Characterization of messenger RNA for aldolase B in adult and fetal human liver

High molecular weight cellular RNA was isolated from adult and fetal human liver tissue by a procedure of ethanol precipitation in concentrated guanidine-HCl solutions. About 5 mg of RNA were obtained from one gram of liver. RNA was fractionated by sucrose gradient ultracentrifugation. Aldolase B neosynthesized in a reticulocyte lysate cell-free system under the direction of total or fractionated RNA was purified by immunoaffinity microchromatography. Messenger RNA specifying synthesis of aldolase B exhibited a sedimentation coefficient of 16 S both in adult and fetal liver. This enzyme represented 1.3 % of the total neosynthesized proteins in adult liver, 0.1 % in the liver of a 6-month-old fetus and less than 0.01 % in the liver of a 4.5 month-old fetus.     

Presence of different types of procollagen messenger RNAs in human hepatoma cell lines

Human hepatoma cell lines were shown for the first time to contain various types of procollagen mRNAs. The amounts and types of procollagen mRNAs differed depending on the cell lines. Pro alpha 1 (III) and pro alpha 1 (IV) collagen mRNAs were present in PLC/PRF/5, a hepatocellular carcinoma cell line, whereas pro alpha 1 (I), pro alpha 2 (I), pro alpha 1 (IV) and pro alpha 2 (V) collagen genes contrast, HepG2 cells derived from hepatoblastoma contained little, if any, mRNAs for these types of procollagens we had examined.     

Structures of human and rabbit beta-globin precursor messenger RNAs in solution

The structures in solution of human and rabbit beta-globin precursor messenger RNAs containing their first intervening sequence have been investigated. This was accomplished by chemical probing experiments to determine sites of potential base pairing, and by cross-linking experiments to determine the sites of long-range interactions. Secondary structures for both molecules were predicted by using this information. Both molecules are arranged into two separate domains. The first domain, consisting of the first exon, contains several long-range interactions between the beginning of the molecule and sites adjacent to the donor splice site and a partially conserved stem/loop structure. The second domain contains part of the intervening sequence and the beginning of the second exon. The secondary structures involved in the second domain are different in the two molecules. These studies indicate a lack of connection between the donor and acceptor splice sites in these two molecules on the level of the secondary structure. Furthermore, given the absence of strongly conserved structures, it is unlikely that there could be any strict requirements for secondary structures that influence splice site usage.     

Expression of intermediate filament proteins in fetal and adult human lung tissues

The expression patterns of intermediate filament proteins in fetal and normal or nonpathological adult human lung tissues are described using (chain-specific) monoclonal antibodies. In early stages of development (9-10 weeks and 25 weeks of gestation) only so-called simple cytokeratins such as cytokeratins 7 (minor amounts). 8, 18 and 19 are detected in bronchial epithelial cells. At later stages of development, the cytokeratin expression patterns become more complex. The number of bronchial cells positive for cytokeratin 7 increases, but basal cells in the bronchial epithelium remain negative. These latter cells show, however, expression of cytokeratin 14 in the third trimester of gestation. Developing alveolar epithelial cells express cytokeratins 7, 8, 18 and 19. In adult human bronchial epithelium cytokeratins 4 (varying amounts), 7, 8, 13 (minor amounts), 14, 18 and 19 can be detected, with the main expression of cytokeratins 7, 8, and 18 in columnar cells and the main expression of cytokeratin 14 in basal cells. Vimentin is detected in all mesenchymal tissues. In addition, fetal lung expresses vimentin in bronchial epithelium, however, to a lesser extent with increasing age, resulting in the expression of vimentin in only few scattered bronchial cells at birth. Also in adult bronchial epithelium the expression of vimentin is noticed in part of the basal and columnar epithelial cells. Desmin filaments, present in smooth muscle cells of the lung, appear to alter their protein structure with age. In early stages of development smooth muscle cells surrounding blood vessels are partly reactive with some cytokeratin antibodies and with a polyclonal desmin antibody. At week 9-10 and week 25 of gestation a monoclonal antibody to desmin, however, is not reactive with blood vessel smooth muscle cells but is only reactive with smooth muscle cells surrounding bronchi. With increasing age the reactivity of cytokeratin antibodies with smooth muscle cells in blood vessels decreases, while the reactivity with the monoclonal desmin antibody increases. Our results show that during differentiation profound changes in the intermediate filament expression patterns occur in the different cell types of the developing lung.     

Immunolocalization of inhibin and activin alpha and betaB subunits and expression of corresponding messenger RNAs in the human adult testis

Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an alpha and a beta(B) subunit. In adult testes, the cellular site of production is still controversial, and it was hypothesized that germ cells contribute to inhibin B production. To determine which cell types in the testes may produce inhibin B, the immunohistochemical localization of the two subunits of inhibin B were examined in adult testicular biopsies with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only (SCO) tubules. Moreover, using in situ hybridization with mRNA probes, the mRNA expression patterns of inhibin alpha and inhibin/activin beta(B) subunits have been investigated. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin alpha subunit and expressed inhibin alpha subunit mRNA. Using inhibin beta(B) subunit immunoserum on testes with normal spermatogenesis and with spermatogenic arrest, intense labeling was located in germ cells from pachytene spermatocytes to round spermatids but not in Sertoli cells. Inhibin beta(B) subunit mRNA expression was intense in germ cells from spermatogonia to round spermatids and in Sertoli cells in these testes. In testes with SCO, high inhibin beta(B) subunit mRNA labeling density was observed in both Sertoli cells and Leydig cells, whereas beta(B) subunit immunostaining was negative for Sertoli cells and faintly positive for Leydig cells. These results agree with the recent opinion that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells.     

Isolation and characterization of human foetal alpha-globulin (alpha 1F) from foetal and hepatoma sera

The alpha 1F present in human foetal sera and the serum from a patient with hepatocellular carcinoma was isolated by immune precipitation using rabbit anti-alpha 1F. After labelling with 125I, some physicochemical properties of alpha 1F were investigated. The molecular weights of 125I-labelled alpha 1F isolated from foetal and hepatoma sera were 61,000 and 63,000, respectively. The protein was not dissociated in 6 M guanidine after complete reduction, showing that it contains a single peptide chain.     

Expression of extracellular matrix genes by cultured human cells: localization of messenger RNAs and antigenic epitopes

Simplified and expedient methodologies for examination of cellular gene expression at the mRNA and protein levels, utilizing in situ hybridization and peroxidase-anti-peroxidase immunodetection, were developed. These techniques were first optimized for the detection of extracellular matrix genes expressed by cultured human skin fibroblasts and keratinocytes, the two principal cell types of human skin. In situ hybridizations and Northern transfer analyses with human-sequence-specific cDNAs encoding collagenous and noncollagenous protein sequences demonstrated selective expression of different matrix genes by these two cell types, indicating different biosynthetic capacities of these cells and attesting to the specificity of the hybridizations. The utility of in situ hybridization was also demonstrated in mixed primary cell cultures established from cutaneous neurofibromas consisting of Schwann cells, perineurial cells, and fibroblasts. The methodologies developed here were further utilized for simultaneous detection of fibronectin mRNA and immunoreactive protein in fibroblast cultures. This procedure allowed detection of grains representative of radioactively labeled cDNA-mRNA hybrids and protein epitopes, as visualized by peroxidase-anti-peroxidase immunodetection on the same cells. This methodology, with appropriate modifications, may be applicable to other cell types as well as tissue specimens.     

Galanin and vasoactive intestinal peptide messenger RNAs increase following axotomy of adult sympathetic neurons

The adult rat superior cervical ganglion (SSG) contains low levels of galanin- and vasoactive intestinal peptide-(VIP) like immunoreactivity, with very few immuno-stained principal neurons. Immunoreactivity for both neuropeptides increases in these neurons after explanation or postganglionic axotomy in vivo. Northern blot analysis had demonstrated concomitant increases in mRNAs encoding these peptides. To localize cells in axotomized ganglia which increase their expression of these mRNAs, we performed in situ hybridization studies. In control SCG, only a few principal neurons contained mRNA for either galanin or VIP. After 48 h in organ culture, galanin mRNA was expressed in the majority of principal neurons. At 48 h after in vivo axotomy of both postganglionic trunks of the SCG, the internal and external carotid nerves, the distribution and number of neurons expressing galanin mRNA increased similarly to that seen in culture. Lesioning either trunk alone produced increases in galanin mRNA localized to those regions of the ganglion containing neurons that project into the lesioned trunk. Transection of the predominantly preganglionic cervical sympathetic trunk increased galanin mRNA expression in a small population of neurons that nerve trunk. The distributions of these labeled neurons, together with previous neuroanatomical studies, suggests that they had been axotomised by the lesions. Similar studies examining VIP mRNA expression demonstrated that although considerably fewer VIP mRNA expressing neurons than galanin mRNA expressing neurons were present after axotomy, the distribution of neuropeptide mRNA-positive cells were similar in both cases. These observations suggest that increases in the peptide galanin and VIP after nerve transection result from changes in the levels of their mRNAs in those neurons that have been axotomized. 1994 John Wiley & Sons, Inc.