Temporin G, an amphibian antimicrobial peptide against influenza and parainfluenza respiratory viruses: Insights into biological activity and mechanism of action.

Treatment of respiratory viral infections remains a global health concern, mainly due to the inefficacy of available drugs. Therefore, the discovery of novel antiviral compounds is needed; in this context, antimicrobial peptides (AMPs) like temporins hold great promise. Here, we discovered that the harmless temporin G (TG) significantly inhibited the early life-cycle phases of influenza virus. The in vitro hemagglutinating test revealed the existence of TG interaction with the viral hemagglutinin (HA) protein. Furthermore, the hemolysis inhibition assay and the molecular docking studies confirmed a TG/HA complex formation at the level of the conserved hydrophobic stem groove of HA. Remarkably, these findings highlight the ability of TG to block the conformational rearrangements of HA2 subunit, which are essential for the viral envelope fusion with intracellular endocytic vesicles, thereby neutralizing the virus entry into the host cell. In comparison, in the case of parainfluenza virus, which penetrates host cells upon a membrane-fusion process, addition of TG to infected cells provoked ~1.2 log reduction of viral titer released in the supernatant. Nevertheless, at the same condition, an immunofluorescent assay showed that the expression of viral hemagglutinin/neuraminidase protein was not significantly reduced. This suggested a peptide-mediated block of some late steps of viral replication and therefore the impairment of the extracellular release of viral particles. Overall, our results are the first demonstration of the ability of an AMP to interfere with the replication of respiratory viruses with a different mechanism of cell entry and will open a new avenue for the development of novel therapeutic approaches against a large variety of respiratory viruses, including the recent SARS-CoV2.

The Amphibian Antimicrobial Peptide Temporin B Inhibits In Vitro Herpes Simplex Virus 1 Infection.

The herpes simplex virus 1 (HSV-1) is widespread in the population, and in most cases its infection is asymptomatic. The currently available anti-HSV-1 drugs are acyclovir and its derivatives, although long-term therapy with these agents can lead to drug resistance. Thus, the discovery of novel antiherpetic compounds deserves additional effort. Naturally occurring antimicrobial peptides (AMPs) represent an interesting class of molecules with potential antiviral properties. To the best of our knowledge, this study is the first demonstration of the in vitro anti-HSV-1 activity of temporin B (TB), a short membrane-active amphibian AMP. In particular, when HSV-1 was preincubated with 20 µg/ml TB, significant antiviral activity was observed (a 5-log reduction of the virus titer). Such an effect was due to the disruption of the viral envelope, as demonstrated by transmission electron microscopy. Moreover, TB partially affected different stages of the HSV-1 life cycle, including the attachment and the entry of the virus into the host cell, as well as the subsequent postinfection phase. Furthermore, its efficacy was confirmed on human epithelial cells, suggesting TB as a novel approach for the prevention and/or treatment of HSV-1 infections.

Bacillomycin D and its combination with amphotericin B: promising antifungal compounds with powerful antibiofilm activity and wound-healing potency.

AIMS: In this study, we evaluated the ability of the lipopeptide bacillomycin D and the antifungal drug amphotericin B as well as their combination, to inhibit Candida albicans biofilm formation and to accelerate keratinocyte cell migration. METHODS AND RESULTS: The antibiofilm activity of bacillomycin D and its combination with amphotericin B was carried out by crystal violet colorimetric method. Our results have shown that, when combined together at low concentrations nontoxic to mammalian cells, corresponding to 1/32 MIC (0·39 µg ml(-1) ) and 1/4 MIC (0·06 µg ml(-1) ) for bacillomycin D and amphotericin B, respectively, a clear antibiofilm activity is manifested (95% inhibition of biofilm formation) along with a clear inhibition of germ tube formation. Moreover, the effect of both drugs on preformed biofilm of C. albicans strain was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The combination of the two antifungal compounds at 0·39 and 1 µg ml(-1) for bacillomycin D and amphotericin B, respectively, resulted in a clear enhancement of biofilm eradication compared to the results obtained with each drug alone. Furthermore, this combination was found to promote the closure of a gap produced in a monolayer of human keratinocytes. CONCLUSIONS: Bacillomycin D and its combination with amphotericin B display impressive anti-biofilm and wound-healing activities. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of the lipopeptide bacillomycin D and the antifungal drug amphotericin B in medical devices may offer a promising alternative for topical treatment of Candida-associated infections in the setting of a wound.

Neoadjuvant oxaliplatin and 5-fluorouracil with concurrent radiotherapy in patients with locally advanced rectal cancer: a single-institution experience.

PURPOSE: The aim of this study was to evaluate the rate of pathological response (PR), disease control and safety of neoadjuvant chemotherapy using oxaliplatin (OX) and 5-fluorouracil (5-FU) with concurrent radiotherapy for treating locally advanced rectal cancer. MATERIALS AND METHODS: Between November 2002 and December 2010, 90 patients with locally advanced rectal cancer treated with neoadjuvant chemoradiotherapy (CRT) were retrospectively analysed. All patients underwent preoperative radiotherapy (45 Gy in 1.8-Gy fractions) with concurrent OX (80 mg/m(2) i.v., day 1) and a 120-h continuous infusion of 5-FU (1,000 mg/m(2) per day). Surgery was performed within 6 weeks after completion of CRT treatment. RESULTS: Complete pathological response was obtained in six patients (6.7%), and 39 (43.3%) had their disease downstaged. The median follow-up period was 4.7 years (6 months to 9 years). Local recurrence occurred in two patients (2.2%), one of whom developed also liver metastases. Distant metastases not associated with local relapse occurred in 23 (25.6%) patients. Overall (OS) and disease-free (DFS) survival were 62.9% and 52.8%, respectively. CRT was well tolerated, with only one grade 3 (1.2%) haematological toxicity (neutropaenia). CONCLUSIONS: Neoadjuvant systemic chemotherapy based on OX and 5-UC associated with radiotherapy is well tolerated, with good results in terms of pathological response, disease control and survival, in rectal cancer patients.

Treatment of invasive male breast cancer: a 40-year single-institution experience.

PURPOSE: We conducted a retrospective analysis to evaluate the management and outcome of invasive male breast cancer treated in a single-institution over a period of 40 years. MATERIALS AND METHODS: We reviewed the clinical and pathological features of 60 male patients affected by breast carcinoma treated at our Radiotherapy Unit between 1971 and 2011. Tumours were classified according to histological type and the updated 2010 TNM classification of malignant tumours. RESULTS: At a median follow-up of 8.9 [range, 0.6-20; standard deviation (SD), 4.98] years, 32 patients (53.3%) were alive and 16 patients died (26.7%) due to disease progression and 12 (20%) due to other causes. At univariate analysis for overall survival, pathological tumour size (p=0.031), histological subtype (p=0.013) and nodal status (p=0.006) emerged as significant predictors of death. At multivariate analysis, independent death predictors were advanced pathological tumour size (p=0.016), positive nodal status (p=0.003) and invasive cribriform histological type (p=0.0003). CONCLUSIONS: In consideration of the rarity of the disease, many issues are still being debated, and future collaborative studies are required. However, our experience confirms the prognostic role of greater pathological tumour size and positive nodal status as unfavourable features for survival in male breast cancer.

Normal tissues toxicities triggered by combined anti-angiogenic and radiation therapies: hurdles might be ahead.

BACKGROUND: Combined-modality therapy is a promising approach to improve the therapeutic index of radiotherapy. However, these improvements could come at the cost of increased toxicities. Clinical trials evaluating anti-tumour efficacy of bevacizumab combined with radiotherapy have encountered unexpected side effects. This study is the first systematic evaluation of normal tissue toxicity triggered by anti-angiogenic agents combined with radiation therapy in mice. METHODS: Effect of a mouse anti-VEGF antibody was monitored on acute toxicity studying radiation-induced intestinal ulceration (12 Gy TBI); on subacute toxicity using a model of oral mucositis (16.5 Gy); on late radiation injuries by monitoring lung fibrosis (bleomycin and 19 Gy). RESULTS: Combination of irradiation with anti-VEGF antibody enhanced intestinal damages with severe epithelial ulcerations, had no adverse impact on oral mucositis and dramatically worsened the fibrotic picture induced by bleomycin and irradiation to the lung. INTERPRETATION: These reports bring to light the important questions about safety and underscore the need for appropriate preclinical modelling of the impact on normal tissues of novel drug-radiation regimens. Our findings also highlight the complexity of anti-VEGF action, which could in defined conditions exert tissue-specific protection. The findings indicate that the combination of targeted drugs with radiotherapy should be approached with caution.

Tumor-associated macrophages (TAMs) modulate response to HER2-targeted agents in a humanized mouse model of breast cancer.

PURPOSE: Tumor-associated macrophages (TAM) may participate to antitumor activity of anti-HER2-targeted therapies (Pertuzumab, Trastuzumab) in breast cancers harbouring HER-2 overexpression through antibody-dependent phagocytosis. Additive antitumor effect of concurrent cytotoxic chemotherapies, including Paclitaxel, may be counterbalanced by alteration in TAM infiltrate. The aim of this study is to evaluate the role of TAM in tumor response to anti-HER2-targeted therapies and chemotherapy in an experimental model of HER2-amplified breast cancer. METHODS: A xenograft mouse model was built by subcutaneous injection of the SKBR-3 human HER2-amplified breast cancer cell line in Hu-CD34+ mice. Animals were randomized to receive weekly administration of Cremophor (control), Trastuzumab+Pertuzumab (TP), and Paclitaxel+Trastuzumab+Pertuzumab (PTP) with or without macrophage depletion with clodronate (C). At week 4, mice were euthanised and tumors were harvested for immunohistochemical analysis of TAM infiltration (RBP-J CD163 and CD68 for M1, M2, and overall TAM, respectively). RESULTS: Tumor size was significantly lower in mice treated with TP, PTP, and PTP+C as compared to control, while no meaningful difference was observed in the TP+C arm. Analysis of TAM infiltrate showed significantly lower CD68 and CD163 expression in PTP, TP+C, and PTP+C as compared to TP and control arm. RBP-J expression was significantly decreased in mice treated with clodronate depletion. CONCLUSIONS: Activity of TP is modulated by TAM infiltrate, that is inhibited by concurrent administration of Paclitaxel. To enhance the effect of anti-HER2-targeted therapies and minimize chemotherapy-related side effects, modulation of TAM should be considered in novel therapeutic combinations.

Prospective assessment of AR splice variant and PSMA detection on circulating tumor cells of mCRPC patients: preliminary analysis of patients enrolled in PRIMERA trial (NCT04188275).

In our institution, a prospective observational trial testing micro-RNA (miRNA) and ARV7 mutational status in metastatic, castration resistant prostate cancer (mCRPC), is currently recruiting (PRIMERA trial, NCT04188275). A pre-planned interim analysis was performed when 50% of the planned accrual was reached. In this report, we explored the predictive value of Circulating Tumor Cell (CTC) detection in mCRPC patients undergoing 1st line therapy. Moreover, ARV7, ARFL, PSMA and PSA expression on CTC was reported to explore potential correlation with patient prognosis and response to therapy. PRIMERA is a prospective observational trial enrolling mCRPC patients undergoing standard treatment (ARTA + ADT) after I line ADT failure. Clinical and pathological features were collected. Outcomes selected for this preliminary analysis were time to castration resistance (TTCR), PSA at 8 weeks after ARTA therapy start, PSA drop at 8 weeks, Overall PSA drop, PSA nadir. Correlation between these outcomes and CTC detection was tested. Expression of ARV7, ARFL, PSA and PSMA was explored in CTC+ patients to assess their prevalence in this cohort and their impact on selected outcomes. Median TTCR was significantly shorter in CTC+ vs CTC- patients (32.3 vs 75 months, respectively, p = 0.03) and in ARFL+ vs ARFL- patients (30.2 vs 51.1 months, respectively, p = 0.02). ARV7, PSMA and PSA expression on CTC had no impact on median TTCR, nor on biochemical response to therapy. Patients in whom CTC and ARFL expression were detected had significant reduced TTCR. However, PSA response was not influenced by CTCs detection and specific biomarkers expression.

Binding of an antimicrobial peptide to bacterial cells: Interaction with different species, strains and cellular components.

Antimicrobial peptides (AMPs) selectively kill bacteria by disrupting their cell membranes, and are promising compounds to fight drug-resistant microbes. Biophysical studies on model membranes have characterized AMP/membrane interactions and the mechanism of bilayer perturbation, showing that accumulation of cationic peptide molecules in the external leaflet leads to the formation of pores ("carpet" mechanism). However, similar quantitative studies on real cells are extremely limited. Here, we investigated the interaction of the dansylated PMAP23 peptide (DNS-PMAP23) with a Gram-positive bacterium, showing that 107 bound peptide molecules per cell are needed to kill it. This result is consistent with our previous finding for Gram-negative strains, where a similar high threshold for killing was determined, demonstrating the general relevance of the carpet model for real bacteria. However, in the case of the Gram-positive strain, this number of molecules even exceeds the total surface available on the bacterial membrane. The high affinity of DNS-PMAP23 for the anionic teichoic acids of the Gram-positive cell wall, but not for the lipopolysaccharides of Gram-negative bacteria, provides a rationale for this finding. To better define the role of anionic lipids in peptide/cell association, we studied DNS-PMAP23 interaction with E. coli mutant strains lacking phosphatidylglycerol and/or cardiolipin. Surprisingly, these strains showed a peptide affinity similar to that of the wild type. This finding was rationalized by observing that these bacteria have an increased content of other anionic lipids, thus maintaining the total membrane charge essentially constant. Finally, studies of DNS-PMAP23 association to dead bacteria showed an affinity an order of magnitude higher compared to that of live cells, suggesting strong peptide binding to intracellular components that become accessible after membrane perturbation. This effect could play a role in population resistance to AMP action, since dead bacteria could protect the surviving cells by sequestering significant amounts of peptide molecules. Overall, our data indicate that quantitative studies of peptide association to bacteria can lead to a better understanding of the mechanism of action of AMPs.

The critical role of SDF-1/CXCR4 axis in cancer and cancer stem cells metastasis.

Chemokines exert their multifunctional role in several physiologic and pathologic processes through interaction with their specific receptors. Much evidence have revealed that metastatic spread tumor cells may use chemokine-mediated mechanisms. In particular, an involvement of stromal cell-derived factor-1 (SDF-1) in growth of primary tumors and in metastatic process has been demonstrated. Indeed, it has been suggested that CXCR4 expression by tumor cells, plays a critical role in cell metastasis by a chemotactic gradient to organs expressing the ligand SDF-1. Moreover, CXCR4 overexpression correlated with poor prognosis in many types of cancer. In physiologic condition, SDF-1 also plays an essential role modulating stem cell proliferation, survival, and homing through its canonical receptor CXCR4. Recently, several studies have demonstrated the existence of a small subset of cancer cells which share many characteristics with stem cells and named cancer stem cells (CSC). They constitute a reservoir of self-sustaining cells with the ability to maintain the tumor growth. In particular, most of them express CXCR4 receptor and respond to a chemotactic gradient of its specific ligand SDF-1, suggesting that CSC probably represent a subpopulation capable of initiating metastasis. This review focuses on the role of SDF-1/CXCR4 axis in cancer and in the metastatic progression by tumoral cells, as well as the role of CSC in tumor pathogenesis and in metastatic process. A better understanding of migratory mechanism involving cancer cells and CSC provides a powerful tool for developing novel therapies reducing both local and distant recurrences.

Use of tamoxifen in pT1a-pT1b, pN0 breast cancer.

PURPOSE: To evaluate if in low-risk breast cancer patients (pT1a-pT1b, pN0) tamoxifen can reduce local recurrence and improve survival. METHODS: Retrospectively 700 patients were analyzed. All patients were treated from 1980 to 2003 with conservative surgery plus radiotherapy at the University of Florence. No patients were treated with adjuvant chemotherapy. Tamoxifen was prescribed in 359 patients (51.3%). The crude probability of survival (or local recurrence) was estimated by using Kaplan-Meier method, and survival (or local recurrence) comparisons were carried out using Cox proportional hazard regression models. RESULTS: The univariate analysis for specific survival showed that only histological type and local recurrence were significant prognostic factors (log rank test: p=0.02 and p<0.0001, respectively). The Cox regression model by stepwise selection confirmed lobular histological type (p=0.008; HR: 3.83, 95% CI: 1.31-11.21) and local recurrence (p<0.001; HR: 9.05, 95% CI: 3.05-26.82) as independent prognostic factors for disease specific survival. For local disease free survival, multivariate analysis did not show any significant parameters. CONCLUSION: In our series tamoxifen did not seem to improve disease specific survival and local disease specific survival. The number of events in terms of death for cancer or in terms of local recurrence is too small in this group of patients. However, according to our results we suggest not to prescribe tamoxifen in patients affected by pT1a-pT1b, pN0 breast cancer.

Adjuvant hypofractionated radiation therapy for breast cancer after conserving surgery.

AIMS: To evaluate the incidence of locoregional recurrence (LRR) and the cosmetic results in a group of patients with breast cancer treated with a hypofractionated schedule of adjuvant radiotherapy after conservative surgery. MATERIALS AND METHODS: In total, 539 patients with pTis-pT1-pT2 breast cancer underwent radiotherapy treatment after conservative surgery at the University of Florence and at the Pistoia Hospital. The dose delivered was 44 Gy (2.75 Gy daily fraction). The tumour bed boost (10 Gy) was given by electrons. RESULTS: At the time of the analysis, 1.8% of patients (10/539) had breast relapse. No patients developed nodal recurrence (supraclavicular, axillary and internal mammary nodes). The 3- and 5-year actuarial rates for LRR were 1.2% (+/- 0.5% standard error) and 2.1% (+/- 0.6% standard error), respectively. Considering the late toxicity, we found that 412 (76.4%) patients had grade 0 or grade 1 late toxicity, 113 patients (20.9%) had grade 2 late toxicity and 14 patients (2.5%) had grade 3 late toxicity. No patients developed grade 4 toxicity. CONCLUSION: This type of approach resulted in an effective treatment in terms of local control in patients with negative or one to three positive axillary nodes and negative surgical margins. Patients treated with a hypofractionated schedule showed very good cosmesis.

Impact of molecular subtypes classification concordance between preoperative core needle biopsy and surgical specimen on early breast cancer management: Single-institution experience and review of published literature.

BACKGROUND: Core needle biopsy (CNB) plays a crucial role as diagnostic tool for breast cancer (BC). The characterization of biomarkers status before surgical treatment is crucial when primary systemic therapy is a therapeutic option. The aim of this analysis was to report concordance between preoperative CNB and surgical specimen (SS) in evaluating biomarkers and molecular subtypes. METHODS: Data have been collected from a cohort of 101 patients affected by early BC treated at Careggi Florence University Hospital, between January 2014 and March 2015. The conformity between molecular subtype classification was tested using kappa (κ) test. RESULTS: Mean age was 57.5 years (range 29-86). There was concordance between the estrogen receptor (ER) assessment on CNB and SS in 95 cases (94.1%). Concordance of the progesterone receptor (PgR) assessment was observed in 89 cases (88.1%). Concordance for detecting immunohistochemistry-assessed BC molecular subtypes was 87.1% (κ = 0.78). Concerning Ki-67 evaluation, we report a concordance rate of 88.1% (κ = 0.68). The evaluation of luminal A plus luminal B/HER negative subgroup showed a κ-value of 0.65. CONCLUSIONS: CNB showed good accuracy in evaluating hormonal receptors status, HER2, and BC molecular subtypes. Evaluation of Ki67 status was less accurate than other biomarkers; therefore, we recommend that it should be detected both on CNB and SS samples, especially in hormonal positive HER2 negative tumors, in order to avoid a misclassification of tumor subtypes that could lead to an omission of potential effective systemic therapy.

Ionic channels underlying cardiac automaticity: new insights from genetically-modified mouse strains.

The spontaneous activity (or pacemaker activity) of the heart constitutes a fundamental physiological function in higher organisms. Pacemaker activity is generated in the sino-atrial node (SAN) by a specialized cell population adapted to the generation of a rhythmic electrical oscillation. The precise ionic mechanisms underlying initiation of pacemaking in automatic cells has not been entirely elucidated. Ionic channels and intracellular Ca2+ signalling in pacemaker cells are both required for the proper setting of pacemaking. Understanding the mechanisms of pacemaker activity is important for developing new therapeutic approaches for controlling the heart rate in the diseased myocardium. Controlling the heart rate in the clinical practice is a promising way to increase cardioprotection and improve patient's survival in cardiac ischemic pathology. We describe here the contribution of several ion channels families into the generation and regulation of the heart rate using new approaches involving genetically modified mouse strains. These studies underline the functional redundancy of mechanisms underlying pacemaking, an important safety parameter for new drugs targeting ion channels to modulate cardiac frequency.

If current inhibition: cellular basis and physiology.

The slow diastolic depolarization phase in cardiac pacemaker cells is the electrical basis of cardiac automaticity. The hyperpolarization-activated current (I(f)) is one of the key mechanisms underlying diastolic depolarization. Particularly, I(f) is unique in being activated on membrane hyperpolarization following the repolarization phase of the action potential. I(f) has adapted biophysical properties and voltage-dependent gating to initiate pacemaker activity. I(f) possibly constitutes the first voltage-dependent trigger of the diastolic depolarization. For these reasons, I(f) is a natural pharmacological target for controlling heart rate in cardiovascular disease. In this view, I(f) inhibitors have been developed in the past, yet the only molecule to have reached the clinical development is ivabradine. At the cellular level, the remarkable success of ivabradine is to be ascribed to its relatively high affinity for f-channels. Furthermore, ivabradine is the most I(f)-specific inhibitor known to date, since moderate inhibition of other voltage-dependent ionic currents involved in automaticity can be observed only at very high concentrations of ivabradine, more than one order of magnitude from that inhibiting I(f). Finally, the mechanism of block of f-channels by ivabradine has particularly favorable properties in light of controlling heart rate under variable physiological conditions. In this article, we will discuss how I(f) inhibition by ivabradine can lead to reduction of heart rate. To this aim, we will comment on the role of I(f) in cardiac automaticity and on the mechanism of action of ivabradine on f-channels. Some aspects of the cardiac pacemaker mechanism that improve the degree of security of ivabradine will also be highlighted.

Properties of the hyperpolarization-activated current (I(f)) in isolated mouse sino-atrial cells.

OBJECTIVE: We have investigated the properties of the hyperpolarization-activated (I(f)) current in pacemaker cells from the mouse sino-atrial node (SAN). METHODS: The I(f) current was studied in cells isolated enzymatically from the SAN region of adult C57BL6/J mice. The whole-cell variation of the patch-clamp technique was employed to investigate the basic properties of I(f). RESULTS: In mouse SAN cells, the I(f) current density at -120 mV was 18+/-2 pA/pF (n=23). I(f) was not detected in cells showing atrial-like morphology that were also found in SAN preparations (n=7). I(f) was blocked by 5 mM Cs(+), was inhibited by application of 5 microM acetylcholine, and was increased by 10 microM noradrenaline. The I(f) current reversal potential was -31+/-2 mV under physiological concentration of Na(+) and K(+) ions. Lowering the extracellular Na(+) concentration reduced I(f) amplitude, while increased when the extracellular K(+) concentration was augmented. I(f) voltage for half activation was -87+/-1 mV (n=6). CONCLUSIONS: We conclude that the native I(f) current in mouse SAN cells shows functional properties that are similar to I(f) described in rabbit SAN tissue. This study opens the possibility of investigating the involvement of I(f) in the regulation of heart rate in genetically modified mice.

Facilitation of the L-type calcium current in rabbit sino-atrial cells: effect on cardiac automaticity.

OBJECTIVE: The L-type Ca(2+) current (I(Ca,L)) contributes to the generation and modulation of the pacemaker action potential (AP). We investigated facilitation of I(Ca,L) in sino-atrial cells. METHODS: Facilitation was studied in regularly-beating cells isolated enzymatically from young albino rabbits (0.8-1 kg). We used the whole-cell patch-clamp technique to vary the frequency of the test depolarizations evoked at -10 mV or the conditioning diastolic membrane potential prior to the test pulse. RESULTS: High frequencies (range 0.2-3.5 Hz) slowed the decay kinetics of I(Ca,L) evoked from a holding potential (HP) of -80 mV in 68% of cells resulting in a larger Ca(2+) influx during the test pulse. The amount of facilitation increased progressively between 0.2 and 3.0 Hz. When the frequency was changed from 0.1 to 1 Hz, the averaged increase in the time integral of I(Ca,L) was 27+/-7% (n=22). Application of conditioning voltages between -80 and -50 mV induced similar facilitation of I(Ca,L) in 73% of cells. The maximal increase of Ca(2+) entry occurred between -60 and -50 mV, and was on average 38+/-14% for conditioning prepulses of 5 s in duration (n=15). Numerical simulations of the pacemaker activity showed that facilitation of I(Ca,L) promotes stability of sino-atrial rate by enhancing Ca(2+) entry, thus establishing a negative feedback control against excessive heart rate slowing. CONCLUSION: Facilitation of I(Ca,L) is present in rabbit sino-atrial cells. The underlying mechanism reflects modulation of I(Ca,L) decay kinetics by diastolic membrane potential and frequency of depolarization. This phenomenon may provide an important regulatory mechanism of sino-atrial automaticity.

Modulation of the alpha 1A Ca2+ channel by beta subunits at physiological Ca2+ concentration.

The class A Ca2+ channel alpha 1 subunit (alpha 1A) was expressed in Xenopus oocytes alone or in combination with the beta 1b, beta 2a, beta 3, or beta 4 subunit. Analysis of voltage-dependent activation and inactivation in the presence of 1.8 mM external Ca2+ showed an hyperpolarising shift of both relations when compared to similar recordings performed in the presence of 40 mM Ba2+. These shifts, which differed for activation and inactivation, were strongly modulated by the nature of the coexpressed beta subunit. On the other hand, for each combination, the kinetics of inactivation were similar in 1.8 mM Ca2+ and 40 mM Ba2+ (for example co-expression of the beta 2a subunit reduced inactivation using either 40 mM Ba2+ or 1.8 mM Ca2+). Thus, modulation of channel properties by the beta subunit is different in physiological Ca2+ or high Ba2+ concentrations. These results must be taken into consideration to extrapolate the role of the beta subunit in native cells.

Effect of glucocorticoids on the synthesis of antimicrobial peptides in amphibian skin.

Gene-encoded peptide antibiotics are widespread in insects, plants and vertebrates and confer protection against bacterial and fungal infections. NF-kappaB is an important transcription factor for many immunity-related mammalian proteins and also for insect immune genes. The activity of NF-kappaB is regulated by the interaction with an inhibitor, I kappaB. It was recently demonstrated that glucocorticoids induce the synthesis of I kappaB in human cell lines. So far, all genes for peptide antibiotics have promoter motifs with NF-kappaB binding sites, but its actual function in peptide regulation has been studied only in insects. Here we show that glucocorticoid treatment of the frog Rana esculenta inhibits the transcription of all genes encoding antibacterial peptides by inducing the synthesis of I kappaB alpha. These results suggest that also in vertebrates peptide-mediated innate immunity is controlled by NF-kappaB-regulated transcription.