High EphA3 expressing ophthalmic trigeminal sensory axons are sensitive to ephrin-A5-Fc: implications for lobe specific axon guidance.

The ophthalmic, maxillary and mandibular axon branches of the trigeminal ganglion provide cutaneous sensory innervation to the vertebrate face. In the chick embryo, the trigeminal ganglion is bilobed, with ophthalmic axons projecting from the ophthalmic lobe, while maxillary and mandibular projections emerge from the maxillomandibular lobe. To date, target tissue specific guidance cues that discriminately guide the axon projections from the two trigeminal ganglion lobes are unknown. EphA receptor tyrosine kinases and ephrin-A ligands are excellent candidates for this process as they are known to mediate axon guidance in the developing nervous system. Accordingly, the expression of EphAs and ephrin-As was investigated at stages 13, 15, 20 of chick embryogenesis when peripheral axons from the trigeminal ganglion are pathfinding. EphA3 is expressed highly in the ophthalmic trigeminal ganglion lobe neurons in comparison to maxillomandibular trigeminal ganglion lobe neurons. Furthermore, from stages 13-20 ephrin-A2 and ephrin-A5 ligands are only localized to the mesenchyme of the first branchial arch (maxillary and mandibular processes), the target fields for maxillomandibular trigeminal ganglion axons. We found that ophthalmic and not maxillomandibular lobe axons were responsive to ephrin-A5-Fc utilizing a substratum choice assay. The implication of these results is that EphA3 forward signaling in ophthalmic sensory axons may be an important mechanism in vivo for lobe specific guidance of trigeminal ganglion ophthalmic projections.

Alternative splicing and expressivity of the Axin(Fu) allele in mice.

Variable expressivity and incomplete penetrance are common for numerous mutations. In most cases the reasons behind these phenomena remain obscure. Caused by the insertion of a murine retrotransposon into intron 6 of the Axin locus, the Axin(Fu) mutation induces alternative splicing and ultimately leads to abnormal tail development in mice. In this investigation RNase protection assay was used to investigate the potential connection between alternative splicing with the expressivity and penetrance of the mutant allele. The results reported here confirm previous observations that alternative splicing occurs in mRNA transcribed from the mutant Axin(Fu) allele. However this investigation also shows that lower levels of alternative splicing commonly take place in the wild type transcript. Correlation analysis reveals a significant connection between tail abnormalities and the ratio of correct to alternatively spliced mRNAs. Overall this paper demonstrates that higher levels of alternatively spliced mRNAs correlate with stronger expression of the mutant trait.

Developmental mosaicism may explain spontaneous reappearance of the Axin(Fu) mutation in mice.

The Axin(Fu) mutation is caused by an IAP insertion. In a small percentage of mice, the Axin(Fu) mutation disappears and is not observed in the next generation. In these mice, [Axin(Fu)]/+, the IAP is absent and Axin(Fu) has reverted to the wild allele. Concomitantly with the loss of IAP, there is widespread reorganisation of numerous microsatellite loci across the surrounding region of chromosome. In rare cases, spontaneous reappearance of the mutation can be observed in progeny of [Axin(Fu)]/+ mice. It is revealed here that reappearance of Axin(Fu) was associated with restoration of the IAP insertion. In such mice, alleles of the surrounding microsatellite loci were identical to the alleles observed on chromosomes that carried Axin(Fu). Developmental mosaicism can potentially explain spontaneous reappearance of the Axin(Fu) mutation. Mosaicism can also explain other observations including postnatal changes at the Axin locus, unusual segregation in progeny, and the appearance of [Axin(Fu)]/+ mice that were phenotypically mutant. 2001.

Unusual inheritance of the AxinFu mutation in mice is associated with widespread rearrangements in the proximal region of chromosome 17.

AxinFu is a mutation in mice that causes fused tails and other developmental abnormalities as a result of insertion of an intracisternal-A particle (IAP), a murine retrotransposon, into intron 6. In a small percentage of offspring we found that the mutant allele reverts to wild-type through loss of the insertion with concomitant disappearance of the mutant phenotype. Investigation of a series of microsatellite loci in the proximal region of chromosome 17 revealed novel alleles which arise simultaneously with disappearance of IAP from AxinFu. These novel microsatellite variants are distinct from the parental alleles and those so far discovered are organized into two haplotypes. Both haplotypes demonstrate stable Mendelian inheritance. Results show that these rearrangements, which are involved in the production of the new haplotypes, exceed millions of base pairs.

Multimodality therapy of patients with locally advanced squamous cell cancer of the head and neck: preliminary results of two pilot trials using paclitaxel and cisplatin.

Chemotherapy has been integrated into the initial treatment of patients with locally advanced squamous cell cancer of the head and neck to improve locoregional control and survival. Two strategies for improving these outcomes are the use of new, potentially more effective drugs either with concurrent radiotherapy or as induction regimens. Because of its inherent activity against squamous cell cancer of the head and neck and its radiation-sensitizing properties, paclitaxel may be a valuable agent in the treatment of this patient population. We describe the preliminary results of two trials that evaluated the combination of paclitaxel and cisplatin in patients with locally advanced disease: a phase I trial of weekly paclitaxel and cisplatin with concurrent postoperative radiation therapy in patients with high-risk disease and a phase I/II trial of paclitaxel as a 96-hour infusion in combination with cisplatin as induction therapy. These studies identified tolerable doses of paclitaxel and cisplatin administered in these settings, with apparent clinical activity. These trials formed the basis for subsequent evaluation of induction paclitaxel and cisplatin followed by definitive radiotherapy and concurrent weekly paclitaxel and cisplatin plus radiotherapy.

A novel subfamily of LINE-derived elements in mice.

Hybrid sequences have been described previously that consist of a 5' region homologous to ORF2 of LINEs and a 3' end that shares homology with a sequence located in the first intron of Cepsilon immunoglobulin. The present investigation has revealed 14 new sequences from seven murine species, that show high homology to those observed earlier. Database search has found several new homologous hybrid sequences including one located in the mouse T-cell receptor (Tcra) locus. Several interesting features of this sequence include identical 15-bp flanking short direct repeats as well as poly-A signal and A-rich sequence at the 3' end. We have classified this set of sequences as LINE-derived elements (LDEs), which constitute a newly observed subfamily. Comparative analysis of these sequences suggests that a single recombination event was responsible for the production of an LDE progenitor. The phylogenetic tree shows a number of elements that pre-existed in the common ancestor of murine species and displays different evolutionary rates. The time of LDE origin is estimated at approximately 10-15 MYA.

Phase I study of paclitaxel on a 3-hour schedule followed by carboplatin in untreated patients with stage IV non-small cell lung cancer.

This study sought to determine the principal toxicities and feasibility of administering paclitaxel as a 3-hour infusion followed by carboplatin without and with granulocyte colony-stimulating factor (G-CSF) in chemotherapy-naive patients with stage IV non-small cell lung carcinoma (NSCLC), and to recommend doses for subsequent clinical trials. Twenty-three patients were treated with paclitaxel at doses ranging from 175 to 225 mg/m2 followed by carboplatin targeting area under the concentration-time curve (AUC) 7 or 9 mg/mL.min every 3 weeks. AUCs were targeted using the Calvert formula with estimated creatinine clearance as a surrogate for the glomerular filtration rate. A high rate of intolerable, mutually exclusive toxicities, consisting primarily of thrombocytopenia, as well as neutropenia, nausea and vomiting, and mucositis, precluded escalation of carboplatin above a targeted AUC of 7 mg/mL.min with paclitaxel 225 mg/m2, which approaches the maximum tolerated dose (MTD) of paclitaxel given as a single agent on a 3-hour schedule. Moderate to severe peripheral neurotoxicity occurred in several patients after multiple courses. Due to the heterogeneous nature of the principal toxicities and the ability to administer clinically-relevant doses of both agents in combination without G-CSF, further dose escalation using G-CSF was not performed. Nine of 23 (39%) total patients and 43% of 21 assessable patients had partial responses (PR). The recommended doses for subsequent clinical trials are paclitaxel 225 mg/m2 as a 3-hour infusion followed by carboplatin at a targeted AUC of 7 mg/mL.min. The ability to administer clinically-relevant single agent doses of paclitaxel and carboplatin in combination, as well as the significant antitumor activity noted in this phase I trial, indicate that further evaluations of this regimen in both advanced and early stage NSCLC are warranted.

Phase I study of paclitaxel as a 3-hour infusion followed by carboplatin in untreated patients with stage IV non-small cell lung cancer.

Preliminary results of a phase I study of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ), given by 3-hour infusion, followed by carboplatin in chemotherapy-naive patients with stage IV non-small cell lung cancer indicate that both agents can be combined at clinically relevant single-agent doses. The paclitaxel (mg/m2)/carboplatin area under the concentration-time curve (mg.min/mL) dose level of 225/7 is projected to be the maximally tolerated and recommended phase II dose level for future evaluations. Dose-limiting neutropenia, thrombocytopenia, and nausea and vomiting preclude treatment with carboplatin doses estimated to target an area under the concentration-time curve of 9 mg.min/mL when given with paclitaxel 225 mg/m2. The heterogeneous nature of the principal toxicities, as well as the ability to administer clinically relevant single-agent doses of both agents in combination, also indicate that further dose escalation of paclitaxel and carboplatin using hematopoietic growth factors would not be feasible. The preliminary antitumor activity noted to data, as well as the safety associated with the clinically relevant single-agent doses that can be given in combination, indicate that phase II/III evaluations of this regimen are warranted in patients with both advanced and early stage non-small cell lung cancer.

Esophageal cancer.

Esophageal cancer is a disease of the developing nations worldwide, and it is associated with nutritional deficiencies and exposure to tobacco and alcohol. In the West, the epidemiology is changing with an unexplained increase in adenocarcinoma of the distal esophagus and gastroesophageal junction, whereas rates for squamous cell carcinoma remain stable. Advances in our understanding of the epidemiology and molecular biology of this disease are being translated into clinical applications in screening and prognosis. Combined-modality therapy with chemotherapy, radiation therapy, and surgery appears promising for improving long-term and disease-free survival. The optimum combinations of modalities and best surgical procedures await to be defined in controlled clinical trials. This article uses the recent literature to illustrate our evolving knowledge of the basic biology and clinical management of esophageal cancer.

Changes in pulmonary lavage fluid of guinea pigs exposed to ultrafine zinc oxide with adsorbed sulfuric acid.

Ultrafine metal oxide particles (diameters less than 0.1 microns) and sulfur dioxide are important products of coal combustion. Interaction of these products in the effluent stream results in formation of ultrafine particles with adsorbed sulfur compounds, including sulfuric acid. The toxicity of ultrafine zinc oxide particles with adsorbed sulfuric acid was evaluated by comparing pulmonary lavage fluid from guinea pigs exposed for 1, 2, 3, 4, or 5 consecutive daily 3-h periods to ultrafine zinc oxide generated in the presence of sulfur dioxide (ZnO + SO2) to pulmonary lavage fluid from guinea pigs exposed to an equivalent concentration of ultrafine ZnO. Two groups of guinea pigs exposed either to SO2 or to particle-free furnace gas served as additional controls. Cells, protein, and activities of lactate dehydrogenase, acid phosphatase, and alkaline phosphatase were increased in lavage fluid obtained from guinea pigs exposed to ZnO + SO2 as compared to guinea pigs exposed to ZnO. These results demonstrate the potential importance of ultrafine metal oxides as carries of sulfuric acid derived from fossil fuel combustion.

Lung injury in guinea pigs caused by multiple exposures to ultrafine zinc oxide: changes in pulmonary lavage fluid.

Metal oxide particles with diameters of less than 0.1 micron (ultrafine particles) are important products of fossil fuel combustion. Pulmonary lavage fluid was obtained from guinea pigs given 1, 2, or 3 consecutive, daily, 3-h, nose-only exposures to 0, 2.3, 5.9, or 12.1 mg/m3 of freshly generated zinc oxide (ZnO) particles with a projected area diameter of 0.05 micron. Exposure to ZnO at 5.9 or 12.1 mg/m3 was associated with increased protein, neutrophils, and activities of angiotensin-converting enzyme, alkaline phosphatase, acid phosphatase and lactate dehydrogenase in lavage fluid, and with histologic evidence of pulmonary damage characterized by centriacinar inflammation. The severity of inflammation, graded by the number of inflammatory foci per square centimeter of lung, correlated with the amount of protein and the activity of angiotensin-converting enzyme and other enzymes in lavage fluid. These results indicate that analysis of pulmonary lavage fluid is a useful and sensitive method for quantitative evaluation of pulmonary damage caused by inhalation of low levels of ultrafine ZnO.

Transformation of NIH 3T3 cells by cotransfection with c-src and nuclear oncogenes.

pp60c-src, the cellular homolog of the Rous sarcoma virus transforming protein, does not completely transform cells even when present at high levels, but has been shown to be involved in polyomavirus-induced transformation when activated by polyomavirus middle T (pmt)-antigen binding. Here we show that cotransfection, but not solo transfection, of expression plasmids for c-src and either adenovirus E1A, v-myc, c-myc, or the 5' half of polyomavirus large T (pltN) antigen into NIH 3T3 cells induces anchorage-independent growth, enhanced focus formation, and, for pltN cotransfection, tumorigenicity in adult NFS mice. Enhancement of transformation was not observed with polyomavirus small t (pst) antigen. Cotransfection of c-src with pltN induced modification of pp60c-src that altered its electrophoretic mobility and in vivo phosphorylation state and stimulated its in vitro kinase activity. Similar alterations were not seen after c-src-E1A cotransfection, suggesting that at least two different mechanisms of enhancement are involved.

Eye and head movements to auditory targets.

Three adult female monkeys were trained to direct their gaze toward auditory targets. When the animals were free to move their heads about the vertical axis, this was accomplished with short-latency, coordinated eye-head movements reminiscent of responses to visual targets. The similarity of response to auditory and visual targets suggests a common motor program elicited by stimuli of different modalities. Since these modalities do not share the same reference system, this implies a remapping between the two reference systems.

150 MeV proton medical cyclotron design study.

A brief design study has been done for a 150 MeV proton sector cyclotron. The object was to minimize cost but maintain good reliability and easy maintenance. The use of the proton beam would be for therapy, radiography and isotope production.