No evidence of PEG1/MEST gene mutations in Silver‐Russell syndrome patients

Silver‐Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (α and β) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST α coding region, and there were no significant mutations in the 5′‐flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST α were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS. © 2001 Wiley‐Liss, Inc.     

Recombinant Sendai viruses with L1618V mutation in their L polymerase protein establish persistent infection, but not temperature sensitivity

The Sendai virus pi strain (SeVpi) isolated from cells persistently infected with SeV shows mainly two phenotypes: (1) temperature sensitivity and (2) an ability of establishing persistent infection (steady state). Three amino acid substitutions are found in the Lpi protein and are located at aa 1088, 1618, and 1664. Recombinant SeV(Lpi) (rSeV(Lpi)) having all these substitutions is temperature sensitive and is capable of establishing persistent infection (steady state). rSeVs carrying the fragment containing L1618V show both phenotypes. rSeV(L1618V), in which leucine at aa 1618 is replaced with valine, has the ability of establishing persistent infection, but is not a temperature-sensitive mutant, indicating that the ability of a virus to establish persistent infection can be separated from temperature sensitivity. The amino acid change at 1618(L-->V) coexisting with aa 1169 threonine is required for acquirement of a temperature-sensitive phenotype. Three amino acid substitutions are also found in the Ppi protein, but rSeV(Ppi) does not show these phenotypes.     

Effects of GM1-ganglioside and α-sialyl cholesterol on amino acid uptake,protein synthesis,and Na+, K+-ATPase activity in superior cervical and nodose ganglia excised from adult rats

We examined the effect of GM₁-ganglioside in combination with cholera toxin B, and synthetic α-sialyl cholesterol (α-SC) on neutral amino acid (tritiated α-aminoisobutyric acid, [³H]AIB) uptake, protein synthesis ([³H]leucine incorporation), and Na⁺, K⁺-ATPase activity in isolated superior cervical ganglia (SCG) and nodose ganglia (NG) from adult rats after aerobic incubation, usually for 2 h at 37°C in vitro. Cholera toxin B, that specifically masks the oligosaccharide chain of GM₁-ganglioside, antagonized the GM₁-induced changes in [³H]AIB uptake, [³H]leucine incorporation, and Na⁺, K⁺-ATPase activity almost completely in SCG, but partially in NG. Although cholesterol itself had little effect on either [³H]AIB uptake and Na⁺, K⁺-ATPase activity both in SCG and NG, α-SC caused considerable reduction of both amino acid uptake and the transport enzyme activity in each ganglia. However, cholesterol was more effective than α-SC in decreasing [³H]leucine incorporation in either ganglia. Whereas addition of EGTA markedly reduced either GM₁-induced or α-SC-induced change in [³H]leucine incorporation into acid-insoluble fraction in both SCG and NG, application of Ca²⁺ ionophore produced considerable recovery of the protein synthesis from the inhibited level by Ca²⁺-deprivation. ATP and creatine phosphate contents in SCG were elevated by the presence of GM₁ or α-SC, whereas [³H]AIB uptake and Na⁺, K⁺-ATPase activity were inhibited, suggesting that utilization for membrane transport was diminished as a result of GM₁- or α-SC-induced decrease of ATPase activity.     

Neurotoxicity and pharmacokinetics of ventriculolumbar perfusion of methyl 6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-alpha-D-gluco-pyranoside (MCNU) in dogs

Summary Ventriculolumbar perfusion of methyl 6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-alpha-D-glucopyranoside (MCNU), a water soluble nitrosourea with log P-0.71, may be efficacious in the treatment of subarachnoid dissemination of malignant glioma. We used 2 dogs to study the neurotoxicity and pharmacokinetics of MCNU. MCNU (1 mg), dissolved in 10 ml of artificial CSF, was administered via the right lateral ventricle during a period of 18 to 42 min and the CSF was drained by lumbar puncture. The perfusion was repeated once a week for 10 consecutive weeks. No neurological and systemic symptoms were noted after perfusion. Histological examination of the brain and spinal cord showed local denudation of the ependyma and local subependymal spongy degeneration and gliosis in the lateral ventricle into which MCNU was administered in one dog and local denudation of the ependyma in the other. When administration was over a period of 21 to 38 min, the MCNU concentration in the lumbar CSF peaked at 11.11 to 50.67 g/ml, in 28 to 78 min. The area under the drug concentration-time curve (AUC) was 1152 g×min/ml on average, significantly larger than that of ACNU. The elimination phase followed linear kinetics and the half-time was 41.1 min on average, significantly longer than that of ACNU. These findings suggest that ventriculolumbar perfusion of MCNU may be effective in the treatment of subarachnoid dissemination of malignant glioma notwithstanding some local histological changes.     

Complete nucleotide sequence of mouse mammary tumor virus from jyg chinese wild mice: Absence of bacterial insertion sequences in the cloned viral gag gene

Mammary tumors of a newly isolated strain of Chinese wild mouse (JYG mouse) harbor exogenous mouse mammary tumor virus (MMTV). The complete nucleotide sequence of exogenous JYG-MMTV was determined on the proviral 5' long terminal repeat (LTR)(partial)-gag-pol-env-3' LTR (partial) fragment cloned into a plasmid vector and the cDNA sequence from JYG-MMTV producing cells. Similarly to the other MMTV species the LTR of JYG-MMTV contains an open reading frame (ORF). The amino acid sequence of the JYG-MMTV ORF resembles that of SW-MMTV (92% identity) and endogenous Mtv-7 (93% identity) especially at the C-terminal region. Thus, a functional similarity in T-cell receptor V beta recognition as a superantigen is implicated among these MMTV species. Analysis of the viral gag nucleotide sequence revealed that this gene is not disrupted by the bacterial insertion sequence IS1 or IS2, which have been reported to be present in the majority of the plasmids containing the gag region. Comparison of amino acid sequences of JYG-MMTV with those of BR6-MMTV showed that over 96% of the amino acids of gag, pol, protease and env products are identical. These results suggest the intact nature of the nucleotide sequence of the near full-length MMTV genome cloned in the plasmid.     

Effects of coronary artery bypass surgery on the stunned and hibernating myocardium in the cases with depressed left ventricular function

Chronic left ventricular (LV) dysfunction may result from irreversible damage (cell death), stunned myocardium (ST), or hibernating myocardium (HB). However, both of ST and HB are expected to be reversible. In this report, the effects of coronary artery bypass grafting on the regions of ST and HB were evaluated in 37 patients with less than 40% of LV ejection fraction. The patients were divided into two groups. Group I consisted of the patients whose postoperative LV ejection fraction rose by more than 10% compared to the preoperative value. Group II included the remaining patients with no significant improvement. After successful revascularization, 61% of HB changed to ST and 52% of ST to normal in group I. These changes were significant in comparison with group II because 48% of HB and 83% of ST in group II remained unchanged. Immediate or rapid recovery of HB hardly occurred in both of the groups. To recover normal function, HB may pass through a stage of ST on reperfusion. On the other hand, it is difficult to determine whether HB and ST with no significant changes after reperfusion are irreversibly damaged or reversible and take time to return to normal.     

Postsynaptic expression of Ca2+-permeable AMPA-type glutamate receptor channels by viral-mediated gene transfer

The ability to artificially express a particular receptor protein in the postsynaptic sites of neurons in the central nervous system (CNS) would be useful for the study of synaptic function of cloned receptor genes as well as for gene therapy of neurological disorders caused by dysfunction of postsynaptic receptors. In this study, we aimed to express the cDNA of unedited GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor that forms inwardly rectifying and Ca2+-permeable channel in CNS neurons by using adenoviral-mediated gene transfer. For this purpose, we have constructed a recombinant adenovirus bearing an expression-switching unit, where the unedited GluR2 cDNA can be activated by the Cre recombinase-mediated excisional deletion of a stuffer DNA interposed between the promotor and the coding region. When PC12 cells were infected with this recombinant adenovirus together with an adenovirus expressing Cre recombinase, the inwardly rectifying and Ca2+-permeable AMPA receptor channels were expressed in nearly 100% of infected cells. Two days after co-infection of cultured rat hippocampal neurons with these adenoviruses, fast excitatory neurotransmission in the glutamatergic synapse was mediated predominantly by the inwardly rectifying and Ca2+-permeable AMPA receptor channels. This indicates that the native AMPA receptors in the postsynaptic sites of the glutamatergic synapse are replaced rapidly with recombinant receptors newly produced by the viral-mediated gene transfer.     

Three-Dimensional Echocardiographic Reconstruction of the Left Ventricle by a Transesophageal Tomographic Technique: In Vitro and In Vivo Validation of its Volume Measurement

Accurate determination of left ventricular (LV) volume has important therapeutic and prognostic implications in patients with cardiac disease. Volume estimations by two-dimensional techniques are not very accurate due to geometric assumptions. OBJECTIVES: To validate LV volume determinations by a new transesophageal three-dimensional echocardiographic technique. We performed three-dimensional reconstruction of the LV using an echo-computed tomographic (CT) technique based on serial pullback parallel slice imaging technique in both in vitro and in vivo settings. Fourteen latex balloons with various sizes (30-235 mL) and shapes (conical, pear shaped, round, elliptical, and aneurysms in various locations) filled with known volumes of water were imaged in a water bath. From the static three-dimensional image, the LV long axis was defined and the LV was sectioned perpendicular to this axis into 2-mm slices. The volume of each slice was calculated with the observer blinded to the actual volume as the product of the slice thickness and the manually traced perimeter of the slice and the LV volume as the sum of the volumes of the slices (Simpson's method). The calculated LV volume closely correlated with the actual volume (r = 0.99, P < 0.0001, calculated volume = 1.06x - 11.3, Deltavolume = -5.7 +/- 10.0 cc). Using the same system, transesophageal echocardiographic (TEE) images of the LV were obtained in 15 patients gated to respiration and ECG. Satisfactory dynamic three-dimensional reconstruction of the LV was possible in ten patients. The three-dimensional LV volumes (systolic and diastolic) using Simpson's method correlated well with those obtained from biplane or multiplane TEE images using the area length method (r = 0.89, p < 0.0001, y = 12.7 + 0.84x, Deltavolume = 1.3 +/- 18.1 cc). The LV major-axis diameters by the two methods showed very close correlations as well (r = 0.86, P < 0.0001, y = 19 + 0.74x, Deltadiameter = 1.0 +/- 7.2 mm). We conclude that three-dimensional LV volume calculation by the echo-CT technique is intrinsically sound, is independent of LV geometry, and with some limitations, is applicable in vivo. (ECHOCARDIOGRAPHY, Volume 13, November 1996)     

Liver Targeting of Interferon Through Pullulan Conjugation

Purpose. The purpose of this study was to actively target interferon (IFN) to the liver through its chemical conjugation with pullulan, a water-soluble polysaccharide with a high affinity for the liver. Methods. Chemical conjugation of IFN with pullulan was achieved by a cyanuric chloride method. Following intravenous injection of the conjugates to mice, their body distribution and the activity of an IFN-induced enzyme, 2,5-oligoadenylate (2-5A) synthetase in the liver and other organs, were evaluated. Results. The cyanuric chloride method enabled us to prepare an IFN-pullulan conjugate that retained approximately 7–9 % of the biological activity of IFN. Pullulan conjugation enhanced the liver accumulation of IFN and the retention period with the results being reproducible. When injected intravenously to mice, the IFN-pullulan conjugate enhanced the activity of 2-5A synthetase in the liver. The activity could be induced at IFN doses much lower than those of free IFN injection. In addition, the liver 2-5A synthetase induced by conjugate injection was retained for 3 days, whereas it was lost within the first day for the free IFN-injected mice. Conclusions. IFN-pullulan conjugation was promising for IFN targeting to the liver with efficient exertion of its antiviral activity therein.