Epitope recognition by anti-cathepsin D autoantibodies in endometrial cancer patients.

OBJECTIVE: This study analyzed a model for the identification of specific epitopes recognized by autologous tumor-reactive humoral responses of endometrial cancer patients as potential markers for the monitoring of cancer. METHODS: The presence of circulating pro- and mature forms of cathepsin D and antibodies reactive with this enzyme were identified by Western immunoblot and quantitated by an enzyme immunoassay. Specific immunoreactivities with 34- and 52-kDa cathepsin D forms were analyzed by Western immunoblot using sera from endometrial cancer patients (n = 40) and normal volunteers (n = 15). Subsequently, reactivities with specific cathepsin D epitopes were defined by a peptide-specific ELISA. RESULTS: Circulating pro-forms of cathepsin D were detected in 31 of 40 endometrial cancer patients tested and none of the control volunteers. Circulating IgG reactive with cathepsin D could be demonstrated in 29/31 patients with circulating procathepsin D, while an anti-cathepsin D response was not detectable in normal controls. This response appeared to be directed against the pro-peptide portion of cathepsin D. Using a peptide-specific ELISA, the frequencies of antibody production against specific epitopes within the pro-peptide were defined. CONCLUSION: There is a demonstrable tumor-reactive immune response elicited in endometrial cancer patients, directed against specific antigenic epitopes, some of which are conserved among these patients. Since these proteins are recognized as non-self, due at least in part to posttranslational processing errors, defining these epitopes will be useful as a means of diagnosis, assessment of therapeutic success, and, ultimately, identification of immunotherapeutic targets.

The role of Ca2+ stores in the muscarinic inhibition of the K+ current IK(SO) in neonatal rat cerebellar granule cells.

Cerebellar granule neurons (CGNs) possess a standing outward potassium current (IK(SO)) which shares many similarities with current through the two-pore domain potassium channel TASK-1 and which is inhibited following activation of muscarinic acetylcholine receptors. The action of muscarine on IK(SO) was unaffected by the M2 receptor antagonist methoctramine (100 nM) but was blocked by the M3 antagonist zamifenacin, which, at a concentration of 100 nM, shifted the muscarine concentration-response curve to the right by around 50-fold. Surprisingly, M3 receptor activation rarely produced a detectable increase in [Ca2+]i unless preceded by depolarization of the cells with 25 mM K+. Experiments with thapsigargin and ionomycin suggested that the endoplasmic reticulum Ca2+ stores in CGNs were depleted at rest. In contrast, cerebellar glial cells in the same fields of cells possessed substantial endoplasmic reticulum Ca2+ stores at rest. Pretreatment of the cells with BAPTA AM, thapsigargin or the phospholipase C (PLC) inhibitor U-73122 all blocked the muscarine-induced Ca2+ signal but had little or no effect on muscarinic inhibition of IK(SO). Raising [Ca2+]i directly with ionomycin caused a small but significant inhibition of IK(SO). It is concluded that muscarine acts on M3 muscarinic acetylcholine receptors both to inhibit IK(SO) and to mobilize Ca2+ from intracellular stores in CGNs. While the mobilization of Ca2+ occurs through activation of PLC, this does not seem to be the primary mechanism underlying muscarinic inhibition of IK(SO).

Modulation of proliferation and chemosensitivity by procathepsin D and its peptides in ovarian cancer.

Since the presence of precursors (pro-forms) of the aspartyl endoprotease, cathepsin D, appears to be linked with tumor progression, their presence was examined in sera and tumor tissues of ovarian cancer patients. The role of cathepsin D pro-forms was further assessed in the dysregulated proliferation and chemoresistance observed in advanced ovarian cancer. Cathepsin D was isolated from sera of ovarian cancer patients (n = 20) and normal volunteers (n = 11), as well as from solubilized normal ovarian epithelium (n = 8) and ovarian epithelial tumor tissue (n = 12). The specific molecular forms of cathepsin D were analyzed in these samples by Western immunoblot. Multiple circulating molecular weight forms of cathepsin D were identified in ovarian cancer patients ranging from 24 to 60 kDa, while in normal controls, a major band was observed at 34 kDa in all samples and minor bands corresponding to 27 and 48 kDa were detected in approximately half of the controls. To assess its consequences on ovarian cancer, the 52-kDa protein was immunoprecipitated from culture medium of an exponentially growing ovarian tumor cell line and was further purified by reverse-phase high-pressure liquid chromatography. Its effect on proliferation was assayed by determining cell doubling times and their chemosensitivity was measured in a standard cytotoxicity assay using cisplatin. In addition, decapeptides corresponding to the pro-portion of cathepsin D were analyzed in parallel. Procathepsin D and one decapeptide, peptide 2, as well as IGF-II (as a known positive) increased cell proliferation, with doubling times of 28.4, 28.8, and 30.3 h, respectively, versus untreated UL-1 cells (36.4 h). Procathepsin D treatment of UL-1 tumor cells significantly increased the cisplatin LD(50) (74.9 microgram/ml) over untreated (33.9 microgram/ml) as well as IGF-II-treated (38.8 microgram/ml) cells. Peptide 2 also showed a significant increase in LD(50) (69.5 microgram/ml) compared to untreated and peptide 1-treated cells (37.1 microgram/ml). There are several unique forms of cathepsin D expressed and accumulated by ovarian tumors and these forms are detectable in the sera of those with ovarian cancer. The presence of these procathepsin D can increase the proliferation of these tumor cells, while decreasing their sensitivity to chemotherapeutic agents. While procathepsin D and IGF-II both enhance proliferation, only procathepsin D (and peptide 2) appears to modulate chemosensitivity, suggesting a separate receptor or pathway for this consequence.

Voltage-activated potassium channels in mammalian neurons and their block by novel pharmacological agents.

1. Electrophysiological studies have shown that a number of different types of potassium (K) channel currents exist in mammalian neurons. Among them are the voltage-gated K channel-currents which have been classified as fast-inactivating A-type currents (KA) and slowly inactivating delayed-rectifier type currents (KDR). 2. Two major molecular superfamilies of K channel have been identified; the KIR superfamily and the Shaker-related superfamily with a number of different pore-forming alpha-subunits in each superfamily. 3. Within the Shaker-related superfamily are the KV family, comprising of at least 18 different alpha-subunits that almost certainly underlie classically defined KA and KDR currents. However, the relationship between each of these cloned alpha-subunits and native voltage-gated K currents remains, for the most part, to be established. 4. Classical pharmacological blockers of voltage-gated K channels such as tetraethylammonium ions (TEA), 4-aminopyridine (4-AP), and certain toxins lack selectivity between different native channel currents and between different cloned K channel currents. 5. A number of other agents block neuronal voltage-gated K channels. All of these compounds are used primarily for other actions they possess. They include organic calcium (Ca) channel blockers, divalent and trivalent metal ions and certain calcium signalling agents such as caffeine. 6. A number of clinically active tricyclic compounds such as imipramine, amitriptyline, and chlorpromazine are also potent inhibitors of neuronal voltage-gated K channels. These compounds are weak bases and it appears that their uncharged form is required for activity. These compounds may provide a useful starting point for the rational design of novel selective K channel blocking agents.

Is EAG the answer to the M-current?

The resemblance between the Drosophila EAG current and the mammalian M-current is very strong, but final confirmation for a member of the extended EAG family encoding the M-current remains to be determined.

Effects on K+ currents in rat cerebellar granule neurones of a membrane-permeable analogue of the calcium chelator BAPTA.

1. Whole cell recordings of voltage-activated K+ currents were made with the amphotericin B perforated patch technique from cerebellar granule (CG) neurones of 6-8 days rats that had been in culture for 1 to 16 days. By use of appropriate voltage protocols, the effects of the membrane-permeant form of BAPTA, 1,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), on the transient A current (IKA), the delayed rectifier current (IKV) and a standing outward current (IKSO) were investigated. 2. Bath application of 25 microM BAPTA-AM inhibited both IKV and IKSO in cultured neurones, but did not seem to affect IKA. Neither 25 microM BAPTA (free acid) nor 25 microM ethylenediaminetetraacetic acid acetoxymethyl ester (EDTA-AM) had any significant effect on the magnitude of IKSO. Similarly in short-term (1-2 days) cultured CG neurones IKV, but not IKA, was inhibited by 25 microM BAPTA-AM. 3. BAPTA-AM (2.5 microM) reduced IKV in short-term culture CG neurones, with further inhibition being seen when the perfusate was changed to one containing 25 microM BAPTA-AM. 4. Tetraethylammonium ions (TEA) (10 mM) reversibly inhibited IKV in these cells with a similar rate of block of IKV to that induced by 25 microM BAPTA-AM. 5. The degree of inhibition of IKV by 25 microM BAPTA-AM was both time- and voltage-dependent, in contrast to the inhibition of this current by TEA. 6. These data indicate that BAPTA-AM reduces K+ currents in cerebellar granule neurones and that this inhibition cannot be explained in terms of intracellular Ca2+ chelation, but is a direct effect on the underlying channels.

A non-inactivating K+ current sensitive to muscarinic receptor activation in rat cultured cerebellar granule neurons.

1. Whole-cell recordings were made from cultured cerebellar granule neurons using perforated patch clamp techniques. The primary cultures were prepared using 6- to 9-day-old Sprague-Dawley rats. 2. Neurons in culture for less than 48 h possessed resting membrane potentials of -29 mV. However, neurons in culture for 7 days had much more hyperpolarized resting membrane potentials (-89 mV). Over the same period, these neurons developed an additional component of outward current. 3. This non-inactivating current was activated by depolarization, exhibited outward rectification and reversed close to the potassium equilibrium potential. The kinetics of activation and deactivation were very rapid. 4. Muscarine ((+)-muscarine chloride) reversibly inhibited the current with an EC50 of 0.17 microM. The inhibition by muscarine was unaffected by pre-incubation for 17-20 h with 120 micrograms ml-1 pertussis toxin. 5. The current and its inhibition by muscarine were unaffected by 100 microM Cd2+. In Ca(2+)-free conditions, the current was significantly larger than in 0.5 mM Ca2+, but inhibition by 10 microM muscarine was significantly reduced. 6. The standing outward current was not obviously affected by 50 microM 5-HT, 50 microM noradrenaline, 50 microM 2-chloroadenosine or 5 mM tetraethylammonium. It was reduced by 10 microM La3+, 10 microM Zn2+ and 1 mM Ba2+. 7. Muscarinic agonists increased the input resistance of neurons and shifted the zero current level in the depolarized direction when voltage clamped. This enhanced excitability was evident under current clamp, where 10 microM muscarine depolarized granule neurons such that action potentials became evident.

Modulation of the gating of the transient outward potassium current of rat isolated cerebellar granule neurons by lanthanum.

The effects of the trivalent cation, lanthanum (La3+) on voltage-dependent K+ conductances were studied in rat isolated cerebellar granule neurons under whole-cell voltage-clamp conditions. La3+ at low micromolar concentrations caused a pronounced enhancement in the outward current evoked by depolarising steps from -50 mV, with the apparent recruitment of an inactivating component. The steady-state inactivation curve for the transient outward current, evoked by depolarising steps from -140 mV, was shifted by approximately 40 mV in the depolarising direction by 10 microM La3+, with a slight increase in the slope factor. The kinetics of activation and inactivation were slowed in the presence of La3+. A shift of 10 mV in the depolarising direction was seen for the activation curve of the delayed rectifier current in the presence of 10 microM La3+. These results indicate that La3+ has a potent effect on the gating characteristics of voltage-activated K+ currents. This effect cannot be explained by surface charge considerations.

Characterization of a new bacteriophage which infects bacteria of the genus Acidiphilium.

A novel bacteriophage, termed phi AC1, that infects strains of the genus Acidiphilium (acidophilic, heterotrophic, aerobic, Gram-negative eubacteria) most commonly isolated from acidic mine drainage environments, has been discovered and several of its properties have been determined. This is the first report of a bacteriophage infecting such cells. The virion has a lambdoid morphology and is larger than lambda, as shown by electron microscopy and sucrose gradient centrifugation. The sedimentation coefficient of the virion is approximately 615S. The nucleic acid of phi Ac1 is dsDNA, approximately 102 kb in length. Several experimental results show that phi Ac1 is a temperate phage. The plaques are turbid, and most cells isolated from plaques produced on sensitive cells by filter-sterilized phage preparations contain the phage and are resistant to further phage infection. Southern blot analysis shows that phi Ac1 prophage DNA is integrated into the bacterial genome during the temperature growth phase.

Beta-lactam antibiotics potentiate magainin 2 antimicrobial activity in vitro and in vivo.

The ability of magainin 2 to augment antibiotic therapy was examined. Susceptibility to magainin 2 was determined on Escherichia coli incubated in the presence and absence of sublethal concentrations of antibiotics both in vitro and in vivo. Experiments in buffer and normal human serum revealed that E. coli exposed to sublethal amounts of cefepime, a beta-lactam antibiotic, was significantly more susceptible to the antimicrobial activity of magainin 2. Bacteria incubated with subinhibitory concentrations of other beta-lactam type antibiotics, but not amikacin (an aminoglycoside) or ciprofloxacin (a quinolone), were also more susceptible to magainin 2 in normal human serum. Bacteria were less susceptible to magainin 2 when they were examined in heat-inactivated serum. Complement was shown to be required for magainin 2 activity in serum by using C8-deficient sera. The combination of magainin 2 and cefepime was shown to be more antimicrobial in normal human serum for a variety of bacterial strains. Magainin 2 was completely inactive as a therapeutic agent when it was administered alone (2 mg per mouse) but significantly increased the survival of mice when it was administered with a low level of cefepime.