Characterization of a cDNA for the unexpressed form of cytochrome P-450g from the (-g) rat and differentiation of its mRNA from that of the (+g) phenotype using specific oligoprobes

Our laboratory recently isolated a cDNA for cytochrome P-450g (IIC13), a male-specific, highly polymorphic P-450 isozyme, from livers of the high phenotype (+g) of Sprague-Dawley rats [McClellan-Green et al. (1989) Biochemistry 28, 5832-5839]. Hybridization studies using a specific oligonucleotide probe for P-450 (+g) indicated that equivalent amounts of P-450g mRNA were present in livers of both the high and low phenotypes (+g and -g) of male Sprague-Dawley, Fischer (inbred -g), or ACI (inbred +g) rats. In the present study, we isolated one full-length and one nearly full-length cDNA clone coding for the unexpressed form of cytochrome P-450g from a cDNA library constructed from mRNA from a (-g) male Sprague-Dawley rat. The longest cDNA had an open reading frame of 1473 nucleotides which coded for a 490 amino acid polypeptide of Mr 55,839. Although the 5'-noncoding leader sequence and the 3'-noncoding region were unchanged, the coding sequence of the (-g) phenotype differed from that of the cDNA isolated from the (+g) phenotype by nine bases changes. These base changes would result in seven amino acid differences between the protein sequences for the two phenotypes. Two specific oligonucleotide probes for (+) P-450g and (-) P-450g containing three base differences between the (+g) and (-g) sequences hybridized differentially to mRNA from the (+g) and (-g) phenotypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apolipoprotein C-I modulates the interaction of apolipoprotein E with beta-migrating very low density lipoproteins (beta-VLDL) and inhibits binding of beta-VLDL to low density lipoprotein receptor-related protein

The binding of native rabbit beta-very low density lipoproteins (beta-VLDL) to the low density lipoprotein receptor-related protein (LRP) requires incubation with exogenous apolipoprotein (apo) E. Inclusion of a mixture of the C apolipoproteins in the incubation inhibits this binding. In the present study, the ability of the individual C apolipoproteins (C-I, C-II, and C-III) to block binding of beta-VLDL to the LRP was examined by measuring cholesteryl ester formation in mutant fibroblasts that lack low density lipoprotein receptors or by measuring binding to the LRP using ligand blotting. In each assay, both apoC-I and apoC-II inhibited binding; apoC-I was the more effective inhibitor. Apolipoprotein C-III had no effect on binding activity, regardless of its sialylation level. Binding of human apoE to rabbit beta-VLDL in the absence or presence of human apoC-I, apoC-II, and monosialo-apoC-III was also determined, by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of these studies are consistent with a mechanism in which exogenous human apoE displaces the endogenous apoE and the beta-VLDL particle becomes enriched with apoE (by 4.2-fold in this study). At this higher apoE content, the beta-VLDL bound to the LRP. Inclusion of apoC-I, apoC-II, or apoC-III in the incubation mixture resulted in a differential displacement of apoE from the beta-VLDL; however, at the concentrations examined, only apoC-I and apoC-II were capable of displacing sufficient apoE to abolish binding to LRP.     

A universal primer mixture for sequence determination at the 3′ ends of cDNAs

We have devised a universal primer which can be used to sequence the 3′-ends of cloned cDNAs containing a polyA tail. The primer consists of an equimolar mixture of three primers: 20 T nucleotides followed by either an A, C, or G nucleotide (5′→3′). With this primer mixture and the dideoxynucleotide chain termination method, we determined the 3′-terminal sequence of human β-actin cDNA in an Okayama-Berg vector, in four parallel sets of reactions containing either a single primer (T₂₀G, T₂₀C, or T₂₀A) or an equimolar mixture of all three primers. Priming with both T₂₀A and the triple mixture gave clearly readable results that agree with the known sequence of the human β-actin gene, and we have applied this method successfully to several other cDNAs in the Okayama-Berg expression vector. Use of this universal primer mixture facilitates determination of sequences at the 3′-ends of cDNAs while by-passing the polyA tail region.     

Identification of conditioned medium proteins from human cancer cells adapted to serum-free conditions

Recent studies have shown that human cancer cell lines can be adapted to grow in serum-free, unsupplemented RPMI-1640 (RO) medium. We have developed similar techniques to rapidly identify proteins of interest in serum-free conditioned medium (CM) of human lung cancer cell lines. Classic and variant small cell lung cancer (SCLC) lines were adapted to growth in RO medium. CM from each line was concentrated and fractionated on an anion-exchange column of a fast protein liquid chromatography system. Concentrates of each fraction were loaded onto lanes of minigels of an automated electrophoresis system. Analysis of the chromatograms reveals peaks seen only in CM of the classic SCLC lines. Electrophoretic analysis of the fractions containing these peaks reveal protein bands distinguishing between the subtypes of human SCLC. One protein was purified to homogeneity with subsequent reversed-phase chromatography and identified by protein microsequencing as histone H2B. These automated techniques have general use in the rapid identification of CM proteins associated with the differentiation or progression of the many types of neoplastic cells which can be adapted to growth in RO medium.     

Dynorphin immunocytochemistry in the rat central nervous system

The distribution of dynorphin in the central nervous system was investigated in rats pretreated with relatively high doses (300–400 μg) of colchicine administered intracerebroventricularly. To circumvent the problems of antibody cross-reactivity, antisera were generated against different portions as well as the full dynorphin molecule (i.e., residues 1–13, 7–17, or 1–17). For comparison, antisera to [Leu]enkephalin (residues 1–5) were also utilized. Dynorphin was found to be widely distributed throughout the neuraxis. Immunoreactive neuronal perikarya exist in hypothalamic magnocellular nuclei, periaqueductal gray, scattered reticular formation sites, and other brain stem nuclei, as well as in spinal cord. Additionally, dynorphin-positive fibers or terminals occur in the cerebral cortex, olfactory bulb, nucleus accumbens, caudate-putamen, globus pallidus, hypothalamus, substantia nigra, periaqueductal gray, many brain stem sties, and the spinal cord. In many areas studied, dynorphin and enkephalin appeared to form parallel but probably separate anatomical systems. The results suggest that dynorphin occurs in neuronal systems that are immunocytochemically distinct from those containing other opioid peptides.     

Interclonal variation in methylation patterns for expressed and non-expressed genes.

A mass culture of human diploid fibroblasts, and eight clones isolated from that mass culture, were examined for methylation patterns in several regions of DNA. Plasmid-inserted cDNA sequences were used as probes for alpha-hCG, beta-globin, A gamma- and G gamma-globin, and beta- and gamma-actin gene regions. Each probe revealed a different clone-specific pattern of DNA methylation, indicating a striking degree of inter-clonal heterogeneity, for those gene regions which are not normally expressed in diploid fibroblasts (alpha-hCG, gamma-globin and beta-globin). Intra-clonal variation was also evident in many instances, implying that heterogeneity could arise de novo in pure cell clones during serial passage. Thus methylation patterns, in particular for repressed genes, appear to be unstably inherited in these cells, and this instability may lead to random derepression in some cell lineages during mitotic growth.     

Similarities and differences in the Fc-binding glycoprotein (gE) of herpes simplex virus types 1 and 2 and tentative mapping of the viral gene for this glycoprotein.

We performed affinity chromatography and immunoprecipitation experiments to determine whether cells infected with herpes simplex virus type 2 (HSV-2) expressed a glycoprotein that was functionally and antigenically related to the HSV-1 Fc-binding glycoprotein designated gE. We found that a protein from extracts of HSV-2-infected HEp-2 cells bound specifically to an Fc affinity column and that the electrophoretic mobility of this protein in sodium dodecyl sulfate-acrylamide gels was slightly less than the mobility of HSV-1 gE. Immunoprecipitation experiments performed with an antiserum prepared against HSV-1 gE revealed that (i) extracts from HSV-2-infected cells contained a glycoprotein that was antigenically related to HSV-1 gE; (ii) the electrophoretic mobility of the HSV-2 gE was indistinguishable from the mobility of the HSV-2 Fc-binding protein; (iii) the antiserum reacted with both newly synthesized transient forms and stable fully processed forms of both HSV-1 gE and HSV-2 gE; and (iv) the transient and stable forms of HSV-2 gE all had lower electrophoretic mobilities than their HSV-1 counterparts. Electrophoretic analyses of gE precipitated from extracts of HEp-2 cells infected with two sets of HSV-1 x HSV-2 intertypic recombinant viruses suggested that the gene for gE is located at the right end of the HSV genome (0.85 to 0.97 map units) in the unique portion of the S component.     

Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells

Human epithelioid carcinoma A-431 cells are known to express unusually large numbers of receptors for the polypeptide hormone epidermal growth factor. The current studies demonstrate that this cell line also expresses 5- to 10-fold more low density lipoprotein (LDL) receptors per cell than either human fibroblasts or Chinese hamster ovary (CHO) cells. As visualized with an LDL-ferritin conjugate, the LDL receptors in A-431 cells appeared in clusters that were distributed uniformly over the cell surface, occurring over flat regions of the membrane as well as over the abundant surface extensions. Only 4% of the LDL receptors were located in coated pits. The LDL receptors in A-431 cells showed the same affinity and specificity as the LDL receptors in human fibroblasts and other cell types. In addition, they were subject to feedback regulation by sterols in the same manner as the LDL receptors in other cells. However, in contrast to other cell types in which the receptor-bound LDL is internalized with high efficiency, in the A-431 cells only a small fraction of the receptor-bound LDL entered the cell. In CHO cells approximately 66% of the LDL receptors were located over coated regions of membrane, and the efficiency of LDL internalization was correspondingly 10-fold higher than in A-431 cells. These findings support the concept that the rate of LDL internalization is proportional to the number of LDL receptors in coated pits and that the inefficiency of internalization in the A-431 cells is caused by a limitation in the ability of these cells to incorporate their LDL receptors into coated pits.     

Functional analysis of human T cell subsets defined by monoclonal antibodies. III. Regulation of helper factor production by T cell subsets

In the present report we extended our previous studies demonstrating that obligatory T-T interactions are important in regulating human immune responses in vitro. Functionally distinct human T cell subsets were isolated by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Evidence was obtained that during allogeneic interactions, OKT4+, but not OKT8+, responder T cells are required to generate helper factor(s) capable of polyclonally activating human B cells independent of additional T cell help. Importantly, the alloantigen-induced helper factor(s) production and/or release was found to be suppressed by addition of graded numbers of radiosensitive OKT8+ cells. On the other hand, no evidence was obtained that supernatant derived from alloactivated OKT8+ cells could counterbalance the helper activity generated in the presence of supernatant from alloactivated OKT4+ cells. Furthermore, OKT8+ cells, known to suppress PWM-driven B cell differentiation in the presence of OKT4+ cells, do not suppress B cell differentiation induced by preformed helper factor even in the presence of OKT4+ cells. These data further underscore the importance of functional T-T interactions in immunoregulation in vitro and support the idea that the target of suppression of B cell differentiation, induced either by alloantigen-triggered helper factor or PWM, are OKT4+ cells and not B cells themselves.