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Biomedical subjects

Lu Wang

Publications and source records attributed to Lu Wang.

At least 91 records · Page 5Linked to original sources

Expression and purification of the carboxyl terminus domain of Schizosaccharomyces pombe dicer in Escherichia coli.

The carboxyl terminus domain of Schizosaccharomyces pombe dicer (yDicerC) was expressed in Escherichia coli as an MBP-fusion protein (MBP-yDicerC). When the E. coli strain was cultured and induced at 25 degrees C, the MBP-yDicerC was partly expressed in the soluble fraction. It was then purified by two step affinity chromatography with amylose resin and Ni-NTA His Bind(R) resin. The purified MBP-yDicerC showed double-strand RNA digestion activity. siRNA-like products about 22-nt in length were generated.

Carrier Proteins↗

[Analysis of spontaneous reperfusion in acute myocardial infarction and its effect on short-term prognosis].

OBJECTIVE: To analyse the clinical and angiographic characteristics of spontaneous reperfusion (SR) in AMI, and to evaluate its effect on short-term prognosis. METHODS: 112 consecutive AMI patients without intravenous thrombolytic therapy received emergent coronary angiography and primary PCI. The patients were divided into SR group (antegrade TIMI grade 2-3 flow) and non-SR group (antegrade TIMI grade 0-1 flow). The clinical, angiographic and prognostic features of the patients were analyzed. RESULTS: 31 patients (27.7%) were in SR group, and there was no significant difference in base-line clinical characteristics between the two groups. Compared with non-SR group, peak values of CK and CK-MB, Ventricular wall motion abnormality and mortality were lower in SR group, ejection fraction was higher in SR group. Logistic regression analysis showed that there was good correlation between SR and peak value of CK, collaterals, ventricular wall motion abnormality and pre-dilation in PCI. CONCLUSION: SR decreased infarction size, improved heart function and reduced 30-day mortality.

Aged↗

Risk factors for postoperative complications after liver resection.

BACKGROUND: Liver resection is still a complicated operation with a high risk of postoperative morbidity. This study was undertaken to analyze the risk factors for postoperative complications after liver resection. METHODS: From 2001 to 2004, a total of 146 patients underwent liver resection for malignant or benign lesions. Postoperative complications after the resection were classified as surgical and medical, their incidences were analyzed retrospectively. The risk factors for both surgical and medical complications were analyzed. To increase the safety of liver resection, surgical techniques were modified after April 2003, including control of inflow or outflow and intra-operative test with methylene blue. RESULTS: Before April 2003, a series of 58 patients received liver resection. Modified surgical techniques were used in liver resections for 88 patients after April 2003. A total of 36 patients (24.7%) had postoperative complications. Surgical and medical complications occurred in 24 and 13 patients respectively (One patient had both surgical and medical complications). Perioperative blood transfusion was related to a higher risk of surgical complications (P < 0.05). Patients with diabetes mellitus were associated with a higher risk of medical complications (P < 0.05). Surgical complications and postoperative hospitalization were decreased after the use of modified surgical techniques (P < 0.05). CONCLUSION: Postoperative surgical complications can be decreased by modified surgical techniques, and careful selection of patients for liver resection may help to decrease postoperative medical complications also.

Adult↗

Interferon alpha 2a down-regulates VEGF expression through PI3 kinase and MAP kinase signaling pathways.

An earlier report demonstrated that interferon alpha (IFN-alpha) inhibited tumor growth and recurrence in an MHCC97 xenograft model in nude mice by suppressing tumor angiogenesis rather than by inhibiting tumor cell proliferation. However, the underlying molecular mechanism was not fully elucidated. In this study, we demonstrated that IFN-alpha 2a could down-regulate VEGF expression both in mRNA and in protein levels, as well as down-regulating HIF-1 alpha mRNA expression in MHCC97 cells in vitro. A cDNA micro array analysis followed by Northern and Western blot analysis revealed that PI3 kinase and MAP kinase signaling pathways might be inhibited by IFN-alpha 2a. Blocking the function of IFN-alpha receptor with a specific peptide could eliminate the inhibitory effects of IFN-alpha 2a on VEGF expression. In addition, wortmannin and PD098059, respective inhibitors of the PI3 kinase and the MAP kinase signaling pathways, when used independently or in combination, could also down-regulate the VEGF synthesis and secretion in a similar pattern of IFN-alpha 2a. These observations may lead to the conclusion that IFN-alpha 2a could suppress VEGF synthesis and secretion by down-regulating HIF-1 alpha expression, via inhibition of the PI3 kinase and/or the MAP kinase signaling pathways.

Angiogenesis Inducing Agents↗

Chlorine dioxide reduction by aqueous iron(II) through outer-sphere and inner-sphere electron-transfer pathways.

The reduction of ClO(2) to ClO(2)(-) by aqueous iron(II) in 0.5 M HClO(4) proceeds by both outer-sphere (86%) and inner-sphere (14%) electron-transfer pathways. The second-order rate constant for the outer-sphere reaction is 1.3 x 10(6) M(-1) s(-1). The inner-sphere electron-transfer reaction takes place via the formation of FeClO(2)(2+) that is observed as an intermediate. The rate constant for the inner-sphere path (2.0 x 10(5) M(-1) s(-1)) is controlled by ClO(2) substitution of a coordinated water to give an inner-sphere complex between ClO(2) and Fe(II) that very rapidly transfers an electron to give (Fe(III)(ClO(2)(-))(H(2)O)(5)(2+))(IS). The composite activation parameters for the ClO(2)/Fe(aq)(2+) reaction (inner-sphere + outer-sphere) are the following: DeltaH(r)++ = 40 kJ mol(-1); DeltaS(r)++ = 1.7 J mol(-1) K(-1). The Fe(III)ClO(2)(2+) inner-sphere complex dissociates to give Fe(aq)(3+) and ClO(2)(-) (39.3 s(-1)). The activation parameters for the dissociation of this complex are the following: DeltaH(d)++= 76 kJ mol(-1); DeltaS(d)++= 32 J K(-1) mol(-1). The reaction of Fe(aq)(2+) with ClO(2)(-) is first order in each species with a second-order rate constant of k(ClO2)- = 2.0 x 10(3) M(-1) s(-1) that is five times larger than the rate constant for the Fe(aq)(2+) reaction with HClO(2) in H(2)SO(4) medium ([H(+)] = 0.01-0.13 M). The composite activation parameters for the Fe(aq)(2+)/Cl(III) reaction in H(2)SO(4) are DeltaH(Cl(III))++ = 41 kJ mol(-1) and DeltaS(Cl(III))++ = 48 J mol(-1) K(-1).

Journal Article↗

Cooperativity between far upstream enhancer and proximal promoter elements of the human {alpha}2(I) collagen (COL1A2) gene instructs tissue specificity in transgenic mice.

Interaction between the proximal (-378) promoter and the far upstream (-20 kb) enhancer is essential for tissue-specific expression of the human alpha2(I) collagen gene (COL1A2) in transgenic mice. Previous in vitro studies have shown that three Sp1 binding sites (around -300) are part of a cytokine-responsive element and that two TC-rich boxes (around -160 and -125) and a CBF/NFY consensus sequence (around -80) confer optimal promoter activity by interacting among themselves and with the upstream Sp1 sites. Here we report that mutations of the Sp1 binding sites, TC-rich boxes or CBF/NFY consensus sequence lead to reduced transgene activity, thus underscoring the functional interdependence of the proximal promoter elements. Loss of the Sp1 binding sites was associated with loss of transgene expression in osteoblasts, whereas elimination of the CBF/NFY binding site (alone or in combination with the TC-rich boxes) was correlated with a lack of activity in the ventral fascia and head dermis and musculature. Additionally, transgene expression in skin fascia fibroblasts depended on the integrity of the Sp1 binding sites and TC-rich boxes, and on their physical configuration. Evidence is also presented suggesting cooperativity between cis-acting elements of the far upstream enhancer and proximal promoter in assembling tissue-specific protein complexes. This study thus reiterates the complex and highly combinatorial nature of the regulatory network governing COL1A2 transcription in vivo.

Animals↗

MRI-based treatment planning for radiotherapy: dosimetric verification for prostate IMRT.

PURPOSE: Magnetic resonance (MR) and computed tomography (CT) image fusion with CT-based dose calculation is the gold standard for prostate treatment planning. MR and CT fusion with CT-based dose calculation has become a routine procedure for intensity-modulated radiation therapy (IMRT) treatment planning at Fox Chase Cancer Center. The use of MRI alone for treatment planning (or MRI simulation) will remove any errors associated with image fusion. Furthermore, it will reduce treatment cost by avoiding redundant CT scans and save patient, staff, and machine time. The purpose of this study is to investigate the dosimetric accuracy of MRI-based treatment planning for prostate IMRT. METHODS AND MATERIALS: A total of 30 IMRT plans for 15 patients were generated using both MRI and CT data. The MRI distortion was corrected using gradient distortion correction (GDC) software provided by the vendor (Philips Medical System, Cleveland, OH). The same internal contours were used for the paired plans. The external contours were drawn separately between CT-based and MR imaging-based plans to evaluate the effect of any residual distortions on dosimetric accuracy. The same energy, beam angles, dose constrains, and optimization parameters were used for dose calculations for each paired plans using a treatment optimization system. The resulting plans were compared in terms of isodose distributions and dose-volume histograms (DVHs). Hybrid phantom plans were generated for both the CT-based plans and the MR-based plans using the same leaf sequences and associated monitor units (MU). The physical phantom was then irradiated using the same leaf sequences to verify the dosimetry accuracy of the treatment plans. RESULTS: Our results show that dose distributions between CT-based and MRI-based plans were equally acceptable based on our clinical criteria. The absolute dose agreement for the planning target volume was within 2% between CT-based and MR-based plans and 3% between measured dose and dose predicted by the planning system in the physical phantom. CONCLUSIONS: Magnetic resonance imaging is a useful tool for radiotherapy simulation. Compared with CT-based treatment planning, MR imaging-based treatment planning meets the accuracy for dose calculation and provides consistent treatment plans for prostate IMRT. Because MR imaging-based digitally reconstructed radiographs do not provide adequate bony structure information, a technique is suggested for producing a wire-frame image that is intended to replace the traditional digitally reconstructed radiographs that are made from CT information.

Humans↗

Ambiguous allele combinations in HLA Class I and Class II sequence-based typing: when precise nucleotide sequencing leads to imprecise allele identification.

Sequence-based typing (SBT) is one of the most comprehensive methods utilized for HLA typing. However, one of the inherent problems with this typing method is the interpretation of ambiguous allele combinations which occur when two or more different allele combinations produce identical sequences. The purpose of this study is to investigate the probability of this occurrence. We performed HLA-A,-B SBT for Exons 2 and 3 on 676 donors. Samples were analyzed with a capillary sequencer. The racial distribution of the donors was as follows: 615-Caucasian, 13-Asian, 23-African American, 17-Hispanic and 8-Unknown. 672 donors were analyzed for HLA-A locus ambiguities and 666 donors were analyzed for HLA-B locus ambiguities. At the HLA-A locus a total of 548 total ambiguous allele combinations were identified (548/1344 = 41%). Most (278/548 = 51%) of these ambiguities were due to the fact that Exon 4 analysis was not performed. At the HLA-B locus 322 total ambiguous allele combinations were found (322/1332 = 24%). The HLA-B*07/08/15/27/35/44 antigens, common in Caucasians, produced a large portion of the ambiguities (279/322 = 87%). A large portion of HLA-A and B ambiguous allele combinations can be addressed by utilizing a group-specific primary amplification approach to produce an unambiguous homozygous sequence. Therefore, although the prevalence of ambiguous allele combinations is high, if the resolution of these ambiguities is clinically warranted, methods exist to compensate for this problem.

Journal Article↗

Pentamidine-induced long QT syndrome and block of hERG trafficking.

The diamidine pentamidine is used to treat leishmaniasis, trypanosomiasis, and Pneumocystis carinii pneumonia. Treatment may be accompanied by prolongation of the QT interval of the electrocardiogram and torsades de pointes tachycardias. Up to now, it has been thought that therapeutic compounds causing QT prolongation are associated with direct block of the cardiac potassium channel human ether a-go-go-related gene (hERG), which encodes the alpha subunit of cardiac I(Kr) currents. We show that pentamidine has no acute effects on currents produced by hERG, KvLQT1/mink, Kv4.3, or SCNA5. Cardiac calcium currents and the guinea pig cardiac action potential were also not affected. After overnight exposure, however, pentamidine reduced hERG currents and inhibited trafficking and maturation of hERG with IC(50) values of 5 to 8 microM similar to therapeutic concentrations. Surface expression determined in a chemiluminescence assay was reduced on exposure to 10, 30, and 100 microM pentamidine by about 30, 40, and 70%, respectively. These effects were specific for hERG since expression of hKv1.5, KvLQT1/minK, and Kv4.3 was not altered. In isolated guinea pig ventricular myocytes, 10 microM pentamidine prolonged action potential duration APD(90) from 374.3 +/- 57.1 to 893.9 +/- 86.2 ms on overnight incubation. I(Kr) tail current density was reduced from 0.61 +/- 0.09 to 0.39 +/- 0.04 pA/pF. We conclude that pentamidine prolongs the cardiac action potential by block of hERG trafficking and reduction of the number of functional hERG channels at the cell surface. We propose that pentamidine, like arsenic trioxide, produces QT prolongation and torsades de pointes in patients by inhibition of hERG trafficking.

Action Potentials↗

Diagnosis and surgical treatments of hepatocellular carcinoma with tumor thrombosis in bile duct: experience of 34 patients.

AIM: Hepatocellular carcinoma (HCC) with bile duct tumor thrombosis (BDT) is a rare event. The prognosis of this type of patients is very dismal. The aim of this study was to share the experience in the diagnosis and treatment of HCC with BDT, to further improve the prognosis of these patients. METHODS: Thirty-four patients of HCC with BDT received surgical treatment in authors' institute from July 1987 to January 2003 were reviewed retrospectively. The experience in the diagnosis and treatment, and the outcome of this type of HCC patients were summarized. RESULTS: Thirty of the 34 patients (88.2%) were positive for alpha-fetoprotein (AFP) (>20 microg/L), and 12 patients (35.3%) were found having obstructive jaundice before operation, 18 cases were suspected of "obstruction of bile duct" preoperatively. The primary tumors were frequently located at the left medial (13 cases) or right anterior lobe (14 cases). Thirty-one patients received liver resections and removal of BDT, while the other 3 patients received removal of BDT combined with hepatic artery ligation and cannulation (HAL+HAI), or only removal of BDT because their liver function reservation and general condition could not tolerate the primary tumor resection. The 1-year survival rate was 71.4%(20/28). The longest disease-free survival was over 15 years. The intrahepatic tumor recurrence within 1 year after operation was found in 14 patients (14/28, 50.0%). CONCLUSION: Surgical removal of primary tumors and BDT is safe and beneficial to the HCC patients with BDT. Early detection, diagnosis, and surgical treatment are the key points to prolong the survival time of patients.

Bile Duct Neoplasms↗

[Study on the inhibitory effect of small tripeptide CMS024 on tumor growth and metastasis in nude mice model of human hepatocellular carcinoma metastasis].

OBJECTIVE: This experiment was designed to study the effect of small molecule tripeptide CMS024 on tumor growth and metastasis in a nude mice model of human hepatocellular carcinoma (HCC) metastasis. METHODS: Highly metastatic human HCC cell strain HCCLM6 was injected into subcutaneous region of nude mice. When the subcutaneous tumor reached 0.8 cm in diameter, the tumor was removed and cut into small pieces about 1 mm in diameter and implanted orthotopically into the liver of 20 BALB/c nude mice, which were randomized into treatment group and control group each with 10 mice. The treatment group received daily CMS024 intraperitoneal injection at the dose of 300 microg/kg beginning from the second postoperative day. The control group received intraperitoneal injection of equal amount of 0.85% sodium chloride solution. The mice were observed for signs of disease development. Thirty-five days later, the mice were sacrificed and abdominal and pulmonary metastases were recorded and peripheral blood hematological and biochemical parameters were determined. RESULTS: After 35 days of intervention, all the 10 mice in the treatment group were alive and well, but there were only 9 living mice in the control group. The mean tumor weights were 1.9 g +/- 0.5 g in the treatment group and 2.3 g +/- 0.8 g in the control group (P > 0.05). The abdominal wall metastases and intraperitoneal metastases were 100% for the control group and only 60% and 50%, respectively, for the treatment group (P < 0.05). Bloody ascites and gross intrahepatic metastatic nodules were found in 70% and 90% of nude mice, respectively, for the control group, and 20% and 40%, respectively, for the treatment group (P > 0.05). The median number of grades I, II, III and IV pulmonary metastases were 92, 24, 15 and 16, respectively, for the control group, and 24, 20, 10 and 8, respectively, for the treatment group. There were statistically significant differences in the number of grade I pulmonary metastases between the treatment group and the control group (24 vs 92, P < 0.05). No statistically significant differences in the body weight, peripheral blood hematological and biochemical parameters were observed between the two groups. No toxic effects were observed during the observation period. CONCLUSIONS: Small molecule tripeptide CMS024 demonstrated positive effects to retard tumor growth and inhibit loco-regional and long distance metastases in a nude mice model of human HCC metastasis, and no obvious toxic or side effects on the tumor bearing nude mice.

Animals↗

Stereotactic IMRT for prostate cancer: setup accuracy of a new stereotactic body localization system.

The purpose of this work is to prospectively assess the setup accuracy that can be achieved with a stereotactic body localizer (SBL) in immobilizing patients for stereotactic intensity-modulated radiotherapy (IMRT) for prostate cancer. By quantifying this important factor and target mobility in the SBL, we expect to provide a guideline for selecting planning target volume margins for stereotactic treatment planning. We analyzed data from 40 computed tomography (CT) studies (with slice thickness of 3 mm) involving 10 patients with prostate cancer. Each patient had four sets of CT scans during the course of radiotherapy. For the purpose of this study, all four sets of CT scans were obtained with the patients immobilized in a customized body pillow formed by vacuum suction. Unlike other immobilization devices, this system consists not only of a customized body pillow, but also of a fixation sheet used to suppress patient respiratory motion, a stereotactic body frame to provide stereotaxy, and a carbon fiber base board to which both the body cushion and the frame are affixed. We identified four bony landmarks and measured their coordinates in the stereotactic body frame on each set of CT scans. The displacements of the bony landmarks from their corresponding positions on the simulation scan (first CT scan) were analyzed in three dimensions in terms of overall, systematic, and random categories. The initial planned isocenter was also marked on the patients' skin with fiducials for each CT study. The distance from each bony landmark to the fiducial-based isocenter was measured and compared among the four sets of CT scans. The deviations in distances were also compared to those measured from the landmarks to the stereotactic frame center, in order to determine the effectiveness of the rigid body frame in positioning patients with prostate cancer. Target inter-fraction motion in this system was also studied for five patients by measuring the deviations in distances from the target geometric center to the bony landmarks. Our results showed that the overall setup accuracy had standard deviations (SDs) of 2.58 mm, 2.41 mm, and 3.51 mm in lateral (LAT), anterior-posterior(AP), and superior-inferior (SI) directions, respectively. The random component had SDs of 1.72 mm, 2.06 mm, and 2.79 mm, and the systematic component showed SDs of 0.92 mm, -0.27 mm, and 0.90 mm in these three directions. In terms of three-dimensional vector, the mean displacement over 116 measurements was 3.0 mm with an SD of 1.29 mm. Compared to the rigid reference, the skin-mark-based reference was less reliable for patient repositioning in terms of reproducing known bony landmark positions. The mean target mobility relative to the bony landmarks was 2.22 +/- 3.45 mm, 0.17 +/- 1.11 mm, and 0.11 +/- 2.69 mm in the AP, LAT, and SI directions, respectively. In conclusion, the body immobilization system has the ability to immobilize prostate cancer patients with satisfactory setup accuracy for fractionated extracranial stereotactic radiotherapy. A rigid frame system serves as a reliable alignment reference in terms of repositioning patients into the planning position, while skin-based reference showed larger deviations in repositioning patients.

Equipment Design↗

An alternative form of paraptosis-like cell death, triggered by TAJ/TROY and enhanced by PDCD5 overexpression.

Accumulating reports demonstrate that apoptosis does not explain all the forms of programmed cell death (PCD), particularly in individual development and neurodegenerative disease. Recently, a novel type of PCD, designated 'paraptosis', was described. Here, we show that overexpression of TAJ/TROY, a member of the tumor necrosis factor receptor superfamily, induces non-apoptotic cell death with paraptosis-like morphology in 293T cells. Transmission electron microscopy studies reveal extensive cytoplasmic vacuolation and mitochondrial swelling in some dying cells and no condensation or fragmentation of the nuclei. Characteristically, cell death triggered by TAJ/TROY was accompanied by phosphatidylserine externalization, loss of the mitochondrial transmembrane potential and independent of caspase activation. In addition, TAJ/TROY suppressed clonogenic growth of HEK293 and HeLa cells. Interestingly, overexpression of Programmed cell death 5 (PDCD5), an apoptosis-promoting protein, enhanced TAJ/TROY-induced paraptotic cell death. Moreover, cellular endogenous PDCD5 protein was significantly upregulated in response to TAJ/TROY overexpression. These results provide novel evidence that TAJ/TROY activates a death pathway distinct from apoptosis and that PDCD5 is an important regulator in both apoptotic and non-apoptotic PCD.

Apoptosis↗

Mechanical strain inhibits airway smooth muscle gene transcription via protein kinase C signaling.

Mechanical strain affects airway myocyte phenotype, cytoskeletal architecture, proliferation, and contractile function. We hypothesized that (i) short-term mechanical strain modulates transcription of smooth muscle-specific gene promoters for SM22 and smooth muscle myosin heavy chain (smMHC); and (ii) strain-induced change is mediated by altered actin polymerization in association with activation of protein kinase C (PKC). Primary cultured canine tracheal myocytes were transiently transfected with luciferase reporter plasmids harboring a murine SM22, human smMHC, or artificial serum response factor (SRF)-specific gene promoter and then subjected to cyclic strain for 48 h. This strain protocol significantly reduced transcriptional activity of SM22 and smMHC promoters and an artificial SRF-dependent promoter by 55 +/- 5.9%, 57 +/- 6.4%, and 75 +/- 7.9%, respectively, with concomitant reduction in F/G actin ratio by 31 +/- 8%. PKC inhibitors, GF109203X or Gö6976, significantly attenuated these affects. Similar to strain, strain-independent activation of PKC inhibited SM22, smMHC, and SRF-dependent promoter activity by 61 +/- 4%, 66 +/- 5%, and 28 +/- 15%, respectively, and reduced the F/G actin ratio by 30 +/- 5%. Gel shift assay revealed that PKC activation led to decreased binding of the required transcription factor, SRF, to CArG elements in the SM22 promoter. These data suggest a previously unknown role for PKC isoforms in mechanosensitive signaling in airway myocytes that is associated with coordinated regulation of actin cytoskeletal dynamics and smooth muscle-specific gene transcription.

Actins↗

Last intron of the chemokine-like factor gene contains a putative promoter for the downstream CKLF super family member 1 gene.

The genes for chemokine-like factor (CKLF) and four chemokine-like factor super family members (CKLFSF1-4) are tightly linked on chromosome 16, with only 325 bp separating CKLF and CKLFSF1. We used Northern blotting and RT-PCR to show that these two genes are expressed independently of one another. We then used a novel computational promoter prediction method based on the interaction among transcription factor binding sites (TFBSs) to identify a putative promoter region for the CKLFSF1 gene. Our method predicted a promoter region in the last intron of the upstream gene, CKLF. We PCR amplified the predicted promoter region and used a luciferase assay to show that the region was able to drive the luciferase gene. DNA decoy experiments indicated that 214 bp fragment neighboring the TATA box markedly inhibited CKLFSF1 gene expression. Sequence analysis of the region revealed a typical TATA box (TATATAA) and multiple potential transcription factor binding sites, providing further evidence for this being a functional promoter for CKLFSF1. This work provides the first evidence of a promoter from one gene located in an intron of another.

Base Sequence↗

Ontogenesis of myosin light chain kinase mRNA and protein content in guinea pig tracheal smooth muscle.

We previously reported in guinea pig tracheal smooth muscle that maximal shortening velocity decreases from 3 weeks of age to adulthood. It is not known whether myosin light chain kinase (MLCK), a key enzyme determining the velocity of smooth muscle contraction, undergoes maturational changes. In the present work, we investigated MLCK protein content and mRNA expression in 1-week-old, 3-week-old, and adult guinea pigs. We extracted either proteins or RNA from isolated tracheal smooth muscle. The content of MLCK was assessed by Western immunoblots. MLCK mRNA was evaluated by Northern analysis and by quantitative real time reverse transcriptase-polymerase chain reaction (RT-PCR). The content of MLCK increased 3-fold at 3 weeks of age and then decreased in adults, being 0.116 +/- 0.042, 0.330 +/- 0.125 (P < 0.05), and 0.153 +/- 0.054 microg/mg of total protein, respectively, in 1-week, 3-week, and adult animals. Quantitative RT-PCR revealed that MLCK mRNA increased with age to 135 +/- 35% and 177 +/- 23% (P < 0.01) in 3-week and adult animals, respectively, compared to 1-week animals. The transient increase of MLCK content in juvenile guinea pig tracheal smooth muscle may contribute to the increased shortening velocity at this age. We suggest that this increased content of MLCK is one of the mechanisms leading to maturation of airway smooth muscle contractility, which in turn contributes to the airway hyperresponsiveness reported in children and young animals.

Age Factors↗

Molecular cloning and characterization of chemokine-like factor super family member 1 (CKLFSF1), a novel human gene with at least 23 alternative splicing isoforms in testis tissue.

Chemokine-like factor (CKLF) was isolated from PHA-stimulated U937 cells. It is composed of 152 amino acids and located on chromosome 16q22. Utilizing bioinformatics, based on CKLF cDNA and protein sequences, in combination with experimental validation, we identified a novel gene designated chemokine-like factor super family member 1 (CKLFSF1). CKLFSF1 maps on chromosome 16q22, and the full-length gene comprises of seven exons and six introns. Using RACE-PCR, we identified two potential alternative transcription start sites, 1A and 1B. Northern blot and RT-PCR analysis demonstrated that CKLFSF1 is predominantly expressed in human testis tissue, with only lower levels of expression in many other human tissues. RT-PCR and cDNA sequencing identified 23 alternatively spliced isoforms of CKLFSF1 in the testis tissue, which encode protein variants ranging from 36 to 169 amino acids in length. Immunohistochemistry analysis demonstrated that CKLFSF1 proteins are highly expressed in spermatocyte and in tissue fluid of human testes tissue. In light of these findings, we propose that CKLFSF1 may play an important role in spermatogenesis or testicular development.

Alternative Splicing↗