3M KCl Research Articles

Dependence of acquired systemic tolerance to rat islet allografts induced by intrathymic soluble alloantigens on host responsiveness, MHC differences, and transient immunosuppression in the high responder recipient.

Recent studies suggest that the adult immune system can be manipulated by intrathymic (IT) inoculation of donor Ag to accept cardiac and islet allografts in the low responder rat combination of Lewis-to-WF. We have now extended this study to examine the effect of IT inoculation of soluble protein Ag obtained from 3M KCl extracts of resting T cells combined with transient ALS immunosuppression on islet allograft survival in the high responder combination of WF-to-Lewis. We first confirmed the earlier observation that IT injection of 2 mg soluble Ag on day -7 led to permanent islet graft survival (>200 days) in the Lewis-to-WF rat combination without the use of recipient immunosuppression and found this to be true in the Lewis-to-ACI rat combination. In the high responder combination of WF-to-Lewis, unmodified Lewis rats pretreated with IT inoculation of 2 mg soluble Ag acutely rejected WF and BN islet allografts. IT inoculation of donor Ag combined with 1 ml ALS transient immunosuppression on day -7 led to a modest graft prolongation [24.8+/-10.1 days as compared with 15.2+/-3.6 days in ALS only treated controls]. Intrathymic injection of soluble Ag on day -7 combined with 1 ml ALS on days -7 and 0 relative to allografting resulted in 100% permanent islet graft survival (>200 days) compared with an MST of 20.6+/-2.3 days in ALS only-treated controls. Similar treatment led to acute rejection of 3rd party (BN) grafts, thus demonstrating donor-specificity. In addition, extrathymic inoculation of donor Ag in similarly immunosuppressed animals did not result in islet graft prolongation, once again confirming the importance of the thymus in tolerance induction. To examine them for donor-specific tolerance, long-term unresponsive (>120 days) Lewis recipients of renal subcapsular islets underwent nephrectomy of the islet bearing kidneys and were challenged with intraportal donor- or third party-type islets after becoming diabetic. All the nonimmunosuppressed recipients of donor-type (WF) islets became permanently normoglycemic (>100 days) while the third-party (BN) grafts were promptly rejected, with an MST of 10.6 days. These findings confirm that acquired thymic tolerance induced by IT inoculation of soluble protein Ag in the low to moderate responder rat combinations is reproducible in the high responder combination provided that adequate peritransplant immunosuppression is used. This study suggests that acquired thymic tolerance in the rat model is dependent on host responsiveness to alloantigens, MHC differences between the donor-recipient pair, and the use of transient immunosuppression in the high responder recipient. This model may have potential clinical application in the development of strategies for specific transplantation tolerance.

EFFECTIVENESS OF INTRATHYMIC INOCULATION OF SOLUBLE ANTIGENS IN THE INDUCTION OF SPECIFIC UNRESPONSIVENESS TO RAT ISLET ALLOGRAFTS WITHOUT TRANSIENT RECIPIENT IMMUNOSUPPRESSION

Our finding that intrathymic inoculation of resting T cells but not dendritic cells induces donor-specific unresponsiveness to organ allografts led us to hypothesize that presentation of MHC class I alloantigens by thymic antigen-presenting cells to T cell precursors during their ontogeny may convey a tolerogenic signal to the recipient. In this study, we examined whether intrathymic inoculation of soluble antigen obtained from 3M KCl extracts of allogeneic T cells could induce donor-specific unresponsiveness to islet allografts in the Lewis-to-WF rat combination. Our results showed that while intrathymic injection of 0.5 mg soluble antigen on day −7 relative to islet transplantation caused acute graft rejection, intrathymic inoculation of 1.0 mg soluble antigen significantly prolonged the survival of Lewis islet allografts from 10.3±1.1 days in controls to 53.5±15.6 days (P< 0.001) in naive (nonimmunosuppressed) STZ (streptozotocin)-induced diabetic WF recipients. In contrast, intrathymic inoculation of 2.0 or 4.0 mg soluble Ag on day −7 led to indefinite Lewis islet survival (>150 days) in all naive diabetic WF recipients; a finding that suggests that 2.0 mg soluble Ag is the optimal effective dose of intrathymic inoculum required to induce donor-specific unresponsiveness in naive recipients in this model. This finding could not be reproduced by intravenous injection of 2.0 mg soluble Ag, thus confirming the privileged position of the thy- mus in the induction of Ag-specific unresponsiveness. Third-party (BN) islet allografts were rejected in an acute fashion in similarly prepared recipients. Our results suggest that (1) intrathymic inoculation of soluble Ag, unlike cellular Ag, induces donor-specific unresponsiveness to islet allografts without the use of transient recipient immunosuppression; (2) induction of specific unresponsiveness appears to be dose dependent; and (3) the tolerogenic effect of soluble Ag is dependent on the indirect pathway of Ag-presentation in the thymus in the absence of donor antigen-presenting cells in the inoculum. This novel approach.

EFFECTIVENESS OF INTRATHYMIC INOCULATION OF SOLUBLE ANTIGENS IN THE INDUCTION OF SPECIFIC UNRESPONSIVENESS TO RAT ISLET ALLOGRAFTS WITHOUT TRANSIENT RECIPIENT IMMUNOSUPPRESSION

Our finding that intrathymic inoculation of resting T cells but not dendritic cells induces donor-specific unresponsiveness to organ allografts led us to hypothesize that presentation of MHC class I alloantigens by thymic antigen-presenting cells to T cell precursors during their ontogeny may convey a tolerogenic signal to the recipient. In this study, we examined whether intrathymic inoculation of soluble antigen obtained from 3M KCl extracts of allogeneic T cells could induce donor-specific unresponsiveness to islet allografts in the Lewis-to-WF rat combination. Our results showed that while intrathymic injection of 0.5 mg soluble antigen on day -7 relative to islet transplantation caused acute graft rejection, intrathymic inoculation of 1.0 mg soluble antigen significantly prolonged the survival of Lewis islet allografts from 10.3 +/- 1.1 days in controls to 53.5 +/- 15.6 days (P < 0.001) in naive (nonimmunosuppressed) STZ (streptozotocin)-induced diabetic WF recipients. In contrast, intrathymic inoculation of 2.0 or 4.0 mg soluble Ag on day -7 led to indefinite Lewis islet survival (> 150 days) in all naive diabetic WF recipients; a finding that suggests that 2.0 mg soluble Ag is the optimal effective dose of intrathymic inoculum required to induce donor-specific unresponsiveness in naive recipients in this model. This finding could not be reproduced by intravenous injection of 2.0 mg soluble Ag, thus confirming the privileged position of the thymus in the induction of Ag-specific unresponsiveness. Third-party (BN) islet allografts were rejected in an acute fashion in similarly prepared recipients. Our results suggest that (1) intrathymic inoculation of soluble Ag, unlike cellular Ag, induces donor-specific unresponsiveness to islet allografts without the use of transient recipient immunosuppression; (2) induction of specific unresponsiveness appears to be dose dependent; and (3) the tolerogenic effect of soluble Ag is dependent on the indirect pathway of Ag-presentation in the thymus in the absence of donor antigen-presenting cells in the inoculum. This novel approach of thymic reeducation of adult animals by the deliberate intrathymic inoculation of soluble major histocompatibility complex Ag without the use of recipient immunosuppression may have therapeutic potential in the induction of transplantation tolerance.

Autolymphocyte therapy —I. In vivo tumour-specific adoptive cellular therapy of murine melanoma and carcinoma using ex vivo activated memory T-lymphocytes

Autolymphocyte therapy (ALT) is tumour-specific adoptive cellular therapy of neoplastic disease based upon non-specific ex vivo activation of autologous peripheral blood lymphocytes (PBL), using the supernatant derived from a previously prepared one-way mixed lymphocyte culture (MLC). To determine the requirement for tumour antigen during the activation process, splenocytes from C57BL/6J healthy syngeneic mice (HSM) and tumourbearing mice (TBM) were activated ex vivo using a MLC-supernatant (MLCS). Ex vivo activation was performed both in the presence (HSM splenocytes) and absence (TBM splenocytes) of a 3M KCl syngeneic tumour-antigen (STA) extract prepared from Lewis lung (3LL) carcinoma, B16 melanoma, or normal lung. Immunophenotyping of splenocytes pre- and post-activation by MLCS plus STA or MLCS only revealed expansion of activated CD44 + (memory) T-cells. Ex vivo tumour-specific cytotoxicity was demonstrated using MLCS-activated (TBM) or MLCS + STA-activated (HSM) splenocytes against 3LL or B16 target cells. CD44 + T-cells (ALT-cells) were then infused into syngeneic 3LL and B16 TBM. Significant antitumour activity was detected in 3LL and B16 TBM receiving cells from normal mice that were activated with MLCS in the presence of 3LL or B16 STA, respectively, and in 3LL and B16 TBM receiving splenocytes from 3LL-TBM and B16-TBM, respectively, activated by MLCS alone. Infusions of 3LL-derived or 1116-derived ALT-cells into HSM provided specific immunity on tumour challenge. No antitumour activity was seen in 3LL and B16 TBM receiving fresh TBM splenocytes, ALT-cells derived from HSM which were activated ex vivo using MLCS without antigen, normal lung tissue as antigen, or using MLCS-activated splenocytes without STA derived from reciprocal TBM. Ex vivo depletion of CD44 + cells or Thy-1.2 + T-cells abrogated all antitumour activity in TBM and in HSM challenged with tumour. Depletion of NK-1.1 + natural killer (NK)-cells had no effect on antitumour efficacy. These data suggest that tumour-specific adoptive cellular therapy is possible using ex vivo activated HSM splenocytes with STA, or TBM splenocytes activated ex vivo without STA, and that these antitumour effects are dependent on CD44 + memory T-cells.

Cellular electrophysiology of WAY-123,398, a new class III antiarrhythmic agent: specificity of IK block and lack of reverse use dependence in cat ventricular myocytes.

The objectives were (a) to evaluate the effects of WAY-123,398, a new class III antiarrhythmic agent, on the action potential of canine Purkinje fibres in comparison with dofetilide, E-4031, and dl-sotalol, and (b) to characterise the mechanism of the class III action by studying its effects on several ionic currents in isolated cat myocytes. Transmembrane potentials in Purkinje fibres were studied with standard microelectrodes filled with 3M KCl. Myocytes were isolated by enzymatic disaggregation with collagenase and current recordings were obtained by voltage clamp with either the nystatin perforated patch technique or the usual whole cell configuration. WAY-123,398 prolonged action potential duration (APD) in Purkinje fibres and in cat ventricular myocytes without altering other variables of the action potential; in Purkinje fibres the concentration producing a 20% prolongation of APD-60 mV at a basic cycle length of 1000 ms was 0.2 microM. After depolarising voltage steps, the delayed rectifier (IK) peak tail currents in cat myocytes were blocked with IC50 = 0.1 microM. The block was unaffected by varying the duration (200 to 500 ms) or the frequency (0.4 to 2.5 Hz) of the depolarising steps. A much higher concentration of WAY-123,398 (10 microM) did not have effects on the L type Ca current (ICa-L), and on the inward rectifier (IK1) and transient outward (I(to)) K currents. The results indicate that WAY-123,398 is an effective and specific class III agent devoid of class I activity, and suggest that WAY-123,398 prolongs cardiac repolarisation by specifically blocking the delayed rectifier current (IK). The block was unchanged over a range of frequencies and duration of depolarisation, showing no evidence of "reverse use dependence" of block.

THE COMBINED EFFECT OF PERIOPERATIVE DONOR SPLEEN CELLS OR KC1-EXTRACTED ANTIGEN AND CYCLOSPORINE ON RENAL ALLOGRAFT SURVIVAL IN THE RAT

The effects of using perioperative cyclosporine in conjunction with pretreatment with donor spleen cells or 3M KCl solubilized extracts of donor antigen were investigated in a LEW-to-DA rat renal allograft model. Cyclosporine given orally in a dose of 10 mg/kg/day around the time of transplantation (days -1, 0, +1), did not prolong renal allograft survival (median survival time [MST]--10 days). However when used in combination with pretreatment with either 10(8) donor spleen cells (1 day before transplantation), or 10(5) donor spleen cells (7 days before transplantation), pretreatment regimens that were in themselves ineffective, DA recipients accepted Lewis renal allografts indefinitely (MST greater than 100 days). Soluble antigen was prepared by 3M KCl extraction from donor spleen cells. Absorption assays were used to quantify the amount of class I major histocompatibility complex antigen in the preparation, and amounts of antigen equivalent to that expressed by 10(6)-10(8) donor spleen cells were used for pretreatment. These soluble antigen preparations given either 1 or 7 days before transplantation with or without perioperative cyclosporine did not prolong allograft survival of either homozygous or heterozygous donors (MST 10 days).

Determination of subnanomolar concentrations of tungsten (VI) by catalytic adsorption polarography

A new differential pulse polarographic method for the determination of W(VI) using a catalytic adsorption wave is described. W(VI) is first chelated by 7-iodo-8-hydroxyquinoline-5-sulfonic acid at pH 0.5. The complex ion formed is strongly adsorbed on the surface of a dropping mercury electrode. At a potential of −0.95 V versus the Ag/AgCl (3M KCl) reference electrode the adsorbed complex is reduced by the polarographic current and oxidized very fast by hydrated hydrogen ions providing the oxidized form of the complex ion for repeated redox cycles. As the redox process taking place in the electric double layer, the diffusion of the complex does not limit the polarographic current. Therefore, high currents occur, and consequently, a very high sensitivity is obtained. The practical detection limit (PDL) is 3.7 ng W/kg solution corresponding to 2 × 10−11M. The standard deviation of single values is 1.2 ng/kg at the concentration of 91 ng/kg lying in the middle of the linear part of the calibration curve. Because Mo (VI) gives a very similar catalytic adsorption wave, serious mutual interferences occur in the analysis of mixtures of both species. An effective separation of Mo(VI) was worked out. Using 1% (w/v) solution of trioctylphosphinoxide in kerosene, Mo(VI) can almost completely be extracted from 1.8M HCl with a threefold extraction resulting in a separation factor of 40000.

Treatment of metastatic renal cell carcinoma with autolymphocyte therapy: Low Toxicity Outpatient Approach to Adoptive Immunotherapy Without Use of In Vivo Interleukin-2

Thirty-six patients with Stage IV renal cell carcinoma were treated with autolymphocyte therapy (ALT). This new form of adoptive immunotherapy is based on the infusion of relatively small numbers of autologous lymphocytes that are depleted of suppressor cells and immunized in vitro by a method designed for antigen-specific activation using a 3M KCl extract of autologous tumor and an autologous lymphokine mixture. Patients received six monthly infusions of immunized lymphocytes, all on an outpatient basis. The majority of patients experienced no toxicicity The few reactions that occurred were minor and self-limiting; none required any medical intervention or subsequent delay in therapy. Patients also received oral cimetidine to reduce in vivo suppressor cell function. Survival at twenty-four months is 36 percent. Median survival is fifteen months, a significant improvement over the natural history of this disease. A multi-site, randomized, controlled trial of ALT in renal cell carcinoma has been initiated to confirm that this treatment causes a significant prolongation of survival with virtually no toxicity in these patients.

Ability of 3M KC1-extracted histocompatibility antigen to potentiate the immunosuppressive effect of cyclosporine to prolong the survival of heterotopic rat cardiac allografts.

A soluble histocompatibility antigen preparation (HAg) derived by 3M KCl extraction of donor spleen cells has been shown to prolong rat renal allografts in CsA-treated hosts. In the present study, the effect of combined soluble antigen-CsA treatment on cardiac allograft survival was studied in WFu hosts grafted with BUF hearts. Cardiac allograft survival was prolonged in WFu recipients treated with both BUF HAg and CsA compared with survival time in untreated controls or controls treated with HAg or CsA alone. In addition, experiments were performed to test the antigenicity of the HAg extract. BUF extract given sc to WFu hosts before grafting specifically sensitized the hosts to BUF grafts, as shown by the accelerated rejection of BUF grafts but not third-party grafts. Assays to determine the major histocompatibility antigenic determinants present in the extract showed that class I and class II determinants were present.

DC potentials of the lateral wall of the scala media.

We recorded DC potential and resistance changes of the cochlear lateral wall in guinea pigs, using (3M KCl) electrolyte glass microelectrodes. The patterns found for slow penetration showed different potential and resistance characteristics at different segments of the lateral wall. In addition, our findings demonstrate that the monitoring of microelectrode tip resistance is a useful procedure for DC cochlear recordings.

LOW-TOXICITY OUTPATIENT ADOPTIVE IMMUNOTHERAPY OF METASTATIC CANCER WITH AUTOLOGOUS LYMPHOCYTES GENERATED BY IN VITRO IMMUNIZATION (IVI)

We have previously described results with a novel form of adoptive immunotherapy using in vitro immunized (IVI) autologous lymphocytes. We now report results from an on-going study using an improved version of that method. Patient lymphocytes are depleted of suppressor T-cells and in vitro immunized for 60 hours in medium containing autologous serum, mixed lymphocyte culture supernatant and a 3M KCl autologous tumor extract. The resultant IVI cells are washed, depleted again of suppressor cells and infused into the patient. All patients also receive cimetidine to block in vivo suppressor cells. Patients will be treated monthly for 6 months. To date, 30 patients have received a total of 113 cell infusions, all delivered on an outpatient basis, (average of 109 IVI cells/infusion). Toxicity has been minimal, consisting only of mild, transient fever and chills accompanying 42 of the infusions (37%). Twenty-one patients have reached the first evaluation timepoint at 3 mqnths.This approach to immunotherapy is safe and appears to be biologically active, in that 7 of 21 evaluable patients (33%) have evidence of stable disease or objective response after only 3 months of treatment.

Biochemical studies of human placental microvillous plasma membrane proteins.

Isolated human syncytiotrophoblast microvillous plasma membranes (StMPM) have been examined by electron microscopy, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional PAGE (2D-PAGE), and immunoblots. Electron microscopy of StMPM pellets revealed populations of membrane-bounded vesicles that disrupted after treatment with the chaotrope 3M KCl for 16 hr; with increasing molarity of another chaotrope (NH4SCN), the vesicles became smaller and more homogeneous. NH4SCN treatment resulted in significant reduction on SDS and 2D-PAGE analysis of only one protein at 80kd, shown by immunoblotting to be transferrin; 3M KCl had little effect and appeared to be a poor chaotrope. Chromogenic silver staining of SDS-PAGE gels demonstrated over 50 StMPM-associated discrete protein components. Immunoblotting revealed transferrin (80kd), albumin (65kd), IgG heavy chain (56kd), and Gc protein (56kd). Alpha-2-macroglobulin (alpha 2M) was identified at 180kd and 95kd; the smaller component may be a proteolytic derivative indicating alpha 2M binding to a trophoblast surface protease. Numerous discrete protein dots, and groups of dots characteristic of charge heterogeneity of individual proteins, were observed on high resolution 2D-PAGE. The most intensely stained proteins were transferrin (80kd), albumin (65kd), placental-type alkaline phosphatase (66kd), and actin (46kd). This 2D-PAGE technique is a superior method for analyzing the trophoblast membrane proteins, and the system described will enable systematic mapping of these components.

Surface enhanced Raman scattering in a nonaqueous electrochemical cell: Pyridine, benzoic acid, and nitrobenzene in methanol

We have studied SERS on the following systems for comparison between aqueous and nonaqueous media: (1a) 0.05M pyridine 2×10 −3fM KCI methanol , (1b) 0.05M pyridine 2×10 −3M KCl water , (2a) 0.05M henzoic acid 2×10 −3M KCI methanol , (2b) 5×10 −3M benzoic acid 2×10 −3M KCl water , and (3) 0.05M nitrobenzene 2×10 −3M KCI methanol . As already reported, the surface adsorbed SERS species seem to be the same, irrespective of whether the solvent is water or methanol. However, considerable differences are found in small details between the nonaqueous and the aqueous SERS spectra, the significance of which is discussed in terms of both electrochemistry and SERS. For nitrobenzene, the observed SERS spectra did not show the Raman lines of the original nitrobenzene but some reduction products of nitrobenzene, probably aniline and phenylhydroxylamine.

Nature of the suppressor cells mediating prolonged graft survival after administration of extracted histocompatibility antigen and cyclosporine.

Antigen-specific suppressor T cells are induced by donor histocompatibility antigen extracted from spleen cells with 3M KCl combined with cyclosporine (Ag-CsA). A single i.v. injection of 5 mg 3M-KCl-extracted donor Buffalo (Buf, RT1b) antigen (Ag) combined with a three day course of CsA prolonged renal allograft survival in Wistar-Furth (WFu, RT1u) hosts to a greater extent (MST 26.5 days) than CsA alone (MST 11.8 days). Peripheral blood lymphocytes (PBL) or spleen cells harvested from Ag-CsA-treated recipients ten days after transplantation inhibited the mixed lymphocyte reaction (MLR) between normal responder WFu cells and irradiated Buf cells (55.6% and 64.4% suppression, respectively, P less than 0.025), but not third-party Brown-Norway (BN, RT1n) stimulator cells (13.6% and -18.3% suppression, respectively, NS). The suppressor effect was not mediated by cytolytic cells; there was neither primary nor secondary cytolytic activity against 51Cr-labeled Con-A blastoid Buf cells. The suppressor cells were neither adherent to plastic dishes nor to nylon-wool columns. PBL irradiated with 800 rads, but not 1500 rads, suppressed the MLR. A single injection of cyclophosphamide (CY, 25 mg/kg) seven days after transplantation abrogated the suppression induced by Ag-CsA treatment. Moreover, PBL from Ag-CsA recipients failed to suppress the MLR, if depleted either of all T cells by treatment with monoclonal antibody (Mab) W3/13 HLK (pan T cells; % suppression -15.8), or of cytotoxic/suppressor cells with Mab OX-8 (-19.3% suppression) together with rabbit antimouse immunoglobulin and complement. On the other hand, PBL treated with the Mab W3/25 (helper) showed suppressor cell activity (+56.4%, P less than 0.001) similar to untreated cells (62.4%, P less than 0.001). Moreover, adoptive transfer of suppressor T cells purified from pooled lymphocytes by rosetting using Mab significantly prolonged the survival of donor-specific, but not third-party, test grafts in naive secondary hosts. Thus, these studies demonstrated antigen-specific suppressor T cells mediate the long-term unresponsiveness induced by the Ag-CsA regimen.

Prolongation of rat kidney allografts by pretransplant administration of donor antigen extract or whole blood transfusion combined with a short course of cyclosporine.

The immunosuppressive effect of the combination of a three day course of cyclosporine with one i.v. injection of 3M KCL-extracted donor antigen or donor blood transfusion was tested across the strong histocompatibility barrier causing rejection within 8 days of kidney transplants from Buffalo (Buf, RT1b) to Wistar-Furth (WFu, RT1u) inbred rats. Administration of 10 mg/kg/day cyclosporine alone for three days (-1, 0, and 1) slightly prolonged graft survival time from 7 to 11 days. The combination of cyclosporine with donor Buf 3M KCl antigen or with a Buf blood transfurion administered one day prior to transplantation caused greater prolongation of graft survival--19 and 25 days, respectively. Neither third-party BN soluble antigen nor BN blood transfustions acted synergistically with cyclosporine to prolong Buf graft survival. Increasing doses of donor-soluble antigen up to an optimal dose of 5 mg proportionately prolonged graft survival; however, administration of 10 mg antigen was less effective than 5 mg. On the other hand, administration of 1 ml of donor blood achieved the maximal effect. Lymphocytes harvested ten days after transplantation from recipients that had received combined therapy with cyclosporine and donor 3M KCl antigen not only displayed specific unresponsiveness to donor stimulator cells in mixed lymphocyte culture, but also specifically suppressed the proliferative response of syngeneic, virgin WFu responder cells to allogeneic donor Buf but not to third-party BN cells. Furthermore, suppressor cell activity was suggested by diminished responses in an in vivo local adoptive mixed lymphocyte culture assay and by prolongation of Buf kidney survival following systemic adoptive transfer. These findings suggest that immunosuppression with cyclosporine permits induction of specific suppressor cells by 3M KCl donor antigen, resulting in specific unresponsiveness to allografts.

Preliminary identification of a tumor-associated glycoprotein in bilharzial bladder cancer urine.

A tumor-associated antigen was preliminarily identified in urine from bilharzial (squamous-cell carcinoma) bladder cancer patients. Monospecific rabbit antisera were made by immunization with concentrated bladder cancer urine and exhaustive absorption with insoluble normal human serum and urine. The urine tumor-associated antigen was identical to an antigen from 3M KCl bladder tumor extract by immunodiffusion. The antigen in urine was found in nine of 10 bladder cancer patients and was absent from normal urine and serum by immunodiffusion and immunoelectrophoresis. The antigen was a concanavalin-A-binding glycoprotein which was anodal on immunoelectrophoresis. It was stable up to 2 years at -20 degrees C and did not cross-react with carcinoembryonic antigen or with Schistosoma haematobium antigens.

KCl leakage from microelectrodes and its impact on the membrane parameters of a nonexcitable cell.

Microcapillary electrodes filled with a variety of salt solutions, including 1m KCl, have been used to measure the membrane potentials and resistances of spherical cells from the mycelial fungusNeurospora (cell diameters 15–25 μm, cell volumes 3–8 pl). During impalements with electrodes containing 0.3–1.0m KCl, membrane potential and resistance decayed over a period of 3–10 min. In contrast, electrodes filled with 0.1m KCl gave stable membrane potentials of −180 mV and membrane resistivities of 40 kΩ cm2, values comparable to earlier results from the fungal hyphae. Salt leakage from 1.0m KCl-filled electrodes (tip diameters 0.2–0.3 μm, resistances 50–75 MΩ) occurred at rates of 4–5 fmol sec−1, as indicated by direct intracellular measurements with ion-sensitive microelectrodes. Depending on cell size, such leakage rates could elevate cytoplasmic KCl content at initial rates of 30–170 mM min−1, and actual values as high as 70mm min−1 were observed. Salt leakage and changes in cytoplasmic KCl concentration were reduced five- to sevenfold when impalements were made with electrodes containing 0.1m KCl. The effects on cell membrane parameters of salt leakage from microelectrodes could be attributed to chloride ions. Substitution of the KCl electrolyte with half-molar K2SO4 or Na2SO4 and molar concentrations of K- and Na-MES [potassium and sodium 2-(N-morpholino)ethanesulfonate] gave stable membrane potentials in excess of −200 mV and membrane resistivities greater than 50 kΩ cm2, while the permeant anions NO 3 − and SCN− depressed the membrane parameters in a manner similar to that observed with 1m KCl. Furthermore, modest elevation of cytoplasmic chloride concentration (below ca. 50 mM) affected both membrane potential and resistance in direct proportion to the concentration, and could be quantitatively described by the Constant Field Theory with a fixed membrane permeability (P Cl∼4×10−8 cm sec−1). Higher cytoplasmic chloride levels produced a collapse of the membrane resistance and drastic depolarization in a fashion requiring large changes of membrane permeability. At least for cells with volumes of 10 pl or less, the standard practice of filling electrodes with 1 or 3m KCl should be abandoned. Half-molar (and lower) concentrations of K2SO4 or Na2SO4 are suggested as satisfactory replacements.

Mathematical analysis of axisymmetric sessile drop: Application to the reduction of bromine in a two-phase electrolyte

The profile of an axisymmetric sessile drop was mathematically analyzed to relate the drop height, H, and equatorial diameter, D, to the contact radius with 180° contact angle, R, and the ratio of buoyant to interfacial tension forces, β. The relationships between ( H D ) and ( R D ) or log 10 β, which are valid over 0.2 < H D < 0.9 , were derived from a numerical integration of the differential equations and fitting of the results to fifth-order polynomials. Asymptotic formulas were also obtained analytically for the cases where H D < 0.2 and H D > 0.9 and all contact angles. The results of the mathematical analysis were used to analyze the data on the enhancement in the bromine reduction current that was observed with a two-phase electrolyte consisting of sessile drops of bromine complexed with trimethylethylammonium bromide less than 0.2 cm high on a ruthenized titanium electrode in an aqueous electrolyte ( 2M ZnBr 2 1M ZnCl 2 3M KCl ) containing dissolved bromine.

Microelectrode studies of Necturus antral mucosa: electrical potentials and resistances.

Intracellular microelectrode techniques were applied to Necturus antral mucosa. Stable intracellular impalements were obtained with 15-50 M omega microelectrodes filled with 3M KCl. It was possible to change rapidly the mucosal bathing solution while maintaining the microelectrode in the cell. With these techniques, we were able to measure the electrical potentials and resistances of the cell membranes and the shunt pathway. The transepithelial potential was -4.9 +/- 1.3 mV, serosal solution reference. Apical cell membrane potential was -43.9 +/- 0.6 mV, cell negative to the mucosal solution. Basolateral cell membrane potential was -48.8 +/- 1.3 mV, cell negative to serosal solution. Transepithelial resistance was 427 +/- 66 omega . cm2. The ratio of apical to basolateral membrane resistances was 3.4 +/- 0.3. The electrical resistances of the transcellular and paracellular pathway were determined by the measurement of the total transepithelial resistance and the ratio of apical to basolateral cell membrane resistances before and after blocking apical membrane sodium permeability with amiloride. The resistances of the apical cell membrane, basolateral cell membrane, and the shunt were 2,203 +/- 585, 1,296 +/- 384, and 604 +/- 81 omega . cm2, respectively (mean +/- SE). Calculations from these measurements indicate that the shunt contribution to transepithelial conductance was approximately 85%.

Hydrophobic chromatographic purification of ethylene-enhanced chlorophyllase from Citrus unshiu fruits

Ethylene-enhanced chlorophyllase from Citrus unshiu fruits was purified to a homogeneous state after solubilization with sodium cholate, using acetone precipitation and hydrophobic chromatography. The enzyme adhered to phenyl Sepharose CL-4B in 3M KCl and was eluted with a linear gradient of Triton X-100 (0–0.5%). Its MW (SDS-PAGE) was 27 000. The enzyme behaved as a protein of MW 110 000 on Sephacryl S-200 gel filtration. The enzyme showed a specific activity of 0.069 μamol chlorophyllide a produced/min/ mg protein. This purification procedure is a rapid method for obtaining pure chlorophyllase.