Polymerase chain reaction in diagnosis of toxoplasmosis of the central nervous system: effect of methods for isolating DNA from samples of cerebrospinal fluid on results of the reaction

We examined influence of the method of isolation of DNA from cerebrospinal fluid samples on results of PCR in the diagnosis of toxoplasmosis of the central nervous system. Three different protocols of DNA isolation were used for DNA extraction from 360 samples made of cerebrospinal fluid spiked with tachyzoites of Toxoplasma gondii: thermic, enzymatic and enzymatic-filtering. Purified DNA samples were tested by PCR with primers T15 and T16 designed for the B1 gene of the parasite. Enzymatic method of DNA isolation appeared most effective allowing detection of T. gondii DNA in 50% of samples containing single parasite cell.     

Synthesis and biological activity of N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid

2-Deamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a potent inhibitor of thymidylate synthase. Its analogue, N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid, containing p-aminophenylacetic acid residue substituting p-aminobenzoic acid residue, was synthesized. The new analogue exhibited a moderately potent thymidylate synthase inhibition, of linear mixed type vs. the cofactor, N(5,10)-methylenetetrahydrofolate. The Ki value of 0.34 microM, determined with a purified recombinant rat hepatoma enzyme, was about 30-fold higher than that reported for inhibition of thymidylate synthase from mouse leukemia L1210 cells by ICI 198583 (Hughes et al., 1990, J. Med. Chem. 33, 3060). Growth of mouse leukemia L5178Y cells was inhibited by the analogue (IC50 = 1.26 mM) 180-fold weaker than by ICI 198583 (IC50 = 6.9 microM).     

AmfS, an extracellular peptidic morphogen in Streptomyces griseus

The amf gene cluster was previously identified as a regulator for the onset of aerial-mycelium formation in Streptomyces griseus. The nucleotide sequences of amf and its counterparts in other species revealed a conserved gene organization consisting of five open reading frames. A nonsense mutation in amfS, encoding a 43-amino-acid peptide, caused significant blocking of aerial-mycelium formation and streptomycin production, suggesting its role as a regulatory molecule. Extracellular-complementation tests for the aerial-mycelium-deficient phenotype of the amfS mutant demonstrated that AmfS was secreted by the wild-type strain. A null mutation in amfBA, encoding HlyB-like membrane translocators, abolished the extracellular AmfS activity without affecting the wild-type morphology, which suggests that AmfBA is involved not in production but in export of AmfS. A synthetic C-terminal octapeptide partially induced aerial-mycelium formation in the amfS mutant, which suggests that an AmfS derivative, but not AmfS itself, serves as an extracellular morphogen.     

Release of salbutamol sulphate and ketoprofen enantiomers from matrices containing HPMC and cellulose derivatives

We studied the release of salbutamol and ketoprofen enantiomers from HPMC K100M matrices containing two types of cellulose derivatives: cellulose tris (3,5-dimethylphenylcarbamate) and cellulose tris (2,3-dichlorophenylcarbamate), chiral excipients used as stationary phases for liquid chromatography. These matrices provided an extended release of both drugs. Ketoprofen release from formulations elaborated with cellulose tris (2,3-dichlorophenylcarbamate) was by anomalous transport, because the value of n (release exponent of the diffusion equation) ranged between 0.60-0.68, whereas for all other formulations the value of exponent n ranged from 0.50-0.54. The drug thus diffuses through the matrix and is released following a quasi-Fickian diffusion mechanism (stereoselective process). The matrices preferentially retained R-salbutamol and S-ketoprofen and cellulose tris (3,5-dimethylphenylcarbamate) showed more capacity of chiral discrimination for both drugs than cellulose tris (2,3-dichlorophenylcarbamate). Moreover, we observed that stereoselectivity is dependent on the amount of chiral excipient in the formulation. Diffusion tests confirmed the chiral interaction between drugs and cellulose derivatives observed in the dissolution assays except for matrices elaborated with ketoprofen and cellulose tris (2,3-dichlorophenylcarbamate), where the low stereoselectivity observed with the matrices is due to the presence of HPMC K100M. We conclude that the inclusion of these cellulose derivatives in HPMC matrices does not result in a relevant stereoselectivity with respect to the two drugs studied.     

Influence of the red fox (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>, Linnaeus 1758) on the distribution and number of breeding birds in an intensively used farmland

This study tested the hypothesis that small birds at their nest sites avoid areas around dens of the red fox (Vulpes vulpes, Linneaus 1758) in an intensively used farmland. Birds were counted at 18 points (radius 100m) located near dens, as well as at 18 control points that were located at least 600m away from the nearest den. These two types of points did not differ with respect to the number of recorded bird species. However, a negative effect of the proximity of fox dens on the total density of the bird community was observed. This effect was also recorded for the most abundant bird species, the skylark (Alauda arvensis, Linneaus 1758). In agreement with our expectations, these results indicate a negative impact of fox presence on a breeding bird community in an open farmland.     

Determination of the absolute configuration of (+)-2,7(14),10-bisabolatrien-1-ol-4-one from Japanese cedar,Cryptomeria japonica

The absolute configuration of (+)-2,7(14),10-bisabolatrien-1-ol-4-one, a peculiar sesquiterpenol in the Japanese cedar, Cryptomeria japonica, was determined as (1S,6R)-2,7(14),10-bisabolatrien-1-ol-4-one by comparing the specific rotation values of cryptomeriones respectively converted from (+)-2,7(14),10-bisabolatrien-1-ol-4-one and synthesized from (R)-(-)-carvone.     

Differential recognition of tyrosine-based basolateral signals by AP-1B subunit mu1B in polarized epithelial cells

To investigate the importance of tyrosine recognition by the AP-1B clathrin adaptor subunit mu1B for basolateral sorting of integral membrane proteins in polarized epithelial cells, we have produced and characterized a mutant form of mu1B. The mutant (M-mu1B) contains alanine substitutions of each of the four conserved residues, which in the AP-2 adaptor subunit micro2 are critical for interacting with tyrosine-based endocytosis signals. We show M-mu1B is defective for tyrosine binding in vitro, but is nevertheless incorporated into AP-1 complexes in transfected cells. Using LLC-PK1 cells expressing either wild type or M-mu1B, we find that there is inefficient basolateral expression of membrane proteins whose basolateral targeting signals share critical tyrosines with signals for endocytosis. In contrast, membrane proteins whose basolateral targeting signals are distinct from their endocytosis signals (transferrin and low-density lipoprotein receptors) accumulate at the basolateral domain normally, although in a manner that is strictly dependent on mu1B or M-mu1B expression. Our results suggest that mu1B interacts with different classes of basolateral targeting signals in distinct ways.     

Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF, identical to prokineticin 1) is a novel peptide recently identified as a selective mitogen for endocrine gland endothelial cells. The present study demonstrates that EG-VEGF/prokineticin 1 and a peptide closely related to EG-VEGF, prokineticin 2, are cognate ligands of two orphan G-protein-coupled receptors designated ZAQ (=EG-VEGF/PK-R1) and I5E (=EG-VEGF/PK-R2). EG-VEGF/prokineticin 1 and prokineticin 2 induced a transient increase in intracellular calcium ion concentration ([Ca(2+)](i)) with nanomolar potency in Chinese hamster ovary (CHO) cells expressing EG-VEGF/PK-R1 and -R2 and bind to these cells with high affinity and with different receptor selectivity. EG-VEGF/prokineticins provoke rapid phosphorylation of p44/42 MAP kinase and DNA synthesis in the bovine adrenal capillary endothelial cells (BACE). The mRNAs of both EG-VEGF/PK-R1 and -R2 were expressed in BACE. The identification of the receptors for EG-VEGF/prokineticins may provide a novel molecular basis for the regulation of angiogenesis in endocrine glands.     

Enhancement of bone repair with a helper-dependent adenoviral transfer of bone morphogenetic protein-2

Regional gene therapy, which involves the delivery of growth factors to a specific anatomic site, has the potential to enhance bone formation in clinical application. Helper-dependent adenoviral vectors, which have deleted all of the viral coding regions, have been shown to be safe and highly efficient with long-lasting transgene expression. In this study, we constructed a helper-dependent adenoviral vector producing bone morphogenetic protein-2 (AdHDBMP-2). The AdHDBMP-2 increased the alkaline phosphatase activity of W-20-17 cells in vitro. In addition, when AdHDBMP-2 infected rat bone marrow cells were implanted into the hindlimbs of SCID mice, orthotopic bone formation was shown at 2 weeks. To our knowledge, this is the first study to demonstrate bone formation with the helper-dependent adenoviral vector with the BMP-2 expression cassette. This type of gene therapy vector could prove to be highly useful for bone augmentation in patients with bone loss associated with trauma, revision total joint arthroplasty, or cancer.