Characterization of the specificities of human blood group H gene-specified alpha 1,2-L-fucosyltransferase toward sulfated/sialylated/fucosylated acceptors: evidence for an inverse relationship between alpha 1,2-L-fucosylation of Gal and alpha 1,6-L-fucosylation of asparagine-linked GlcNAc

The assembly of complex structures bearing the H determinant was examined by characterizing the specificities of a cloned blood group H gene-specified alpha 1,2-L-fucosyltransferase (FT) toward a variety of sulfated, sialylated, or fucosylated Gal beta 1,3/4GlcNAc beta- or Gal beta 1,3GalNAc alpha-based acceptor structures. (a) As compared to the basic type 2, Gal beta 1,4GlcNAc beta-(K(m) = 1.67 mM), the basic type 1 was 137% active (K(m) = 0.83 mM). (b) On C-6 sulfation of Gal, type 1 became 142.1% active and type 2 became 223.0% active (K(m) = 0.45 mM). (c) On C-6 sulfation of GlcNAc, type 2 showed 33.7% activity. (d) On C-3 or C-4 fucosylation of GlcNAc, both types 1 and 2 lost activity. (e) Type 1 showed 70.8% and 5.8% activity, respectively, on C-6 and C-4 O-methylation of GlcNAc. (f) Type 1 retained 18.8% activity on alpha 2,6-sialylation of GlcNAc. (g) Terminal type 1 or 2 of extended chain had lower activity. (h) With Gal in place of GlcNAc in type 1, the activity became 43.2%. (i) Compounds with terminal alpha 1,3-linked Gal were inactive. (j) Gal beta 1,3GalNAc alpha- (the T-hapten) was approximately 0.4-fold as active as Gal beta 1,4GlcNAc beta-. (k) C-6 sulfation of Gal on the T-hapten did not affect the acceptor activity. (l) C-6 sulfation of GalNAc decreased the activity to 70%, whereas on C-6 sulfation of both Gal and GalNAc the T-hapten lost the acceptor ability. (m) C-6 sialylation of GalNAc also led to inactivity. (n) beta 1,6 branching from GalNAc of the T-hapten by a GlcNAc residue or by units such as Gal beta 1, 4GlcNAc-, Gal beta 1,4(Fuc alpha 1,3)GlcNAc-, or 3-sulfoGal beta 1,4GlcNAc- resulted in 111.9%, 282.8%, 48.3%, and 75.3% activities, respectively. (o) The enhancement of enzyme affinity by a sulfo group on C-6 of Gal was demonstrated by an increase (approximately 5-fold) in the K(m) for Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn in presence of 6-sulfoGal beta 1,- 4GlcNAc beta-O-Me (3.0 mM). (p) Among the two sites in Gal beta 1, 4GlcNAc beta 1,6(Gal beta 1,3) GalNAc alpha-O-Bn, the enzyme had a higher affinity ( > 3-fold) for the Gal linked to GlcNAc. (q) With respect to Gal beta 1,- 3GlcNAc beta-O-Bn (3.0 mM), fetuin triantennary asialo glycopeptide (2.4 mM), bovine IgG diantennary glycopeptide (2.8 mM), asialo Cowper's gland mucin (0.06 mM), and the acrylamide copolymers (0.125 mM each) containing Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, Gal beta 1,3GalNAc alpha-, Gal beta 1,3Gal beta-, or Gal alpha 1,3Gal beta- units were 153.6%, 43.0%, 6.2%, 52.5%, 94.9%, 14.7%, 23.6%, and 15.6% active, respectively. (r) Fucosylation by alpha 1,2-L-FT of the galactosyl residue which occurs on the antennary structure of the bovine IgG glycopeptide was adversely affected by the presence of an alpha 1,6-L-fucosyl residue located on the distant glucosaminyl residue that is directly attached to the asparagine of the protein backbone. This became evident from the 4-fold activity of alpha 1,2-L-FT toward bovine IgG glycopeptide after approximately 5% removal of alpha 1,6-linked Fuo.     

Down-regulation of glial fibrillary acidic protein expression during acute lytic varicella-zoster virus infection of cultured human astrocytes

The effects of the varicella-zoster virus (VZV) OKA vaccine strain in producing morphologic and antigenic changes in dissociated cultures of human fetal brain was investigated. Cultures containing 80% glial acidic fibrillary protein (GFAP), GFAP+ (positive) astrocytes and 20% GFAP- (negative) fibroblastic-like cells were infected with cell-free VZV OKA at a multiplicity of infection of 0.1 plaque-forming units per cell. Cytopathic effects and significant viral antigen labeling with antibodies against VZV gpl and immediate-early (IE) protein 62 were first detected 6 to 7 days postinfection. Several observations indicated that astrocyte GFAP expression was altered and diminished as a result of VZV infection itself, thereby raising doubts about the utility of combining cell markers and viral antigenic labeling in assessing the susceptibility of neural cell types to viral infection. The down-regulation of GFAP expression by VZV appears to be mediated by early rather than late events in the viral replication cycle and may not be the result of virally induced global shut-off of host cell protein synthesis. Similar observations were made using VZV Ellen, a multipassaged, nonvaccine strain. These observations have potential in vivo implications related to histologic analysis of VZV-infected tissues and disease pathogenesis.     

Value of image-guided needle aspiration cytology in the assessment of thoracolumbar and sacrococcygeal masses

We have used a primary cloning assay to determine the frequency of 6-thioguanine (TG)-resistant tubular epithelial cells in kidney tissue from 72 human donors ranging in age from 2 to 94 years. The frequency of TG-resistant mutants ranged from approximately 5 x 10(-5) for donors in the first decade of life to approximately 2.5 x 10(-4) for donors in the eighth and later decades of life. Two different statistical analyses indicated that this increase in mutant frequency is exponential with age. We also observed a 2-fold higher TG-resistant mutant frequency in nephrectomy kidneys containing a coincident renal carcinoma. DNA sequence analyses revealed HPRT gene mutations in each of 14 TG-resistant mutants from seven unrelated donors. Thirteen of these 14 mutants resulted from independent mutational events. These results suggest that somatic mutations are common in renal--and perhaps in other human--epithelia, and thus could play an important role in the genesis of age-associated disease.     

System considerations favoring rotary artificial hearts with blood-immersed bearings

Hydrodynamic blood pumps provide such advantages as not requiring an air vent or compliance chamber as well as a great reduction in mechanical complexity with the potential for very long durability. The detailed design of their bearings is emerging as the single most important determinant of long-term success. Three categories of bearings include remote force, such as magnetic suspension; blood-isolated, which require a shaft seal; and blood-immersed using either mechanical or hydrodynamic support. Blood-immersed bearings permit maximum simplification and miniaturization of the entire system, require no flush fluid, and require no electronics as with magnetic suspension. The Jarvik 2000 heart represents an example of their potential. The intraventricular titanium pump (25 mm diameter, 25 cc, 85 g), uses blood flow through the motor air gap with blood-immersed bearings. The longest in vitro bearing durability test is ongoing at 20,000,000,000 revolutions with minimal wear (3 years at 15,000 rpm). In vivo results include 5-month calf survival, no thromboembolism, plasma Hb 2-5 mg%, and power under 10 W.     

Using sequence logos and information analysis of Lrp DNA binding sites to investigate discrepanciesbetween natural selection and SELEX

In vitro experiments that characterize DNA-protein interactions by artificial selection, such as SELEX,are often performed with the assumption that the experimental conditions are equivalent to natural ones. To test whether SELEX gives natural results, we compared sequence logos composed from naturally occurring leucine-responsive regulatory protein (Lrp) binding sites with those composed from SELEX-generated binding sites. The sequence logos were significantly different, indicating that the binding conditions are disparate. A likely explanation is that the SELEX experiment selected for a dimeric or trimeric Lrp complex bound to DNA. In contrast, natural sites appear to be bound by a monomer. This discrepancy suggests that in vitro selections do not necessarily give binding site sets comparable with the natural binding sites.     

Using fluorescence-based human androgen receptor gene assay to analyze the clonality of microdissected dendritic cell tumors

The AGT1 permease is a alpha-glucoside-H+ symporter responsible for the active transport of maltose, trehalose, maltotriose, alpha-methylglucoside, melezitose and sucrose. In wild-type as well as in MAL constitutive strains, alpha-methylglucoside seemed to be the best inducer of transport activity, while trehalose had no inducing effect. Based on the initial rates of transport it seems that the sugar preferentially transported by this permease is trehalose, followed by sucrose.     

Creatine and cyclocreatine treatment of human colon adenocarcinoma xenografts: 31P and 1H magnetic resonance spectroscopic studies

Creatine (Cr) and cyclocreatine (cyCr) have been shown to inhibit the growth of a variety of human and murine tumours. The purpose of this study was to evaluate the anti-tumour effect of these molecules in relation to drug accumulation, energy metabolism, tumour water accumulation and toxicity. Nude mice carrying a human colon adenocarcinoma (LS174T) with a creatine kinase (CK) activity of 2.12 units mg(-1) protein were fed Cr (2.5% or 5%) or cyCr (0.025%, 0.1% or 0.5%) for 2 weeks and compared with controls fed standard diet. Cr concentrations of 2.5% and 5% significantly inhibited tumour growth, as did 0.1% and 0.5% cyCr. In vivo 31P magnetic resonance spectroscopy (MRS) after 2 weeks of treatment showed an increase in [phosphocreatine (PCr)+phosphocyclocreatine (PcyCr)]/nucleoside triphosphate (NTP) with increasing concentrations of dietary Cr and cyCr, without changes in absolute NTP contents. The antiproliferative effect of the substrates of CK was not related to energy deficiency but was associated with acidosis. Intratumoral substrate concentrations (measured by 1H-MRS) of 4.8 micromol g(-1) wet weight Cr (mice fed 2.5% Cr) and 6.2 micromol g(-1) cyCr (mice fed 0.1% cyCr) induced a similar decrease in growth rate, indicating that both substrates were equally potent in tumour growth inhibition. The best correlant of growth inhibition was the total Cr or (cyCr+Cr) concentrations in the tissue. In vivo, these agents did not induce excessive water accumulation and had no systemic effects on the mice (weight loss, hypoglycaemia) that may have caused growth inhibition.