Efficient infection of tumor endothelial cells by a capsid-modified adenovirus.

Targeted antiangiogenic gene therapy is an attractive approach to treat metastatic cancer. However, the relative paucity of the receptors of the commonly used adenovirus serotype 5 in endothelial cells as compared with liver cells undermines the use of this vector for targeting the endothelial cells in tumors. To overcome this problem, we analyzed the ability of a hybrid Ad5/35 virus, where the serotype 5 fiber has been replaced with the fiber from serotype 35, to target tumor vasculature. Infection of human umbilical vein endothelial cells (HUVECs) with Ad5/35 at MOI 120 infected 100% of cells. In contrast, infection with Ad5 at the same MOI infected only 10% HUVECs. Ad5/35 was even more effective in transducing human aortic endothelial cells (HAECs), as infection with Ad5/35 at MOI 3.6 was sufficient to transduce 95% of cells. Gene expression analyses demonstrated that infection of HUVECs and HAECs with Ad5/35 resulted in between 1 and 3 orders of magnitude higher gene expression than infection with Ad5. Furthermore, various liver-derived cells were less infectable with Ad5/35 than Ad5, indicating a favorable toxicity profile for this virus. In a rat colon carcinoma tumor model, Ad5 was located mainly in the liver parenchyma after hepatic artery administration. In contrast, Ad5/35 was found only in the angiogenesis-rich border region of the tumor. Double immunostaining revealed that Ad5/35 colocalized with CD31 and Flk-1 positive endothelial cells. These results indicate that Ad5/35 may be useful in anticancer strategies targeting tumor endothelial cells.

Interaction of insulin-like growth factor (IGF)-I and -II with IGF binding protein-2: mapping the binding surfaces by nuclear magnetic resonance.

The interaction of IGF binding protein-2 (IGFBP-2) with IGF-I and -II has been investigated in solution using nuclear magnetic resonance (NMR) spectroscopy. Chemical shift perturbations in 15N- and 2H/15N-labelled IGF-I or -II upon binding to unlabelled thioredoxin-tagged bovine IGFBP-2 (Trx(1-279)IGFBP-2) have been monitored to identify residues involved directly in the binding interaction as well as any affected by conformational changes associated with the interaction. A key step in obtaining high-quality spectra of the complexes was the use of transverse relaxation optimised spectroscopy (TROSY) methods with partially deuterated ligands. Indeed, because the effects of conformational averaging and aggregation are eliminated in IGF-I and -II bound to IGFBP-2, the spectra of the complexes are actually superior to those of the free ligands. Comparison of our results with the crystal structure of the complex between IGF-I and an N-terminal fragment of IGFBP-5 allowed identification of those residues perturbed by the C-domain of IGFBP-2. Other perturbations, such as those of Gly 19 and Asp 20 of IGF-I (and the corresponding residues in IGF-II) - which are located in a reverse turn linking N-domain and C-domain interactive surfaces - are due to local conformational changes in the IGF-I and -II. Our results confirm that the C-domain of IGFBP-2 plays a key role in binding regions of IGF-I and -II that are also involved in binding to the type-1 IGF receptor and thereby blocking ligand binding to this receptor.

Isolation of dopamine D(2) receptor (D (2)R) promoters in Mugil cephalus.

This paper reports the isolation of two putative D(2)R promoters from grey mullet, one 5'flanking and the other an intronic sequence immediately upstream of the first coding exon. Promoter activity of the intronic sequence was confirmed in vitro through functional analysis using luciferase as reporter gene. The functional characteristics of the region flanking the 5'UTR is currently under investigation.

BIAcore analysis of bovine insulin-like growth factor (IGF)-binding protein-2 identifies major IGF binding site determinants in both the amino- and carboxyl-terminal domains.

In the absence of a complete tertiary structure to define the molecular basis of the high affinity binding interaction between insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs), we have investigated binding of IGFs by discrete amino-terminal domains (amino acid residues 1-93, 1-104, 1-132, and 1-185) and carboxyl-terminal domains (amino acid residues 96-279, 136-279, and 182-284) of bovine IGFBP-2 (bIGFBP-2). Both halves of bIGFBP-2 bound IGF-I and IGF-II in BIAcore studies, albeit with different affinities ((1-132)IGFBP-2, K(D) = 36.3 and 51.8 nm; (136-279)IGFBP-2HIS, K(D) = 23.8 and 16.3 nm, respectively). The amino-terminal half appears to contain components responsible for fast association. In contrast, IGF binding by the carboxyl-terminal fragment results in a more stable complex as reflected by its K(D). Furthermore, des(1-3)IGF-I and des(1-6)IGF-II exhibited reduced binding affinity to (1-279)IGFBP-2HIS, (1-132)IGFBP-2, and (136-279)IGFBP-2HIS biosensor surfaces compared with wild-type IGF. A charge reversal at positions 3 and 6 of IGF-I and IGF-II, respectively, affects binding interactions with the amino-terminal fragment and full-length bIGFBP-2 but not the carboxyl-terminal fragment.

Single DNA sequence common to all chlamydial species employed for PCR detection of these organisms.

Chlamydial infection is responsible for a wide spectrum of diseases of the eye, genitourinary tract, and lung. This group of organisms is also implicated in the pathogenesis of coronary artery disease as well as arthritis. Since cross-species infection is widely reported (though probably underestimated), it is an advantage to have a rapid and reliable method to detect all forms of chlamydiae in patient samples. We have identified a 160/163-bp DNA fragment in Chlamydia which is highly conserved in all chlamydial species. A polymerase chain reaction method based on this sequence has been developed to detect, in clinical samples, chlamydiae which have been shown to be positive by fluorescent-staining immunoassay; this method can be utilized in combination with restriction endonuclease cleavage to identify individual chlamydial species. Thus we have developed a sensitive and rapid detection method and have used it on samples from patients with respiratory and genital infections.

Phylogeographic differentiation in the mitochondrial control region in the koala, Phascolarctos cinereus (Goldfuss 1817).

The koala, Phascolarctos cinereus, is a geographically widespread species endemic to Australia, with three currently recognized subspecies: P.c. adustus, P.c. cinereus, and P.c. victor. Intraspecific variation in the mitochondrial DNA (mtDNA) control region was examined in over 200 animals from 16 representative populations throughout the species' range. Eighteen different haplotypes were defined in the approximately 860 bp mtDNA control region, as determined by heteroduplex analysis/temperature gradient gel electrophoresis (HDA/TGGE). Any single population typically possessed only one or two haplotypes yielding an average within-population haplotypic diversity of 0.180 +/- 0.003, and nucleotide diversity of 0.16%. Overall, mtDNA control region sequence diversity between populations averaged 0.67%, and ranged from 0% to 1.56%. Nucleotide divergence between populations averaged 0.51%, and ranged from 0% to 1.53%. Neighbour-joining methods revealed limited phylogenetic distinction between geographically distant populations of koalas, and tentative support for a single evolutionarily significant unit (ESU). This is consistent with previous suggestions that the morphological differences formalized by subspecific taxonomy may be interpreted as clinal variation. Significant differentiation in mtDNA-haplotype frequencies between localities suggested that little gene flow currently exists among populations. When combined with microsatellite analysis, which has revealed substantial differentiation among koala populations, we conclude that the appropriate short-term management unit (MU) for koalas is the local population.

Morphology of the thyroid in coastal and noncoastal populations of the koala (Phascolarctos cinereus) in Queensland.

The gross morphology, histology, and ultrastructure of the thyroid gland of the koala, Phascolarctos cinereus, is described. Generally, the glands were found to contain large-diameter follicles in association with an epithelium of low height. Morphometric analysis demonstrated a high relative thyroid weight (0.3 +/- 0.2 g/kg) for koalas compared with the 0.07-0.24 g/kg typical of eutherian mammals and 0.03-0.1 g/kg found in other marsupials. The relative thyroid weight of glands (0.33 +/- 0.21 g/kg) from the coastal population (less than 28 km from the coastline) was found to be significantly higher (ANOVA: P = 0.007, significant at the 1% level) than that for glands (0.21 +/- 0.11 g/kg) of noncoastal koalas (greater than 28 km from the coastline). Follicle size was positively correlated (at the 0.1% level) with relative thyroid weight in the overall koala sample. The presence of C cells, occurring singly in the epithelial layer, was demonstrated in electron micrographs. Structural features such as low epithelial height, large follicle length and width, and large intercellular spaces in association with low concentrations of free T4 (3.3 +/- 2.1 pM) and free T3 (1.4 +/- 0.9 pM) as reported previously (Lawson et al., 1996) are consistent with an unusually low level of glandular activity in the koala thyroid even though iodine concentrations in the thyroid gland [4.7 +/- 1.6 mg/g (dry weight)] as well as leaf [0.8 +/- 0.3 microg/g (dry weight)] and soil samples [3.8 microg/g (dry weight)] from the koalas' habitat appear unremarkable.

Changes in brain function after manipulation of the cervical spine.

OBJECTIVE: To ascertain whether manipulation of the cervical spine is associated with changes in brain function. DESIGN: Physiological cortical maps were used as an integer of brain activity before and after manipulation of the cervical spine in a large (500 subjects), double-blind controlled study. SETTING: Institutional clinic Participants: Adult volunteers. INTERVENTION: Five hundred subjects were divided into six comparative groups and underwent specific manipulation of the second cervical motion segment. Blinded examiners obtained reproducible pre- and postmanipulative cortical maps, which were subjected to statistical analysis. MAIN OUTCOME MEASURES: Brain activity was demonstrated by reproducible circumferential measurements of cortical hemispheric blind-spot maps before and after manipulation of the second cervical motion segment. Twelve null hypotheses were developed. The critical alpha level was adjusted in accordance with Bonferroni's theorem to .004 (.05 divided by 12) to reduce the likelihood of wrongly rejecting the null hypothesis (i.e., committing a Type I error). RESULTS: Manipulation of the cervical spine on the side of an enlarged cortical map is associated with increased contralateral cortical activity with strong statistical significance (p < .001). Manipulation of the cervical spine on the side opposite an enlarged cortical map is associated with decreased cortical activity with strong statistical significance (p < .001). Manipulation of the cervical spine was specific for changes in only one cortical hemisphere with strong statistical significance (p < .001). CONCLUSIONS: Accurate reproducible maps of cortical responses can be used to measure the neurological consequences of spinal joint manipulation. Cervical manipulation activates specific neurological pathways. Manipulation of the cervical spine may be associated with an increase or a decrease in brain function depending upon the side of the manipulation and the cortical hemisphericity of a patient.

Lipopolysaccharide biosynthesis genes in koala type I Chlamydia: cloning and characterization.

We showed in 1988 that there are two strains of Chlamydia psittaci which infect the koala (Phascolarctos cinereus). In order to further investigate the role of these chlamydial strains in pathogenesis, we have attempted to identify genes of koala type I strain chlamydia which are involved in the immunogenic response. Transformation of Escherichia coli with a plasmid containing a 6.3-kb fragment (pKOC-10) of C. psittaci DNA caused the appearance of a specific chlamydial lipopolysaccharide (LPS) epitope on the host strain. The smallest DNA fragment capable of inducing the expression of chlamydial LPS was an XbaI fragment, 2.4 kb in size (pKOC-5). DNA sequence analysis of the complete fragment revealed regions of high identity, at the amino acid level, to the gseA genes of C. pneumoniae, C. psittaci 6BC and C. trachomatis, and the kdtA gene of E. coli which code for transferases catalysing the addition of 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A. Two open reading frames (ORFs) of 1,314 and 501 nucleotides in size, within the 2.4-kb fragment, were evident, and mRNA species corresponding to these ORFs were detected by Northern analysis. Both ORF1 and ORF2 are required for the appearance of chlamydia-specific LPS on the surface of recombinant E. coli.

Concentrations of thyroid hormones and iodothyronine binding proteins in serum of the koala (Phascolarctos cinereus).

OBJECTIVE: To examine circulating total and free thyroid hormone (T3 and T4) concentrations, determine serum iodothyronine binding characteristics and estimate thyroid stimulating hormone (TSH) activity in sera of coastal and inland koalas. DESIGN: A prospective study. PROCEDURE: Koala serum T3 and T4 were measured by radioimmunoassay. T4 binding parameters were determined by radioligand binding and electrophoresis. Koala TSH values were determined by bioassay. RESULTS: Mean total T4 concentrations were 3.2 +/- 2.1 nM although values were significantly higher in inland-dwelling females in comparison to coastal-dwelling males. Free T4 was 3.3 +/- 2.1 pM. Total and free T3 were 0.4 +/- 0.2 nM and 1.4 +/- 0.9 pM respectively, although these values were at the lower end of the assay detection limit and should be viewed with reservation. Electrophoresis of [125I]-T4-labelled serum revealed only two proteins of electrophoretic mobility similar to human transthyretin (TTR) and albumin. Scatchard analysis of T4 binding to serum gave a curvilinear plot, which could be resolved into two binding sites with affinities identical to that of TTR and albumin but both of low concentration. The bioactivity of the TSH present in the sera was measured using a cell line (JP09) transfected with the human TSH receptor. The mean level of stimulation found in the sera corresponded to a bovine TSH activity of less than 10 mU/L. CONCLUSION: These results suggest that the serum concentrations of free and total thyroid hormones in koalas are low compared to other marsupials and very low compared to eutherian mammals. The mechanism of maintenance of euthyroidism in this species remains to be determined.

Remarkable sequence relatedness in the DNA encoding the major outer membrane protein of Chlamydia psittaci (koala type I) and Chlamydia pneumoniae.

DNA encoding the major outer membrane protein (MOMP) of the koala type-I strain of Chlamydia psittaci (pathogen responsible for blindness and infertility in koalas) was cloned and sequenced. Comparison with momp gene sequences from other chlamydial species revealed a remarkable degree of homology (> 97%) with that of the human pathogen, Chlamydia pneumoniae. In comparison, the sequence only shared 75% DNA sequence homology with other C. psittaci members and 69% homology with C. trachomatis. The open reading frame consisted of 1167 bp encoding a 389-amino acid (aa) pre-MOMP including a leader sequence of 23 aa, similar to the C. pneumoniae gene. These genes were closely related even within the four variable domains (86-100% homology). Specific antibodies were capable of distinguishing between koala type I and C. pneumoniae. This very high degree of relatedness between C. pneumoniae, a human pathogen, and an individual strain of C. psittaci in the momp gene raises further questions on the host specificity, classification and evolutionary relationships of the different chlamydial species.

Seminal characteristics and spermatozoal morphology of captive Queensland koalas (Phascolarctos cinereus adustus).

Viable koala spermatozoa were successfully collected from 8 of 11 captive Queensland koalas on 52 of 76 attempts using electroejaculation under general anesthesia. Semen characteristics, including sperm concentration, percent progressive sperm motility and nuclear heterogeneity appeared to be similar to those of free-ranging Victoria koalas (P.c . victor). Two new head morphotypes (Type XI and teratoid) were identified, and the incidence of midpiece and principal piece spermatozoal abnormalities were recorded.

Immuno-dot blot as a rapid diagnostic method for detection of chlamydial infection in koalas (Phasolarctos cinereus).

The sensitivity and specificity of an immunoscreening test for anti-chlamydial antibodies in koala (Phasolarctos cinereus) serum were determined after the adsorption of non-specific antibodies. The results of the test were compared with complement fixation tests, tissue culture, gene probe analysis and dot blot immunoscreening for host-borne chlamydial antigen. The immunoscreening test was the most sensitive test for the identification of chlamydial infection in koala serum samples, furthermore it was rapid, taking approximately 16 hours to complete, and inexpensive. However, for the assay of swab material from koalas, gene probe analysis remains the most sensitive method of detection of chlamydiae.

Some aspects of the immune response of koalas (Phascolarctos cinereus) and in vitro neutralization of Chlamydia psittaci (koala strains).

Western-blot analysis was used to study the reaction of koala antisera, two specific polyclonal antibodies and one monoclonal antibody, with chlamydial antigens in koalas infected with Chlamydia psittaci. The koala sera recognized four C. psittaci surface antigens, corresponding to the major outer membrane protein (39.5 kDa), 31 kDa protein, 18 kDa protein and lipopolysaccharide. The S25-23 LPS specific monoclonal antibody inhibited chlamydial infection (55-67%) with both koala strains (type I and type II). Both koala antiserum and rabbit polyclonal antibodies against either type of chlamydia significantly reduced the number of infected cells resulting from type II infections at a dilution of 1 in 20. Rabbit antiserum against type II was effective in neutralizing infection by type II elementary bodies, but was less effective against type I infection. In addition, no koala antiserum was effective in neutralizing type I infection.

Detection of Chlamydia psittaci in free-ranging koalas (Phascolarctos cinereus): DNA hybridization and immuno-slot blot analyses.

DNA-slot hybridization and immuno-slot blot analyses were compared for the detection of Chlamydia psittaci in crude swab material from free-ranging koalas. Immuno-slot blot analysis detected chlamydiae in 43 out of 68 koalas, with the sensitivity of the assay varying from 52 to 73% depending on the site of infection. Gene probe analysis was also used employing a genus-specific probe pCKO-10 isolated from a koala chlamydial gene library (ocular strain) and a plasmid probe pCKU cloned from a urogenital strain. The sensitivity of these two assays was comparable and they were considerably more efficient than the immuno-slot blot method for the detection of chlamydiae. Comparison of these data with a cell-culture method of detection, previously used with the same samples, demonstrated that gene probe analysis detected more positives than observed with cell culture. However, this appears to reflect more on the condition of the swab material rather than the sensitivity of the method.