Thermoacoustic molecular imaging of small animals

We have designed, constructed, and tested a thermoacoustic computed tomography (TCT) scanner for imaging optical absorption in small animals in three dimensions. The device utilizes pulsed laser irradiation (680-1064 nm) and a unique, 128-element transducer array. We quantified the isotropic spatial resolution of this scanner to be 0.35 mm. We describe a dual-wavelength subtraction technique for isolating optical dyes with TCT. Phantom experiments demonstrate that we can detect 5 fmol of a near-infrared dye (indocyanine green, ICG) in a 1-microL volume using dual-wavelength subtraction. Initial TCT imaging in phantoms and in two sacrificed mice suggests that three-dimensional, optical absorption patterns in small animals can be detected with an order of magnitude better spatial resolution and an order of magnitude better low-contrast detectability in small animals when compared to fluorescence imaging or diffusion optical tomography.     

Comparison of the Escherichia coli proteomes for recombinant human growth hormone producing and nonproducing fermentations

Two-dimensional electrophoretic analyses of Escherichia coli cells producing recombinant human growth hormone (Nutropin) in fermentations were conducted. The resulting two-dimensional protein profiles were compared with those of nonproducing (blank) cells. A qualitative comparison was performed to address regulatory issues in the biopharmaceutical industry, and a semiquantitative comparison was performed to reveal information about the physiological state of the cells. The protein spots unique to production fermentation profiles were all related to recombinant human growth hormone (hGH); these included intact hGH, charge variants of hGH, and a proteolytically cleaved form of hGH, as expected. There were no E. coli host cell proteins unique to either the production or blank fermentation profiles. Rather, all detectable differences in E. coli proteins were quantitative in nature. Specifically, the levels of IbpA (inclusion body binding protein A), Ivy (inhibitor of vertebrate lysozyme), and a cleaved form of GroEL (Hsp60 homolog) were higher in hGH production profiles, whereas the levels of GlmU protein and PspA (phage shock protein A) were higher in blank profiles. In general, the high degree of similarity between proteomes for hGH-producing and nonproducing cells suggests that E. coli proteins from a nonproducing (blank) fermentation are appropriate for eliciting antibodies that are then used in immunoassays to measure host cell proteins in samples from production fermentations.     

Detection of Naegleria sp. in a thermal, acidic stream in Yellowstone National Park

An initial survey of sequences of PCR-amplified portions of the 18S rRNA genes from a community DNA clone library, prepared from an algal mat in a thermal, acidic stream in Yellowstone National Park, WY, USA, revealed among other sequences, several that matched Vahlkampfia. This finding prompted further investigation using primers specific for Naegleria. Sequences from a subsequent DNA clone library, prepared from the 5.8S rRNA gene and the adjacent internal transcribed spacer (ITS) regions of the rRNA, closely matched Naegleria and formed an independent lineage within a clade containing Naegleria sturti and Naegleria niuginiensis. The sequences may represent a new Naegleria species.     

A high-throughput method for enzyme kinetic studies

A simple and flexible setup for conducting drug metabolism studies is described in this report. A heating block was designed for the Multimek liquid handler platform for incubation of multiple samples at 37 degrees C in a 96-well format. This setup enables the rapid performance of drug metabolism experiments on a large number of samples. In this report, the authors present the validation of the system by 1) showing reproducible and consistent determination of the in vitro half-life of midazolam in every well across the entire plate and 2) determination of metabolic parameter values of midazolam, testosterone, diclofenac, warfarin, and dextromethorphan and inhibition parameter values of quinidine and ketoconazole, all comparable to literature values. In addition, the authors demonstrate the application of the setup to determining the metabolic stability of a set of proprietary compounds, the inhibition of activity of cytochrome P450 (CYP) enzymes, and the conduct of a single combination experiment that can simultaneously determine the metabolic stability and CYP inhibition activity. Overall, the system represents a simple, high-throughput and useful tool for drug metabolism screening in drug discovery.     

The two alpha-tubulin isotypes in budding yeast have opposing effects on microtubule dynamics in vitro

The yeast Saccharomyces cerevisiae has two genes for α-tubulin, TUB1 and TUB3, and one β-tubulin gene, TUB2. The gene product of TUB3, Tub3, represents ~10% of α-tubulin in the cell. We determined the effects of the two α-tubulin isotypes on microtubule dynamics in vitro. Tubulin was purified from wild-type and deletion strains lacking either Tub1 or Tub3, and parameters of microtubule dynamics were examined. Microtubules containing Tub3 as the only α-tubulin isotype were less dynamic than wild-type microtubules, as shown by a shrinkage rate and catastrophe frequency that were about one-third of that for wild-type microtubules. Conversely, microtubules containing Tub1 as the only α-tubulin isotype were more dynamic than wild-type microtubules, as shown by a shrinkage rate that was 50% higher and a catastrophe frequency that was 30% higher than those of wild-type microtubules. The results suggest that a role of Tub3 in budding yeast is to control microtubule dynamics.     

Pyk2- and Src-dependent tyrosine phosphorylation of PDK1 regulates focal adhesions

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a signal integrator that activates the AGC superfamily of serine/threonine kinases. PDK1 is phosphorylated on tyrosine by oxidants, although its regulation by agonists that stimulate G-protein-coupled receptor signaling pathways and the physiological consequences of tyrosine phosphorylation in this setting have not been fully identified. We found that angiotensin II stimulates the tyrosine phosphorylation of PDK1 in vascular smooth muscle in a calcium- and c-Src-dependent manner. The calcium-activated tyrosine kinase Pyk2 acts as a scaffold for Src-dependent phosphorylation of PDK1 on Tyr9, which permits phosphorylation of Tyr373 and -376 by Src. This critical function of Pyk2 is further supported by the observation that Pyk2 and tyrosine-phosphorylated PDK1 colocalize in focal adhesions after angiotensin II stimulation. Importantly, infection of smooth muscle cells with a Tyr9 mutant of PDK1 inhibits angiotensin II-induced tyrosine phosphorylation of paxillin and focal adhesion formation. These observations identify a novel interaction between PDK1 and Pyk2 that regulates the integrity of focal adhesions, which are major compartments for integrating signals for cell growth, apoptosis, and migration.     

Synthesis of radiolabeled biphenylsulfonamide matrix metalloproteinase inhibitors as new potential PET cancer imaging agents

Novel matrix metalloproteinase (MMP) inhibitor radiotracers, (S)-3-methyl-2-(2',3',4'-methoxybiphenyl-4-sulfonylamino)-butyric acid [(11)C]methyl ester (1a-c), (S)-3-methyl-2-(2',3',4'-fluorobiphenyl-4-sulfonylamino)-butyric acid [(11)C]methyl ester (1d-f), and (S)-3-methyl-2-(4'-nitrobiphenyl-4-sulfonylamino)-butyric acid [(11)C]methyl ester (1g), a series of substituted biphenylsulfonamide derivatives, have been synthesized for evaluation as new potential positron emission tomography (PET) cancer imaging agents.     

Synthesis and SAR of aminoalkoxy-biaryl-4-carboxamides: novel and selective histamine H3 receptor antagonists

Novel 4'-[(NR1R2-1-yl)]-propoxy-biaryl-4-carboxamides were designed and synthesized. All compounds were tested for affinity at histamine H(3)receptors. Most compounds were highly potent and selective for human and rat H(3) receptors and selected examples such as A-349821 showed functional antagonism of H(3) receptors in vitro and in a mouse dipsogenia model.