Real-time PCR methods for independent quantitation of TTV and TLMV

There is considerable interest in the possible clinical effects of the human circoviruses TT virus (TTV) and TTV-like mini virus (TLMV). Most people appear to have at least one of these viruses replicating actively in their bodies, thus mere correlation of the presence of virus and disease states are probably less informative than a quantitative analysis of viraemia. Real-time PCR based methods, with either SYBR Green or TaqMan probe, designed to quantitate selectively TTV and TLMV are described. The suggested TaqMan-based protocols were suitable for quantitation of viruses in the range of 10(2)-10(9) copies/ml of sample; and proved, by sequencing of PCR products, to be specific for each of the two viruses.     

Complete Sequence of a 93.4-kb Contig from Chromosome 3 of Trypanosoma cruzi Containing a Strand-Switch Region

We have initiated large-scale sequencing of the third smallest chromosome of the CL Brener strain of Trypanosoma cruzi and we report here the complete sequence of a contig consisting of three cosmids. This contig covers 93.4 kb and has been found to contain 20–30 novel genes and several repeat elements, including a novel chromosome 3-specific 400-bp repeat sequence. The intergenic sequences were found to be rich in di- and trinucleotide repeats of varying lengths and also contained several known T. cruzi repeat elements. The sequence contains 29 open reading frames (ORFs) longer than 700 bp, the longest being 5157 bp, and a large number of shorter ORFs. Of the long ORFs, seven show homology to known genes in parasites and other organisms, whereas four ORFs were confirmed by sequencing of cDNA clones. Two shorter ORFs were confirmed by a database homology and a cDNA clone, respectively, and one RNA gene was identified. The identified genes include two copies of the gene for alanine-aminotransferase as well as genes for glucose-6-phosphate isomerase, protein kinases and phosphatases, and an ATP synthase subunit. An interesting feature of the sequence was that the genes appear to be organized in two long clusters containing multiple genes on the same strand. The two clusters are transcribed in opposite directions and they are separated by an ~20-kb long, relatively GC-rich sequence, that contains two large repetitive elements as well as a pseudogene for cruzipain and a gene for U2snRNA. It is likely that this strand switch region contains one or more regulatory and promoter regions. The reported sequence provides the first insight into the genome organization of T. cruzi and shows the potential of this approach for rapid identification of novel genes.     

Microcarrier cell culture: Vero cells on cytodex® 1

Summary All the necessary steps required for the cultivation of Vero cells on microcarriers are described. These procedures are used routinely in our laboratories for the growth of Vero cells on Cytodex 1. Consistent, high density, final yields of more than 10⁶ cells/ml of culture medium are obtained. The protocols described here can be modified for the growth of a variety of anchorage-dependent cell types. Culture parameters important for the successful exploitation of microcarrier cell culture technology are discussed.     

Manipulations in Maternal Environment Reverse Periodontitis in Genetically Predisposed Rats

The predisposition to develop periodontitis is partly genetically determined in humans as well as in animals. Here we demonstrate, however, that early manipulations in the maternal environment of an animal (rat) model of periodontitis can fully reverse the genetic predisposition to develop periodontitis at adult age.     

Characterization of the major secreted zinc metalloprotease- dependent glycerophospholipid:cholesterol acyltransferase, PlaC, of Legionella pneumophila

Legionella pneumophila, an intracellular pathogen causing a severe pneumonia, possesses distinct lipolytic activities which have not been completely assigned to specific enzymes so far. We cloned and characterized a gene, plaC, encoding a protein with high homology to PlaA, the major secreted lysophospholipase A of L. pneumophila and to other hydrolytic enzymes belonging to the GDSL family. Here we show that L. pneumophila plaC mutants possessed reduced phospholipase A and lysophospholipase A activities and lacked glycerophospholipid:cholesterol acyltransferase activity in their culture supernatants. The mutants' reduced phospholipase A and acyltransferase activities were complemented by reintroduction of an intact copy of plaC. Additionally, plaC conferred increased lysophospholipase A and glycerophospholipid:cholesterol acytransferase activities to recombinant Escherichia coli. Furthermore, PlaC was shown to be another candidate exported by the L. pneumophila type II secretion system and was activated by a factor present in the bacterial culture supernatant dependent on the zinc metalloprotease. Finally, the role of plaC in intracellular infection of Acanthamoeba castellanii and U937 macrophages with L. pneumophila was assessed, and plaC was found to be dispensable. Thus, L. pneumophila possesses another secreted lipolytic enzyme, a protein with acyltransferase, phospholipase A, and lysophospholipase A activities. This enzyme is distinguished from the previously characterized phospholipases A and lysophospholipases A by its capacity not only to cleave fatty acids from lipids but to transfer them to cholesterol. Cholesterol is an important compound of eukaryotic membranes, and an acyltransferase might be a tool for host cell modification to fit the needs of the bacterium.     

Intestinal fluid transport in the small intestine of normotensive and spontaneously hypertensive rats: the importance of enteric nerves, chloride and bicarbonate secretion

Fluid transport was studied in periarterially denervated jejunal segments of spontaneously hypertensive rats (SHR) of the Okamoto strain and as a control also in Wistar-Kyoto rats (WKR). In agreement with the findings of an earlier report a 'spontaneous' fluid secretion was observed in SHR whereas the intestinal segments of WKR absorbed fluid. The fluid secretion in SHR was inhibited by tetrodotoxin or lidocaine placed on the serosal surface of the intestinal segment under study. These observations confirm our earlier proposal that secretory nervous pathways in the enteric nervous system evoke the fluid secretion in SHR. In an attempt to analyse the cellular mechanisms that underlie the fluid secretion in SHR the animals were given loop diuretics in doses that evoked diuresis. No effect on intestinal fluid transport was seen in SHR or WKR. Furthermore, to study the importance of bicarbonate transport alkaline secretion was monitored with a pH-stat technique. In the initial part of the experiments the alkaline secretion in SHR and WKR was similar. In half of the SHR experiments alkaline secretion increased with time. This increase could be completely reversed with hexamethonium and atropine (only tested in three experiments). The time course of the alkaline and fluid secretion in SHR did not coincide, indicating that bicarbonate ion transport was not the major cause of fluid secretion in SHR. In agreement with this conclusion it was observed that acetazolamide (a blocker of carbonic anhydrase) did not influence rate of fluid transport in SHR or WKR.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Effects of “Immunosympathectomy” on Blood Pressure and Vascular “Reactivity” in Normal and Spontaneously Hypertensive Rats

Newborn litters of spontaneously hypertensive rats (SHR) and normotensive control rats (NCR) were identically treated with sympathetic nerve growth factor antiserum (Wellcome) which markedly interferes with adrenergic cardiovascular control (Zaimis 1967). Blood pressure, measured intermittently during 8 months, was in treated SHR (SHR_is) about 25 % higher than in NCR_is, their respective pressures being about 40 % and 25 % lower than those of sham-treated SHR and NCR.–The hindquarters of one SHR_is, or NCR_is, were then perfused at constant flow in parallel with those of ordinary NCR. Starting from maximal vasodilatation, resistance increases were induced by graded noradrenaline (NA) infusions, from “threshold” to maximal pressor responses. Compared to NCR_is, SHR_is showed an increased resistance at maximal dilatation, an increased slope of the NA dose-response curve and an increased maximal pressor response, while their NA “thresholds” did not differ significantly. Thus, the structurally determined hemodynamic differences between ordinary SHR and NCR (Folkow et al. 1970 b) characterize also SHR_is and NCR_is, though to a reduced extent. Even when comparing SHR_is with ordinary- NCR, which exhibited similar “resting” pressures, these differences partly remain, suggesting that the SHR resistance vessels might, for genetic reasons, be more prone to adapt structurally to pressure loads than those of NCR.     

Trisomy 7 in nonneoplastic kidney tissue cultured with and without epidermal growth factor.

A 7-month-old infant with Klinefelter's syndrome was diagnosed as having acute monoblastic leukemia (AMoL). Chromosome studies of bone marrow at diagnosis showed the karyotype 46,XXY,-Y,t(10;11)(p13;q14). This is the first report of M5A leukemia associated with Klinefelter's syndrome.