Representation of auditory signals in the M-cell: role of electrical synapses.

The teleost Mauthner (M-) cell mediates a sound-evoked escape behavior. A major component of the auditory input is transmitted by large myelinated club endings of the posterior VIIIth nerve. Paradoxically, although nerve stimulations revealed these afferents have mixed electrical and glutamatergic synapses on the M-cell's distal lateral dendrite, paired pre- and postsynaptic recordings indicated most individual connections are chemically silent. To determine the sensory information encoded and the relative contributions of these two transmission modes, M-cell responses to acoustic stimuli in air were recorded intracellularly. Excitatory postsynaptic potentials (EPSPs) evoked by both short 100- to 900-Hz "pips" and longer-lasting amplitude- and frequency-modulated sounds were dominated by fast, repetitive EPSPs superimposed on an underlying slow depolarization. Fast EPSPs 1) have kinetics comparable to presynaptic action potentials, 2) are maximal on the distal lateral dendrite, and 3) are insensitive to GluR antagonists. They presumably are coupling potentials, and power spectral analysis indicated they constitute a high-pass signal that accurately tracks sound frequency and amplitude. The spatial profile of the slow EPSP suggests both proximal and distal dendritic sources, a result supported by predictions of a multicompartmental model and the effects of AMPAR antagonists, which preferentially reduced the proximal component. Thus a second class of afferents generates a portion of the slow EPSP that, with sound stimuli, demonstrate that the dominant mode of transmission at LMCE synapses is electrical. The slow EPSP is a dynamic, low-pass representation of stimulus strength. Accordingly, amplitude and phase information, which are segregated in other systems, are faithfully represented in the M-cell.

Baculovirus-mediated large-scale expression and purification of a polyhistidine-tagged rubella virus capsid protein.

The capsid protein of rubella virus was produced in baculovirus-infected Spodoptera frugiperda insect cells, with a polyhistidine affinity tag at the carboxy terminus. The RV capsid recombinant protein was produced in a 10-liter bioreactor and purified, under nondenaturing conditions, using immobilized metal-ion affinity chromatography. Immunoblot analyses indicated that the purified recombinant protein was intact and migrated with the expected molecular weight. The final yield was 5 mg of purified protein per liter of cell culture. Surface plasmon resonance was used to investigate the antigenic potential of the histidine tagged capsid protein in an antigen-antibody interaction study. A specific interaction between the two proteins was shown. Our results suggest that this strategy should be useful in interaction studies of other virus-specific proteins and antibodies.

Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: large scale production of the E1 protein.

The two envelope glycoproteins of rubella virus (RV), E1 of 58 kDa and E2 of 42-47 kDa, were individually expressed in lepidopteran Spodoptera frugiperda as well as in Trichoplusia ni insect cells using baculovirus vectors. The authentic signal sequences of E1 and E2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E1 and E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes. Synthesis of the recombinant proteins in the absence and presence of tunicamycin revealed that both E1 and E2 were glycosylated with apparent molecular weights of 52 kDa and 37 kDa, respectively. Recombinant E2 appeared to be partially secreted, whereas E1 was essentially found inside the infected insect cell. The E1 protein was produced in large scale using a 10-1 bioreactor and serum-free medium (SFM). Purification of the recombinant protein product was performed from cytoplasmic extracts by ammonium sulphate precipitation followed by Concanavalin A affinity chromatography. This type of purified recombinant viral glycoproteins may be useful not only in diagnostic medicine or for immunization, but should enable studies designed to solve the structure of the virus particle.

Phantom limb pain and etiology of amputation in unilateral lower extremity amputees.

The relationships between phantom limb pain and both the etiology of amputation (blood clots, nonclot diabetes, and miscellaneous) and the occurrence of gangrene and/or infection were investigated in 92 unilateral lower extremity (LE) amputees who were seen sequentially. There were 55 above-knee (AK) and 37 below-knee (BK) amputations in 61 men and 31 women. The mean age was 51.4 years. The blood-clot etiology had the highest levels of phantom pain both pre- and postrehabilitation, the longest time interval between amputation and prosthetic fitting, and the greatest number of medical conditions. The nonclot diabetes and miscellaneous etiologies followed in order. A history of gangrene and/or infection was associated with higher levels of pain and longer time interval between amputation and prosthetic fitting.

Carpal tunnel syndrome: a clinicopathologic study.

We evaluated 44 patients with carpal tunnel syndrome and performed histological evaluations of tenosynovium taken at the time of carpal tunnel release. Age, disease and work history, radiographs, and outcome were assessed. The clinical parameters were then compared with the histologic features to determine if the histology was predictive of the clinical course of carpal tunnel syndrome. We found no significance in the histologic changes in patients with carpal tunnel syndrome when age, duration of symptoms, work history, radiographs, and outcome were evaluated.

Preoperative radiation therapy and iododeoxyuridine for large retroperitoneal sarcomas.

PURPOSE: Local failure is frequent after conventional therapy for patients with retroperitoneal sarcomas. A Phase I/II multimodality approach was used, combining iododeoxyuridine (IdUrd) and radiation therapy, followed by attempted surgical resection, with the goal of improving local control. METHODS AND MATERIALS: Patients with retroperitoneal sarcomas were treated with three to five consecutive cycles of treatment. Each 14-day cycle consisted of a continuous intravenous infusion of IdUrd on days 1-5, twice a day radiation therapy (1.25 Gy/fraction) on days 8-12, and a break on day 13 and 14. Surgical resection was attempted after three or five cycles. Patients resected after three cycles received an additional two cycles of treatment with radiation directed to the tumor bed. IdUrd dose was escalated in Phase I fashion (1000 mg/m2/day, 1333 mg/m2/day, and 1600 mg/m2/day). The median potential follow-up was 31 months. RESULTS: Sixteen patients (13 with high grade tumors) were treated. The median maximum tumor size was 17 cm. Resection margins were negative in four patients, microscopically positive in four patients, and grossly positive in three patients. Five patients were not resected. The only grade 4 acute toxicity observed was vomiting which occurred in three patients receiving upper abdominal radiation. Postsurgical and long-term complications were rare. Median survival overall and for resected patients were 18 and 32 months, respectively. Local control was observed in three out of four patients with negative margins (9, 40+, and 51+ months), two out of four patients with microscopically positive margins (4 and 22 months), and one out of three patients with grossly positive margins (46+ months). The overall freedom from local progression was 45% at 24 months. CONCLUSION: Retroperitoneal sarcomas can be resected after preoperative radiation therapy and IdUrd, with encouraging local control in patients resected with negative or microscopically positive margins. The recommended dose using this drug and radiation schedule appears to be 1600 mg/m2/day, which will form the basis for a Phase II trial.

Phase II trial of ifosfamide and cisplatin in the treatment of metastatic sarcomas: a Southwest Oncology Group study.

The Southwest Oncology Group (SWOG) performed a phase II trial of a combination of ifosfamide/mesna/cisplatin in patients with metastatic soft-tissue sarcoma who had previously received one chemotherapeutic regimen. A total of 39 patients were registered in the study, including 7 treated during a limited-institution pilot phase; 38 patients were fully eligible and evaluable. During the pilot phase, patients were treated with 2.5 g/m2 ifosfamide daily on days 1-3, 2.5 g/m2 mesna daily on days 1-4, and 100 mg/m2 cisplatin on days 2 and 9. Due to excessive myelosuppression, the day-9 cisplatin dose was dropped when the study was opened groupwide, and the subsequent 32 patients were treated at 3- to 4-week intervals with 2.5 g/m2 ifosfamide daily on days 1-3, 2.5 g/m2 mesna daily on days 1-4, and 100 mg/m2 cisplatin on day 2. Myelosuppression was severe, with granulocytopenia (< 0.5 x 10(9)/l) being observed in 26 of 38 patients. Three cases of National Cancer Institute grade 3 or 4 nephrotoxicity (serum creatinine, > 3 times the normal value) and three cases of grade 3 or 4 central nervous system toxicity were reported. Overall, three complete and five partial responses were achieved, for a major response rate of 21%. The median survival of all patients was 11 months. We conclude that ifosfamide-based chemotherapy can produce objective responses in previously treated patients with metastatic soft-tissue sarcoma but that cisplatin increases the toxicity of therapy. Phase II trials of new agents are needed to identify drugs with clinical activity in the treatment of soft-tissue sarcomas.

Large-scale propagation of insect cells.

Cultured insect cells have many uses in agriculture and medicine. They can be used in the diagnosis and isolation of a number of viruses infecting both animals and plants and for the laboratory study of these viruses. Large-volume culture of insect cells has been envisioned as a way of producing viruses for use in controlling insect pests and for the production of viral antigens for vaccine preparations. Recently they have become a potentially valuable way of producing a variety of proteins for human and veterinary medicine using the genetically engineered baculovirus expression vectors. The development of satisfactory cell lines and culture methods has proceeded at a slow, irregular pace, inhibited by the lack of knowledge of the physiology of the insect, its small size, and often by the lack of consistent, adequate support for the necessary developmental research. However, now that the basic culture systems are available, cell lines have been developed or can easily be developed for most needs. Suitable media are available and recent developments in refining existing media formations have resulted in low-cost media containing little protein to interfere with down-stream processing of cellular metabolites. Future developments are likely to further improve the media formulations and lower the cost. Technical problems relating to oxygen demand and cell fragility that inhibited the continued development of large-volume culture systems beyond the laboratory a few years ago now appear to be solved or at least are solvable. The successful culture of the Spodoptera cells in bioreactors of 40-liter capacity indicates that means of producing insect cells or their metabolic products on a commercial scale can be made economically feasible.

Insect cells as substrates for biologicals.

Various methods for the growth of insect cells and the production of recombinant human beta-interferon (rHu beta-IFN) by insects cells, Spodoptera frugiperda, IPL-Sf-21AE after infection with recombinant AcNPV are described. Using a suspension perfusion Biospin filter culture system, an IPL-Sf-21AE cell line was propagated in a semi-controlled environment. To obtain and maintain high cell densities and viabilities in this system, the culture medium for suspension methods was modified by supplementing with ZnSO4, ALCl3, methyl cellulose and Darvan #2. Using these improved conditions, multiple harvests of rHu beta-IFN were collected in the cell culture supernatant for downstream processing.

Herpesvirus simiae (B virus): replication of the virus and identification of viral polypeptides in infected cells.

The events and products of replication of Herpesvirus simiae (B virus) in Vero cells were studied. The time course of the synthetic events of DNA replication and protein synthesis were found to be similar to the processes of the herpes simplex viruses and SA 8. Infectious progeny virus were detected by 4 hours post infection and were first found extracellularly between 6 and 8 hours post infection (PI). As in the case of SA 8, all cell lines tested were permissive for lytic infection by B virus. Analyses of B virus-infected cells by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed approximately 50 infected cell polypeptides (ICP) ranging in molecular weight from about 26,000 to 239,000 daltons. The kinetics of synthesis of the ICPs were also identified. At least nine glucosamine-containing glycopeptides were noted ranging from 133,000 to 29,000 daltons.

Effect of aluminum chloride and zinc sulfate on Autographa california nuclear polyhedrosis virus (ACNPV) replication in cell culture.

When IPL-SF-21AE III continuous insect cell line was grown and maintained in IPL-41 insect cell culture medium supplemented with 16 microM of AlCl3 or 0.24 microM of ZnSO4 . 7H2O, or both metallic salts, and then infected with Autographa california nuclear polyhedrosis virus, virus replication was increased significantly. The yield of polyhedral inclusion bodies (PIB) was enhanced up to 121%. Synthesis of cell-free nonoccluded virus was increased to 365% when infectivity was assayed by the plaque method. Newly applied electron microscopic quantitation and stereological techniques also revealed a significant increase in virus particles (VP) and in amount and size of PIB as well as number of VP per PIB.

Quantitative measurement of oxygen consumption in insect cell culture infected with polyhedrosis virus.

A simple and reproducible quantitative method for measuring dissolved oxygen (DO) in uninfected and baculovirus infected cells in culture is described. To establish this method, an industrial DO measuring system for fermentation was employed. During this process the physical characteristics of the cell culture vessel were taken into account permitting a direct readout of DO in microliters per vessel. During these studies, it was experimentally documented that insect cells, particularly baculovirus infected cells, in culture from 1 to 14 days utilize an appreciable level of DO.

Chemical inhibition of foamyvirus in primary baboon (Papio cynocephalus) kidney cells.

This report describes the conditions for the use of aluminum chloride (AlCl3) in growth and maintenance media for the suppression or inhibition of simian foamyviruses (SFV) in primary baboon kidney (BAK) and rabbit kidney (RK) cell cultures. When RK cells were planted in medium containing AlCl3, infected with SFV, and passaged, the growth of SFV was suppressed or inhibited by the presence of AlCl3. With this method, BAK cells yielded higher viral titers after infection with various viruses, thus making these cells more suitable for virological applications.