Patrick C. D'Haese

The functional organization of the nopaline A. tumefaciens plasmid pTiC58

Abstract We have employed the P type plasmid RP4 and the transposons Tn1 and Tn7 to isolate insertion and deletion mutations in the nopaline Ti-plasmid pTiC58. Mutations that inactivate all known Ti phenotypes have been located on the physical map. Most importantly, we have positioned several regions involved in the determination of oncogenicity. They correspond to regions of homology between octopine and nopaline plasmids. One of these regions is part of the T-DNA, the Ti-plasmid DNA present in transformed plant cells. There are also segments of the T-DNA that are not essential for oncogenicity. One of these determines the biosynthesis of nopaline in tumors. The latter regions might allow insertion of foreign DNA that can then be introduced into plant cells.

Hypozincemia in depression

This study investigates serum levels of zinc in 48 unipolar depressed subjects (16 minor, 14 simple major and 18 melancholic subjects) and 32 normal volunteers, and the relationships between zincemia and plasma neopterin levels, postdexamethasone adrenocorticotropic hormone and cortisol values, and anorexia-weight loss. Serum zinc levels were significantly lower in major depressed subjects than in normal controls, whereas minor depressed subjects showed intermediate values. There were significant negative correlations between serum zinc, and severity of depression and plasma neopterin concentrations. No significant relationships between zincemia and either postdexamethasone hormone values or anorexia/weight loss were found. The findings suggest that hypozincemia in major depression may be related to activation of cell-mediated immunity in that illness.

Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii

Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.

Size, Location and Polarity of T-DNA-Encoded Transcripts in Nopaline Crown Gall Tumors - Common Transcripts in Octopine and Nopaline Tumors

Up to thirteen T-DNA-encoded, polyadenylated transcripts of different relative abundance were detected by Northern blot hybridization in the tobacco nopaline BT37 crown gall teratoma tissue. Their sizes range from 900 to 2,700 bases. The polarity of eight of the thirteen transcripts was assigned by hybridization of labeled RNA to single-stranded DNA fragments of the T-region obtained by cloning in an M13 vector. Both strands of the T-DNA are transcribed. Our data indicate that most, if not all, transcripts are generated via independent promoter and poly(A)-addition sites on the T-DNA. Comparison of T-DNA-encoded transcripts present in crown gall tumors showing teratoma-like growth (BT37) with those from an unorganized tumor line (W38C58) reveals that this difference in phenotype is accompanied by a difference in the expression of the T-DNA. T-DNA sequences common to both octopine and nopaline tumors encode at least five, and probably six, cross-hybridizing transcripts of the same size, location, polarity and function. These transcripts are involved in the process of plant tumor formation and maintenance.

Ultrastructural Analysis of Vascular Calcifications in Uremia

Accelerated intimal and medial calcification and sclerosis accompany the increased cardiovascular mortality of dialysis patients, but the pathomechanisms initiating microcalcifications of the media are largely unknown. In this study, we systematically investigated the ultrastructural properties of medial calcifications from patients with uremia. We collected iliac artery segments from 30 dialysis patients before kidney transplantation and studied them by radiography, microcomputed tomography, light microscopy, and transmission electron microscopy including electron energy loss spectrometry, energy dispersive spectroscopy, and electron diffraction. In addition, we performed synchrotron x-ray analyses and immunogold labeling to detect inhibitors of calcification. Von Kossa staining revealed calcification of 53% of the arteries. The diameter of these microcalcifications ranged from 20 to 500 nm, with a core-shell structure consisting of up to three layers (subshells). Many of the calcifications consisted of 2- to 10-nm nanocrystals and showed a hydroxyapatite and whitlockite crystalline structure and mineral phase. Immunogold labeling of calcification foci revealed the calcification inhibitors fetuin-A, osteopontin, and matrix gla protein. These observations suggest that uremic microcalcifications originate from nanocrystals, are chemically diverse, and intimately associate with proteinaceous inhibitors of calcification. Furthermore, considering the core-shell structure of the calcifications, apoptotic bodies or matrix vesicles may serve as a calcification nidus.

New occupational risk factors for chronic renal failure

Occupational pollutants may have a role in development of chronic renal failure (CRF). Most epidemiological studies have been cross-sectional, limited to certain renal diagnoses, or concentrated on early transient renal effects. In a case-control study, we examined the association between CRF and occupational exposure. Occupational histories of 272 men and women with CRF (of all types) were compared with those of 272 controls matched for age, sex, and region of residence. Exposures were assessed and degree and frequency were scored independently by three industrial hygienists unaware of case/control status. Significantly increased risks of CRF were found for exposure to lead (odds ratio 2.11 [95% CI 1.23-4.36]), copper (2.54 [1.16-5.53]), chromium (2.77 [1.21-6.33]), tin (3.72 [1.22-11.3]), mercury (5.13 [1.02-25.7]), welding fumes (2.06 [1.05-4.04]), silicon-containing compounds (2.51 [1.37-4.60]), grain dust (2.96 [1.24-7.04]), and oxygenated hydrocarbons (5.45 [1.84-16.2]). The frequencies of various occupational exposures were high among patients with diabetic nephropathy. This epidemiological study confirms previously identified risk factors and suggests that additional occupational exposures, for which there is some other experimental evidence, may be important in the development of CRF. The role of grain dust and the association between occupational exposure and diabetic nephropathy merit further investigation.

Does the Phosphate Binder Lanthanum Carbonate Affect Bone in Rats with Chronic Renal Failure

Adequate control of phosphate levels remains an important issue in patients with chronic renal failure (CRF). Lanthanum carbonate has been proposed as a new phosphate binder. Previous studies have shown a high phosphate binding capacity (>97%) and low gastrointestinal absorption of lanthanum, without serious toxic side effects in the presence of a normal renal function (NRF). Because of lanthanums physicochemical resemblance to calcium, the possible effects of it on bone have to be considered. The aim of this study was to investigate the effects of lanthanum carbonate on bone histology in NRF and CRF rats after oral administration of the compound with doses of 100, 500, or 1000 mg/kg per d for 12 wk. Bone histomorphometry showed that CRF animals that received vehicle developed secondary hyperparathyroidism. Urinalysis of lanthanum-loaded CRF animals showed a dose-dependent decrease in urinary phosphorus excretion, which was clearly more pronounced in the CRF groups compared with NRF animals. Phosphatemia, however, remained normal. Lanthanum carbonate administration induced a dose-dependent decrease in bone formation rate and increase in osteoid area in CRF animals. Three of seven animals in the CRF-1000 group and one of eight animals in the NRF-100 group were classified as having a mineralization defect. The number of cuboidal osteoblasts, however, was not affected, indicating that bone changes were not due to a toxic effect of lanthanum on the osteoblast. Furthermore, lanthanum concentrations in the femur remained low and did not correlate with histomorphometric parameters. These findings suggest that the administration of high doses of phosphate binder (1000 mg/kg per d lanthanum carbonate), in combination with decreased 25-(OH) vitamin D(3) in the uremic state, resulted in phosphate depletion and followed by an increased mobilization of phosphorus out of bone and/or reduced incorporation into bone. There was no evidence that lanthanum had a direct toxic effect on osteoblasts.

DNA Flux Across Genetic Barriers: The Crown Gall Phenomenon

Crown gall tumors are neoplastic proliferations induced by the soil bacterium Agrobacterium tumefaciens on wounded dicotyledonous plants (for recent reviews, see Kahl and Schell, 1982; Caplan et al, 1983; Depicker et al, 1983; Zambryski et al 1983a). In nature, the infection is often located at or near the junction of the root and the stem, the crown of the plant. Since the turn of the century, plant pathologists have been interested in this malignant transformation not only because of its agricultural consequences, but also for the unusual observation that a bacterium induces plant neoplasia.

Identification of sequences involved in the polyadenylation of higher plant nuclear transcripts using Agrobacterium T-DNA genes as models.

Sequences in the 3′‐untranslated region of two different octopine T‐DNA genes were analyzed with regard to their significance in polyadenylation. Poly(A) addition sites were localized precisely by S1 nuclease mapping with T‐DNA‐derived mRNAs isolated from tobacco. The gene encoding transcript 7’ contains two AATAAA hexanucleotides, respectively 119 bp and 170 bp downstream of the TAA stop codon. A single poly(A) site was mapped 24‐25 bp downstream of the first AATAAA. Further, we show that a mutant octopine synthase gene, which has lost part of its 3′‐untranslated region by deletion, is still active. This mutant gene terminates 19 bp upstream from the major wild‐type polyadenylation site. The deletion also removes the AATAAT signal preceding this site. The mutant octopine synthase gene contains a minimum of four different poly(A) sites. The most prominent of these sites is identical to the minor poly(A) site of the wild‐type gene, and is preceded by a sequence AATGAATATA. Three other sites are located within the adjacent plant DNA, giving rise to hybrid T‐DNA/plant DNA transcripts. The two most distal sites are probably dependent on a motif AATAAATAAA, found 29 bp away from the T‐DNA/plant DNA junction.

Lanthanum carbonate: a new phosphate binder

Purpose of reviewHyperphosphatemia remains an important aspect in the management of end-stage renal disease patients. Consequently, there is a need for new, efficient and well-tolerated phosphate binders. In this review, a new phosphate-binding drug, lanthanum carbonate, with an attractive preclinical efficacy profile compared with existing binders, is discussed. Although the available human efficacy and safety data over 3 years are encouraging, the consequences of low-level tissue deposition continue to be evaluated in longer-term clinical studies. Recent findingsLanthanum carbonate has been shown in clinical studies of up to 3 years to be an effective, well-tolerated phosphate binder. Reported adverse effects are mainly gastrointestinal, and do not differ from those of calcium carbonate. The gastrointestinal absorption of lanthanum is very low. Whereas the element is mainly excreted by the liver, renal excretion of the absorbed fraction is less than 2%. Bone lanthanum levels seen after long-term treatment (up to 4 years) seem not to affect the physicochemical process of mineralization, or osteoblast number/function. Preliminary data on the localization of lanthanum in bone have shown the element to be present at both active and quiescent sites of bone mineralization, independent of the type of renal osteodystrophy, a profile distinct from aluminum, as well as diffusely distributed throughout the mineralized bone matrix especially in rats/humans with an increased bone turnover. A randomized, comparator-controlled, parallel group, open-label study comparing lanthanum carbonate with calcium carbonate in dialysis patients showed no evolution towards low bone turnover in the lanthanum group, and no aluminum-like effect on bone. SummaryLanthanum carbonate seems to be a potent phosphate-binding drug, minimally absorbed from the gut, with an encouraging safety profile, and no deleterious effects on bone.