Effect of the multidrug inhibitor GG918 on drug sensitivity of human leukemic cells

The drug GG918 has been specifically developed for overcoming MDR phenotype and is now in use in clinical trials. In this study, the effects of GG918 on leukemic cell were investigated using a 3 day MTT assay. Results showed that, in a highly resistant P-gp(+) leukemic cell line, 0.1 microM of GG918 gives rise to a 40-fold sensitization to daunorubicin (DNR) (residual resistance: 2.1), a 57-fold sensitization to mitoxantrone (residual resistance: 1.5), and a 3.3-fold sensitization to idarubicin (residual resistance: 2.9). When human AB serum was added to the incubation medium, 1 microM of GG918 was needed to observe the full P-gp modulation potency described above. The effect of 1 microM of GG918 was tested on 27 samples of poor prognosis acute leukemia (25 AML, two ALL). DNR sensitization (using the MTT assay) and modulation of rhodamine 123 uptake were monitored and used as criteria for comparing the in vitro modulation potency of this new compound to the potency of 10 microM of verapamil, which was used as reference. A good correlation (r = 0.8, P = 0.001) was observed between the results of the two tests. Eleven out of the 26 cases tested were MDR1(+) (42%), and showed a higher IC50 for DNR than the negative cases (861 +/- 1284 nM vs 187 +/- 246 nM, P = 0.05). GG918 was able to modulate the in vitro resistance to DNR in eight cases (seven MDR1(+), no MDR1(-), one non-tested). Verapamil did not increase DNR toxicity in four of these eight cases, but was more efficient in one other MDR1(+) case. In conclusion, the DNR sensitivity of the majority of the fresh AML samples expressing P-gp could be modulated in vitro by 1 microM of GG918.     

Isolation of human mannan binding lectin, serum amyloid P component and related factors from Cohn fraction III

BACKGROUND: The suicide gene and prodrug, herpes simplex thymidine kinase (HStk) and ganciclovir (GCV), are now in clinical trials for recurrent malignancies. METHODS: We evaluated in vitro and in vivo efficacy of HStk gene transfer and GCV treatment of colonic adenocarcinoma in a syngeneic murine model. RESULTS: In vitro analysis demonstrated that CT-26 adenocarcinoma cells transduced with LTKOSN.2 retroviral vector inhibited the proliferation of wild-type CT-26 (nontransduced) cells after GCV exposure. Cooperative killing with HStk gene therapy was shown in vivo, mixtures of HStk CT-26 transduced cells (CT-26 TK), and nontransduced (CT-26 NV) cells and tumors containing only 9% CT-26 TK cells demonstrated complete regression after GCV (100 mg/kg). CONCLUSIONS: This in vitro and in vivo demonstration suggests that metabolic cooperation permits destruction of tumors even when gene transfer is effective only to a relatively small portion of the tumor. These important results suggest new avenues can be developed for the treatment of this lethal malignancy.     

Polarization-enhanced NMR spectroscopy of biomolecules in frozen solution

Large dynamic nuclear polarization signal enhancements (up to a factor of 100) were obtained in the solid-state magic-angle spinning nuclear magnetic resonance (NMR) spectra of arginine and the protein T4 lysozyme in frozen glycerol-water solutions with the use of dynamic nuclear polarization. Polarization was transferred from the unpaired electrons of nitroxide free radicals to nuclear spins through microwave irradiation near the electron paramagnetic resonance frequency. This approach may be a generally applicable signal enhancement scheme for the high-resolution solid-state NMR spectroscopy of biomolecules.     

The kleeblattsch?del anomaly in Apert syndrome: intracranial anatomy, surgical correction, and subsequent cranial vault development

We present a case of Apert syndrome in which intracranial anomalies of the cranial base were localized to the lesser wings of the sphenoid and sphenoid ridge. The lesser wings of the sphenoid were displaced superiorly to follow the fused coronal sutures bilaterally, where they met at a single point on the skull vertex. Careful preoperative study of the intracranial anatomy in the kleeblattsch?del anomaly led to a surgical plan for early correction of the anomaly. The present report indicates that an aggressive approach to the correction of the kleeblattsch?del anomaly beginning early in infancy can result in normalization of the trilobar skull configuration. Although this approach can correct the kleeblattsch?del anomaly, 3.5-year follow-up in this patient with Apert syndrome demonstrates progressive turricephaly despite repeated cranial vault remodeling. Although the trilobar skull configuration can be corrected through early surgical intervention, the long-term correction of progressive turricephaly in patients with Apert syndrome remains an unsolved problem.     

The importance of MHC-I and MHC-II responses in vaccine efficacy against lethal herpes simplex virus type 1 challenge

To investigate the importance of major histocompatability complex (MHC) class I- and MHC class II-dependent immune responses in herpes simplex virus-1 (HSV-1) vaccine efficacy, groups of beta 2% (MHC I-) and Ab% (MHC II-) mice were inoculated with various vaccines, and then challenged intraperitoneally with HSV-1. Following vaccination with either live avirulent HSV-1, expressed HSV-1 glycoprotein D (gD), or a mixture of seven expressed HSV-1 glycoproteins (7gPs), Ab% (MHC-II-) mice developed no enzyme-linked immunosorbent assay (ELISA) or neutralizing antibody titres. In contrast, significant ELISA and neutralizing antibody titres were induced in beta 2m% (MHC-I-) mice by all three vaccines. The neutralizing antibody titres were similar for all three vaccines, but were only approximately 1/4 to 1/3 of that developed in C57BL/6 (parental) mice vaccinated with the same antigens. All three vaccines protected 100% of the wild-type C57BL/6 mice against lethal challenge with 2 x 10(7) plaque-forming units (PFU) of HSV-1. The live virus vaccine and the 7gPs vaccine also protected 80% of the beta 2m% mice against the same lethal HSV-1 challenge dose. In contrast, in Abo/o mice, none of the vaccines provided significant protection against the same lethal challenge dose of HSV-1. However, at a lower challenge dose of 2 x 10(6) PFU, all three vaccines protected 70-80% of the vaccinated Ab% mice (compared to only 10% survival in mock vaccinated controls). Thus, vaccination provided some protection against lethal HSV-1 challenge in both beta 2m% and Ab% mice; however, the protection was less than that seen in the parental C57BL/6 mice. In addition, Ab% mice were less well protected by vaccination than were beta 2m% mice. Our results suggest that (1) both MHC-I and MHC-II are involved in vaccine efficacy against HSV-1 challenge; (2) both types of responses must be present for maximum vaccine efficacy: and (3) the MHC-II-dependent immune response appeared to be more important than the MHC-I-dependent immune response for vaccine efficacy against HSV-I challenge.     

Sarcomeric gene expression and contractility in myofibroblasts

Myofibroblasts are unusual cells that share morphological and functional features of muscle and nonmuscle cells. Such cells are thought to control liver blood flow and kidney glomerular filtration rate by having unique contractile properties. To determine how these cells achieve their contractile properties and their resemblance to muscle cells, we have characterized two myofibroblast cell lines. Here, we demonstrate that myofibroblast cell lines from kidney mesangial cells (BHK) and liver stellate cells activate extensive programs of muscle gene expression including a wide variety of muscle structural proteins. In BHK cells, six different striated myosin heavy chain isoforms and many thin filament proteins, including troponin T and tropomyosin are expressed. Liver stellate cells express a limited subset of the muscle thick filament proteins expressed in BHK cells. Although these cells are mitotically active and do not morphologically differentiate into myotubes, we show that MyoD and myogenin are expressed and functional in both cell types. Finally, these cells contract in response to endothelin-1 (ET-1); and we show that ET-1 treatment increases the expression of sarcomeric myosin.     

Regulation of tissue-specific P-element pre-mRNA splicing requires the RNA-binding protein PSI

Binding of a multiprotein complex to a 5' exon inhibitory element appears to repress splicing of the Drosophila P-element third intron (IVS3) in the soma. We have purified 97- and 50-kD proteins that interact specifically with the inhibitory element using RNA affinity chromatography. Antibodies specific for the 97-kD protein relieve inhibition of IVS3 splicing in somatic extracts, providing direct evidence that inhibition requires this protein, P-element somatic inhibitor (PSI). We identify the 50-kD protein as hrp48, a protein similar to the mammalian splicing factor hnRNP A1, and show that hrp48 recognizes specific nucleotides in a pseudo-5' splice site within the inhibitory element. The results indicate that PSI is an alternative splicing factor that regulates tissue-specific splicing, probably through interactions with generally expressed factors such as hrp48.