José Luis Millán

Comparison of mouse and human keratin 18 a component of intermediate filaments expressed prior to implantation

Keratin 18 is a type-I keratin that is found in a variety of simple epithelial tissues. In mice, the corresponding protein, called Endo B, is expressed at the 4- to 8-cell stage of mouse development and may be one of the first intermediate-filament proteins synthesized after fertilization. A cDNA clone for keratin 18, designated pK18, was isolated from a human placental cDNA library by hybridization with the mouse Endo-B probe. It was characterized by hybridization selection of RNA, translation, immunoprecipitation, Northern blotting, and sequence analysis. Synthetic T7 polymerase transcripts of the cDNA were indistinguishable in size from keratin-18 mRNA, suggesting that pK18 represents a full-length copy of the RNA. The cDNA insert is 1,428 nucleotides long and contains a single open reading frame of 1,342 nucleotides coding for 429 amino acids. The deduced amino acid sequence is 89.7% identical with that of Endo B. The only extensive difference between the two sequences is due to 9 additional amino acids being present in the last half of the N-terminal domain of keratin 18. The 38-nucleotide-long 3 noncoding region of the cDNA is 75% identical with the corresponding portion of Endo B. The 5 noncoding regions are 59% identical. The expression of keratin-18 mRNA was found to vary more than tenfold when HeLa cells and BeWo trophoblastic cells were compared.

MUSEAP, a novel reporter gene for the study of long-term gene expression in immunocompetent mice.

The improvement of gene therapy vectors would benefit from the availability of a reporter gene that can be used for long-term studies in immunocompetent laboratory animals. We describe the construction and characterization of a novel reporter gene, murine secreted embryonic alkaline phosphatase (MUSEAP). We demonstrate by gene transfer in skeletal muscle of immunocompetent mice that MUSEAP is efficiently secreted and detected in the bloodstream and that injection of an increasing dose of DNA leads to a dose-dependent increase of plasma MUSEAP activity. We also show that the expression of MUSEAP under the control of a constitutive promoter is stable for 1 year and that the activity of MUSEAP in the bloodstream reflects the changes in the transcription rate of its gene. These properties make MUSEAP the only reporter gene that can be used for somatic gene transfer into immunocompetent mice in order to study the impact of gene transfer vectors of metabolic, developmental or environmental factors on long-term gene expression.

Monoclonal antibodies block the bromelain-mediated release of human placental alkaline phosphatase from cultured cancer cells

Certain monoclonal antibodies (mAbs) to human placental alkaline phosphatase (PLAP) block bromelain cleavage of a 2‐kDa segment from each of the two polypeptide chains of PLAP. These mAbs also prevent the release of PLAP from cultured cancer cell surfaces by bromelain. Such proteolysis‐blocking mAbs serve as tools to specifically modify the molecular topography of cell surfaces by protease treatment.

Evidence for the Expression of a Primitive Intestinal-like Alkaline Phosphatase in the Intestinal 407 Cell Line.

Intestinal-like alkaline phosphatase was found to be expressed in the intestinal 407 cell line. This enzyme was identified by use of monoclonal antibodies specific for human placental (H7 and HPMS-1) and intestinal alkaline phosphatase (2HIMS-1 and 2HIMS-3) separately. Purification of this isozyme by use of two different monoclonal antibody immunoaffinity chromatographies demonstrates a single protein band on SDS-polyacrylamide gel electrophoresis indicating that this enzyme is not formed as a heterodimer. The apparent monomer subunit molecular weight and the dimer molecular weight of this isozyme were determined to 70000 and 160000, respectively. The enzyme is a homodimer according to molecular weight determinations. Furthermore, this isozyme is neuraminidase sensitive and comparatively heat stable, properties also characteristic for the placental enzyme. Our data suggest that the intestinal-like alkaline phosphatase in the intestinal 407 cell line displays properties intermediate of the intestinal and placental isozymes which may reflect the existence and reexpression of a new primitive isozyme.

Establishment of Meiotic Germ-Cell Lines and Their Use to Study Spermatogenesis In Vitro

In vertebrates, the germ-cell lineage first arises in the early embryo as a small migratory population of cells, the primordial germ cells. These cells, first identified by their high content of alkaline phosphatase in the yolk sac, near the root of the developing allantois (1, 2), proliferate and migrate to the genital ridges where they will undergo gametogenesis. Once in the gonads these primordial germ cells or gonocytes undertake markedly different pathways in the female vs. the male gonad (3–7). In the male, gonocytes undergo mitotic arrest almost immediately after colonizing the fetal testis. These arrested gonocytes will restart mitosis during puberty to proliferate and differentiate through different stages of spermatogonia, and undergo meiosis I and II to generate spermatids that differentiate into spermatozoa through the process known as spermiogenesis. The molecular signals and genetic controls that cue spermatogonia to enter the meiotic pathway, rather than to continue proliferation, remain unclear, as do the regulatory mechanisms that oversee the progression of spermatocytes through the first and second meiotic divisions and spermatid differentiation. Thus, spermatogenesis presents us with an excellent developmental model system to ask questions aimed at understanding the cellular decision between proliferation and differentiation.