Expression of Smad4 in the FaDu cell line partially restores TGF-beta growth inhibition but is not sufficient to regulate fibronectin expression or suppress tumorigenicity

Mutations of the Smad4 gene, a member of a group of TGF-beta signal transduction components, occur in several types of cancer suggesting that its inactivation significantly affects TGF-beta responsiveness in these tumors. To further investigate the role of Smad4 with respect to TGF-beta signaling and carcinogenesis, we re-expressed the Smad4 gene in the Smad4-deficient cancer cell line FaDu by microcell-mediated chromosome transfer (MMCT) and retroviral infection to closely approximate physiological protein levels. The Smad4-expressing FaDu clones were then evaluated for TGF-beta responsiveness to assess the role of Smad4 in TGF-beta-induced growth inhibition and target gene regulation. We found that the re-expression of the Smad4 gene by either method partially restored TGF-beta responsiveness in FaDu cells with respect to both growth inhibition and expression of p21WAF1/CIP1 and p15INK4B. However, only the microcell hybrids showed growth retardation in organotypic raft culture and an enhanced ability to upregulate fibronectin. In contrast, the re-expression of Smad4 by either method failed to suppress tumorigenicity. These results suggest that in addition to a homozygous deletion of Smad4, FaDu cells contain additional defects within the TGF-beta signaling pathway, thereby limiting the extent of TGF-beta responsiveness upon Smad4 re-expression and perhaps accounting for the inability to induce p15INK4B to a high level. They also demonstrate the advantages of providing a physiological extracellular environment, when assessing TGFbeta responsiveness.     

Honey bee (Hymenoptera: Apidae) foraging in response to preconditioning with onion flower scent compounds

Onion (Allium cepa L.) seed production has long been plagued with yield problems because of lack of pollination by the honey bee, Apis mellifera L. To attempt to attract more pollinators to the onion seed production field, honey bees were conditioned to associate onion floral odor components with a reward. Isolated nucleus hives of honey bees were fed 30% sucrose solutions scented with a 0.2% solution of onion floral odor compounds. After feeding on these solutions for 6 wk, bees were not found to prefer onion flowers to two competing food sources, carrot and alfalfa flowers, at the 5% significance level. However, there was an overall trend indicating a change in honey bee behavior, with fewer "trained" bees visiting alfalfa and carrot and more visiting onion. Thus, it may be possible to alter honey bee behavior with preconditioning but probably not to a degree that would be economically significant.     

Noninvasive evaluation of liver metabolism by 2H and 13C NMR isotopomer analysis of human urine

Mammalian liver disposes of acetaminophen and other ingested xenobiotics by forming soluble glucuronides that are subsequently removed via renal filtration. When given in combination with the stable isotopes 2H and 13C, the glucuronide of acetaminophen isolated from urine provides a convenient "chemical biopsy" for evaluating intermediary metabolism in the liver. Here, we describe isolation and purification of urinary acetaminophen glucuronide and its conversion to monoacetone glucose (MAG). Subsequent 2H and 13C NMR analysis of MAG from normal volunteers after ingestion of 2H2O and [U-13C3]propionate allowed a noninvasive profiling of hepatic gluconeogenic pathways. The method should find use in metabolic studies of infants and other populations where blood sampling is either limited or problematic.     

Interdependent folding of the N- and C-terminal domains defines the cooperative folding of alpha-lytic protease

Alpha-lytic protease (alphaLP) serves as an important model in achieving a quantitative and physical understanding of protein folding reactions. Synthesized as a pro-protease, alphaLP belongs to an interesting class of proteins that require pro regions to facilitate their proper folding. alphaLP's pro region (Pro) acts as a potent folding catalyst for the protease, accelerating alphaLP folding to its native conformation nearly 10(10)-fold. Structural and mutational studies suggested that Pro's considerable foldase activity is directed toward structuring the alphaLP C-terminal domain (CalphaLP), a seemingly folding-impaired domain, which is believed to contribute significantly to the high-energy folding and unfolding transition states of alphaLP. Pro-mediated nucleation of alphaLP folding within CalphaLP was hypothesized to subsequently enable the alphaLP N-terminal domain (NalphaLP) to dock and fold, completing the formation of native protease. In this paper, we find that ternary folding reactions of Pro and noncovalent NalphaLP and CalphaLP domains are unaffected by the order in which the components are added or by the relative concentrations of the alphaLP domains, indicating that neither discrete CalphaLP structuring nor docking of the two alphaLP domains is involved in the folding transition state. Instead, the rate-limiting step of these folding reactions appears to be a slow and concerted rearrangement of the NalphaLP and CalphaLP domains to form active protease. This cooperative and interdependent folding of both protease domains defines the large alphaLP folding barrier and is an apparent extension of the highly cooperative alphaLP unfolding transition that imparts the protease with remarkable kinetic stability and functional longevity.     

The wandering carpel mutation of Zea mays (Gramineae) causes misorientation and loss of zygomorphy in flowers and two-seeded kernels

We have isolated a new mutation, wandering carpel (wcr), which affects polarity of the maize flower, altering its orientation or converting it from zygomorphy to radial symmetry. These changes result in the development of embryos on locations other than the normal, acropetal side of the kernel. More than two carpels can develop into silks. More rarely, two ovules develop in a single ovary, giving rise to kernels with two seeds. The wcr mutation is a maternal-sporophyte-effect, semidominant mutation whose expression is background dependent. As spikelets with abnormal flowers are almost always paired with a normal spikelet, we hypothesize that WCR+ is required for establishing polarity in spikelet meristems during inflorescence development.     

Association of specific language impairment (SLI) to the region of 7q31

FOXP2 (forkhead box P2) was the first gene characterized in which a mutation affects human speech and language abilities. A common developmental language disorder, specific language impairment (SLI), affects 6%-7% of children with normal nonverbal intelligence and has evidence of a genetic basis in familial and twin studies. FOXP2 is located on chromosome 7q31, and studies of other disorders with speech and language impairment, including autism, have found linkage to this region. In the present study, samples from children with SLI and their family members were used to study linkage and association of SLI to markers within and around FOXP2, and samples from 96 probands with SLI were directly sequenced for the mutation in exon 14 of FOXP2. No mutations were found in exon 14 of FOXP2, but strong association was found to a marker within the CFTR gene and another marker on 7q31, D7S3052, both adjacent to FOXP2, suggesting that genetic factors for regulation of common language impairment reside in the vicinity of FOXP2.     

The J-domain of Hsp40 couples ATP hydrolysis to substrate capture in Hsp70

The Escherichia coli Hsp40 DnaJ uses its J-domain to target substrate polypeptides for binding to the Hsp70 DnaK, but the mechanism of J-domain function has been obscured by a substrate-like interaction between DnaJ and DnaK. ATP hydrolysis in DnaK is associated with a conformational change that captures the substrate, and both DnaJ and substrate can stimulate ATP hydrolysis. However, substrates cannot trigger capture by DnaK in the presence of ATP, and substrates stimulate a DnaK conformational change that is uncoupled from ATP hydrolysis. The role of the J-domain was examined using the fluorescent derivative of a fusion protein composed of the J-domain and a DnaK-binding peptide. In the absence of ATP, DnaK-binding affinity of the fusion protein is similar to that of the unfused peptide. However, in the presence of ATP, the affinity of the fusion protein is dramatically increased, which is opposite to the decrease in DnaK affinity typically exhibited by peptides. Binding of a fusion protein that contains a defective J-domain is insensitive to ATP. According to results from isothermal titration calorimetry, the J-domain binds to the DnaK ATPase domain with weak affinity (K(D) = 23 microM at 20 degrees C). The interaction is characterized by a positive enthalpy, small heat capacity change (DeltaC(p)= -33 kcal mol(-1)), and increasing binding affinity for increasing temperatures in the physiological range. In conditions that support binding of the J-domain to the ATPase domain, the J-domain accelerates ATP hydrolysis and a simultaneous conformational change in DnaK that is associated with peptide capture. The defective J-domain is inactive, despite the fact that it binds to the DnaK ATPase domain with higher than wild-type affinity. The results are most consistent with an allosteric mechanism of J-domain action in which the J-domain couples ATP hydrolysis to peptide capture by accelerating ATP hydrolysis and delaying DnaK closure until ATP is hydrolyzed.     

Regulation of the calcium release channel from skeletal muscle by suramin and the disulfonated stilbene derivatives DIDS,DBDS, and DNDS

Activation of skeletal muscle ryanodine receptors (RyRs) by suramin and disulfonic stilbene derivatives (Diisothiocyanostilbene-2',2'-disulfonic acid (DIDS), 4,4'-dibenzamidostilbene-2,2'-disulfonic acid (DBDS),and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)) was investigated using planar bilayers. One reversible and two nonreversible mechanisms were identified. K(a) for reversible activation (approximately 100 micro M) depended on cytoplasmic [Ca(2+)] and the bilayer composition. Replacement of neutral lipids by negative phosphatidylserine increased K(a) fourfold, suggesting that reversible binding sites are near the bilayer surface. Suramin and the stilbene derivatives adsorbed to neutral bilayers with maximal mole fractions between 1-8% and with affinities approximately 100 micro M but did not adsorb to negative lipids. DIDS activated RyRs by two nonreversible mechanisms, distinguishable by their disparate DIDS binding rates (10(5) and 60 M(-1) s(-1)) and actions. Both mechanisms activated RyRs via several jumps in open probability, indicating several DIDS binding events. The fast and slow mechanisms are independent of each other, the reversible mechanism and ATP binding. The fast mechanism confers DIDS sensitivity approximately 1000-fold greater than previously reported, increases Ca(2+) activation and increases K(i) for Ca(2+)/Mg(2+) inhibition 10-fold. The slow mechanism activates RyRs in the absence of Ca(2+) and ATP, increases ATP activation without altering K(a), and slightly increases activity at pH < 6.5. These findings explain how different types of DIDS activation are observed under different conditions.     

Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells

The main potential of intrinsically fluorescent proteins (IFPs), as noninvasive and site-specific markers, lies in biological applications such as intracellular visualization and molecular genetics. However, photophysical studies of IFPs have been carried out mainly in aqueous solution. Here, we provide a comprehensive analysis of the intracellular environmental effects on the steady-state spectroscopy and excited-state dynamics of green (EGFP) and red (DsRed) fluorescent proteins, using both one- and two-photon excitation. EGFP and DsRed are expressed either in the cytoplasm of rat basophilic leukemia (RBL-2H3) mucosal mast cells or anchored (via LynB protein) to the inner leaflet of the plasma membrane. The fluorescence lifetimes (within approximately 10%) and spectra in live cells are basically the same as in aqueous solution, which indicate the absence of both IFP aggregation and cellular environmental effects on the protein folding under our experimental conditions. However, comparative time-resolved anisotropy measurements of EGFP reveal a cytoplasmic viscosity 2.5 +/- 0.3 times larger than that of aqueous solution at room temperature, and also provide some insights into the LynB-EGFP structure and the heterogeneity of the cytoplasmic viscosity. Further, the oligomer configuration and internal depolarization of DsRed, previously observed in solution, persists upon expression in these cells. DsRed also undergoes an instantaneous three-photon induced color change under 740-nm excitation, with efficiently nonradiative green species. These results confirm the implicit assumption that in vitro fluorescence properties of IFPs are essentially valid for in vivo applications, presumably due to the beta-barrel protection of the embodied chromophore. We also discuss the relevance of LynB-EGFP anisotropy for specialized domains studies in plasma membranes.     

Membrane protein folding: beyond the two stage model

The folding of alpha-helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable alpha-helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization. Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process. Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure.