Evaluation of Cisplatin-Induced Pathology in the Larval Zebrafish Lateral Line

Cisplatin is an effective anticancer agent, but also causes permanent hearing loss by damaging hair cells—the sensory receptors essential for hearing. There is an urgent clinical need to protect cochlear hair cells in patients undergoing cisplatin chemotherapy. The zebrafish lateral line organ contains hair cells and has been frequently used in studies to screen for otoprotective compounds. However, these studies have employed a wide range of cisplatin dosages and exposure times. We therefore performed a comprehensive evaluation of cisplatin ototoxicity in the zebrafish lateral line with the goal of producing a standardized, clinically relevant protocol for future studies. To define the dose- and time-response patterns of cisplatin-induced hair-cell death, we treated 6-day-old larvae for 2 h in 50 µM–1 mM cisplatin and allowed them to recover. We observed delayed hair cell death, which peaked at 4–8 h post-exposure. Cisplatin also activated a robust inflammatory response, as determined by macrophage recruitment and phagocytosis of hair cells. However, selective depletion of macrophages did not affect hair cell loss. We also examined the effect of cisplatin treatment on fish behavior and found that cisplatin-induced lateral line injury measurably impaired rheotaxis. Finally, we examined the function of remaining hair cells that appeared resistant to cisplatin treatment. We observed significantly reduced uptake of the cationic dye FM1-43 in these cells relative to untreated controls, indicating that surviving hair cells may be functionally impaired. Cumulatively, these results indicate that relatively brief exposures to cisplatin can produce hair cell damage and delayed hair cell death. Our observations provide guidance on standardizing methods for the use of the zebrafish model in studies of cisplatin ototoxicity.     

In vitro translation of a 2.3-kb splicing variant of the hamster pgp1 gene whose presence in transfectants is associated with decreased drug resistance

Adherent macrophage populations derived from monocytes isolated from peripheral blood were evaluated for their ability to "shed" the membrane-associated receptor for TNF-alpha (TNFR) following exposure to a calcium ionophor (A23187) and a synthetic chemotactic peptide (fMLP) reagent. A soluble fraction of TNFR was detected in "cell-free" supernatant produced by stimulated macrophage populations applying 125I-TNF-alpha and biotinylated TNF-alpha ligand-binding analysis (96-well format) in combination with conventional autoradiographic techniques. Approximate molecular weight of the shed TNFR glycoprotein fraction was estimated to be 75 kDa based on interpretation of nondenaturing PAGE gels transferred laterally onto sheets of nitrocellulose membrane subsequently probed by ligand-binding analysis applying 125I-TNF-alpha and biotinylated TNF-alpha as detection modalities. Immunorecognition techniques were also employed to detect TNFR fragments shed from macrophages using biotinylated anti-TNFR Type II (75 kDa) monoclonal antibody in combination with conjugated strepavidin:HRPO and a chemiluminescent substrate reagent. In an effort to identify the class of enzyme directly mediating TNFR Type II (75 kDa) shedding, a spectrum of carboxyl- (e.g., aspartate), hydroxyl- (e.g., serine), thiol (e.g., cysteine), and metalo- (e.g., Ca2+, Mg2+) protease-inhibiting agents were evaluated. Experimental findings implied that a carboxy (aspartate) peptidase, and possibly to a lesser extent, serine (hydroxyl), and thiol (cysteine) peptidases participate in macrophage TNFR Type II (75 kDa) shedding phenomena. Subsequent investigations demonstrated that the carboxy (aspartate) peptidase cathepsin-D promoted liberation of TNFR Type II (75 kDa) in unactivated populations of adherent macrophages. In an effort to complement these observations, a protein fraction with presumed carboxy (aspartate) protease activity was isolated from the cell-free supernatant generated by activated populations of adherent macrophages using immobilized pepstatin-A beaded agarose. Exposure of unstimulated populations of adherent macrophages to the partially purified pepstatin-A binding protein fractions resulted in the liberation of a soluble TNFR Type II (75 kDa) fragment based on interpretation of ligand-binding and immunorecognition analysis of samples developed by SDS-PAGE/PAGE format and transferred onto sheets of nitrocellulose membrane. The molecular weight of the macrophage pepstatin-A binding protein fraction was estimated to be 47-52 kDa with lesser bands also visible at approximately 26-32 kDa, and 100 kDa based on SDS-PAGE analysis. Nondenaturing hemoglobin-PAGE substrate gel analysis of protein fractions possessing pepstatin-A binding-avidity detected a protease with a molecular weight of approximately 47-52 kDa that proteolytically digested hemoglobin, in addition to a synthetic cathepsin-D specific peptide substrate. Collective interpretation of these experimental findings directly corresponds with many of the physical (molecular) and functional (biochemical) characteristics known to be associated with the leukocyte carboxy (aspartate) peptidase cathepsin-D, which is a non-metaloprotease known to exert relatively limited proteolytic activity.     

Coding mammograms using the classification "probably benign finding--short interval follow-up suggested"

OBJECTIVE: Many benign breast lesions revealed by mammography show features indicating that the lesions have a high, but not complete, likelihood of being benign. The Breast Imaging Reporting and Data System (BI-RADS) allows radiologists to classify these mammograms as "probably benign finding-short interval follow-up suggested" (category 3). We explored whether certain factors are associated with the use of category 3 in a national cancer detection program. MATERIALS AND METHODS: We analyzed data from the National Breast and Cervical Cancer Early Detection Program, a comprehensive nationwide program that provides cancer screening for low-income and medically underserved women. The study population included all women at least 40 years old who had undergone mammography on or before September 30, 1996 (n = 372,760). RESULTS: Of the 372,760 mammograms, 7.7% were classified as category 3. The probability of receiving a category 3 classification decreased as patients' ages increased. Women who were symptomatic were nearly twice as likely as women who were asymptomatic to receive a category 3 classification, and women whose clinical breast examinations had abnormal findings were more than twice as likely as women with examinations having normal findings to receive a category 3 classification. The percentage of mammograms classified as category 3 by state or tribal organization ranged from 1.4% to 14.0%. CONCLUSION: Several patient variables, including patient symptomatology, were associated with the probability of having a mammogram classified as category 3. One of the most important determinants was where the patient underwent mammography, which suggests that variability exists among radiologists themselves in using this BI-RADS code for "probably benign" mammographic lesions.     

Laboratory investigation of the mass stability of sampling cassettes from inhalable aerosol samplers

A study was conducted to evaluate the mass stability of the materials used in the construction of samplers with internal cassettes for the gravimetric measurement of inhalable aerosol exposures. The internal cassettes from IOM samplers were studied. Results indicate that the mass stability of filters is uniform, but the mass stability of the cassette material may dramatically affect the results of the measurement. Cassettes constructed from plastic exhibited drastic shifts in mass depending on the environmental conditions of their storage. Under room humidity, the plastic cassettes absorbed 1 to 2 mg of water over several days. When these cassettes were placed in a desiccator, they lost mass consistently but did not approach a stable mass. Studies repeated with cassettes made of stainless steel showed negligible mass variability. Based on this study, the use of stainless steel cassettes is recommended for gravimetric determinations of aerosol exposure, although field blanks may in some cases be used for correction of data from plastic cassettes. This study shows the need to evaluate the mass stability of the cassette material of any sampling device where an internal cassette is weighed together with the filter.     

Surface topography of phytochrome A deduced from specific chemical modification with iodoacetamide

Phytochromes are a photoreversible photochromic light switch for photomorphogenesis in plants. The molecular structure and functional mechanism of phytochromes are not fully understood. On the basis of complete mapping of total tryptic digest of the iodoacetamide-modified oat phytochrome A (phyA), the molecular surface topography of phyA was probed by specific chemical modification of cysteine residues with [14C]iodoacetamide. Under native conditions, only two cysteines (Cys-158 and Cys-311) of eleven half-cystines of the N-terminal chromophore binding domain were modified to a significant extent. In the C-terminal domain, six cysteine residues (Cys-715, Cys-774, Cys-809, Cys-869, Cys-961, Cys-995) were readily accessible to iodoacetamide. Among the reactive cysteine residues, only cysteine-311 displayed reactivity that was dependent on the photochromic form (Pr left arrow over right arrow Pfr) of the photoreceptor. Surprisingly, the modification of Cys-311 in the vicinity of the chromophore attachment site (Cys-321) did not have any detectable effect on spectral properties of phyA. Most of the cysteines of the N-terminal domain (Cys-83, Cys-175, Cys-291, Cys-370, Cys-386, Cys-445, Cys-506) are deeply buried in the core of the chromophore binding domain, as they can be modified only after denaturation of the chromoprotein. In the C-terminal domain, modification of only one cysteine residue (Cys-939) required protein denaturation. Since all 22 half-cystines can be modified with iodoacetamide without reduction of the chromoprotein, it follows that oat phyA does not have any disulfide bonds. We found that Cys-311, Cys-774, Cys-961, and Cys-995 could be easily partially oxidized under the conditions used for phytochrome isolation. The surface topography/conformation of oat phyA and its role in protein-protein recognition in phytochrome-mediated signal transduction are discussed in terms of the relative reactivity of cysteine residues.     

Connexin43 is highly localized to sites of disturbed flow in rat aortic endothelium but connexin37 and connexin40 are more uniformly distributed

Vascular endothelial cells are linked by gap junctions, which facilitate the propagation of electrical and chemical signals along the vessel wall. The aim of this study was to determine the distribution and identity of the gap junction structural proteins (connexins) expressed by endothelial cells in situ. Connexin expression in different regions of the rat aortic endothelium was analyzed with the use of indirect immunofluorescence microscopy and Western blotting. Connexin40 and connexin37 were present in most, if not all, of the thoracic and abdominal aortic endothelia in the form of maculae at cell-cell appositions. In contrast, connexin43 was undetectable in most endothelia but extremely abundant in small numbers of cells localized at the downstream edge of the ostia of branching vessels and at flow dividers, regions that experience turbulent shear stress from disturbed blood flow. To examine the relationship of shear stress and connexin43 expression, localized stress was induced by surgical coarctation of the aorta, which was sufficient to cause striking local upregulation of connexin43 within 8 days. Thus, increases in connexin43 levels are an endothelial response to mechanical stress.     

The association of complete laryngotracheoesophageal cleft with left lung agenesis: pathophysiological clues provided by an experiment of nature

The authors present a patient with complete laryngotracheoesophageal cleft and concurrent left lung agenesis and microgastria. Prenatal ultrasound scan showed polyhydramnios and a hypertrophic right lung. The authors propose that the combination of right lung hypertrophy, polyhydramnios, and microgastria in the absence of a competent laryngeal mechanism may suggest that the preferential path for swallowed amniotic fluid was into the lung, rather than the normal route through the stomach. This case illustrates the prenatal findings suggestive of complete laryngotracheoesophageal cleft and lung agenesis, and suggests a potential causal relationship between shunting of swallowed amniotic fluid into the bronchial tree and prenatal lung hypertrophy and microgastria.