Comparison of pixantrone-based regimen (CPOP-R) with doxorubicin-based therapy (CHOP-R) for treatment of diffuse large B-cell lymphoma.

BACKGROUND: Pixantrone is an aza-anthracenedione with enhanced, preclinical antitumor activity and reduced cardiotoxicity compared with doxorubicin. PATIENTS AND METHODS: We compared the efficacy and toxic effect of CPOP-R (substituting pixantrone for doxorubicin) against CHOP-R in untreated, diffuse large B-cell lymphoma (DLBCL) patients. The primary objective was to demonstrate non-inferiority of CPOP-R by complete response/complete response unconfirmed (CR/CRu) rate. RESULTS: The CR/CRu rate for CPOP-R was 75% versus 84% for CHOP-R. Three-year overall survival was lower for CPOP-R (69% versus 85%) (P = 0.029). Median progression-free survival (PFS) was not reached for CPOP-R and was 40 months for CHOP-R [HR 95% confidence interval (CI) = 1.02 (0.60, 1.76), P = 0.934]. Fewer CPOP-R patients developed congestive heart failure (CHF) (0% versus 6%, P = 0.120), ≥ 20% declines in ejection fraction (2% versus 17%, P = 0.004), or elevations in troponin-T (P = 0.003). CONCLUSIONS: CPOP-R is an active regimen with modestly lower response rates than CHOP-R but similar PFS and event-free survival. This study demonstrates a substantially lower cardiotoxicity of pixantrone compared with doxorubicin when used as first-line therapy in DLBCL.

A subtilisin-like serine protease is required for epidermal surface formation in Arabidopsis embryos and juvenile plants.

The surfaces of land plants are covered with a cuticle that is essential for retention of water. Epidermal surfaces of Arabidopsis thaliana embryos and juvenile plants that were homozygous for abnormal leaf shape1 (ale1) mutations were defective, resulting in excessive water loss and organ fusion in young plants. In ale1 embryos, the cuticle was rudimentary and remnants of the endosperm remained attached to developing embryos. Juvenile plants had a similar abnormal cuticle. The ALE1 gene was isolated using a transposon-tagged allele ale1-1. The predicted ALE1 amino acid sequence was homologous to those of subtilisin-like serine proteases. The ALE1 gene was found to be expressed within certain endosperm cells adjacent to the embryo and within the young embryo. Expression was not detected after germination. Our results suggest that the putative protease ALE1 affects the formation of cuticle on embryos and juvenile plants and that an appropriate cuticle is required for separation of the endosperm from the embryo and for prevention of organ fusion.

Agonist-mediated down-regulation of rat beta1-adrenergic receptor transcripts: role of potential post-transcriptional degradation factors.

The human beta1-adrenergic receptor (AR) and hamster beta2-AR transcripts can be post-transcriptionally regulated at the level of mRNA stability and undergo accelerated agonist-mediated degradation via interaction of their 3' untranslated regions (UTR) with RNA binding proteins. Using RNase protection assays, we have determined that chronic isoproterenol exposure of rat C6 glioma cells results in the accelerated reduction of beta1-AR mRNAs. To determine the role of cellular environment on the agonist-independent and agonist-mediated degradation of beta1-AR mRNAs, we transfected rat beta1-AR expression recombinants into both hamster DDT1MF2 cells and rat L6 cells. The rat beta1-AR mRNAs in the two transfectant cell pools retain longer agonist-independent half-lives than in the C6 environment and undergo accelerated degradation upon chronic agonist exposure. Using UV-cross-linking/immunoblot and immunoprecipitation analyses, we have determined that the rat beta1-AR 3' UTR recognizes a predominant M(r) 39,000 component, identified as the mammalian elav-like protein HuR, and several other minor components, including the heteronuclear protein hnRNP A1. HuR levels are more highly expressed in C6 cells than in DDT1MF2 and L6 cells and are induced after chronic isoproterenol treatment. Furthermore, C6 transfectants containing an HuR expression recombinant exhibit reduced beta1-AR mRNA half-lives that were statistically comparable with half-lives identified in isoproterenol-treated C6 cells. These results imply that HuR plays a potential role in the agonist-independent and agonist-mediated down-regulation of beta1-AR mRNAs.

The transposition pattern of the Ac element in tobacco cultured cells.

We investigated physical distances and directions of transposition of the maize transposable element Ac in tobacco cultured cells. We introduced a T-DNA construct that carried a non-autonomous derivative of Ac (designated dAc-I-RS) that included sites for cleavage by restriction endonuclease MluI. Another cleavage site was also introduced into the T-DNA region outside of the dAc-I-RS transposable element. The tobacco cultured cell line BY-2 was transformed with the T-DNA and several transformed lines that had a single copy of the T-DNA at a different chromosomal location were isolated. These lines were co-cultured with Agrobacterium tumefaciens cells that carried a cDNA for the Ac transposase gene under the control of various promoters. Sublines of cultured cells in which dAc-I-RS had been transposed, were isolated. The genomic DNAs of these sublines were isolated and digested with MluI. Sizes of DNA segments generated by digestion were determined by pulse-field gel electrophoresis. Our results showed that 20 to 70% of transposition events had occurred within several hundreds kilo-base pairs (kb) on the same chromosome. These results demonstrate that the Ac-Ds element preferentially transposed to regions near the original site in a tobacco chromosome. In addition, the present results are an example of asymmetric transposition as demonstrated by the distance of transposition on the chromosome.

The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana regulates formation of a symmetric lamina, establishment of venation and repression of meristem-related homeobox genes in leaves.

The asymmetric leaves2 (as2) mutant of Arabidopsis thaliana generated leaf lobes and leaflet-like structures from the petioles of leaves in a bilaterally asymmetric manner. Both the delayed formation of the primary vein and the asymmetric formation of secondary veins were apparent in leaf primordia of as2 plants. A distinct midvein, which is the thickest vein and is located in the longitudinal center of the leaf lamina of wild-type plants, was often rudimentary even in mature as2 leaves. However, several parallel veins of very similar thickness were evident in such leaves. The complexity of venation patterns in all leaf-like organs of as2 plants was reduced. The malformed veins were visible before the development of asymmetry of the leaf lamina and were maintained in mature as2 leaves. In vitro culture on phytohormone-free medium of leaf sections from as2 mutants and from the asymmetric leaves1 (as1) mutant, which has a phenotype similar to that of as2, revealed an elevated potential in both cases for regeneration of shoots from leaf cells. Analysis by the reverse transcription-polymerase chain reaction showed that transcripts of the KNAT1, KNAT2 and KNAT6 (a recently identified member of the class 1 knox family) genes accumulated in the leaves of both as2 and as1 plants but not of wild type. Transcripts of the STM gene also accumulated in as1 leaves. These findings suggest that, in leaves, the AS2 and AS1 genes repress the expression of these homeobox genes, which are thought to maintain the indeterminate cell state in the shoot apical meristem. Taken together, our results suggest that AS2 and AS1 might be involved in establishment of a prominent midvein and of networks of other veins as well as in the formation of the symmetric leaf lamina, which might be related to repression of class 1 knox homeobox genes in leaves.

Simian retrovirus vectors for gene transfer in nonhuman primate cells.

We have recently identified and sequenced a molecular clone of the serogroup 2 simian retrovirus (SRV), D2/RHE/OR/V1, that retains an enhanced ability to infect specific T cell lines. In this report, using deletion mutagenesis, we localized the psi packaging signal, necessary for packaging of D2/RHE/OR/V1 particles, to the genomic region 345-650, which comprises the 5' intergenic region (IR) and the extreme 5' portion of the gag gene. To build an SRV-based gene transfer system and to reduce the possibility of recombination and regeneration of replication-competent viruses, we constructed split-genome D2/RHE/OR/V1 plasmid recombinants containing distinct and non-overlapping retroviral gene regions and several replacement components. For the retrovirus gene transfer vehicle, we deleted the D2/RHE/OR/V1 structural genes and substituted a cassette including the psi-packaging region, the beta-galactosidase reporter gene, and the 3' IR. Both packaging cell recombinants were used to generate stable monkey packaging cell lines; the gene transfer vehicle was subsequently transfected into the packaging cell lines, and replication-defective viruses were recovered for subsequent infection into fresh monkey cells. Successful infection by the recovered viruses verifies the potential efficacy of the SRV-based system as a research tool for gene transfer of heterologous genes into nonhuman primate cells.

Simian retrovirus serogroup 5: partial gag-prt sequence and viral RNA distribution in an infected rhesus macaque.

The simian type D retroviruses (SRVs) are one of the causative agents of simian acquired immunodeficiency syndrome (SAIDS) in Asian macaques. In this report, we describe the infection of a rhesus macaque with the SRV serogroup 5 isolate, D5/RHE/OR. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and dot blot hybridization analyses, we have determined the tissue distribution of D5/RHE/OR in this infected rhesus macaque, and have demonstrated viral mRNA in the majority of the surveyed tissues, including robust loads in the bone marrow, seminal vesicle, submaxillary salivary gland, prostate, and skeletal muscle. Microscopic examination of necropsy tissues revealed generalized lymphoid hyperplasia that was most severe in the salivary gland, bone marrow, kidney, and spleen. We also describe the first sequence analyses of portions of the D5/RHE/OR gag-prt region, obtained as a RT-PCR amplification product from infected rhesus macaque tissue, and report the first confirmation using Northern blot analyses that the SRV serogroups, including D5/RHE/OR, express similarly-sized genomic and subgenomic env mRNAs.

The Alaska Blind Child Discovery project: rationale, methods and results of 4000 screenings.

BACKGROUND: Photoscreening allows lay persons to adapt the Enhanced Brückner Test to preschoolers in an attempt to identify refractive amblyopia. The Alaska Blind Child Discovery (ABCD) project is charitably funded and administered. METHODS: MTI photoscreening was offered to children in rural and urban communities in southern Alaska from 1996 through June 1999. Parents answered questions concerning the child's health, family ocular history and whether the child had any eye "Warning Signs." The MTI images were interpreted by two eye doctors using a modification in MTI published guidelines. RESULTS: Out of 4000 screenings performed on 3930 children, there was an overall "not normal" interpretation of 9% and an inconclusive rate of 1%. The mean S.D. age was 3.9 2 years. Only 6% had had a prior eye exam. The average number of Polaroid pictures per screening was 1.16. Follow-up data on "not normal" results was obtained on just over 50%. The positive predictive value during the first two years was 77% but improved to 92% from 1998-1999. Affirmative answers to the questions concerning previous eye exam, child's health, siblings eye health and positive "Warning Signs" were significantly associated with "not normal" interpretations but affirmative answers about eye health of mother, father and relatives were not. Community penetrance of photoscreening to the target age-group ranged from only 5% for Anchorage to almost 100% for the Bristol Bay public health nurses. Five percent of parents of "positive" results surveyed would not have recommended screening for their friends. Equipment functioned dependably even in remote Alaska. CONCLUSION: Charitable volunteer Polaroid photoscreening detected amblyopia and significant pediatric eye disease in over 300 children during the first 3.5 years of ABCD.

Mutations in the WUSCHEL gene of Arabidopsis thaliana result in the development of shoots without juvenile leaves.

The vegetative growth of Arabidopsis thaliana can be divided into two phases. The transition from the juvenile (early) phase to the adult (later) phase is associated with changes in several morphological features of leaves, such as the shape of leaf blades, the number of trichomes and patterns of venation. In a screening of mutants with altered morphological identities of leaves, we found one which we named juvenile leafless and misshapen shoot apical meristem (jam). The mutation represented a new allele of the WUSCHEL (WUS) gene, and, in its presence, plants produced no juvenile leaves. Analysis of the morphology of mutant plants revealed that all the rosette leaves had characteristics of adult leaves. The formation of the first rosette leaf in the wus(jam) mutant was markedly delayed, and occurred at the almost same time as formation of the third or fourth leaf in wild-type plants. In the wild-type, these leaves correspond to the first adult leaves. Analysis by RT-PCR showed that transcripts of WUS accumulated in shoot apices and roots, but not in cotyledons and leaves. The present results suggest that the WUS gene controls the morphological traits of rosette leaves either directly or indirectly. In view of the predicted function of the WUS gene, namely maintenance of stem cells within the shoot apical meristem, we suggest that the lack of juvenile leaves in the mutant might have been caused by interruption of leaf initiation during the juvenile phase or by halting of an entire process of formation of juvenile leaves.

Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants.

Two kinds of T-DNA constructs, I-RS/dAc-I-RS and Hm(R)Ds, carrying a non-autonomous transposable element of Ac of maize were introduced into rice plants by Agrobacterium-mediated gene transfer. Six transgenic rice plants identified as containing a single copy of the element were crossed with two transgenic rice plants carrying a gene for Ac transposase under the control of the cauliflower mosaic virus 35S promoter. In F2 progenies, excision of the element was detected by PCR analysis and re-integration of the element was investigated by Southern blot analysis. The frequency of the excision of the element was found to vary from 0 to 70% depending on the crossing combination. The frequency of the number of individual transposition events out of the total number of F2 plants with germinal excision was 44% in one crossing combination and 38% in the other. In the most efficient case, 10 plants with independent transposition were obtained out of the 49 F2 plants tested. Linkage analysis of the empty donor site and the transposed Ds-insertion site in F3 plants demonstrated that one of five Ds-insertion sites was not linked to the empty donor site. The transgenic rice obtained in this study can be used for functional genomics of rice.

Rat beta 1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins.

beta 1-Adrenergic receptors (beta1-ARs) serve as important regulators of central nervous system (CNS)-mediated behavior and several neural functions, including mood, memory, neuroendocrine control, and stimulation of autonomic function. Using beta 1-AR-luciferase reporter recombinants, we have previously determined that important beta 1-AR genetic elements controlling expression within the C6 glioma cell line are contained within the region -396 to -299, relative to the translational start site. By conducting progressive internal deletions of the rat beta 1-AR 5' flanking region and with the use of beta 1-AR-luciferase recombinants, we have verified that this region contains the primary beta 1-AR promoter and/or major regulatory elements. To begin the identification of protein factors involved in beta 1-AR transcriptional activity conferred by this beta 1-AR region and flanking sequences, we conducted electrophoretic mobility shift assays using defined beta 1-AR DNA subregion probes. One probe (GS-1), encompassing the region -396 to -367, was found to produce two major and two minor mobility shift complexes when bound to nuclear extracts from the beta 1-AR expresser C6 cell line. UV-crosslinking of DNA-protein complexes, coupled with DNase I digestion, indicated that this beta 1-AR region interacts with one major protein of approximately 117 kDa molecular weight and additional minor proteins. GS-1 DNA-protein complexes were observed using beta 1-AR expresser tissues in the CNS, including cortex, hippocampus, and olfactory bulb. No DNA-protein complexes were observed when using nuclear extracts from beta 1-AR nonexpresser tissues; in some cases, using L6 cells, previously characterized to express little or no beta1-ARs, a reduction in intensities of the DNA-protein complexes was observed. Competition experiments indicate that nuclear protein binds to one of two subregions within the GS-1 sequence that contain AP-2-like consensus elements. Recombinant AP-2 protein will bind to both the beta 1-AR GS-1 promoter fragment and commercially available AP-2 consensus element control probes. Interestingly, using antibody supershift and immunoblotting experiments, no supershifts were observed and the major 117-kDa protein was not immunoreactive to antibodies recognizing either AP-2 alpha or AP-2 beta. These results support our contention that this beta 1-AR regulatory region contains AP-2 consensus elements that recognize novel transactivator proteins.

Nucleocytoplasmic export of type D simian retrovirus genomic RNA: identification of important genetic subregions and interacting cellular proteins.

The simian retrovirus (SRV) genome contains a constitutive transport element (CTE) within its 3' intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. The serogroup 2 SRV CTE is predicted to form a stable stem-loop structure containing two major internal loops exhibiting 180 degrees inverse symmetry, with loop face sequences A, A', B, and B' and additional minor internal and terminal loops. To begin the identification of potential CTE-interacting proteins and to assess structural requirements for protein interaction, we conducted RNA mobility shift assays using IR fragments that obliterated this region's known stable stem-loop structure. Using immunoblotting assays, we have determined that RNA helicase A, implicated in the nuclear export of unspliced SRV genomic RNA, does not appear to interact directly with either the complete serogroup 2 SRV 3' IR or the subregion RNAs and that formation of RNA-protein complexes is conferred by interaction with other novel proteins. UV crosslinking of RNA-protein complexes, coupled with RNase T1/A digestion, indicates that a novel protein of 120 kDa molecular weight interacts with the complete CTE or with individual subregion RNAs. Transfection analyses indicate that SRV recombinants containing A, A', B, or B' sequences forming the faces for two open loops undergo RNA export; only the complete sense CTE recombinant or a second recombinant containing two subregions in sense orientation that reconstitute the 3' two-thirds of the 3' IR, and contain only A' and B that form the faces for two terminal loops, are capable of SRV RNA export. These experiments indicate that secondary structural determinants of the 3' IR and multiple protein interactions may be important factors in the nuclear export of unspliced SRV RNA.

Promoter-activated expression of nerve growth factor for treatment of neurodegenerative diseases.

Genetic transfer approaches have received recent consideration as potential treatment modalities for human central and peripheral nervous system (CNS and PNS, respectively) neurodegenerative disorders, including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. Transplantation of genetically modified cells into the brain represents a promising strategy for the delivery and expression of specific neurotrophic factors, neurotransmitter-synthesizing enzymes, and cellular regulatory proteins for intervention in neurodegenerative diseases. The use of specific regulatable promoters may also provide potential control of gene expression required for dose-specific or time-specific therapeutic strategies. In this article, we review the potential use of activated promoters in ex vivo systems for the potential genetic therapy of neurodegenerative disorders, and then describe our own studies using the zinc-inducible metallothionein promoter for the regulated expression of nerve growth factor (NGF) in rodent brain transplants.

Molecular cloning and cell-specific growth characterization of polymorphic variants of type D serogroup 2 simian retroviruses.

Simian retroviruses (SRVs), the etiological agent of a spontaneous Simian acquired immunodeficiency syndrome, endemically infects large percentages of Asian macaques housed in biomedical research colonies and severely compromises the effective use of these species as a viable research animal. We recently described the molecular cloning of a serogroup 2 SRV, D2/RHE/OR, which causes mild immunosuppression in rhesus macaques. A restriction site variant, D2/RHE/OR/V1, has also been recovered from severely ill animals endemically infected with D2/RHE/OR. We now report the complete nucleotide sequences of D2/RHE/OR and D2/RHE/OR/V1. Both infectious molecular clones retain the genetic structure typical of type D SRVs (5' LTR-gag-prt-pol-env-3'LTR) and encode identically sized 8105-bp proviruses. D2/RHE/OR and D2/RHE/OR/V1 are 99.3% similar at the amino acid level, exhibiting only 17 residue differences, of which 10 are located in the envelope glycoproteins. The molecular clones and reciprocal chimeric viruses were used to assess the contribution of different genetic domains to virus infectivity in a T cell infection assay. These experiments indicate that D2/RHE/OR has a reduced ability to infect specific T cell lines, especially Hut-78 and MT-4 cells, and that the envelope gene is not the sole determinant of in vitro tropism.

Protein kinase C-mediated down-regulation of beta1-adrenergic receptor gene expression in rat C6 glioma cells.

In the current study, we investigated the mechanism by which protein kinase C (PKC) regulates the expression of beta1-adrenergic receptor (beta1AR) mRNA in rat C6 glioma cells. Exposure of the cells to 4beta-phorbol-12-myristate-13-acetate (PMA), an activator PKC, resulted in a down-regulation of both beta1AR binding sites and mRNA levels in a time- and concentration-dependent manner. This effect was not observed with phorbol esters that do not activate PKC and was blocked by bisindolylmaleimide, a specific PKC inhibitor. Activation of PKC did not reduce the half-life of beta1AR mRNA but significantly decreased the activity of the beta1AR promoter, as determined by reporter analysis. A putative response element, with partial homology to a consensus cAMP response element, was identified by mutation analysis of the promoter at positions -343 to -336, relative to the translational start site. Mutation of this putative regulatory element, referred to as a beta1AR-PKC response element, completely blocked the PKC-mediated down-regulation of beta1AR promoter activity. Gel mobility shift analysis detected two specific bands when C6 cell extracts were incubated with a labeled DNA probe containing the beta1AR-PKC response element sequence. Formation of one of these bands was inhibited by an oligonucleotide probe containing a consensus CRE and disrupted by an antibody for cAMP response element binding protein. Based on these studies, we propose that the PKC-induced down-regulation of beta1AR gene transcription in C6 cells is mediated in part by a cAMP response element binding protein-dependent mechanism acting on a novel response element.

Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox).

An NADPH oxidase analogous to that in mammalian phagocytes has been hypothesized to produce reactive oxygen species (ROS) in the plant defence response. A. thaliana contains at least six gp91phox homologues, designated AtrbohA-F (A. thaliana Respiratory Burst Oxidase Homologues), which map to different positions. Transcripts of three of these genes can be detected in healthy plants by RNA gel blot analyses. The Atrboh gene products are closely related to gp91phox and the intron locations suggest a common evolutionary origin. A putative EF-hand Ca(2+)-binding motif in the extended N-terminal region of the Atrboh proteins suggests a direct regulatory effect of Ca2+ on the activity of the NADPH oxidase in plants.