以酶标免疫吸附法测定大鼠肝脏精氨酸酶含量

本文利用丙酮沉淀、凝胶过滤、离子交换层析等方法提纯了大鼠肝脏精氨酸酶,在SDS-PAGE中表现为单一的蛋白质带,其亚基分子量为37,000.建立了大鼠肝精氨酸酶的酶标免疫吸附测定法(ELISA),比较了两种常规的精氨酸酶活性测定法与ELISA法,并初步探讨了ELISA法在精氨酸酶测定方面的一些应用。     

Spectral Properties of Acid Denaturation of Rat Liver Arginase

Circular dichroism spectra in the ultraviolet region, similar to the spectrum of the collagen helix, were regenerated when various grades of industrial gelatin were allowed to stand at 5°C in a concentration low enough that they did not form gels. Spectrum generation was decreased by an addition of salt or alcohol and by raising the temperature. Maximal regeneration took place at the respective iso-ionic points of the acid- and alkali-processed gelatins. As judged from these spectra, 60% of the helical structure of collagen was regenerated from the industrial gelatin that had the highest α-chain content.     

Isolation and Biochemical Characterization of Peroxisomes from Cultured Rat Glial Cells

Peroxisomes are now recognized to play important cellular functions and its dysfunction leads to a group of neurological disorders. This study reports peroxisomal enzyme activities in cultured glial cells and peroxisomes isolated from cultured oligodendrocytes and C₆ glial cells. Peroxisomal enzyme activities were found to be higher in oligodendroglial cells than in astrocytes or mixed glial cells. We also developed a method for the isolation of peroxisomes from glial cells by a combination of differential and density gradient centrifugation techniques. Peroxisomes from oligodendrocytes in nycodenz gradient were isolated at a density of 1.165 g/ml ± 0.011. Activities of dihydroxyacetone phosphate acyl transferase, -oxidation of lignoceric acid and -oxidation of phytanic acid were almost exclusively associated with the distribution of catalase activity (a marker enzyme for peroxisomes) in the gradient. This protocol should be a resource for studies designed to investigate the structure and function of peroxisomes in brain cells.     

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.     

Characterization of Chromatin Subfractions Enriched in RNA from Lactating Mammary Gland of the Rat

Mammary gland chromatin from lactating rats was fractionated according to the Mg²⁺-solubility method after mild digestion with DNase II. The chromatin properties were compared between the Mg²⁺-soluble (S₂) and the Mg²⁺-insoluble (P₂) fractions. The weight ratio of RNA to DNA was much greater in the S₂ fraction than in the P₂ fraction (S₂, 0.92; P₂, 0.03). The DNA repeat length of the nucleosome was very similar between the two fractions (S₂, 193 ± 7; P₂, 1% ± 8 nucleotide pairs). A significant difference was seen in electrophoretic pattern of H1 histone between the two fractions in the acid-urea gel system. Nonhistone proteins with molecular weights higher than about 40,000 dalton were found to be enriched in the S₂ fraction.     

Isolation of Three Novel Rat and Mouse Papillomaviruses and Their Genomic Characterization

Despite a growing knowledge about the biological diversity of papillomaviruses (PV), only little is known about non-human PV in general and about PV mice models in particular. We cloned and sequenced the complete genomes of two novel PV types from the Norway rat (Rattus norvegicus; RnPV2) and the wood mouse (Apodemus sylvaticus; AsPV1) as well as a novel variant of the recently described MmuPV1 (originally designated as MusPV) from a house mouse (Mus musculus; MmuPV1 variant). In addition, we conducted phylogenetic analyses using a systematically representative set of 79 PV types, including the novel sequences. As inferred from concatenated amino acid sequences of six proteins, MmuPV1 variant and AsPV1 nested within the Beta+Xi-PV super taxon as members of the Pi-PV. RnPV2 is a member of the Iota-PV that has a distant phylogenetic position from Pi-PV. The phylogenetic results support a complex scenario of PV diversification driven by different evolutionary forces including co-divergence with hosts and adaptive radiations to new environments. PV types particularly isolated from mice and rats are the basis for new animal models, which are valuable to study PV induced tumors and new treatment options.     

Isolation, Characterization, and Synthesis of AlphaFetoprotein from Neonatal Rat Brain

Monospecific anti-rat serum alpha-fetoprotein (AFP) IgG was coupled to cyanogen bromide-activated Sepharose-4B (4.5 mg/ml packed volume of gel) to yield an immunoaffinity matrix. The immunoaffinity column was used to isolate AFP from feto-neonatal rat brain. The purified AFP was immunologically and electrophoretically similar to serum AFP. It yielded a single band with a molecular weight of 70,000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the protein under nondenaturing conditions yielded two charge variants of AFP, reminiscent of AFP from feto-neonatal rat serum. The AFP was observed to bind estradiol with Ka = 5.8 X 10(8) M -1 and 1.3 X 10(8) M -1 by dextran-coated charcoal adsorption and Sephadex gel filtration techniques, respectively. Newborn rat brain cells linearly incorporated [14C]leucine into immunoprecipitable AFP during 6 h in culture. It is, therefore, concluded that feto-neonatal rat brain contains AFP similar to that present in fetal serum and that it may arise in brain as a result of its in situ synthesis.     

Isolation and Sequence Characterization of Mammary Derived Growth Inhibitor Gene of Riverine Buffalo (Bubalus Bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5′ UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3′ UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI//fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

Cecropia Vitellogenin: Isolation and Characterization

The sex-limited blood protein, vitellogenin, of the silkmothHyalophora cecropia was isolated by chromatography on DEAE-celluloseemploying a Tris-citrate buffer system, and chemical and physicalmeasurements have been performed on the purified product. Itwas found to be a lipophosphoprotein with a molecular weightof 500,000.     

生长抑素在大鼠乳腺组织中的分布和定位

目的研究生长抑素在大鼠乳腺组织中的分布和定位。方法本实验应用即用型快速免疫组化方法对处女期、妊娠6 d、12 d、18 d和泌乳6 d、12 d、18 d的SD大鼠的乳腺进行生长抑素检测。结果发现从处女期到泌乳期大鼠乳腺组织中均有生长抑素的表达,且主要分布于上皮细胞的胞质和腺泡的分泌物中。结论大鼠乳腺上皮细胞的胞质和腺泡的分泌物中有生长抑素的分布。     

Isolation and Characterization of an Enriched Golgi Fraction from Neurons of Developing Rat Brains

We report a method for the isolation of enriched fractions of intact Golgi apparatus from neurons of 10- to 12-day-old rat brains. Neurons were prepared according to a modified method of Farooq and Norton [J. Neurochem. 31, 887-894 (1978)]. Golgi-enriched fractions were obtained after centrifugation of postmitochondrial supernatants in a discontinuous sucrose gradient. Golgi fractions 1 and 2, recovered at the interfaces of 28-34% and 34-36% sucrose densities, respectively, were examined with morphometric and enzymatic methods. Morphometric analyses showed that 21-34% of fraction 1 and 11-29% of fraction 2 consisted of intact Golgi apparatus. Lysosomes, mitochondria, ribosomes, and rough endoplasmic reticulum contaminated fraction 1 (6-10%) and fraction 2 (14-26%). Golgi fraction 1 showed a 25- to 65-fold enrichment over neurons of UDP Gal:GlcNAc galactosyltransferase, CMP-sialic acid:lactosylceramide sialyltransferase, and PAPS:cerebroside sulfotransferase activities. Golgi fraction 2 showed a 8- to 23-fold enrichment over neurons of the activities of the above glycolipid- and glycoprotein-synthesizing enzymes. The activities of the possible marker enzymes rotenone-insensitive NADH-cytochrome c reductase, succinate-cytochrome c reductase, and arylsulfatase were low or minimally elevated in the Golgi fractions. A sevenfold enrichment of Na+, K+-ATPase activities was found in the Golgi fractions. This is consistent either with significant plasma membrane contamination or with the presence of this enzyme in the neuronal Golgi apparatus.     

新生大鼠脊髓神经干细胞的分离培养及鉴定

目的　从新生大鼠的脊髓中分离培养神经干细胞并观察其增殖和分化能力。方法　采用细胞培养技术结合间接免疫荧光细胞化学法。结果　分离的细胞生长旺盛 ,单克隆化生成的细胞团 ,BrdU掺入呈强阳性。分离培养获得的细胞团呈Nestin强阳性 ,至今已在体外连续传代 8个月。培养的细胞团经 1%小牛血清诱导可分化为神经元和星形胶质细胞。结论　成功分离培养了新生大鼠脊髓神经干细胞     

Isolation and Characterization of Rat and Human cDNAs Encoding a Novel Putative Peroxisomal Enoyl-CoA Hydratase

We have used a PCR-based subtractive hybridization method to identify upregulated cDNAs in the livers of rats treated with a peroxisome proliferator [clofibrate or di(2-ethylhexyl) phthalate]. After four rounds of subtractive hybridization 62 differentially hybridizing clones were partially sequenced and analyzed by sequence homology searching. Of 62, 49 were identical to 14 different upregulated rat sequences in the databank (mostly genes encoding microsomal or peroxisomal enzymes), 4 of 62 were fragments of three previously unknown genes, and 9 of 62 were false positives. Two of the unknown fragments hybridized to a single novel cDNA that was found to be more than 20-fold induced by both peroxisome proliferators. The 36-kDa predicted protein product of this cDNA shows a high degree of sequence homology to enoyl-CoA hydratases of several different species and has a C-terminal peroxisomal targeting sequence. An epitope-tagged protein product of a full-length cDNA was targeted to peroxisomes in a human cell line. We named this gene, which encodes an apparent peroxisomal enoyl-CoA hydratase, ECH1. We have also identified human ECH1 cDNA and mapped its structural gene to 19q13, 3′ to the ryanodine receptor, by hybridization to somatic cell hybrid DNA and chromosome 19-specific cosmid arrays. Possible roles for the ECH1 protein product in peroxisomal β-oxidation are discussed.     

Mammary Fat of Breast Cancer: Gene Expression Profiling and Functional Characterization

Mammary fat is the main composition of breast, and is the most probable candidate to affect tumor behavior because the fat produces hormones, growth factors and adipokines, a heterogeneous group of signaling molecules. Gene expression profiling and functional characterization of mammary fat in Chinese women has not been reported. Thus, we collected the mammary fat tissues adjacent to breast tumors from 60 subjects, among which 30 subjects had breast cancer and 30 had benign lesions. We isolated and cultured the stromal vascular cell fraction from mammary fat. The expression of genes related to adipose function (including adipogenesis and secretion) was detected at both the tissue and the cellular level. We also studied mammary fat browning. The results indicated that fat tissue close to malignant and benign lesions exhibited distinctive gene expression profiles and functional characteristics. Although the mammary fat of breast tumors atrophied, it secreted tumor growth stimulatory factors. Browning of mammary fat was observed and browning activity of fat close to malignant breast tumors was greater than that close to benign lesions. Understanding the diversity between these two fat depots may possibly help us improve our understanding of breast cancer pathogenesis and find the key to unlock new anticancer therapies.     

Immunohistochemical Localization of Arginase II and Other Enzymes of Arginine Metabolism in Rat Kidney and Liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were collocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.     

Rat Kidney Histamine N-Methyltransferase: Purification and Partial Characterization

Abstract

Histamine N-methyltransferase (HMT, EC 2.1.1.8) was purified 8,420-fold In 44% yield from rat kidney. The basic steps in the purification included differential centrlfugation, calcium phosphate adsorption, DEAE cellulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous as determined by gel electrophoresis and was stable for at least five months at ?80°C. The apparent molecular weight of the enzyme was found to be 31,500 as determined by gel filtration through Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Isoelectric point of the enzyme was determined to be 5.4. The K_m's for histamine and S-adenosyl-L-methionine were 12.4 ± 1.3 μM and 10.2 ±0.5 μM, respectively. When S-adenosyl-L-methionlne was the variable substrate, the K₁'s for S-adenosyl-L-homocysteine and S-adenosyl-D-homocys-teine were 31.9 ± 3.4 μM and 32.0 ± 3.5 μM, respectively. When histamine was the variable substrate, the K₁ for S-adenosyl-L-homocysteine was 11.8 ± 0.6 μM. Comparison of physico-chemical and catalytic properties of the rat kidney and the guinea pig enzymes suggest that these proteins have similar structural and catalytic characteristics.     

Charge Variants of an Avastin Biosimilar Isolation,Characterization, In Vitro Properties and Pharmacokinetics in Rat

The similarity between a proposed biosimilar product and the reference product can be affected by many factors. This study is designed to examine whether any subtle difference in the distribution of the charge variants of an Avastin biosimilar can affect its in vitro potency and in vivo PK. Here, the acidic, basic and main peak fractions of a biosimilar product were isolated using high-performance cation-exchange chromatography and were subjected to various studies to compare their in vitro properties and in vivo PK profile. A serial of analytical methods, including size exclusion chromatography (SEC), imaged capillary isoelectric focusing (icIEF) capillary zone electrophoresis (CZE) and cation-exchange chromatography (CEX-HPLC) were also used to characterize the isolated charge variants. The kinetics constant was measured using a Biacore X100 system. The study indicates the biosimilar product has a high similarity with avastin in physicochemical properties. The potency in vitro and PK profile in rat of charge variants and biosimilar product are consistent with avastin.     

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for α-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.