Susan A. Tarlé

Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse Minute.

Ribosomal protein mutations, termed Minutes, have been instrumental in studying the coordination of cell and tissue growth in Drosophila. Although abundant in flies, equivalent defects in mammals are relatively unknown. Belly spot and tail (Bst) is a semidominant mouse mutation that disrupts pigmentation, somitogenesis and retinal cell fate determination. Here, we identify Bst as a deletion within the Rpl24 riboprotein gene. Bst significantly impairs Rpl24 splicing and ribosome biogenesis. Bst/+ cells have decreased rates of protein synthesis and proliferation, and are outcompeted by wild-type cells in C57BLKS↔ROSA26 chimeras. Bacterial artificial chromosome (BAC) and cDNA transgenes correct the mutant phenotypes. Our findings establish Bst as a mouse Minute and provide the first detailed characterization of a mammalian ribosomal protein mutation.

Molecular basis of hypoxanthine-guanine phosphoribosyltransferase deficiency in ten subjects determined by direct sequencing of amplified transcripts.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency is an inborn error of purine metabolism. Mutant HPRT gene sequences from patients deficient in enzyme activity have previously been characterized by cDNA cloning or amino acid sequencing techniques. The presence of HPRT-specific mRNA in nearly all deficient subjects, as well as the small size of the HPRT mRNA (1,400 bp), make the polymerase chain reaction (PCR) an alternative for the identification of mutations at this locus. In this report we use the PCR to identify previously undetermined mutations in HPRT mRNA from B lymphoblasts derived from 10 deficient individuals. Six of these variants contain single point mutations, three contain deletions, and one contains a single nucleotide insertion. Several of these mutations map near previously identified HPRT variants, and are located in evolutionarily conserved regions of the molecule.

Stem cell therapy for craniofacial bone regeneration: a randomized, controlled feasibility trial.

Stem cell therapy offers potential in the regeneration of craniofacial bone defects; however, it has not been studied clinically. Tissue repair cells (TRCs) isolated from bone marrow represent a mixed stem and progenitor population enriched in CD90- and CD14-positive cells. In this phase I/II, randomized, controlled feasibility trial, we investigated TRC cell therapy to reconstruct localized craniofacial bone defects. Twenty-four patients requiring localized reconstruction of jawbone defects participated in this longitudinal trial. For regenerative therapy, patients were randomized to receive either guided bone regeneration (GBR) or TRC transplantation. At 6 or 12 weeks following treatment, clinical and radiographic assessments of bone repair were performed. Bone biopsies were harvested and underwent quantitative micro-computed tomographic (μCT) and bone histomorphometric analyses. Oral implants were installed, subsequently restored, and functionally loaded with tooth restorations. Reconstructed sites were assessed for 1 year following therapy. No study-related, serious adverse events were reported. Following therapy, clinical, radiographic, tomographic, and histological measures demonstrated that TRC therapy accelerated alveolar bone regeneration compared to GBR therapy. Additionally, TRC treatment significantly reduced the need for secondary bone grafting at the time of oral implant placement with a fivefold decrease in implant bony dehiscence exposure (residual bone defects) as compared to GBR-treated sites (p < 0.01). Transplantation of TRCs for treatment of alveolar bone defects appears safe and accelerates bone regeneration, enabling jawbone reconstruction with oral implants. The results from this trial support expanded studies of TRC therapy in the treatment of craniofacial deformities (ClinicalTrials.gov number CT00755911).

The human ZBP-89 homolog, located at Chromosome 3q21, represses gastrin gene expression

Rat ZBP-89cDNA encodes an 89-kDa, zinc finger protein thatbinds a GC-rich element in the human gastrin promoter. Thiselement modulates both basal- and epidermal growth factor(EGF)-induction of gastrin gene expression (Merchant et al. 1995).Coexpression of ZBP-89 and gastrin reporter constructs blocksEGF induction and represses basal gastrin gene expression (Mer-chant et al. 1996). Rat ZBP-89similarly binds to and represses theactivity of the ornithine decarboxylase (ODC) promoter, concomi-tantly inhibiting cell proliferation (Remington et al. 1997). ZBP-89shares 99% amino acid sequence identity with two closely relatedmouse homologs, G-rich box-binding protein (GRBBP; Passantinoet al. 1996) and BFCOL1 (Hasegawa and de Crombrugghe 1997).Mutations of the BFCOL1 binding site in the pro a2(I) collagenpromoter inhibit BFCOL1 binding and result in increased pro-moter activity (Hasegawa and de Crombrugghe, 1997). Collec-tively, these data show that rodent ZBP-89-related genes act astranscriptional repressors by binding to specific GC-rich promoterelements, and, at least in some cases, this activity exerts a regu-latory effect on cell proliferation.The human cDNA, htb, shares about 90% DNA sequenceidentity with the open reading frames of rodent ZBP-89homologs.However, two single-nucleotide deletions in htb cDNA, 38 of thezinc finger domain, cause premature stop codons. As a result, htbencodes a 494 rather than a 794-amino acid protein (Merchant etal. 1996; Wang et al. 1993). The truncated, 49-kDa, htb proteinacts as a transcriptional activator, moderately activating T-cellreceptor promoters (Wang et al. 1993). The structural and func-tional comparisons of rodent ZBP-89homologs with htb suggestedto us that the point deletions that distinguish htb may have orig-inated as somatic mutations of wild type human ZBP-89 in theJurkat cells from which the htb cDNA was isolated. To test thishypothesis, we isolated a full-length (∼89 kDa) human ZBP-89cDNA from a normal leukocyte library, compared genomic local-ization of the corresponding gene with that of htb, and determinedits effects on gastrin gene expression. We report that full-lengthZBP-89 maps to Chr 3q21 where the htb cDNA was mapped(Schuler et al. 1996). Furthermore, like its rat homolog, humanZBP-89 functions as a repressor of gastrin gene expression.The human ZBP-89 cDNA clone hZBP-89-16 contains a 2.4-kb insert and was isolated from a SuperScript (Life Technologies)leukocyte library by positive selection (GeneTrapper, Life Tech-nologies) with oligonucleotides from htb. The open reading frameof hZBP-89-16 encodes a 794-amino acid protein, similar to the ratand mouse homologs, and lacks the internal stop codons of htb.A600-bp,EcoRV fragment from the 58-UTR of ZBP-89-16 cDNAwas used to screen a human BAC library (Genome Systems, St.Louis, Mo.), and overlapping clones B469N19, B460C18, andB502J22 were isolated (Fig. 1). A subcloned 1.2-kbEcoRI frag-ment from B469N19 was sequenced and used to develop an STS(374F/794R) for PCR-based screening of the human CEPH YACDNA matrix pools (Research Genetics, Huntsville, Ala.). The re-sulting YAC/BAC contig shows that the human ZBP-89 genemaps to chromosome band 3q21 (144–146 cM on the genetic map)and, more precisely, is within 100 kb of D3S1551. This agrees withour FISH data showing hybridization to 3q21 using BACB469N19 as a probe (data not shown). This localization is also thesame as reported for htb (Schuler et al. 1996), suggesting that twoalternative forms of human ZBP-89-related protein are derivedfrom a single locus. To better assess this possibility, we analyzedthe coding sequences contained in the ZBP-89 BAC clones.The ZBP-89 DNA sequence derived from BAC B469N19 wascompared with those of related genes (Fig. 2). The human se-quence shares 97% amino acid sequence identity with correspond-

An OmpA-like protein from Acinetobacter spp. stimulates gastrin and interleukin-8 promoters.

ABSTRACT Bacterial overgrowth in the stomach may occur under conditions of diminished or absent acid secretion. Under these conditions, secretion of the hormone gastrin is elevated. Alternatively, bacterial factors may directly stimulate gastrin. Consistent with this hypothesis, we found that mice colonized for 2 months with a mixed bacterial culture of opportunistic pathogens showed an increase in serum gastrin. To examine regulation of gene expression by bacterial proteins, stable transformants of AGS cells expressing gastrin or interleukin-8 (IL-8) promoters were cocultured with live organisms. Both whole-cell sonicates and a heat-stable fraction were also coincubated with the cells. A level of 108 organisms per ml stimulated both the gastrin and IL-8 promoters. Heat-stable proteins prepared from these bacterial sonicates stimulated the promoter significantly more than the live organism or unheated sonicates. A 38-kDa heat-stable protein stimulating the gastrin and IL-8 promoters was cloned and found to be an OmpA-related protein. Immunoblotting using antibody to the OmpA-like protein identified an Acinetobacter sp. as the bacterial species that expressed this protein and colonized the mouse stomach. Moreover, reintubation of mice with a pure culture of theAcinetobacter sp. caused gastritis. We conclude that bacterial colonization of the stomach may increase serum gastrin levels in part through the ability of the bacteria to produce OmpA-like proteins that directly stimulate gastrin and IL-8 gene expression. These results implicate OmpA-secreting bacteria in the activation of gastrin gene expression and raise the possibility that a variety of organisms may contribute to the increase in serum gastrin and subsequent epithelial cell proliferation in the hypochlorhydric stomach.

Biochemical Basis for Dominant Inheritance, Variable Penetrance, and Maternal Effects in RBP4 Congenital Eye Disease.

Gestational vitamin A (retinol) deficiency poses a risk for ocular birth defects and blindness. We identified missense mutations in RBP4, encoding serum retinol binding protein, in three families with eye malformations of differing severity, including bilateral anophthalmia. The mutant phenotypes exhibit dominant inheritance, but incomplete penetrance. Maternal transmission significantly increases the probability of phenotypic expression. RBP normally delivers retinol from hepatic stores to peripheral tissues, including the placenta and fetal eye. The disease mutations greatly reduce retinol binding to RBP, yet paradoxically increase the affinity of RBP for its cell surface receptor, STRA6. By occupying STRA6 nonproductively, the dominant-negative proteins disrupt vitamin A delivery from wild-type proteins within the fetus, but also, in the case of maternal transmission, at the placenta. These findings establish a previously uncharacterized mode of maternal inheritance, distinct from imprinting and oocyte-derived mRNA, and define a group of hereditary disorders plausibly modulated by dietary vitamin A.

Tooth Storage, Dental Pulp Stem Cell Isolation, and Clinical Scale Expansion without Animal Serum

INTRODUCTION Dental pulp stem cells (DPSCs) have therapeutic potential for dentin and dental pulp regeneration. For regenerative approaches to gain clinical acceptance, protocols are needed to determine feasible ways to store teeth, isolate DPSCs, and expand them to clinical scale numbers. METHODS In this study, 32 third molars were obtained from patients and immediately placed in saline or tissue culture medium followed by overnight storage at 4°C or immediate isolation of DPSCs. Upon isolation, cells were expanded in medium containing either fetal bovine serum (FBS) or human serum (HS). Cell proliferation (population doubling time [PDT]), cell surface marker expression, and multipotency were compared between DPSCs in FBS and DPSCs in HS. RESULTS The time frame of storage and storage medium did not affect the ability to isolate DPSCs. However, using HS instead of FBS in the initial isolation of DPSCs significantly decreased (P < .01) the isolation success rate from 89% (FBS) to 23% (HS). Yet, incorporating fibronectin in the DPSC initial isolation (using HS) significantly (P < .01) increased the isolation success rate to 83%. Interestingly, it was found that the proliferation rate was significantly (P < .05) higher for DPSCs in HS (PDT = 1.59 ± 0.46) than that for DPSCs in FBS (PDT = 2.84 ± 2.5). Finally, there was no difference in the expression of CD73, CD90, CD105, or multipotency (as measured by osteogenic, adipogenic, and chondrogenic differentiation) between DPSCs in FBS and DPSCs in HS. CONCLUSIONS These findings show a clinically feasible method of storing third molars for the isolation of DPSCs. Additionally, DPSCs can be isolated and expanded to clinical scale numbers in media devoid of FBS and still maintain their phenotypic properties.

Dental Pulp Tissue Regeneration Using Dental Pulp Stem Cells Isolated and Expanded in Human Serum

Introduction Dental pulp–derived stem cells (DPSCs) have the potential to regenerate dentin and dental pulp tissue because of their differentiation capacity and angiogenic properties. However, for regenerative approaches to gain regulatory and clinical acceptance, protocols are needed to determine more feasible ways to cultivate DPSCs, namely, without the use of xenogeneic‐derived components (animal sera) and exogenous growth factors. Methods In this study, human DPSCs were isolated from third molars and expanded in standard culture conditions containing fetal bovine serum (DPSCs‐FBS) or conditions containing human serum (DPSCs‐HS). After cell characterization and evaluation of their angiogenic secretome, DPSCs were seeded in tooth slice/scaffolds and implanted subcutaneously into immunodeficient mice. After 30 days, tooth slices were retrieved and evaluated for dental pulp tissue regeneration. Immunohistochemistry and confocal microscopy were used to quantify blood vessel formation and evaluate predentin and dentin formation. Results After culture, DPSCs‐HS produced concentrations of angiogenic growth factors equivalent to DPSCs‐FBS. Additionally, in DPSCs‐HS, several angiogenic factors were produced in at least 1‐fold higher concentrations than in DPSCs‐FBS. In vivo, it was determined that DPSCs‐HS produced a robust angiogenic response and regeneration of dentin equivalent to DPSCs‐FBS. Conclusions These findings show that DPSCs can be isolated and expanded to clinical scale numbers in media devoid of animal serum or exogenous growth factors and still maintain their pulp regenerative properties. The implications of these findings are significant for further development of clinical protocols using DPSCs in cell therapies.

Human adenine phosphoribosyltransferase (APRT) deficiency: single mutant allele common to the Japanese.

Adenine phosphoribosyltransferase (APRT) is a purine salvage enzyme, which catalyzes the conversion of adenine to adenylic acid in the presence of phosphoribosylpyrophosphate (PRPP). In a complete APRT deficient subject, accumulated adenine is oxidized to 2, 8-dihydroxyadenine (2, 8-DHA) by xanthine oxidase. 2, 8-DHA excreted into urine precipitates and then forms a urinary stone.1 Partial deficiency develops no clinical symptoms. APRT deficiency is a common genetic disorder caused by a defective APRT gene on chromosome 16 and inherited in an autosomal recessive manner. The APRT gene, which is approximately 2.6 kb in length, consists of five exons and four introns. The frequency of heterozygotes has been estimated to be 0.4% – 1.2%.