Research progress on natural inhibitors towards phosphodiesterases / 中草药

Phosphodiesterases (PDEs) represent an important enzyme class constituted by 11 isozymes (PDE1 to PDE11). They can regulate gene expression and nucleotide signaling pathway through the hydrolysis of cAMP and cGMP. Studies showed that PDEs have become one of the most popular research topics in the new drug development because they involved in the development of many pathological processes, such as inflammation, depression, asthma and so on. In recent years, many specific and efficient PDEs inhibitors derived from natural products have been reported. This paper reviews the biochemical properties, physiological functions, and their natural inhibitors of the PDEs families for providing a solid support for the further new drug development of selective PDEs inhibitors.

Relationship between phosphodiesterase 4D gene rs966221 single nucleotide polymorphisms and ischemic stroke / 中华老年心脑血管病杂志

Objective To study the relationship between phosphodiesterase 4D (PDE4D) gene rs966221 single nucleotide polymorphisms (SNPs) and ischemic stroke (IS) in Guangxi Zhuang population.Methods One hundred and one IS patients from Guangxi Zhuang Autonomous Region served as IS pgroup and 104 healthy subjects undergoing physical ecamination served as control group in this study.Their PDE4D gene rs966221 SNPs were detected by SNaPshot technique.The genotypes and frequencies of alleles were compared between the two groups and the relationship between PDE4D gene rs966221 SNPs and IS was analyzed.Results No significant difference was found in the GG,GA,AA genotypes and in the frequencies of G and A alleles between the two groups (0.99％ vs 3.85％,29.70％ vs 21.15％,69.31％ vs 75.00％,P＞0.05;15.84％ vs 14.42％,84.16％ vs 85.58％,P＞0.05).Univariate and multivariate logistic regression analysis showed that the PDE4D gene rs966221 SNPs were not related with the risk of IS in dominant AA vs GG+GA,recessive GG vs AA+GA and additive GG vs AA genetic models (P＞0.05).Conclusion The PDE4D gene rs966221 SNPs are not related with IS in Guangxi Zhuang population.

Daily use of phosphodiesterase type 5 inhibitors as prevention for recurrent priapism / Prevenção do priapismo recorrente com a utilização diária de inibidores da fosfodiesterase tipo 5

Summary Objective: The pathogenesis of recurrent priapism is currently being investigated based on the regulation of the phosphodiesterase 5 (PDE5) enzyme. We explored the daily use of PDE5 inhibitors to treat and prevent priapism recurrences. Method: We administered PDE5 inhibitors using a long-term therapeutic regimen in seven men with recurrent priapism, with a mean age of 29.2 years (range 21 to 35 years). Six men (85.7%) had idiopathic priapism recurrences and one man (24.3%) had sickle cell disease-associated priapism recurrences. Tadalafil 5 mg was administered daily. The mean follow-up was 6.6 months (range 3 to 12 months). Results: Daily long-term oral PDE5 inhibitor therapy alleviated priapism recurrences in all patients. Five (71.4%) had no episodes of priapism and two (28.6%) referred decrease in their episodes of priapism. All patients referred improvement in erectile function. Conclusion: These findings suggest the hypothesis that PDE5 dysregulation exerts a pathogenic role for both sickle cell disease-associated priapism and for idiopathic priapism, and that it offers a molecular target for the therapeutic management of priapism. These preliminary observations suggest that continuous long-term oral PDE5 inhibitor therapy may treat and prevent recurrent priapism.

Resumo Objetivo: Uma das teorias propostas para explicar a etiologia do priapismo recorrente está baseada no mecanismo de regulação da fosfodiesterase tipo 5. Estudamos o uso diário dos inibidores de fosfodiesterase tipo 5 no tratamento e na prevenção do priapismo recorrente. Método: Sete homens com diagnóstico de priapismo recorrente, com idade média de 29,5 anos (21 a 35 anos), utilizaram inibidor de fosfodiesterase tipo 5 em dose diária (tadalafila 5 mg/dia) por período prolongado. Seis homens (85,7%) apresentavam priapismo recorrente de etiologia idiopática, e um homem (24,3%), de etiologia associada à anemia falciforme. O seguimento médio foi de 6,6 meses (3 a 12 meses). Resultados: Todos os pacientes se beneficiaram com a utilização de inibidores de fosfodiesterase tipo 5. Cinco (71,4%) não apresentaram nenhum episódio de priapismo e dois (28,6%) relataram diminuição dos episódios. Todos os pacientes relataram melhora da função erétil. Conclusão: Estes achados sugerem que a hipótese do mecanismo de regulação da fosfodiesterase tipo 5 exerce papel importante na patogenia do priapismo recorrente. O uso contínuo e diário de inibidores da fosfodiesterase tipo 5 pode ser uma opção no tratamento do priapismo recorrente.

Progress in c-di-GMP inhibitors / 生物工程学报

The cyclic dinucleotide c-di-GMP is known as an important second messenger in bacteria, which controls various important cellular processes, such as cell differentiation, biofilm formation and virulence factors production. It is extremely vital for the development of new antibacterial agents by virtue of blocking c-di-GMP signal conduction. Current research indicates that there are three potential targets for discovering new antibacterial agents based on c-di-GMP regulated signal pathway, which are c-di-GMP synthases, c-di-GMP degrading enzymes and c-di-GMP receptors. Herein, we review small molecules that have been developed to inhibit c-di-GMP related enzymes and indicate perspectives of c-di-GMP inhibitors.

Protective effects of adipose?derived stem cells with phosphodiesterase 5 inhibition by lentivirus?mediated stable gene silencing on ischemia?reperfusion injury of renal tubular epithelial cells / 中华肾脏病杂志

Objective To explore the protective effects of adipose - derived stem cells (ADSCs) with phosphodiesterase 5 inhibition by lentivirus-mediated stable gene silencing on the proliferation and apoptosis of renal tubular epithelial cells induced by ischemia-reperfusion injury in vitro. Methods To isolate cultivate and indentify ADSCs from rats. Lentiviral expression vector of carrying PDE5 shRNA gene was transfected into ADSCs, and a negative control group was set up.Western blotting was used to detect PDE5 protein expression levels. ADSCs were co-cultured with NRK-52E in a transwell system, and NRK-52E cells were treated with ischemia/reoxygenation protocol. Edu assay was performed to evaluate the proliferation of NRK cells, flow cytometry to detect the apoptosis of NRK cells, and ELISA to quantify the protein expressions of fibroblast growth factor (FGF) and hepatocyte growth factor (HGF). The expression of E - cadherin and cytokeratin 18 (CK18) was quantified by real time PCR and flow cytometry. Results Western blotting for PDE5 protein indicated a significant reduction of PDE5 protein levels in PDE5 shRNA transduced population. After the treatment of ischemia/reoxygenation in vitro, the proliferative viability and apoptosis of NRK-52E cells co-cultured with ADSCs induced by PDE5 gene inhibition were significantly improved, compared to the normal group (all P<0.05). And the release of HGF, FGF were markedly enhanced (all P<0.05). Moreover, the NRK-52E cells survival, the expression of E-cadherin and CK18 on PDE5 inhibited ADSCs co-cultured with I/R injured NRK cells was significantly increased compared to that in the negative control group (all P<0.05). Conclusion ADSCs preconditioned by inhibition of PDE5 can be a powerful novel approach to improve the survival of renal tubular cells following ischemia-reperfusion injury, and have an obvious tendency to transform epithelial cells.

Relationship of the phosphodiesterase 4D gene rs153031 polymorphism and the susceptibility of unstable angina pectoris / 重庆医学

Objective To investigate the association between the phosphodiesterase 4D(PDE4D) gene rs153031 polymorphism and the susceptibility of unstable angina pectoris(UAP) in Chinese Han population of Changwu region .Methods The PDE4D gene rs153031 polymorphism was genotyped by Taqman probe in 172 UAP patients(UAP group) ,as well as in gender-and-age-matched 220 subjects without coronary heart disease(CHD)(control group) .Results In this crowd ,there was PDE4D gene rs153031 poly-morphism in patients with UAP and in subjects without CHD .Compared with control group ,frequencies of GG ,GA ,AA genotypes and G allele of rs153031 in UAP group showed no statistical differences (P> 0 .05) .Conclusion In Chinese Han population of Changwu region ,PDE4D gene rs153031 polymorphism shows no association with the susceptibility of UAP .

Significance of salivary phosphodiesterase level in oral squamous cell carcinoma patients.

Oral cancer, more specifically oral squamous cell carcinoma (OSCC) consider as common cancer that 300,000 people diagnosed per year worldwide. The only effective treatment for OSCC is surgical intervention. Over the past two decades, overall disease condition has not improved although advancement of treatment has considerably increased. The phosphodiesterase (PDEs) are responsible for the hydrolysis of the second messengers with a fundamental role in the transduction of the intracellular signals. In numerous pathological conditions such as cellular differentiation, apoptosis, and tumor invasivity the different PDF activity has been observed that shown role in pathophysiological mechanism. The role of PDEs as an intervention factor for activation of angiogenesis by influencing a tumor growth has been shown. The objective of this study was to estimate and compare salivary PDEs levels in healthy controls and biopsy-proven oral cancer patients before definitive therapy. Study was done in patients age between 25-65 years biopsy proven oral cancer patients and control group. After obtaining prior consent from biopsy-proven oral cancer patients (n=26) (before onset of any definitive treatment) and age- and sex-matched healthy controls (n=29), salivary sample was collected for estimation of the activity of phosphodiesterases (PDEs).

Role of phosphodiesterase 4B in lipopolysaccharide-induced release of inflammatory factors in rat microglias / 中华麻醉学杂志

Objective To evaluate the role of phosphodiesterase 4B (PDE4B) in lipopolysaccharide (LPS)-induced release of inflammatory factors in rat microglias.Methods The pri mary cultured microglial cells were randomly divided into 5 groups (n =24 each) using a random number table:control group (group C),LPS group,vehicle group,mismatch small interfering RNA (siRNA) group and PDE4B-siRNA group.The cells were incubated for 48 h in C and LPS groups.The cells were transfected with lipofectaminTM RNAiMAX 1 μl for 48 h in vehicle group.In mismatch siRNA and PDE4B-siRNA groups,mismatch siRNA 2 μl and PDE4B-siRNA 2 μl were added to 100μl serum-free culture medium (final concentration of siRNA 20 nmol/L),respectively,lipofectaminTM RNAiMAX 1 μl was added simultaneously and the cells were then transfected for 48 h.The expression of PDE4B protein and mRNA was determined by Western blot and real-time PCR,respectively.The cells were cultured for 24 h in serum-free culture medium containing LPS 100 ng/ml in LPS,vehicle,mismatch siRNA and PDF4B-siRNA groups.Then the release of TNF-α and IL-1β was measured by ELISA and the expression of extracellular signalregulated protein kinase (ERK) and phosphorylated ERK (p-ERK) was detected by Western blot.Results Compared with C group,the expression of PDE4B protein and mRNA was significantly down-regulated in group PDE4BsiRNA (P ＜ 0.05),no significant changes were found in LPS,vehicle and mismatch siRNA groups (P ＞ 0.05),and the release of TNF-α and IU1β was increased and the expression of p-ERK was up-regulated in the other four groups (P ＜ 0.05).Compared with LPS group,the release of TNF-α and IL-1β was decreased and the expression of p-ERK was down-regulated in PDE4B-siRNA group,and no significant changes were found in vehicle and mismatch siRNA groups (P ＞ 0.05).There was no significant difference in ERK expression between the five groups (P ＞ 0.05).Conclusion PDF4B can promote LPS-induced release of inflammatory factors in rat microglias and activation of ERK is involved in the mechanism.

Role of expression of phosphodiesterase 4B in spinal cord in inflammatory responses in a rat model of neuropathic pain: relationship with extracellular signal-regulated kinase / 中华麻醉学杂志

Objective To evaluate the role of expression of phosphodiesterase 4B (PDE4B) in the spinal cord in inflammatory responses in a rat model of neuropathic pain and the relationship with extracellular signal-regulated kinase (ERK).Methods Sixty healthy male Sprague-Dawley rats,weighing 180-220 g,in which intrathecal catheters were implanted at L5,6 interspace,were used.The location of catheters was confirmed 6 days later.The rats were randomly divided into 5 groups (n =12 each):sham operation group (group Sham),normal saline (NS) group,vehicle group (group Ⅴ),mismatch siRNA group (group siR-M),and PDE4B-siRNA group (group siR-B).Neuropathic pain was induced by ligation of L5 spinal nerve (SNL).In Sham group,the L5 spinal nerve was only exposed,but not ligated.Immediately after ligation and on 1,3,5,and 7 days after ligation,10 μl NS,10 μl NS,LipofectaminTM RNAiMAX,PDE4B-siRNA (2 μg/10 μl) encapsulated in mismatch siRNA and PDE4B-siRNA (2 μg/10 μl) encapsulated in LipofectaminTM RNAiMAX were injected intrathecally in Sham,NS,V,siR-M,and siR-B groups,respectively.The mechanical pain threshold was measured at 1 day before and 2,4,6 and 8 days after operation.After behavioral testing on 8th day after operation,the rats were sacrificed and the lumbar segment of the spinal cord was removed for determination of PDE4B protein,ERK and phosphor-ERK (p-ERK)expression,and TNF-α,IL-1β and IL-6 levels.Results Compared with Sham group,the mechanical pain threshold was significantly decreased at 2,4,6 and 8 days after operation in NS,V,siR-M and siR-B groups (P ＜0.05),and no significant change was found in the mechanical pain threshold at 2,4,6 and 8 days after operation (P ＞ 0.05) and the expression of p-ERK and PDE4B protein,and levels of TNF-α,IL-1β and IL-6 were increased at 2,4,6 and 8 days after operation in V and siR-M groups (P ＜ 0.05).Compared with NS group,the mechanical pain threshold was significantly increased,and the expression of p-ERK and PDE4B protein and levels of TNF-α,IL-1β and IL-6 were decreased at 2,4,6 and 8 days after operation (P ＜ 0.05),and no significant change was found in the parameters mentioned above in V and siR-M groups (P ＞ 0.05).Conclusion Up-regulation of the expression of PDE4B protein in the spinal cord is involved in the development of neuropathic pain in rats,which may be related to promoted phosphorylation of ERK in the spinal cord and enhanced inflammatory responses.

ALOX5AP and PDE4D gene polymorphisms and ischemic stroke / 国际脑血管病杂志

Cerebral infarction is a polygenic disease caused by genetic factors and environmental factors.The first discovery in the Icelanders is that the ALOX5AP and PDE4D gene polymorphism may be associated with cerebral infarction.So far,many conclusions of foreign studies are still controversial.This article will summarize the research status and the progress of these two genes.

Aspirin attenuates the anti-inflammatory effects of theophylline via inhibition of cAMP production in mice with non-eosinophilic asthma

Theophylline is commonly used to treat severe asthma and chronic obstructive pulmonary disease (COPD) characterized by non-eosinophilic inflammation. Acetyl salicylic acid (ASA) is one of the most widely used medications worldwide, but up to 20% of patients with asthma experience aggravated respiratory symptoms after taking ASA. Here we evaluated the adverse effect of ASA on the therapeutic effect of theophylline in mice with non-eosinophilic asthma. A non-eosinophilic asthma mouse model was induced by airway sensitization with lipopolysaccharide-containing allergen and then challenged with allergen alone. Therapeutic intervention was performed during allergen challenge. Theophylline inhibited lung inflammation partly induced by Th1 immune response. ASA attenuated the beneficial effects of theophylline. However, co-administration of the ASA metabolite salicylic acid (SA) showed no attenuating effect on theophylline treatment. The therapeutic effect of theophylline was associated with increase in cAMP levels, which was blocked by co-treatment of theophylline and ASA. ASA co-treatment also attenuated the anti-inflammatory effects of a specific phosphodiesterase 4 inhibitor. These results demonstrate that ASA reverses anti-inflammatory effects of theophylline, and that ASA exerts its adverse effects through the inhibition of cAMP production. Our data suggest that ASA reverses lung inflammation in patients taking theophylline, although clinical evidence will be needed.

Phosphodiesterase 4D Gene and Risk of Noncardiogenic Ischemic Stroke in a Korean Population

Recently published studies from different populations provide apparently conflicting evidence on the association between the phosphodiesterase 4D (PDE4D) gene and ischemic stroke. The relationship between a representative PDE4D genotype and ischemic stroke was explored in a case-control study of 205 consecutive Korean patients with noncardiogenic ischemic stroke and 103 healthy controls who were neurologically and radiologically proven to be stroke-free. We selected and genotyped a PDE4D single nucleotide polymorphism (SNP 41, rs152312) as a candidate marker for susceptibility to ischemic stroke because SNP 41 has shown the most significant association with stroke in both a meta-analysis and the original Icelandic study of the PDE4D gene. No significant difference was observed between the cases and controls in the distribution of the PDE4D SNP 41 genotypes. The results from the adjusted conditional logistic regression analysis (adjusted for age, hypertension, diabetes and smoking status) showed no significant association between PDE4D SNP 41 genotypes and an increased risk of noncardiogenic ischemic stroke. The PDE4D gene is not a major risk factor for noncardiogenic ischemic stroke in a Korean population, which supports the recent evidence suggesting that the causative genetic variants of ischemic stroke may differ across populations.

The Analysis of Clinical Efficacy of Phosphodiesterase 5 Inhibitors for Erectile Dysfunction using SS-Penogram / 대한남성과학회지

PURPOSE: As the first-line pharmacotherapy for erectile dysfunction (ED), phosphodiesterase type 5 (PDE5) inhibitors are widely used and their clinical efficacy is well established. But up to now, most of these comparative studies have been based on questionnaires, and Udenafil and Mirodenafil, which have recently been developed in South Korea, have not yet to be compared in clinical studies. We compared and analyzed the efficacy of five PDE5 inhibitors using the sexual stimulated (SS)-Penogram. MATERIALS AND METHODS: A total of 347 patients taking PDE5 inhibitors for the treatment of ED and underwent SS-Penogram was enrolled in this study. AUC (area under the curve) of SS-Penogram was compared in each PDE5 inhibitors. Differences in AUC according to age, combined disorders, serum testostereone and prolactin, cholesterol and IIEF were also analysed using Chi-square and Kruskal-Wallis test. P-value less than 0.05 was considered as statistically insignificant. RESULTS: Ratios of Type I curves in medication groups with normal erection were not statistically different before and after medication. Ratios of the excellent response (>50 % increase in area under the curve) and non-response groups (no increase or even decrease in area under the curve) in each medication group were not statistically different. CONCLUSIONS: In this clinical comparative study of the efficacy of PDE5 inhibitors using the SS-Penogram, there were no definite differences in efficacy among PDE5 inhibitors.

Past, Present and Future of PDE5 Inhibitor / 대한남성과학회지

The phosphodiesterase type-5 inhibitors (PDE5-Is) sildenafil, vardenafil and tadalafil are widely used as first-line therapy for erectile dysfunction (ED). Since the approval of sildenafil in 1998, more than 40 million men worldwide have been successfully treated with PDE5-Is. Pharmacologically, the proven safety and high tolerance of PDE5-Is is an attractive tool to investigate further physiological functions of PDE5, for example the modulation of intracellular cyclic GMP (cGMP) pools. As cGMP is a key component of intracellular signaling this may provide novel therapeutic opportunities beyond ED even for indications in which chronic administration is necessary. The approval of sildenafil for the treatment of pulmonary hypertension in 2005 was a notable success in this area of research. A number of other potential new indications are currently in various phases of preclinical research and development. In recent years, extensive but very heterogeneous information has been published in this field. The aim of this review is to summarize existing preclinical and clinical knowledge and critically discuss the evidence to support potential future indications for PDE5 inhibitors.

Effect of sildenafil citrate on interleukin-1beta-induced nitric oxide synthesis and iNOS expression in SW982 cells

The purpose of this study was to identify the effect of sildenafil citrate on IL-1 beta induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1 beta stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1 beta -induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1 beta treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1 beta -induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1 beta -induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines.

Time-Phasic Development of Nitrate Tolerance According to the Hemodynamic Responses and the Expression of Phosphodiesterase 1A1

BACKGROUND AND OBJECTIVES: Time-phasic development of nitrate tolerance in cardiovascular diseases is very important because it can contribute to the advent of blunted vasodilation or rebound ischemia even during continuous NTG treatment. In such a condition, we should change the therapeutic regimen of nitrate treatment to prevent the worsening of symptoms. MATERIALS AND METHODS: We created a nitrate-tolerant rat model using an osmotic minipump, and we examined the hemodynamic response to bolus NTG infusion in vivo. We checked the phosphodiesterase (PDE) 1A1 mRNA and protein level by relative quantitative RT-PCR and western blot analysis. We used 8-cpt-cGMP for investigating the development of a time-phasic nitrate tolerance mechanism after nitrate infusion. RESULTS: NTG-treated rats revealed a significant decrease in NTG-induced MAP drop (nitrate tolerance) from 1-day and this continued to the third day. The mRNA and protein levels of PDE1A1 similarly increased during these periods. CONCLUSION: This study revealed the development of time-phasic nitrate tolerance from the the aspects of in vivo hemodynamic responses and PDE 1A1 gene expression, and our work supports the need for further investigation to come up with a different therapeutic strategy and new drugs.

Changes of adenyl cyclase and phosphodiesterase activities at different time point in hypothalamus of rats with fever and hypothermia / 中国病理生理杂志

AIM: To observe the changes of adengl cyclase(AC) and phosphodiesterase(PDE) activities of at different time point in hypothalamus of rats with fever and hypothermia. METHODS: Radioisotope method was used to measure the activity of AC and PDE. RESULTS:The fresh yeast caused rats fever after subcutaneous injection 4h( P

Astragali radix extract ameliorates renal resistance to atrial natriuretic peptide in rats with experimental nephrotic syndrome / 中国病理生理杂志

AIM:To study the effects of astragali radix extract(ARE)on renal resistance to atrial natriuretic peptide(ANP)in rats with experimental nephrotic syndrome.METHODS:Male Sprague-Dawley rats were randomly divided into normal control,adriamycin nephropathy(ADR),ADR treated with ARE(2.5 g? kg-1? d-1)and ADR treated with benazepril(10 mg? kg-1? d-1).After 6 weeks,rats received intravenous infusion of 2% body weight isotonic saline.Urinary cGMP excretion(UcGMPV),plasma ANP level,renal PDE5 activity and protein expression were also detected.RESULTS:ARE increased UNaV while ACEI was not natriuretic.Nephrotic rats had a blunted natriuretic response and reduced rate of UcGMPV after volume expansion despite higher plasma ANP concentration.ARE increased UcGMPV and restored partly natriuretic response to volume expansion.The activity and protein abundance of renal PDE5 were high in nephrotic rats.ARE significantly reduced the PDE5 activity and protein expression.CONCLUSION:ARE may ameliorate the renal resistance to ANP in rats with adriamycin nephropathy by inhibiting the PDE5.

Observation on the change of activites of adenyl cyclase and phosphodiesterase in the hypothalamus of rabbits during fever response and electric acupuncture / 中国病理生理杂志

24 white big ear female rabbits were divided into 3 groups at random, i. e. control group (CG), fever group (FG) and electric acupuncture group (EAG). CG were not given any treatment. Endotonin (ET) was i. v. injected into animals of FG and fever ensued. 80 min after the injection ET the body temperature of the animals raised 1.18℃. 80 min after the ET injection into animals of EAG. the body temperature of animals raise 0.35℃. The body temperature of CG was compared with that of FG an obvious difference (P

A study on the mechanism of insulin resistance of adipose tissue after scalding in rats / 第三军医大学学报

The possible mechanism of insulin resistance of the adipose tissue after burn injury was studied.The binding activity of the insulin receptors,the insulin effects on insulin-sensitive phosphodiesterase(PDE)and the level of free fatty acid(FFA)and cAMP were observed for 4 d in the adipose tissue of the rats inflicted with 30% TBSA full thickness scalding. It was found that the maximal binding capacity of the insulin receptors was greatly decreased(P