Engineered herpes simplex virus expressing bacterial cytosine deaminase for experimental therapy of brain tumors.

Lack of effective therapy of primary brain tumors has promoted the development of novel experimental approaches utilizing oncolytic viruses combined with gene therapy. Towards this end, we have assessed a conditionally replication-competent, gamma(1)34.5-deleted herpes simplex virus type 1 (HSV-1) expressing cytosine deaminase (CD) for treatment of malignant brain tumors. Our results are summarized as follows: (i) a recombinant HSV (M012) was constructed in which both copies of the gamma(1)34.5 gene were replaced with the bacterial CD gene, under the control of the cellular promoter Egr-1; (ii) M012-infected cells in vitro efficiently convert 5-fluorocytosine (5-FC) to 5-fluorouracil, thereby enhancing cytotoxicity of neighboring, uninfected cells; (iii) both direct and bystander cytotoxicity of murine neuroblastoma and human glioma cell lines after infection with M012 were demonstrated; (iv) direct intracerebral inoculation of A/J mice demonstrated lack of neurotoxicity at doses similar to G207, a gamma(1)34.5-deleted HSV with demonstrated safety in human patient trials and (v) intratumoral injection of M012 into Neuro-2a flank tumors in combination with 5-FC administration significantly reduced tumor growth versus tumors treated with R3659 combined with 5-FC, or treated with M012 alone. Thus, M012 is a promising new oncolytic HSV vector with an enhanced prodrug-mediated, antineoplastic effect that is safe for intracranial administration.

Killed but metabolically active microbes: a new vaccine paradigm for eliciting effector T-cell responses and protective immunity.

We developed a new class of vaccines, based on killed but metabolically active (KBMA) bacteria, that simultaneously takes advantage of the potency of live vaccines and the safety of killed vaccines. We removed genes required for nucleotide excision repair (uvrAB), rendering microbial-based vaccines exquisitely sensitive to photochemical inactivation with psoralen and long-wavelength ultraviolet light. Colony formation of the nucleotide excision repair mutants was blocked by infrequent, randomly distributed psoralen crosslinks, but the bacterial population was able to express its genes, synthesize and secrete proteins. Using the intracellular pathogen Listeria monocytogenes as a model platform, recombinant psoralen-inactivated Lm DeltauvrAB vaccines induced potent CD4(+) and CD8(+) T-cell responses and protected mice against virus challenge in an infectious disease model and provided therapeutic benefit in a mouse cancer model. Microbial KBMA vaccines used either as a recombinant vaccine platform or as a modified form of the pathogen itself may have broad use for the treatment of infectious disease and cancer.

Uses and limitations of mammalian fetal membranes and placenta for phylogenetic reconstruction.

Comparative studies on the development of the mammalian fetal membranes and placenta, and discussions of the possible evolutionary significance of these data, have been carried out for more than a century. Throughout much of this period, however, such data were considered in isolation from cranioskeletal and other biological findings, so that only rarely were placental features incorporated into comparative assessments of mammalian evolution. Evolutionary interpretations of the fetal membrane evidence were commonly based on the concept of overall similarity, and most placentologists were strongly influenced by the widely accepted hypothesis that living "insectivores" were closely related to the ancestral eutherian mammals. Mossman's emphasis on the use of all available developmental data on the fetal membranes and placenta, rather than simply the nature of the definitive placenta, marked a turning point in the history of using such findings for assessing eutherian mammalian evolution. Recent advances in the methodology of phylogenetic inference, with emphasis on evaluating convergence versus homology, and on distinguishing between relatively primitive versus derived homologous similarities shared by two or more taxa, have provided a sound framework for the phylogenetic analysis of fetal membrane data. Character analysis of mammalian fetal membrane traits provides a mechanism for testing phylogenetic hypotheses formulated by cranioskeletal features. It is emphasized in this study that fetal membrane data have their greatest input into assessing mammalian phylogeny when they are evaluated in conjunction with the available cranioskeletal and molecular evidence.

Amplification and characterization of an inverted repeat from the Chlamydomonas reinhardtii mitochondrial genome.

Based on the nucleotide (nt) sequences of cob and L2a, two oligodeoxyribonucleotides (oligos) were synthesized and used in the polymerase chain reaction (PCR) to amplify the termini of the Chlamydomonas reinhardtii mitochondrial (mt) genome. A 0.8-kb PCR product was detected by agarose-gel electrophoresis when using unligated mt DNA as the template for PCR. This may have indicated the presence of a naturally occurring circular mt DNA molecule that acted as the PCR template. The 0.8-kb DNA could also be amplified from the linear mt DNA via an intramolecular jump during PCR. The sequence data from the 0.8-kb PCR product, and the right 0.6-kb and left 1-kb terminal fragments of the linear mt DNA, along with Southern hybridization analysis, indicated that a 0.49-kb inverted repeat (IR) sequence is present at the right and left termini of the linear mt DNA. The IR contains A+T-rich clusters, as well as numerous short direct repeats (DR) and IR, and might be involved in the recombination, replication and expression of the C. reinhardtii mt genome.

The group I intron of apocytochrome b gene from Chlamydomonas smithii encodes a site-specific endonuclease.

The mitochondrial DNA molecules of two interfertile algal species, Chlamydomonas smithii and C. reinhardtii, are co-linear except for a 1075 bp intron (the alpha-insert) that is present in the cob gene of C. smithii. The alpha-insert, a group I intron (Cs cob.1) containing an open reading frame (ORF) which encodes a basic, hydrophilic protein of 237 amino acids, is unidirectionally transmitted to all diploid progeny during interspecific crosses. In this report, we show that the Cs cob.1-encoded protein is a site-specific endonuclease (I-Csm I) which could mediate the intron transfer via the gene conversion mechanism. The Cs cob.1 ORF was cloned into the vector pMALcr1 and over-expressed as a hybrid protein fused to maltose-binding protein (MBP). This fusion protein exhibited an in vivo endonuclease activity which specifically cleaved the intron homing site within the intronless cob gene.

Development of deciduous and permanent dentition in Tarsius and its phylogenetic significance.

The earliest stages of development and differentiation of deciduous and permanent teeth were studied in prenatal and perinatal tarsiers. Both deciduous and successional teeth develop at loci for I1, I2, C, P2-4. Relationships among the dental lamina, enamel organs, and successional lamina were used to determine tooth homologies. During the second half of prenatal life, dP2 and dI2 undergo abnormal development. Their dentin becomes fragmented, and these teeth are shed or resorbed without eruption during the perinatal period. Our study provides evidence of an intermediate phase in evolutionary tooth loss. These findings do not corroborate recent hypotheses of primate phylogeny based mainly on tooth eruption patterns.

Development and phylogenetic significance of the fetal membranes and placenta of the African hystricognathous rodents Bathyergus and Hystrix.

Morphogenesis of fetal membranes in African and American hystricognathous rodents is essentially the same, even in the possession of such uniquely derived features as a subplacenta and a capillary ring on the inverted yolk sac, features that are unknown in any other rodent group. This is good evidence that the African and American hystricognaths comprise a monophyletic group. Certain interesting features of the decidua, decidual giant cells, periplacental bilaminar omphalopleure, and epamnion are described in Bathyergus and Hystrix. Of special interest in Bathyergus is the widespread decidualization of the uterine connective tissue, even that of the myometrium and perimetrium; and the presence of numerous, usually binucleate, decidual giant cells.

Origin and differentiation of the yolk sac and extraembryonic mesoderm in presomite human and rhesus monkey embryos.

Reexamination of presomite human and rhesus monkey embryos in the Carnegie Collection provides no evidence to corroborate the hypothesis that the trophoblast is the source of all extraembryonic tissues in these embryos. Instead, the present study indicates that the developmental pattern of the yolk sac and extraembryonic mesoderm is homologous to that in other eutharian mammals. The primary yolk sac of 10- to 11-day human blastocysts is partially filled with a meshwork of extraembryonic endoderm, whereas such a meshwork is absent in the rhesus monkey. It is suggested that this endodermal meshwork develops as the result of interstitial implantation in the human embryo. A small secondary yolk sac develops in 12- to 13-day human and macaque embryos as the result of pinching off of a portion of the larger primary yolk sac. Development of a secondary yolk sac in higher primates appears to be related causally to differential rates of expansion of the blastocyst and primary yolk sac within the simplex uterus. The caudal margin of the primitive streak develops precociously in 12- to 14-day human and macaque embryos, and this appears to be the source of all the extraembryonic mesoderm of the chorion, chorionic villi, and body stalk. It is suggested that the peripheral spread of extraembryonic mesoderm plays in inductive role in the development of chorionic villi, similar to other types of epithelial-mesenchymal inductive interactions. In contrast to previous hypotheses, the human and macaque trophoblasts appear to give rise only to additional trophoblast.

Cladistic relationships among primate higher categories: evidence of the fetal membranes and placenta.

Cladistic analysis of the total ontogenetic pattern of the fetal membranes and placenta in all extant primate superfamilies provides clear evidence of a strepsirhine-haplorhine dichotomy in the order Primates. The suborder Prosimii appears to be a paraphyletic taxon, based on the retention of numerous primitive character states in tarsiers and strepsirhines. Fetal membrane evidence supports the sister group relationship of Tarsiiformes and Anthropoidea in the suborder Haplorhini, based on their possession of shared derived characters. Morphogenetic patterns of the fetal membranes and placenta in haplorhines are consistent with the concept of a monophyletic origin of Anthropoidea from an ancestral tarsilform stock.

The development of primordial and definitive amniotic cavities in early Rhesus monkey and human embryos.

Re-examination of early rhesus monkey and human embryos in the collection of the Carnegie Institution of Washington suggests that the mechanism of amniogenesis in both is basically similar to that of the hedgehog and vespertilionid bats. A primordial amniotic cavity develops by cavitation within the embryonic mass of 10-day rhesus monkey, and 7-day human, blastocysts. This primordial cavity has no relationship initially with the overlying trophoblast, contrary to earlier reports. Subsequently, there is a thinning and peripheral spreading of the epiblastic roof of the primordial cavity, resulting in partial opening of the roof and formation of a slightly cupped embryonic disc. The resulting space is not homologous with the primordial amniotic cavity; instead, it is a transitory tropho-epiblastic cavity. The definitive amniotic epithelium forms by the upfolding and mitotic proliferation of the margins of the epiblastic disc; this process is completed in 11-day rhesus, and 9-day human, blastocysts. Amniogenesis by cavitation is associated with the persistence of polar trophoblast following implantation, and it is suggested that this cavitation process may be essential for providing a free epithelial surface for the morphogenetic movement of epiblastic cells during subsequent formation of the primitive streak.