Sensory and Sympathetic Nerve Sprouting in the Rat Cornea Following Neonatal Administration of Capsaicin

Corneal sensory and sympathetic nerves exert opposing actions on corneal mitogenesis and wound healing. The mechanisms by which these nerves exert their actions are unknown; however, the release of axonally transported neuropeptides has been postulated. In the present study, we investigated changes in innervation densities of calcitonin gene-related peptide (CGRP-) and tyrosine hydroxylase (TH-)immunoreactive (IR) nerves of the rat cornea following neonatal capsaicin administration, and the relationships between these changes and the development of neuroparalytic keratitis. Newborn rats were injected with capsaicin on each of the first 3 days of life. Forty-eight hours after the last injection, corneal CGRP immunostaining had totally disappeared from the cornea, whereas TH immunostaining was relatively unaffected. Over the next several weeks, a dramatic reinnervation of the cornea took place. By 6–8 weeks both the CGRP-and TH-IR corneal innervation density in the capsaicin-treated animals exceeded that of age-matched control or normal animals; that is, the corneas had become “hyper-reinnervated”. The pattern of innervation that returned was grossly abnormal and was characterized by the presence of a bizarre subepithelial plexus of fine stromal sprouts; an abundance of myelinated axons; and complex, atypical, epithelial leash morphologies. Retrograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) from the central cornea in control and capsaicin-treated adult animals labeled an average of 143 and 47 trigeminal ganglion cells, respectively (with mean diameters of 25.7 × 0.49 μm and 34.3 × 0.72 μm), suggesting a 67% decrease in corneal afferent neurons in the capsaicin-treated animals. Transection of the ophthalmomaxillary nerve in adult capsaicin-treated animals completely eliminated corneal CGRP-IR staining, and extirpation of the superior cervical ganglion resulted in the loss of 70–80% of corneal TH-IR nerves, thus demonstrating the sensory and predominantly sympathetic origins, respectively, of these fiber populations. Chronic keratitis and neovascularization developed in the capsaicin-treated animals by approximately 3 weeks of age, achieved a maximum intensity between 4 and 6 weeks, and showed some gradual improvement thereafter. However, the keratitis never completely disappeared, even after 13 months. In conclusion, these data show that corneal sensory (CGRP-IR) and sympathetic (TH-IR) nerve fibers undergo extensive sprouting following partial corneal sensory denervation with the neurotoxin capsaicin. However, the resultant “hyper-reinnervation” is morphologically abnormal and, for reasons unknown, functionally incapable of preventing or totally reversing the keratitis.     

Assay for and enzymatic formation of an ethylene precursor, 1-aminocyclopropane-1-carboxylic acid

A simple and sensitive chemical assay was developed for 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene. The assay is based on the liberation of ethylene from ACC at pH 11.5 in the presence of pyridoxal phosphate, MnCl₂ and H₂O₂. This assay was used to detect ACC in extracts of tomato fruits (Lycopersicon esculentum Mill.) and to measure the activity of a soluble enzyme from tomato fruit that converted S-adenosylmethionine (SAM) to ACC. The enzyme had a K_m of 13 M for SAM, and conversion of SAM to ACC was competitively and reversibly inhibited by aminoethoxyvinylglycine (AVG), an analog of rhizobitoxine. The K_i value for AVG was 0.2 M. The level of the ACC-forming enzyme activity was positively correlated with the content of ACC and the rate of ethylene formation in wild-type tomatoes of different developmental stages. Mature fruits of the rin (non-ripening) mutant of tomato, which only produce low levels of ethylene, contained much lower levels of ACC and of the ACC-forming enzyme activity than wild-type tomato fruits of comparable age.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG ammoethoxyvinylglycine, the aminoethoxy analog of rhizobitoxine L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid - SAM S-adenosyl-L-methionine Michigan Agricultural Experiment Station No. 8876     

小粒野生稻STK类抗病基因同源序列的克隆与分析

根据丝氨酸/苏氨酸蛋白激酶类(serine-threonine kinase,STK)抗病基因结构中保守结构域,设计引物,以小粒野生稻基因组DNA为模板,扩增获得10条STK类抗病基因同源序列.同源性分析表明其都具有STK保守结构域,与已克隆的STK类抗病基因有不同程度的相似性,为进一步克隆小粒野生稻中的STK类抗病基因提供依据.     

Structure, dynamics and mapping of membrane-binding residues of micelle-bound antimicrobial peptides by natural abundance C NMR spectroscopy

Worldwide bacterial resistance to traditional antibiotics has drawn much research attention to naturally occurring antimicrobial peptides (AMPs) owing to their potential as alternative antimicrobials. Structural studies of AMPs are essential for an in-depth understanding of their activity, mechanism of action, and in guiding peptide design. Two-dimensional solution proton NMR spectroscopy has been the major tool. In this article, we describe the applications of natural abundance ¹³C NMR spectroscopy that provides complementary information to 2D ¹H NMR. The correlation of ¹³Cα secondary shifts with both 3D structure and heteronuclear ¹⁵N NOE values indicates that natural abundance carbon chemical shifts are useful probes for backbone structure and dynamics of membrane peptides. Using human LL-37-derived peptides (GF-17, KR-12, and RI-10), as well as amphibian antimicrobial and anticancer peptide aurein 1.2 and its analog LLAA, as models, we show that the cross peak intensity plots of 2D ¹H-¹³Cα HSQC spectra versus residue number present a wave-like pattern (HSQC wave) where key hydrophobic residues of micelle-bound peptides are located in the troughs with weaker intensities, probably due to fast exchange between the free and bound forms. In all the cases, the identification of aromatic phenylalanines as a key membrane-binding residue is consistent with previous intermolecular Phe-lipid NOE observations. Furthermore, mutation of one of the key hydrophobic residues of KR-12 to Ala significantly reduced the antibacterial activity of the peptide mutants. These results illustrate that natural abundance heteronuclear-correlated NMR spectroscopy can be utilized to probe backbone structure and dynamics, and perhaps to map key membrane-binding residues of peptides in complex with micelles. ¹H-¹³Cα HSQC wave, along with other NMR waves such as dipolar wave and chemical shift wave, offers novel insights into peptide-membrane interactions from different angles.     

Preparation of bromo[1-14C]acetyl-coenzyme A as an affinity label for acetyl-coenzyme A binding sites

Bromo[1-14C]acetyl-CoA has been prepared from CoASH and the N-hydroxysuccinimide ester of bromo[1-14C]acetic acid, and unlabeled bromoacetyl-CoA by reaction of CoASH with bromoacetyl bromide. The products were purified by high-pressure liquid chromatography. Purified bromoacetyl-CoA was characterized, and found to be a potent alkylating agent with a substantial stability in aqueous solution: it decomposed at 30 degrees C and pH 6.6 and 8.0 with halftimes of 3.3 and 2.5 h, respectively. The major breakdown products were CoASH and CoAS X CO X CH2 X SCoA. Bromo[1-14C]acetyl-CoA has been used to affinity label the acetyl-CoA binding site of 3-hydroxy-3-methylglutaryl-CoA synthase from ox liver. It was found to irreversibly inhibit the enzyme activity and bind covalently with a stoichiometry for complete inhibition of about 0.8 mol/mol enzyme dimer.     

Regulation of PKR by HCV IRES RNA: Importance of Domain II and NS5A

Protein kinase R (PKR) is an essential component of the innate immune response. In the presence of double-stranded RNA (dsRNA), PKR is autophosphorylated, which enables it to phosphorylate its substrate, eukaryotic initiation factor 2α, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5′-untranslated region of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and noncanonical features of domain II cumulate to a total of ∼ 33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV nonstructural protein 5A (NS5A) to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection.     

Metabolism of A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 by CYP24A1

The metabolism of 1alpha,25(OH)(2)D(3) (1alpha,3beta) and its A-ring diastereomers, 1beta,25(OH)(2)D(3) (1beta,3beta), 1alpha,25(OH)(2)-3-epi-D(3) (1alpha,3alpha), and 1beta,25(OH)(2)-3-epi-D(3) (1beta,3alpha), was examined to compare the substrate specificity and reaction specificity of CYP24A1 between humans and rats. The ratio between C-23 and C-24 oxidation pathways in human CYP24A1-dependent metabolism of (1alpha,3alpha) and (1beta,3alpha) was 1:1, although the ratio for (1alpha,3beta) and (1beta,3beta) was 1:4. These results indicate that the orientation of the hydroxyl group at the C-3 position determines the ratio between C-23 and C-24 oxidation pathways. A remarkable increase of metabolites in the C-23 oxidation pathway was also observed in rat CYP24A1-dependent metabolism. The binding affinity of human CYP24A1 for A-ring diastereomers was (1alpha,3beta)>(1alpha,3alpha)>(1beta,3beta)>(1beta,3alpha), indicating that both hydroxyl groups at C-1 and C-3 positions significantly affect substrate-binding. The information obtained in this study is quite useful for understanding substrate recognition of CYP24A1 and designing new vitamin D analogs.     

小剂量辣椒素对豚鼠心室肌细胞L-型钙电流的影响

应用全细胞膜片钳技术研究低浓度辣椒素(capsaicin,CAP)对单个豚鼠心室肌细胞L-型钙电流的影响及其作用机制.CAP(1～25 nmol/L)可浓度依赖性增加电压依赖性的ICa-L的峰值并下移I-V曲线.CAPl,10,25 nmol/L使ICa-L最大峰值分别由-9.67±0.7pA/pF增至-10.21±0.8pA/pF(P＞0.05),-11.37±0.8pA/pF和-12.84±0.9pA/pF(P＜0.05).CAP25nmol/L可明显使稳态激活曲线左移,激活中点电压(V0.5)由-20.76±2.0mV变至-26.71±3.0mV(P＜0.05),表明低浓度CAP改变了钙通道激活的电压依赖性.CAP25nmol/L对电压依赖性稳态失活曲线和ICa-L从失活状态下复活过程无明显影响.辣椒素受体(VR1)阻断剂钌红(RR,10μmol/L)可阻断低浓度辣椒素的效应.以上结果表明,低浓度辣椒素使钙通道稳态激活曲线左移,增加ICa-L,这一效应可能由VRl介导.     

Synthesis and opioid activity of new fentanyl analogs

Three new fentanyl analogs (compounds 3-4-5) have been synthesized and evaluated for antinociceptive properties using the writhing test. The analgesic property of the active compound, N-[1-phenylpyrazol-3-yl]-N-[1-(2-phenethyl)-4-piperidyl)] propenamide (compound 4), was tested using the hot plate test in mice. Its opioid agonistic activity was characterized using three isolated tissues: guinea pig ileum, mouse vas deferens, and rabbit vas deferens. Compound 4 was as effective as fentanyl or morphine and it showed less antinociceptive potency than fentanyl but it was more potent than morphine. The duration of the antinociception was similar to that of fentanyl. This compound inhibited the electrically evoked contractions of myenteric plexus-longitudinal muscle strips of guinea pig ileum and of mouse vas deferens but not those of rabbit vas deferens. These effects could be reversed by micro selective antagonists (naloxone and/or CTOP) but not by the delta selective antagonist naltrindole, thus indicating that the compound acted as a micro opioid agonist. Finally, the binding data confirmed that compound 4 had high affinity and selectivity for the micro-receptor.     

药用真菌金耳的rDNA ITS序列分析与鉴别

研究了两个居群的金耳Tremella aurantialba以及近似品的rDNAITS区碱基全序列的特征及其差异,首次报道了金耳的ITS和5.8SrDNA完整序列,序列总长度为467~468,长度变异较少。聚类分析表明两个居群金耳亲缘关系非常密切,金耳药材和近似品金黄银耳形成一个稳定的独立分支,近似品黄金银耳形成另一个分支。ITS序列的差异为金耳的鉴别提供了可靠的分子标记,为金耳菌类药材基原入药建立了遗传基础。