Induction of p21(CIP1/Waf1) and activation of p34(cdc2) involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells

The biological activity of retinoic acid (RA) was examined in human hepatoma Hep3B cells. Under serum-deprived conditions, RA induced S/M-phase elevation and mitotic index increase within 24 h, followed by apoptosis. This RA-induced apoptosis was accompanied by p53-independent up-regulation of endogenous p21(CIPI/Waf1) and Bax proteins, as well as activation of p34(cdc2) kinase, and increase of Rb2 protein level and phosphorylation pattern. In addition, RA had no effect on the levels of Bcl-XL; Bcl-XS; cyclins A, B, D1, D3, or E; or Rb1 expression but markedly down-modulated Cdk2 kinase activity and reduced Cdk4 expression. RA also slightly delayed p27(Kip1) expression. Olomoucine, a potent p34(cdc2) and Cdk2 inhibitor, effectively blocked RA-mediated p34(cdc2) kinase activation and prevented RA-induced apoptosis. Furthermore, antisense oligonucleotide complementary to p21(CIP2/Waf1) and p34(cdc2) mRNA significantly rescued RA-induced apoptosis. Our data indicate that p21(CIP2/Waf1) overexpression may not be the only regulatory factor necessary for RA-induced apoptosis in human hepatoma Hep3B cells. RA treatment leads to Rb2 hyperphosphorylation, and p34(cdc2) kinase activation is coincident with an aberrant mitotic progression, followed by appearance of abnormal nucleus. This aberrant cell cycle progression appeared requisite for RA-induced cell death. These findings suggest that inappropriate regulation of the cell cycle regulators p21(CIP2/Waf1) and p34(cdc2) is coupled with induction of Bax and involved in cell death with apoptosis when Hep3B cells are exposed to RA.     

Inactivation of the glucose 6-phosphate transporter causes glycogen storage disease type 1b

Glycogen storage disease type 1b (GSD-1b) is proposed to be caused by a deficiency in microsomal glucose 6-phosphate (G6P) transport, causing a loss of glucose-6-phosphatase activity and glucose homeostasis. However, for decades, this disorder has defied molecular characterization. In this study, we characterize the structural organization of the G6P transporter gene and identify mutations in the gene that segregate with the GSD-1b disorder. We report the functional characterization of the recombinant G6P transporter and demonstrate that mutations uncovered in GSD-1b patients disrupt G6P transport. Our results, for the first time, define a molecular basis for functional deficiency in GSD-1b and raise the possibility that the defective G6P transporter contributes to neutropenia and neutrophil/monocyte dysfunctions characteristic of GSD-1b patients.     

Type-1c glycogen storage disease is not caused by mutations in the glucose-6-phosphate transporter gene

Glycogen storage disease type 1 (GSD-1) is a group of autosomal recessive disorders caused by deficiencies in glucose-6-phosphatase (G6Pase) and the associated substrate/product transporters. Molecular genetic studies have demonstrated that GSD-1a and GSD-1b are caused by mutations in the G6Pase enzyme and a glucose-6-phosphate transporter (G6PT), respectively. While kinetic studies of G6Pase catalysis predict that the index GSD-1c patient is deficient in a pyrophosphate/phosphate transporter, the existence of a separate locus for GSD-1c remains unclear. We have previously shown that the G6Pase gene of the index GSD-1c patient is intact; we now show that the G6PT gene of this patient is normal, strongly suggesting the existence of a distinct GSD-1c locus.     

Determination of albumin concentration by MIP-QCM sensor

An MIP-QCM sensor able to detect micro-determine albumin concentrations was prepared by imprinting albumin with 3-dimethylaminopropyl methacrylamide-acrylate. The albumin MIP was coated on a QCM Au electrode. The adsorption characteristics of different electrodes, such as Au-OH, Au-COOH, Au-NH2 and Au electrodes, with albumin or mixture was examined. In the tetraethyleneglycol dimethacrylate crosslinking agent system, the adsorption capacity of different Au electrodes is in the order Au-OH > Au-COOH > Au-NH2 > Au. Additionally, the time taken to receive a steady-state frequency is in the order Au-NH2 < Au-OH < Au-COOH < Au. However, in the trimethylolpropane trimethacrylate crosslinking agent system, the adsorption capacity is in the order Au > Au-NH2 > Au-OH > Au-COOH electrode. Hence, the crosslinking agent had a significant effect on the MIP-QCM. On the adsorption selectivity, the albumin MIP-QCM exhibited higher response to albumin, the adsorption mass ratio of cytochrome c:lysozyme:albumin:myoglobin was 160:1:1942:30. On the other hand, in the non-MIP-QCM, the adsorption mass ratio of cytochrome c:lysozyme:albumin:myoglobin was 13:1:249:86. Additionally, a linear fitting was established and a clinical real sample was tested. This novel potential application of molecular imprinting to the recognition element of an MIP-QCM sensor appears to be promising.     

Troglitazone administration limits infarct size by reduced phosphorylation of canine myocardial connexin43 proteins

Troglitazone, an antidiabetic thiazolidinedione, has been shown to have a scavenging effect on reactive oxygen species, which can modulate expression of connexin43. The study purpose was to evaluate whether troglitazone provides cardioprotection and to assess whether the cardioprotection is associated with an attenuated expression of connexin43 at the border of infarction in a canine model of acute myocardial infarction. Vehicle or troglitazone (1, 5, and 50 mg/kg; n = 14 for each group) was given intravenously 15 min before the coronary artery occlusion. Among the survivors, infarct size was significantly larger in the control than in the supplemented groups. There was a significantly lower infarct size in the high-dose group compared with that in the low-dose group (15 +/- 7% vs. 23 +/- 10% of the risk region in the low-dose group, P = 0.04). Reperfusion caused a significant elevation in superoxide anions as measured by lucigenin-derived chemiluminescence, which was significantly inhibited in animals treated with troglitazone. Connexin43 underwent dephosphorylation in response to ischemia-reperfusion measured by Western blot in control hearts at the border zone; these changes were significantly enhanced by troglitazone administration. Confocal microscopy confirmed the changes of junctional complexes. The magnitude of infarct size positively correlated with the magnitude of phosphorylated connexin43 expression assessed by Western blot analysis (r = 0.73, P < 0.0001). This result demonstrated that the cardioprotective effect of troglitazone as an antioxidant may be associated with reduced phosphorylation of myocardial connexin43 protein.     

KCl cotransport is an important modulator of human cervical cancer growth and invasion

Cervical cancer is a major world health problem for women, but the pathophysiology of this disease has received scant attention. Here we show that the growth and invasion of cervical cancer cells are strongly linked the expression and activity of the KCl cotransporter (KCC), an important regulator of the ionic and cellular osmotic homeostasis. Functional assays of KCl cotransport activation by osmotic swelling, staurosporine, and N-ethylmaleimide indicate that removal of the N-terminal 117 amino acids from KCC1 produces a dominant-negative loss-of-function phenotype for KCl cotransport in human cervical cancer cells. The capability for regulatory volume decrease is much attenuated in the loss-of-function KCC mutant cervical cancer cells. The loss-of-function KCC mutant cervical cancer cells exhibit inhibited cell growth accompanied by decreased activity of the cell cycle gene products retinoblastoma and cdc2 kinase. Reduced cellular invasiveness is in parallel by reduced expression of alpha v beta 3 and alpha 6 beta 4 integrins, accompanied by decreased activity of matrix metalloproteinase 2 and 9. Inhibition of tumor growth in SCID mice confirms the crucial role of KCC in promoting cervical cancer growth and invasion. Thus, blockade of KCl cotransport may be a useful therapeutic adjunctive strategy to retard or prevent cervical cancer invasion.     

Organelle DNA phylogeography of Cycas taitungensis, a relict species in Taiwan

The phylogegraphic pattern of Cycas taitungensis, an endemic species with two remaining populations in Taiwan, was investigated based on genetic variability and phylogeny of the atpB-rbcL noncoding spacer of chloroplast DNA (cpDNA) and the ribosomal DNA (rDNA) internal transcribed spacer (ITS) of mitochondrial DNA (mtDNA). High levels of genetic variation at both organelle loci, due to frequent intramolecular recombination, and low levels of genetic differentiation were detected in the relict gymnosperm. The apportionment of genetic variation within and between populations agreed with a migrant-pool model, which describes a migratory pattern with colonists recruited from a random sample of earlier existing populations. Phylogenies obtained from cpDNA and mtDNA were discordant according to neighbour-joining analyses. In total four chlorotypes (clades I-IV) and five mitotypes (clades A-E) were identified based on minimum spanning networks of each locus. Significant linkage disequilibrium in mitotype-chlorotype associations excluded the possibility of the recurrent homoplasious mutations as the major force causing phylogenetic inconsistency. The most abundant chlorotype I was associated with all mitotypes and the most abundant mitotype C with all chlorotypes; no combinations of rare mitotypes with rare chlorotypes were found. According to nested clade analyses, such nonrandom associations may be ascribed to relative ages among alleles associated with the geological history through which cycads evolved. Nested in networks as interior nodes coupled with wide geographical distribution, the most dominant cytotypes of CI and EI may represent ancestral haplotypes of C. taitungensis with a possible long existence prior to the Pleistocene glacial maximum. In contrast, rare chlorotypes and mitotypes with restricted and patchy distribution may have relatively recent origins. Newly evolved genetic elements of mtDNA, with a low frequency, were likely to be associated with the dominant chlorotype, and vice versa, resulting in the nonrandom mitotype-chlorotype associations. Paraphyly of CI and EI cytotypes, leading to the low level of genetic differentiation between cycad populations, indicated a short period for isolation, which allowed low possibilities of the attainment of coalescence at polymorphic ancestral alleles.     

Utilization ofDrosophila eye to probe the functions of two mammalian serine/threonine kinases, Snk and HsHPK

Here we report a quick functional analysis of two mammalian serine/threonine kinases, a serum inducible kinase (Snk) and Homo sapiens hepatoma protein kinase (HsHPK), using Drosophila eye as a model system. We generated transgenic fly lines carrying constructs of both kinases under control of the GAL upstream activating sequence (UAS). Each UAS line was then crossed to a line in which GAL4 expression was driven by one of the following promoters, eyeless (ey), glass or decapentaplegic. Thus, different kinase mutants can be ectopically expressed in a promoter-dependent manner. We observed that the ectopic expression of either the wild-type or active form of Snk driven by the glass promoter resulted in a rough-eye phenotype. Nevertheless, the ectopic expression of HsHPK under the control of the ey promoter resulted in a small-eye phenotype. The results of this study demonstrated that ectopic expression of these two mammalian genes could be achieved by the regulation of Drosophila promoters. In addition, the effects of these ectopically expressed genes on eye development could be an implication of their functions with respect to cell proliferation and differentiation. Thus, Drosophila eye, with the powerful genetic tools and vast information on eye development available, can be a useful system to probe the functions of mammalian genes in the postgenome era.     

W206R]-procaspase 3: an inactivatable substrate for caspase 8.

We report here the cloning and high-level expression of a soluble proform of human caspase 3 (Ser(24)-H(277)) engineered to contain a short stretch of N-terminal sequence (MTISDSPREQD) from the prosegment of procaspase 8 and a C-terminal heptahistidine tag. The precursor protein isolated from extracts of recombinant Escherichia coli by immobilized metal-ion affinity chromatography was predominantly unprocessed and migrated as a 32-kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gels. Incubation of this protein with recombinant human caspase 8 produced fragments characteristic of the properly processed caspase 3, but the product was inactive. Amino-terminal sequence analysis of the caspase 3 polypeptides proved that caspase 8 had specifically cleaved the Asp(175)-Ser(176) bond to yield the expected p18 and p12 subunits, with partial cleavage at the Asp(28)-Ser(29) bond to release the prosegment. The lack of caspase 3 activity was found to be the result of a fortuitous mutation in which Trp(206) in the S4 subsite was replaced by arginine (W206R). This mutant procaspase 3, which we call m-pro3, serves as a useful reagent with which to test the efficacy of caspase 8 inhibitors in blocking processing of the natural polypeptide substrate of this enzyme and may be valuable as a source of "proenzyme" for crystallographic analysis.     

Passive immunization against oral AIDS virus transmission: An approach to prevent mother‐to‐infant HIV‐1 transmission?

To develop immunoprophylaxis regimens against mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission, we established a simian-human immunodeficiency virus (SHIV) model in neonatal macaques that mimics intrapartum mucosal virus exposure (T.W. Baba, J. Koch, E.S. Mittler et al: AIDS Res Hum Retroviruses 10:351-357, 1994). We protected four neonates from oral SHIV-vpu+ challenge by ante- and postpartum treatment with a synergistic triple combination of immunoglobulin (Ig) G1 human anti-HIV-1 neutralizing monoclonal antibodies (mAbs) (T.W. Baba, V. Liska, R. Hofmann-Lehmann et al: Nature Med 6:200-206, 2000), which recognize the CD4-binding site of Env, a glycosylation-dependent gp120, or a linear gp41 epitope. Two neonates that received only postpartum mAbs were also protected from oral SHIV-vpu+ challenge, indicating that postpartum treatment alone is sufficient. Next, we evaluated a similar mAb combination against SHIV89.6P, which encodes env of primary HIV89.6. One of four mAb-treated neonates was protected from infection and two maintained normal CD4+ T-cell counts. We conclude that the epitopes recognized by the three mAbs are important determinants for achieving protection. Combination immunoprophylaxis with synergistic mAbs seems promising to prevent maternal HIV-1 transmission in humans.