The use of constitutive nuclear oncoproteins to count neurons in the enteric nervous system of the guinea pig

Immunohistochemical double labelling of the enteric nervous system of the guinea pig ileum was performed with a monoclonal antibody (anti-MYC 033) directed against a peptide sequence of the human c-Myc protein together with antibodies directed against either the neuron-specific antigens neuron-specific enolase or PGP 9.5 or the glia-specific marker S-100 to demonstrate that anti-MYC 033 labelled the nuclei of all enteric neurons but not glia. This strategy was also employed to demonstrate that another anti-c-Myc monoclonal anti-body, anti-MYC 070, labelled the nuclei of all neurons and glia, as well as perhaps all other cells in these preparations. A polyclonal antiserum raised against a peptide sequence of the human c-Fos protein (anti-FOS 4) was shown to label the identical nuclei as anti-MYC 033. The ganglionic density of nuclei labelled by anti-FOS 4 was found to be similar to previous measures of the ganglionic density of neurons. Double labelling with anti-MYC 033 and an antiserum directed against vasoactive intestinal polypeptide was performed to reexamine the ganglionic density of neurons that express this neuropeptide. Our results suggest that the ganglionic density of these neurons might be less than previously determined.     

The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish,Carassius auratus

Goldfish Carassius auratus were acclimated to either 10 or 30°C for a minimum of 5 weeks. A 65-kDa protein specific to warm-temperature-acclimated fish was extracted from the gel with 70% formic acid after two-dimensional electrophoresis of the muscle cytoplasmic protein fraction. The 65-kDa protein thus prepared to homogeneity was used to raise specific antibodies in rabbit by conventional methods. The antibody produced exhibited specific reaction with a protein having the same molecular weight from brain and liver tissue, suggesting that the 65-kDa protein is a ubiquitous cytosolic component in warm-acclimated goldfish. When water temperature was increased from 20 to 30°C over a 20-h period, a prominent amount of the 65-kDa protein was observed in muscle tissue extracts within 5 days of additional rearing; this was demonstrated by immunoblotting with the specific antibody. The N-terminal amino acid sequence of the 65-kDa protein was determined as Asp-Glu-Pro-Gln-Gly-His-Gln-His (or Asp)-Glu-Leu, differing from that of a family of known heat-shock proteins having about 70 kDa in molecular mass (hsp 70). No interaction between ATP and the 65-kDa protein revealed by ATP-agarose affinity chromatography further confirmed the different properties of the 65-kDa protein from those of hsp 70.Abbreviations ATP adenosine 5-triphosphate - hsp heat-shock protein(s) - IgG immunoglobulin G - mRNA messenger ribonucleic acid - PMSF phenylmethylsulphonyl fluoride - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulphate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Functions of milk protein gene 5′ flanking regions on human growth hormone gene

Fragments containing 5′ flanking regions of four bovine milk protein genes—alpha lactalbumin (bαLA), alpha S₁ casein (bαS₁CN), beta casein (bβCN), kappa casein (b_kCN)—and mouse whey acidic protein (mWAP) gene were prepared by PCR and ligated to human growth hormone (hGH) gene. These recombinant DNAs were microinjected into rat embryos to produce transgenic rats, and the functions of the 5′ regions to direct secretion of hGH in the milk were tested. Although milk was obtained only in 5 of 19 mWAP/hGH rat lines, more than two-thirds of the rats carrying the other four DNAs produced milk. More than 80% of the lactated rats carrying bαLA/, bβCN/, and mWAP/hGH, and 33% of the laclated bαS₁CN/hGH rats secreted detectable amounts of hGH (> 0.05 μg/ml) in the milk. In some rats, the hGH concentrations in the milk were comparable to or more than that of the corresponding milk protein in bovine milk. The ranges of hGH concentrations in the milk of bαLA/, bβCN/, bαS₁CN/, and mWAP/hGH rats were 1.13–4,360 μg/ml, 0.11–10,900 μg/ml, 86.8–6,480 μg/ml, and 6.87–151 μg/ml, respectively. HGH was also detected in the sera of these rats, and some abnormalities of growth and reproduction were observed. All but one virgin mWAP/hGH rat secreted up to 0.0722 μg/ml of hGH in the serum, and more than half of them showed abnormal fat accumulations at their abdomen. None of the bαCN/hGH rats secreted detectable amount of hGH into their milk, whereas 8 of the 11 lines secreted hGH into their sera. For the production of hGH in transgenic rat milk, the 5′ region of bαS₁CN was shown most suitable, because the bαS₁CN/hGH rat secreted > 6,000 μg/ml of hGH into the milk and could be reproduced. © 1994 Wiley-Liss, Inc.     

Identification of a cDNA/Protein Leading to an Increased P i -uptake in Xenopus laevis Oocytes

In a previous report we documented an increased Na⁺-dependent transport of inorganic phosphate (P _i ) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211–216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P _i -uptake stimulator) lead to a 3-4-fold stimulation of Na⁺-dependent P _i -uptake (10ng cRNA injected, 3–5 days of expression). Na⁺-independent uptake of P _i was also affected but transport of sulphate and l-arginine (in the presence or absence of sodium) remained unchanged. The apparent K _m -values for the induced Na⁺-dependent uptake were 0.26 ± 0.04 mm for P _i and 14.8 ± 3.0 mm for Na⁺. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of ∼2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P _i -uptake into Xenopus laevis oocytes, but which is not a P _i -transporter itself. Received: 31 July 1996/Revised: 16 October 1996     

哺乳动物乳蛋白基因的表达与调控

本文在介绍了乳蛋白基因组结构及进化关系的基础上,就其乳蛋白基因表达的影响因素：顺式调控成分,反式作用因子及激素对表达的诱导进行了讨论,最后就该系统作为生物反应器的开发前景作了展望。     

Cloning and Characterization of the cDNA Encoding a Novel Brain-Specific 14-kDa Protein

A new acidic protein with a molecular weight of 14,000 was purified from rat brain, in which it was specifically expressed, and partially sequenced by protein sequencing. On the basis of results obtained from the amino acid sequences, mixed oligonucleotides were synthesized and used as probes to clone a cDNA from a rat brain cDNA library. The cloned cDNA provided the full-length sequence of the 14-kDa protein. Northern blot hybridization using total RNA from several tissues of the rat provided evidence that the 14-kDa protein was expressed specifically in rat brain. Transfection of this cDNA into mammalian cells resulted in expression of the 14-kDa protein. The amino acid sequence predicted from the cDNA of the rat brain 14-kDa protein contained 137 amino acid residues. A hydropathy profile revealed a hydrophobic domain (amino acids 60-80) flanked by highly hydrophilic stretches on both sides. Whereas the N-terminal region of the 14-kDa protein contained four repeating motifs, EKTKEGV, the C-terminal domain was rich in glutamic acid and proline. A computer search of the amino acid sequence of the 14-kDa protein indicated no homology to any other protein reported so far.