Molecular Cloning and Characterization of a cDNA Clone That Encodes a Cdc2 Homolog from Nicotiana tabacum

We have isolated a cDNA clone (cdc2Nt1) that encodes a homologof p34^cdc2/CDC28 kinase from tobacco (Nicotiana tabacum). Thecdc2Ntl protein showed extensive similarity to other homologsof Cdc2 from plants. Complementation studies showed that thecdc2Ntl gene was able to overcome cell cycle arrest at boththe G1/S and the G2/M transitions of cdc28^ts mutants of buddingyeast, demonstrating that the cdc2Ntl protein was able to replacethe Cdc28 kinase at both the G1/S and the G2/M transitions.Analysis of gene expression demonstrated that the cdc2Ntl genewas transcribed constitutively throughout the cell cycle butthat it was preferentially expressed in actively dividing tobaccoBY-2 cells. (Received July 13, 1995; Accepted February 15, 1996)     

Interstellar dust,chirality, comets and the origins of life: Life from dead stars?

The physical, chemical and astrophysical processes by which chiral prebiotic molecules can be produced in interstellar dust and later delivered safely to the earth are considered. A laboratory analog experiment on the irradiation by circularly polarized UV light of mirror image molecules at the low temperatures of interstellar dust demonstrates that a substantial degree of chirality can be produced by irradiation of the dust by circularly polarized light from pulsars whose mean brightness and distribution in the Milky Way provide the energetic photons. The chirality is then preserved by cold aggregation of the dust into low density fragile nuclei. The thermal evolution of comets following them from birth through billions of years in the Oort cloud and back to the inner solar system results in preservation of dust organics in largely pristine form — even including effects of radiogenic heating. Physical justification for the cushioned transfer of fragments of the fluffy comets impacting the earth's atmosphere provides a conceptual basis for depositing significant concentrations of interstellar prebiotic molecules. Chiral amplification in water on the earth is presumed to be enhanced by this local concentration. If chiral molecules are discovered in comet nucleus material which will some day be returned to the laboratory, we may have in our hands the same building blocks from which we evolved.     

Hepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro

Bone marrow (BM) cells originally include alpha-fetoprotein (AFP)- and c-Met [a receptor for hepatocyte growth factor (HGF)]-expressing cells. In vitro treatment of BM cells with HGF induced albumin-expressing hepatocyte-like cells. Furthermore, those hepatocyte-like cells expressed cytokeratins 8 and 18, which are typically expressed in normal adult hepatocytes. These findings demonstrate that BM cells include AFP-expressing hepatic progenitor cells that can be differentiated into hepatocytes by HGF in culture, indicating that such cultures are useful resources for cell transplantation therapy for liver diseases.     

Proteolytic activation of protein kinase C delta and epsilon by caspase-3 in U937 cells during chemotherapeutic agent-induced apoptosis

Protein kinase C (PKC) family members play pivotal roles in cellular signal transduction and nPKCdelta and theta are known to be subjected to restrictive proteolysis during apoptosis. Here we show that nPKCepsilon was specifically cleaved and generates 43-kDa and 36-kDa C-terminal fragments during chemotherapeutic drug-induced apoptosis. The proteolytic cleavage of nPKCdelta and epsilon was completely inhibited by pretreatment with Ac-DEVD-cho, a specific inhibitor of caspase-3 family enzymes. Furthermore, nPKCepsilon in non-treated U937 cell lysates was cleaved by purified recombinant caspase-3 to generate the 43-kDa fragment, identical in size to the fragment observed in vivo. This cleavage was prevented by the addition of Ac-DEVD-cho. These results suggest that caspase-3 specifically cleaves nPKCepsilon. These findings suggest the possibility that nPKC subfamily members are generally involved in the execution of apoptosis but they are regulated diversely depending on the different apoptotic stimuli.     

Thimet oligopeptidase cleaves the full-length Alzheimer amyloid precursor protein at a beta-secretase cleavage site in COS cells.

We developed an assay method using a novel quenched fluorescent substrate (QFS) flanking the beta-cleavage site of amyloid precursor protein (APP), and purified a candidate beta-secretase from bovine brain. N-terminal amino acid analysis showed the candidate to be thimet oligopeptidase (TOP). The cDNA for human TOP was cloned from a human brain cDNA library and expressed in COS cells. The enzyme was further purified on a Ni2+-agarose column. TOP cleaved the Swedish Alzheimer's substrate (SEVNLDAEFR) as well as the normal substrate (SEVKMDAEFR). We then coexpressed TOP with APP695 in COS cells, collected transfected cells and conditioned media, and analyzed them by immunoblotting. The antibody against the specific secreted APP cleaved by beta-secretase (sAPPbeta) detected the secretion of sAPPbeta only from APP/hTOP-overexpressing cells, and not from cells overexpressing of antisense hTOP cDNA. Finally, we analyzed the immunolocalization of overexpressed hTOP in COS cells. Most hTOP was localized in the nuclei, but a small amount was localized in the Golgi or other organelles around the nuclei. These results suggest that TOP has a beta-secretase-like activity responsible for the processing of APP.     

Onset of subacute aggravation of chronic thyroiditis followed immediately by transient hypothyroidism during antithyroid drug therapy for Graves' hyperthyroidism.

A 56-year-old man presented with clinical and biochemical hyperthyroidism with high thyroid 99mTc uptake, positive result for antimicrosomal antibody (MCHA; 1:8,100) and markedly high activities of thyrotropin-binding inhibitory immunoglobulin (TBII; 90.0%) and thyroid-stimulating antibody (TSAb; 2,400%). Fifty days after the initiation of antithyroid drug therapy, he developed a painful tender enlarged thyroid and an accelerated erythrocyte sedimentation rate (ESR), which were followed immediately by hypothyroidism with a transient increase in MCHA titer (peak; 1:218,700) despite of maintenance of high TBII and TSAb activities. Two and a half months after the recovery from hypothyroidism, recurrent hyperfunction was observed with further elevation of TSAb activity (4,643%). After about 2 weeks, recurrences of a painful tender enlarged thyroid and an accelerated ESR, which were followed by abrupt progression to hypothyroidism, were found. Specimens obtained when he had still slightly tender goiter after the first and second episodes of neck pain showed microscopically extremely extended interstitial fibrosis with collapsed follicles and moderate lymphocytic infiltration. Thyroid-stimulation-blocking antibody was not detected at either onset of hypothyroidism. Thus, it is possible that Graves' disease, subacute aggravation of chronic thyroiditis and hypothyroidism coexist in the same individual. In such patients, thyroid status may be determined by the degree of each of the stimulating factors (TSH, TSAb and/or unknown factors) and suppressive or destructive factors (humoral and/or cellular) and may be changed in a very short interval.