Non-specific Gene Research Articles

Novel Mouse Model of Autosomal Semidominant Adult Hypophosphatasia Has a Splice Site Mutation in the Tissue Nonspecific Alkaline Phosphatase Gene Akp2

Deactivating mutations in the TNSALP gene cause HPP. Akp2(-/-) mice model severe infantile HPP, but there is no model for the relatively mild adult form. Here we report on mice with an induced mutation in Akp2 that affects splicing. The phenotype of homozygotes mirror aspects of the adult form of HPP. Hypophosphatasia (HPP) is a clinically varied skeletal disorder resulting from deficiency of tissue nonspecific alkaline phosphatase (TNSALP). Mice lacking Akp2 model infantile HPP characterized by skeletal hypomineralization, impaired growth, seizures, and perinatal mortality. No animal model exists to study the less severe forms of the disease that typically present in later life. N-ethyl-N-nitrosourea (ENU) mutagenesis was used to generate mouse models of human disease. A mouse with low plasma alkaline phosphatase (ALP) activity was identified by our clinical chemistry screen. Its offspring were used for inheritance studies and subjected to biochemical, histological, and radiological phenotyping. DNA was extracted for mapping and osteoblasts harvested for functional studies. We showed semidominant inheritance of the low ALP phenotype and mapped the underlying point mutation to Akp2. Affected offspring bear the splice site mutation 862 + 5G>A-a hypomorphic allele named Akp2(Hpp). The same mutation has been reported in a patient. Akp2(Hpp/+) mice have approximately 50% of normal plasma ALP but display no other biochemical or skeletal abnormalities. Unlike Akp2(-/-) mice, Akp2(Hpp/Hpp) mice have normal initial skeletal development and growth, a normal lifespan and do not have seizures. TNSALP is low but detectable in Akp2(Hpp/Hpp) plasma. Osteoblasts display approximately 10% of normal ALP activity and reduced intracellular inorganic phosphate levels, yet are capable of normal mineralization in vitro. TNSALP substrates are significantly elevated in urine (inorganic pyrophosphate and phosphoethanolamine) and plasma (pyridoxal 5'-phosphate), whereas plasma inorganic pyrophosphate levels are normal. Akp2(Hpp/Hpp) mice develop late-onset skeletal disease, notably defective endochondral ossification and bone mineralization that leads to arthropathies of knees and shoulders. Akp2(Hpp/Hpp) mice mirror a number of clinical features of the human adult form of HPP. These mice provide for the first time an animal model of late onset HPP that will be valuable in future mechanistic studies and for the evaluation of therapies such as those aimed at HPP.

A protocol for designing siRNAs with high functionality and specificity

Effective gene silencing by the RNA interference (RNAi) pathway requires a comprehensive understanding of the elements that influence small interfering RNA (siRNA) functionality and specificity. These include (i) sequence space restrictions that define the boundaries of siRNA targeting, (ii) structural and sequence features required for efficient siRNA performance, (iii) mechanisms that underlie nonspecific gene modulation and (iv) additional features specific to the intended use (i.e., inclusion of native sugar or base chemical modifications for increased stability or specificity, vector design, etc.). Attention to each of these factors enhances siRNA performance and heightens overall confidence in the output of RNAi-mediated functional genomic studies. Here, we provide a detailed protocol explaining the methodologies used for manual and web-based design of siRNAs.

Non-specific effects of siRNAs on tumor cells with implications on therapeutic applicability using RNA interference

Elimination of protein expression using RNA interference (RNAi) significantly improves the understanding of gene function and represents a promising technique for the treatment of diseases such as cancer and neurological disorders. Accumulating evidence suggests the so-called interferon-independent non-specific gene silencing of short interfering RNA (siRNA); however, its biological and functional cellular consequences are largely unidentified. We therefore analyzed the effects of different nonsense siRNAs on characteristic bio-parameters such as cell viability, proliferation, cell cycle distribution, apoptosis, and migration of tumor cells. All analyzed cellular aspects have been observed to be significantly affected by the presence of siRNA in an interferon-independent manner: viability, mitosis, and motility were significantly diminished and programmed cell death was significantly elevated. Moreover, all cell cycle stages (G0/G1-, G2/M-, and S-phase) were moderately shifted. Together, these results support the hypothesis that siRNA, due to sequence-specific cellular consequences, modulate bio-functionality independent of the target sequence. This phenomenon affects the design of siRNA experiments for future in vitro but also for in vivo tests as well as for potential therapeutic and preventive strategies. Moreover, monitoring interferon response after transfection of siRNAs is necessary but not sufficient to exclude potential off-target effects in non-diseased cells.

Mutations of the tissue non-specific alkaline phosphatase gene (TNAP) causing a non-lethal case of perinatal hypophosphatasia

Mutations of the tissue non-specific alkaline phosphatase gene (TNAP) causing a non-lethal case of perinatal hypophosphatasia

Gene therapy targeting to tumor endothelium

Tumor-associated vasculature is a relatively accessible component of solid cancers that is essential for tumor survival and growth, providing a vulnerable target for cancer gene therapy administered by intravenous injection. Several features of tumor-associated vasculature are different from normal vasculature, including overexpression of receptors for angiogenic growth factors, markers of vasculogenesis, upregulation of coagulation cascades, aberrant expression of adhesion molecules and molecular consequences of hypoxia. Many of these differences provide candidate targets for tumor-selective 'transductional targeting' of genetically- or chemically modified vectors and upregulated gene expression can also enable 'transcriptional targeting', regulating tumor endothelia-selective expression of transgenes following nonspecific gene delivery. Tumor vasculature also represents an important site of therapeutic action by the secreted products of antiangiogenic gene therapies that are expressed in non-endothelial cells. In this review we assess the challenges faced and the vectors that may be suitable for gene delivery to exploit these targets. We also overview some of the strategies that have been developed to date and highlight the most promising areas of research.

Association Between Nonspecific Airway Hyperresponsiveness and Arg16Gly β2-Adrenergic Receptor Gene Polymorphism in Asymptomatic Healthy Japanese Subjects

Nonspecific airway hyperresponsiveness (AHR), a cardinal feature of asthma, is thought to result from several genetic and environmental factors. Asymptomatic AHR in nonasthmatic healthy subjects might be a risk factor for the development of asthma. Genetic variations in codons 16 and 27 of the human beta(2)-adrenergic receptor (beta(2)-AR) alter receptor function in vitro and are associated with various asthma-related phenotypes, including asthma severity and AHR. To date, however, few reports have examined the impact of beta(2)-AR gene polymorphism on AHR in asymptomatic healthy subjects.To determine whether polymorphism of the beta(2)-AR gene (Arg16Gly and Gln27Glu) might influence nonspecific AHR in asymptomatic healthy Japanese subjects.A cohort of 120 asymptomatic healthy subjects was analyzed using a stepwise linear regression model. Nonspecific airway responsiveness was measured using a continuous methacholine inhalation method (Astograph; Chest; Tokyo, Japan). We used values of the cumulative dose of inhaled methacholine measured at the inflection point at which respiratory conductance starts to decrease (Dmin) as an index of AHR. Genotyping to identify polymorphisms at codons 16 and 27 was conducted using an assay combining kinetic real-time quantitative polymerase chain reaction with allele-specific amplification.The Gly16Gly genotype was associated with lower Dmin values. The log Dmin value of asymptomatic healthy subjects carrying the Arg16 allele (Arg16/Arg or Arg16/Gly, n = 90) was 1.09 +/- 0.56 (mean +/- SD), while those homozygous for the Gly16 allele (n = 30) yielded a log Dmin value of 0.85 +/- 0.56 (p < 0.05).This study indicates that a specific beta(2)-AR polymorphism at codon 16 might be a genetic determinant of AHR, as judged by methacholine-induced bronchoconstriction in asymptomatic healthy subjects.

Posttranscriptional modulation of the human tissue–nonspecific alkaline phosphatase gene expression by 1,25-dihydroxyvitamin D 3 in MG-63 osteoblastic osteosarcoma cells

Although tissue-nonspecific alkaline phosphatase (TNSALP) plays a key role in bone mineralization and 1,25-dihydroxyvitamin D 3 is an important dietary regulator of bone mineralization, relationships between TNSALP and vitamin D have not been well characterized. We studied the effect of 1,25-dihydroxyvitamin D 3 on the expression of the human TNSALP gene in a cell culture system. We tested several osteoblastic osteosarcoma cell lines but found that only MG-63 cells responded to 1,25-dihydroxyvitamin D 3. When MG-63 cells were cultured for 48 hours with 1,25-dihydroxyvitamin D 3 in a serum-free medium, vitamin D treatment prevented a decrease in alkaline phosphatase activity observed in nontreated cells. Real-time reverse transcriptase polymerase chain reaction showed that 1,25-dihydroxyvitamin D 3 caused conserved expression of TNSALP messenger RNA (mRNA) levels compared with nontreated cells. Despite this conserved expression of TNSALP mRNA levels, luciferase assays revealed that reporter plasmid containing a vitamin D response element-like sequence in the upstream region of the human TNSALP gene did not activate transcription. However, mRNA stability assay using 5,6-dichlorobenzimidazole 1- β- d-ribofuranoside as a transcription inhibitor showed that 1,25-dihydroxyvitamin D 3 increased the half-life of TNSALP mRNA approximately 2.4-fold. Thus, 1,25-dihydroxyvitamin D 3 modulated posttranscriptionally the human TNSALP activity and the mRNA expression by increasing mRNA stability in MG-63 osteoblastic osteosarcoma cells.

Interstitial cells and cholinergic signalling in the outer muscle layers of the guinea‐pig bladder

To explore the relationship between cholinergic mechanisms and interstitial cells (ICs) in the outer muscle layer of the bladder. In bladder tissue from male guinea-pigs, ICs were identified by their response to nitric oxide (NO) with a rise in cGMP. Sections of the lateral bladder wall were incubated in Krebs' solution containing 1 mm of the nonspecific phosphodiesterase inhibitor isobutyl-methyl-xanthene. Tissues were then exposed to 100 microm of the NO donor NONOate for 10 min, control tissues remained in Krebs' solution. Tissues were then processed for immunohistochemistry for cGMP, choline acetyltransferase (ChAT), neurofilament protein, and the nonspecific neuronal marker protein gene product (PGP) 9.5. cGMP-positive ICs were found mainly in the outer muscle layers of the bladder wall. Three types were identified based on location; on the outer surface of the bladder wall, on the surface of the muscle bundles, and within the muscle bundles. Some of the intramuscular ICs stained for ChAT, but they did not stain with PGP 9.5. Nerve fibres were seen in close contact with the ChAT-positive intramuscular ICs, and these nerves expressed ChAT and neurofilament protein. A subpopulation of intramuscular ICs can synthesise acetylcholine, and might release acetylcholine onto the underlying muscle. These cells are in close contact with nerves, suggesting that they might be activated by neural inputs. Thus there may be a system in the detrusor involving cholinergic nerves acting on ICs which can activate the smooth muscle via a complex cholinergic input.

60. Radiation Improves Local Delivery of a Virus-Mimicking Nanostructure Complex in Tumors

Efficiency and specificity of gene delivery to tumors are major concerns in cancer gene therapy. We have previously used regional ionizing radiation to improve adenovirus uptake and to enhance gene expression in intrahepatic tumors (Zhang, M, et al. Mol Ther. 2003, 8:21-28; Qian, J, et al. Cancer Res. 2005, 65:5493-5497). However, non-specific gene expression and cytotoxicity in normal liver is still inevitable. Here, we explore the potentials of radiation in improving gene delivery by a virus-mimicking nanostructure complex, transferrin (Tf)-cationic liposome-DNA complex (Tf- lipoplex) (Liang, Xu, et al.

84. RNA Silencing: Role of Interferon System Components in Non_Specific Effects Induced by Chemically Synthesized Small Interfering RNAs, Enzymatically Synthesized Small Interfering RNAs and Short Hairpin RNAs

In mammalian cells, long double-stranded RNAs (dsRNAs) activate interferon (IFN) signaling as well as interferon inducible RNA-dependent enzymes that affect translational activity including the protein kinase (PKR) and 2',5'-oligoadenylate synthetases (2',5'-OAS). DsRNA also is the mediator of RNA interference (RNAi), a widely used gene silencing strategy with utility both as a basic research tool and potential therapeutic. RNAi mediated by short dsRNA molecules (|[sim]|20-bp) was originally thought to circumvent the IFN response to achieve specific target silencing without triggering potentially problematic non-specific effects. However, reports suggest that some small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) produced from transfected plasmids can induce cytokine signaling and cause other non-specific effects. We therefore undertook a further combined genetic and biochemical examination of the involvement of IFN system components in RNAi to directly address the potential involvement of IFN-regulated gene products in non-specific responses to siRNAs. We examined the contribution of IFN signaling, PKR and RNase L to both on-target and non-specific gene silencing as well as induction of cytokines induced by three RNAi strategies. We show, through the use of cell lines genetically deficient in signaling and action components of the IFN system, that chemically synthesized siRNAs, T7 RNA polymerase-synthesized siRNAs, and shRNAs differentially activate IFN signaling and non-specific gene silencing. ShRNAs with up to 50 nucleotides complementary to a target gene mediated specific silencing and did not trigger nonspecific gene silencing. While the plasmids that delivered the shRNA constructs induced IFN-|[beta]| in a wild type cell line sensitive to CpG DNA, there was no differential induction of IFN-|[beta]| caused by the shRNAs in this cell type or other cell types not sensitive to CpG DNA. By contrast, both 20-bp and 50-bp T7 synthesized siRNAs caused significant non-specific gene silencing and induced higher levels of IFN-|[beta]| in wild type cells than chemically synthesized siRNAs. The IFN-|[beta]| induction capacity of the T7 synthesized siRNAs varied between preparations and could be reduced by gel purification of the siRNA. Chemically synthesized siRNAs caused non-specific gene silencing only at extremely high concentration and did not induce significant IFN-|[beta]| production in any cell type tested. Non-specific siRNA-mediated silencing was independent of PKR and RNase L, and only partially dependent upon IFN receptormediated signaling. We also found a relationship between the nonspecific gene silencing and the induction of apoptosis by the different RNAi mediators in different cell lines.

Haloperidol-associated Stealth Liposomes: A POTENT CARRIER FOR DELIVERING GENES TO HUMAN BREAST CANCER CELLS

Sigma receptors are membrane-bound proteins that are overexpressed in certain human malignancies including breast cancer. These receptors show very high affinity for various sigma ligands including neuroleptics like haloperidol. We hypothesized that in associating haloperidol-linked lipid into the cationic lipid-DNA complex, we can specifically target and deliver genes to breast cancer cells that overexpress sigma receptors. In the present study, haloperidol was chemically modified to conjugate at the distal end of the polyethylene glycollinked phospholipid, which was then incorporated into the cationic liposome known to condense and deliver genes inside cells. The resulting haloperidol-conjugated targeted lipoplex showed at least 10-fold higher (p < 0.001) reporter gene expression in MCF-7 cells than control lipoplex. The reporter gene expression of the targeted lipoplex was significantly blocked by haloperidol (p < 0.001) and by another sigma ligand, 1,3-ditolylguanidine (p < 0.001) in the majority of cationic lipid to DNA charge ratios (+/-). Spironolactone-mediated sigma receptor down-regulation enabled MCF-7 to show 10-fold lower transgene expression with targeted lipoplex compared with that obtained in spironolactone-untreated cells. The targeted lipoplex generated nonspecific gene expression in sigma receptor-nonexpressing human cancer cells such as Hela, KB, HepG2, and Chinese hamster ovary cells. Moreover, the transgene expression remained unabated in physiologically relevant serum concentrations. This is the first study to demonstrate that haloperidol-targeted gene delivery systems can mediate efficient targeting of genes to sigma receptor-overexpressing breast cancer cells, thereby becoming a novel class of therapeutics for the treatment of human cancers.

Optimization of radiation controlled gene expression by adenoviral vectors in vitro

The radiation-inducible EGR-1-promoter has been used in different gene therapy approaches in order to enhance and locally restrict therapeutic efficacy. The aim of this study was to reduce nonspecific gene expression in the absence of irradiation (IR) in an adenoviral vector. Rat rhabdomyosarcoma R1H tumor cells were infected with adenoviral vectors expressing either EGFP or HSV-TK under control of the murine EGR-1 promoter/enhancer. Cells were irradiated at 0-6 Gy. Gene expression was determined by FACS-analysis (EGFP), or crystal violet staining (HSV-TK). The bovine growth hormone polyadenylation signal (BGH pA) was used as insulating sequence and was introduced upstream or upstream and downstream of the expression cassette. Infected R1H cells displayed IR dose-dependent EGFP expression. Cells treated with IR, AdEGR.TK and ganciclovir displayed a survival of 17.3% (6 Gy). However, significant gene expression was observed in the absence of IR with EGR.TK and EGR.EGFP constructs. Introduction of BGHpA upstream or upstream and downstream of expression cassette resulted in decreased nonspecific cytotoxicity by a factor of 1.6-2.3 with minor influence on the induced level of cytotoxicity. Introduction of insulating sequences in adenoviral vectors might allow tighter temporospatial control of gene expression by the radiation-inducible EGR-1 promoter.

Characterization of 11 novel mutations in the tissue non-specific alkaline phosphatase gene responsible for hypophosphatasia and genotype–phenotype correlations

Hypophosphatasia is an inherited disorder caused by mutations in the bone alkaline phosphatase gene. We report here 11 new mutations responsible for hypophosphatasia. Four of them were deletions or insertions resulting in frameshift, two affected a donor splice site and five were missense mutations. Site-directed mutagenesis and transfection experiments of missense mutations showed that the mutations resulted in loss of most enzymatic activity, confirming the disease-causing role of these mutations. Analysis of the 3D model of tissue non-specific alkaline phosphatase showed that among the five missense mutations, one affected a residue in the crown domain and four affected residues located in the calcium-binding region. Alignment of the protein sequences of the calcium-binding region from 11 species showed that the four residues coordinating the calcium ion and the residues affected by the missense mutations described here are conserved in vertebrates. Together, our results confirm the functional role of the calcium site and suggest that its function is likely to be specific to vertebrates.

Asymmetric Allosteric Activation of the Symmetric ArgR Hexamer

Hexameric arginine repressor, ArgR, bound to L-arginine serves both as the master transcriptional repressor/activator at diverse regulons in a wide range of bacteria and as a required cofactor for resolution of ColE1 plasmid multimers. Multifunctional ArgR is thus unusual in possessing features of specific gene regulators, global regulators, and non-specific gene organizers; its closest functional analog is probably CAP, the cyclic AMP receptor/activator protein. Isothermal titration calorimetry, surface plasmon resonance, and proteolysis indicate that binding of a single L-argine [corrected] per ArgR hexamer triggers a global conformation [corrected] change and resets the affinities of the remaining five sites, making them 100-fold weaker. The analysis suggests a novel thermodynamic signature for this mechanism of activation.

Mesothelin-mediated targeting of adenoviral vectors for ovarian cancer gene therapy

Adenoviruses (Ads) are efficient gene transfer vehicles, but Ad-mediated gene therapy for ovarian cancer remains limited in vivo by inefficient and nonspecific gene transfer. Mesothelin (MSLN), a cell surface glycoprotein, is overexpressed in ovarian cancer but not in normal tissues except mesothelial cells. Therefore, MSLN is an attractive candidate for transcriptional and transductional targeting in the context of ovarian cancer gene therapy. We evaluated the expression of MSLN mRNA and MSLN surface protein in ovarian cancer cells. Ads containing the MSLN promoter driving reporter gene expression were created and tested in ovarian cancer cell lines and purified ovarian cancer cells isolated from patients. To evaluate transductional targeting, we used an Ad vector containing an Fc-binding domain within the fiber protein, which served as a docking domain for binding with anti-MSLN immunoglobulins. Both RT-PCR and flow cytometry revealed high MSLN gene and protein expression in ovarian cancer cells. The MSLN promoter was activated in ovarian cancer cells, but showed significantly reduced activity in normal control cells. Transductional targeting of Ads via anti-MSLN antibody increased transgene expression in ovarian cancer cells. This report describes the use of MSLN for transcriptional as well as transductional targeting strategies for ovarian cancer gene therapy.

Identification of a 49-bp fragment of the HvLTP2 promoter directing aleurone cell specific expression

Identification of regulatory elements directing definite and specific spatiotemporal expression patterns is a prerequisite to the next generation of transgenic plants with commercial and ethical feasibility for producing plantibodies or other pharmaceutically important compounds. Here we describe the functional dissection of the barley nonspecific lipid transfer protein gene promoter, HvLTP2. The gene is specifically expressed in aleurone cells of cereals and used as an aleurone marker in maize and rice. The transcript is uniformly localised in the barley aleurone cells from around 10 DAP. Patchy expression in aleurone cells of transgenic rice has been reported and explained by silencing of transgenes. We have performed deletion analyses of the 801-bp HvLTP2 promoter to gain insight into the molecular basis of its regulation and the presence of putative regulatory elements. From the deletion studies, a 49-bp promoter region directing aleurone-specific expression was identified. Simultaneously, in vivo footprinting was carried out to identify promoter elements bound by putative regulatory proteins. Within the 49-bp fragment, the most promising candidate for a minimal cis-acting regulatory region directing aleurone specificity is the ds-sequence. Based on our results, we hypothesise that the ds-sequence directs aleurone specificity, possibly through a concerted action with elements directing general expression in the seed. Moreover, we present an overview of LTP2 elements putatively involved in directing seed, endosperm, and aleurone expression. Additionally, we report HvLTP2 expression in the embryo, not previously detected. The regulatory element(s) directing expression in embryo is located downstream of the 49-bp fragment directing aleurone specificity, thus demonstrating independent control of aleurone and embryo-localised expression. Finally, we discuss the existence of several endosperm-specific boxes and whether alternative promoter elements and combinations of them may direct aleurone expression, explaining why comparing different genes expressed in aleurone fail to identify only one required, common promoter element.

DNA methylation of multiple genes in gastric carcinoma: association with histological type and CpG island methylator phenotype.

Hypermethylation of CpG islands is associated with silencing of various tumor suppressor genes. Recent studies on colorectal and gastric cancer have identified a CpG island methylator phenotype (CIMP), which involves the targeting of multiple genes by promoter hypermethylation. For determination of association between DNA methylation pattern or histological type and CIMP status in gastric carcinoma, CpG islands in the promoters of hMLH1 and CDH1 genes, CpG islands overlapping exon 1 of MGMT and p16(INK4a) genes, and a non-CpG island in exon 1 of the RAR-beta gene were studied. The presence of the CIMP was determined by monitoring five methylated in tumor (MINT ) loci in 103 gastric carcinomas. Among the 103 gastric carcinomas, DNA hypermethylation was detected in the following frequencies: 14 (14%) for hMLH1, 26 (25%) for MGMT, 26 (25%) for p16(INK4a), 54 (52%) for CDH1, and 53 (52%) for RAR-beta. Forty-two (41%) of 103 gastric carcinomas were positive for the CIMP. CIMP and hypermethylation of p16(INK4a) gene were found more frequently in intestinal and diffuse-adherent types than in diffuse-scattered type (P = 0.013 and 0.017, respectively). In contrast, hypermethylation of the CDH1 and RAR-beta genes was more common in the diffuse-scattered type than in the other types (P = 0.008 and 0.007, respectively). In intestinal- and diffuse-adherent-type gastric carcinomas, we found significant associations between the presence of the CIMP and hypermethylation of several genes: hMLH1 (P = 0.006), p16(INK4a) (P = 0.018), CDH1 (P = 0.024), and RAR-beta (P = 0.044). Our overall results suggest that in some intestinal- and diffuse-adherent-type gastric carcinomas, DNA hypermethylation affects non-specific gene promoters concordantly, at least in part, whereas in diffuse-scattered-type gastric carcinoma, DNA hypermethylation affects specific genes such as CDH1 and RAR-beta.

Development of Respiratory Syncytial Virus “Bronchiolitis” in Guinea Pigs Does Not Reflect an Allergic Predisposition in the Host

Respiratory syncytial virus (RSV) infection causes bronchiolitis in a minority of children. Using a guinea pig model to determine if an allergic predisposition in the host increases permissiveness to RSV infection or severity of experimental "bronchiolitis," we compared the effects of RSV inoculation between strain 2 (allergy-resistant) and strain 13 (allergy-susceptible) inbred animals. One month-old, juvenile guinea pigs were classified into four groups (eight guinea pigs per group): (group 1) strain 2, uninfected; (group 2) strain 13, uninfected; (group 3) strain 2, RSV infected; and (group 4) strain 13, RSV infected. Seven days after inoculation, the animals were studied by the following: viral plaque assays for quantification of intrapulmonary RSV; immunohistochemical localization of RSV antigens in lung tissue sections; physiologic assessment of airway obstruction and nonspecific bronchial hyperresponsiveness; quantitative histology of airway T lymphocytes, neutrophils, and eosinophils; and semiquantitative reverse transcriptase-polymerase chain reaction for levels of messenger RNA expression of a panel of proinflammatory cytokines and chemokines. Significantly higher titers of replicating RSV were isolated from the lungs of strain 13 vs strain 2 animals (p < or = 0.001). The two guinea pig strains showed similar cell types with positive viral immunostaining; RSV-associated changes in airway obstruction and nonspecific bronchial hyperresponsiveness; airway T cells, neutrophils, and eosinophils; and messenger RNA expression of cytokines and chemokines. Strain 13 guinea pigs show increased pulmonary RSV replication than strain 2 animals, but this increased permissiveness to the virus is not reflected by more severe virus-induced changes in airway obstruction, nonspecific bronchial hyperresponsiveness, airway inflammation, or gene expression of proinflammatory cytokines and chemokines.

Overcoming obstacles in DNA sequencing of expression plasmids for short interfering RNAs.

Overcoming obstacles in DNA sequencing of expression plasmids for short interfering RNAs.

Gonadoblastoma in a patient with del(9)(p22) and sex reversal: report of a case and review of the literature

Studies of distinct clinical prototypes have significantly contributed to our understanding of evolutionary abnormalities and their association with neoplasia. We describe a phenotypic female, aged 20 years at report, who was examined as an infant for developmental retardation. The clinical characteristics of the 9p− syndrome were present and the external genitalia were those of a normal female. The karyotype was 46XY,del(9)(p22). The parental karyotypes were normal. No SRY deletion or mutation was detected. Sonography showed the presence of a uterus. Basal luteinizing hormone values were normal; follicle stimulating hormone values were high (40 IU/L). Stimulation with human chorionic gonadotropin did not produce any rise in testosterone. The gonads were removed and histologic analysis disclosed dysgenetic gonads with gonadoblastoma in situ. This case constitutes the fourth case of gonadoblastoma developing in an individual with 9p− syndrome and sex reversal. This and analogous prototypes point to a locus (or loci) on the short arm of chromosome 9, which either constitutes a nonspecific suppressor gene or a gonadoblastoma suppressor gene. An alternative hypothesis would be that a gonad not normally differentiated is more prone to gonadoblastoma development without any specific gene involvement.