Cloning and characterization of keratin D,a murine endodermal cytoskeletal protein induced during in vitro differentiation of F9 teratocarcinoma cells

Summary We have identified a cDNA coding for the murine keratin D from a collection of clones representing F9 teratocarcinoma stem cell mRNA sequences. These sequences are synthesized specifically after the addition of retinoic acid and cAMP to the culture medium. The clone is 1,382 nucleotides long and contains the entire information for the active polypeptide, the complete 3 end and most, if not all, of the 5 non-coding region. The mRNA is found in hepatocytes, in PYS-2 cells (an endodermal cell line) and in differentiated (retinoic-acid-treated) F9 cells, but not in untreated F9 cells. The length of the mRNA is 1.4 kb, as estimated by Northern blot hybridization. Southern hybridization performed under very stringent conditions detects a single fragment hybridizing strongly with the cloned cDNA, suggesting that the mouse genome contains only one or very few copies of this gene. We present the first complete sequence of a keratin expressed in simple epithelia, i.e. keratin D, and discuss its structural features.     

Filipin-sterol complexes in golden hamster sperm membranes with special reference to epididymal maturation

Summary The distribution of membrane filipin-sterol complexes (FSCs) was qualitatively surveyed on freeze-fracture replicas of spermatozoa from the male reproductive tract and ejaculates of golden hamster. In the head, the acrosomal plasma membrane showed the strongest filipin labeling on the principal segment, but it was absent in the quilt-like pattern areas. These latter were observed in both caput and corpus epididymal spermatozoa, but were absent in mature spermatozoa. The postacrosomal plasma membrane had few FSCs and both the outer and inner acrosomal membranes were always negative to filipin. The nuclear membrane of the principal segment was constantly filipinpositive. The nuclear membrane of the postacrosomal region had more FSCs than that of the principal segment, particularly in mature spermatozoa. Many linear, rod-like FSCs were observed on the postacrosomal nuclear membrane of mature spermatozoa, especially in the uterine spermatozoan samples. In the neck, the plasma membrane had only a few FSCs. The redundant nuclear membrane was slightly filipin-positive, while the membrane scroll of mature spermatozoa was heavily labeled. In the tail, the plasma membrane of both the middle and principal piece was moderately labeled.     

Neuronal Intermediate Filament Expression During Neurite Outgrowth from Explanted Goldfish Retina: Effect of Retinoic Acid

Regulation of the goldfish neuronal intermediate filament proteins ON1 and ON2 was investigated in a retinal explant system. The synthesis of these proteins in explanted retina decreased with increasing time in culture, despite continuing neurite outgrowth. Thus, ON1/ON2 neurofilament expression is regulated independently from neurite outgrowth. During regeneration of the goldfish optic nerve in vivo, the expression of these proteins increased during the later phase of the process, when growing axons make contact with the optic tectum. The declining synthesis of ON1 and ON2 during neurite outgrowth in culture suggests that factors extrinsic to the retina are necessary to support synthesis of these proteins. Treating retinal explants with retinoic acid stimulated the synthesis of the ON1/ON2 proteins in a dose-dependent manner. This stimulation was effective during a period of declining synthesis of the ON1/ON2 proteins, restoring their synthesis towards initial levels of expression. These results show that retinoic acid serves as a modulator of neurofilament expression in this in vitro model of nerve regeneration.     

Rat epididymis-specific sperm maturation antigens. I. Evidence that the 26 kD 4E9 antigen found on rat caudal epididymal sperm tail is derived from a protein secreted by the epididymis

Monoclonal antibody 4E9, which was raised against a partially purified detergent extract of rat caudal epididymal sperm, recognizes the tail of sperm from the cauda, but not from caput epididymidis, as well as epithelial cells in a restricted region of the distal caput/corpus epididymidis and proteins in epididymal fluid from corpus and cauda epididymidis. The antigen is apparently a glycoprotein, since it is retained on a Ricinus communis agglutinin l lectin column. Epididymal fluid antigens have apparent M_rS of 38–26 kD, whereas the memrane-associated form of the molecule has an M_r of 26 kD. Immunocytochemical data and Western immunoblot data suggest that the membrane antigen is derived from the fluid antigen, which, in turn, is secrteted by the epididymal epithelium. Characterization of the membrane antigen indicates that it is tightly associated with the sperm surface, behaving as though it is an integral membrane protein. The antigen persists on ejaculated sperm. © 1994 Wiley-Liss, Inc.     

几种药物对HL-60细胞的诱导分化作用及其过程中蛋白激酶C活力分布的变化

本文就HHT、RA、WB_(852)对HL-60细胞的诱导分化作用及此过程中PKC活力在细胞胞浆部分及膜溶脱部分的变化进行研究。结果表明,在适当的用药浓度下,从细胞生长抑制情况、形态学观察及NBT还原能力测定判断,三种药物对HL-60细胞有明显的诱导分化作用。PKC活力分布变化的研究结果表明,用药组细胞胞浆部分酶活力有不同程度的下降,尤在用药早期(约6h以前)下降显著;而膜部分PKC活力则表现上升、或下降,或活力相差不大的结果。暗示在信息传递过程中起核心作用的PKC对不同的胞外刺激可能采取不同的应答方式。PKC的作用可能主要发生在信息传递的早期。     

视黄酸对人肝癌细胞表面N糖链类型及天线数的影响

本文采用系列凝集素柱层析法,并配合外切糖苷酶处理研究了在视黄酸(RA)作用1—5天过程中人肝癌细胞株SMMC-7721细胞表面N糖链结构的变化。结果表明,RA促进3~H-甘露糖(Man)参入细胞表面N糖链,使高甘露糖型N糖链的百分比下降,复杂型百分比上升,并促进二天线N糖链的生物合成,使多天线特别是四天线和C_2,C_(21)b三天线N糖链的合成减少。结果提示,N糖链结构的这些变化可能是RA诱导SMMC-7721细胞向正常方向分化的结果。     

Interactions between interferon γ and retinoic acid with transforming growth factor β in the induction of immune recognition molecules

The cell-surface expression of major histocompatibility (MHC) antigens and the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) is essential for target cell recognition by T lymphocytes. The expression of both classes of molecule is induced by various cytokines, notably interferon (IFN). Since transforming growth factor (TGF) has been recently reported to antagonise HLA-DR induction by IFN we have examined, using a number of murine and human cell lines, the effect of TGF on IFN-induced MHC class I and class II and ICAM-1 expression. All of the cell lines tested expressed elevated class I MHC following IFN treatment. Class II MHC induction was seen on most but not all of the cells, the exceptions being among a panel of human colorectal carcinoma cell lines. A striking difference between cells of different origin was noted in the response to TGF. TGF was found to antagonise IFN-induced class I and class II MHC expression on C3H 10T1/2 murine fibroblasts, early-passage BALB/c mouse embryo fibroblasts, a murine oligodendroglioma cell line, and on MRC5 human fibroblasts and two human glioblastoma cell lines. Class II MHC was much more strongly inhibited (sometimes completely) than class I MHC. TGF also inhibited induction of class I MHC expression by IFN. However, TGF did not inhibit class I or class II MHC induction by IFN in any of the nine colorectal carcinoma cell lines, although two of five of the lines tested were growth-inhibited by TGF. On the other hand, human ICAM-1 induction by IFN was not affected by simultaneous treatment with TGF in any of the cell lines. The down-regulation of IFN-induced MHC antigens by TGF is not, therefore, the result of a general antagonism of IFN. Retinoic acid has recently been reported to induce ICAM-1 expression on human tumour cells. We have confirmed this observation on MRC5, and the two human glioblastoma cell lines, however six colorectal carcinoma cell lines tested did not respond. In contrast to IFN-induced ICAM-1 expression, retinoic-acid-induced ICAM-1 expression was inhibited by TGF on two of the three responsive lines.     

Pertussis Toxin-Sensitive G Protein α-Subunits: Production of Monoclonal Antibodies and Detection of Differential Increases on Differentiation of PC12 and LA-N-5 Cells

Abstract: Monoclonal antibodies were produced that are specific for the three major pertussis toxin-sensitive G protein α-subunits present in mammalian brain—αo, αi1, and αi2—using purified bovine brain G proteins, purified rat brain G proteins, and purified recombinant αi2, respectively. These monoclonal antibodies were used to monitor changes in the concentrations of the three G protein α-subunits during differentiation of PC12 cells and human neuroblastoma LA-N-5 cells. In PC12 cells, levels of αi1 but not αi2 increased during nerve growth factor-induced differentiation. In contrast, αi2 but not αi1 increased when LA-N-5 cells were differentiated with retinoic acid. The concentration of αo increased in both cell lines during differentiation. Electrophoretic resolution of αo subtypes revealed that although αo2 was the major subtype in undifferentiated cells, only the concentration of αo1 increased during differentiation of both PC12 and LA-N-5 cells. The level of 43-kDa growth-associated protein, a protein known to associate with αo, increased similarly to that of αo1. ADP-ribosylation of αo, αi1, and αi2 with pertussis toxin did not alter the reactivities of the monoclonal antibodies, but toxin treatment of cells reduced the concentrations of each protein after 24 h. There was no change in the concentration of αq, which is not ADP-ribosylated by pertussis toxin. Thus, these new monoclonal antibodies enabled the detection of differential increases in subtypes of αi and αo associated with neuronal differentiation.     

Epididymal histoplasmosis diagnosed by isolation ofHistoplasma capsulatum from semen

An autochthonous case of epididymal histoplasmosis masquerading as tuberculosis in a 55-year-old male patient is reported from India. It was diagnosed by culture ofHistoplasma capsulatum from semen and by demonstration of the fungus upon re-examination of epididymal biopsy sections previously misinterpreted as tuberculous granuloma. The patient's main complaints were painful epididymal swelling, occasional fever and cough. He was treated successfully by excision of epididymis and vas deferens combined with amphotericin B therapy. This is believed to be the first case of epididymal histoplasmosis to be reported outside the American continent and the fourth of its type reported in the English literature. The case is also noteworthy in thatH. capsulatum was isolated for the first time from semen, and it underlines the importance of mycological culture of semen specimens for diagnosis of genitourinary infections of obscure etiology.Presented at the XII Congress of the International Society for Human and Animal Mycology, Adelaide, Australia, March 13–18, 1994.