Widened clinical spectrum of the Q128P MECP2 mutation in Rett syndrome.

CASE REPORT: We describe a female patient with Arnold Chiari type I malformation, atypical Rett syndrome characterized by postnatal onset microcephaly, stereotypic hand movements, ataxia, severe developmental delay, intractable tonic-clonic seizures, and a MECP2 mutation with a unique set of clinical findings. Implementation of a ketogenic diet resulted in decreased seizure activity and an improvement in the patient's degree of social relatedness with her family members. DISCUSSION: An early diagnosis of Rett syndrome allows families to maximize utilization of existing treatment modalities and seek appropriate genetic counseling and prenatal diagnoses. This case also provides further evidence for the treatment benefit of ketogenic diets for seizures in patients with Rett syndrome.

Implication of interfering antibody formation and apoptosis as two different mechanisms leading to variable duration of adenovirus-mediated transgene expression in immune-competent mice.

This study explores the genetic and immunologic factors involved in the differences in duration of transgene expression following in vivo transduction with recombinant adenoviruses. Different strains of mice (C3H/HeJ [C3H], C57BL/6J [B6], BALB/cJ [Balb/c], C. B10-H2(b)/LiMcdJ [Balb.B], CB6F1/J [(Balb/c x B6)F1], B6C3F1/J [(B6 x C3H)F1], and BALB/cj SCID) received 5 x 10(9) PFU of the first-generation adenovirus, which expresses human alpha1-antitrypsin (Ad/RSVhAAT). While all strains studied showed similar patterns of anti-adenovirus antibody formation, only Balb/c and C3H mice developed significant levels of anti-hAAT antibodies by 8 weeks posttransduction. In addition, while all strains had quantitatively comparable amounts of adenovirus genomes and hAAT mRNA transcripts in the liver 9 days posttransduction, only Balb/c mice had undetectable adenovirus vector genomes and hAAT mRNA in the liver 40 days posttransduction. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining of liver sections from control and Ad/RSVhAAT-infected mice 5, 9, and 40 days posttransduction suggested that apoptosis was involved in the rapid elimination of transduced hepatocytes in Balb/c mice. Persistent expression of hAAT protein observed in BALB/cj SCID mice suggests that antigen-dependent immunity was essential for this apoptotic process in transduced Balb/c hepatocytes. In contrast to Balb/c mice, the loss of expression in C3H mice did not correlate with the loss of vector genomes or hAAT mRNA. Instead, the anti-hAAT antibodies in C3H but not Balb/c mice were found to interfere with detection of hAAT in the serum. In Balb. B and B6 mice, vector genome, hAAT mRNA transcripts, and hAAT protein levels persisted for at least 40 days posttransduction. This persistence correlated with poor anti-hAAT antibody formation and minimal hepatocyte toxicity. The expression of hAAT in (Balb/c x B6)F1 pups was found to be intermediate between the duration observed in the parental strains, while in (C3H x B6)F1 pups hAAT expression was similar to that seen in the B6 parents, which together support polygenic control of the immune responses in these mice. In summary, these findings suggest that there are three different profiles and at least two defined immune system-mediated mechanisms resulting in the loss of hAAT expression in mice and that different strains differ in the capacity to utilize these mechanisms.

Hepatocyte growth factor induces hepatocyte proliferation in vivo and allows for efficient retroviral-mediated gene transfer in mice.

Recombinant retroviral vectors are an attractive means of transferring genes into the liver because they integrate into the host cell genome and result in permanent gene expression. However, efficient in vivo gene transfer is limited by the requirement of active cell division for integration. Traditional approaches to induce liver proliferation have the disadvantage of inducing hepatocellular injury by delivery of toxins or by surgical partial hepatectomy. As a nontraumatic alternative, we show that exogenous hepatocyte growth factor (HGF) is a powerful and safe mitogen for the mature intact murine liver when delivered continuously into the portal vein. A 5-day infusion of human HGF (5 mg/kg/d) resulted in > 140% increase in relative liver mass, which returned to normal in 4 to 5 weeks. This clearly shows that an exogenous growth factor can induce robust liver proliferation in vivo. In addition, we show that the HGF-induced proliferation was independent of interleukin-6, an essential cytokine involved in liver regeneration after partial hepatectomy. When recombinant retroviral vectors were infused in combination with HGF, 30% of hepatocytes were stably transduced with no indication of hepatic injury or histopathology. These results show the ability to obtain a clinically relevant transduction efficiency with retroviral vectors in vivo without the prior induction of liver injury. The level of hepatic gene transfer achieved has the potential to be curative for a large number of genetic liver diseases.

Constitutive expression of murine CTLA4Ig from a recombinant adenovirus vector results in prolonged transgene expression.

The administration of soluble muCTLA4Ig around the time of adenovirus vector mediated gene transfer into murine hepatocytes has been shown to markedly prolong transgene expression, diminish the formation of adenovirus neutralizing antibody, decrease T cell proliferative response and infiltration into the liver without causing irreversible systemic immunosuppression. In this study, an E1/E3-deleted adenovirus vector constitutively expressing murine CTLA4Ig (Ad.RSV-muCTLA4Ig) was constructed in order to determine if production of muCTLA4Ig from within transduced cells (i.e. hepatocytes) would provide a more specific/localized interference with the CD28/B7-1 and B7-2 signaling pathways, and thus result in prolonged transgene expression in vivo at nonimmunosuppressive serum concentrations. In contrast to C3H mice receiving a control adenovirus, transduction with 6 x 10(9) p.f.u. of Ad.RSV-muCTLA4Ig and a reporter adenovirus (2 x 10(9) p.f.u. of Ad.PGK-hAAT) resulted in prolonged reporter gene expression, reduced anti-adenovirus and anti-hAAT antibody production, and attenuated T cell proliferation and IFN-gamma production in response to adenoviral vector. Mice given a constant total amount of adenovirus with diminishing amounts of Ad.RSV-muCTLA4Ig and a constant amount of reporter virus (2 x 10(9) p.f.u. of Ad.PGK-hAAT) demonstrated prolonged reporter gene expression and decreased anti-adenovirus and anti-hAAT antibody production only when high serum levels of muCTLA4Ig were produced. Taken together, these findings suggest that a certain threshold of muCTLA4Ig must be achieved to alter the immune responses and prolong transgene expression from adenoviral vectors.

Heterologous expression of adenovirus E3-gp19K in an E1a-deleted adenovirus vector inhibits MHC I expression in vitro, but does not prolong transgene expression in vivo.

An E1a-deleted adenovirus vector constitutively expressing native adenovirus E3-gp19K (Ad.RSV-gp19K) was constructed in order to determine whether or not E3-gp19K mediated interference with antigen presentation would result in prolonged transgene expression in vivo. Cultured fibroblasts infected with Ad.RSV-gp19K produced a native size gp19K protein and had decreased cell surface levels of MHC I as shown by immunoprecipitation and flow cytometry. The congenic mouse strains Balb/b (H-2b MHC I with high gp19K affinity), Balb/k (H-2k MHC I with no gp19K affinity), and Balb/c (H-2d MHC I with moderate gp19K affinity) were chosen for in vivo experiments because of their range of gp19K affinities. Following transduction of mice form each strain with Ad.RSV-gp19K and AD/RSV-hAAT (a reporter adenovirus), or Ad/RSV-cFIX (control adenovirus) and Ad/RSV-hAAT, the level and duration of serum hAAT protein were unrelated to gp19K protein expression. Evaluation of MHC I abundance on hepatocytes following in vivo transduction demonstrated that recombinant adenovirus rapidly increased the abundance of surface MHC I molecules on hepatocytes, and surface MHC I molecules were reduced earlier and to a greater extent following wild-type adenovirus infection compared with hepatocytes transduced with control or Ad.RSV-gp19K recombinant adenovirus. This difference in surface MHC I down-regulation may be related to the different promoters (RSV-LTR versus the native E3 promoter) and will be an important consideration in the development of newer generation adenovirus vectors designed to evade host immune responses.

Facio-oculo-acoustico-renal (FOAR) syndrome: case report and review.

We report on an 11-year-old boy with distinct facial anomalies, iris coloboma, iris hypoplasia, cataract, high myopia, retinal detachment, moderate sensorineural hearing loss, and proteinuria. He appears to have the facio-oculo-acoustico-renal (FOAR) syndrome, a rare familial disorder reported only 4 times previously. In contrast to the other patients, he has normal intellect.

Characterization of progesterone receptor binding to the 90- and 70-kDa heat shock proteins.

In this study a model system for expression of the chicken progesterone receptor in cultured cells was developed using a quail fibroblast cell line, QT6. The chicken progesterone receptor form A expressed in QT6 cells was evaluated and determined to have a number of similarities to receptor isolated from chicken oviduct. These include hormone binding, sedimentation profile, phosphorylation pattern, heat shock protein (hsp) 70 and hsp90 associations and the ability to stimulate a reporter gene construct. Therefore, the receptor expressed in this system functioned adequately for further evaluation of the particular region (or regions) involved in hsp70 and hsp90 binding. Several receptor deletion mutants were tested for hsp70/hsp90 binding; only the d369-659 mutant, which has the entire steroid-binding domain deleted, was unable to bind hsp90 and hsp70. Three separate regions of the steroid-binding domain were found to partially restore hsp90 and hsp70 binding to the d369-659 mutant protein. However, hsp binding was not abolished when these or other regions of the steroid binding domain were deleted individually. These findings indicate that hsp90 and hsp70 both bind to the steroid-binding domain of the receptor through interactions at multiple locations or through some structural quality that is distributed throughout this region of the protein.

Reconstitution of progesterone receptor with heat shock proteins.

Nonactivated chick progesterone receptor from hypotonic tissue extracts exists in a large complex containing the heat shock proteins hsp90 and hsp70 plus additional smaller proteins; activation of receptor to a DNA-binding form involves the dissociation of proteins from the complex. Whereas numerous attempts to reversibly bind components to the activated receptor have been unsuccessful, we now report conditions that promote the reassociation of hsp90 and hsp70 to progesterone receptor. Cytosolic receptor was dissociated from hsp90 and hsp70 by treatment with 0.5 M KCl and 10 mM ATP in the absence of progesterone. It was then purified by binding to immunoaffinity resins. After wash steps, the receptor-resin complex was incubated in rabbit reticulocyte lysate at 30 C, rewashed, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Saturable binding of rabbit hsp90 and hsp70 to chick receptor was found after incubation with reticulocyte lysate; hsp binding was temperature dependent, but not dependent on exogenous ATP. Incubation of dissolved receptor with oviduct cytosol, from which receptor was obtained, or with purified hsp did not result in hsp binding. Furthermore, mixing oviduct cytosol with lysate inhibited hsp reconstitution, suggesting negative factors for hsp binding in oviduct cytosol. The steroid-binding domain of the receptor was required, since no hsp binding was observed in the reconstitution system using a receptor mutant lacking this domain. When the receptor was isolated in the presence of progesterone, reconstitution with hsp90 and hsp70 did not occur. This is consistent with the in vivo effects of progesterone in promoting hsp dissociation.

A method of sequencing without subcloning and its application to the identification of a novel ORF with a sequence suggestive of a transcriptional regulator in the water mold Achlya ambisexualis.

Genomic amplification with transcript sequencing (GAWTS) is a method of direct sequencing that involves amplification with PCR using primers containing phage promoters, transcription of the amplified product, and sequencing with reverse transcriptase. GAWTS requires the generation of PCR primers that are specific for the sequences on both sides of a region. Here we describe promoter ligation and transcript sequencing (PLATS), a direct method for rapidly obtaining novel sequences that utilizes generic primers and only requires knowledge of the sequence on one side of a region. PLATS involves restriction digestion of the amplified vector insert, ligation with a phage promoter, and then GAWTS using phage promoter sequences as the PCR primers. The method is rapid and economical because it uses a limited set of oligonucleotides, and it is potentially amenable to automation because it does not require in vivo manipulations. PLATS facilitates the determination of a genomic sequence responsible for cross-hybridization in a Southern blot. Using PLATS, sequence has been obtained from a 1.1-kb segment in Achlya ambisexualis, which cross-hybridizes to the DNA-binding region of the chicken and Xenopus estrogen receptors. To our knowledge, this represents the first sequence reported from the Oomycetes, a large and widely distributed group of fungi. The sequence reveals a large, transcribed open reading frame that is markedly deficient in the dinucleotide TpA. A putative zinc finger containing three cysteines and one histidine (C-X2-C-X12-H-X3-C) and an acidic segment hint that this clone may be a member of a novel class of transcriptional regulators.

The generation of radiolabeled DNA and RNA probes with polymerase chain reaction.

By including a radioactive triphosphate during polymerase chain reaction (PCR), probes of very high specific activity can be generated. The advantages of PCR labeling include (1) uniform labeling with a specific activity of 5 X 10(9) cpm/micrograms or higher (sensitivity of detection: 0.028 pg of target DNA per 24 h); (2) ease of regulation of both the specific activity and the amount of labeled probe produced; (3) efficient labeling of fragments less than 500 bp; (4) efficient incorporation over a wide range of input DNA template; (5) labeling with subnanogram amounts of input DNA; and (6) direct labeling of genomic DNA. The minimal amount of input DNA allows a virtually unlimited number of PCR labeling reactions to be performed on DNA generated by one amplification under the previously described nonlabeling conditions. This obviates the need for CsCl gradients or other large scale methods of DNA preparation. The above advantages except for the very high specific activity can also be achieved by transcript labeling after an amplification where one or both of PCR primers contain a phage promoter sequence.

Chemical and catalytic properties of the peroxisomal acyl-coenzyme A oxidase from Candida tropicalis.

The peroxisomal acyl-CoA oxidase has been purified from extracts of the yeast Candida tropicalis grown with alkanes as the principal energy source. The enzyme has a molecular weight of 552,000 and a subunit molecular weight of 72,100. Using an experimentally determined molar extinction coefficient for the enzyme-bound flavin, a minimum molecular weight of 146,700 was determined. Based on these data, the oxidase contains eight perhaps identical subunits and four equivalents of FAD. No other beta-oxidation enzyme activities are detected in purified preparations of the oxidase. The oxidase flavin does not react with sulfite to form an N(5) flavin-sulfite complex. Photochemical reduction of the oxidase flavin yields a red semiquinone; however, the yield of semiquinone is strongly pH dependent. The yield of semiquinone is significantly reduced below pH 7.5. The flavin semiquinone can be further reduced to the hydroquinone. The behavior of the oxidase flavin during photoreduction and its reactivity toward sulfite are interpreted to reflect the interaction in the N(1)-C(2)O region of the flavin with a group on the protein which acts as a hydrogen-bond acceptor. Like the acyl-CoA dehydrogenases which catalyze the same transformation of acyl-CoA substrates, the oxidase is inactivated by the acetylenic substrate analog, 3-octynoyl-CoA, which acts as an active site-directed inhibitor.

A serological survey of Leptospira interrogans serotype pomona in Alberta and Saskatchewan striped skunks and possible transmission between cattle and skunks.

The range of known occurrence of Leptospira interrogans serotype pomona is extended to Alberta in striped skunks (Mephitis mephitis); no evidence of L. sejroe was found. Reacting sera from skunks were confined to the southern portion of Alberta and adjacent Saskatchewan, although a number of reactors were found sufficiently further north in Saskatchewan suggesting that a different mode of infection may be functioning there. Of 95 skunk sera from near a dairy farm infected with serotype pomona 40% were reactors. Of 438 skunk sera from other areas only 5.7% were reactors; that difference was suggestive of transmission from cattle to skunks on the dairy farm. Of 22 skunk sera collected near the dairy farm in summer none were reactors, whereas 52% of skunk sera taken the previous and following winters were. That seasonal difference was not evident among sera from other locations.

Prevalence of antibodies to Toxoplasma gondii in striped skunks from Saskatchewan and Alberta.

Fifteen percent (81 of 542) of striped skunks, Mephitis mephitis, collected in the prairie of Alberta and Saskatchewan during 1974 to 1978, were positive for antibodies against Toxoplasma gondii. The seropositive rates varied from 8% (6 of 78) for skunks less than six months of age to 47% (9 of 19) in animals three or more years old. Spring and summer transmission was indicated by a preponderance of high titres (greater than or equal to 1:1024) in seropositive skunks collected April through September (22 of 40, 55%) compared to seropositives collected October through MFarch (10 of 38, 26%) (P = < 0.05). Prevalence was significantly greater among skunks collected in the relatively humid parkland (63 of 286, 22%) than in the arid prairie grassland biome (20 of 225, 8%) (P = < 0.01). The results indicate that T. gondii is focally enzootic in Alberta and Saskatchewan.

Characteristics of bat rabies in Alberta.

Rabies in bats was monitored in Alberta from 1971 to 1978 Big brown bats replaced silver-haired bats as the species most frequently reported rabid during these years. Rabies infection was comparatively high among little brown bats in central Alberta in 1973 and has subsequently declined. Only one rabid little brown bat was discovered in southern Alberta which is populated by a different subspecies. Outbreaks of rabies in little brown and big brown bat colonies tended to be brief events. Observations of free-ranging bats with probable furious rabies suggested that bats do not generally identify humans as targets for attack. Independent trends in infection rates suggested that spread of rabies is primarily intraspecific but there is evidence that migratory bats play a role in introduction and maintenance of rabies in northern temperate bat communities. The dynamics of bat rabies in Alberta are discussed.