[Investigation of Anisakis infections in coastal marine fishes and awareness of anisakiasis control knowledge among residents in Yantai City in 2021].

OBJECTIVE: To investigate the prevalence of Anisakis infections in coastal marine fishes and awareness of anisakiasis control knowledge among local residents in Yantai City, Shandong Province in 2021, so as to provide insights into formulation of anisakiasis control interventions. METHODS: Marine fishes were purchased from Shunxin Port, Yantai City, Shandong Province in November 2021, and the presence of Anisakis was detected in different species of fishes and different fish sites. The correlations between body length and weight of marine fish and intensity of Anisakis infections were examined using Spearman's rank correlation analysis, and the dietary habits and anisakiasis control knowledge were investigated using questionnaire surveys among local residents. RESULTS: A total of 201 marine fishes belonging to 20 species were dissected, and Anisakis was detected in 77 marine fishes (38.31%) belonging to 11 species (55.00%), with a mean infection intensity of 45.04 parasites per fish (3 468/77). Spearman's rank correlation analysis revealed that the body length (rs = 0.74, P < 0.05) and weight (rs = 0.79, P < 0.01) of the monkfish correlated positively with the intensity of Anisakis infections, and the body length (rs = 0.68, P < 0.05) of the flatfish correlated positively with the intensity of Anisakis infections, while no correlations were examined between the body length or weight of other marine fishes and the intensity of Anisakis infections. Of all respondents, 53.38% men and 56.67% women did not know anisakiasis control knowledge at all, and there was a significant difference in the proportion of respondents using separate chopping boards for raw and cooked food from different villages (χ2 = 17.89, P < 0.01), while there was an age-specific proportion of respondents with habitats of eating raw or semi-raw seafood (χ2 = 28.27, P < 0.01). CONCLUSIONS: The prevalence and intensity of Anisakis infections were high in coastal marine fishes in Yantai City in 2021, and the awareness of anisakiasis control knowledge was low among local residents. Intensified health education pertaining to anisakiasis control knowledge is recommended to reduce the risk of Anisakis infections.

Effects of Breathing Patterns on Amide Proton Transfer MRI in the Kidney: A Preliminary Comparative Study in Healthy Volunteers and Patients With Tumors.

BACKGROUND: The amide proton transfer-weighted (APTw) imaging for kidney diseases is important. However, the breathing patterns on APTw imaging remains unexplored. PURPOSE: This study aimed to investigate the effects of intermittent breath-hold (IBH) and free breathing (FB) on renal 3D-APTw imaging. STUDY TYPE: Healthy volunteers were enrolled prospectively, and renal clear cell carcinoma (RCCC) patients were included retrospectively. POPULATION: 58 healthy volunteers and 10 RCCC patients. FIELD STRENGTH/SEQUENCE: 3-T, turbo spin echo, and fast field echo. ASSESSMENT: 3D-APTw imaging was scanned using the IBH and FB methods in volunteers and using the IBH method in RCCC patients. The image quality was evaluated by three observers according to the 5-point Likert scale. Optimal images rated at three points or higher were used to measure the APT values. STATISTICAL ANALYSIS: The measurement repeatability was assessed using the intraclass correlation coefficient (ICC) and the Bland-Altman plot. The APT values were analyzed using McNemar's test, one-way analysis of variance, and t test. RESULTS: 50 healthy volunteers and 8 RCCC patients were enrolled. Renal 3D-APTw imaging using the IBH method revealed a higher success rate (88% vs 78%). The ICCs were excellent in the IBH group (ICCs > 0.74) and were good in the FB group (ICCs < 0.74). No significant differences in the APT values among various zones using the IBH (P = 0.263) or FB method (P = 0.506). The mean APT value using the IBH method (2.091% ± 0.388%) was slightly lower than the FB method (2.176% ± 0.292%), but no significant difference (P = 0.233). The APT value of RCCC (4.832% ± 1.361%) was considerably higher than normal renal using the IBH method. CONCLUSIONS: The study demonstrated that the IBH method substantially increased the image quality of renal 3D-APTw imaging. Furthermore, APT values may vary between normal and tumor tissues. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 2.

Feature pyramid network-based computer-aided detection and monitoring treatment response of brain metastases on contrast-enhanced MRI.

AIM: To investigate the value of feature pyramid network (FPN)-based computer-aided detection (CAD) of brain metastases (BMs) before and after non-surgical treatment, and to evaluate its performance in monitoring treatment response of BM on contrast-enhanced (CE) magnetic resonance imaging (MRI). MATERIAL AND METHODS: Eighty-five cancer patients newly diagnosed with BM who had undergone initial and follow-up three-dimensional (3D) CE MRI at Liaocheng People's Hospital were included retrospectively in this study. Manual detection (MD) was performed by reviewer 1. Computer-aided detection (CAD) was performed by reviewer 2 using uAI Discover-BMs software. The treatment response was assessed by the two reviewers for each patient separately. A paired chi-square test was used to compare the differences in the detection of BM between MD and CAD. Agreement between MD and CAD in monitoring treatment response was assessed by kappa test. RESULTS: The sensitivities of MD and CAD on initial 3D CE MRI were 78.65% and 99.13%, respectively. The sensitivities of MD and CAD on follow-up 3D CE MRI were 76.32% and 98.24%, respectively. There was a very good agreement between Reviewer 1 and Reviewer 2 in evaluating the treatment response of BM. CONCLUSION: FPN-based CAD has a higher sensitivity of close to 100% and lower false negatives (FNs) for BM detection, compared to MD. Although CAD had a few shortcomings in reflecting changes of BMs after treatment, it had high performance in monitoring treatment response of BM on CE MRI.

[Correlation between balloon volume and Meckel's cave size and its influence of percutaneous microballoon compression for trigeminal neuralgia].

Objective: To investigate the correlation between balloon volume and Meckel's cave size during percutaneous puncture microballoon compression (PMC) for trigeminal neuralgia and the influence of the compression coefficient (the ratio of balloon volume/Meckel's cave size) on the prognosis. Methods: Seventy-two patients (28 males and 44 females) aged (62±11) years who underwent PMC under general anesthesia for trigeminal neuralgia in the First Affiliated Hospital of Zhengzhou University from February 2018 to October 2020 were retrospectively collected. All patients underwent preoperative cranial magnetic resonance imaging (MRI) to measure Meckel's cave size, intraoperative balloon volume was recorded, and the compression coefficient was calculated. Follow-up visits were performed preoperatively (T0) and 1 d (T1), 1 month (T2), 3 months (T3), and 6 months (T4) postoperatively, either in the outpatient clinic or by telephone, and the Barrow Neurological Institute pain scale (BNI-P) score, the Barrow Neurological Institute facial numbness (BNI-N) score and the occurrence of complications were recorded and compared at each time point. Patients were divided into 3 groups according to different prognoses: patients in group A (n=48) were with no recurrence of pain and mild facial numbness, patients in group B (n=19) were with no recurrence of pain but severe facial numbness, while those in group C (n=5) had recurrence of pain. The differences in balloon volume, Meckel's cave size, and compression coefficient were compared among the three groups, and the correlation between balloon volume and Meckel's cave size in each group was analyzed by Pearson correlation. Results: The effective rate of PMC for trigeminal neuralgia was 93.1% (67/72). At time points from T0 to T4, patients had BNI-P scores [M (Q1, Q3)] of 4.5 (4.0, 5.0), 1.0 (1.0, 1.0), 1.0 (1.0, 1.0), 1.0 (1.0, 1.0) and 1.0 (1.0, 1.0), and BNI-N scores [M (Q1, Q3)] of 1.0 (1.0, 1.0), 4.0 (3.0, 4.0), 3.0 (3.0, 4.0), 3.0 (2.0, 4.0) and 2.0 (2.0, 3.0), respectively. Compared with those at T0, patients had lower BNI-P scores and higher BNI-N scores from T1 to T4 (all P<0.05). In all patients, group A, group B, and group C, the balloon volume was (0.65±0.15), (0.67±0.15), (0.59±0.15) and (0.67±0.17) cm3, respectively, with no statistically significant difference (P>0.05), while the Meckel's cave size was (0.42±0.12), (0.44±0.11), (0.32±0.07), and (0.57±0.11) cm3, with a statistically significant difference (P<0.001). The balloon volumes and Meckel's cave sizes were all linearly and positively correlated (r=0.852, 0.924, 0.937 and 0.969, all P<0.05). The compression coefficient in group A, B and C was (1.54±0.14), (1.84±0.18) and (1.18±0.10), respectively, with a statistically significant difference (P<0.001). There were no serious intraoperative complications such as death, diplopia, arteriovenous fistula, cerebrospinal fluid leak, and subarachnoid hemorrhage. Conclusions: Intraoperative balloon volume during PMC for trigeminal neuralgia is linearly and positively correlated with the volume of the patient's Meckel's cave. The compression coefficient varies among patients with different prognoses and the compression coefficient may be a factor affecting the patient's prognosis.

Space Weathering Effects in Troilite by Simulated Solar-Wind Hydrogen and Helium Ion Irradiation.

Space weathering is a key process in the interpretation of airless planetary surfaces. As we engage new missions to planetary objects with potentially novel surfaces such as 16 Psyche, there is renewed interest in expanding our knowledge of space weathering effects to a wider variety of analog materials, including the physical/chemical effects of solar-wind ions on planetary regoliths. We have experimentally simulated the effects of solar ions on two polished thick sections of meteoritic troilite (FeS) via irradiation with 1 keV hydrogen (H+) and 4 keV helium (He+), to investigate effects resulting from different ion species. We detected depletion of sulfur over the course of each irradiation using in situ X-ray photoelectron spectroscopy. Sulfur depletion rates were surprisingly similar for H+ and He+, interpreted as a function of subsurface ion-activated diffusion. By comparing XPS-derived elemental abundances with SDTrimSP computer simulations, we further quantified sulfur diffusion, sputtering yield, and altered-layer composition with respect to incident-ion fluence, and accounted for the influence of surface oxidation due to atmospheric sample storage. Using scanning electron microscopy, we detected an increase in nanoscale surface roughness resulting from the irradiation, which we quantified using atomic force microscopy. Based on these results, we estimate that an exposure time of order 103 Earth-years is required for troilite on Psyche to reach equilibrium sulfur depletion within the first atomic layer.

Meta-analysis of the association between PADI4 -92C/G polymorphism and rheumatoid arthritis in the Chinese population.

Many studies have evaluated the correlation between peptidylarginine deiminase 4 (PADI4) -92C/G polymorphism and rheumatoid arthritis (RA), but the results remain inconclusive. Therefore, we performed a meta-analysis in the Chinese population to provide comprehensive data on the association between PADI4 -92C/G polymorphism and RA. Eligible studies published before May 2016 were identified in PubMed and Chinese databases. The strengths of these associations were assessed by pooled odds ratios (OR) and 95% confidence interval (CI). Eight studies documenting a total of 1351 RA cases and 1585 controls were included in this meta-analysis. In the overall analysis, a significant association between the PADI4 -92C/G polymorphism and RA was found in the Chinese population (G vs C: OR=1.32, 95%CI=1.02-1.71; GG+CG vs CC: OR=1.75, 95%CI=1.20-2.53). The subgroup analyses stratified by geographic area(s) and source of controls revealed significant results in South China, in hospital-based studies and population-based studies. In summary, this meta-analysis suggested that PADI4 -92C/G polymorphism may be associated with the RA incidence in the Chinese population, especially for South China. Further studies conducted on other ethnic groups are required for definite conclusions.

Meta-analysis of the association between PADI4 -92C/G polymorphism and rheumatoid arthritis in the Chinese population

Many studies have evaluated the correlation between peptidylarginine deiminase 4 (PADI4) -92C/G polymorphism and rheumatoid arthritis (RA), but the results remain inconclusive. Therefore, we performed a meta-analysis in the Chinese population to provide comprehensive data on the association between PADI4 -92C/G polymorphism and RA. Eligible studies published before May 2016 were identified in PubMed and Chinese databases. The strengths of these associations were assessed by pooled odds ratios (OR) and 95% confidence interval (CI). Eight studies documenting a total of 1351 RA cases and 1585 controls were included in this meta-analysis. In the overall analysis, a significant association between the PADI4 -92C/G polymorphism and RA was found in the Chinese population (G vs C: OR=1.32, 95%CI=1.02-1.71; GG+CG vs CC: OR=1.75, 95%CI=1.20-2.53). The subgroup analyses stratified by geographic area(s) and source of controls revealed significant results in South China, in hospital-based studies and population-based studies. In summary, this meta-analysis suggested that PADI4 -92C/G polymorphism may be associated with the RA incidence in the Chinese population, especially for South China. Further studies conducted on other ethnic groups are required for definite conclusions.

[Utility of texture analysis of magnetic resonance imaging in differential diagnosis of common pediatric cerebellar tumors in children].

OBJECTIVE: To analyze magnetic resonance images of the posterior cranial fossa tumors, and evaluate the texture characteristics of three kinds of children's posterior cranial fossa tumors by support vector machine (SVM). METHODS: From May 2008 to August 2015, a total of 76 patients with children's posterior cranial fossa tumors in First Affiliated Hospital of Zhengzhou University were retrospectively analyzed, texture analysis was performed on the three types of tumors. RESULTS: The 5 texture parameters extracted from the gray level co-occurrence matrix had at least 3 parameters which had statistically significant difference between the two different classes of data sets (P<0.05). For all kinds of data sets, the variance parameters of gray value of ROI region had statistically significant differences.The accuracy rate of SVM test in the remarkable difference of the ependymomas and medulloblastomas, the ependymomas and astrocytomas and the medulloblastomas and astrocytomas was 86.15%±4.16%, 73.63%±5.82% and 74.32%±5.85%, respectively. CONCLUSION: The analysis of texture features can provide more quantitative information which could provide a new thought and method for the differential diagnosis of tumors in the posterior cranial fossa.

Isolation and identification of acaricidal compounds in Inula japonica (Asteraceae).

We identified acaricidal compounds in Inula japonica Thunberg (Asteraceae) that were effective against carmine spider mite, Tetranychus cinnabarinus (Boisduval). Petroleum-ether extracts from I. japonica were toxic to T. cinnabarinus, with a 50% lethal concentration (LC50) value of 1.18 mg/ml. Silica gel column chromatography was used to separate the acaricidal components. Seventeen of 38 fraction groups had mite mortality rates >50%, nine fraction groups had rates >60%, and three fraction groups had rates >80% at 2 mg/ml. The major volatile compounds in the bioactive fraction groups were identified by gas chromatography-mass spectroscopy, and beta-sitosterol (1), stigmasterol (2), lupeol (3), and alpha-amyrin (4) were identified and determined to have acaricidal activity against T. cinnabarinus in vitro.

Assessment of the contact toxicity of methyl palmitate on Tetranychus viennensis (Acari: Tetranychidae).

Previous work demonstrated that methyl palmitate possesses acaricidal activity against Tetranychus viennensis Boisduval (Acari: Tetranychidae) via an unknown mechanism. Here, the symptoms of methyl palmitate toxicity to T. viennensi were studied to determine the acaricidal mechanism of action of this fatty acid methyl ester. Methyl palmitate caused concentration-dependent mortality of T. viennensis, with a moderate concentration (5 mg/ml) eliciting excitement and premature oviposition without spinning shortly after exposure. Tremors of the appendages were subsequently observed, followed by quiescence after approximately 5 h. Mites developed dorsal fluid exosmosis at 15-20 h posttreatment with reduced egg production, followed shortly thereafter by death. Some typical neurotoxic symptoms such as excitement and convulsions were observed in methyl palmitate-exposed mites, suggesting that methyl palmitate may be a neurotoxin. Compared with other neurotoxic acaricides, methyl palmitate poisoning is a slow process in mites. Transmission electron microscopy revealed serious ultrastructural damage in response to 5 mg/ml methyl palmitate exposure. Autolysis of membranous structures was also observed, especially in the mitochondria, suggesting a novel mode of action for methyl palmitate-induced toxicity.

Autoantibodies to plasminogen and tissue plasminogen activator in women with recurrent pregnancy loss.

Reduced fibrinolytic activity has been described in primary anti-phospholipid syndrome (PAPS), and may be responsible for thrombotic events. Antibodies to tissue type plasminogen activator (t-PA) or plasminogen (PLG) might contribute to the hypofibrinolytic state in autoimmune diseases, but the clinical significance of these antibodies is still unclear in recurrent pregnancy loss (RPL). The aim of this study is to evaluate the prevalence and clinical significance of anti-PLG and anti-t-PA antibodies in 87 patients with a history of RPL: 54 women with well-defined PAPS (mean age 32.5 years; range 26-38) and 33 women with unexplained RPL (mean age 30 years; range 24-39). IgG anti-PLG antibodies were found in 20 and four patients from the group with RPL/PAPS and unexplained RPL, respectively; IgG anti-t-PA antibodies were found in 11 and two patients from the above two groups, respectively. IgG anti-PLG antibodies were associated with the high risk of RPL (OR 7.2, P = 0.004), especially with RPL/PAPS (OR 11.2, P < 0.001) evaluated by Fisher's exact test, while IgG anti-t-PA were associated with RPL/PAPS (OR 10.0, P = 0.01) but not with RPL (OR 6.8, P = 0.06). A significant inhibition of exogenous fibrinolysis was observed by IgG fractions from patients with anti-PLG or anti-t-PA antibodies on microplates and on the human umbilical vein endothelial cells, compared with those from healthy controls. The prevalence of IgG anti-PLG antibodies was high in RPL patients, especially in RPL/PAPS, while the prevalence of IgG anti-t-PA antibodies was high in RPL/PAPS but not in RPL, and some of them might inhibit fibrinolysis in patients.

Concurrent chemoradiotherapy for locally advanced esophageal cancer--a pilot study by using daily low-dose cisplatin and continuous infusion of 5-fluorouracil.

BACKGROUND: Concurrent chemoradiotherapy (CCRT) has recently become a promising treatment for esophageal cancer. However, most investigators have adopted the conventional or modified Wayne-State PF (cisplatin plus 5-fluorouracil) regimen, which is inevitably associated with moderate to severe treatment-related toxicities. In this pilot study, we incorporated a daily low-dose regimen of cisplatin and 5-fluorouracil into CCRT in order to improve the compliance of the patients. PATIENTS AND METHODS: Between July 1993 and Dec. 1997, 25 patients with locally advanced esophageal cancer (T3, or N1 disease), received CCRT which consisted of daily low-dose cisplatin (6 mg/m2/day) and continuous infusion of 5-FU (225 mg/m2/day) with radiotherapy (fraction size = 200-250 cGy/day). Except for the initial 9 patients, for whom post-CCRT esophagectomy was compulsory, all subsequent patients underwent esophagectomy only when inadequate response to CCRT was noted. The scheduled radiation dose was 50 Gy for the first 9 patients, and 60 Gy for the rest of the patients. RESULTS: Eighteen patients (72%) completed the CCRT without interruption. Clinically, there were 8 CR and 9 PR, with a total response rate of 68% (47-87%, 95% C.I.). All patients were evaluable for toxicity. Grade 3/4 leukopenia and thrombo-cytopenia developed in 14 (56%) and 7 (28%) patients, respectively. Grade 3/4 non-hematologic toxicity was seen in 4 (16%) patients. The median survival of the whole group was 8 months (range: 2-59+). The projected 3-year overall survival was 24%. CONCLUSION: We suggest that for locally advanced esophageal cancer CCRT with the aforementioned daily low-dose regimen, is a treatment with good patient compliance.

[Clinical applications of island and myocutaneous flaps].

OBJECTIVE: This is to introduce the experience in clinical applications of the island and myocutaneous flaps. METHODS: Since 1985 fifteen types of vascularized island and myocutaneous flaps have been applied in 52 cases (21 positions) for the following purposes: repairing traumatic soft tissue defects on the limb, scar contracture, congenital recto-vaginal fistula, reconstructing congenital nasal deformity and rehabilitating function. The non-major artery island flap was used in 47 cases (90.38%) and the retrograde island flap was used in 24 cases (46.15%). The size of the flap ranged from 2.5 cm x 1.5 cm to 15 cm x 12 cm. RESULTS: All the flaps survived with primary healing. Follow-up for 8 months to 11 years showed aesthetically and functionally satisfactory results in all cases. CONCLUSION: These island and myocutaneous flaps have rich blood supply and are highly anti-infective. The operation is easy and the success rate is high.

Mechanism of Ca2+-dependent activity of human neutrophil gelatinase B.

Progelatinase B can be activated in vitro by organomercurial compounds and by proteolytic enzymes such as trypsin, chymotrypsin, and stromelysin. Activation of the proenzyme by either 4-aminophenylmercuric acetate or chymotrypsin yielded proteins that absolutely required Ca2+ for activity, regardless of the pH of the reaction mixture. The trypsin- and stromelysin-activated gelatinases, on the other hand, did not require Ca2+ for activity at pH 7.5, but the activity of the trypsin-activated enzyme became Ca2+ dependent as the pH increased. The pH study revealed that an amino acid residue with an apparent pKa of 8.8 was involved in this process. The NH2-terminal analyses showed that trypsin- and stromelysin-activated enzymes had the same NH2 termini (Phe88), but 4-aminophenylmercuric acetate- and chymotrypsin-activated enzymes had Met75 and Gln89 or Glu92 as the NH2-terminal amino acid, respectively. These data, in conjunction with the x-ray crystal structure of collagenase, suggest that a salt linkage involving Phe88 is responsible for the Ca2+-independent activity of trypsin- and stromelysin-activated gelatinase. Replacing Asp432 in progelatinase with either Glu, Asn, Gly, or Lys resulted in the proteins that, upon activation by trypsin, required Ca2+ for activity. These substitutions did not significantly affect Km for the synthetic substrate but decreased the kcat and increased the half-maximal Ca2+ concentration required for enzyme activity (KCa) by severalfold. The effects on kcat and KCa depended on both charge and size of the side chains of the substituted amino acids. The decrease in kcat correlated well with the increase in KCa of the mutants. The orders of decrease in kcat and increase in KCa were wild type >/= D432E > D432N > D432G > D432K and wild type

Role of the conserved histidine and aspartic acid residues in activity and stabilization of human gelatinase B: an example of matrix metalloproteinases.

Gelatinase B (MMP-9), a member of the matrix metalloproteinase family, is a zinc- and calcium-dependent endopeptidase that is known to play a role in tumor cell invasion and in destruction of cartilage in arthritis. It contains a conserved sequence. 400His-(X)3-His-(X)28-Asp-Asp-(X)2-436Gly, the function of which is under investigation. The conserved Asp-432 and Asp-433 residues were individually replaced with Gly; these substitutions reduced the gelatinolytic activity of the enzyme to 23% and 0%, respectively. Replacing Asp-433 with Glu, however, decreased the gelatinolytic activity of the enzyme by 93% and proteolytic activity of the enzyme for the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 substrate by 79%. The wild-type and D432G and D433E, mutant enzymes had similar Km values for the synthetic substrate and similar Ki values for the competitive inhibitor, GM6001. The kcat/Km values for D432G and D433E mutant enzymes, however, were reduced by a factor of approximately 4 and their KaCa values were increased by four- and sixfold, respectively. The significance of His-400 in the activity of the enzyme was assessed by replacing this residue with Ala and Phe. Both H400A and H400F mutants were inactive toward gelatin substrate. These data demonstrate that Asp-432, Asp-433, and His-400 residues are important for the activity of gelatinase B. His-400 may act as a zinc-binding ligand similar to the His-197 in interstitial collagenase (MMP-7) and Asp-432 and Asp-433 residues are probably involved in stabilization of the active site of the enzyme. The His-400 and Asp-433 residues are conserved in all members of the MMP family. Therefore, our results are relevant to this group as a whole.

Mechanism of activation of human neutrophil gelatinase B. Discriminating between the role of Ca2+ in activation and catalysis.

Gelatinase B is a Zn(2+)- and Ca(2+)-dependent endopeptidase that is secreted from cells as an inactive proenzyme. The enzyme can be activated in vitro by organomercurial compounds and by trypsin. The role of Ca2+ in autoproteolytic processing initiated by 4-aminophenylmercuric acetate and trypsin and in catalytic activity of the activated enzyme was investigated by zymography and by kinetic analysis. Treatment of unglycosylated 57.5-kDa pro-gelatinase B with 4-aminophenylmercuric acetate (1 mM) in the absence of Ca2+ generated a 49-kDa inactive intermediate (E'), whereas a 41.5-kDa active species (E") was generated in the presence of Ca2+ (5 mM). Upon addition of Ca2+ to the reaction mixture of Ca(2+)-depleted E' or E" at 37 degrees C, E' showed a lag period in generation of the product as a function of time, but E" presented an immediate activity. The appearance of enzymatic activity of E' correlated with the generation of the E" species. NH2-terminal sequence analyses showed that E' and E" had the same NH2 termini, i.e. Met-75, suggesting that Ca(2+)-dependent removal of COOH terminus of E' is required for activation of the enzyme. Treatment of pro-gelatinase B with trypsin in the absence of Ca2+, led to degradation of the enzyme. In the presence of Ca2+, trypsin processed the pro-enzyme to a 40-kDa active species. In contrast to E", this active species did not require Ca2+ for activity. The Ca2+ dependence of E" activity was also abolished by treatment of the enzyme with trypsin. NH2-terminal sequence analysis indicated that amino acid residues 75-87 had been removed from the NH2 terminus of E" by trypsin, suggesting that these residues are responsible for the Ca(2+)-dependent activity of the enzyme. Removal of Ca2+ and catalytic Zn2+ inhibited the activities of both E" and trypsin-treated E". In the absence of Ca2+, either Zn2+, Co2+, Mn2+, or Cd2+ was able to restore the activity of trypsin-treated E". None of the divalent cations tested however, was able to stimulate the activity of E" in the absence of Ca2+. These experiments further suggest that binding of Ca2+ to E" or removal of the NH2-terminal residues of the enzyme by trypsin induces a conformational change in the protein and makes the active site of the enzyme accessible to various metal ions rendering the enzyme active.

Stimulation of myosin light-chain kinase by Cd2+ and Pb2+.

The effect of Cd2+ on myosin light chain kinase (MLCK) reported in the literature is controversial, apparently because the level of Ca2+ contaminating the reaction mixture could not be accurately controlled by the addition of a metal chelator when Cd2+ was also present. In the present study, we have reduced the contaminating Ca2+ to a trace level that did not interfere with the enzyme activity; thus the use of a metal chelator was not necessary. We showed that Cd2+, or Pb2+ had a biphasic effect on MLCK isolated from chicken gizzard: stimulation at low and inhibition at high concentrations. (The stimulatory effect of on the enzyme activity isolated from chicken gizzard: stimulation at low and inhibition at high concentrations). The stimulatory effect of Cd2+ or Pb2+ on MLCK activity was not seen in the absence of calmodulin, and was abolished by trifluoperazine, a calmodulin antagonist, indicating that the heavy metals exert their activation via calmodulin. The inhibition of the enzyme activity by Cd2+ or Pb2+ at higher concentrations was also seen with the calmodulin-independent catalytic fragment of MLCK, suggesting that the inhibition is probably through their binding to sulfhydryl groups that are essential for catalytic activity. Pb2+ was more effective than Cd2+ in stimulating the enzyme activity, but less potent in inhibition. The extent of stimulation by heavy metals most likely resulted from a combination of the biphasic effects. Dithiothreitol and N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine selectively chelated Cd2+ and Pb2+ over Ca2+, and reversed their stimulatory or inhibitory effect on MLCK.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of troponin C by Cd2+ and Pb2+.

Certain heavy metal actions such as Cd2+ and Pb2+ mimic Ca2+ effectively in stimulating calmodulin (CaM). We now show that these cations also activate skeletal muscle troponin C (TnC), a Ca2(+)-binding protein highly homologous to CaM. Like Ca2+, these cations allow TnC to alter its electrophoretic mobility on polyacrylamide gels, and to bind to phenyl-Sepharose. Moreover, they activate TnC to stimulate myofibrillar ATPase. When TnC was removed from the skeletal myofibrils by treatment with trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CDTA), the ATPase activity was no longer stimulated by the cations. However, after reconstitution of CDTA-treated skeletal myofibril with TnC, the response of ATPase to Ca2+, Cd2+ or Pb2+ was restored. These findings suggest that the activation of myofibrillar ATPase by Cd2+ and Pb2+ is mediated through TnC. The ability of the heavy metals to stimulate TnC-supported ATPase activity correlated quite well with the ability to increase the extent of the myofibrillar superprecipitation. The activation of TnC by Cd2+ or Pb2+ could constitute a possible molecular basis for their toxicity.