Modular microfluidic system as a model of cystic fibrosis airways.

A modular microfluidic airways model system that can simulate the changes in oxygen tension in different compartments of the cystic fibrosis (CF) airways was designed, developed, and tested. The fully reconfigurable system composed of modules with different functionalities: multichannel peristaltic pumps, bubble traps, gas exchange chip, and cell culture chambers. We have successfully applied this system for studying the antibiotic therapy of Pseudomonas aeruginosa, the bacteria mainly responsible for morbidity and mortality in cystic fibrosis, in different oxygen environments. Furthermore, we have mimicked the bacterial reinoculation of the aerobic compartments (lower respiratory tract) from the anaerobic compartments (cystic fibrosis sinuses) following an antibiotic treatment. This effect is hypothesised as the one on the main reasons for recurrent lung infections in cystic fibrosis patients.

Xenopus laevis TRK-fused gene (TFG) is an SH3 domain binding protein highly expressed in the cement gland.

TRK-fused gene (TFG) was originally identified in humans as the N-terminus of an oncogenic fusion protein TRK-T3, associated with papillary thyroid carcinoma. An amino-terminal coiled coil domain of TFG is responsible for mediating oligomerization of the TRK-T3 oncoprotein, resulting in constitutive activation of the TRK protein tyrosine kinase and oncogenesis. We have cloned the Xenopus laevis homologue of TFG and demonstrated that xTFG was highly expressed in the cement gland of tailbud embryos. Overexpression of xTFG2-136 (including the coiled coil domain) in early embryos, via mRNA microinjection as well as transgenic expression using the recently described restriction enzyme mediated integration (REMI) transgenesis, did not alter embryonic development or development of a functional cement gland, despite clear evidence that xTFG2-136 strongly interacted with endogenous xTFG. Finally, we have identified a potential SH3 binding motif in xTFG (and in TFG) and have demonstrated that xTFG selectively interacted with SH3 domains of Src, PLCgamma, and the p85 phosphatidylinositol 3-kinase subunit.

Development of an ED teaching program aimed at reducing prehospital delays for patients with chest pain.

OBJECTIVE: Delays in providing thrombolytic agents to patients with chest pain occur mainly in the prehospital arena. To reduce prehospital delay in treating patients with chest pain, we created a discharge teaching video that emphasized calling 911 in the event of a possible heart attack and a written action plan to be posted near the telephone. We also gave patients their EKG readings to bring with them on their next visit to the emergency department. SETTING AND SAMPLE: All patients with chest pain admitted to the Chest Pain Observation Unit at Baystate Medical Center, Springfield, Mass, were eligible for this teaching effort. We tracked 127 nonconsecutive patients from January 1997 to May 1997. Of these patients, 108 were included in the study. RESULTS: We interviewed 102 patients (94%) 3 days after they were discharged from the Chest Pain Observation Unit. Within this group, 92% were able to describe what a heart attack might feel like, and 81.4% said they would call 911 or go to the hospital if they had symptoms of a heart attack. If they thought that their symptoms might be indigestion, 69% said they would take an antacid, then go to the hospital if they did not feel better. Fifty-one percent remembered what to do with their EKG readings, and 60.7% knew how to take their nitroglycerin correctly. CONCLUSION: We concluded that patients understood the message they were given and retained some of the material 3 days after discharge from the Chest Pain Observation Unit. The follow-up telephone calls revealed areas for improvement in the discharge teaching tools.

Possible relationship between conditions associated with chronic hypoxia and brain mitochondrial DNA deletions.

The brain relies heavily on aerobic metabolism which requires functional mitochondria. Mitochondria are subcellular organelles with their own genome which codes for 13 essential protein subunits. By employing PCR assays to examine brain tissue from 43 age-comparable individuals (between ages 34 and 73), we found a correlation between mitochondrial DNA deletion mutations, mtDNA4977 deletions, and conditions associated with chronic hypoxia. In prior studies, utilizing only 6 to 12 clinical samples, mtDNA4977 deletions were reported to increase in specific regions of the brain with aging. However, we found 12-fold and 5-fold higher levels of mtDNA4977 deletions in the putamen and the superior frontal gyrus of the cortex, respectively, from individuals who had conditions associated with chronic hypoxia when compared with individuals without evidence of such conditions. These findings suggest that chronic hypoxia should be more closely examined in the pathophysiology of central nervous system diseases.

Allelopathic potential ofIpomoea tricolor (Convolvulaceae) in a greenhouse experiment.

The allelopathic potential ofIpomoea tricolor, a plant used in Mexican agriculture to control weeds, and tricolorin A, the major phytogrowth inhibitor present in the so-called "resin glycosides" of this plant, have been evaluated by testing leachates of the plant and the compound on the germination and radicle growth ofAmaranthus hypochondriacus, Echinochloa crusgalli, Senna uniflora, I. tricolor, andI. purpurea. The allelopathic potential ofI. tricolor was evaluated in a greenhouse experiment with dryI. tricolor mixed with sterile and nonsterile soil in pots.A. hypochondriacus was sown in pots containingI. tricolor, 2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5 triazine (Gesaprim) or 1-glyphosphate, and the glyphosphate salt of isopropylamine (Faena), two different commercial herbicides used as a comparison toI. tricolor. Number and dry weights of different monocotyledonous and dicotyledonous weeds andA. hypochondriacus growing in the different treatments were measured.Ipomoea and Faena herbicide had a similar inhibitory effect on monocots.

Genetic analysis of temperature-sensitive mutants which define the genes for the major herpes simplex virus type 2 DNA-binding protein and a new late function.

Eleven temperature-sensitive mutants of herpes simplex virus type 2 (HSV-2) exhibit overlapping patterns of complementation that define four functional groups. Recombination tests confirmed the assignment of mutants to complementation groups 1 through 4 and permitted the four groups to be ordered in an unambiguous linear array. Combined recombination and marker rescue tests (A. E. Spang, P. J. Godowski, and D. M. Knipe, J. Virol. 45:332-342, 1983) indicate that the mutations lie in a tight cluster near the center of UL to the left of the gene for DNA polymerase in the order 4-3-2-1-polymerase. The seven mutants that make up groups 1 and 2 fail to complement each other and mutants in HSV-1 complementation group 1-1, the group thought to define the structural gene for the major HSV-1 DNA-binding protein with a molecular weight of 130,000. At 38 degrees C, mutants in groups 1 and 2 synthesize little or no viral DNA, and unlike cells infected with the wild-type virus, mutant-infected cells exhibit no detectable nuclear antigen reactive with monoclonal or polypeptide-specific antibody to the major HSV-2 DNA-binding protein. The four mutants that make up groups 3 and 4 do not complement each other, nor do they complement mutants in group 2. They do, however, complement mutants in group 1 as well as representative mutants of HSV-1 complementation group 1-1. At 38 degrees C, mutants in groups 3 and 4 are phenotypically DNA+, and nuclei of mutant-infected cells contain the HSV-2 DNA-binding protein. Thus, the four functional groups appear to define two closely linked genes, one encoding an early viral function affecting both viral DNA synthesis and expression of the DNA-binding protein with a molecular weight of 130,000 (groups 1 and 2), and the other encoding a previously unidentified late viral function (groups 3 and 4). The former gene presumably represents the structural gene for the major HSV-2 DNA-binding protein.

Genetic analysis of temperature-sensitive mutants which define the gene for the major herpes simplex virus type 1 DNA-binding protein.

We have assigned eight temperature-sensitive mutants of herpes simplex virus type 1 to complementation group 1-1. Members of this group fail to complement mutants in herpes simplex virus type 2 complementation group 2-2. The mutation of one member of group 1-1, tsHA1 of strain mP, has been shown to map in or near the sequence which encodes the major herpes simplex virus type 1 DNA-binding protein (Conley et al., J. Virol. 37:191-206, 1981). The mutations of five other members of group 1-1 map in or near the sequence in which the tsHA1 mutation maps, a sequence which lies near the center of UL between the genes for the viral DNA polymerase and viral glycoprotein gAgB. These mutants can be divided into two groups; the mutations of one group map between coordinates 0.385 and 0.398, and the mutations of the other group map between coordinates 0.398 and 0.413. At the nonpermissive temperature mutants in group 1-1 are viral DNA negative, and mutant-infected cells fail to react with monoclonal antibody to the 130,000-dalton DNA-binding protein. Taken together, these data indicate that mutants in complementation groups 1-1 and 2-2 define the gene for the major herpes simplex virus DNA-binding protein, an early gene product required for viral DNA synthesis.

Mapping of the herpes simplex virus DNA sequences present in herpes simplex virus type-1 thymidine kinase-transformed cells.

Analyses of the herpes simplex virus (HSV) DNA sequences which are present in three HSV thymidine kinase-transformed (HSVtk+) mouse cell lines have revealed that these cells contain relatively large and variable portions of the viral genome. Two of these cell lines do not contain the viral DNA sequences known to encode the early viral genes normally responsible for regulating tk gene expression during lytic HSV infections. This finding suggests that cell-associated viral tk gene expression may be regulated by cellular rather than viral control mechanisms. In addition, we have compared the viral DNA sequences present in one unstable HSVtk+ cell line to those present in tk- revertant and tk+ rerevertant cell lines sequentially derived from it. Our results have shown that within the limits of sensitivity of our mapping approach, these three related cell lines contain the same set of viral DNA sequences. Thus, gross changes in viral DNA content do not appear to be responsible for the different tk phenotypes of these cells.

Transfer of the herpes simplex thymidine kinase gene from human cells to mouse cells by means of metaphase chromosomes.

Thymidine kinase (TK)-deficient human cells were infected with ultraviolet light-inactivated Herpes simplex virus type 1, and "transformed" cells that expressed Herpes TK activity were isolated. Purified metaphase chromosomes were isolated from the transformed human line and incubated with TK-deficient mouse cells. TK+ cells were selected, and it was shown that these cells were gene transferents which expressed Herpes TK activity, identical to that found in the transformed human cells. The gene transferents contained no intact human chromosomes. When removed from selective pressure, the gene transferents rapidly lost the TK+ phenotype. However, upon continued growth in nonselective medium, a subpopulation in which the TK+ phenotype had become more stabilized appeared. These results suggest that the Herpes gene for thymidine kinase has integrated into the genome of the HSV-transformed human cells and that it can be transferred to other cells by means of purified metaphase chromosomes.

Deletion of the Escherichia coli crp gene.

Spontaneous crp mutants Escherichia coli were selected from a strain that does not require 3',5'-cyclic adenosine monophosphate for CAP activity. Several deletions of the crp gene were characterized. The crp gene was not essential for growth of E. coli. crp mutations reduced the donor ability of Hfr strains.