The prognosis for patients with osteosarcoma who have received prior manipulative therapy.

We evaluated the long-term outcome of patients with an osteosarcoma who had undergone prior manipulative therapy, a popular treatment in Asia, and investigated its effects on several prognostic factors. Of the 134 patients in this study, 70 (52%) patients had manipulative therapy and 64 (48%) did not. The age, location, and size of tumour were not significantly different between the groups. The five-year overall survival rate was 58% and 92% in the groups with and without manipulative therapy (p = 0.004). Both the primary and overall rates of lung metastasis were significantly higher in the manipulative group (primary: 32% vs 3%, p = 0.003; overall lung metastasis rate: 51.4% vs 18.8%, p < 0.001). Patients who had manipulative therapy had higher local recurrence rates in comparison to patients who did not (29% vs 6%, p = 0.011). The prognosis for patients with osteosarcoma who had manipulative therapy was significantly poorer than those who had not. Manipulative therapy was an independent factor for survival. This form of therapy may serve as a mechanism to accelerate the spread of tumour cells, and therefore must be avoided in order to improve the outcome for patients with an osteosarcoma.

Development of human umbilical vein endothelial cell (HUVEC) and human umbilical vein smooth muscle cell (HUVSMC) branch/stem structures on hydrogel layers via biological laser printing (BioLP).

Angiogenesis is one of the prerequisite steps for viable tissue formation. The ability to influence the direction and structure in the formation of a vascular system is crucial in engineering tissue. Using biological laser printing (BioLP), we fabricated branch/stem structures of human umbilical vein endothelial cells (HUVEC) and human umbilical vein smooth muscle cells (HUVSMC). The structure is simple as to mimic vascular networks in natural tissue but also allow cells to develop new, finer structures away from the stem and branches. Additionally, we printed co-culture structures by first depositing only HUVECs, followed by 24 h incubation to allow for adequate cell-cell communication and differentiation into lumina; these cell printed scaffold layers were then removed from incubation and inserted into the BioLP apparatus so that HUVSMCs could be directly deposited on top and around the previously printed HUVEC structures. The growth and differentiation of these co-culture structures was then compared to the growth of printed samples with either HUVECs or HUVSMCs alone. Lumen formation was found to closely mimic the original branch and stem structure. The beginning of a network structure is observed. HUVSMCs acted to limit HUVEC over-growth and migration when compared to printed HUVEC structures alone. HUVSMCs and HUVECS, when printed in close contact, appear to form cell-cell junctions around lumen-like structures. They demonstrate a symbiotic relationship which affects their development of phenotype when in close proximity of each other. Our results indicate that it is possible to direct the formation and growth of lumen and lumen network using BioLP.

The angiosuppressive effects of 20(R)- ginsenoside Rg3.

Aberrant angiogenesis is an essential step for the progression of solid tumors. Thus anti-angiogenic therapy is one of the most promising approaches to control tumor growth. In this study, we examined the ability of 20(R)-ginsenoside Rg3 (Rg3), one of the active compounds present in ginseng root, to interfere with the various steps of angiogenesis. Rg3 was found to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) with an IC50 of 10 nM in Trypan blue exclusion assay. Rg3 (1-10(3) nM) also dose dependently suppressed the capillary tube formation of HUVEC on the Matrigel in the presence or absence of 20 ng/ml vascular endothelial growth factor (VEGF). The VEGF-induced chemoinvasion of HUVEC and ex vivo microvascular sprouting in rat aortic ring assay were both significantly attenuated by Rg3. In addition, Rg3 (150 and 600 nM) remarkably abolished the basic fibroblast growth factor (bFGF)-induced angiogenesis in an in vivo Matrigel plug assay. The Matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, which play an important role in the degradation of basement membrane in angiogenesis and tumor metastasis present in the culture supernatant of Rg3-treated aortic ring culture were found to decrease in their gelatinolytic activities. Taken together, these data underpin the anti-tumor property of Rg3 through its angiosuppressive activity.

Picoliter-scale protein microarrays by laser direct write.

We demonstrate the accurate picoliter-scale dispensing of active proteins using a novel laser transfer technique. Droplets of protein solution are dispensed onto functionalized glass slides and into plastic microwells, activating as small as 50-microm diameter areas on these surfaces. Protein microarrays fabricated by laser transfer were assayed using standard fluorescent labeling techniques to demonstrate successful protein and antigen binding. These results indicate that laser transfer does not damage the active site of the dispensed protein and that this technique can be used to successfully fabricate a functioning protein microarray. Also, as a result of the efficient nature of the process, material usage is reduced by two to four orders of magnitude compared to conventional pin dispensing methods for protein spotting.

alpha-Adrenoceptor mechanisms of thermoregulation during cold challenge in humans.

1. Previous studies on the thermoregulatory effects of alpha-adrenoceptor antagonists have been performed primarily in animals and the findings have been inconsistent. There is evidence for thermoregulatory impairment by alpha-adrenergic antagonists in humans not exposed to cold, but the effects of alpha-adrenergic blockade during cold challenge have not been investigated. 2. Fourteen healthy human volunteers (seven elderly, aged 55-68 years and seven young, aged 19-27 years) were studied on three separate days and received three randomly assigned treatments; (i) control (no drug), (ii) low-dose phentolamine and (iii) high-dose phentolamine. On each day cold intravenous saline (4 degrees C) was given until both vasoconstriction and shivering were triggered or a maximum fluid volume (40 ml/kg) was delivered. Core temperature, peripheral vasoconstriction and metabolic heat production were measured. 3. The alpha-adrenoceptor antagonist caused a dose-dependent inhibition of vasoconstriction in the elderly but did not impair vasoconstriction in the young subjects at the doses that were given. Shivering and metabolic heat production were unaffected by alpha-adrenergic blockade in the elderly or in the young. 4. These findings illustrate the selective inhibition of vasoconstriction (but not shivering) by alpha-adrenoceptor antagonism in elderly individuals. Compared with the young, the elderly are more sensitive to the effects of alpha-antagonists, perhaps due to downregulation of the alpha-adrenoceptor. These findings lead us to conclude that thermoregulatory vasoconstriction is alpha-adrenergically mediated, and this response is attenuated by alpha-adrenoceptor blockade in elderly humans.

A circular dichroism study of ethidium bromide binding to Z-DNA induced by dinuclear platinum complexes.

Dinuclear bis(platinum) complexes have been shown previously to induce the B-->Z transition in synthetic DNAs (Nucleic Acids Res. 7, 1697-1703, J. Inorganic Biochem. 54, 207-220). In this paper, the reversibility of the Z conformation back to the B form was assessed by treatment of the induced Z form in poly(dG-dC).poly(dG-dC) with ethidium bromide (Etd). Z-DNA induced by the tetra-amine cations [{Pt(NH3)3}2(H2N(CH2)nNH2)]4+, which are capable of only electrostatic interactions with the polynucleotide, was readily reversible. The spectroscopic data mirrored that of ethidium bromide/poly(dG-dC).poly(dG-dC) in the presence of 4.4 M NaCl. In contrast, Z-DNA induced by the bifunctional complexes [{trans-PtCl(NH3)2}2(H2N(CH2)nNH2)]2+ did not produce spectra typical of Etd intercalation and reversal to B-form DNA. The original Z-form CD spectra of DNA treated with the bifunctional complexes could be reobtained following removal of Etd by extensive dialysis. The bifunctional complexes are very effective interstrand cross-linking agents. The data suggest that interstrand cross-linking by dinuclear complexes can stabilize or "lock" the Z-conformation prohibiting its reversal to the B-form. The implications for the biological activity of the dinuclear complexes are briefly discussed.

Role of alpha-adrenoceptors in the maintenance of core temperature in humans.

1. Although alpha-adrenoceptor antagonists have been shown to induce core hypothermia in animals, it is unclear whether the primary mechanism is increased heat loss or decreased heat production. Furthermore, studies have not been performed in humans to determine the role of alpha-adrenoceptors in the maintenance of core temperature. 2. alpha-Adrenoceptor blockade was achieved with three doses of phentolamine given by random assignment on three different study days in five male and five female healthy subjects. Core temperature, mean skin-surface temperature, fingertip capillary blood flow and metabolic heat production were measured. Dose-response curves were plotted for all measured variables, and males and females were compared to identify potential gender differences. 3. Core temperature decreased with all doses of phentolamine. At the completion of the phentolamine infusion, the decrease in core temperature was more significant with high-dose (0.3 +/- 0.1 degrees C, P = 0.03) and with medium-dose (0.2 +/- 0.0 degrees C, P = 0.05) phentolamine than with low-dose phentolamine (0.1 +/- 0.0 degrees C). The maximum core temperature decrease during the study was more significant with high dose (0.6 +/- 1 degrees C) than with medium (0.3 +/- 1 degrees C, P = 0.04) or low (0.3 +/- 1 degrees C, P = 0.005) doses. Mean skin-surface temperature was increased with all doses. Fingertip blood flow was increased (approximately 60% above baseline) with the medium and high doses, but was unchanged with the low dose. Total body oxygen consumption was unchanged regardless of dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical reactivity and DNA sequence specificity of formally monofunctional and bifunctional bis(platinum) complexes.

The synthesis of the formally monofunctional bis(platinum) complex [(Pt(NH3)3)mu-H2N(CH2)4NH2-(trans-PtCl(NH3)2)]3+ (1,0/t) is reported. The interactions of this species and the formally bifunctional bis(platinum) complex [(trans-PtCl(NH3)2)2H2N(CH2)4NH2]Cl2(1,1/t,t) with DNA were investigated. Comparison was made with the monomeric [PtCl(dien)]Cl, (Pt(DIEN)), and cis-[PtCl2(NH3)2], (cis-DDP). The initial rates of reaction with small self-complementary oligonucleotides 5'-ATATATN4ATATAT-3' (N4 = GCGC and N4 = GGCC) were calculated. For all compounds, the GGCC oligonucleotide reacted faster than the GCGC counterpart. The order of reactivity of the platinum compounds for the GCGC oligonucleotide was 1,1/t,t > 1,0/t > Pt(DIEN) > cis-DDP. The reaction of 1,0/t and 1,1/t,t with poly(dG-dC).poly(dG-dC) was also investigated using circular dichroism (CD) spectroscopy where both compounds were shown to induce a B-->Z conformational change.