Biomedical subjects

K Guo

Publications and source records attributed to K Guo.

At least 19 recordsLinked to original sources

PubMed2001

A Dictyostelium nuclear phosphatidylinositol phosphate kinase required for developmental gene expression.

The generation of diacylglycerol (DAG) in response to receptor stimulation is a well-documented signalling mechanism that leads to activation of protein kinase C (PKC). Putative alternative effectors contain sequences that interact with DAGs, but the mechanisms of signal transduction are unknown. We have identified a Dictyostelium gene encoding a novel protein which contains a domain with high identity to the DAG-binding domain of PKC. It does not encode a PKC homologue as the conservation does not extend outside this region. We confirm that the proposed DAG-binding domain is sufficient to mediate interaction of a fusion protein with vesicles containing DAG. The protein also shows significant homology to mammalian phosphatidylinositol phosphate (PIP) kinases and we show that this domain has PIP kinase activity. The protein, PIPkinA, is enriched in the nucleus and abrogation of gene function by homologous recombination inhibits early developmental gene expression, blocking development at an early stage. Thus, we have identified a PIP kinase from Dictyostelium which is required for development, is a candidate effector for DAG and has the potential to synthesize nuclear PIP(2).

Animals↗

PubMed2001

Hypoxia induces the expression of the pro-apoptotic gene BNIP3.

It has been shown that oxygen deprivation results in apoptotic cell death, and that hypoxia inducible factor 1 (HIF1) and the tumor suppressor p53 play key roles in this process. However, the molecular mechanism through which hypoxia and HIF1 induce apoptosis is not clear. Here we show that the expression of pro-apoptotic gene BNIP3 is dramatically induced by hypoxia in various cell types, including primary rat neonatal cardiomyocytes. Overexpression of HIF1alpha, but not p53, induces the expression of BNIP3. Overexpression of BNIP3 leads to a rather unusual type of apoptosis, as no cytochrome c leakage from mitochondria was detected and inhibitors of caspases were unable to prevent cell death. Taken together, these data suggest that HIF1-dependent induction of BNIP3 may play a significant role during hypoxia-induced cell death.

Animals↗

PubMed2001

[Analysis of short tandem repeats with fluorescence labeling polymerase chain reaction to document engraftment following allogeneic bone marrow transplantation].

OBJECTIVE: In order to determine the status of engraftment following allogeneic bone marrow transplantation (allo-BMT) and predict the outcome, short tandem repeats (STR) were analysed by fluorescence labeling polymerase chain reaction (PCR). METHOD: Fluorescence labeling PCR detecting STR 9 loci was established. DNA was extracted by monoclonal magnetic beads from recipients peripheral blood nucleated cells and labeled with four color fluorescences before and from 7 days to six months after allogeneic bone marrow transplantation. RESULTS: 1. STR and clinical feature of 12 patients after allogeneic bone marrow transplantation were analysed. In the ten patients with full donor chimeras, 8 received marrow grafts from HLA identical sibling donors, 1 from HLA identical unrelated donor and 1 from HLA haploidentical mother. All of the 10 patients had durable engraftment and survived disease-freely for 3-25 months of follow-up. 2. One patient received a HLA-AB loci mismatched graft and his STR showed mixed chimeras at day 30, and converted into full donor chimeras at day 60 after transplantation. 3. One patient received HLA haploidentical sibling donor stem cells failed to be engrafted and died 4 months later. CONCLUSION: Analysis of multi-loci of STR after allo-BMT provided a real indication of engraftment. Mixed chimeras after transplantation suggested that donor lymphocytes or bone marrow cells be re-infused to enhance the conversion from mixed chimeras to full donor chimeras. For the documentation of engraftment, analysis of STR was better than any other available methods.

Adolescent↗

PubMed2000

Inhibition of HIV-1 protease by a boron-modified polypeptide.

Six boronated tetrapeptides with the carboxy moiety of phenylalanine replaced by dihydroxyboron were synthesized, and their activities against human immunodeficiency virus 1 (HIV-1) protease subsequently investigated. The sequences of these peptides were derived from HIV-1 protease substrates, which included the C-terminal part of the scissile bond (Phe-Pro) within the gag-pol polyprotein. Enzymatic studies showed that these compounds were competitive inhibitors of HIV-1 protease with K(i) values ranging from 5 to 18 microM when experiments were performed at high enzyme concentrations (above 5 x 10(-8) M); however, at low protease concentrations inhibition was due in part to an increase of the association constants of the protease subunits. Ac-Thr-Leu-Asn-PheB inhibited HIV-1 protease with a K(i) of 5 microM, whereas the non-boronated parental compound was inactive at concentrations up to 400 microM, which indicates the significance of boronation in enzyme inhibition. The boronated tetrapeptides were inhibitory to an HIV-1 protease variant that is resistant to several HIV-1 protease inhibitors. Finally, fluorescence analysis showed that the interactions between the boronated peptide Ac-Thr-Leu-Asn-PheB and HIV-1 protease resulted in a rapid decrease of fluorescence emission at 360 nm, which suggests the formation of a compound/enzyme complex. Boronated peptides may provide useful reagents for studying protease biochemistry and yield valuable information toward the development of protease dimerization inhibitors.

Boron Compounds↗

PubMed2000

Pyridoxal kinase knockout of Dictyostelium complemented by the human homologue.

The gene (pykA) encoding pyridoxal kinase which converts pyridoxal (vitamin B(6)) to pyridoxal phosphate was isolated from Dictyostelium discoideum using insertional mutagenesis. Cells of a pykA gene knockout grew poorly in axenic medium with low yield but growth was restored by the addition of pyridoxal phosphate. Sequencing indicated a gene, with one intron, encoding a predicted protein of 301 amino acids that was 42% identical in amino acid sequence to human pyridoxal kinase. After expression of the wild-type gene in Escherichia coli, the purified PykA protein product was shown to have pyridoxal kinase enzymatic activity with a K(m) of 8.7 microM for pyridoxal. Transformation of the Dictyostelium knockout mutant with the human pyridoxal kinase gene gave almost the same level of complementation as that seen using transformation with the wild-type Dictyostelium gene. Phylogenetic analysis indicated that the Dictyostelium amino acid sequence was closer to human pyridoxal kinase than to pyridoxal kinases of lower eukaryotes.

Amino Acid Sequence↗

PubMed2000

Mitochondria localization and dimerization are required for CIDE-B to induce apoptosis.

Cell death-inducing DFF45-like effector (CIDE)-B is a member of the novel family of apoptosis-inducing factors that share homology with the N-terminal region of DFF, the DNA fragmentation factor. The molecular mechanism of CIDE-B-induced apoptosis is unclear. We have shown here that CIDE-B protein is localized in mitochondria and forms homodimers and heterodimers with other family members. Serial deletion analyses suggest that the mitochondria localization signal and dimerization interface are overlapped and localized to the 30 amino acid residues at the C-terminal region of CIDE-B. Mitochondria localization and dimerization are both required for CIDE-B-induced apoptosis. Our study has thus revealed a mechanism for CIDE-B-induced apoptosis by localization to mitochondria and the formation of a high affinity homo- or heterodimeric complex.

Animals↗

PubMed2000

A temperature-sensitive adenylyl cyclase mutant of Dictyostelium.

Dictyostelium development starts with the chemotactic aggregation of up to 10(6) amoebae in response to propagating cAMP waves. cAMP is produced by the aggregation stage adenylyl cyclase (ACA) and cells lacking ACA (aca null) cannot aggregate. Temperature-sensitive mutants of ACA were selected from a population of aca null cells transformed with a library of ACA genes, a major segment of which had been amplified by error-prone PCR. One mutant (tsaca2) that can complement the aggregation null phenotype of aca null cells at 22 degrees C but not at 28 degrees C was characterized in detail. The basal catalytic activity of the enzyme in this mutant was rapidly and reversibly inactivated at 28 degrees C. Using this mutant strain we show that cell movement in aggregates and mounds is organized by propagating waves of cAMP. Synergy experiments between wild-type and tsaca2 cells, shifted to the restrictive temperature at various stages of development, showed that ACA plays an important role in the control of cell sorting and tip formation.

Adenylyl Cyclases↗

PubMed2000

Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases.

Caspases, a unique family of cysteine proteases involved in cytokine activation and in the execution of apoptosis can be sub-grouped according to the length of their prodomain. Long prodomain caspases such as caspase-8 and caspase-9 are believed to act mainly as upstream caspases to cleave downstream short prodomain caspases such as caspases-3 and -7. We report here the identification of caspases as direct substrates of calcium-activated proteases, calpains. Calpains cleave caspase-7 at sites distinct from those of the upstream caspases, generating proteolytically inactive fragments. Caspase-8 and caspase-9 can also be directly cleaved by calpains. Two calpain cleavage sites in caspase-9 have been identified by N-terminal sequencing of the cleaved products. Cleavage of caspase-9 by calpain generates truncated caspase-9 that is unable to activate caspase-3 in cell lysates. Furthermore, direct cleavage of caspase-9 by calpain blocks dATP and cytochrome-c induced caspase-3 activation. Therefore our results suggest that calpains may act as negative regulators of caspase processing and apoptosis by effectively inactivating upstream caspases.

Animals↗

PubMed2000

[Human Fab antibodies derived from phage display library neutralize hepatitis A virus in vitro].

OBJECTIVE: Development of recombinant human monoclonal antibody to hepatitis A virus as a emergent measure for prevention of hepatitis A virus infection. METHODS: Human neutralizing monoclonal antibody Fab fragments to HAV have been developed by using phage display technique. The heavy and light chains of human IgG Fab genes were amplified from a HAV patient in convalescent stage. The combinatorial phage antibody library was established by inserting both heavy and light chains of Fab genes into phage mid-vector pComb3 and followed by help phage infection after 4 rounds of panning with purified HAV as coated antigen. RESULTS: The human Fab fragments to HAV were selected and expressed in bacteria. CONCLUSIONS: The specific binding of Fab antibodies to HAV were demonstrated by their reaction with HAV antigen in ELISA. These results provide the basis for further development of a neutralizing recombinant human whole IgG molecule and hold promise for future use in the prophylaxis of HAV infection.

Antibodies, Monoclonal↗

PubMed1999

Stages in motion processing revealed by the ocular following response.

Motion perception and associated involuntary eye movements depend on factors such as the physical attributes of the stimulus and visual attention. Cues from spatial changes in luminance (first-order motion in the Fourier domain) or more complicated transitions involving two-dimensional patterns (second-order, non-Fourier) require rather different kinds of analyses to detect their net motion. During a fixation task we monitored eye movements induced by the onset of motion to examine the functional properties of the monkey cortical motion processing system. Eye movement velocity was indistinguishable to first- and second-order motion; concomitant response latency confirmed an additional calculation is required to detect the direction and velocity of second-order motion.

Analysis of Variance↗

PubMed1999

Cortical evoked potentials due to motion contrast in the blind hemifield.

Subcortical visual inputs to motion-selective cortex in primates survive after damage to the primary visual cortex (area 17/V1). Activation of human motion cortex was examined using scalp electrodes in a V1-damaged hemianope. Blind field motion-onset visual evoked potentials (VEPs) shared many of the characteristics associated with sighted vision but were smaller in amplitude and had longer latencies. The representative negative wave (C(II) peak) showed typical dependency on stimulus contrast, its peak latency increased and amplitude decreased as contrast decreased, reflecting the difficulty with which directional information could be detected. VEPs were present at contrasts below 25% when blind field motion was imperceptible even though direction guessing was paradoxically accurate. Subcortical inputs to motion cortex contribute to visual experience but not to conscious perception.

Adult↗

PubMed1999

The membrane association domain of RGS16 contains unique amphipathic features that are conserved in RGS4 and RGS5.

Regulators of G protein signaling (RGS proteins) modulate G protein-mediated signaling pathways by acting as GTPase-activating proteins for Gi, Gq, and G12 alpha-subunits of heterotrimeric G proteins. Although it is known that membrane association is critical for the biological activities of many RGS proteins, the mechanism underlying this requirement remains unclear. We reported recently that the NH2 terminus of RGS16 is required for its function in vivo. In this study, we show that RGS16 lacking the NH2 terminus is no longer localized to the plasma membrane as is the wild type protein, suggesting that membrane association is important for biological function. The region of amino acids 7-32 is sufficient to confer the membrane-targeting activity, of which amino acids 12-30 are predicted to adopt an amphipathic alpha-helix. Site-directed mutagenesis experiments showed that the hydrophobic residues of the nonpolar face of the helix and the strips of positively charged side chains positioned along the polar/nonpolar interface of the helix are crucial for membrane association. Subcellular fractionation by differential centrifugation followed by conditions that distinguish peripheral membrane proteins from integral ones indicate that RGS16 is a peripheral membrane protein. We show further that RGS16 membrane association does not require palmitoylation. Our results, together with other recent findings, have defined a unique membrane association domain with amphipathic features. We believe that these structural features and the mechanism of membrane association of RGS16 are likely to apply to the homologous domains in RGS4 and RGS5.

Amino Acid Sequence↗

PubMed1999

Isolation of spermidine synthase gene (spsA) of Dictyostelium discoideum.

The gene encoding spermidine synthase (spsA) was isolated from Dictyostelium discoideum using the technique of insertional mutagenesis. Northern blot analysis showed that the spsA mRNA is expressed maximally during the vegetative stage and decreases gradually during the 24 h of development. Sequencing of the genomic DNA and a full-length cDNA clone indicated the presence of one intron in a gene coding for a predicted protein (SpsA) with 284 amino acids. The sequence is highly conserved, with amino acid identities compared to spermidine synthases of humans, 59.5%, to mouse, 61.3%, and to yeast, 58.1%. A null mutant of the spsA gene is unable to grow in the absence of exogenous spermidine. Development of spsA null cells grown in the absence of spermidine produced fruiting bodies that have abnormally short stalks.

Amino Acid Sequence↗

PubMed1999

Grating and plaid chrominance motion influences the suppressed ocular following response.

Involuntary eye movements to foveal stimulation were measured in a monkey while it performed a fixation task. Second-order plaid motion generated higher velocities of eye movements than did first-order gratings, yet the latency of the early following response was no different for grating or plaid motion. Nevertheless, early suppressed ocular following responses to isoluminant motion continue to be titrated by stimulus velocity and spatial frequency. Motion defined by 60% luminance contrast gratings and plaids generated a motion signal gain of 60% over chrominance motion. The 20% longer latency of eye movements to chrominance motion may reflect the longer conduction latency of the parvocellular channel and an additional stage in cortical processing en route to motion areas and eye movement control.

Analysis of Variance↗

PubMed1999

Cell cycle withdrawal promotes myogenic induction of Akt, a positive modulator of myocyte survival.

During myogenesis, proliferating myoblasts withdraw from the cell cycle, acquire an apoptosis-resistant phenotype, and differentiate into myotubes. Previous studies indicate that myogenic induction of the cyclin-dependent kinase inhibitor p21 results in an inhibition of apoptotic cell death in addition to its role as a negative cell cycle regulator. Here we demonstrate that the protein encoded by the Akt proto-oncogene is induced in C2C12 cells during myogenic differentiation with a corresponding increase in kinase activity. In differentiating cultures, expression of dominant-negative forms of Akt increase the frequency of cell death whereas expression of wild-type Akt protects against death, indicating that Akt is a positive modulator of myocyte survival. Antisense oligonucleotides against p21 block cell cycle withdrawal, inhibit Akt induction, and enhance cell death in differentiating myocyte cultures. Adenovirus-mediated transfer of wild-type or constitutively active Akt constructs confer partial resistance to cell death under conditions where cell cycle exit is blocked by the antisense oligonucleotides. Collectively, these data indicate that cell cycle withdrawal facilitates the induction of Akt during myogenesis, promoting myocyte survival.

Adenoviruses, Human↗

PubMed1999

Vascular smooth muscle cell growth arrest on blockade of thrombospondin-1 requires p21(Cip1/WAF1).

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is thought to play an important role in the pathogenesis of atherosclerosis and restenosis. Previous studies have implicated the extracellular matrix protein thrombospondin-1 (TSP1) in mitogen-dependent proliferation of VSMCs. In this study, we investigated the molecular mechanisms involved in TSP1-mediated regulation of VSMC growth. Neutralizing A4.1 anti-TSP1 antibody inhibited the activity of the G(1)/S cyclin-dependent kinase 2 (cdk2) and blocked the induction of S-phase entry, which normally occurs in serum-stimulated VSMCs. This growth-inhibitory effect was associated with a marked induction of p21(Cip1/WAF1) (p21) expression in A4.1-treated VSMCs. Moreover, addition of A4.1 antibody to VSMCs markedly increased the level of p21 bound to cdk2. Thus growth arrest on antibody blockade of TSP1 may be mediated by the cdk inhibitory protein p21. Consistent with this notion, anti-TSP1 antibody inhibited [(3)H]-thymidine incorporation in wild-type but not in p21-deficient mouse embryonic fibroblasts (MEFs). Together, these data suggest that p21 plays an important role in TSP1-mediated control of cellular proliferation.

Animals↗

PubMed1999

A myb-related protein required for culmination in Dictyostelium.

The avian retroviral v-myb gene and its cellular homologues throughout the animal and plant kingdoms contain a conserved DNA binding domain. We have isolated an insertional mutant of Dictyostelium unable to switch from slug migration to fruiting body formation i.e. unable to culminate. The gene that is disrupted, mybC, codes for a protein with a myb-like domain that is recognized by an antibody against the v-myb repeat domain. During development of myb+ cells, mybC is expressed only in prestalk cells. When developed together with wild-type cells mybC- cells are able to form both spores and stalk cells very efficiently. Their developmental defect is also bypassed by overexpressing cAMP-dependent protein kinase. However even when their defect is bypassed, mybC null slugs and culminates produce little if any of the intercellular signalling peptides SDF-1 and SDF-2 that are believed to be released by prestalk cells at culmination. We propose that the mybC gene product is required for an intercellular signaling process controlling maturation of stalk cells and spores and that SDF-1 and/or SDF-2 may be implicated in this process.

Amino Acid Sequence↗

PubMed1999

U.S. academic medical centers under the managed health care environment.

This research investigates the impact of managed health care on academic medical centers in the United States. Academic medical centers hold a unique position in the U.S. health care system through their missions of conducting cutting-edge biomedical research, pursuing clinical and technological innovations, providing state-of-the-art medical care and producing highly qualified health professionals. However, policies to control costs through the use of managed care and limiting resources are detrimental to academic medical centers and impede the advancement of medical science. To survive the threats of managed care in the health care environment, academic medical centers must rely on their upper level managers to derive successful strategies. The methods used in this study include qualitative approaches in the form of key informants and case studies. In addition, a survey questionnaire was sent to 108 CEOs in all the academic medical centers in the U.S. The findings revealed that managers who perform the liaison, monitor, entrepreneur and resource allocator roles are crucial to ensure the survival of academic medical centers, so that academic medical centers can continue their missions to serve the general public and promote their well-being.

Academic Medical Centers↗