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Expression from the human parvovirus B19p6 promoter fused to the firefly luciferase ('Luc') reporter gene was evaluated in a non-erythroid human nasopharyngeal carcinoma cell line, KB, and a human megakaryocytic leukaemia cell line, MB-02, known to become permissive for B19 replication following erythroid-differentiation. The B19p6-Luc construct was introduced into KB and MB-02 cells, both in undifferentiated and differentiated states, either via DNA-mediated transfection, or via infection with recombinant adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus known to possess a broad host-range. Although Luc activity was readily detected in KB cells following transfection of the B19p6-Luc plasmid DNA, no expression from the B19p6 promoter was observed following infection with recombinant virus. In addition, transfection of the reporter plasmid resulted in high-level expression of Luc in differentiated but not in undifferentiated MB-02 cells. However, no Luc activity was detected, even in differentiated MB-02 cells, following infection with recombinant virus. Further studies with an additional recombinant as well as wild-type (wt) AAV revealed that MB-02 cells were non-permissive for AAV infection. A second human megakaryocytic leukaemia cell line, M07e, was likewise resistant to infection by recombinant as well as wt AAV. Taken together, these studies identify the first human cell type that cannot be infected by AAV. They indicate that expression from the B19p6 promoter, in the context of an AAV genome, is restricted to primary human haematopoietic cells, perhaps because parvoviral DNA replication and transcription are intrinsically coupled.

Recombinant adeno-associated virus 2 (AAV) virions were constructed that contained the genomic copy of a normal human beta-globin gene marked with a 4-bp Clal linker, and the herpesvirus thymidine kinase (TK) promoter-driven bacterial gene for resistance to neomycin (v beta m-globin), as well as those containing the DNase l-hypersensitive site 2 (HS-2) from the locus control region (LCR) of the human beta-globin gene cluster (vHS2-beta m-globin). These recombinant virions were used to infect a human erythroleukemia cell line which normally does not express the beta-globin gene (K562), or a human nasopharyngeal carcinoma cell line (KB). Cell populations resistant to G418, a neomycin analogue, were obtained following infections with the recombinant virions, indicating high-efficiency transduction of the chimeric gene as well as functional activity of the transduced neo gene in both cell types. Southern blot analysis using a human beta-globin DNA probe substantiated stable integration of the exogenous beta-globin allele in these cells. There was no expression of the transduced beta-globin gene in K562 or KB cells infected with the v beta m-globin virus. High-level expression of the transduced beta-globin gene occurred only in the vHS2-beta m-globin virus-infected K562 cells, but not in KB cells, as determined by Northern blot as well as RNase protection analyses. Expression of the human beta-globin protein could also be detected in approximately 10-20% of the vHS2-beta m-globin virus-infected K562 cells. These studies suggest that the AAV-based vector system may prove useful for high-efficiency globin gene transfer in human hematopoietic cells.

It is found that minor changes around the basic motif (88-160) and the acidic motif (771-810) of the PHO81 protein can lead to the constitutive expression of the acid phosphatase gene (PHO5), and the two motifs work cooperatively. The PHO81 protein has six ankyrin repeats, which are the recognition sites of PHO81 protein with the PHO80-PHO85 protein complex. It is found that the ankyrin repeats 1,2,4,5 and 6 are important for the PHO81 protein, but the deletion of Pro(509) and Leu(510) in ankyrin repeat 3 does not affect the PHO81 protein. We have found a candidate nucleoplasmin-like nuclear location sequence at 701-719 of the PHO81 protein and the deletion of the corresponding DNA fragment inactivates the PHO81 protein. However, when the conservative amino acids Arg701 and Lys702 are constituted by Ser and Gln, or Lys717 replaced by Asn, or Arg719 is turned to Ser, the function PHO81 protein is not affected.

Yeast PHO2 protein plays a role in the expression of several different genes and acts as a multiple global activator. Here we report the comparison of the effect of PHO2 protein on the expression of PHO5, HIS4 and Ho genes. In the PHO2 defective yeast strain, PHO5 gene could not be depressed in low Pi and the expression of the HIS4 and HO genes was decrease to 25% and 40% of the normal level respectively. When the PHO2 gene carried by low copy shuttle vector was transformed into this strain, the expression of the three genes could be restored. Previously the PHO2 gene was mutated on its conspicuous regions with site-directed mutagenesis, deletion and linker insertion. Here the effects of these mutations on these genes were compared.

The ciliary neurotrophic factor (CNTF) plays a very important role in the development and regeneration of the nervous system. In this study, the prediction of secondary structure and the hydrophobicity analysis of human CNTF were performed according to the amino acid sequence deduced from the nucleotide sequence of the cDNA. Based on the results of the prediction of structure, the human CNTF gene was modified by insertion and deletion mutagenesis. The various mutants were all highly expressed in E. coli. The recombinant proteins were purified from bacterial via DEAE A-50 and Sephacryl S-200 chromatography, and their survival-promoting activities were determined by using cultures of the dorsal root ganglion neurons of embryonic chick. The results showed that the alpha-helixes in CNTF were critical for the biological activity and the flexible C-terminus of human CNTF was not essential. Our data also indicated that the middle and the tail part of the D-helix might play crucial roles in the biological functions of CNTF.

The GST gene fusion system was used to express PHO2 gene and its mutants in E. coli Gel retardation assays showed that PHO2 fusion protein can bind to the upstream activation sequence (UAS) of the acid phosphatase gene PHO5. The homeodomain of PHO2 protein has such structure as alpha-helix 2-beta-turn-alpha-helix 3, which acts as the DNA binding domain of the transcriptional factor. The Mutation of lle 123 to Pro in helix 3 or the insertion of 4 amino acids (PDPD) between 112 and 113 in alpha-helix 2 led to the complete loss of DNA-binding activity of PHO2, while the mutation of Pro 117 to Ala in beta-turn did not affect the binding activity significantly. Deletions of the PHO80 homologous region, acidic region or C-terminal 132 residues had no great effect on the DNA-binding activity, although these mutants had lost the ability to activate PHO5 expression in vivo.

We report here that PHO2 protein is also phosphorylated by an unidentified protein kinase. A Ser-230 to Ala mutation in the consensus sequence (SPIK) recognized by cc2/CDC28-related kinase in the PHO2 protein led to the complete loss of its ability to activate the transcription of PHO5 gene. Further work showed that Pro-231 to Ser mutation inactivated PHO2 protein as well, while Ser-230 to Asp mutation did not affect PHO2 activity. Since PHO2 Asp-230 mutant mimics Ser-230 phosphorylated PHO2, we postulate that only phosphorylated PHO2 protein could activate the transcription of the PHO5 gene. The results of in vitro phospho-labelling experiments showed that the whole cell extract of the YPH499 strain grown under low phosphate conditions phosphorylated GST (glutathione S-transferase)- PHO2 (wild type) fusion protein, but not the GST-PHO2 mutant (Pro-231 to Ser) protein in which the putative phosphorylation sequence was destroyed. We therefore propose that the PHO2 protein may also be phosphorylated in vitro at Ser-230, and the phosphorylation of this site may be necessary for its function in controlling PHO5 gene expression.

The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies.

The pathogenic human parvovirus B19 contains a promoter at map unit 6 (B19p6) of the viral genome, expression from which is largely restricted to human cells in the erythroid lineage, whereas a putative promoter at map unit 44 (B19p44) is inactive during a natural viral infection. Although nonerythroid human cells, such as HeLa and KB, allow expression from the B19p6 promoter but not from the B19p44 promoter following DNA-mediated transfection, little expression from the B19p6 promoter occurs following recombinant virus infection (S. Ponnazhagan, X.-S. Wang, M.J. Woody, F. Luo, L.Y. Kang, M.L. Nallari, N.C. Munshi, S.Z. Zhou, and A. Srivastava, submitted for publication). However, significant expression from the B19p6 promoter as well as the B19p44 promoter could be detected in a human 293 cells line that expresses the adenovirus early gene products, suggesting that coinfection with adenovirus might mediate transcriptional transactivation of the B19 promoters in nonpermissive cells. Expression of the firefly luciferase reporter gene from the B19 promoters was evaluated either following plasmid transfection or following infection with the recombinant adeno-associated virus type 2 vectors. Both B19p6 and B19p44 promoters could be transactivated by coinfection with adenovirus in nonpermissive human cells, although the extent of transactivation of the B19p44 promoter was significantly lower than that of the B19p6 promoter. Expression of the adenovirus E1A proteins was necessary and sufficient for the observed transactivation of the B19 promoters. These studies further illustrate that the underlying molecular mechanisms of transactivation of parvovirus promoters in general by the adenovirus early proteins have similarities with those of the well-documented transactivation of the adeno-associated virus type 2 promoters.

It is becoming increasingly clear that the adeno-associated virus 2 (AAV)-based vector system may prove to be useful for high-efficiency gene transfer in human cells as well as for potential gene therapy in humans. A recombinant AAV vector containing the gene for a human hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), was constructed and used to infect COS-1 cells, a monkey kidney cell line. COS-1 cells infected with the recombinant virus, but not mock-infected cells, expressed high levels of the human GM-CSF gene transcripts. Furthermore, in co-cultivation experiments with the recombinant virus-infected cells, but not in those with mock-infected cells, active proliferation of a GM-CSF-dependent human megakaryocytic leukemia cell line, M07e, could be obtained in the absence of exogenously added GM-CSF. The recombinant GM-CSF protein released into the supernatant was biologically active in progenitor cell assays carried out with primary human hematopoietic cells, and this activity was specifically abrogated by treatment of the supernatant with anti-GM-CSF antibodies. This recombinant virus may be potentially useful in the management and gene therapy of a variety of malignant disorders in the human hematopoietic system.

Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU-granulocyte erythroid macrophage megakaryocyte colonies from mock-infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these cells before transplantation.

Ocular biometry in 255 eyes (131 subjects) with CT scan revealed that the length of the antero-posterior axis was 23.63 +/- 0.92 mm in hyperopia, 24.62 +/- 0.38 mm in emmetropia and 26.68 +/- 0.75 mm in myopia, and the length of the horizontal transverse axis was correspondingly 24.61 +/- 0.53 mm, 24.91 +/- 0.37 mm and 25.12 +/- 0.73 mm. There were significant differences among groups of different refractions, and the axial length increased with the severity of myopia. It was evident that refractive errors were chiefly caused by the varying axial lengths. The authors propose the parameter of the ocular diametrical ratio which is the ratio of the anteroposterior axis to the horizontal transverse axis, and accordingly hyperopia has a ratio under 1, myopia a ratio over 1 and an emmetropic eye is apparently spherical with the ratio in the vicinity of 1. The authors point out that the refractive state is determined by the ocular diametrical ratio.

Recombinant human adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) promoter (vTK-Neo), the murine colony-stimulating factor-1 (CSF-1) promoter (vCSF1-Neo) or the CSF-1 promoter plus an upstream human erythroid cell-specific enhancer, HS-2 (vHS2-CSF1-Neo). Recombinant virions were used to infect low-density murine primary bone marrow cells. In hematopoietic progenitor cell assays initiated with cells infected with these recombinant virions, myeloid as well as erythroid cell colonies resistant to the drug G418, a neomycin analogue, were readily obtained, indicating that the murine hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions and that the transduced neo gene was functionally active in these cells. Whereas only approximately 10% of the colony-forming unit-granulocyte/macrophage (CFU-GM) colonies cloned from mock-infected cells survived the G418-selection at a final active concentration of 250 micrograms/mL of the drug, the extent of the CFU-GM colony formation initiated with the recombinant AAV-Neo virions was as follows: 15% with vTK-Neo, 22% with vCSF1-Neo and 49% with vHS2-CSF1-Neo. In addition, only 14% of the burst-forming unit-erythroid (BFU-E) colonies from mock-infected cells were resistant to G418, whereas 82% of the BFU-E colonies initiated with cells infected with vHS2-CSF1-Neo virions survived the drug selection, suggesting that a human erythroid cell-specific enhancer was able to potentiate expression of the transduced neoR gene from a murine promoter. Individual CFU-GM and BFU-E colonies from mock-infected or recombinant AAV-Neo virus-infected cultures were subjected to polymerase chain reaction (PCR) analysis using a neo-specific synthetic oligonucleotide primer-pair. A 276 bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was detected only in colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest the feasibility of using the AAV-based vector system in an animal model as a prelude to evaluating its safety and efficacy in human gene therapy.

We compared electrocardiographic abnormalities and plasma norepinephrine concentration in 40 patients with subarachnoid hemorrhage within the first 24 hours, at 72 hours, and after 1 week. In the 20 patients with high plasma norepinephrine concentrations within the first 24 hours, sinus tachycardia and negative T waves were more frequently seen than in the 20 patients with normal plasma norepinephrine concentrations. After 72 hours, only sinus tachycardia was found with increased frequency in the 26 patients with high plasma norepinephrine concentrations. Although 24 patients had high plasma norepinephrine concentrations after 1 week, we found no difference in the frequency of electrocardiographic abnormalities as compared with patients with normal plasma norepinephrine. QT prolongation, U waves, ST depression, and arrhythmias were found with similar frequency in patients with high and normal plasma norepinephrine concentrations. We conclude that, with the exception of sinus tachycardia and negative T waves, other electrocardiographic changes in patients with subarachnoid hemorrhage do not depend on elevated plasma norepinephrine concentrations.

We have constructed several plasmid vectors with which a more efficient molecular cloning, followed by rescue, replication, and packaging of DNA fragments, can be achieved. The availability of these vectors should facilitate construction of a variety of recombinant adeno-associated virus 2 (AAV)-based virions containing therapeutic genes for potential use in human gene therapy.

Macrophage colony-stimulating factor (M-CSF, CSF-1) and whole-body hyperthermia (WBH) were evaluated, alone or in combination, for their capability to influence disease progression in mice inoculated with the polycythemia-inducing strain of the Friend virus complex (FVC-P). DBA/2 mice were injected i.v. with FVC-P and were treated with 20 micrograms/dose M-CSF s.c. twice a day for 5 days beginning 6 days after injection of FVC-P and/or with WBH (between 38.8 degrees C and 40.2 degrees C) given on days 5 and 12 after FVC-P injection. Fourteen days after viral inoculation, mice were sacrificed and spleen cells evaluated for: 1) spleen focus-forming virus (SFFV), by the spleen focus-forming unit assay (SFFU); 2) SFFV mRNA and genomic DNA using, respectively, Northern and Southern analysis with a B-E-SFFV DNA probe; and 3) natural killer (NK) cell activity, by 51Cr-release assay. Treatment with M-CSF or WBH alone had a small effect on SFFU numbers but little or no effect on SFFV mRNA expression and SFFV-specific DNA. However, dramatically decreased levels of SFFU and SFFV mRNA and specific DNA fragments were observed in mice treated with M-CSF in combination with WBH, and NK cell activity was restored to normal. These results suggest the possibility that M-CSF may have a therapeutic effect in combination with WBH in the in vivo treatment of certain hematologic malignancies and/or retroviral infections.

Mice infected with the polycythemia-inducing strain of the Friend virus complex (FVC-P) have been used as a leukemic mouse model. In the present study, purified iron-saturated human lactoferrin (LF) and recombinant murine (rmu) interferon-gamma (IFN-gamma), alone or in combination, were used to influence disease progression in virally infected mice. DBA/2 mice were injected i.v. with FVC-P, and were treated s.c. with 100 micrograms LF at day 7, and/or rmuIFN-gamma at 5 x 10(4) units/day for 3 days beginning at day 6 after viral infection. Mice were assessed for survival, and also 14 days after virus inoculation, the mice were killed and spleen extracts were assessed for spleen focus forming virus (SFFV) titers by spleen focus forming unit (SFFU) assay, SFFV mRNA and genomic DNA expression, and natural killer (NK) cell activity. Treatment with LF or rmuIFN-gamma alone had little or no effect on SFFU numbers or SFFV mRNA or genomic DNA expression. However, dramatically decreased SFFV titers and levels of SFFV mRNA and genomic DNA were observed in mice treated with the combination of LF and rmuIFN-gamma. NK cell activity decreased by FVC-P was returned to normal levels by LF and rmuIFN-gamma. The combined treatment also enhanced the survival rates of FVC-P-infected mice. The results suggest synergistic suppressive effects of LF with rmuIFN-gamma on disease progression in FVC-P-infected mice. This information might be of significance as a potential therapy for patients with leukemia and those infected with retroviruses.

DBA/2 mice infected with lethal dosages of Friend virus complex (FVC) can be 100% cured by split-dose total body irradiation (TBI) at 150 cGy, an effect associated with the restoration of the cellular immunity which is compromised by the virus. The exact mechanism underlying the curative effect is unknown, but it may involve the interferon (IFN) system and interleukin-2 (IL-2) production. Initial studies indicated that TBI did not directly inactivate the virus, suggesting that irradiation either acted on the target cells for virus replication or on other cells mediating the effect. We have now examined the effect of this relatively low dose TBI on replication, transcription, and protein expression of the Friend virus. Northern blot analysis revealed that in FVC infected mice treated with curative low dose TBI, no spleen focus-forming virus (SFFV)-specific mRNA species were detected. Southern blot analysis revealed that a 6.0 kb SFFV fragment could be detected in infected, untreated spleen cells, but not in cells from FVC-infected mice treated with TBI, or in uninfected spleen cells. Western blot analysis revealed that the SFFV envelope glycoprotein was expressed in the spleen cells from untreated FVC infected mice, but not in the cells from TBI treated FVC infected mice. These results, consistent with our previous findings of greatly reduced spleen focus forming units in mice with FVC which had been treated with this regimen of TBI, suggest the possibility of using such treatments in other retroviral associated disorders.

A method is described which can be used to extract common spatial patterns underlying the EEGs from two human populations. These spatial patterns account, in the least-squares sense, maximally for the variance in the EEGs from one population and minimally for the variance in the other population and therefore would seem to be optimal for quantitatively discriminating between the individual EEGs in the two populations. By using this method, it is suggested that the problems associated with the more common approach to discriminating EEGs, significance probability mapping, can be avoided. The method is tested using EEGs from a population of normal subjects and using the EEGs from a population of patients with neurologic disorders. The results in most cases are excellent and the misclassification which occurs in some cases is attributed to the nonhomogeneity of the patient population particularly. The advantages of the method for feature selection, for automatically classifying the clinical EEG, and with respect to the reference-free nature of the selected features are discussed.