Equilibrative nucleoside transporter 3 depletion in ß-cells impairs mitochondrial function and promotes apoptosis: Relationship to pigmented hypertrichotic dermatosis with insulin-dependent diabetes.

Loss of function recessive mutations in the SLC29A3 gene that encodes human equilibrative nucleoside transporter 3 (ENT3) have been identified in patients with pigmented hypertrichotic dermatosis with insulin-dependent diabetes (PHID). ENT3 is a member of the equilibrative nucleoside transporter (ENT) family whose primary function is mediating transport of nucleosides and nucleobases. The aims of this study were to characterise ENT3 expression in islet ß-cells and identify the effects of its depletion on ß-cell mitochondrial activity and apoptosis. RT-PCR amplification identified ENT3 expression in human and mouse islets and exocrine pancreas, and in MIN6 ß-cells. Immunohistochemistry using human and mouse pancreas sections exhibited extensive ENT3 immunostaining of ß-cells, which was confirmed by co-staining with an anti-insulin antibody. In addition, exposure of dispersed human islet cells and MIN6 ß-cells to MitoTracker and an ENT3 antibody showed co-localisation of ENT3 to ß-cell mitochondria. Consistent with this, Western blot analysis confirmed enhanced ENT3 immunoreactivity in ß-cell mitochondria-enriched fractions. Furthermore, ENT3 depletion in ß-cells increased mitochondrial DNA content and promoted an energy crisis characterised by enhanced ATP-linked respiration and proton leak. Finally, inhibition of ENT3 activity by dypridamole and depletion of ENT3 by siRNA-induced knockdown resulted in increased caspase 3/7 activities in ß-cells. These observations demonstrate that ENT3 is predominantly expressed by islet ß-cells where it co-localises with mitochondria. Depletion of ENT3 causes mitochondrial dysfunction which is associated with enhanced ß-cell apoptosis. Thus, apoptotic loss of islet ß-cells may contribute to the occurrence of autoantibody-negative insulin-dependent diabetes in individuals with non-functional ENT3 mutations.

Elevated levels of renal and circulating Nop-7-associated 2 (NSA2) in rat and mouse models of diabetes, in mesangial cells in vitro and in patients with diabetic nephropathy.

AIMS/HYPOTHESIS: We previously found that Nop-7-associated 2 (NSA2), which is involved in ribosomal biogenesis in yeast and is a putative cell cycle regulator in mammalian cells, is elevated in the kidney of Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes. Here we tested the hypothesis that elevated NSA2 is involved in diabetic nephropathy (DN). METHODS: We examined Nsa2/NSA2 expression and NSA2 production in two rodent models of diabetes, in cultured renal glomerular cells, and in diabetic patients with and without nephropathy. Patients with nephropathy who had a history of albuminuria were further divided as responders (DN-NA; DN patients normoalbuminuric at the time of this study with a history of albuminuria) and non-responders (DN-A; diabetic nephropathy patients with albuminuria) to current treatment for albuminuria. RESULTS: Renal Nsa2/NSA2 mRNA increased in tandem with hyperglycaemia in GK rats, in a streptozotocin-induced mouse model of diabetes, and in human mesangial cells (HMCs) grown in high glucose (p < 0.05). In the mouse model of diabetes, hyperglycaemia resulted in increased Nsa2 expression and NSA2 levels in tubular and glomerular cells and in circulating cells; this increase was normalised by diabetes treatment. Circulating NSA2 mRNA levels were elevated in patients with DN independently of body weight (BMI), glycaemic (HbA(1c)) and haemodynamic (blood pressure) control, and showed an inverse correlation with renal function (GFR, p < 0.05). NSA2 levels were the only variable that showed a significant difference between patients with albuminuria (DN-A) compared with non-albuminuric patients (DN-NA) and diabetic controls (p < 0.05), this increase being independent of all other variables, including GFR. CONCLUSION: We show for the first time that renal and circulating NSA2/NSA2 levels are increased in hyperglycaemia in experimental models of diabetes, and that circulating NSA2 is elevated in DN patients with albuminuria. Further studies will be required to assess whether NSA2 plays a role in the pathogenesis of DN.

The prevalence of ocular complications in leprosy patients seen in the United Kingdom over a period of 21 years.

OBJECTIVE: The objective of this study was to determine the prevalence of ocular complications and blindness among leprosy patients presenting in the United Kingdom. METHODS: Observational prospective study. RESULTS: A total of 126 consecutive leprosy patients attending their ophthalmic visit were examined, out of which 18 patients were blind in one eye (14.3%) and five patients were blind in both the eyes (4.0%). Visual acuity of ≥ 6/18 was present in 96 patients (76.2%). A total of 65 patients (51.6%) had an ocular complication and 28 patients (22.2%) had a sight-threatening leprosy complication (lagophthalmos, severe corneal, or iris disease). The most common ocular complications were impaired lid closure (24 patients, 19%), impaired corneal sensation (20 patients, 15.9%), cataract (20 patients, 15.9%), mild corneal opacity (17 patients, 13.5%), and iris atrophy (17 patients, 13.5%). Impaired corneal sensation was associated with vision <6/18 (P<0.001, OR 13.5, 95% CI 5.14-35.44) and vision <3/60 (P=0.01 OR 6.42, 95% CI 2.15-19.15). Impaired lid closure was significantly associated with increasing age (P=0.029, OR 1.039, 95% CI 1.0-1.08) and vision <3/60 (P=0.03, OR 6.06, 95% CI 1.81-20.24). CONCLUSION: There is a significant rate of ocular complications and blindness seen in leprosy patients in the United Kingdom, and over one in five had a potentially sight-threatening ocular complication. Health professionals and all leprosy patients, including those cured of the disease, need to be aware that new eye symptoms and signs require prompt ophthalmology review to prevent avoidable blindness, due to the life-long risk of sight-threatening ocular complications.

Use of rapid diagnostic tests for diagnosis of malaria in the UK.

BACKGROUND: Malaria is currently diagnosed almost exclusively by microscopy in clinical laboratories. The introduction of rapid diagnostic tests (RDTs) may be useful in achieving rapid detection of malaria parasites, especially in situations where malaria is not often seen or where staff are inexperienced. AIM: To explore the use of RDT in UK laboratories. METHODS: The current use of RDTs was surveyed in UK laboratories subscribing to the United Kingdom National External Quality Assessment Scheme blood parasitology and haematology schemes. RESULTS: An overall survey response rate of 60.3% was seen. RDTs were found to be the preferred choice, either alone or in conjunction with microscopy in 31.2% of the samples examined during normal working hours and in 44.3% of the specimens examined on call. CONCLUSIONS: During on-call hours, the use of RDTs was observed to increase and RDTs changed the diagnosis in 12% of laboratories. No established protocol for RDT use was, however, observed in the UK. A protocol that needs to be validated in the laboratory setting is suggested.

An actin-mediated two-step mechanism is required for ventral enclosure of the C. elegans hypodermis.

The epiboly of the Caenorhabditis elegans hypodermis involves the bilateral spreading of a thin epithelial sheet from the dorsal side around the embryo to meet at the ventral midline in a process known as ventral enclosure. We present evidence that ventral enclosure occurs in two major steps. The initial migration of the hypodermis is led by a quartet of cells, which exhibit protrusive activity at their medial tips and are required to pull the hypodermis around the equator of the embryo. These cells display actin-rich filopodia and treatment with cytochalasin D immediately halts ventral enclosure, as does laser inactivation of all four cells. Once the quartet of cells has migrated around the equator of the embryo and approaches the ventral midline, the remainder of the leading edge becomes visible on the ventral surface and exhibits a localization of actin microfilaments along the free edges of the cells, forming an actin ring. Cytochalasin D and laser inactivation block ventral enclosure at this later stage as well and, based upon phalloidin staining, we propose that the second half of enclosure is dependent upon a purse string mechanism, in which the actin ring contracts and pulls together the edges of the hypodermal sheet at the ventral midline. The ventral cells then form junctions with their contralateral neighbors to complete ventral enclosure.

Isolation of diabetes-associated kidney genes using differential display.

Differential Display was used to isolate genes that show transcriptional changes in the kidney during the development of diabetes in the GK rat. Eight candidate diabetes-associated cDNA fragments, CDK1-8, were isolated and characterised. cDNA sequencing and subsequent database analysis revealed that CDK2, 4, 5 and 6 showed no significant sequence similarity to previously reported genes, suggesting that they represent novel genes, whereas CDK 1, 3, 7 and 8 showed significant similarity with rat lactate dehydrogenase, rat amiloride sensitive sodium channel, EST109013 and mouse ubiquitin-like protein respectively. The differential mRNA expression of CDK1-8 was confirmed using differential screening of slot blots. CDK1, 2, 4 and 8 mRNAs appeared to increase whereas CDK3, 5, 6 and 7 mRNAs decreased in the kidneys of GK rats with increasing hyperglycaemia. The altered renal mRNA expression of these genes in association with increased hyperglycemia in the GK rat suggest that they are candidates for a role in the development of diabetic nephropathy.

Molecular characterisation of the Xenopus laevis chaperonin gene Cctg.

By library screening and PCR we have obtained cDNA clones which encode the gamma subunit of the CCT chaperonin complex from Xenopus laevis. The gene (XlCctg), which encodes the CCT gamma subunit contains an open reading frame which codes for 547 amino acid residues (60 kDa) and the predicted amino acid sequence shares a high degree of sequence identity with other CCT gamma homologues. The XlCctg mRNA measures 2.1 kb and is expressed ubiquitously in all of the X. laevis tissues examined. The mRNA levels of XlCctg are significantly higher in the ovary compared with other tissues.

Drosophila melanogaster P1 genomic clone DS05563 contains the chaperonin-encoding gene Cctg.

We report the sequence analysis of a Drosophila melanogaster (Dm) P1 genomic clone (DS05563) which contains the gamma-chaperonin-encoding gene, Cctg. The (Hs) Cctg orthologue was found to share strong sequence identity with a 1603-bp region of DS05563, suggesting that Dm Cctg is located within this region. Detailed analysis has shown that Dm Cctg comprises four exons and is interrupted by three introns of 55, 85 and 66 bp. Dm Cctg encodes a predicted peptide of 545 amino acids (aa) (approx. 60 kDa). The predicted Dm CCT gamma aa sequence shares a high degree of sequence identity with gamma-orthologues from human (70%), mouse (70%), protozoa (60%) and yeast (60%), and also contains domains found in other chaperonins including bacterial GroEL, mitochondrial Hsp60 and plant Rubisco large subunit-binding protein. These data support the conclusion that the DS05563 clone contains the Dm Cctg gene.

Cloning, structure and mRNA expression of human Cctg, which encodes the chaperonin subunit CCT gamma.

We describe the cloning, DNA sequence analysis and mRNA expression analysis of human Cctg (HsCctg), a gene that encodes the gamma-subunit of the eukaryotic cytosolic 'chaperonin-containing TCP-1' (CCT). Partial clones representing the 3' region of HsCctg cDNA were isolated from a human kidney cDNA library, and the missing 5' region was amplified directly from human kidney cDNA. The Cctg mRNA transcript is expressed in numerous human and mouse tissues and, like Tcp-1/Ccta, Cctg mRNA is expressed at higher levels in mouse testis when compared with kidney and brain. Southern-blot analysis has also revealed the Cctg gene to be highly conserved in mouse, rat, sheep and frog. The 1901 bp HsCctg cDNA has a coding region of 1635 bp and encodes a predicted 60 kDa protein (544 amino acids). The predicted HsCCT gamma amino acid sequence shares a high degree of sequence similarity with gamma-subunits from the mouse Mus musculus (98% similarity), the yeast Saccharomyces cerevisiae (75% similarity) and the protozoan Tetrahymena pyriformis (76% similarity) as well as with other members of the TF55/TCP-1 family, such as human TCP-1/CCT alpha (55% similarity) and TCP-20/CCT zeta (54% similarity). HsCCT gamma also shares conserved domains previously identified in the TF55/TCP-1 family of chaperonins and more distantly related chaperonins such as GroEL and Hsp60.

Cloning and nucleotide sequence of a full length cDNA encoding ribosomal protein L27 from human fetal kidney.

Differential screening of a human fetal kidney cDNA library resulted in the isolation of D69, eventually renamed HumRPL27, which was expressed at higher levels in fetal kidney than in adult kidney. The 476 bp cDNA insert from HumRPL27 contains an open reading frame of 135 amino acids displaying 100% identity to rat RPL27 and chicken RPL27 predicted protein sequences although 64 and 38 silent base pairs changes respectively are found at the DNA level. In Northern blots, a 1.0 kb HumRPL27 mRNA transcript is expressed abundantly in all fetal tissues examined and at lower abundance in adult tissues. Southern analysis of HumRPL27 suggests the presence of multiple copies of the gene in human, rat, mouse and hamster DNA.

Parathyroid hormone related peptide gene expression in human fetal and adult heart.

OBJECTIVE: The aim was to investigate the expression of parathyroid hormone related peptide (PTHrP) gene in the human fetal and adult heart. METHODS: Molecular biological techniques were employed as well as immunocytochemistry and western blot analysis using rabbit polyclonal anti-PTHrP(1-34) and anti-PTHrP (56-86) on normal human fetal and adult heart tissues. Northern blot analysis of both normal human fetal and adult heart total RNA, using a human full length cDNA probe, and polymerase chain reaction analysis of normal human fetal and adult heart cDNAs with exon specific oligonucleotides were carried out. RESULTS: Positive staining was detected with both anti-PTHrP(1-34) and anti-PTHrP(56-86) in fetal heart at 12 weeks of gestation. In both fetal and adult hearts, multiple putative PTHrP proteins were observed with apparent molecular mass of 14-125 kDa. Multiple hybridising PTHrP mRNA isoforms (1.4, 2.1, 3.2, and 4.5 kb) were detected in both fetal and adult heart total RNAs. The fetal and adult heart cDNAs amplified from the cDNA libraries showed the presence of the 5' non-coding exon II and coding exons III-IV but not the 5' non-coding exon Ic. CONCLUSIONS: PTHrP is expressed in normal human fetal and adult hearts suggesting that it has a function as an endogenous modulator of the cardiovascular system.

Molecular genetics of Pseudomonas syringae pathovar pisi: plasmid involvement in cultivar-specific incompatibility.

A mutant (PF24) of the race 1 strain, 299A, of Pseudomonas syringae pv. pisi has been characterized in terms of its interactions with pea (Pisum sativum) cultivars. The mutant showed a changed reaction (avirulence to virulence) with a group of pea cultivars, including cvs. Belinda and Puget, previously thought to contain resistance genes R1 and R3. Avirulence towards cv. Puget was restored by transfer of any one of five cosmid clones from a race 3 (strain 870A) gene library to a rifampicin-resistant derivative of PF24. These observations were in agreement with a revised race-specific resistance genotype for Belinda and similar cultivars comprising a single resistance gene, R3. An incompatible interaction was observed between strain PF24 and cvs. Vinco (postulated to harbour race-specific resistance genes R1, R2, R3 and R5) and Hurst's Greenshaft (R4 and possibly R1), indicating that the mutant retains at least one avirulence gene (A1 or A1 and A4). Mutant PF24 showed loss of a cryptic plasmid (pAV212) compared with its progenitor, strain 299A. A subclone (pAV233) of one of the race 3 restoration clones showed strong hybridization with similar-sized digestion fragments in race 3 plasmid DNA, confirming the A3 gene to be plasmid-borne. Strong cross-hybridization was also observed with a single 3.27 kb EcoRI fragment of plasmid DNA present in strain 299A but absent from strain PF24. This is consistent with the corresponding A3 determinant being located on pAV212 in the race 1 strain 299A. The novel avirulence gene corresponding to A3 in strain 870A is provisionally designated avrPpi3.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone-related peptide in normal human fetal development.

Parathyroid hormone-related peptide (PTHrP) has been detected in fetal serum and amniotic fluid. Using a combination of immunocytochemistry and molecular biology we have detected the peptide and its mRNA in a variety of fetal tissues throughout gestation. Tissue-specific mRNA isoforms were observed, the pattern of hybridization of which changed throughout gestation. In addition, the intensity and pattern of immunocytochemical localization of the peptide was found to vary over the time-period studied (8-30 weeks). PTHrP is expressed by a variety of tumours associated with the syndrome of humoral hypercalcaemia of malignancy and probably accounts for the hypercalcaemia by virtue of its limited amino acid homology with parathyroid hormone. These data demonstrate for the first time that PTHrP, a tumour-related peptide, is expressed during normal human fetal development, and suggest the possibility that it may function to regulate fetal calcium balance and growth in utero.