Comparative teratogenic activity of cancer chemopreventive retinoidal benzoic acid congeners (arotinoids).

The benzoic acid derivatives of retinoic acid, often referred to as arotinoids, are synthetic retinoids that possess some of the properties of vitamin A. In general, these retinoids have more favorable therapeutic ratios, based on acute toxicity in adults, than all-trans-retinoic acid in cancer chemoprevention. In the present study, a single dose of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]benzoic acid (Ro 13-7410; arotinoic acid), ethyl-(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphthalenyl)-1-propen-1-yl]benzoate (Ro 13-6298; arotinoid ethyl ester), (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]phenylmethanol (Ro 13-8320; arotinoic methanol), or (E)-1,2,3,4-tetrahydro-1,1,4, 4-tetramethyl-6-[1-(4-methylphenyl)-1-propen-2-yl]naphthalene (Ro 13-9272; methyl arotinoid) was administered to pregnant Syrian golden hamsters during the early primitive streak stage of gestation. A significant increase in the numbers of litters containing one or more malformed offspring occurred at all doses of each retinoid studied. The types of malformations induced by oral arotinoid treatment were essentially identical to those found after maternal treatment with all-trans-retinoic acid or other teratogenic retinoids during the same gestational age. The results indicate that the alcohol congener was approximately 400 times more potent on a milligram per kilogram basis than all-trans-retinoic acid as a teratogen, and it was 70 times as embryolethal as all-trans-retinoic acid. The ethyl ester congener was 132 times, and the free acid 123 times, as embryolethal as all-trans-retinoic acid. On a molar basis, the arotinoic acid was at least 375 times as teratogenic as all-trans-retinoic acid and at least 140 times as teratogenic as etretinate in hamsters. Because the dose-response curves for arotinoids were significantly parallel to that for all-trans-retinoic acid, and because the spectrum of congenital defects induced by arotinoids was identical to that induced by all-trans-retinoic acid and other teratogenic retinoids, the mechanism of embryopathic action of these conformationally restricted retinoids was concluded to be similar to that of all-trans-retinoic acid.

Phase I and pharmacokinetic evaluation of the combination of orally administered docetaxel and cyclosporin A in tumor-bearing cats.

Intravenously administered docetaxel (DT) is problematic in cats because of the requirement for premedication to ameliorate acute vehicle-induced hypersensitivity reactions. Previously we have revealed that therapeutic plasma concentrations of DT can be achieved in normal and tumor-bearing dogs when DT is administered PO in combination with oral cyclosporin A (CSA). The purpose of this study was to identify the maximally tolerated dosage and characterize the pharmacokinetic disposition of oral DT combined with CSA in cats with tumors. Eighteen tumor-bearing cats were enrolled in this phase I dose escalation and pharmacokinetic study. DT was administered by gavage with CSA (5 mg/kg) twice over a 3-week period. The starting dose of DT was 1.0 mg/kg. Based on the clinical toxicity profile, with gastrointestinal adverse effects and hematologic toxicity the maximal tolerated dose of oral DT was 1.75 mg/kg in combination with 5 mg/kg CSA. Additional studies are necessary to determine the efficacy of DT/CSA in cats with epithelial tumors.

Transcriptional activation of the human osteocalcin gene by basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) has been detected in bone cells and stimulates osteoblast proliferation; however, its role in the regulation of bone metabolism remains speculative. We demonstrated that the human osteocalcin promoter is activated by bFGF when transfected into rat osteoblastic (ROS 17/2.8) cells. This effect is concentration dependent, with a twofold induction at 10 ng/ml detected after 20 h. The bFGF response is independent of both the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and retinoic acid activation of the osteocalcin promoter. To identify the promoter sequences through which bFGF exerts its effect, we tested a series of promoter deletion constructs for their response to bFGF. Deletion of the upstream region between -673 and -588 bp results in a significant loss of induction. Gel-shift analysis demonstrates that proteins present in ROS 17/2.8 nuclear extracts bind specifically to these sequences. This region alone was unable to confer the bFGF response on a minimal osteocalcin or an heterologous promoter. However, sequences between -678 and -476 bp, which also includes the vitamin D response element (VDRE), were able to confer bFGF inducibility on both a minimal osteocalcin and a heterologous promoter. These data suggest that induction of the human osteocalcin promoter by bFGF requires the interaction of more than one sequence element.

Test-retest reliability of three aphasia tests: performance of non-brain-damaged older adults.

This study was designed to assess the test-retest reliability of three aphasia tests. The Auditory Comprehension Test for Sentences (ACTS), the Boston Naming Test (BNT), and the Reading Comprehension Battery for Aphasia (RCBA) were administered on two separate occasions to 31 non-brain-damaged adults aged 50 to 76 years. The results showed acceptable score stability for all three aphasia tests. Sixty-eight percent of the time, older non-brain-damaged adults would be expected to score within 4.0% or less of the total number of test items on repeated testing.

Role of lactoferrin in the tear film.

The surface of the eye provides an inert barrier against infection. Through its unique combination of antimicrobial action and anti-inflammatory activities lactoferrin (Lf) in the tear film plays an important role in the maintenance of ocular health. In order to maintain clarity the eye must provide immunological defense without immunopathology. Along with physical barriers, soluble plasma factors and other proteins such as lysozyme, Lf produced by the acinar cells of the lacrimal gland serves a number of roles in defense for this purpose. Lf in tears provides antimicrobial efficacy by binding free iron thus reducing the availability of iron necessary for microbial growth and survival as well as pathogenesis. Lf has been shown to inhibit biofilm formation and thus may play a role in protecting contact lens surfaces from colonization. Virus particles' entry into epithelial cells is inhibited by Lf while an excess of Lf in tear film is thought to limit the opportunistic Lf-mediated bridging of adenovirus and host cell that occurs in other tissues. Lf dampens the classical complement activation pathway by binding to markers of inflammation and immune activation while pathogen-associated molecular patterns such as lipopolysaccharide (LPS) are targeted by Lf for removal through tears and hydrodynamic flushing. This review focuses on the role of Lf in human tear film and its contribution to ocular health during contact lens wear.

Presence or absence of lipopolysaccharide O antigens affects type III secretion by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the major causative agents of mortality and morbidity in hospitalized patients due to a multiplicity of virulence factors associated with both chronic and acute infections. Acute P. aeruginosa infection is primarily mediated by planktonic bacteria expressing the type III secretion system (TTSS), a surface-attached needle-like complex that injects cytotoxins directly into eukaryotic cells, causing cellular damage. Lipopolysaccharide (LPS) is the principal surface-associated virulence factor of P. aeruginosa. This molecule is known to undergo structural modification (primarily alterations in the A- and B-band O antigen) in response to changes in the mode of life (e.g., from biofilm to planktonic). Given that LPS exhibits structural plasticity, we hypothesized that the presence of LPS lacking O antigen would facilitate eukaryotic intoxication and that a correlation between the LPS O-antigen serotype and TTSS-mediated cytotoxicity would exist. Therefore, strain PAO1 (A+ B+ O-antigen serotype) and isogenic mutants with specific O-antigen defects (A+ B-, A- B+, and A- B-) were examined for TTSS expression and cytotoxicity. A strong association existed in vitro between the absence of the large, structured B-band O antigen and increased cytotoxicity of these strains. In vivo, all three LPS mutant strains demonstrated significantly increased lung injury compared to PAO1. Clinical strains lacking the B-band O antigen also demonstrated increased TTSS secretion. These results suggest the existence of a cooperative association between LPS O-antigen structure and the TTSS in both laboratory and clinical isolates of P. aeruginosa.

Loss of bacterial diversity during antibiotic treatment of intubated patients colonized with Pseudomonas aeruginosa.

Management of airway infections caused by Pseudomonas aeruginosa is a serious clinical challenge, but little is known about the microbial ecology of airway infections in intubated patients. We analyzed bacterial diversity in endotracheal aspirates obtained from intubated patients colonized by P. aeruginosa by using 16S rRNA clone libraries and microarrays (PhyloChip) to determine changes in bacterial community compositions during antibiotic treatment. Bacterial 16S rRNA genes were absent from aspirates obtained from patients briefly intubated for elective surgery but were detected by PCR in samples from all patients intubated for longer periods. Sequencing of 16S rRNA clone libraries demonstrated the presence of many orally, nasally, and gastrointestinally associated bacteria, including known pathogens, in the lungs of patients colonized with P. aeruginosa. PhyloChip analysis detected the same organisms and many additional bacterial groups present at low abundance that were not detected in clone libraries. For each patient, both culture-independent methods showed that bacterial diversity decreased following the administration of antibiotics, and communities became dominated by a pulmonary pathogen. P. aeruginosa became the dominant species in six of seven patients studied, despite treatment of five of these six with antibiotics to which it was sensitive in vitro. Our data demonstrate that the loss of bacterial diversity under antibiotic selection is highly associated with the development of pneumonia in ventilated patients colonized with P. aeruginosa. Interestingly, PhyloChip analysis demonstrated reciprocal changes in abundance between P. aeruginosa and the class Bacilli, suggesting that these groups may compete for a similar ecological niche and suggesting possible mechanisms through which the loss of microbial diversity may directly contribute to pathogen selection and persistence.

Computer model to characterize the air volume displaced by the vibrating vocal cords.

We use a vocal-cord/vocal-tract model devised earlier to study acoustic effects of the air volume displaced by the vibrating vocal cords. We consider both lateral and longitudinal motion of the cords. We make computer simulations to establish quantitatively the contribution of the displacement current to the total glottal volume velocity (and hence to the total sound excitation for voiced sounds). The displacement current from both lateral and longitudinal motion is found to be second-order by comparison to the total glottal flow. The displacement contribution can, however, be identified in the time waveforms and spectra of the glottal and mouth volume velocities. We use the computer formulation to synthesize speech sounds with and without the displacement current. Auditory observations and spectral calculations on the synthetic output show the displacement current to be discriminable only in close differential comparisons.

Parathyroid hormone-stimulated cadmium accumulation in Madin-Darby canine kidney cells.

Although most renal cadmium transport occurs in proximal tubules indirect evidence suggests that distal tubules may also transport this heavy metal. Since the distal nephron is the site at which parathyroid hormone (PTH) regulates calcium absorption, we evaluated the effects of PTH on Cd2+ accumulation in Madin-Darby canine kidney (MDCK) cells. MDCK cells express a distal-like phenotype including PTH-sensitive adenylyl cyclase and stimulation of calcium transport. MDCK cells were grown to confluence in phenol red-free Dulbecco's modified Eagle's medium. PTH increased 109CdCl2 accumulation in a concentration-dependent manner over the range of 10(-11)-10(-9) M bPTH[1-34]. At 10(-9) M, PTH increased Cd2+ accumulation maximally by 205%. The PTH antagonist, bPTH[3-34], failed to augment 109Cd2+ accumulation. The dihydropyridine agonist, Bay k 8644, in the presence of PTH, increased 109Cd2+ uptake by 200% over vehicle-treated controls and by approximately 100% over PTH or Bay k 8644 alone. The apparent Km for Bay k 8644 activation was 1.3 microM. Bay k 8644-augmented 109Cd2+ uptake was competitively inhibited by the calcium channel antagonist nifedipine. No voltage dependence of Bay k 8644-amplified 109Cd2+ uptake could be detected. Based on these observations we conclude: (1) MDCK cells accumulate Cd2+; (2) PTH increases Cd2+ uptake into MDCK cells; and (3) Cd2+ entry in kidney epithelial cells is mediated, at least in part, by dihydropyridine-sensitive calcium channels.

Polymerase chain reaction for fast, nonradioactive detection of high- and low-risk papillomavirus types in routine cervical specimens and in biopsies.

A polymerase chain reaction (PCR) procedure capable of amplifying specific DNA sequences by up to a millionfold was developed for detection of infection by human papillomaviruses (HPVs) of low (HPV6, HPV11) or high (HPV16, HPV18, HPV33) oncogenic potential. For high-risk HPVs the region chosen was within the E6 open reading frame, which can become integrated into genomic DNA. A region corresponding to this was chosen for low-risk HPVs. After repeated cycles of specific oligonucleotide-primed extension of viral DNA with Klenow or thermophilic DNA polymerase, the type of HPV present was then determined on the basis of the size of the ethidium-bromide-stained band visible after polyacrylamide gel electrophoresis: for HPV6 or 11 the band was approximately 120 bp, for HPV 16 or 33 it was approximately 200 bp, and for HPV18 it was approximately 100 bp. Specific hybridization to the relevant band was seen using radioactive or nonradioactive (alkaline-phosphatase-linked) target oligonucleotide probes. Using the PCR method, we have determined, within as little as a few hours, the infection status of a variety of clinical specimens, including cervical scrapes and lavages, anal scrapes, and anogenital biopsies. The PCR steps can be automated, adding to the potential of PCR for widespread use in the detection of HPV, which is becoming increasingly popular in cervical screening.

Stimulation by parathyroid hormone of calcium absorption in confluent Madin-Darby canine kidney cells.

Parathyroid hormone (PTH) increases renal calcium absorption exclusively in cortical thick limbs and distal tubules. Lack of sufficient tissue has precluded detailed biochemical study of the mechanisms responsible for the hypercalcemic effect of PTH. Therefore, we assessed PTH action on calcium transport in Madin-Darby canine kidney (MDCK) cells, a cell line expressing distal characteristics, to determine its suitability as a model for analyzing PTH action. Calcium transport across MDCK cells grown to confluence on porous filters was measured at 37 degrees C in Ussing chambers. Mucosal-to-serosal calcium fluxes (JCa, mol/min cm-2 x 10(-9)) were measured with 45Ca at -3, -1, 5, 10, and 20 min; agonist was added at 0 min. Basal JCa averaged 0.98. PTH at 0.2 microM increased JCa by 12% (P less than 0.05) and 1 microM PTH by 70% (P less than 0.01). Calcitonin (1 microM) had no effect on JCa. The fact that high concentrations of dibutyryl cAMP (1 mM) and forskolin (10 microM) increased JCa by only 37% and 22%, respectively, suggested that cAMP-independent mechanisms may participate in PTH-stimulated JCa. Therefore we examined the effect of other putative second messengers. In the presence of 2 mM external [Ca], 10 nM A23187 increased JCa by 88%, and 10 microM A23187 increased JCa by 121%. Addition of 10 microM phorbol 12-myristate 13-acetate (PMA) increased JCa by 60%. We conclude that: 1) PTH specifically stimulates unidirectional calcium absorption in MDCK cells; 2) both adenylate cyclase-coupled and calcium-coupled receptors may participate in signaling the response to PTH; and 3) confluent MDCK cells represent a useful experimental model for elucidating the biochemical mechanisms involved in the renal hypercalcemic action of PTH.

Ski-interacting protein interacts with Smad proteins to augment transforming growth factor-beta-dependent transcription.

Transforming growth factor-beta (TGF-beta) signaling requires the action of Smad proteins in association with other DNA-binding factors and coactivator and corepressor proteins to modulate target gene transcription. Smad2 and Smad3 both associate with the c-Ski and Sno oncoproteins to repress transcription of Smad target genes via recruitment of a nuclear corepressor complex. Ski-interacting protein (SKIP), a nuclear hormone receptor coactivator, was examined as a possible modulator of transcriptional regulation of the TGF-beta-responsive promoter from the plasminogen activator inhibitor gene-1. SKIP augmented TGF-beta-dependent transactivation in contrast to Ski/Sno-dependent repression of this reporter. SKIP interacted with Smad2 and Smad3 proteins in vivo in yeast and in mammalian cells through a region of SKIP between amino acids 201-333. In vitro, deletion of the Mad homology domain 2 (MH2) domain of Smad3 abrogated SKIP binding, like Ski/Sno, but the MH2 domain of Smad3 alone was not sufficient for protein-protein interaction. Overexpression of SKIP partially overcame Ski/Sno-dependent repression, whereas Ski/Sno overexpression attenuated SKIP augmentation of TGF-beta-dependent transcription. Our results suggest a potential mechanism for transcriptional control of TGF-beta signaling that involves the opposing and competitive actions of SKIP and Smad MH2-interacting factors, such as Ski and/or Sno. Thus, SKIP appears to modulate both TGF-beta and nuclear hormone receptor signaling pathways.

Cross-talk between 1,25-dihydroxyvitamin D3 and transforming growth factor-beta signaling requires binding of VDR and Smad3 proteins to their cognate DNA recognition elements.

1,25-Dihydroxyvitamin D(3) (vitamin D) and transforming growth factor-beta (TGF-beta) regulate diverse biological processes including cell proliferation and differentiation through modulation of the expression of target genes. Members of the Smad family of proteins function as effectors of TGF-beta signaling pathways whereas the vitamin D receptor (VDR) confers vitamin D signaling. We investigated the molecular mechanisms by which TGF-beta and vitamin D signaling pathways interact in the regulation of the human osteocalcin promoter. Synergistic activation of the osteocalcin gene promoter by TGF-beta and vitamin D was observed in transient transfection experiments. However, in contrast to a previous report by Yanagisawa, J., Yanagi, Y., Masuhiro, Y., Suzawa, M., Watanabe, M., Kashiwagi, K., Toriyabe, T., Kawabata, M., Miyazono, K., and Kato, S. (1999) Science, 283, 1317-1321, synergistic activation was not detectable when the osteocalcin vitamin D response element (VDRE) alone was linked to a heterologous promoter. Inclusion of the Smad binding elements (SBEs) with the VDRE in the heterologous promoter restored synergistic activation. Furthermore, this synergy was dependent on the spacing between VDRE and SBEs. The Smad3-Smad4 heterodimer was found to bind in gel shift assay to two distinct DNA segments of the osteocalcin promoter: -1030 to -989 (SBE3) and -418 to -349 (SBE1). Deletion of SBE1, which is proximal to the VDRE, but not the distal SBE3 in this promoter reporter abolished TGF-beta responsiveness and eliminated synergistic co-activation with vitamin D. Thus the molecular mechanism, whereby Smad3 and VDR mediate cross-talk between the TGF-beta and vitamin D signaling pathways, requires both a VDRE and a SBE located in close proximity to the target promoter.

Automated polymerase chain reaction for papillomavirus screening of cervicovaginal lavages: comparison with dot-blot hybridization in a sexually transmitted diseases clinic population.

The aim of the present study was to compare the recently developed polymerase chain reaction (PCR) technique with conventional dot-blot DNA hybridization for human papillomavirus (HPV) detection. Cells were collected by cervicovaginal lavage from a study group of 109 women attending a sexually transmitted diseases clinic. Using a machine that we developed for alternation of temperature cycles, HPV was detected in 51% of patients by PCR. By dot-blot hybridization, 44% of the patients were positive. Concordance of combined positive and negative results between PCR and dot blot was 69%. The greater sensitivity of PCR may have accounted for 19% of specimens that were PCR positive but dot-blot negative. Unexpectedly, however, 12% of specimens were dot-blot positive but negative by PCR, and several specimens were discordant for type of HPV. Both HPV DNA tests agreed with cytology in 41% of women, and in 33% cytology was negative in the face of positive PCR and dot blot. Concordance of cytology with just PCR was 59%, and only with dot blot was 56%. Cervicography agreed with both HPV DNA tests in 41% of patients, with PCR alone in 55%, and with dot blot alone in 58%. Biopsy results did not reveal a strong correlation between histopathological criteria of HPV infection and detection of HPV DNA by either PCR or dot-blot hybridization. Thus the present study has shown that PCR is a slightly more sensitive indicator of HPV infection than dot-blot hybridization. Agreement of HPV DNA results with conventional screening tests was not strong, an observation consistent with many comparative studies by others. In conclusion, PCR is slightly more sensitive than DNA hybridization for detection of HPV, it can be used in conjunction with specimen collection by gentle lavage of the cervicovaginal epithelium, and the possibility remains that it may prove suitable as a screening test.