[Coiled tube of femoral anterolateral flap for repair of circumferential defect after operation of advanced hypopharyngeal carcinoma].

Objective: To investigate the clinical application value of coiled tube of femoral anterolateral flap in the repair of circumferential defect after resection of advanced hypopharyngeal carcinoma. Methods: Clinical data of 42 patients with advanced hypopharyngeal cancer admitted to the Second Affiliated Hospital of Fujian Medical University from January 2016 to April 2022 were retrospectively analyzed, including 41 males and 1 female, aged from 33 to 82 years old. All patients received surgical treatment, including total laryngectomy plus total laryngopharyngectomy in 20 cases, total laryngectomy, total laryngopharyngectomy and resection of partial tongue base in 8 cases, total laryngectomy, total laryngopharyngectomy and resection of cervical esophagus in 9 cases, total laryngectomy, total laryngopharyngectomy, and resection of partial tongue base and cervical esophagus in 5 cases. The postoperative circumferential defects were repaired with the coil tube of anterolateral femoral skin flap in phase Ⅰ, and the healing status of the flap, wound healing and swallowing function were observed. All cases were followed up. Results: The lengths of the hypopharyngeal defects were 7-18 cm and the sizes of the harvested flaps were 6 cm×9.5 cm-10 cm×20 cm. Flaps survived in 41 cases, flap necrosis occurred in one case, and the survival rate of flaps was 97.6%. One artery and one vein were anastomosed in 40 cases, and one artery and two veins were anastomosed in 2 cases. Postoperative cervical wound infection occurred in 5 cases, and pharyngeal fistula occurred in 2 cases. Three months of follow-up after surgery, 31 cases had normal diet, 9 cases presented with semi-liquid diet and 2 cases with liquid diet. Following up for 6-65 months, recurrence and metastasis occurred in 14 patients (33.3%), including primary site recurrence in 4 cases (9.5%), cervical lymph node recurrence in 6 cases (14.3%), and distant metastasis in 4 cases (9.5%). The 1-year and 3-year overall survival rates were respectively 79.4% and 60.5%. Conclusion: Coiled tube of femoral anterolateral flap is an ideal skin flap for repair of circumferential defects after resection of advanced hypopharyngeal carcinoma.

Significance of the expression of integrin ß1, VEGF and MVD in hypopharyngeal squamous cell carcinoma.

This study aimed to determine the expression of integrin ß1 and vascular endothelial growth factor (VEGF) and microvascular density (MVD) by CD105 staining in hypopharyngeal squamous cell carcinoma to determine their association with clinicopathologic characteristics, and to determine their role and the effects of their interactions in the development and progression of hypopharyngeal squamous cell carcinomas. The expression of integrin ß1 and VEGF and MVD in hypopharyngeal squamous cell carcinomas and normal hypopharyngeal tissues were evaluated using immunohistochemistry. The Image-Pro Plus software was used to determine the mean optical density of the immunohistochemical images. Integrin ß1 expression was significantly higher in hypopharyngeal squamous cell carcinoma tissues (78.00%) than in normal hypopharyngeal tissues (35.00%; P = 0.001) and significantly differed across pathologic grades and different T stages, and regarding the presence of cervical lymph node metastasis (P < 0.05). VEGF expression was significantly higher in hypopharyngeal squamous cell carcinoma tissues (74.00%) than in normal hypopharyngeal tissues (30.00%; P = 0.002), VEGF overexpression differed significantly across different pathologic grades and different T stages, and regarding the presence of cervical lymph node metastasis (P < 0.05). The MVD count was significantly higher in hypopharyngeal squamous cell carcinoma tissues (37.10 ± 5.95) than in normal hypopharyngeal tissues (8.70 ± 3.34; P = 0.000). MVD differed significantly across different pathologic grades and different T stages, and regarding the presence of cervical lymph node metastasis (P < 0.05). The expression of integrin ß1 and VEGF and the MVD count exhibited no significant differences in terms of age, gender, history of smoking, and clinical stages (P > 0.05). VEGF expression was positively associated with the MVD count of hypopharyngeal squamous cell carcinomas (r = 0.582, P = 0.000); however, integrin ß1 was not associated with VEGF or MVD (P > 0.05). Integrin ß1 and VEGF are overexpressed and MVD increased in hypopharyngeal squamous cell carcinomas. VEGF is positively correlated with MVD.

Spontaneous resolution of acute T cell-mediated rejection in a renal transplant patient.

This case report presents spontaneous resolution of acute rejection in a 66-year-old man who underwent a kidney transplant and developed acute rejection and pneumonia. Our main concern in this case was how to treat the concurrent infection while maintaining the immunosuppressive therapy with a narrow available therapeutic range, in order to save the renal allograft without increasing antirejection therapy.

Association between human adenovirus-36 and lipid disorders in Korean schoolchildren.

BACKGROUND: Although the human adenovirus-36 (Ad-36) has been associated with obesity and related lipid disorders in the United States, this association has yet to be identified in other countries. Therefore, we tried to determine whether Ad-36 is associated with obesity or lipid disorders in Korean schoolchildren. METHOD: A total of 318 Korean schoolchildren aged 6-15 years, who participated in the Ewha Womans University Obesity Research Study, were selected for a community-based cohort study. Non-obese and obese were defined as body mass index (BMI) <85th and > or = 95th percentiles of the Korean reference BMI-for-age curves, respectively, according to International Obesity Task Force definitions. The cutoff points for lipid disorders were modified from the age-modified standards of the National Cholesterol Education Program (NCEP)-Adult Treatment Panel (ATP) III metabolic syndrome criteria. The Ad-36 antibody was measured using a serum neutralization assay. RESULTS: More obese participants than non-obese participants tested positive for the Ad-36 antibody (28.57 vs 13.56%, respectively; P = 0.0174). Within the obese group, the participants who tested positive for the Ad-36 antibody had higher levels of triglycerides (TG) and total cholesterol than those who tested negative for the Ad-36 antibody (P<0.001). However, these associations were not present in the non-obese group. The unadjusted odds ratio (OR) for Ad-36 antibody positivity was greater in obese participants than non-obese participants (OR = 2.550, 95% confidence interval (CI): 1.154-5.633). However, this OR seemed to be nonsignificant when age, sex and lipid variables were included in the analysis (OR = 1.752, 95% CI: 0.763-4.020). The unadjusted OR for the elevated TG was significantly higher in participants who were Ad-36 antibody-positive than those who were Ad-36 antibody-negative (OR = 2.511, 95% CI: 1.448-4.353). This trend remained constant even after adjustment for age, sex and obesity (OR = 2.328, 95% CI: 1.296-4.181). CONCLUSION: Ad-36 seems to be strongly associated with lipid disorders in Korean schoolchildren regardless of obesity.

Spinal deformities in Noonan syndrome: a clinical review of sixty cases.

BACKGROUND: Skeletal abnormalities, including spinal deformities, in Noonan syndrome have been described, but no detailed and systematic study of such spinal deformities has been presented in the literature. METHODS: The cases of sixty patients with Noonan syndrome were reviewed retrospectively, and the general appearance, growth disturbance, and mental status of the patients were documented. Spinal deformities were evaluated radiographically, and the frequency, pattern, and severity of the curves were documented. RESULTS: Spinal deformity was present in eighteen (30%) of the sixty patients. Two patients had congenital spinal deformity. Of the remaining sixteen patients with scoliosis, nine had a single thoracic curve, four had a single thoracolumbar curve, and three had a double major curve. Thoracic lordosis was also present in three of these sixteen patients. No patient had only increased kyphosis or lordosis. The mean age when the spinal deformities were detected was nine years; seven deformities were detected before the age of seven years. Overall, surgery was recommended to eleven of the eighteen patients; it was recommended for the treatment of scoliosis (mean, 68.5 degrees; range, 45 degrees to 125 degrees ) in eight patients and for the treatment of an associated thoracic lordosis (8 degrees, 15 degrees, and 18 degrees ) in three. Seven of the eleven patients underwent spinal arthrodesis. The operation was deferred in one patient because malignant hyperthermia developed during the induction of anesthesia. CONCLUSIONS: Scoliosis with an associated thoracic lordosis occurs more frequently in Noonan syndrome than has been reported previously. Since the deformities tend to develop early and are relatively severe, a clinical and, if necessary, radiographic assessment of the spine with careful follow-up should be performed for early detection and treatment of spinal deformity. Although malignant hyperthermia is rare, all patients with Noonan syndrome should be considered to be at risk for the development of this complication before operative treatment.

Dynamic sagittal imbalance of the spine in degenerative flat back: significance of pelvic tilt in surgical treatment.

STUDY DESIGN: A retrospective study of 26 patients with degenerative flat back treated with corrective osteotomy. OBJECTIVE: To analyze dynamic sagittal imbalance and to elucidate the cause of postoperative persistent stooping in degenerative flat back. SUMMARY OF BACKGROUND DATA: Sagittal spinal imbalance in degenerative flat back was more evident on walking, suggesting its dynamic nature. The most puzzling complication in its surgical treatment was postoperative persistent stooping. METHODS: This study analyzed 26 surgically treated patients with preoperative gait analysis. Patients were divided into two groups according to postoperative improvement in stooping: Group 1 with marked improvement in stooping and Group 2 with persistent stooping. Various radiographic and gait parameters were compared between the two groups. RESULTS: Comparison of radiographic parameters, representing the static status of the spine, did not indicate any clue to the mechanism for persistent stooping. However, comparison of gait parameters, representing the dynamic status of the spine, revealed meaningful differences between the two groups. Among various gait parameters compared, pelvic tilt seemed to be the most important clue. Patients in Group 1 showed posterior pelvic tilt, whereas those in Group 2 showed marked anterior pelvic tilt. CONCLUSION: Degenerative flat back could be classified into two types based on pelvic position during walking: one with posterior pelvic tilt and the other with marked anterior pelvic tilt. In the former type, corrective surgery improved the stooping. In the latter, corrective surgery was ineffective, resulting in postoperative persistent stooping.

Impact response of the intervertebral disc in a finite-element model.

STUDY DESIGN: A three-dimensional nonlinear poroelastic finite-element model of a vertebra disc was used to analyze the biomechanical effects of impact loading on the spinal segment. OBJECTIVES: To predict changes in biomechanical parameters such as intradiscal pressure, dynamic stiffness, stresses in the endplate region, and the shock-absorbing mechanism of the spine under different impact duration/loading rates, and to investigate the relation between the rate of loading and the fracture potential of the vertebral body. SUMMARY OF BACKGROUND DATA: It is not practical to discern the role of impact duration using experimental protocols. Analytical studies are better suited to this purpose. However, previous poroelastic finite-element models of the motion segments have dealt mostly with creep phenomena. METHODS: A three-dimensional, L3-L4 motion-segment, finite-element model was modified to incorporate the poroelastic properties of the disc, endplate, and cancellous core, and thus simulate the shock-absorbing phenomena. The results were analyzed under variable impact durations for a constant maximum compressive impact load of 3 kN. RESULTS: For a shorter impact duration and a given F(max), relatively high cancellous core pressure was generated as compared with a case of long impact duration, although the amount of impulse was increased. In contrast, relatively constant pore pressures were generated in the nucleus regardless of the impact duration. The changes in spinal segment stiffness as a function of impact duration indicated that for a shorter duration of impact, high dynamic stiffness increases the stability of the spinal segment against the impact load. However, the corresponding increase in stresses within the vertebral body and endplate may produce fractures. CONCLUSIONS: The finite-element technique was used to address the role of impact duration in producing trauma to the spinal motion segment. Within the limitations of the model, the results suggest that fractures are likely to occur under shorter impact duration conditions. Depending on the strength of the region, a fracture may be initiated in the endplate region or the posterior wall of the cortical shell. The nucleus pressure is independent of the impact duration and depends only on the magnitude of the impact force.

Pulmonary venous flow from fetal to neonatal period.

There are few reports about the effect of fetal or transitional circulation on the pulmonary venous flow. The purposes of this study were to investigate flow patterns of the pulmonary vein serially from fetal to neonatal period and to determine the relationship between pulmonary venous flow and other parameters from aortic and mitral valve. Pulmonary venous flow velocity was analyzed in 21 normal term human fetuses. Postnatal follow-up studies were performed at 1, 6, 24 h, 3 days, 1 week and 1 month. In each time point, pulsed Doppler echocardiography was used to interrogate right upper pulmonary vein, mitral and aortic valve. The measured parameters of pulmonary vein were heart rate, velocity time integral (VTI), and velocities at systolic peak (S), at diastolic peak (D), at nadir between S and D (O), and at nadir between D and the next S (X). E/A ratio and VTI were measured for mitral valve and peak systolic velocity and VTI for aortic valve. Pulmonary venous flow in fetus was phasic and continuous with low velocity. One hour after birth, without a change of flow pattern, all velocities increased dramatically. These high velocities showed a significant decrease during 24 h after birth. Three days after birth, the velocity decreased slightly and flow pattern changed from continuous to interrupted pattern with or without atrial reversal. No Doppler parameters from aortic or mitral valve showed any correlation with parameters from pulmonary vein. In conclusion, the flow pattern of the pulmonary vein in fetus may result from low pulmonary flow and decreased capacitance of the pulmonary venous system. Sudden increase in the pulmonary flow after birth is likely to be responsible for the highest velocities recorded immediately after birth. Left to right shunt through the ductus arteriosus may also contribute to the flow pattern observed in the first several days, as do changes in reservoir function of the pulmonary vein.

Effect of high blood flow on the expression of endothelial constitutive nitric oxide synthase in rats with femoral arteriovenous shunts.

The effect of high blood flow on the expression of endothelial nitric oxide synthase has been investigated in the femoral arteriovenous shunt (AVS) rats created by inserting U-shaped polyurethane tubes in the left femoral arteries and veins. Three days after inserting the femoral AVS, the mean aortic blood flow rate in the abdominal aorta of the AVS rats was about 2.0 times higher than that in the control rats (110.0 +/- 8.4 ml/min vs 52.7 +/- 2.7 ml/min, p < 0.001). The competitive reverse transcriptase-polymerase chain reaction (RT-PCR) data revealed that the mRNA expression level of the endothelial constitutive nitric oxide synthase (ecNOS) was increased in the aortas of the femoral AVS rats compared to that in the control rats. Western blot analysis using a monoclonal antibody against ecNOS revealed that the ecNOS protein levels were markedly increased in the aortas of femoral AVS rats, but ecNOS protein levels in aortas without endothelium were not significantly increased. Inducible nitric oxide synthase (iNOS) protein was not expressed in the aortic tissues with and without endothelium in the control rats. This iNOS expression was not increased by the high blood flow in the femoral AVS rats. These findings suggest that high blood flow could up-regulate the expression levels of ecNOS mRNA and proteins in femoral arteriovenous shunt rats.

Asymmetric mRNA expression in the retinal neuroepithelium of the chick embryo assessed by differential display polymerase chain reaction.

In the normal retinotectal topography established during the embryonic development of the chick visual system, retinal ganglion cell axons from the nasal retina connect to the posterior part and temporal retinal axons connect to the anterior part of the optic tectum. For the investigation of position-specific gene expression along the nasal-temporal axis of the retinal neuroepithelium (RN), differential display PCR was carried out from the nasal or temporal part of the RN at HH11 (E2). We found several genes that are differentially expressed either in the nasal or in the temporal part of the RN and the analysis of the asymmetrically expressed fragments showed at least one cDNA fragment to be exclusively expressed in the nasal RN. This fragment was 550 bp in size.

Isolation of cDNA clones encoding human histone macroH2A1 subtypes.

cDNA clones encoding two different subtypes of histone macroH2A1, macroH2A1.1 and macroH2A1.2, have been isolated from human liver tissue. The open reading frames in the isolated clones predicted proteins comprising 368 and 371 amino acids respectively. Estimated molecular masses of the two proteins were 39.0 kDa and 39.4 kDa. Human histone macroH2A1.1 and macroH2A1.2 showed about 98% identity with their counterparts isolated from rat. The features of the nucleotide sequences of the two macroH2A1 subtypes in human were the same as in the rat system. Northern blot analysis showed that the macroH2A1.1 and macroH2A1.2 subtypes were expressed as mRNA species with a size of 1.5 and 4.4 kb, respectively. They were expressed in all human tissues examined.

Expression of the CIP1 gene induced under cadmium stress in Candida sp.

Cadmium is a toxic heavy metal with no known biological activity. Differential display of mRNA was employed to isolate cDNA corresponding to the transcript that is induced in cadmium-treated Candida sp. In this report we describe the molecular characterization of the CIP1 gene, which was shown to be rapidly induced after cadmium treatment. Northern blot analysis showed that the CIP1 transcript was not present in normal cells, but accumulated at higher levels in cadmium-treated cells. Treatment of other heavy metals such as copper, mercury, lead, zinc, or heat-shock had no effect on the expression of the CIP1 gene. Sequence analysis of CIP1 revealed that it encodes a 32 kDa hydrophobic protein that contains a putative transmembrane domain. The deduced amino acid sequence of CIP1 showed a little homology with isoflavone reductase of plants. From the promoter sequence analysis, we also identified a sequence similar to pas, a cadmium-responsive element of the ParA gene in tobacco. Our results suggest that Candida CIP1 may play a crucial role in the establishment of specific cellular response to stress evolved by the cadmium treatment.

Cloning and sequencing of cDNA encoding 4-aminobenzoate hydroxylase from Agaricus bisporus.

A cDNA clone encoding 4-aminobenzoate hydroxylase (EC 1.14.13.27) has been isolated using a probe prepared by PCR on the basis of partially determined amino acid sequences of the enzyme. The cDNA contained 1380-base pair open reading frame encoding 460 amino acid residues (M(r) 50974), 14-base pair 5'-untranslated region and 123-base pair 3'-untranslated region including a poly(A) tail of 20 nucleotides. All of the partially determined amino acid sequences were shown to be included in the deduced amino acid sequence. Homology analyses showed that the two regions on the enzyme share other flavoproteins such as salicylate hydroxylase and p-hydroxybenzoate hydroxylase.

A quantitative approach to identify and isolate pure populations of fluorescently labeled adult retinal ganglion cells using a pressure-driven microaspiration technique.

We developed a technique to obtain pure subsets of neuronal populations for specific analysis at the molecular level. The fact that most areas of the brain consist of mixed types of neuronal and non-neuronal cells was circumvented by retrograde labeling of retinal ganglion cells (RGCs) either from the superior colliculus (SC) or the optic nerve (ON) using fluorescent dyes (4Di-10ASP or Rhodamine). Subsequent enzymatic dissociation of the retina with papain allowed to identify and collect pure populations of RGC by using the pressure-driven microaspiration technique. Enzymatic treatment and additional mechanical dissociation destroy most of the vulnerable ganglion cells leaving between 1.8% and 6% of the labeled cells intact. This low outcome was consistent among the techniques used to apply the dye and the two different dyes used in the study. However, the total numbers of cells obtained from each retina were sufficient to collect about 200 cells within 1 day and process these cells for molecular biology. As an example of further processing of collected cells, the expression of beta-actin gene was analyzed.

Purification and characterization of protease Ci, a cytoplasmic metalloendoprotease in Escherichia coli.

Protease Ci, a cytoplasmic metalloprotease in Escherichia coli, has been purified to apparent homogeneity by conventional chromatographic procedures using 125I-labeled oxidized insulin B-chain as a substrate. The purified enzyme behaves as a 54-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. It is inhibited by metal-chelating agents, including o-phenanthroline and NaCN, but not by inhibitors of serine proteases or thiol-blocking agents. Furthermore, protease Ci was found to contain 1.1 mol of zinc per mol of the enzyme upon analysis by HR ICP mass spectroscopy. Thus, protease Ci must be a zinc metalloprotease. Among the polypeptides tested as substrates, oxidized insulin B-chain and glucagon are most rapidly hydrolyzed. Intact insulin is a much poorer substrate than oxidized insulin B-chain, even though the affinity of the enzyme to intact insulin is approximately 100-fold greater than that to the B-chain. Since unlabeled oxidized insulin A-chain is capable of inhibiting the hydrolysis of 125I-labeled insulin B-chain, it also appears to be a substrate. Protease Ci also degrades lysozyme and lactalbumin, although to a much lesser extent than oxidized insulin B-chain. However, it shows little or no activity against proteins larger than 15 kDa (e.g. ovalbumin and denatured bovine serum albumin). Hydrolysis of oxidized insulin B-chain followed by amino acid composition analyses of the cleavage products reveals that as many as 10 of its 29 peptide bonds are hydrolyzed by protease Ci. This ability to hydrolyze relatively small polypeptides suggests that protease Ci may catalyze the later steps in the pathway for intracellular protein breakdown.

Isolation and characterization of a novel perchloric acid-soluble protein inhibiting cell-free protein synthesis.

We found a novel protein in the postmitochondria supernatant fraction of rat liver, which is soluble in 5% perchloric acid and strongly inhibits protein synthesis in a rabbit reticulocyte lysate system. The protein extracted from the supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The protein was shown to consist of two identical subunits with a molecular mass of 14 kDa. By immunoscreening with the rabbit antisera against the protein, a cDNA encoding the protein was cloned and sequenced. The cDNA contained an open reading frame of 411 base pairs encoding a 136-amino acid protein with a molecular mass of 14,149 Da. The deduced amino acid sequence was completely identical with that constructed from all of the above peptides. Interestingly, the perchloric acid-soluble protein inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner from RNase A. The protein is likely to inhibit an initiation stage of cell-free protein synthesis. Among the rat tissues tested, the protein was located only in liver and kidney. These findings are the first report on a new inhibitor that may be involved in the regulation of protein synthesis in those tissues.

Stabilities of the fluorescent SH-reagent eosin-5-maleimide and its adducts with sulfhydryl compounds.

The stabilities of the SH-reagent eosin-5-maleimide (EMA) and its adducts with the SH-compounds L-cysteine, N-acetyl-L-cysteine and glutathione (reduced form) were studied under various conditions in comparison with those of the adducts of N-ethylmaleimide (NEM). Studies by reversed-phase high performance liquid chromatography and mass spectrometry showed that EMA was less stable than NEM at neutral and moderately alkaline pH values. EMA formed a succinimide-type adduct with SH-compounds, and then underwent further modification by nucleophilic attack of OH- or an amino group. The succinimide-type adducts with acetylcysteine and glutathione were converted to open-type adducts, in which the succinimide ring was cleaved, whereas the adduct with cysteine was modified to a thiazine-type adduct. Kinetic analyses showed that these open-type and thiazine-type adducts were readily formed and were stable at moderately alkaline pH values such as pH 8.0 or 9.0.

Identification of endo-beta-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine amidase as cluster-dispersing enzymes in Staphylococcus aureus.

Two proteins which are capable of dispersing cell clusters of Staphylococcus aureus have been purified from a S. aureus FDA209P culture supernatant. Both of them were found to have bacteriolytic activity. From the elution profile of column chromatography and Western blot (immunoblot) analysis, one of them was identified as a 51-kDa endo-beta-N-acetylglucosaminidase (GL). The other was a 62-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis. Analysis of the peptidoglycan fragments following treatment with the 62-kDa protein indicated that this protein is an N-acetylmuramyl-L-alanine amidase (AM). In vitro studies of cluster dispersion activities using S. aureus mutant strains Lyt66 or S. aureus Wood46 grown as clusters demonstrated that these two enzymes act synergistically to disperse clusters into single cells. Antiserum against the 51-kDa GL cross-reacted with the 62-kDa AM, and S. aureus FDA209P grown in the presence of anti-51-kDa-GL immunoglobulin G induced giant clusters. Clusters induced by anti-51-kDa GL and by Cibacron blue F3G-A were dispersed by coincubation with the 51-kDa GL and the 62-kDa AM. Western blot analysis demonstrated that the 51-kDa GL and the 62-kDa AM were missing in culture supernatants of S. aureus Lyt66, Wood46, and RUSAL2 (Tn551 autolysin-defective mutant), which grow in clusters. These results strongly suggest that the 51-kDa GL and 62-kDa AM are involved in cell separation of daughter cells after cell division.

Trypsin and elastase inhibitors from bitter gourd (Momordica charantia LINN.) seeds: purification, amino acid sequences, and inhibitory activities of four new inhibitors.

Serine proteinase inhibitors of the squash family were isolated from bitter gourd (Momordica charantia LINN.) seeds by the conventional purification method. Heat treatment of the extract of the seeds allowed removal of large amounts of protein without loss of trypsin and elastase inhibitory activities. From the supernatants thus obtained, the inhibitors were isolated to homogeneity by ion-exchange chromatography, gel filtration, and reversed phase chromatography. One trypsin inhibitor (Momordica charantia trypsin inhibitor-III; MCTI-III) and three elastase inhibitors (Momordica charantia elastase inhibitor-II, -III, and -IV; MCEI-II, -III, and -IV) were newly isolated in addition to trypsin inhibitors MCTI-I and -II and elastase inhibitor MCEI-I previously reported [Hara, S. et. al. (1989). J. Biochem. 105, 88-92]. The primary structures of the four new inhibitors were determined as follows. [sequence: see text] The dissociation constants, Ki, of MCTI-III complex with bovine beta-trypsin, and of MCEI-II, -III, -IV with porcine elastase were determined to be 1.9 x 10(-7) M, 9.4 x 10(-9) M, 4.0 x 10(-9) M, and 4.7 x 10(-9) M, respectively. Although MCTI-III differed from MCTI-I in only two amino acids, having Gly(3) and Gln(13) in place of Arg(3) and Arg(13), the Ki value of MCTI-III was 20-fold larger than that of MCTI-I. Addition of an amino terminal Glu residue, a dipeptide (Glu-Glu-), and a tripeptide (Glu-Glu-Glu-) to MCEI-I strengthened its elastase inhibitory activity by 200-fold.

A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and characterization.

The Tn551 insertion site of the autolysis-deficient Staphylococcus aureus mutant RUSAL2 was cloned and used to identify the autolysis gene atl in the parent strain, RN450. The open reading frame for atl was 3768 bp in length, encoding a deduced protein of 1256 amino acids and molecular size of 137,381 Da. The atl gene product is a bifunctional protein that has an amidase domain and an endo-beta-N-acetylglucosaminidase domain which must undergo proteolytic processing to generate the two extracellular lytic enzymes found in the culture broth of S. aureus.