Proteomic insights into the regulatory function of ARID1A in colon cancer cells.

The AT-rich interacting domain-containing protein 1A (ARID1A) is a tumor suppressor gene that has been implicated in several cancers, including colorectal cancer (CRC). The present study used a proteomic approach to elucidate the molecular mechanisms of ARID1A in CRC carcinogenesis. Stable ARID1A-overexpressing SW48 colon cancer cells were established using lentivirus transduction and the successful overexpression of ARID1A was confirmed by western blotting. Label-free quantitative proteomic analysis using liquid chromatography-tandem mass spectrometry identified 705 differentially altered proteins in the ARID1A-overexpressing cells, with 310 proteins significantly increased and 395 significantly decreased compared with empty vector control cells. Gene Ontology enrichment analysis highlighted the involvement of the altered proteins mainly in the Wnt signaling pathway. Western blotting supported these findings, as a decreased protein expression of Wnt target genes, including c-Myc, transcription factor T cell factor-1/7 and cyclin D1, were observed in ARID1A-overexpressing cells. Among the altered proteins involved in the Wnt signaling pathway, the interaction network analysis revealed that ARID1A exhibited a direct interaction with E3 ubiquitin-protein ligase zinc and ring finger 3 (ZNRF3), a negative regulator of the Wnt signaling pathway. Further analyses using the The Cancer Genome Atlas colon adenocarcinoma public dataset revealed that ZNRF3 expression significantly impacted the overall survival of patients with CRC and was positively correlated with ARID1A expression. Finally, an increased level of ZNRF3 in ARID1A-overexpressing cells was confirmed by western blotting. In conclusion, the findings of the present study suggest that ARID1A negatively regulates the Wnt signaling pathway through ZNRF3, which may contribute to CRC carcinogenesis.

Reduction of lauric acid content in virgin coconut oil improved plasma lipid profile in high-fat diet-induced hypercholesterolemic mice.

Virgin coconut oil (VCO) is claimed to have various health benefits, but favorable effects of its major component (∼50%), lauric acid, are controversial. Therefore, we aimed to reduce lauric acid content (∼30%) in VCO and evaluate its effect compared to VCO and medium-chain triglycerides (MCT), on food intake, bodyweight (BW), lipid profiles, and hepatic histology. Female C57BL/6 mice were treated with different diets for 3 months: control (normal diet), high-fat diet (HF), HF + VCO, HF + MCT, HF + low lauric acid VCO (LLA), and normal diet + LLA (C + LLA). LLA was prepared by enzymatic interesterification of VCO with methyl octanoate (methyl caprylate) and methyl decanoate (methyl caprate). Plasma and liver lipids, including total cholesterol (TC), high-density lipoprotein (HDL), and triglyceride, were measured by colorimetric assay, and hepatic fat accumulation was examined by oil-red-O staining. HF mice exhibited high plasma and liver TC and low-density lipoprotein (LDL). VCO or MCT treatment lowered liver TC and LDL, whereas LLA increased plasma HDL and markedly improved TC:HDL ratio. The HF-induced hepatic fat accumulation was attenuated by all treatments, of which VCO was the most effective. Control mice administered with LLA demonstrated lower liver TC and LDL, but higher plasma TC and HDL compared to controls. Lowest BW gain and food intake were found in mice treated with LLA. In conclusion, VCO, MCT, and LLA ameliorated hepatic histopathology caused by HF. VCO and MCT improved liver lipid profiles, whereas LLA has more beneficial effect on plasma lipids via a better TC:HDL ratio and showed promise for BW control.

Increased expression of apoptotic markers in human full-term placenta after exposure to elevated environmental cadmium levels during pregnancy.

Cadmium (Cd), a toxic heavy metal, produces various forms of environmental contaminations and health problems in human. In this study, we aimed to examine the localization of several apoptotic markers in human placentas from pregnant women who were environmentally exposed to Cd. Twelve pregnant women participated in this analysis and they were divided into 2 groups according to their living areas: high-Cd (H-Cd) and low-Cd (L-Cd) groups. After delivery, the placentas were immediately harvested, and the placental width, length, and weight were measured. The placental Cd concentration was determined by using ICP-MS. The expression of three apoptotic markers, cleaved caspase-3, cleaved lamin A/C, and TUNEL, was examined in immunohistochemistry. In results, the placental Cd concentration in the H-Cd group was higher than that in the L-Cd group. In contrast, a significant decrease in the BW (birth weight):PW (placenta weight) ratio representing the placental nutrient transport function was found in the H-Cd group, and an inverse correlation between placental Cd concentration and BW:PW ratio was demonstrated. Additionally, significant elevations in the expression of cleaved caspase-3, cleaved lamin A/C proteins, and TUNEL were shown in the H-Cd placenta. Moreover, positive correlations were found between the placental Cd concentration and the expression of cleaved caspase-3 and TUNEL. Collectively, our findings suggest that the exposure of pregnant women to environmental Cd might induce Cd to be transferred to the body and then accumulated in the placenta, resulting in disturbance of the placental function and eventual apoptosis.

RAGE signaling antagonist suppresses mouse macrophage foam cell formation.

The engagement of the receptor for advanced glycation end-products (receptor for AGEs, RAGE) with diverse ligands could elicit chronic vascular inflammation, such as atherosclerosis. Binding of cytoplasmic tail RAGE (ctRAGE) to diaphanous-related formin 1 (Diaph1) is known to yield RAGE intracellular signal transduction and subsequent cellular responses. However, the effectiveness of an inhibitor of the ctRAGE/Diaph1 interaction in attenuating the development of atherosclerosis is unclear. In this study, using macrophages from Ager+/+ and Ager-/- mice, we validated the effects of an inhibitor on AGEs-RAGE-induced foam cell formation. The inhibitor significantly suppressed AGEs-RAGE-evoked Rac1 activity, cell invasion, and uptake of oxidized low-density lipoprotein, as well as AGEs-induced NF-κB activation and upregulation of proinflammatory gene expression. Moreover, expression of Il-10, an anti-inflammatory gene, was restored by this antagonist. These findings suggest that the RAGE-Diaph1 inhibitor could be a potential therapeutic drug against RAGE-related diseases, such as chronic inflammation and atherosclerosis.

An unusual topographic relationship between high origin of superficial ulnar artery and bitendinous palmaris longus.

The ulnar artery usually arises from the brachial artery at the cubital fossa. It descends beneath the flexor carpi ulnaris in the forearm and then terminates at the wrist joint by forming the superficial palmar arch with the contributory radial artery. In the present study, we report a more proximal origin of ulnar artery presenting a superficial course in the lower portion of the upper extremity, termed superficial ulnar artery. Interestingly, this artery lies superficially to a bitendinous palmaris longus, a variant muscle in the forearm. The relation between arterial and muscular variations may be useful during clinical procedures such as angiography, forearm flap and tendon grafting as well as avoiding accidental intra-arterial injection.

Anatomical Variation of the Auriculotemporal Nerve in Thai Cadavers / Variación Anatómica del Nervio Auriculotemporal en Cadáveres Tailandeses

SUMMARY: The auriculotemporal nerve (ATN) is an important structure lying within a limited area of an infratemporal region (ITR). The ATN is originated from the posterior branch of the mandibular division of the trigeminal nerve (V3). The ATN conveys somatosensory, secretomotor, and parasympathetic fibres of the V3 and gustatory nerve (CN IX). In literature, the most common pattern of the ATN is composed of 2 roots that encloses the middle meningeal artery (MMA). However, in many studies, it has been reported that there are many variations in ATN pattern formation. To study the variation of ATN pattern formation in Thai cadavers, 73 hemifaces from 39 Thai embalmed cadavers were dissected and the relations of the ATN to the MMA were recorded. This study concluded that there were 4 patterns observed in Thai cadavers. The common pattern is 2 roots (67.1 %), 3 roots (15.1 %), 1 root (9.6 %), and 4 roots (8.2 %). Knowledge of this variation in the ATN may be useful in understanding the symptoms of temporo-orofacial pain, paresthesia of temporomandibular joint (TMJ), possible side effects from the TMJ surgery and the efficiency of auriculotemporal nerve block for regional anesthesia of the temporomandibular joint in Thai people.

RESUMEN: El nervio auriculotemporal (NAT) es una estructura importante que se encuentra dentro de la región infratemporal (ITR). El NAT se origina en la rama posterior de la división mandibular del nervio trigémino (V3), y transmite fibras somato sensoriales, secreto motoras y parasimpáticas del V3 y del nervio gustativo (CN IX). En la literatura, se reporta que el patrón más común de NAT está compuesto por 2 raíces que contienen la arteria meníngea media (AMM). Sin embargo, en diversos estudios, se ha informado que existen múltiples variaciones en la for- mación de patrones NAT. Con el objetivo de estudiar la variación de la formación del patrón NAT en cadáveres tailandeses, se disecaron 73 estructuras hemi faciales de 39 cadáveres tailandeses y se registraron las relaciones del NAT con el AMM. En conclusión, se observaron 4 patrones en los cadáveres tailandeses. El patrón común de 2 raíces (67,1 %), 3 raíces (15,1 %), 1 raíz (9,6 %) y 4 raíces (8,2 %). El conocimiento de esta variación en el NAT puede ser útil para comprender los síntomas de dolor temporo-orofacial, parestesia de la articulación temporomandibular (ATM), posibles efectos secundarios de la cirugía de ATM y la eficacia del bloqueo del nervio auriculo-temporal para la anestesia regional de la articulación temporomandibular en Tailandeses.

ARID1A knockdown triggers epithelial-mesenchymal transition and carcinogenesis features of renal cells: role in renal cell carcinoma.

Down-regulation/mutation of AT-rich interactive domain 1A (ARID1A), a novel tumor suppressor gene, has been reported in various cancers. Nevertheless, its role in renal cell carcinoma (RCC) remained unclear and underinvestigated. We thus evaluated carcinogenesis effects of ARID1A knockdown in nonmalignant Madin-Darby canine kidney (MDCK) renal cells using small interfering RNA (siRNA) against ARID1A (siARID1A). The siARID1A-transfected cells had decreased cell death, increased cell proliferation, and cell cycle shift (from G0/G1 to G2/M) compared with those transfected with controlled siRNA (siControl). Additionally, the siARID1A-transfected cells exhibited epithelial-mesenchymal transition (EMT) shown by greater spindle index, increased mesenchymal markers (fibronectin/vimentin), and decreased epithelial markers (E-cadherin/zonula occludens-1). Moreover, the siARID1A-transfected cells had increases in migratory activity, nuclear size, self-aggregated multicellular spheroid size, invasion capability, chemoresistance (to docetaxel), Snail family transcriptional repressor 1 expression, and TGF-ß1 secretion. All of these siARID1A-knockdown effects on the carcinogenic features were reproducible in malignant RCC (786-O) cells, which exhibited a higher degree of carcinogenic phenotypes compared with the nonmalignant MDCK cells. Finally, immunohistochemistry showed obvious decrease in ARID1A protein expression in human RCC tissues (n = 23) compared with adjacent normal renal tissues (n = 23). These data indicate that ARID1A down-regulation triggers EMT and carcinogenesis features of renal cells in vitro, and its role in RCC could be proven in human tissues.-Somsuan, K., Peerapen, P., Boonmark, W., Plumworasawat, S., Samol, R., Sakulsak, N., Thongboonkerd, V. ARID1A knockdown triggers epithelial-mesenchymal transition and carcinogenesis features of renal cells: role in renal cell carcinoma.

Increased DMT-1 expression in placentas of women living in high-Cd-contaminated areas of Thailand.

Cadmium (Cd) is a toxic heavy metal and contamination was reported in soil and rice in several areas of Thailand. Humans are normally exposed to environmental Cd, leading to gradual Cd accumulation in their bodies, including the placenta. DMT-1 is a divalent metal transporter which is found in placental tissue and plays a vital role in the transportation of Fe2+ and Cd2+. This study investigated DMT-1 protein and mRNA expressions in full term human placentas comparing those from high-Cd-contaminated areas (high-Cd group) and low-Cd-contaminated areas (low-Cd group), n = 6 per group. The maternal blood Cd (B-Cd) and placental Cd (P-Cd) of the high-Cd group was significantly raised in comparison with those in the low-Cd group. DMT-1 in the fetal portion of the placentas was localized in the apical and basal portions of the cytoplasm of the syncytiotrophoblastic cells, the endothelium of fetal capillaries which is functional structure of the placental barrier, and was also found in the cytoplasm of Hofbauer cells. Moreover, DMT-1 localization in the maternal portion was also detected in most decidual cells. In addition, the DMT-1 protein and mRNA expressions in the high-Cd group were significantly higher than those in the low-Cd group. Therefore, we suggest that pregnant women, who are exposed to environmental Cd, show an increased level of Cd in their maternal blood and this Cd can accumulate in the placenta. Intracellular Cd may induce DMT-1 mRNA transcription which further translates into DMT-1 protein, which can then function as a reciprocal Cd transporter in placental tissue.

Increasing CACNA1C expression in placenta containing high Cd level: an implication of Cd toxicity.

Cadmium (Cd) has known to produce many adverse effects on organs including placenta. Many essential transporters are involved in Cd transport pathways such as DMT-1, ZIP as well as L-VDCC. Fourteen pregnant women participated and were divided into two groups: high and low Cd-exposed (H-Cd, L-Cd) groups on the basis of their residential areas, Cd concentrations in the blood (B-Cd), urine (U-Cd), and placenta (P-Cd). The results showed that the B-Cd and U-Cd were significantly increased in H-Cd group (p < 0.05). Interestingly, the P-Cd in H-Cd group was elevated (p < 0.05) and positively related to their B-Cd and U-Cd values (p < 0.05). However, the mean cord blood Cd (C-Cd) concentration in H-Cd group was not significantly increased about 2.5-fold when comparing to L-Cd group. To determine the Cd accumulation in placental tissues, metallothionein-1A (MT-1A) and metallothionein-2A (MT-2A) expressions were used as biomarkers. The results revealed that mean MT-1A and MT-2A mRNAs and MT-1/2 proteins were up-regulated in H-Cd group (p < 0.05). In addition, the Ca channel alpha 1C (CACNA1C) mRNA and protein expressions were noticeably elevated in H-Cd group (p < 0.05). From these findings, we suggested that CACNA1C might be implicated in Cd transport in human placenta.

Expression and localization of calpain 3 in the submandibular gland of mice.

OBJECTIVES: Calpains comprise a family of intracellular Ca2+-dependent cysteine proteases and are considered to play roles in various physiological phenomena with limited proteolytic activities against specific substrates. We herein revealed the expression and localization of calpain 3, the muscle-type calpain, in the submandibular gland (SMG) of mice. DESIGN: The expression of the mRNA for conventional, ubiquitous calpains 1 and 2 and skeletal muscle-specific calpain 3 was examined in the major salivary glands of mice using RT-PCR, and the expression and localization of calpain 3 protein was examined in the SMG of mice using immunohistochemistry and Western blotting. RESULTS: The large catalytic subunits of calpains 1 and 2 and the small regulatory subunit common to calpains 1 and 2 were weakly expressed in the parotid gland, sublingual gland, and SMG at similar levels in males and females. In contrast, the single large catalytic subunit of calpain 3 was expressed predominantly in the SMG at markedly higher levels in males than in females and in a manner dependent on androgens. Immunoreactivity for calpain 3 was mainly localized in cells of the granular convoluted tubules (GCT) that developed preferentially in the male SMG. In GCT cells, calpain 3 immunoreactivity was localized predominantly in the cytosolic region and was absent in the secretory granules. CONCLUSIONS: These results revealed that the GCT is the primary site of production of calpain 3 in the mouse SMG.

Myeloid Differentiation Factor 88 Signaling in Bone Marrow-Derived Cells Promotes Gastric Tumorigenesis by Generation of Inflammatory Microenvironment.

It has been established that COX-2 and downstream signaling by prostaglandin E2 (PGE2) play a key role in tumorigenesis through generation of inflammatory microenvironment. Toll-like receptor (TLR) signaling through myeloid differentiation factor 88 (MyD88) also regulates inflammatory responses in tumors. However, the relationship between these distinct pathways in tumorigenesis is not yet fully understood. We herein investigated the role of MyD88 in gastric tumorigenesis using Gan mice, which develop inflammation-associated gastric tumors due to the simultaneous activation of the COX-2/PGE2 pathway and Wnt signaling. Notably, the disruption of Myd88 in Gan mice resulted in the significant suppression of gastric tumorigenesis with the inhibition of inflammatory responses, even though COX-2/PGE2 pathway is constitutively activated. Moreover, Myd88 disruption in bone marrow-derived cells (BMDCs) in Gan mice also suppressed inflammation and tumorigenesis, indicating that MyD88 signaling in BMDCs regulates the inflammatory microenvironment. We also found that expression of Tlr2 and its coreceptor Cd14 was induced in tumor epithelial cells in Gan mice, which was suppressed by the disruption of Myd88. It has already been shown that TLR2/CD14 signaling is important for stemness of intestinal epithelial cells. These results indicate that MyD88 in BMDCs, together with COX-2/PGE2 pathway, plays an essential role in the generation of the inflammatory microenvironment, which may promote tumorigenesis through induction of TLR2/CD14 pathway in tumor epithelial cells. These results suggest that inhibition of TLR/MyD88 signaling together with COX-2/PGE2 pathway will be an effective preventive strategy for gastric cancer.

Immunohistochemical localization of keratin 5 in the submandibular gland in adult and postnatal developing mice.

Keratin 5 (K5) is a marker of basal progenitor cells in the epithelia of a number of organs. During prenatal development of the submandibular gland (SMG) in mice, K5(+) progenitor cells in the developing epithelia play important roles in its organogenesis. Although K5(+) cells are also present in the adult mouse SMG and may function in tissue regeneration, their histological localization has not yet investigated in detail. In the present study, we examined the immunohistochemical localization of K5 in the SMG in adult and postnatal developing mice. At birth, K5 immunoreactivity was detected in the entire duct system, in which it was localized in the basal cells of a double-layered epithelium, but was not detected in the terminal tubule or myoepithelial cells. At postnatal weeks 1-3, with the development of intercalated ducts (ID), striated ducts (SD), and excretory ducts (ED), K5-immunoreactive basal cells were gradually restricted to the ED and the proximal double-layered portions of the ID connecting to the SD. At the same time, K5 immunoreactivity appeared in myoepithelial cells, in which its positive ratio gradually increased. In adults, K5 immunoreactivity was localized to most myoepithelial cells, most basal cells in the ED, and a small number of ID cells at the boundary between the ID and SD in the female SMG or between the ID and granular convoluted tubules in the male SMG. These results suggest that K5 is a marker of differentiated myoepithelial cells and duct progenitor cells in the mouse SMG.

Accessory hepatic artery arising from celiac trunk: an incidence in a Thai cadaver / Arteria hepática accesoria derivada del tronco celiaco: hallazgo en un cadaver tailandés

An exhaustive knowledge of the liver vascular patterns as well as possible anatomical variations is significant in the planning and performance of all liver surgical procedures in order for the vascularity not to be disturbed or not causing necrosis of the liver parenchyma postoperatively. The celiac trunk usually provides three branches; left gastric, splenic and common hepatic arteries. The left and right hepatic arteries generally derive from proper hepatic artery which is a branch of common hepatic artery. To study the incidence of celiac trunk ramification, the branching patterns of the celiac trunk of 23 Thai cadavers (17 males, 6 females) were documented during routine dissection by medical students at the Department of Anatomy, Faculty of Medical Science, Naresuan University, Thailand. The clinically important variations of the celiac trunk were noted. The results showed that all celiac trunks arose from each aortas at the T12 vertebra (17.39%, 4 cases), intervertebral disc between T12 and L1 vertebra (78.26%, 18 cases) and upper 1/3rd of L1 vertebra (4.35%, 1 case). We found 95.65% (22 cases) normal celiac trunk trifurcation; whereas, 4.35% (1 case) was abnormal quadrifurcation of the trunk. The accessory hepatic artery (aHA) was presented as an additional branch of celiac trunk because the conventional pattern of the left and right hepatic arteries was presented. This finding is one of the rare anatomical variations which is reported in available literatures. The awareness of celiac trunk and its stems aberrant is important in procedures such as liver transplant for appropriate vascular ligation and anastomosis.

Un conocimiento exhaustivo de los patrones vasculares del hígado, así como sus posibles variaciones anatómicas son importantes en la planificación y realización de todos los procedimientos quirúrgicos hepáticos para evitar comprometer la vascularización y posible necrosis del parénquima después de la cirugía. El tronco celíaco, por lo general, proporciona tres ramas: gástrica izquierda, esplénica y arteria hepática común. Las arterias hepáticas izquierda y derecha en general derivan de la arteria hepática propia, que es una rama de la arteria hepática común. El objetivo de este trabajo fue estudiar la incidencia de distribución del tronco celíaco mediante la documentación de patrones de ramificación en 23 cadáveres de Tailandia (17 hombres y 6 mujeres). El estudio se efectuó durante la disección de rutina realizada por los estudiantes de medicina en el Departamento de Anatomía de la Facultad de Ciencias Médicas, Universidad de Naresuan, Tailandia. Se observaron las variaciones clínicamente importantes del tronco celíaco. Los resultados mostraron que todos los troncos celíacos surgieron desde la aorta a nivel de la vértebra T12 (17,39%, 4 casos), a nivel del disco intervertebral entre T12 y L1 vértebra (78,26%, 18 casos) y a nivel del tercio superior de la vértebra L1 (4,35%, 1 caso). Encontramos un 95,65% (22 casos) de troncos celíacos normales, es decir, con trifurcación; mientras que un 4,35% (1 caso) era anormal, con 4 ramos terminales. La arteria hepática accesoria (AHA) se presentó como una rama accesoria del tronco celíaco, ya que existía un patrón convencional de las arterias hepáticas izquierda y derecha. Este hallazgo representa una de las raras variaciones anatómicas informada en la literatura. El conocimiento del tronco celíaco y sus ramas aberrantes son importantes en procedimientos como el trasplante hepático, la anastomosis y una ligadura vascular adecuada.

Increasing of syncytial knot and fibrinoid deposit in high-Cd accumulated human placentas / Aumento del nodo sincicial y depósitos de fibrinoide en placenta humanas con alta acumulación de cadmio

A toxic metal, cadmium (Cd), can accumulate in human organs. Placenta is usually used as indicator organ for Cd exposure. Therefore, we aim to investigate the different of placental morphology between the low- and high-Cd accumulated placentas. The samples were collected from 14 pregnant women who resided in low-Cd contaminated (L-Cd group) and high-Cd contaminated (H-Cd group) areas. The concentrations of Cd in blood (B-Cd), urine (U-Cd) and placentas (P-Cd) were measured by ICP-MS and AAS. The morphological appearance of placentas was examined by using routine paraffin section and H & E staining. The results showed that levels of B-Cd, U-Cd and P-Cd were significantly higher in H-Cd group than in L-Cd group (p= 0.001). Moreover, the B-Cd was positively correlated with U-Cd (rs= 0.823, p= 0.000) and P-Cd concentrations (rs= 0.854, p= 0.000). The appearances of syncytial knot (STK) and fibrinoid deposit (Fd) were obviously greater in H-Cd group than in L-Cd group (p= 0.007, p= 0.026). The STK was positively correlated with both Fd (rs= 0.572, p= 0.032) and P-Cd concentration (rs= 0.766, p= 0.001). Although the chorioamnitis and decidual inflammation features were found in both groups but the appearance in H-Cd group seems to be more severe than in L-Cd group. From these results, we suggested that high Cd level in placenta may be involved in morphological changes, especially STK and Fd increasing and probably disturb the connection between maternal and fetal circulation.

Un metal tóxico, el cadmio (Cd), se puede acumular en órganos humanos. La placenta se utiliza, por lo general, como órgano indicador de la exposición a Cd. Nuestro objetivo fue investigar la diferente morfología placentaria entre las placentas con baja y alta acumulación de Cd. Las muestras fueron recolectadas de 14 mujeres embarazadas que residían áreas con alta (grupo H-Cd) y baja contaminación por Cd (grupo L-Cd). Las concentraciones de Cd en la sangre (B- Cd), orina (U-Cd) y placentas (P-Cd) se midieron por ICP-MS y AAS. La apariencia morfológica de las placentas fue examinada usando cortes histológicos teñidos con H-E. Los resultados mostraron que los niveles de B-Cd, U-Cd y P-Cd fueron significativamente mayores en el grupo H-Cd (p= 0,001). Por otra parte, el B-Cd se correlacionó positivamente con las concentraciones de U-Cd (rs= 0,854, p = 0,000 ) y P-Cd (rs= 0,823, p = 0,000). Las apariciones de nodos sinciciales (NS) y depósitos fibrinoides (Fd) fueron mayores en el grupo H-Cd (rs= 0,007, p= 0,026). Los ND se correlacionaron positivamente con los Fd (rs= 0,572, p= 0,032) y la concentración de P-Cd (rs= 0,766, p = 0,001). Aunque características de corioamnitis e inflamación de la decidua se encontraron en ambos grupos, su aparición en el grupo H-Cd pareció ser más grave que en el grupo L-Cd. A partir de estos resultados, sugerimos que el nivel alto de Cd en la placenta puede estar involucrado en los cambios morfológicos, especialmente el aumento de NS y Fd, los que probablemente alteran la relación entre la circulación materna y fetal.

Metallothionein: an overview on its metal homeostatic regulation in mammals / Metalotioneina: una visión general de su regulación homeostática de metales en mamíferos

Metallothionein (MT) is a ubiquitous protein with a low molecular weight of 6-7 kDa weight and it was first identified in the kidney cortex of equines as a cadmium (Cd)-binding protein responsible for the natural accumulation of Cd in the tissue. The mammalian MT contains 61 to 68 amino acid residues, in which 18 to 23 cysteine residues are present. The expression of MT starts by binding of metal transcription factor-1 (MTF-1) to the regulative region of MT gene called metal responsive elements (MREs). The induction of MT through the MREs region can be initiated by several metal ions such as zinc (Zn), copper (Cu) and Cd. However, Zn is the only heavy metal which can reversibly and directly activate the DNA-binding activity of MTF-1. In mammals four types of MT are expressed and they are termed metallothionein-1 (MT1), metallothionein-2 (MT2), metallothionein-3 (MT3), and metallothionein-4 (MT4). MT1 and MT2 are expressed in almost all tissues while MT3 and MT4 are tissue-specific. MT is a key compound involved in the intracellular handling of a variety of essential and nonessential post-transitional metal ions. In order to the heavy metal binding ability of MT, it is suggested to play roles both in the intracellular fixation of essential trace elements Zn and Cu, in controlling the concentrations, and in neutralizing the harmful influences of exposure to toxic elements...

Metalotioneina (MT) es una proteína, con bajo peso molecular de kDa 6-7 y que fue primero identificada en la corteza renal de equinos como cadmio (Cd)-proteína responsable por la acumulación natural de Cd en los tejidos. La MT en mamíferos contiene 61 a 68 residuos de aminoácidos, de los cuales están presentes 18 a 23 residuos de cisteína. La expresión de MT se inicia por la unión del factor-1 de transcripción de metal (MTF-1) a la región reguladora del gen de la MT llamado elementos metálicos responsable (MREs). La inducción de MT a través de la región MREs puede ser iniciada por varios iones metálicos tales como zinc (Zn), cobre (Cu) y Cd. Sin embargo, el Zn es el único metal pesado que puede revertir y activar directamente la unión ADN de MTF-1. En los mamíferos se expresan cuatro tipos de MT y ellos se denominan metalotioneína-1 (MT1), metalotioneína-2 (MT2), metalotioneína-3 (MT3), y metalotioneína-4 (MT4). MT1 y MT2 se expresan en casi todos los tejidos mientras que MT3 y MT4 son tejido-específico. La MT es un compuesto clave implicado en la manipulación intracelular de una variedad de iones metálicos esenciales y no esenciales post-transicionales. Con el fin de evaluar la capacidad de unión de metales pesados de MT, se sugiere que éste desempeña ambos roles tanto en la fijación intracelular de trazas de elementos de Zn y Cu como en el control de las concentraciones, y neutralizando las influencias perjudiciales a la exposición de elementos tóxicos...

Expression of cAMP response element-binding protein in the duct system of the mouse submandibular gland.

The submandibular gland (SMG) of mice shows a marked sexual dimorphism in which a duct portion called the granular convoluted tubule (GCT) is developed preferentially in males during puberty. The administration of testosterone to female mice causes the conversion of striated duct (SD) cells into GCT cells, but the underlying molecular mechanisms are unclear. Cyclic AMP response element-binding protein (CREB) is a transcription factor functioning downstream of a variety of signal transduction pathways. In the present study, we examined the expression, activation and cellular localization of CREB in the mouse SMG using Western blotting and immunohistochemistry. Both total CREB (t-CREB) and phosphorylated CREB (p-CREB) were significantly more abundant in the female than in the male gland and were localized to the nuclei of intercalated duct cells and a subpopulation of SD cells. In contrast, the GCT cells in males were negative for t- and p-CREB. The levels of CREB in the SMG were increased by castration in males and decreased by repeated administration of testosterone to females or castrated males. From 3 h after a single administration of testosterone to females, many SD cells temporarily gained nuclear immunoreactivity for both t- and p-CREB, which was lost as the cells were converted to GCT cells by 24 h. These results suggest the involvement of CREB in the androgen-dependent differentiation of the duct system in the mouse SMG.

Statin-induced bone morphogenetic protein (BMP) 2 expression during bone regeneration: an immunohistochemical study.

OBJECTIVES: The purpose of this study was to investigate bone morphogenetic protein (BMP) 2 expression after implantation of a statin and recombinant human BMP-2 (rhBMP-2) and to compare the bone regeneration capability of these substances in the rabbit nasal bone using immunohistologic methods. STUDY DESIGN: Twelve adult male Japanese white rabbits (n = 12; age 12-16 weeks, weight 2.5-3.0 kg) were divided into 3 experimental groups and 1 control group. A total of 48 bone defects, 4 per rabbit, were created in the nasal bone while preserving the nasal membrane. In the experimental groups, 1 group was implanted with 10 mg of a statin dissolved in 0.2 mL water with an atelocollagen sponge (ACS); the second group was implanted with 5 microg rhBMP-2 with an ACS; and in the third group only the ACS was implanted. No material was implanted in the control group. Animals were killed at 1, 2, and 4 weeks after surgery. The parts that had been operated on were removed and prepared for histologic assessment. The expression of BMP-2 was evaluated using immunohistochemistry, and double-immunostaining for BMP-2 and Ki-67 was observed by fluorescent microscopy. RESULTS: No significant differences were observed between the statin/ACS group and rhBMP-2/ACS group at 1, 2, and 4 weeks after surgery. The number of cells which stained positively for BMP-2 increased significantly in both of the implanted groups compared with the control group (P < .0001). The positive fluorescent double-immunostaining for BMP-2 and Ki-67 was similar in both implanted groups. CONCLUSION: This study suggests that statin/ACS implants show BMP-2 expression and osteoinductive activity that is similar to those of rhBMP-2/ACS implants.

Coexpression of menin and JunD during the duct cell differentiation in mouse submandibular gland.

In the submandibular gland (SMG) of mice, a duct portion called the granular convoluted tubule (GCT) is developed preferentially in males with puberty. This sexual dimorphism is androgen-dependent, but the underlying molecular mechanisms are unclear. We have demonstrated that the expression of a transcription factor JunD is regulated in association with the androgen-induced differentiation of GCT cells from striated duct (SD) cells. Menin, a nuclear protein encoded by the MEN1 tumor-suppressor gene, is known to bind JunD, thereby inhibiting its activity. In the present study, we examined the expression of menin in the mouse SMG by use of Northern blotting, Western blotting, and immunohistochemistry. Immunoreactivity for menin was higher in the female than male gland, and localized to the nuclei of intercalated duct cells and a subpopulation of SD cells. In contrast, GCT cells in males appeared negative for menin. The levels of menin in the SMG were increased with castration in males and decreased by repeated administration of testosterone to females or to castrated males. After a single administration of testosterone to females, many SD cells newly gained nuclear menin, which was lost as the cells converted to GCT cells by 48 hrs. These patterns of the expression and localization of menin were quite similar to those of JunD. Furthermore, the coimmunoprecipitation analysis of the SMG homogenates indicated that menin binds JunD in vivo. The present study suggests that the JunD-menin complex plays significant roles in the androgen-dependent differentiation of the duct system in the mouse SMG.

A novel mouse protein differentially regulated by androgens in the submandibular and lacrimal glands.

We characterized a cDNA clone derived from the female mouse submandibular gland (SMG). The transcript of this cDNA was approximately 1.2kb in size and predicted to code a 165-amino acid protein with a putative signal peptide for a secretory pathway. This protein, named submandibular androgen-repressed protein (SMARP), had homology in the N-terminal region with members of the glutamine/glutamic acid-rich protein (GRP) family from rats. Northern blot analysis revealed that SMARP mRNA is expressed, out of the major mouse organs, only in the SMG and exorbital lacrimal gland (LG), with much more abundance in the former. For the SMG, the level of SMARP mRNA was 36 times higher in females than males, whereas for the LG it was 28 times higher in males than females. Furthermore, the level of SMARP mRNA was increased in the SMG but reduced in the LG with castration in males, whereas it was reduced in SMG but increased in LG after administration of testosterone in females or castrated males. In situ hybridization detected the signal for SMARP mRNA in the female SMG, and immunohistochemistry detected the signal for SMARP protein in the female SMG and male LG. In the female SMG, SMARP mRNA, and protein were localized intensively in a subpopulation of acinar cells, whereas in the male LG, SMARP protein was distributed diffusely in all acinar cells. These results suggested that SMARP is a secretory protein whose expression is regulated by androgens negatively in the SMG and positively in the LG.

Cloning and characterization of a novel animal lectin expressed in the rat sublingual gland.

We cloned a rat gene that is expressed primarily in the sublingual gland and named the predicted 503 amino-acid protein SLAMP (sublingual acinar membrane protein). SLAMP has 63% homology with human ERGIC-53-like protein, a member of the family of animal L-type lectins. Using a cDNA probe for SLAMP mRNA and rabbit antisera against SLAMP, we examined the expression and localization of SLAMP in major rat organs and tissues. With both Northern and Western blot analyses, abundant expression of SLAMP was demonstrated predominantly in the sublingual gland, with single sizes of the mRNA and protein 1.8 kb and 50 kDa, respectively, but not in other organs or tissues, including the parotid and submandibular glands. With immunohistochemistry, SLAMP was localized to the mucous acinar cells, but not to the serous demilunes or the duct system. With immunoelectron microscopy, SLAMP was localized predominantly to regions corresponding to the ER-Golgi intermediate compartment. Besides the sublingual gland, SLAMP immunoreactivity was also demonstrated in mucous cells of the minor salivary glands in oral cavity and of Brunner's glands in the duodenum. These results suggested that rat SLAMP plays a specific role in the early secretory pathway of glycoproteins in specific types of mucous cells.