First Report of Pepino mosaic virus Infecting Tomato in South Africa.

Pepino mosaic virus (PepMV) (genus Potexvirus) is a highly infectious virus that is responsible for significant losses in yield of tomato fruit (Solanum lycopersicum) across Europe, Asia, and the Americas in the last decade (1). During the winter growing season of 2008, uneven discoloration of tomato fruit from farms in Limpopo Province, South Africa, was detected at the Pretoria fresh produce market. Twenty fruit were randomly selected from five different suppliers in this region and the 100 samples were batched into subsamples of five fruit. Leaves with suspect mosaic and bubbling symptoms were also detected from farms in Limpopo and were thus sampled. Leaf and fruit samples were tested by double antibody-sandwich (DAS)-ELISA (2) using polyclonal antibodies against PepMV (Agdia, Elkhart, IN) combined with appropriate positive and negative controls. Fruit samples from two of the suppliers, and all leaf samples tested, reacted strongly with PepMV antibodies. Inoculum was prepared from pooled DAS-ELISA-positive leaf samples and inoculated onto 10, 4-week-old, susceptible S. lycopersicum cv. Rooikhaki seedlings. After 3 weeks, all inoculated plants had developed characteristic PepMV symptoms (2) including leaf bubbling, distortion, and curled leaves. Older leaves developed yellow spots and light/dark green leaf mosaic while apical regions were stunted and branches were distorted to form 'nettle-head' symptoms. Fruit surfaces were marbled or displayed flaming and uneven discoloration. Leaves from symptomatic plants were sampled for confirmation of PepMV infection by DAS-ELISA and all samples reacted positively with PepMV antibodies. Total RNA was extracted from 500-µg replicates of pooled leaf samples from infected plants with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), and amplified by conventional two-step reverse-transcription-PCR using a PepMV-specific primer set: Ker 1 (2) and PepCP-R (4) for a 986-bp region, including the coat protein, of the PepMV genome. PCR products were cloned into pTZ57R/T vector (Fermentas, Vilnius, Lithuania [UAB]) and six clones were purified and sequenced using universal M13 primers (3). Phylogenetic analysis clustered the sequence with EU (European), LP (Peruvian), US1 (United States)/CH1 (Chilean) and US2/CH2 PepMV isolates. The PepMV isolate accessions for US2/CH2 (AY509927, FJ612601, EF408821, FJ212288, and DQ000985) were identified as the closest relatives based on 98 to 99% nucleotide similarity obtained using BLASTN. The coat protein sequence of the South African isolate was submitted to GenBank (Accession No. HQ872607). To our knowledge, this is the first confirmed report of PepMV in South Africa. Further studies are necessary to determine its incidence and spread in this country. The presence of PepMV signals the urgent need for adoption of appropriate phytosanitary measures to restrict the spread and impact of this virus. References: (1) I. M. Hanssen and B. P. H. J. Thomma. Mol. Plant Pathol. 11:179, 2010. (2) I. M. Hanssen et al. Plant Pathol. 58:450, 2009. (3) J. Messing. Method Enzymol. 101:20, 1983. (4) I. Pagán et al. Phytopathology 96:274, 2006.

The collagenous matrix of bovine predentine.

Predentine obtained from bovine teeth by microdissection was solubilized by cyanogen bromide cleavage. The electrophoretic mobility of the resultant peptides was established on polyacrylamide gel and the amino acid composition of several peptides was determined. The data clearly indicated that this collagen is entirely of the Type I genetic species. No differences were detected between the predentine and dentine collagens except that the mature tissue was more highly crosslinked. Nevertheless the amount of stable cross-link formed in the predentine was higher than expected for an immature tissue.

The solubilization of bone and dentin collagens by pepsin. Effect of cross-linkages and non-collagen components.

Bone and dentin collagen are less susceptible to solubilization by pepsin digestion then is skin collagen. Digestion at 4 degrees C for 72 h solubilized only 35.3% of bovine cortical bone and 5.6% of bovine dentin compared with nearly 100% dissolution of bovine skin. Sodium dodecyl sulfate-acrylamide gel electrophoresis and molecular sieve chromatography showed that, for bone and dentin, intact alpha chains and cross-linked aggregates of beta, gamma and higher weight remained intact after pepsin solubilization but lower molecular weight fragments also were prevalent indicating chain scission in helical regions. Electron microscopic examination of segment long spacing precipitates of the soluble collagens confirmed the presence of solubilized polymerized collagen. The principal reducible cross-link in both bone and dentin was the precursor of dihydroxylsinonorleucine and this cross-link was also present in the solubilized collagens. Small amounts of non-collagenous proteins and glycosaminoglycans of different compositions in dentin and bone resisted extraction before pepsin digestion. However, the differences in solubilization of the collagens have been related to differences in cross-linkage placement.

Active and inactive renin in anephric man: a comparison of molecular weight studies with normal human plasma and the effect of a specific monoclonal anti-renin antibody.

Measurements of active and inactive renin have been made in a group of six anephric patients and in 30 normal healthy volunteers. Plasma renin activity (PRA) was detected in all of the anephric patients but values were below the normal range. A trypsin-activatable totally inactive renin was present in the plasma of all six patients. In three patients, levels of inactive renin were greater than that found for the mean value of the control group. Gel filtration studies of anephric plasma revealed a degree of heterogeneity of molecular forms of active high molecular weight (HMW) renin. However, an active form of approximately 65,000 daltons was usually present. Inactive renin, partially purified from anephric plasma had an apparent molecular weight (MW) of 59,000 daltons. Affinity chromatography with H.77 Sepharose was used to purify active renin from anephric and normal plasma following treatment with trypsin. The increased PRA in plasma from both groups following trypsin was associated with a 56,000 daltons form indicating little, if any, change in MW upon activation. The active HMW forms and the activated inactive renin were all totally inhibited by a renin-specific monoclonal antibody raised against human kidney renin.

Studies of circulating parathyroid hormone following parathyroidectomy in renal osteodystrophy.

A study has been undertaken to document in detail some of the changes that occur following parathyroidectomy in chronic renal failure patients. In an attempt to predict more precisely the timing of the post-operative hypocalcaemia, serum parathyroid hormone (PTH) concentrations were measured in six patients with renal osteodystrophy undergoing subtotal parathyroidectomy [5] and total parathyroidectomy [1]. The values obtained were related to post-operative changes in serum calcium and albumin concentrations. The intact-PTH concentration in serum was shown to decline rapidly post-operatively while the C-terminal PTH followed a more gradual course. Neither total nor ionised serum calcium concentrations exhibited a correspondingly dramatic fall after parathyroidectomy and the serum albumin concentration remained unaltered.

Calcium metabolism following renal transplantation.

Abnormalities of calcium homeostasis are a recognized feature of end-stage renal disease. The treatment of choice is renal transplantation, but this does not always result in normalization of the biochemical profile. Persistent hypercalcaemia is well documented and our study was undertaken to investigate the status of the calcium regulating hormones in renal patients post-transplantation. Serum calcium, parathyroid hormone, 1,25-dihydroxyvitamin D (1,25(OH)2D) and osteocalcin concentrations were measured in post-transplant patients. Twenty per cent of the patients had subnormal 1,25(OH)2D concentrations while 55% had biochemical evidence of hyperparathyroidism but only 5% were hypercalcaemic. Time elapsed since transplantation was not correlated with any of the analytes investigated and there was no relationship between persistent impairment of renal function and abnormalities of calcium homeostasis.

A biochemical and clinical comparison of two commercially available creatine kinase iso-enzyme MB assay kits suitable for use in the routine medical laboratory.

Two methods for the measurement of plasma creatine kinase MB (CK-MB) activity were compared for analytical performance, cost, practicality, and diagnostic correlation with clinical and electrocardiographic findings in patients admitted to the coronary care unit of a district general hospital. The methods were column chromatography and immunoinhibition. Both methods were found acceptable, and the method to be adopted would depend on the staff arrangements and resources available in the laboratory.

Tuberculosis in renal failure: a high incidence in patients born in the Third World.

We report 11 cases of tuberculosis among patients with chronic renal failure being treated in our unit. All were born outside the United Kingdom, and represent 25% of the non-europid patients in the unit. This is about a seventy-fold increased incidence of tuberculosis in this group. Diagnosis is difficult and mortality high if the diagnosis is delayed. Culture of tissue biopsies is the most reliable investigation and chemotherapy should be commenced promptly or a therapeutic trial considered in doubtful cases. Chemoprophylaxis may be beneficial in patients from these ethnic groups or in any uremic patient with evidence of old tuberculosis. Three patients with advanced disease died despite antibiotic treatment, but in the remainder, triple therapy with normal doses of rifampicin, isoniazid and pyrazinamide proved safe and effective.

Biochemical characterization of guanidinium chloride-soluble dentine collagen from lathyritic-rat incisors.

alpha- and beta-Chains were isolated by sequential ion-exchange and gel-filtration chromatography of guanidinium chloride-soluble dentine collagen obtained from Tris/NaCl-extracted EDTA-demineralized lathyritic-rat incisors. The alpha-chains were identified as alpha 1 I and alpha 2 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and amino acid analysis of the intact chains and their CNBr peptides. The dentine alpha-chains exhibited higher lysine hydroxylation and phosphate content, but lower hydroxylysine glycosylation, than alpha-chains from skin. Increased lysine hydroxylation was observed in the helical sequences. The alpha 1 I/alpha 2 ratio was approx. 3:1, and was presumably due to the presence of (alpha 1 I)3 molecules along with (alpha 1 I)2 alpha 2 molecules as shown recently for neutral-salt-soluble dentine collagen [Wohllebe & Carmichael (1978) Eur. J. Biochem. 92, 183--188]. In the borohydride-reduced beta 11- and beta 12-chains from guanidinium chloride-soluble dentine collagen, the reduced cross-links hydroxylysinohydroxynorleucine and hydroxylysinonorleucine were present. A higher proportion of hydroxylysinonorleucine in the reduced beta 12-chain probably reflects differences in extent of hydroxylation of specific lysine residues of the alpha 1 I- and alpha 2-chains.

Type-I trimer and type-I collagen in neutral-salt-soluble lathyritic-rat dentine.

Triple-helical collagen molecules have been obtained from EDTA-demineralized lathyritic rat incisors by neutral buffer extraction. Component alpha chains, isolated by sequential ion-exchange and gel-filtration chromatography, were shown to be alpha1 I and alpha2 chains by cyanogen bromide peptide analysis. The alpha1 I:alpha2 chain ratio was approximately 3:1, which is greater than expected for type I collagen. The excess of alpha1 I chains over that required for type I collagen was due to the presence of type I trimer molecules. Fractional salt precipitation separated type I collagen from type I trimer. It is not known at present if type I trimer synthesis also occurs in normal rat tissues.

The composition of the insoluble collagenous matrix of bovine predentine.

Predentine obtained from bovine teeth by microdissection was extracted with EDTA and tris-NaCl solutions. The insoluble residue consisted mainly of collagen and resembled dentine collagen in overall amino acid composition. The residue differed in containing no detectable phosphoprotein, a much larger amount of collagen hexose and more non-collagenous glycoprotein, the neutral sugar composition of which was determined. Differences were also observed in the contents of reducible collagen cross-links. Half of the total phosphorus found in the predentine could not be accounted for solely by hydroxyapatite. The remainder was partly soluble and dialysable.

Detection and investigation of renal artery stenosis.

A retrospective analysis was done on 235 hypertensive patients undergoing renal arteriography. Of the 85 patients with renal artery stenosis 50 underwent 56 operations or angioplasties and have been followed up for at least a year. 41 (73%) of these procedures were curative or led to improved blood-pressure control. These results make it worthwhile identifying hypertensive patients with renal artery stenosis who may benefit from surgery or angioplasty. Vascular disease, epigastric bruit, and impaired renal function were commoner in the renal artery stenosis patients than in the 81 with normal arteriograms, but there were no features pathognomonic of stenosis. Intravenous urography had a sensitivity of 83% and a specificity of 69.5% in identifying renal artery stenosis; those for isotope renography were 90.5% and 38.5%, respectively. Divided renal vein renins did not predict the outcome of intervention. Arteriography should, if there are no contraindications to intervention, be the first and definitive investigation when renal artery stenosis is suspected--for instance, in hypertensive patients with accelerated or malignant hypertension, those whose blood pressure is poorly controlled by multiple therapy, and those who have had recent deterioration in blood-pressure control or renal function.