Biochimica et biophysica acta
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Biochim. Biophys. Acta · Nov 2000
The PCR plateau phase - towards an understanding of its limitations.
The DNA polymerases from Thermus aquaticus and Thermus flavus were recently found to bind to short double-stranded DNA fragments without sequence specificity [Kainz et al. (2000) Biotechniques 28, 278-82]. In the present study, it is shown that the accumulation of amplification products during later PCR cycles also exerts an inhibitory effect on several enzymes tested. To simulate later cycle conditions, a 1.7 kb sequence from phage lambda DNA was amplified in the presence of various amounts of a 1 kb double-stranded DNA fragment. ⋯ PCR mixtures still in quasi-linear phase partially extended the hairpins. In both cases, a further addition of polymerase significantly improved their function. These results indicate that the main factor contributing to the plateau phase in PCR consists of binding of DNA polymerase to its amplification products.
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Biochim. Biophys. Acta · Jun 2000
Selective targeting of immunoliposomal doxorubicin against human multiple myeloma in vitro and ex vivo.
Circulating malignant CD19(+) B cells have been implicated in the pathogenesis and relapse of multiple myeloma (MM). This study investigated the therapeutic applicability of using long-circulating liposome-encapsulated doxorubicin (DXR) targeted against the internalizing CD19 antigens present on human MM cells. In vitro binding studies using the CD19(+) MM cell line ARH77 demonstrated that CD19-directed immunoliposomes (SIL[anti-CD19]) specifically attached to these cells. ⋯ A decrease in cellular DNA (which is an indicator of apoptosis) caused by the cytotoxicity of DXR-SIL[anti-CD19] to myeloma PBMC was determined by using flow cytometry. While PBMC treatment with free DXR resulted in non-specific cytotoxicity to both B and T cells, DXR-SL were only minimally cytotoxic to either. In contrast, DXR-SIL[anti-CD19] were selectively cytotoxic for B cells in PBMC, indicating that this treatment may be effective in eliminating circulating malignant B cells in MM patients.
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Biochim. Biophys. Acta · Jan 2000
Chemical characterization of pyridoxalated hemoglobin polyoxyethylene conjugate.
Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) was developed in the 1980s as an oxygen carrier and is now under development for treatment of nitric oxide-dependent, volume refractory shock. PHP is made by derivatizing human stroma-free hemoglobin with pyridoxal-5-phosphate and polyoxyethylene (POE). A unique aspect of using POE for modification is that unlike its mono-methoxy polyethylene glycol (PEG) relatives, POE is bifunctional. ⋯ The isoelectric focusing profile is broad, demonstrating a pI range of 5.0-6.5. The hydrodynamic size of PHP was determined to be approximately 7.2 nm by dynamic light scattering. Soluble red blood cell proteins, such as catalase, superoxide dismutase, and carbonic anhydrase, are present in PHP and are also modified by POE.
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Biochim. Biophys. Acta · Aug 1999
Is gut the major source of proinflammatory cytokine release during polymicrobial sepsis?
Although studies have shown that the gut is capable of being a cytokine-producing organ and that the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 are upregulated following the onset of sepsis, it remains unknown whether the gut is indeed the major source of the increased cytokine production under such conditions. To determine this, male rats were subjected to cecal ligation and puncture (CLP, a model of polymicrobial sepsis) or sham operation followed by the administration of normal saline solution subcutaneously (i.e., fluid resuscitation). Systemic and portal blood samples were taken simultaneously at 2, 5, 10, or 20 h after CLP or sham operation. ⋯ However, there were no significant differences in the levels of those proinflammatory cytokines between systemic and portal blood at any points after the onset of sepsis. Moreover, there were no significant alterations in the proinflammatory cytokine gene expression in the small intestine at 10 h after CLP. Since the levels of TNF-alpha, IL-1beta, and IL-6 were not significantly increased in portal blood as compared to systemic blood and since there was no upregulation of gene expression for these cytokines, it appears that organs other than the gut are responsible for the upregulated proinflammatory cytokines during polymicrobial sepsis.
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Biochim. Biophys. Acta · Jul 1999
Inhibitory effect of KW-3902, an adenosine A(1) receptor antagonist, on p-aminohippurate transport in OK cells.
KW-3902 (8-(noradamantan-3-yl)-1,3-dipropylxanthine) is a novel potent and selective adenosine A(1) receptor antagonist. We examined the effect of KW-3902 on p-aminohippurate (PAH) transport in opossum kidney (OK) epithelial cells. Pretreatment for 3 h with KW-3902 inhibited the transcellular transport of PAH across OK cell monolayers from the basal to the apical side. ⋯ Pretreatment with adenosine deaminase or 8-cyclopentyl-1,3-dipropylxanthine, another selective adenosine A(1) receptor antagonist, also decreased the basolateral PAH uptake. KW-3902 pretreatment had no effect on the concentration of intracellular alpha-ketoglutarate which exchanges for PAH across the basolateral membrane of OK cells. These results suggest that KW-3902 has an inhibitory effect on PAH transport in OK epithelial cells.