Ischemia impairs the association between connexin 43 and M3 subtype of acetylcholine muscarinic receptor (M3-mAChR) in ventricular myocytes.

We used Western blot analysis to examine the expression of connexin 43 and M2/M3 acetylcholine muscarinic receptors (mAChR) and their interaction in ventricular myocytes from control and the ischemic heart. We confirmed that the connexin 43 and M2/ M3-mAChR were expressed in ventricular myocytes. Moreover, we showed that M3-mAChR was expressed in non-glycosylated (72 kDa) and glycosylated forms (115 kDa). Immunostaining showed that connexin 43 is closely associated with M3-mAChR in parts of cell membranes of myocytes. Immunoprecipitation of lysate of cardiac myocytes with M2/M3-mAChR antibody pulled down a 44 kDa protein recognized by connexin 43 antibody. Ischemia increased the expression of M3-mAChR in myocytes. The ischemiainduced increase in the M3-mAChR expression was specific because ischemia did not affect the expression of M1, M2, M4 and M5- mAChR in the heart. On the other hand, ischemia decreased the expression of connexin 43 in myocardium. We also examined the effect of ischemia on the interaction between M2/M3-mAChR and connexin 43. Ischemia suppressed the association of M3-mAChR with connexin 43 but did not affect the association of connexin 43 with M2-mAChR. Administration of choline before ischemia not only partially restored the expression of connexin 43 but also attenuated the ischemia-induced suppression of the association between connexin 43 and M3-mAChR. We conclude that connexin 43 interacts with M2/M3-mAChR and that ischemia specifically impairs the association between M3-mAChR and connexin 43.     

Cloned endangered species takin (Budorcas taxicolor) by inter-species nuclear transfer and comparison of the blastocyst development with yak (Bos grunniens) and bovine

Interspecies cloning might be used as an effective method to conserve endangered species and to support the study of nuclear-cytoplasm interaction. In this study, we describe the development of takin-bovine embryos in vitro produced by fusing takin ear fibroblasts with enucleated bovine oocytes and examine the fate of mitochondrial DNA in these embryos. We also compare the blastocyst development of takin-bovine embryos with yak-bovine and bovine-bovine embryos and compare the cell numbers of the blastocyst. Our results indicate that: (1) takin-bovine cloned embryos can develop to the blastocyst stage in vitro (5%), (2) blastocyst mitochondria DNA are derived primarily from bovine oocytes in spite of a little takin donor cell mitochondrial DNA, (3) using the same cloned protocol, development efficiency is significantly different between bovine-bovine cloning, yak-bovine, and takin-bovine cloning (48 vs. 28% vs. 5%, P < 0.01), and (4) cell numbers in the blastocysts of the three species of embryos were not different. These results suggest that the bovine oocytes can reprogram the takin, yak, and bovine fibroblast nuclei. However, the development efficiency of intra-species cloning tends to be higher than inter-species cloning; the more close the species of the donor cell is to the recipient oocyte (yak versus takin), the greater the blastocyst development in vitro.     

Characterization of the structures involved in localization of the SUN proteins to the nuclear envelope and the centrosome

The nuclear envelope forms a selective barrier that separates the cytoplasm from the nucleus. During mitosis the nuclear envelope breaks down so that the microtubule network can form contacts with the kinetochore and guide chromosome segregation. Previous studies have suggested a model in which the centrosome and the microtubule network may play a role in nuclear envelope breakdown through as yet unidentified interactions with proteins localized to the nuclear envelope. In the current study we characterized a nuclear envelope protein SUN2 and identified a substructure involved in its localization to the nuclear envelope. We found that a structurally related protein, SUN1, may be localized to the nuclear envelope through a different mechanism. Furthermore, the SUN2 protein can form different assemblies, including homodimers and heterodimers with SUN1. Finally, we provide evidence indicating that SUN1 and SUN2 may form a physical interaction between the nuclear envelope and the centrosome.     

First record of the raccoon dog (Nyctereutes procyonoides Gray, 1834) in the former Yugoslav Republic of Macedonia

An adult male raccoon dog (Nyctereutes procyonoides Gray, 1834) run over by a car was found on September 6, 2002, in the central part of the Former Yugoslav Republic of Macedonia (UTM: EM 54). This is the first record of this species on the territory of the FYRM, and the southernmost point of its European range so far. This finding indicates a range extension of the raccoon dog on the Balkan Peninsula.     

Cell detachment model for an antibody-based microfluidic cancer screening system

We consider cells bound to the floor of a microfluidic channel and present a model of their flow-induced detachment. We approximate hydrodynamic force and cell elastic response using static finite-element simulation of a single cell. Detachment is assumed to occur when hydrodynamic and adhesive forces are roughly equal. The result is extended to multiple cells at the device level using a sigmoidal curve fit. The model is applied to a microfluidic cancer-screening device that discriminates between normal epithelial cells and cells infected with human papillomavirus (HPV), on the basis of increased expression of the transmembrane protein alpha6 integrin in the latter. Here, the cells to be tested are bound to a microchannel floor coated with anti alpha6 integrin antibodies. In an appropriate flow rate range, normal cells are washed away while HPV-infected cells remain bound. The model allows interpolation between data points to choose the optimal flow rate and provides insight into interaction of cell mechanical properties and the flow-induced detachment mechanism. Notably, the results suggest a significant influence of cell elastic response on detachment.     

3' end cDNA amplification using classic RACE

Having knowledge of the entire 3' sequence of a cDNA is often important because the non-coding terminal region can contain signals that regulate the stability or subcellular localization of the mRNA. Also, some messages use alternative genomic sites for cleavage and polyadenylation that can alter the above properties, or change the encoded protein. Full-length cDNAs can be obtained from complex mixtures of cellular mRNA using rapid amplification of cDNA ends (RACE) PCR as long as part of the mRNA sequence is known; adding non-specific tags to the ends of the cDNA allows the regions between the known parts of the sequence and the ends to be amplified. In 3' RACE, the poly(A) tail functions as a non-specific tag at the 3' end of the mRNA. cDNA ends can be obtained in 1-3 days using this protocol.     

Exchange of Proton and Major Elements in Two-Layer Canopies Under Acid Rain in a Subtropical Evergreen Forest in Central-South China

Canopy exchanges of H^＋ and N （NH4^＋-N, NO3^--N） and other major ions were evaluated and quantified In twolayer canopies based on throughfall measurements in Shaoshan Forest during the period 2000-2002, central-south China, The collected annual rainfall, throughfall, and sub-throughfall were 1 401, 1 191, and 1 084 mm/year, respectively. Fifteen percent and 8% of rainfall （or 9% of throughfall） were intercepted by the top canopy and sub-canopy layers, respectively, The foliar leaching of base cations from the top canopy was significantly higher than that from the sub-canopy, and the latter accounted for 25% of the former. The uptake of H^＋ and NH4^＋ was significantly higher in the top canopy than in the sub-canopy, indicating that the canopy buffering capacity in the top canopy was stronger than the sub-canopy; Mg^2＋ can be absorbed from water flux on the sub-canopy foliar surfaces to compensate for the Mg deficit in the forest soil during the growing season,     

Store-operated Ca2+ channel in renal microcirculation and glomeruli

Store-operated Ca2+ channel (SOC) is defined as a channel that opens in response to depletion of the internal Ca2+ stores. During the last decade, many investigators have made a great effort to identify and characterize SOC, and to evaluate its physiologic function and pathophysiologic relevance in a variety of cell lines, primary cultures, and native tissues. To date, accumulating evidence has demonstrated that SOC is an essential Ca2+ entry mechanism in vascular smooth-muscle cells of renal microvasculature and glomerular mesangial cells, both of which tightly control glomerular hemodynamics and filtration. Store-operated Ca2+, combined with other types of Ca2+ entry channels, constitutes a profile of Ca2+ changes in response to physiologic vasoconstrictors and, thereby, regulates renal microcirculation and mesangial function. In addition, SOC is associated with altered Ca2+ signaling occurring in diseased kidneys, such as diabetic nephropathy. Although the gating mechanism and molecular identity of SOC are still enigmatic and may be cell-type and tissue specific, data from several independent groups suggest that protein kinase C plays an important role in SOC activation and that certain isoforms of canonical transient receptor potential (TRPC) proteins are candidates of SOC in renal microvessels and mesangial cells.     

Successful low dose insemination of flow cytometrically sorted Sika (Cervus nippon) sperm in Wapiti (Cervus elaphus)

The purpose of this study was to determine a practical method in Wapiti (Cervus elaphus) of using predetermined sexed Sika (Cervus nippon) semen. Semen was collected by electro-ejaculation from one stag of proven fertility and transported to the laboratory where it was retained as unsorted (control) or was separated into X- and Y-chromosome-bearing sperm using a modified high-speed cell sorter. Wapiti hinds (n = 81) were inseminated into the uterus by rectum manipulation with 1 × 10⁶ (X1 and Y1 group, respectively) or 2 × 10⁶ (X2 and Y2 group, respectively) of sorted frozen-thawed and 1 × 10⁷ non-sorted frozen-thawed (a commercial dose control) Sika motile sperm 60–66 h after removal of intra-vaginal progesterone-impregnated CIDR devices and administration of 700 IU of PMSG at the time of CIDR removal. The percentage of hinds calving after insemination was similar for X1 (38.5%), X2 (41.7%), Y1 (44.4%), Y2 (38.9%) groups (P > 0.05), but higher for control (75%) treatment (P < 0.05). Ultimately 15 out of the 16 Sika and Wapiti-hybrid calves produced by Wapiti hinds inseminated with Y-sorted sperm were male (93.7%) and 10/10 (100%) Sika and Wapiti-hybrid calves from hinds inseminated with X-sorted sperm were female. The sex ratio of the Sika and Wapiti-hybrid calves born to hinds inseminated with sex-sorted sperm deviated significantly (P < 0.05) from 50% and 50.0% in the control group. All Sika and Wapiti-hybrid calves were born between 237 and 250 d of gestation. Male and female calves in the control group had similar birth weights and weaning weights as calves from hinds inseminated with X- or Y-sorted sperm. In conclusion it can be said that normal Sika and Wapiti-hybrid calves of predicted sex can be produced after artificial insemination of Wapiti does with low numbers of sex-sorted cryopreserved Sika sperm.