Functional and molecular evidence for Na+-HCO3- cotransporter in porcine vas deferens epithelia

This study focused on the role ofsodium-bicarbonate cotransporter (NBC1) in cAMP-stimulated iontransport in porcine vas deferens epithelium. Ion substitutionexperiments in modified Ussing chambers revealed that cAMP-mediatedstimulation was dependent on the presence of Na⁺,HCO, and Cl for a full response.HCO-dependent current was unaffected byacetazolamide, bumetanide, or amiloride but was inhibited bybasolateral 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.Na⁺-driven, HCO-dependent,stilbene-inhibitable anion flux was observed across the basolateralmembrane of selectively permeabilized monolayers. Results ofradiotracer flux studies suggest a4,4'-dinitrostilbene-2,2'-disulfonate-sensitive stoichiometry of 2 baseequivalents per Na⁺. Antibodies raised against rat kidneyNBC epitopes (rkNBC; amino acids 338-391 and 928-1035)identified a single band of ~145 kDa. RT-PCR detected NBC1 message inporcine vas deferens epithelia. These results demonstrate that vasdeferens epithelial cells possess the proteins necessary for thevectoral transport of HCO and that these mechanismsare maintained in primary culture. Taken together, the results indicatethat vas deferens epithelia play an active role in male fertility andhave implications for our understanding of the relationship betweencystic fibrosis and congenital bilateral absence of the vas deferens.

     

Functional studies of the kidney of living animals using multicolor two-photon microscopy

Optical microscopy, when applied to livinganimals, provides a powerful means of studying cell biology in the mostphysiologically relevant setting. The ability of two-photon microscopyto collect optical sections deep into biological tissues has opened upthe field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way inwhich two-photon microscopy can be applied to intravital studies ofkidney physiology. Because the kidney is easily externalized withoutcompromising its function, microscopy can be used to evaluate variousaspects of renal function in vivo. These include cell vitality andapoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes andsimultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of imagescontaining uncharacterized autofluorescence. The studies described heredemonstrate the way in which two-photon microscopy can provide a levelof resolution previously unattainable in intravital microscopy,enabling kinetic analyses and physiological studies of the organs ofliving animals with subcellular resolution.

     

Growth pH and transient increases in amino acid availability influence polyglucose synthesis by Fusobacterium nucleatum grown in continuous culture

When exponential phase cultures of Lactococcus lactis were directly exposed to severe stresses (acid, bile salt, heat, and hydrogen peroxide) for a prolonged period, most of the cells were quickly killed, however, a small number of the cells, approximately 0.01% of the population, was found to survive. How these 'survivor' cells might have survived the stresses, when other supposedly-the-same cells could not, was investigated. The cultures were not exposed to any mild stresses prior to the exposure to the severe stresses, and therefore adaptation can be ruled out as the cause of survival. When the survivor cells were re-cultured and re-exposed to the same severe stresses a similar pattern of survival was displayed, indicating that the survivor cells were not stress-resistant mutants. Furthermore, the survivor cells displayed typical growth kinetics once they were freed of the stresses. The survivor cells appear to be in a distinct physiological state, because when they were tested against a second stress they exhibited significantly greater survival against that stress than the normal cells exposed to the same stress. Also, cells at different time points of synchronously growing culture displayed different levels of survival against stress. It is proposed that the difference in survival of exponential phase cells is due to the difference in the protein makeup of cells at different stages of the cell cycle.     

What's in a name? Are MSG and umami the same?

The Japanese word `umami' has a long past. It was already inuse during the Edo period (Tokugawa Shogunate) of Japanese history,which ended in 1868 (Mason, 1993). In Japanese, `umami' oftenconnotes a cognitive category (Yamaguchi and Ninomiya, 1998) oftaste, or perhaps flavor, with definitions that include deliciousness, flavor,relish,     

The role of hepatocyte growth factor (scatter factor) in epithelial-mesenchymal transition and breast cancer

North American women have a one in eight lifetime risk of developing breast cancer, and approximately one in three women with breast cancer will die of metastases. We, and others, have recently shown that high levels of expression of hepatocyte growth factor (HGF) and its receptor Met are associated with invasive human breast cancer and may be causally linked to metastasis. This high level of HGF and Met expression has been considered as a possible indicator of earlier recurrence and shortened survival in breast cancer patients. In contrast, HGF expression (but not Met) is strongly suppressed in normal breast epithelial cells. HGF and Met are therefore candidate targets for therapeutic intervention in the treatment of breast cancer. We have recently demonstrated that sustained activation or hyper-activation of c-Src and Stat3, which occurs in invasive breast cancer, can stimulate strong expression of HGF in carcinoma cells. In contrast, transient induction of Stat3 occurs in normal epithelium and promotes mammary tubulogenesis. We hypothesize that increased autocrine HGF-Met signaling is a critical downstream function of c-Src-Stat3 activation in mammary tumorigenesis. Future studies will identify novel Stat3 consensus sites that regulate HGF promoter activity and HGF expression preferentially in carcinoma cells and could lead to novel therapeutic drugs that specifically block HGF expression in mammary carcinoma cells, and which could be used in combined treatments to abrogate metastasis.     

Fe(2+) induces a transient Ca(2+) release from rat liver mitochondria

Isolated mitochondria loaded with Ca(2+) and then exposed to Fe(2+) show a transient release of Ca(2+). The magnitude of this response depends on the Ca(2+) loading and the kinetics of the response depends on the concentration of added Fe(2+). We investigated the Fe(2+)-induced Ca(2+) release mechanism by measuring mitochondrial Ca(2+) uptake in the presence of Fe(2+). The presence of Fe(2+) inhibits Ca(2+) uptake two times. Since mitochondria can cycle Ca(2+) across their inner membrane, the suppression of Ca(2+) uptake, but not release, results in an elevation of the extramitochondrial Ca(2+), thereby varying the steady state. The transient release of Ca(2+) initially observed from mitochondria appears to occur via the electroneutral 2H(+)/Ca(2+)-exchange mechanism, since it can be markedly decreased by cyclosporin A and does not involve lipid peroxidation. When Fe(2+) accumulation is completed, reuptake of released Ca(2+) into mitochondria resumes. Finally, we propose that Fe(2+) either inhibits Ca(2+) entry at the uniporter or is transported by it into the matrix.     

PDZ domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator

The C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-DeltaTRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-DeltaTRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from approximately 24 to approximately 13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-DeltaTRL did not differ. However, endocytic recycling of CFTR-DeltaTRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells.     

Design and synthesis of 4,5-disubstituted-thiophene-2-amidines as potent urokinase inhibitors

A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility.     

Comparative population structure and genetic diversity of Arceuthobium americanum (Viscaceae) and its Pinus host species: insight into host-parasite evolution in parasitic angiosperms

In a recent study we revealed that the parasitic angiosperm Arceuthobium americanum is comprised of three distinct genetic races, each associated with a different host in regions of allopatry. In order to assess the role of host identity and geographical isolation on race formation in A. americanum, we compared the genetic population structure of this parasite with that of its three principal hosts, Pinus banksiana, Pinus contorta var. latifolia and Pinus contorta var. murrayana. Despite the fact that A. americanum was divided into three genetic races, hosts were divided into only two genetic groups: (i) Pinus banksiana and hybrids, and (ii) P. contorta var. latifolia and var. murrayana. These findings suggest that factors such as geographical isolation and adaptation to different environmental conditions are important for race formation in the absence of host-driven selection pressures. To assess factors impacting population structure at the fine-scale, genetic and geographical distance matrices of host and parasite were compared within A. americanum races. The lack of a relationship between genetic and geographical distance matrices suggests that isolation-by-distance plays a negligible role at this level. The effect of geographical isolation may have been diminished because of the influence of factors such as random seed dispersal by animal vectors or adaptation to nongeographically patterned environmental conditions. Host-parasite interactions might also have impacted the fine-scale structure of A. americanum because the parasite and host were found to have similar patterns of gene flow.