[Comparison of three-dimensional position of maxillary dentition model treated with two digital transfer methods].

Objective: To compare and evaluate the difference in maxillary dentition position using an anatomical facebow and jaw movement analyzer. Methods: From March to May 2023, 15 medical interns from Yantai Stomatological Hospital were recruited, including 9 males and 6 females, aged 20-25 years. Digital models and plaster models of maxillary dentition were obtained from the 15 medical interns. The anatomical facebow group (AFB) and jaw movement analyzer group (JMA) were used to transfer the position of the maxillary dentition to the virtual articulator. The virtual occlusal articulator module of exocad denture design software was used to measure the inclination angle of the occlusal plane of the two groups, the distance between the mesio-incisal angle of the left maxillary central incisor and the lateral center point of the lateral condylar sphere of the virtual occlusal articulator, the distance between the mesial buccal cusp of the maxillary first molar and the lateral center point of the lateral condyle sphere of the virtual articulator. The same marks (mesial incisor point of left maxillary central incisor and mesial buccal cusp point of both maxillary first molars) were measured in two groups of maxillary dentition, and the root-mean-square error between 3 points was calculated. Results: The occlusal plane inclination angle in AFB group (9.11°±3.85°) was significantly larger than that in JMA group (4.94°±2.69°) (t=10.45, P<0.001). There were significant differences between AFB and JMA groups. The distances from the mesial cusp of the left first molar to the lateral center of the left condylar, from the mesial cusp of the left maxillary central incisor to the lateral center of the left condylar[(91.75±3.05), (129.09±4.60) mm]were significantly smaller than those in the JMA group[(95.68±5.45), (132.41±5.64) mm](t=-4.48, P=0.001; t=-4.21, P=0.001). In both groups of models, the distance of the mesial cusp of the left maxillary central incisor was (8.81±2.56) mm, and the distance between mesial buccal cusp of maxillary left first molar was (7.56±2.49) mm, the distance between mesial buccal cusp of maxillary right first molar was (7.13±2.77) mm; the root mean square error was (7.93± 2.94) mm. Compared with 0, the difference was statistically significant (t=10.45, P<0.001). Conclusions: There were differences between the two methods (anatomical facebow and the jaw movement analyzer) for transferring the maxillary dentition position to the three-dimensional space position of the virtual articulator.

[Expression and protective effect of chemerin in idiopathic pulmonary fibrosis].

Objective: To explore the expression and the role of chemerin in idiopathic pulmonary fibrosis (IPF). Methods: Quantitative PCR and Western blotting were used to determine the mRNA and protein levels of chemerin in lung tissues from IPF patients and the controls. Clinical serum level of chemerin was analyzed by enzyme-linked immunosorbent assay. The mouse lung fibroblasts isolated and cultured in vitro were divided into the control, TGF-ß, TGF-ß+chemerin and chemerin groups. Immunofluorescence staining was used to observe the expression of α-smooth muscle actin (α-SMA). C57BL/6 mice were randomly divided into the control, bleomycin, bleomycin+chemerin, and chemerin groups. Masson and immunohistochemical staining were performed to evaluate the severity of pulmonary fibrosis. Expression of epithelial to mesenchymal transition (EMT) markers was detected by quantitative PCR and immunohistochemical staining in the in vitro and in vivo models of pulmonary fibrosis, respectively. Results: Compared with the control group, the expression of chemerin was downregulated in both the lung tissue and the serum of IPF patients. Immunofluorescence showed that treatment of fibroblasts with TGF-ß alone resulted in a robust expression of α-SMA, whereas treatment with TGF-ß and chemerin together exhibited the similar expression levels of α-SMA as the control group. Masson staining indicated that the bleomycin-induced pulmonary fibrosis model was constructed successfully, while treatment of chemerin partially alleviated the damage of lung tissue. Immunohistochemical staining showed that the expression of chemerin in the lung tissue was significantly decreased in the bleomycin group. Quantitative PCR and immunohistochemistry showed that chemerin attenuated EMT induced by TGF-ß and bleomycin both in vitro and in vivo. Conclusions: The expression of chemerin was reduced in patients with IPF. Chemerin may play a protective role in the development of IPF by regulating EMT, providing a new idea for the clinical treatment of IPF.

[The fundus autofluorescence of retinal astrocytic hamartomas in tuberous sclerosis complex].

Objective: To investigate the autofluorescence findings of retinal astrocytic hamartoma (RAH) in patients with tuberous sclerosis complex (TSC). Methods: It was a retrospective case series study. Twenty-three patients (35 eyes) who were referred to Department of Internal Medicine and Department of Ophthalmology, Peking Union Medical College Hospital between November 2012 and June 2018 with established TSC-associated RAH diagnosis were included. The findings of fundus autofluorescence, fundus photos and spectral-domain optical coherence tomography (SD-OCT) were retrospectively reviewed. RAH lesions were classified into three types based on the morphology shown in fundus photos. The fundus autofluorescence features of TSC-associated RAH were described. The Welch's test and Fisher's exact test were used for statistical analysis. Results: The patients were 8 males and 15 females aged (28±9) years old (range, 15-55 years). Seventy-two RAH lesions were examined, including 59 type 1 RAHs, 7 type 2 RAHs and 6 type 3 RAHs. According to fundus autofluorescence, type 1 RAHs could be further divided into reduced, speckled and background autofluorescence patterns, among which the hypoautofluorescence pattern accounted for the majority (69.5%, 41/59), while the speckled pattern was usually accompanied by outer retinal disorganization and discontinuation of photoreceptor outer segment as revealed by SD-OCT. No significant difference was revealed in tumor thickness for reduced, speckled and background autofluorescence patterns of type 1 RAHs [(490.2±97.9) vs. (589.2±221.6) vs. (463.0±76.2) µm respectively, F=1.426, P=0.283]. Among type 1 RAHs, the number of reduced autofluorescence pattern lesions found in perifoveal, peripapillary, inferonasal, inferotemporal, superonasal, superotemporal quadrants were 9, 4, 4, 7, 4, 13 respectively, while that of speckled autofluorescence pattern lesions were 3, 0, 3, 2, 3, 2 and background autoflurorescence pattern lesions 3, 0, 1, 1, 0, 0. No significant difference was revealed in location distribution (P=0.452) either. Type 2 RAHs featured numerous hyperautofluorescent spots or plaques, and calcification in type 2 RAHs varied in autofluorescence intensity. Type 3 RAHs, combining the features of type 1 and 2 RAHs, were characterized by central hyperautofluorescent spots and hypoautoflurescent rim, but the area of hyperautofluorescence was smaller than that of calcification as shown in fundus photos. Conclusions: In TSC, the fundus autofluorescence of RAHs varies from hypoautofluorescence to hyperautofluorescence patterns according to RAH types. The retinal involvement and calcification degree of TSC-associated RAHs could be reflected on the autofluorescence, which was beneficial to the full assessment. (Chin J Ophthalmol, 2020, 56: 211-216).

[Clinical features and pathological analysis of lacrimal gland occupying lesions in 91 cases].

Objective: To analyze the pathogenesis, histopathological classification and clinical features of lacrimal gland occupying lesions. Methods: This was a retrospective case series study. Clinical data of 91 patients (102 eyes) with lacrimal gland area occupying diseases who received ophthalmic surgery in the Second People's Hospital of Yunnan Province from January 2014 to November 2018 were retrospectively analyzed, including patients' age, reasons for treatment, gender, imageological examination data and pathological diagnosis results. All patients had more than one medical imaging examination results and histopathological diagnosis results. Results: Among 91 cases, 46 patients (50.5%) were male and 45 (49.5%) were female. The age distribution ranged from 1.1 years to 72 years old, with an average age of 43 years. All of benign tumors added up to 58 cases (63.7%). Pleomorphic adenoma (43 cases, 47.3%), dermoid cyst (6 cases, 6.6%), and inflammatory pseudotumor (6 cases, 6.6%) were the most common cases in the benign lacrimal gland occupying tumors. There were 33 cases (36.3%) of malignant tumors. Adenoid cystic carcinoma (15 cases, 16.5%), adenocarcinoma (6 cases, 6.6%) and lymphoma (5 cases, 5.5%) had the highest incidence among the malignant lacrimal gland occupying tumors. The most common reason for seeking medical treatment was exophthalmos (50 cases, 54.9%; 30 cases were pleomorphic adenoma). Brow arch mass (22 cases, 24.2%) and pain in and around the eye (9 cases, 9.9%; 5 cases were adenoid cystic carcinoma) were also major reasons. Conclusions: The most common benign lacrimal gland area occupying lesion in surgery patients of Yunnan is pleomorphic adenoma, which more occurred in patients with exophthalmos as the main symptoms. The most common malignant tumor in the lacrimal gland area is adenoid cystic carcinoma and the most common reason to seek medical advice was pain in and around the eye. (Chin J Ophthalmol, 2019, 55:842-846).

[Analysis of unplanned reoperation following vitreoretinal surgery].

Objective: To determine the incidence of unplanned reoperation following vitreoretinal surgery and identify the reasons for unplanned reoperations. Methods: Case records of all patients undergoing vitreoretinal surgery at Peking Union Medical College Hospital between June 2014 and June 2017 were reviewed to determine the incidence of unplanned reoperations during the primary admission and within 90 days following vitreoretinal surgery. Results: A total of 3 356 case records were reviewed. During the primary admission, 97 times of unplanned reoperation occurred in 81 cases [45 males and 36 females, aged (47.3±16.8) years].The incidence of unplanned reoperation was 2.4% (81/3 356). The most common primary diseases were retinal detachment (25 cases, 30.9%), proliferative diabetic retinopathy (23 cases, 28.4%) and silicone oil filled eye (17 cases, 21.0%). The most common reasons for unplanned reoperation were new onset or recurrent retinal detachment (33 times, 34.0%), increased intraocular pressure (23 times, 23.7%), as well as hyphema and inflammation (16 times, 16.5%). The percentages of the primary diseases and reasons for unplanned reoperation within 90 days following vitreoretinal surgery were not significantly different when compared with those during the primary admission. Desired results could be achieved in all cases after unplanned reoperation. Conclusions: The primary diseases of unplanned reoperation for vitreoretinal surgery are complicated retinal detachment, diabetic retinopathy and silicone oil filled eyes. New onset or recurrent retinal detachment, increased intraocular pressure, hyphema and inflammation are common causes of reoperation.

The Populus holobiont: dissecting the effects of plant niches and genotype on the microbiome.

BACKGROUND: Microorganisms serve important functions within numerous eukaryotic host organisms. An understanding of the variation in the plant niche-level microbiome, from rhizosphere soils to plant canopies, is imperative to gain a better understanding of how both the structural and functional processes of microbiomes impact the health of the overall plant holobiome. Using Populus trees as a model ecosystem, we characterized the archaeal/bacterial and fungal microbiome across 30 different tissue-level niches within replicated Populus deltoides and hybrid Populus trichocarpa × deltoides individuals using 16S and ITS2 rRNA gene analyses. RESULTS: Our analyses indicate that archaeal/bacterial and fungal microbiomes varied primarily across broader plant habitat classes (leaves, stems, roots, soils) regardless of plant genotype, except for fungal communities within leaf niches, which were greatly impacted by the host genotype. Differences between tree genotypes are evident in the elevated presence of two potential fungal pathogens, Marssonina brunnea and Septoria sp., on hybrid P. trichocarpa × deltoides trees which may in turn be contributing to divergence in overall microbiome composition. Archaeal/bacterial diversity increased from leaves, to stem, to root, and to soil habitats, whereas fungal diversity was the greatest in stems and soils. CONCLUSIONS: This study provides a holistic understanding of microbiome structure within a bioenergy relevant plant host, one of the most complete niche-level analyses of any plant. As such, it constitutes a detailed atlas or map for further hypothesis testing on the significance of individual microbial taxa within specific niches and habitats of Populus and a baseline for comparisons to other plant species.

[Diagnosis and thoracoscopic treatment for pleuroperitoneal communication complicating peritoneal dialysis].

Objective: To evaluate the safety and efficacy of diagnosis and treatment of pleuroperitoneal communication in patients on peritoneal dialysis by computerized tomography (CT) peritoneography and thoracoscopic surgery. Methods: The records of 8 pleuroperitoneal communication patients treated by thoracoscopic surgery in Peking University First Hospital from January 2012 to March 2017 were retrospectively reviewed.CT peritoneography was performed before surgical treatment. Iopamiro (50 ml∶15 g) was mixed with 2 L of peritoneal dialysate solution, and then was instilled into the peritoneal cavity through the Tenckhoff catheter. After dwelling for 1 hour, CT scan was taken from thorax to upper abdomen.The thoracic cavity was filled with sterile saline, and then carbon dioxide was inflated to the peritoneal cavity via Tenckhoff catheter.Air bubbles leaking from the diaphragmatic defect were identified during thoracoscopic operation.Then defect was repaired and pleurodesis was performed. Results: The CT value of pleural effusion increased to (155±57) HU.Pleuroperitoneal communication was clearly diagnosed.The diaphragmatic defects in 5 cases were detected and repaired.However, defects in the other 3 cases were not found.The mean operative time was (152±50) minutes. All patients had uneventful recovery and reinstated normal peritoneal dialysis 3 weeks after the operation.The median follow-up time was 14.5 months (1-68 months) and no recurrence occurred. Conclusions: CT peritoneography could identify pleuroperitoneal communication clearly. Thoracoscopic surgery is a safe and reliable method for pleuroperitoneal communication and could restore patients to peritoneal dialysis.

microRNA-21 Contributes to Orthodontic Tooth Movement.

microRNAs could be mechanosensitive and emerge as critical posttranscriptional regulators in the bone-remodeling process. During orthodontic tooth movement (OTM), the application of mechanical force induces alveolar bone remodeling, but whether microRNAs respond to orthodontic force and contribute to OTM is unknown. microRNA-21 (miR-21) has been previously reported in vitro to mediate stretch-induced osteogenic differentiation of periodontal ligament stem cells and support osteoclast differentiation. In this study, the authors show that miR-21 responded to orthodontic force in periodontal tissue in a dose- and time-dependent manner and regulated the osteogenesis of human periodontal ligament stem cells following OTM. Using mmu-miR-21-deficient (miR-21-/-) mice, the authors discovered that mmu-miR-21 deficiency inhibited OTM and prevented force-induced maxillary bone loss. The authors found that miR-21-/- mice showed a normal skeletal phenotype in development and a similar alveolar bone formation rate to wild-type mice postnatally. During OTM, mmu-miR-21 regulated force-induced alveolar osteoblastogenesis in the tensile side, while no effects were detected in the compressive side. However, miR-21-/- mice showed inhibited alveolar osteoclastogenesis when compared with wild-type mice. During OTM, mmu-miR-21 deficiency blocked alveolar bone resorption in both the compressive and tensile sides. To dissect the mechanism by which miR-21 regulates alveolar bone remodeling, the authors screened the reported functional targets of miR-21 and found that periodontal expression of programmed cell death 4 ( Pdcd4) was inhibited following OTM. Furthermore, mmu-miR-21 deficiency removed the suppression of Pdcd4 at both the mRNA and protein levels in the periodontium, resulting in upregulation of the downstream effector C-fos. Further analysis of OTM under lipopolysaccharide-induced periodontal inflammation showed that mmu-miR-21 mediated lipopolysaccharide (LPS)-accelerated OTM and that mmu-miR-21 deficiency blocked lipopolysaccharide-induced maxillary bone loss. In summary, these findings reveal a previously unrecognized mechanism that a microRNA can modulate OTM and alveolar bone remodeling under both normal and inflammatory microenvironments in vivo.

[Relationship and interaction between folate and expression of methyl-CpG-binding protein 2 in cervical cancerization].

OBJECTIVE: To explore the interaction between folate and the expression of methyl-CpG-binding protein 2(MeCP2)in cervical cancerization. METHODS: Forty one patients diagnosed with cervical squamous cell carcinoma(SCC), 71 patients diagnosed with cervical intraepithelial neoplasm(CIN1, n=34; CIN2 +, n=37)and 61 women with normal cervix(NC)were recruited in this study. Microbiological assay was conducted to detect the levels of serum folate and RBC folate, Western blot assay and real-time PCR were performed to detect the expression levels of MeCP2 protein and mRNA, respectively. The data were analyzed by Kruskal-Wallis H test, χ(2) test, trend χ(2) test and Spearman correlation with SPSS statistical software(version 20.0), and the interaction were evaluated by using generalized multifactor dimensionality reduction(GMDR)model. RESULTS: The levels of serum folate(H=44.71, P<0.001; trend χ(2)=24.48, P<0.001)and RBC folate(H=5.28, P<0.001; trend χ(2)=3.83, P<0.05)decreased gradually along with the severity of cervical lesions. There was a positive correlation between serum folate level and RBC folate level(r=0.270, P< 0.001). The expression levels of MeCP2 protein(H=33.72, P<0.001; trend χ(2)=14.74, P<0.001)and mRNA(H=19.50, P<0.001; trend χ(2)=10.74, P<0.001)increased gradually along with the severity of cervical lesions. There were negative correlation between folate level and the expression level of MeCP2 protein(serum folate: r=-0.226, P=0.003; RBC folate: r=-0.164, P=0.004). Moreover, the results by GMDR model revealed there were interaction among serum folate deficiency, RBC folate deficiency, MeCP2 protein high expression and MeCP2 mRNA high expression in SCC and CIN2 + patients. CONCLUSION: Folate deficiency and high expression of MeCP2 gene might increase the risk of cervical cancer and its precancerous lesions through interaction among serum folate deficiency, RBC folate deficiency, MeCP2 protein high expression and mRNA high expression in the progression of cervical cancerization.

[The application of full thicknes skin graft inpartial laryngectomy for glottic carcinoma].

Objective:The aim of this study is to explore the experience and advantages of the application of full thicknes skin graft in glottic carcinoma.partial laryngectomy for glottic carcinoma.Method:One hundred and forty-three patients with glottic cancer were treated with partial laryngectomy.Among those,78 cases were repaired with full-thickness skin graft and 65 cases were repaired with sternohyoid muscular fasciae.Compared the time of extubation and the formation of granulation in laryngeal cavity after operation between the two groups.Result:In the group of full-thickness skin graft,the mean time of decannulation was 6.8 days,5 cases with growth of granulation after operation.In other group,the mean time of decannulation was 10.7 days,16 cases with growth of granulation after operation.The mean time of decannulation(t=-4.739,P<0.01) and the growth of granulation(χ²=9.379,P<0.01) are significantly different between the two groups.No laryngostenosis was found in all patients.Conclusion:The application of full-thicknes skin graft in partial laryngectomy for glottic carcinoma.can shortthe time of extubation and reduce the formation of granulation.

First Report of Actinidia virus A and Actinidia virus B on Kiwifruit in China.

At present, two viruses affecting kiwifruit (Actinidia spp.), Actinidia virus A (AcVA) and Actinidia virus B (AcVB), both belonging to the genus Vitivirus in the family Betaflexiviridae, have been reported from New Zealand (2). The infected trees showed leaf vein chlorosis, flecking, and ringspots. China is the largest commercial kiwifruit producer. During field investigations in the growing season of 2013, symptoms of leaf chlorosis or ringspots, similar to those caused by AcVA and AcVB (1), were observed on some kiwifruit (Actinidia chinensis) plants in Hubei Province in the central China. Leaf samples were collected from three symptomatic and two symptomless plants of two A. chinensis cultivars. Total nucleic acids were extracted from the samples using a CTAB-based protocol described by Li et al. (3) and used as template in RT-PCR for the detection of AcVA and AcVB. Each virus was detected using two sets of primers reported by Blouin et al. (1). Primer sets AcVA 1F/1R and AcVA5F/5R were used for the AcVA detection, and AcVB1F/1R and AcVB5F/Viti3'R were used for the AcVB detection. AcVA was detected in three symptomatic plants (ID: Ac-HN-1, Ac-HN-3, and Ac-HN-5), and AcVB was detected in two symptomatic plants (ID: Ac-HN-1 and Ac-HN-3) and in one symptomless plant (ID: Ac-HN-2). Neither virus was detected in the second symptomless plant (ID: Ac-HN-4). Samples Ac-HN-1 and Ac-HN-3 had mixed infection of AcVA and AcVB, and sample Ac-HN-2 had the latent infection of AcVB. The sequenced 283-bp RT-PCR amplicons of the replicase-encoding gene from AcVA isolates AC-HN-3 and AC-HN-5 using AcVA1F/1R shared 90.8% nucleotide (nt) identity with the corresponding sequence of the New Zealand AcVA isolate (GenBank Accession No. JN427014.1). The 269-bp fragments of the RNA-binding protein-encoding gene obtained by using AcVA5F/5R shared 85.5 to 85.9% nt identities with the corresponding sequence of JN427014.1. The AcVB5F/Viti3'R products of 365 to 369 bp from three AcVB isolates shared 85.5 to 88.6% nt identities with the corresponding sequence of the New Zealand AcVB isolate. The representative sequences were submitted to GenBank with accession numbers KJ696776 and KJ696777 for the 269-bp fragments of AcVA-HN-1 and AcVA-HN-3, and KJ696778 and KJ696779 for the 365-bp and 369-bp fragments of AcVB-HN-1 and AcVB-HN-2, respectively. In addition, 12 and 14 out of 42 kiwi samples (excluding HN-1 to HN-5) collected randomly were positive for AcVA and AcVB as detected by RT-PCR. Meanwhile, the sample affected by AcVA-HN-5 was subjected to deep sequencing of the small RNAs (sRNAs) for complete survey of the infecting viruses. De novo assembly of sRNAs generated four sequence contigs, with lengths ranging from 161 to 285 nt, matching to ORFs 1 to 3 of the genome of the New Zealand AcVA isolate with significant nucleotide (91 to 95%) and amino acid (80 to 94%) similarities, and some other contigs from a new virus (unpublished). The result further confirmed AcVA infection in the kiwi plant. To our knowledge, this is the first report of both AcVA and AcVB outside of New Zealand. The Chinese isolates of the two viruses are distinct from those reported from New Zealand. The results provide valuable information for improving the viral sanitary status of the kiwifruit germplasm in China. References: (1) A. G. Blouin et al. Arch. Virol. 157:713, 2012. (2) A. G. Blouin et al. J. Plant Pathol. 95:221, 2013. (3) R. Li et al. J. Virol. Methods 154:48, 2008.

Inter-field variability in the microbial communities of hydrothermal vent deposits from a back-arc basin.

Diverse microbial communities thrive on and in deep-sea hydrothermal vent mineral deposits. However, our understanding of the inter-field variability in these communities is poor, as limited sampling and sequencing efforts have hampered most previous studies. To explore the inter-field variability in these communities, we used barcoded pyrosequencing of the variable region 4 (V4) of the 16S rRNA gene to characterize the archaeal and bacterial communities of over 30 hydrothermal deposit samples from six vent fields located along the Eastern Lau Spreading Center. Overall, the bacterial and archaeal communities of the Eastern Lau Spreading Center are similar to other active vent deposits, with a high diversity of Epsilonproteobacteria and thermophilic Archaea. However, the archaeal and bacterial communities from the southernmost vent field, Mariner, were significantly different from the other vent fields. At Mariner, the epsilonproteobacterial genus Nautilia and the archaeal family Thermococcaceae were prevalent in most samples, while Lebetimonas and Thermofilaceae were more abundant at the other vent fields. These differences appear to be influenced in part by the unique geochemistry of the Mariner fluids resulting from active degassing of a subsurface magma chamber. These results show that microbial communities associated with hydrothermal vent deposits in back-arc basins are taxonomically similar to those from mid-ocean ridge systems, but differences in geologic processes between vent fields in a back-arc basin can influence microbial community structure.

Cardiac contractile impairment associated with increased phosphorylation of troponin I in endotoxemic rats.

The subcellular mechanisms underlying intrinsic myocardial depression during sepsis remain poorly defined, in particular the relative roles of altered intracellular Ca2+ transients versus changes in myofilament properties. We studied contractile function of cardiac myocytes isolated 12 h after induction of endotoxemia (5 mg/kg intravenous E. coli lipopolysaccharide [LPS]) in conscious rats. Cardiomyocytes from LPS-injected rats had depressed twitch shortening compared with control cells (4.10.2% versus 7.80.3%; P2+ transients (peak indo-1 ratio 1.130.02 versus 1.120.02; P = NS). Contractile depression was unaffected by inhibitors of nitric oxide synthase. Steady-state myofilament response to Ca2+, assessed by tetanization of intact cells over a range of [Ca2+], was reduced significantly in the LPS group (P2+ was unaffected by isoproterenol (3 nmol/L) in endotoxemic cells, whereas there was a rightward shift in control cells. A reduction in myofilament response to Ca2+ is the major determinant of intrinsic cardiac depression in systemic endotoxemia. This condition appears to be related to an increase in myocardial troponin I phosphorylation.

Ca2+-independent inhibition of myocardial contraction by coronary effluent of hypoxic rat hearts.

Endothelial cells release agents that influence cardiac contraction. We recently reported that cultured hypoxic endothelial cells release an unidentified factor(s) that inhibits myocardial contraction. In this study, we investigated the effects of coronary effluent of isolated hypoxic rat hearts on isolated rat ventricular myocyte contraction. Coronary effluent collected during brief moderate hypoxia significantly depressed myocyte twitch shortening and decreased diastolic length, with only minor reduction in intracellular Ca2+ transients. These effects were similar to those of hypoxic rat coronary microvascular endothelial cell superfusates and were reversed by reoxygenation of hearts. "Hypoxic" coronary effluent exerted essentially Ca2+-independent effects on myofilament interaction in intact myocytes, as assessed by 1) peak Ca2+-shortening relations, 2) phase-plane analysis of instantaneous Ca2+-cell length relations, and 3) "steady-state" myofilament responses in tetanized, sarcoplasmic reticulum-disabled cells. Thus an unidentified substance(s) that inhibits myocyte shortening predominantly via effects on the myofilaments is reversibly released during acute moderate hypoxia of isolated hearts, presumably from coronary endothelial cells. Release of such an agent may be relevant to the cardiac contractile response to hypoxia.

Selective dysregulation of nitric oxide synthase type 3 in cardiac myocytes but not coronary microvascular endothelial cells of spontaneously hypertensive rat.

OBJECTIVE: Recent studies indicate that endothelial type nitric oxide synthase (NOS3) modulates cardiac systolic and diastolic function and the inotropic responsiveness to beta-adrenergic agonists, and may affect myocardial oxygen consumption. Although NOS3 is a constitutive protein, its levels of expression can be modified by various physiological and pathophysiological stimuli. We investigated whether the cell-specific expression of NOS3 mRNA and protein are altered in cardiac hypertrophy. METHODS: Left ventricular cardiac myocytes and coronary microvascular endothelial cells were freshly isolated from 12 week old male spontaneously hypertensive rat (SHR) and matched normotensive Wistar rat hearts. NOS3 protein levels were assessed by Western analysis, and mRNA levels by RT-PCR and Southern blotting. RESULTS: Left ventricular/body weight ratios were significantly increased in SHR compared to Wistar controls, indicating significant hypertrophy. The levels of NOS3 protein were markedly decreased in SHR compared to Wistar cardiac myocytes (by approximately 85%). By contrast, the expression of NOS3 mRNA normalized for GAPDH was increased approximately 3 fold in SHR cardiac myocytes relative to Wistar controls. In freshly isolated microvascular endothelial cells, however, levels of NOS3 protein and NOS3 mRNA were similar between the two groups. CONCLUSIONS: The expression of NOS3 is selectively altered in cardiac myocytes but not coronary microvascular endothelial cells of young SHR hearts, with a marked decrease in NOS3 protein but an increase in NOS3 mRNA. This dysregulation of NOS3 could contribute to contractile dysfunction in left ventricular hypertrophy.

Inhibition of myocardial crossbridge cycling by hypoxic endothelial cells: a potential mechanism for matching oxygen supply and demand?

Previous studies have shown that cardiac endothelial cells release substances that influence myocardial contraction. Since PO2 is an important stimulus that modulates endothelial function, we investigated the effects of acute moderate hypoxia and reoxygenation on the release of cardioactive factors by endothelial cells. Endothelial cells cultured from several vascular beds were superfused with normoxic (equilibrated with room air; PO2, approximately 160 mm Hg) or hypoxic (PO2, 40 to 50 mm Hg) physiological buffer solution, and the superfusates were reequilibrated to a PO2 of approximately 160 mm Hg and then tested for their effects on various myocardial assays. Endothelial cell viability and buffer ionic composition were unaltered after the superfusion procedures. The superfusates of hypoxic endothelial cells induced rapid, potent, reversible inhibition of isolated cardiac myocyte contraction without reducing cytosolic Ca2+ transients. This activity was not lost after heating (95 degrees C) and was present in low molecular weight (Mr, <500) superfusate fractions. Hypoxic endothelial superfusate reduced unloaded shortening velocity of human skinned soleus muscle fibers. It markedly depressed in vitro actin motility over cardiac myosin and reduced the rate of actin-activated cardiac myosin ATPase activity but had no effect on corresponding smooth muscle myosin assays. Reoxygenation of hypoxic endothelial cells resulted in loss of this inhibitory activity. These data indicate that cultured endothelial cells respond to acute moderate hypoxia by releasing an unidentified substance(s) that inhibits myocardial crossbridge cycling, independent of Ca2+ or other second messenger signaling pathways. Such a mechanism could have important implications for the regulation of oxygen supply-demand balance in the heart and be relevant to conditions such as myocardial hibernation.

Regional differences in the negative inotropic effect of acetylcholine within the canine ventricle.

1. Regional differences in the effects of ACh on sub-epicardial, mid-wall and sub-endocardial cells of the dog left ventricle have been studied. 2. ACh produced a dose-dependent, atropine-sensitive negative inotropic effect that was greatest in sub-epicardial cells and small or absent in sub-endocardial cells. 3. In sub-epicardial (but not sub-endocardial) cells, ACh also resulted in a dose-dependent decrease in action potential duration. The inotropic effect of ACh on sub-epicardial cells was primarily the result of the decrease of action potential duration, because during trains of voltage clamp pulses the inotropic effect of ACh was reduced or abolished. At a holding potential of -80 mV, 10(-5)M ACh decreased L-type Ca2+ current by approximately 8% and this is thought to be responsible for the small inotropic effect during trains of pulses. 4. Although 4-AP, a blocker of the transient outward current (I(to)), abolished the "spike and dome' morphology of the sub-epicardial action potential, it had little or no effect on the actions of ACh on sub-epicardial cells. ACh had no effect on I(to) in sub-epicardial cells in voltage clamp experiments. 5. ACh activated a Ba(2+)-sensitive outward current (IK,ACh) in sub-epicardial cells, but little or no such current in sub-endocardial cells. In sub-epicardial cells, ACh also inhibited the inward rectifier current, IK,1. 6. It is concluded that in left ventricular sub-epicardial cells, ACh activates IK,ACh. This results in a shortening of the action potential and, therefore, a negative inotropic effect. In subendocardial cells, ACh activates little or no IK,ACh and, therefore, it has little or no negative inotropic effect. This may result from a regional variation in the expression of the muscarinic K+ channel.

A transposon for green fluorescent protein transcriptional fusions: application for bacterial transport experiments.

The movement of bacteria through groundwater is a poorly understood process. Factors such as soil porosity and mineralogy, heterogeneity of soil particle size, and response of the bacteria to their environment contribute to the pattern of bacterial flow. The identification of transported bacteria is often a limiting factor in both laboratory and field transport experiments. Two bacterial strains were modified for use in bacterial transport experiments: a strain of Escherichia coli harboring the pGFP plasmid and a strain of Pseudomonas putida modified with a Tn5 derivative, Tn5GFP1. The Tn5GFP1 transposon incorporates the gene (gfp) encoding green fluorescent protein (GFP) and can be used to mutagenize Gram-bacteria. Fluorescent colonies were suspended in phosphate-buffered saline (PBS) at a concentration of approx. 10(9) bacteria/ml. A 10-cm glass column packed with quartz sand (diameter range 177-250 microns) was equilibrated with PBS prior to the forced flow introduction of the bacteria. Collected fractions were analyzed and the bacteria quantitated using a fluorescence spectrometer. Results demonstrate that the bacteria can be accurately tracked using their fluorescence, and that the intensity of the signal can be used to determine a C/Co ratio for the transported bacteria. The data show a rapid breakthrough of the bacteria followed by a characteristic curve pattern. A lower limit of detection of 10(5) cells was estimated based on these experiments. The Tn5GFP1 transposon should become a valuable tool for labeling bacteria.

Enzymatic histochemistry of retina with experimental intraocular pressure elevation in rabbits.

To investigate the pathogenesis of retina lesions caused by intraocular pressure elevation, activities and distribution of enzymes in retina including lactic dehydrogenase (LDH), succinate dehydrogenase (SDH), adenosinetriphosphatase (AT-Pase), acid phosphatase (ACP), cholinesterase (ChE), cytochrome oxidase (CCO), nucleotidase (5'-Nase) and glucose-6-phosphatase (G6Pase) were determined histochemically in 30 rabbits. It was found that 1) in the early stage of intraocular pressure elevation, the activities of LDH, SDH, ATPase, ACP, and ChE in retina were increased, while the activities of CCO, 5'-Nase decreased; 2) in the late stage of intraocular pressure elevation, the activities of all these enzymes but ACP, which showed a reduced activity, were close to the normal level; 3) in superoxide dismutase.(SOD-CCE) treated group, except the slight increase of LDH and G6Pase activities, the activities of the remaining enzymes were near to normal. Our results suggest that the various histochemical changes in retina induced by intraocular pressure elevation were compensatory in the early stage and were beneficial to the supply of energy needed in retinal tissue and cellular metabolism; while in the late stage, the lesion of retina cells developed due to decompensation. SOD-CCE could alleviate the retinal lesions caused by intraocular pressure elevation, and can be used as auxiliary drug for the treatment of intraocular pressure elevation.