BACKGROUNDWe investigated the co-expression of calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV, a combination of the three is referred to as CaBPs) with gamma-aminobutyric acid (GABA) or glycine in neurons of the rat medullary dorsal horn (MDH).

METHODSImmunofluorescence histochemical double-staining for CaBPs and GABA or glycine was performed on the sections from rat MDH.

RESULTSCB-, CR-, PV-, GABA- and glycine-like immunoreactive (LI) neurons were differentially observed in all layers of the MDH, but particularly in lamina II. Neurons that exhibited immunoreactivity for both CaBPs and GABA or glycine were also observed mainly in lamina II. A few of them were found in laminae I and III. The percentages of neurons which co-expressed CB/GABA or CB/glycine out of the total numbers of CB- and GABA-LI neurons or CB- and glycine-LI neurons were 5.3% and 12.1% or 4.1% and 10.0%, respectively. The ratios of CR/GABA or CR/glycine co-existing neurons out of the total numbers of CR- and GABA-LI neurons or CR- and glycine-LI neurons were 5.8% and 7.6% or 4.4% and 7.1%, respectively. The rates of PV/GABA or PV/glycine co-localized neurons out of the total numbers of PV- and GABA-LI neurons or PV- and glycine-LI neurons were 11.1% and 5.1% or 9.9% and 5.1%, respectively.

CONCLUSIONThe results indicate that some neurons in the MDH contain both CaBPs and GABA or glycine.

Animals ; Calbindin 1 ; Calbindin 2 ; Calbindins ; Calcium-Binding Proteins ; analysis ; Fluorescent Antibody Technique ; Glycine ; analysis ; Immunohistochemistry ; Medulla Oblongata ; cytology ; Parvalbumins ; analysis ; Posterior Horn Cells ; chemistry ; Rats ; S100 Calcium Binding Protein G ; analysis ; gamma-Aminobutyric Acid ; analysis

OBJECTIVETo study the related proteins of apoptosis initiation induced by homoharringtonine (HHT) in HL-60 cells.

METHODSAfter establishment of an apoptosis initiation model induced by HHT in HL-60 cells, proteins of untreated and HHT treated HL-60 cells were extracted, and the two-dimensional polyacrylamide gel electrophoresis (2-DE) maps of the extracted proteins were established by using the immobilized pH gradient (IPG) two-dimensional electrophoresis respectively. The alteration protein spots were identified with assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching.

RESULTSProteomics analysis showed that proteins including MHC class I antigen, calbindin D-28K, chloride channel protein 6, oncoprotein 18, zinc finger protein Helios and apoptosis inhibitor like protein 2 were involved in apoptosis initiation induced by HHT.

CONCLUSIONThe present study might conduce to the researches of HL-60 cells carcinogenesis and pave the way to exploit drug precursor related to HHT and initiation of apoptosis in HL-60 cells.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Calbindins ; Chloride Channels ; analysis ; DNA-Binding Proteins ; analysis ; Electrophoresis, Gel, Two-Dimensional ; methods ; HL-60 Cells ; Harringtonines ; pharmacology ; Histocompatibility Antigens Class I ; analysis ; Humans ; Ikaros Transcription Factor ; Inhibitor of Apoptosis Proteins ; Microtubule Proteins ; Phosphoproteins ; analysis ; Proteins ; analysis ; Proteome ; analysis ; S100 Calcium Binding Protein G ; analysis ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Stathmin ; Transcription Factors ; analysis

The interstitial nucleus of the spinal trigeminal tract (INV) contains many calbindin-D28k-containing neurons (CB-neurons) receiving convergence information from the somatic and visceral structures. The purpose of the present study was to confirm whether the primary afferent terminals from the inferior alveolar nerve (IAN) make close contact and synaptic connections with the same CB-neurons receiving visceral nociceptive signals in INV. Biotinylated dextran amine (BDA) and horseradish peroxidase (HRP) tracing combined with CB and Fos proteins immunohistochemistry were used. After injections of BDA and formalin into unilateral IAN and upper alimentary tract, respectively, the transganglionic labeled afferent fibers and terminals from IAN were observed in the ipsilateral INV, especially in its enlarged part. A large number of CB- and Fos-like immunoreactive (LI) neurons were found in bilateral INV. These CB- and Fos-LI neurons mostly overlapped with BDA-labeled terminals in the enlarged part of INV. About one half of the CB-LI neurons were double labeled with Fos-LI nuclei (74/153). The terminals from IAN were to made close contacts with many CB/Fos-double labeled or CB-single labeled neurons. After injection of HRP into IAN, HRP-labeled fibers and terminals in INV were similar to that labeled with BDA. Under the electron microscope, a large number of CB-LI dendrites and a few soma in the enlarged part of INV were found to form asymmetrical axo-dendritic and axo-somal synapses with the HRP-labeled axon terminals. These results indicate that the orofacial somatic inputs from IAN and the visceral nociceptive inputs from the upper alimentary tract converge onto the same CB-containing neurons in INV. These CB-containing neurons in INV probably play an important role in information integration as well as visceral and cardiovascular activity.
Animals ; Calbindin 1 ; Calbindins ; Face ; innervation ; Male ; Microscopy, Confocal ; Neural Pathways ; cytology ; physiology ; Neurons ; physiology ; Nociceptors ; physiology ; Presynaptic Terminals ; physiology ; Proto-Oncogene Proteins c-fos ; physiology ; Rats ; Rats, Sprague-Dawley ; S100 Calcium Binding Protein G ; metabolism ; physiology ; Trigeminal Nuclei ; physiology ; Viscera ; innervation

The calbindin D-28k (CB)-containing neurons in the interstitial nucleus of the spinal trigeminal tract (INV) that receive visceral and orofacial somatic nociceptive information and emanate projections to the parabrachial nuclei (PB) were investigated by the triple-labeled methods of fluorogold (FG) retrograde tracing combined with Fos and CB proteins immunofluorescence histochemistry in the rat. The results showed (1) in the perioral stimulation group, a large number of FG-retrograde labeled and Fos-immunoreactive neurons were found in the paratrigeminal nucleus (PaV) and the dorsal paramarginal nucleus (PaMd) of the INV ipsilateral to FG and formalin injection made to the PB and lips, respectively, while a lot of CB-immunoreactive neurons were distributed in the INV bilaterally; (2) a majority of the FG-retrograde labeled neurons (77.3%) were double-labeled with CB, and 40.7% of them were double-labeled with Fos; about 38.5% of FG/CB double-labeled neurons were FG/CB/Fos triple-labeled in the INV; and (3) in the upper alimentary tract stimulation group, the distribution and the numbers of FG-retrograde labeled, CB-immunoreactive neurons and FG/CB double-labeled neurons in the INV were similar to those of the perioral stimulation group as described above, except that the Fos immunoreactive neurons were distributed in the INV bilaterally, approximately 41.9% of the FG-retrograde labeled neurons were FG/Fos double-labeled, and over half (52.0%) of those double-labeled neurons were FG/CB/Fos triple-labeled. The results indicate that a part of CB-containing neurons in the INV receive orofacial somatic and visceral nociceptive information and that these neurons sent projections directly to the PB. The CB-containing neurons might play an important role in the transmission of the peripheral nociceptive information from INV to PB.
Animals ; Calbindins ; Face ; innervation ; Male ; Medulla Oblongata ; physiology ; Nerve Tissue Proteins ; metabolism ; Neural Pathways ; physiology ; Neurons ; metabolism ; physiology ; Nociceptors ; physiology ; Pain ; metabolism ; physiopathology ; Pons ; physiology ; Rats ; Rats, Sprague-Dawley ; S100 Calcium Binding Protein G ; metabolism ; Trigeminal Nucleus, Spinal ; physiology ; Viscera ; innervation

OBJECTIVETo test whether in the absence of actin, actin-binding proteins such as caldesmon, calponin, and tropomyosin interact with the myosin of unphosphorylation, Ca2+-dependent phosphorylation (CDP), and Ca2+-independent phosphorylation (CIP) and stimulate myosin Mg2+-ATPase activities.

METHODSMg2+-ATPase activities were measured to evaluate the effects of caldesmon, calponin, and tropomyosin on the myosin in unphosphorylation, CDP by myosin light chain kinase (MLCK), and CIP by MLCK.

RESULTS(1) At different incubation-time, i.e., 5, 10, 20, 40, and 60 minutes, the highest Mg2+-ATPase activity was observed when myosin was in the state of CDP, the medium was CIP of myosin, and the lowest was the unphosphorylated myosin. (2) In the absence of caldesmon, calponin, and tropomyosin, the Mg2+-ATPase activities from high to low were in the following order: CDP, CIP, and unphosphorylated myosin. However, in the presence of caldesmon, calponin, and tropomyosin, the order of relative value of Mg2+-ATPase activities from high to low was unphosphorylated, CIP, and CDP of myosin respectively compared to the corresponding controls.

CONCLUSIONSThe results propose that caldesmon, calponin, and tropomyosin are capable of stimulating Mg2+-ATPase activity of smooth muscle myosin in Ca2+-independent manner, since Ca2+ is not obligating for the stimulating effects of the three proteins. The common characteristic of the three proteins is that when myosin activities are low, their activations are relatively strong and this property might be involved in smooth muscle tension keeping.

Animals ; Ca(2+) Mg(2+)-ATPase ; drug effects ; metabolism ; Calcium ; pharmacology ; Calcium-Binding Proteins ; pharmacology ; Calmodulin-Binding Proteins ; pharmacology ; Chickens ; Microfilament Proteins ; Muscle, Smooth ; enzymology ; Myosins ; metabolism ; Phosphorylation ; Tropomyosin ; pharmacology

12E7 Antigen ; Antigens, CD ; metabolism ; CD56 Antigen ; metabolism ; Calbindin 2 ; Carcinoma, Endometrioid ; metabolism ; pathology ; Cell Adhesion Molecules ; metabolism ; Cystadenocarcinoma, Serous ; metabolism ; pathology ; Diagnosis, Differential ; Female ; Humans ; Immunohistochemistry ; Inhibins ; metabolism ; MART-1 Antigen ; metabolism ; Neprilysin ; metabolism ; Ovarian Neoplasms ; diagnosis ; metabolism ; S100 Calcium Binding Protein G ; metabolism ; Sertoli-Leydig Cell Tumor ; metabolism ; pathology ; Sex Cord-Gonadal Stromal Tumors ; diagnosis ; metabolism ; Steroidogenic Factor 1 ; metabolism ; WT1 Proteins ; metabolism

OBJECTIVETo study the morphological characteristics and immunophenotype of highly cellular leiomyoma (HCL) of uterus, compared with that of uterine endometrial stromal tumors (EST).

METHODSHE and immuno-stained sections EnVision method from 20 cases of HCL, 21 cases of EST and 1 case of stromomyoma were reviewed. Monoclonal antibodies against h-caldesmon, calponin, CD10, desmin and smooth muscle actin (SMA) were used for immunohistochemistry studies.

RESULTSOn microscopic examination, HCL were densely cellular and composed of cells that ranged from spindle-shaped to round with scanty cytoplasm. A focal fascicular pattern was present in all cases. Blood vessels with large, thick muscular walls were a conspicuous feature of the majority of tumors. Cleft-like spaces were present in 9 tumors and 15 cases exhibited irregular focal extensions into the adjacent myometrium. ESTs were composed of cells that resembled endometrial stromal cells of proliferative endometrium. These cases included a significant component of delicate blood vessels similar to spiral arterioles. All 20 low grade endometrial stromal sarcoma cases had infiltrative growth to adjacent myometrium. Immunoreactivities of HCL for h-caldesmon, calponin, CD10, Desmin and SMA were 80.0% (16/20), 100% (20/20), 0 (0/20), 95.0% (19/20) and 100% (20/20), respectively, whereas the positive rates of EST were 4.7% (1/21), 23.8% (5/21), 66.7% (14/21), 23.8% (5/21) and 19.0% (4/21), respectively (P = 0.001).

CONCLUSIONSHighly cellular leiomyomas have distinct morphologic features. H-caldesmon, calponin, CD10, desmin and SMA are helpful in the differential diagnosis of HCL and EST.

Adult ; Calcium-Binding Proteins ; analysis ; Calmodulin-Binding Proteins ; analysis ; Desmin ; analysis ; Endometrial Neoplasms ; metabolism ; pathology ; Endometrial Stromal Tumors ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Leiomyoma ; metabolism ; pathology ; Microfilament Proteins ; Middle Aged ; Neprilysin ; analysis ; Uterine Neoplasms ; metabolism ; pathology

OBJECTIVETo study the effect of hyperthermia on anti-invasion of Tca8113 and the expression change of matrix metalloproteinase-13 (MMP-13) and calcium-binding protein S100A4 (S100A4).

METHODSTca8113 cell pools were incubated at 43 degrees C for 0, 40, 80, 120 min, respectively, and at 37, 41, 43, 45 degrees C respectively for 80 min. The effect of high temperatures on the invasion ability of Tca8113 was measured in vitro. The slides of cells were made and incubated at 43 degrees C for 0, 40, 80, 120 min, respectively. Immunocytochemical method was employed for detecting the expression change of MMP-13 and S100A4 protein. Tca8113 cells were incubated at 43 degrees C for 0, 40, 80, 120 min respectively and at 37, 41, 43, 45 degrees C respectively for 80 min. Western blot method was conducted for detecting the expressionchange of MMP-13 and S100A4 protein.

RESULTSAs incubating time at higher temperature lasted, the proportion of the cells with invasion ability decreased. Except groups of 40 min and 80 min at 43 degrees C and 41, 43 degrees C for 80 min, the rest groups show significant statistic differences (P < 0.05). The expression intensity of MMP-13 and S100A4 proteins in Tca8113 cells would decrease as incubating time at higher temperature lasted. The content of MMP-13 and S100A4 proteins would decrease as incubating time at higher temperature lasted or incubating temperature increased. Except the groups of 40, 80 min at 43 degrees C and 41, 43 degrees C for 80 min, statistic differences were identified (P < 0.05).

CONCLUSIONThe invasion of Tca8113 could be inhibited by hyperthermia. The mechanism of this effect may be due to protein expression inhibition of MMP-13 and S100A4.

Calcium-Binding Proteins ; Cell Line, Tumor ; Humans ; Hyperthermia, Induced ; Matrix Metalloproteinase 13 ; S100 Proteins

OBJECTIVETo investigate the effects of NADPH oxidase inhibition on cardiac function and myocardial calcium regulatory proteins mRNA expressions in rabbits with heart failure (HF).

METHODSHF was induced by experimental aortic insufficiency and abdominal aortic constriction, HF animals were treated with oral apocynin (15 mg/d), a NADPH oxidase inhibitor or equal dose placebo. Eight weeks later, cardiac function was measured by echocardiography. Myocardial NADPH oxidase activity was evaluated by NADPH dependent superoxide production examined using superoxide dismutase-inhibitable cytochrome c reduction. Sarcoplasmic reticulum Ca(2+)ATPase (SERCA2a), ryanodine receptor 2 (RyR2), phospholamban (PLB) and sodium-calcium exchanger (NCX) were determined by RT-PCR.

RESULTSRabbits with HF developed ventricular dilatation and cardiac dysfunction, as well as increase in myocardial NADPH oxidase activity, decreases in mRNA expression of SERCA2a, RyR2 and PLB, and increase in mRNA expression of NCX. Apocynin significantly reduced NADPH oxidase activity (P < 0.05), upregulated SERCA2a, RyR2 and PLB mRNA expressions (SERCA2a/GAPDH: 0.63 +/- 0.11 vs. 0.34 +/- 0.08, RyR2/GAPDH: 0.23 +/- 0.04 vs. 0.17 +/- 0.06, PLB/GAPDH:1.28 +/- 0.13 vs. 0.95 +/- 0.09, P < 0.05), downregulated NCX mRNA expression (NCX/GAPDH: 0.67 +/- 0.10 vs. 0.95 +/- 0.12, P < 0.05), and improved cardiac function [LVEF: (60.06 +/- 10.07)% vs. (38.87 +/- 3.31)%, LVFS: (30.12 +/- 6.56)% vs. (17.40 +/- 2.45)%, P < 0.05] in rabbits with HF.

CONCLUSIONNADPH oxidase inhibition improves cardiac function possibly by preventing abnormal alterations in myocardial calcium regulatory proteins in failing heart.

Acetophenones ; pharmacology ; Animals ; Calcium ; metabolism ; Calcium-Binding Proteins ; metabolism ; Calmodulin ; biosynthesis ; Enzyme Inhibitors ; pharmacology ; Female ; Heart Failure ; metabolism ; physiopathology ; Male ; Myocardium ; metabolism ; NADPH Oxidases ; antagonists & inhibitors ; metabolism ; Rabbits ; Ryanodine Receptor Calcium Release Channel ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Sodium-Calcium Exchanger ; metabolism ; Ventricular Function, Left

Lipocortin 1 has been proposed as a putative mediator of anti-inflammatory actions of glucocorticoids. We investigated the role of lipocortin 1 in the effect of dexamethasone using rat splenic leukocytes. Concanavalin A(ConA; 1-microgram/ml) increased the leukocyte proliferation and nitric oxide(NO) generation, which were measured as (3H)-thymidine uptake by the cells and nitrite accumulation in the culture media, respectively. Dexamethasone suppressed CoNinduced cell proliferation, in a concentration-dependent manner with EC-50 around 50nM. The addition of anti-lipocortin 1 (Anti-LC1) reversed dexamethasone effects: 0.24, 1.2, 6 microgram/ml of Anti-LC1 reversed dexamethasone(50nM)-induced suppression of thymidine uptake by 9+/-3%, 16+/-3%, 36+/-5%, respectively; 0.24, 1.2, and 6-microgram/ml of Anti-LC1 reversed dexamethasone-induced decrease of nitrite concentration by 49 +/- 16%, 61 +/- 20%, 77 +/- 19 %, respectively. The present data indicate that lipocortin 1 mediates, at least in part, glucocorticoids-induced suppression of leukocyte proliferation and blockade of NO generation.
Animals ; Annexin A1* ; Annexins* ; Cell Proliferation ; Culture Media ; Dexamethasone* ; Glucocorticoids ; Leukocytes* ; Nitric Oxide* ; Rats* ; Thymidine