 12 (8.33%) pancreata processed with Pefabloc in TLM were transplanted, in contrast with 15/36 TLM (41.67%) pancreata processed without it. Islet yields were not significantly different among pancreata preserved in UW and processed with Pefabloc (n = 17) versus without Pefabloc (n = 23): 342,693 +/- 45,588 versus 266,609 +/- 29,006 IEQ (P = .149). The number of transplants from UW-preserved pancreata was 3/17 (17.65%) when processed with Pefabloc and 4/23 (17.39%) without. Among the HTK group, there was no significant difference in islet yields between pancreata processed with (n = 7) versus without Pefabloc (n = 3): 248,227 +/- 65,294 versus 483,555 +/- 144,070 IEQ (P = .118). CONCLUSIONS: Pefabloc showed no benefit to improve islet yields. Pancreata preserved in TLM provided better transplant quality islets when processed in the absence of Pefabloc.

Human Ca2+/calmodulin-dependent phosphodiesterase PDE1A: novel splice variants, their specific expression, genomic organization, and chromosomal localization.

We report here the identification of novel human PDE1A splice variants, their tissue distribution patterns, genomic structure, and chromosomal localization of the gene. We identified one N-terminus (N3) and one C-terminus (C3) by cDNA library screening and dbEST database search. These N- and C-termini, including the reported N-termini (N1 and N2) and C-termini (C1 and C2), combined to generate nine different PDE1A cDNAs. N1 and N2 are similar to the 5' ends of the bovine PDE1A proteins of 61 kDa and 59 kDa, respectively, and C1 and C2 are the 3' ends of the reported human PDE1A variants. The results of PCR and Southern blot analysis show that nine PDE1A splice variants exhibit distinctive tissue distribution patterns by the difference of the N-terminus. PDE1As with N2 were widely expressed in various tissues, mainly in the kidney, liver, and pancreas. On the other hand, PDE1As with N1 and N3 were particularly expressed at a high level in the brain and testis, respectively. These findings suggest that the distinct expression patterns among PDE1A variants depend on the several promoters situated upstream of exons encoding 5' ends of the variants. The PDE1A gene spans over 120 kb of genomic DNA, and consists of at least 17 exons and 16 introns. The PDE1A gene was located on human chromosome 2q32 by fluorescent in situ hybridization analysis.

Isolation and characterization of Na+/Ca2+ exchanger gene and splicing isoforms in mice.

The Na+/Ca2+ exchanger gene NCX1 is ubiquitously expressed in mammalian tissues, and encodes several isoforms through alternative RNA splicing. In this report, we describe the gene structure that gives rise to the multiple isoforms, and the tissue-specific expression of these isoforms in mice. The mouse NCX1 gene contains a cluster of six exons (A, B, C, D, E, and F) which encode a variable region in the large intracellular loop of the protein, as previously reported in rabbits and humans. Using reverse transcription-polymerase chain reaction (RT-PCR), expression of the isoforms was examined in several tissues. We also identified a novel splice variant, which originate from exons A, C, D, and F. These findings provide new insights into the significance of the large repertoire of NCX1 isoforms.

Isolation and characterization of two novel phosphodiesterase PDE11A variants showing unique structure and tissue-specific expression.

cDNAs encoding a novel phosphodiesterase, phosphodiesterase 11A (PDE11A), were isolated by a combination of reverse transcriptase-polymerase chain reaction using degenerate oligonucleotide primers and rapid amplification of cDNA ends. Their catalytic domain was identical to that of PDE11A1 (490 amino acids) reported during the course of this study. However, the cDNAs we isolated had N termini distinct from PDE11A1, indicating two novel N-terminal variants of PDE11A. PDE11A3 cDNA encoded a 684-amino acid protein including one complete and one incomplete GAF domain in the N-terminal region. PDE11A4 was composed of 934 amino acids including two complete GAF domains and shared 630 C-terminal amino acids with PDE11A3 but had a distinct N terminus containing the putative phosphorylation sites for cAMP- and cGMP-dependent protein kinases. PDE11A3 transcripts were specifically expressed in testis, whereas PDE11A4 transcripts were particularly abundant in prostate. Recombinant PDE11A4 expressed in COS-7 cells hydrolyzed cAMP and cGMP with K(m) values of 3.0 and 1.4 microm, respectively, and the V(max) value with cAMP was almost twice that with cGMP. Although PDE11A3 showed the same K(m) values as PDE11A4, the relative V(max) values of PDE11A3 were approximately one-sixth of those of PDE11A4. PDE11A4, but not PDE11A3, was phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro. Thus, the PDE11A gene undergoes tissue-specific alternative splicing that generates structurally and functionally distinct gene products.

Videoendoscopic laryngeal surgery.

This paper introduces videoendoscope-assisted laryngeal surgery with office-based equipment. With this technique, a patient is seated and the nose, pharynx, and larynx are topically anesthetized. A flexible videoendoscope with a light-sensitive charge-coupled device chip built into the tip is transnasally inserted by an assistant. Specially designed fine-tipped forceps and scalpels were developed for removal of laryngeal lesions. Videoendoscopic laryngeal surgery was undertaken in 114 cases of laryngeal lesions such as polyps, granuloma, and cancer. For benign vocal fold lesions, postoperative vocal function was shown to be improved on aerodynamic and perceptual analyses. For laryngeal tumors, biopsy of the lesion was easily undertaken. Videoendoscopic laryngeal surgery presents the following advantages. It is applicable to outpatients not requiring general anesthesia, it enables functional monitoring of the patient's voice and vocal fold during phonation, it allows for delicate manipulations with both hands, and it gives high-resolution images in comparison to conventional fiberscopy.

Inwardly rectifying potassium channels expressed by gene transfection into the green Monkey kidney cell line COS-1.

1. cDNA encoding a functional inwardly rectifying K+ (IRK1) channel was transfected into COS-1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage clamp experiments were done after 48-72 h. 2. Transfected cells showed inward rectification under whole-cell recording. The unitary current-voltage relationships in the inside-out configuration were almost linear in the absence of internal Mg2+ and polyamines, and the channel conductance averaged 34.1 +/- 2.0 pS (n = 15) at 23-26 degrees C. 3. Internal Mg2+ (2-10 microM) induced sublevels in the outward current with one-third and two-thirds of the unitary amplitude as in native channels. 4. To determine the subunit stoichiometry, we constructed tandem multimeric cDNAs consisting of the coding sequences of the IRK1 gene linked in a head-to-tail fashion. Cells transfected with tandem homomultimers up to octamers showed similar inwardly rectifying K+ channels. 5. A mutation (E138Q) eliminated the ionic conductance of the channel. Channels expressed by dimeric constructs containing a single mutant have a conductance ranging between 5 and 35 pS. 6. The E138Q mutant cotransfected with a wild-type dimeric, trimeric or tetrameric construct did not alter the channel conductance. The results do not support the notion that IRK1 channel proteins consist of four subunits.

Electrical pacing for dynamic treatment of unilateral vocal cord paralysis. Experiment in long-denervated muscle.

In order to explore the possibility of clinical application of laryngeal pacing as a treatment for unilateral vocal cord paralysis, we examined the reactivity of atrophic muscle to electrical stimulation in dogs whose recurrent laryngeal nerves were damaged by crushing, dissection followed by resuturing, or a 3-cm neurectomy. The threshold level to induce enough vocal cord adduction reached the maximum at 2 weeks after nerve injury, decreased with time, and never surpassed 7 V in each case. On the basis of results of these preliminary probings, laryngeal pacing was conducted on a dog 15 months after resection of the laryngeal nerve. Adduction of the paralyzed vocal cord for synchrony with the intact cord was achieved by 7 V of electrical stimulation of the thyroarytenoid muscle that was triggered by signals from the cricothyroid muscle.

Laryngeal pacing in unilateral vocal cord paralysis. An experimental study.

In an attempt to remobilize a unilaterally paralyzed vocal cord, experiments were made in five adult dogs. Adduction of the paralyzed vocal cord for phonation in synchrony with the intact cord was achieved by electrical stimulation of the adductor muscles that was triggered by the signals from the cricothyroid muscle. The difference in the level of action potentials of the cricothyroid muscle between during phonation and during respiration was used for setting the threshold so as to differentiate phonation from respiration. Nearly synchronous movement of the two vocal cords thereby attained during phonation greatly improved the voice quality that was hoarse without the paced stimulation.