Biomedical subjects
Publications and source records attributed to S Ali.
The ToxB gene was cloned and characterized from a race 5 isolate of Pyrenophora tritici-repentis from North Dakota. ToxB contains a 261-bp open reading frame that encodes a 23 amino acid putative signal peptide and a 64 amino acid host-selective toxin, Ptr ToxB. Analysis of Ptr ToxB from heterologous expression in Pichia pastoris confirms that ToxB encodes a host-selective toxin.
A series of taxane prodrugs with 2-bromoacyl chains attached at the 2'-position of the paclitaxel side chain, varying from six, eight, 12, 14 to 16 carbons in length, were synthesized, characterized and evaluated against human breast MCF-7 cancer cell line for their growth inhibitory (GI50) activities. The GI50 is the drug concentration required to inhibit cell growth by 50%. For comparison, hydrophobic taxanes varying in acyl chain lengths from six to 16 carbons were also synthesized and compared for their G050s with taxanes having equivalent bromoacyl chain lengths. The bromoacyl taxanes bearing six, eight and 12 carbon acyl chain lengths had GI50 values very similar to parent paclitaxel. The GI50 was 3 nM for three taxanes versus 1 nM for paclitaxel on the MCF-7 cell line. Increasing the acyl chain length to 14 or 16 carbons resulted in a significant decrease in cytotoxicity and an increase in the GI50 to 20 or 70 nM, respectively. In general, the GI50 values were directly related to the bromoacyl chain lengths in cultured tumor cells. Unlike bromoacyl taxanes, the taxanes lacking bromine in their acyl chain composition were 50- to 250-fold less active, suggesting that the heteroatom facilitated the hydrolysis of acyl chains to yield free paclitaxel. These differences in growth inhibitory activities may indirectly reflect differences in the susceptibility of the acyl chain to bromine-induced hydrolysis after association of the derivative with cell membranes. Liposome formulations of 2-bromoacyl taxanes bearing six, eight, 12 and 16 carbons were prepared and tested in SCID mice against a xenografted human ovcar-3 ovarian tumor. In vivo results showed that bromoacyl taxanes with a longer chain were therapeutically more efficacious than those with a short chain, presumably due to slow hydrolysis of the prodrug followed by sustained delivery of paclitaxel to the tumor.
We have used the patch-clamp technique to explore the role of A Kinase Anchor Proteins (AKAP) in mediating the effect of cAMP on ROMK1 channels expressed in the Xenopus oocytes. Addition of membrane permeant cAMP analogs increased channel activity only in oocytes injected with ROMK1 and AKAP79 cRNA but had no effect on channel activity in oocytes injected with ROMK1 alone. Using the two-electrode voltage clamp technique, we determined that application of H89, a potent inhibitor of protein kinase A (PKA), abolished the stimulatory effect of cAMP/forskolin. To investigate the role of AKAP specificity in conferring cAMP responses to ROMK1 channels, we examined channel activity in oocytes expressing ROMK1 and either AKAP18, AKAP-KL or AKAP75. Addition of cAMP failed to increase channel current in oocytes expressing ROMK1 and either AKAP18 or AKAP-KL. In contrast, cAMP increased ROMK1 channel activity by 33% in oocytes coexpressing AKAP75, the bovine homologue of AKAP79. The effect of cAMP on ROMK1 in oocytes coexpressing AKAP75 is inhibited by H89. Since all three AKAPs bind PKAII, the results suggest that a unique structural domain in AKAP75/79 collaborates with the PKAII binding site and enables a productive association of PKA with ROMK1 channels. Deletion of either the membrane targeting region of AKAP75 (AKAP45) or PKAII binding domain of AKAP75 (AKAP75DeltaC) abolished the effects of forskolin on ROMK1 channels. This suggests that the membrane targeting and the PKA binding domains of AKAP75 are essential for the effect of cAMP. However, the nature of the AKAP, that interacts with ROMK1 in the native tissue, remains to be determined because AKAP75/79 are not expressed in the kidney. We conclude that the regulation of ROMK1 channels by PKA requires the involvement of the cell membrane-directed AKAPs that are able to specifically link PKA to the target channel protein.
EPI-2010 is a respirable antisense oligonucleotide (RASON), which selectively attenuates discordantly overexpressed adenosine A(1) receptors in allergic lung (Nature 1997;385:721). In the present study, aerosolized [(35)S]-labeled EPI-2010 (5 mg exposure; specific activity 0.055 Ci/mmol) was administered to normal rabbits by endotracheal tube to assess biodistribution, route of elimination, and potential cardiovascular toxicity. The animals were killed at 0, 6, 24, 48, and 72 h after inhalation of EPI-2010. Duplicate aliquots from different tissues and samples were solubilized and assessed for radioactivity. Approximately 1.4% of the total aerosolized EPI-2010 was deposited into the lung. The concentration of the drug in the lung at 0, 6, 24, 48, and 72 h was 64.0 +/- 1.5, 67.0 +/- 4.4, 32.0 +/- 3.7, 23.4 +/- 1.4, and 2.1 +/- 0.5 microg equivalents, respectively. Only a small amount of the radioactivity was detected in extrapulmonary tissues. By 72 h, 67.5% of the administered dose was excreted in the urine, which represented the major pathway of elimination. In postlabeling studies, intact full-length EPI-2010 could only be detected in the lung. Autoradiographic analysis after inhalation of [(35)S]-labeled EPI-2010 showed a relatively uniform deposition of drug throughout the lung. The aerosolized EPI-2010 did not have any significant systemic effects on the cardiovascular system as determined by Cardiomax-II analysis. This pattern of distribution and the lack of effect on cardiovascular function support the concept that RASONs offer the potential to safely address respiratory targets for which systemic distribution and systemic bioavailability may be contraindicated.
In today's troubled healthcare climate, it is not uncommon to run across headlines like: 'Health insurance premiums increasing by 10 percent to 30 percent across the country.' This particular New York Times article went on to explain that this premium price hike, the third consecutive double digit increase in 3 years, is driven largely by escalating pharmaceutical costs. The pharmaceutical industry has largely been vilified in the media and in the recent presidential debates, for fueling healthcare inflation and setting what many perceive to be 'unfair' prices in light of the profit margins on their life-saving products. A report released by the Congressional Research Service found that after tax, profits for the pharmaceutical industry averaged 17% of sales, compared with 5% for all other industries. The White House has added its voice to the popular discontent with notices such as this one reported in the New York Times: 'There is a rising tide politically in this country of strong antagonism against the pharmaceutical industry on the dimension of prices. (Without expanded access to insurance) price controls are an inevitable outcome.' Although the prospect of price control remains dubious in America's entrenched laissez-faire economy, David Kessler, former head of the FDA and the Dean of the Yale School of Medicine, described the situation as a 'powder keg,' stating 'the current system is simply not sustainable'. Although there does not appear to be an immediate solution to this escalating crisis, this editorial will examine pharmaceutical pricing, industry profits and the role of pharmacoeconomic analyses amidst the chaos.
We report the case of an infant with congenital colonic stenosis in the ascending colon and review the literature regarding this uncommon condition.
PURPOSE: Trichostatin A (TSA), an antifungal antibiotic with cytostatic and differentiating properties in mammalian cell culture, is a potent and specific inhibitor of histone deacetylase (HDAC) activity. The purpose of this study was to evaluate the antiproliferative and HDAC inhibitory activity of TSA in vitro in human breast cancer cell lines and to assess its antitumor efficacy and toxicity in vivo in a carcinogen-induced rat mammary cancer model. EXPERIMENTAL DESIGN AND RESULTS: TSA inhibited proliferation of eight breast carcinoma cell lines with mean +/- SD IC(50) of 124.4 +/- 120.4 nM (range, 26.4-308.1 nM). HDAC inhibitory activity of TSA was similar in all cell lines with mean +/- SD IC(50) of 2.4 +/- 0.5 nM (range, 1.5-2.9 nM), and TSA treatment resulted in pronounced histone H4 hyperacetylation. In randomized controlled efficacy studies using the N-methyl-N-nitrosourea carcinogen-induced rat mammary carcinoma model, TSA had pronounced antitumor activity in vivo when administered to 16 animals at a dose of 500 microg/kg by s.c. injection daily for 4 weeks compared with 14 control animals. Furthermore, TSA did not cause any measurable toxicity in doses of up to 5 mg/kg by s.c. injection. Forty-one tumors from 26 animals were examined by histology. Six tumors from 3 rats treated with TSA and 14 tumors from 9 control animals were adenocarcinomas. In contrast, 19 tumors from 12 TSA-treated rats had a benign phenotype, either fibroadenoma or tubular adenoma, suggesting that the antitumor activity of TSA may be attributable to induction of differentiation. Two control rats each had tumors with benign histology. CONCLUSIONS: The present studies confirm the potent dose-dependent antitumor activity of TSA against breast cancer in vitro and in vivo, strongly supporting HDAC as a molecular target for anticancer therapy in breast cancer.
Little is known about which patients miss appointments or why they do so. Using routinely collected data from four practices, we aimed to determine whether patients who missed appointments differed in terms of their age, sex, and deprivation scores from those who did not, and to examine differences between the practices with respect to missed appointments. The likelihood of someone missing at least one appointment was independently associated with being female, living in a deprived area, and being a young adult. Living in a deprived area was associated with a threefold increase in the likelihood of missing an appointment, and the extent of this association was the same across all four practices. Interventions aimed at reducing missed appointments need to be based upon these findings.
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The protein-tyrosine phosphatase SHP-2 modulates signaling events through receptor tyrosine kinases and cytokine receptors including the receptor for prolactin (PRLR). Here we investigated mechanisms of SHP-2 recruitment within the PRLR signaling complex. Using SHP-2 and PRLR immunoprecipitation studies in 293 cells and in the mouse mammary epithelial cell line HC11, we found that SHP-2 co-immunoprecipitates with the PRLR and that the C-terminal tyrosine of the PRLR plays a regulatory role in both the tyrosine phosphorylation and the recruitment of SHP-2. Our results further indicate that SHP-2 association to the PRLR occurs via the C-terminal SH2 domain of the phosphatase. In addition, we determined that the newly identified adaptor protein Gab2, but not Gab1, is specifically tyrosine phosphorylated and is able to recruit SHP-2 and phosphatidyinositol 3-kinase in response to PRLR activation. Together, these studies suggest the presence of dual recruitment sites for SHP-2; the first is to the C-terminal tyrosine of the PRLR and the second is to the adaptor protein Gab2.
We have used the two-electrode voltage clamp technique and the patch clamp technique to investigate the regulation of ROMK1 channels by protein-tyrosine phosphatase (PTP) and protein-tyrosine kinase (PTK) in oocytes coexpressing ROMK1 and cSrc. Western blot analysis detected the presence of the endogenous PTP-1D isoform in the oocytes. Addition of phenylarsine oxide (PAO), an inhibitor of PTP, reversibly reduced K(+) current by 55% in oocytes coinjected with ROMK1 and cSrc. In contrast, PAO had no significant effect on K(+) current in oocytes injected with ROMK1 alone. Moreover, application of herbimycin A, an inhibitor of PTK, increased K(+) current by 120% and completely abolished the effect of PAO in oocytes coexpressing ROMK1 and cSrc. The effects of herbimycin A and PAO were absent in oocytes expressing the ROMK1 mutant R1Y337A in which the tyrosine residue at position 337 was mutated to alanine. However, addition of exogenous cSrc had no significant effect on the activity of ROMK1 channels in inside-out patches. Moreover, the effect of PAO was completely abolished by treatment of oocytes with 20% sucrose and 250 microg/ml concanavalin A, agents that inhibit the endocytosis of ROMK1 channels. Furthermore, the effect of herbimycin A is absent in the oocytes pretreated with either colchicine, an inhibitor of microtubules, or taxol, an agent that freezes microtubules. We conclude that PTP and PTK play an important role in regulating ROMK1 channels. Inhibiting PTP increases the internalization of ROMK1 channels, whereas blocking PTK stimulates the insertion of ROMK1 channels.
The colon has a circular and longitudinal muscle layers and possesses an electric activity. The current study investigated the spread of this electric activity in the colonic musculature. The tests were performed in 16 patients (mean age 40.6 +/- 10.8 SD years, 10 men, 6 women) during operative correction of their incisional hernias in the anterior abdominal wall. Three electrodes were applied to the longitudinal muscle of the descending colon, and 3 to the circular muscle. The colonic pressure was monitored by a 10-F tube inserted per anus and connected to a pressure transducer. Electric waves in the form of pacesetter potentials (PPs) and action potentials (APs) were recorded from the longitudinal but not the circular muscle coat. The PPs and APs had the same frequency, amplitude and conduction velocity from the 3 electrodes of the same patient. The APs were associated with a rise of the intracolonic pressure. The results were reproducible in the individual subject. Being of the visceral type, the colonic smooth muscle fibers are controlled by non-nervous stimuli. Colonic motility is suggested to be effected by means of 2 mechanisms: the longitudinal muscle fibers activity through the action of the electric waves and the circular muscle fibers through the stretch reflex.
Heterotrimeric G proteins transduce signals from cell surface receptors to modulate the activity of cellular effectors. Src, the product of the first characterized proto-oncogene and the first identified protein tyrosine kinase, plays a critical role in the signal transduction of G protein-coupled receptors. However, the mechanism of biochemical regulation of Src by G proteins is not known. Here we demonstrate that Galphas and Galphai, but neither Galphaq, Galpha12 nor Gbetay, directly stimulate the kinase activity of downregulated c-Src. Galphas and Galphai similarly modulate Hck, another member of Src-family tyrosine kinases. Galphas and Galphai bind to the catalytic domain and change the conformation of Src, leading to increased accessibility of the active site to substrates. These data demonstrate that the Src family tyrosine kinases are direct effectors of G proteins.
The ability to specifically monitor the behavior of the inner monolayer lipids of membranous vesicles during the membrane fusion process is useful technically and experimentally. In this study, we have identified N-NBD-phosphatidylserine as a reducible probe particularly suitable for inner monolayer fusion assays because of its low rate of membrane translocation after reduction of the outer monolayer probes by dithionite. Data are presented on translocation as a function of temperature, vesicle size, membrane composition, and serum protein concentration. Translocation as a result of the fusion event itself was also characterized. We further show here that a second membrane-localized probe, a long wavelength carbocyanine dye referred to a diI(5)C18ds, appears to form a membrane-bound resonance energy transfer pair with N-NBD-PS, and its outer monolayer fluorescence can also be eliminated by dithionite treatment. Lipid dilution of these probes upon fusion with unlabeled membranes leads to an increase in NBD donor fluorescence, and hence is a new type of inner monolayer fusion assay. These inner monolayer probe mixing assays were compared to random lipid labeling and aqueous contents mixing assays for cation-dependent fusion of liposomes composed of phosphatidylserine and phosphatidylethanolamine. The results showed that the inner monolayer fusion assay eliminates certain artifacts and reflects fairly closely the rate of non-leaky mixing of aqueous contents due to fusion, while outer monolayer mixing always precedes mixing of aqueous contents. In fact, vesicle aggregation and outer monolayer lipid mixing were found to occur over very long periods of time without inner monolayer mixing at low cation concentrations. Externally added lysophosphatidylcholine inhibited vesicle aggregation, outer monolayer mixing and any subsequent fusion. The state of vesicle aggregation and outer monolayer exchange that occurs below the fusion threshold may represent a metastable intermediate state that may be useful for further studies of the mechanism of membrane fusion.
Thermophilic catechol 2,3-dioxygenase (EC 1.13.11.2) from Bacillus stearothermophilus has been immobilized on highly activated glyoxyl agarose beads. The enzyme could be fully immobilized at 4 degrees C and pH 10.05 with a high retention of activity (around 80%). Enzyme immobilized under these conditions showed little increase in thermostability compared with the soluble enzyme, but further incubation of immobilized enzyme at 25 degrees C and pH 10.05 for 3 h before borohydride reduction resulted in conjugates exhibiting a 100-fold increase in stability (c.f. the free enzyme). The stability of catechol 2,3-dioxygenase immobilized under these conditions was essentially independent of protein concentration whereas free enzyme was rapidly inactivated at low protein concentrations. An apparent stabilization factor of over 700-fold was recorded in the comparison of free and immobilized catechol 2,3-dioxygenases at protein concentrations of 10 µg/ml. Immobilization increased the 'optimum temperature' for activity by 20 degrees C, retained activity at substrate concentrations where the soluble enzyme was fully inactivated and enhanced the resistance to inactivation during catalysis. These results suggest that the immobilization of the enzyme under controlled conditions with the generation of multiple covalent links between the enzyme and matrix both stabilized the quaternary structure of the protein and increased the rigidity of the subunit structures.
The development of stable spherical lipid-coated drug particles that are termed 'lipocores' is reported here. Unlike conventional lipid-based particles (i.e. liposomes, emulsions, micelles), these particles are comprised solely of a core of a poorly water soluble drug surrounded by polyethyleneglycol conjugated lipid (PEG-lipid) and are formed via a 'kinetic' trapping process. These lipocore particles were made with the acyl chain of 16 carbon length (C16) acyl-chain derivatives of paclitaxel or vinblastine and with the polyene antifungal hamycin. Formation of the particles occurred regardless of the type of PEG-phospholipid used (i.e. acyl chain length, chain saturation, and polymer length) and could also be formed with the negatively charged lipid N-glutaryl-dioleoyl-phosphatidylethanolamine (DOPE-GA). Images from both freeze-fracture electron microscopy and electron cryo-microscopy revealed solid spherical structures with no internal lamellae for the PEG-lipid particles made with the C16 derivatives of paclitaxel (BrC16-T) or vinblastine (C16-Vin). From a solute distribution study of lipocores made with BrC16-T and distearoyl-phosphatidylethanolamine-PEG2000 (DSPE-PEG2000), the particles were found to have no measurable aqueous captured volume. Fluorescence anisotropy and order parameter measurements revealed the core material of these particles to be highly immobilized. The mole ratio of BrC16-T:lipid in the lipocores was typically > 90: < 10 and as high as 98:2, and the refrigerated lipocores were stable for several months. BrC16-T/DSPE-PEG2000 lipocores of 50-100 nm particle size were far less toxic than paclitaxel (Taxol) after intraperitoneally (i.p.) or intravenously (i.v.) administration in mice and were active against i.p. and subcutaneously (s.c.) planted human (OvCar3) ovarian carcinoma grown in SCID mice. It is believed the high drug:lipid ratio, the stability, and therapeutic efficacy of these novel particles make them a paradigm for delivery of poorly water soluble drugs and/or their hydrophobic derivatives.
Chemokines are a group of small proteins that have a variety of functions, including the activation and recruitment of immune cells during episodes of inflammation. In common with many cytokines, it has been observed that chemokines have the potential to bind heparin-like glycosaminoglycan molecules, which are normally expressed on proteoglycan components of the cell surface and extracellular matrix. The significance of this interaction for chemokine activity remains a subject of debate. In this study, Chinese hamster ovary cells were transfected separately with the human chemokine receptors CCR1 and CCR5, and these receptors were shown to induce an intracytoplasmic Ca(2+) flux and cellular chemotaxis following stimulation with the natural CC chemokine ligands (MIP-1alpha, RANTES (regulated on activation normal T cell expressed), and MIP-1beta). In further experiments, mutant CHO cells, with a defect in normal glycosaminoglycan (GAG) expression, were also transfected with, and shown to express similar levels of, CCR1 and CCR5. Although these receptors were functional, it was found that the mutant cells required exposure to higher concentrations of ligands than the wild-type cells in order to produce the same intracytoplasmic Ca(2+) flux. Radioligand binding experiments demonstrated that specific chemokine receptors expressed by wild-type cells had a significantly greater affinity for MIP-1alpha than similar receptors expressed by GAG-deficient mutants. However, there was no significant difference between these cells in their affinity for RANTES or MIP-1beta. In conclusion, it has been demonstrated clearly that GAG expression is not necessary for the biological activity of the chemokines MIP-1alpha, RANTES, or MIP-1beta. However, the presence of cell surface GAGs does enhance the activity of low concentrations of these chemokines by a mechanism that appears to involve sequestration onto the cell surface.
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