Estimations of intra- and extracellular volume and pH by 31P magnetic resonance spectroscopy: effect of therapy on RIF-1 tumours

Quantification of metabolite or drug concentrations in living tissues requires determination of intra- and extracellular volumes. This study demonstrates how this can be achieved non-invasively by 31P magnetic resonance spectroscopy (MRS) employing dimethyl methylphosphonate (DMMP) as a marker of total water space, 3-aminopropylphosphonate (3-APP) as a marker of extracellular space and P and 3-APP as markers of intracellular pH (pH) and extracellular pH (pHe) respectively. The MRS measurements of the tumour volumes were validated by classic radiolabelling methods using 3H2O and [14C]inulin as markers of total and extracellular space respectively. The extracellular volume fraction measured by radiolabelling of RIF-1 tumours was 23 +/- 0.83% (mean +/- s.e.m. n = 9), not significantly different (P > 0.1) from that found by MRS (27 +/- 2.9%, n = 9, London, and 35 +/- 6.7, n = 14, Baltimore). In untreated RIF-1 tumours, pH was about 0.2 units higher than pHe (P < 0.01). 5-Fluorouracil (5FU) treatment (165 mg kg(-1)) caused no significant changes in either pHe or per cent extracellular volume. However significant increases in pH, 48 h after treatment (P < 0.01) correlated with decreased tumour size and improved bioenergetic status [NTP/inorganic phosphate (Pi) ratio]. This study shows the feasibility of an MR method (verified by a 'gold standard') for studying the effects of drug treatment on intra- and extracellular spaces and pH in solid tumours in vivo.     

High doses of a helper-dependent adenoviral vector yield supraphysiological levels of alpha1-antitrypsin with negligible toxicity

Optimal gene therapy for many disorders will require efficient transfer to cells in vivo, high-level and long-term expression, and tissue-specific regulation, all in the absence of significant toxicity or inflammatory responses. While recombinant adenoviral vectors are efficient for gene transfer to hepatocytes, their usefulness is limited by short duration of expression related, at least in part, to immune responses to viral proteins and by a low capacity for foreign DNA. A number of systems have been developed for producing adenoviral vectors devoid of all viral coding sequences. Using AdSTK109, a vector lacking all viral coding sequences and carrying the complete human alpha1-antitrypsin (hAAT) genomic DNA locus, we have demonstrated sustained expression for longer than 10 months in mice. Utilizing high doses of this vector for hepatic gene transfer in mice, we find that supraphysiological levels of hAAT can be achieved without hepatotoxicity.     

Phase I study of the orally administered butyrate prodrug, tributyrin, in patients with solid tumors

Butyrates have been studied as cancer differentiation agents in vitro and as a treatment for hemoglobinopathies. Tributyrin, a triglyceride with butyrate molecules esterified at the 1, 2, and 3 positions, induces differentiation and/or growth inhibition of a number of cell lines in vitro. When given p.o. to rodents, tributyrin produces substantial plasma butyrate concentrations. We treated 13 patients with escalating doses of tributyrin from 50 to 400 mg/kg/day. Doses were administered p.o. after an overnight fast, once daily for 3 weeks, followed by a 1-week rest. Intrapatient dose escalation occurred after two courses without toxicity greater than grade 2. The time course of butyrate in plasma was assessed on days 1 and 15 and after any dose escalation. Grade 3 toxicities consisted of nausea, vomiting, and myalgia. Grades 1 and 2 toxicities included diarrhea, headache, abdominal cramping, nausea, anemia, constipation, azotemia, lightheadedness, fatigue, rash, alopecia, odor, dysphoria, and clumsiness. There was no consistent increase in hemoglobin F with tributyrin treatment. Peak plasma butyrate concentrations occurred between 0.25 and 3 h after dose, increased with dose, and ranged from 0 to 0.45 mM. Peak concentrations did not increase in three patients who had dose escalation. Butyrate pharmacokinetics were not different on days 1 and 15. Because peak plasma concentrations near those effective in vitro (0.5-1 mM) were achieved, but butyrate disappeared from plasma by 5 h after dose, we are now pursuing dose escalation with dosing three times daily, beginning at a dose of 450 mg/kg/day.     

Predominant interactions between mu-conotoxin Arg-13 and the skeletal muscle Na+ channel localized by mutant cycle analysis

High-affinity mu-conotoxin block of skeletal muscle Na+ channels depends on an arginine at position 13 (Arg-13). To understand both the mechanism of toxin interaction and the general structure of its binding site in the channel mouth, we examined by thermodynamic mutant cycle analysis the interaction between the critical Arg-13 and amino acid residues known to be in the channel's outer vestibule. Arg-13 interacts specifically with domain II Glu-758 with energy of about -3.0 kcal/mol, including both electrostatic and nonelectrostatic components, and with Glu-403 with energy of about -2.0 kcal/mol. Interactions with the other charged residues in the outer vestibule were shown to be almost entirely electrostatic, because these interactions were maintained when Arg-13 was replaced by lysine. These results place the bound Arg-13 at the channel mouth adjacent to the P (pore) loops of domains I and II. Distance estimates based on interaction energies suggest that the charged vestibule residues are in relative positions similar to those of the Lipkind-Fozzard vestibule model [Lipkind, G. M., and Fozzard, H. A. (1994) Biophys. J. 66, 1-13]. Kinetic analysis suggests that Arg-13 interactions are partially formed in the ligand-channel transition state.     

Phenylalanine 30 plays an important role in receptor binding of verotoxin-1

The homopentameric B subunit of verotoxin 1 (VT1) binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). We produced mutants with alanine substitutions for residues found near the cleft between adjacent subunits. Substitution of alanine for phenylalanine 30 (Phe-30) resulted in a fourfold reduction in B subunit binding affinity for Gb3 and a 10-fold reduction in receptor density in a solid-phase binding assay. The interaction of wild-type and mutant B subunits with Pk trisaccharide in solution was examined by titration microcalorimetry. The carbohydrate binding of the mutant was markedly impaired compared with that of the wild type and was too weak to allow calculation of a binding constant. These results demonstrate that the mutation significantly impaired the carbohydrate-binding function of the B subunit. To ensure that the mutation had not caused a significant change in structure, the mutant B subunit was crystallized and its structure was determined by X-ray diffraction. Difference Fourier analysis showed that its structure was identical to that of the wild type, except for the substitution of alanine for Phe-30. The mutation was also produced in the VT1 operon, and mutant holotoxin was purified to homogeneity. The cytotoxicity of the mutant holotoxin was reduced by a factor of 10(5) compared to that of the wild type in the Vero cell cytotoxicity assay. The results suggest that the aromatic ring of Phe-30 plays a major role in binding of the B subunit to the Galalpha1-4Galbeta1-4Glc trisaccharide portion of Gb3. Examination of the VT1 B crystal structure suggests two potential carbohydrate-binding sites which lie on either side of Phe-30.     

Functional role of hepatitis C virus chimeric glycoproteins in the infectivity of pseudotyped virus

The putative envelope glycoproteins of hepatitis C virus (HCV) likely play an important role in the initiation of viral infection. Available information suggests that the genomic regions encoding the putative envelope glycoproteins, when expressed as recombinant proteins in mammalian cells, largely accumulate in the endoplasmic reticulum. In this study, genomic regions which include the putative ectodomain of the E1 (amino acids 174 to 359) and E2 (amino acids 371 to 742) glycoproteins were appended to the transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein. This provided a membrane anchor signal and the VSV incorporation signal at the carboxy termini of the E1 and E2 glycoproteins. The chimeric gene constructs exhibited expression of the recombinant proteins on the cell surface in a transient expression assay. When infected with a temperature-sensitive VSV mutant (ts045) and grown at the nonpermissive temperature (40.5 degrees C), cells transiently expressing the E1 or E2 chimeric glycoprotein generated VSV/HCV pseudotyped virus. The resulting pseudotyped virus generated from E1 or E2 surprisingly exhibited the ability to infect mammalian cells and sera derived from chimpanzees immunized with the homologous HCV envelope glycoproteins neutralized pseudotyped virus infectivity. Results from this study suggested a potential functional role for both the E1 and E2 glycoproteins in the infectivity of VSV/HCV pseudotyped virus in mammalian cells. These observations further suggest the importance of using both viral glycoproteins in a candidate subunit vaccine and the potential for using a VSV/HCV pseudotyped virus to determine HCV neutralizing antibodies.     

Fracture-dislocation of the lunotriquetral coalition

Bony fusions involving the carpus have a much higher prevalence in blacks relative to whites. This article describes a case of lunotriquetral coalition fracture-dislocation in an African American. This lesion is best treated through open reduction and pin fixation.     

Peripheral somatic nerve function in relation to glucose tolerance in an elderly Caucasian population: the Hoorn study

Only sparse and contradictory data are available on peripheral somatic nerve function in relation to the total range of glucose tolerance. A random sample (n = 708) of people, stratified by age, sex, and glucose tolerance, from a Caucasian population aged 50 to 74 years was invited to undergo an examination including measures of large-fibre nerve function (ankle and knee reflexes, vibration sense, vibratory perception threshold (VPT) at the foot) and one measure of small-fibre function (thermal discrimination threshold (TDT) at the foot). A total of 267 subjects with a normal glucose tolerance (NGT), 167 with impaired glucose tolerance (IGT), 90 with newly diagnosed diabetes mellitus (NDM), and 73 with previously known diabetes (KDM) were included. KDM was associated with the highest prevalence of large-fibre nerve dysfunction. Within the range from NGT to NDM, most large-fibre function measures showed a decline with decreasing glucose tolerance. The TDT showed a decrease with an increase in fasting and post-load insulin levels (p < 0.05). We conclude that glucose intolerance is associated with impaired peripheral large-fibre nerve function, an association which seems to apply even in the non-diabetic range. Higher insulin levels were associated with a better small-fibre nerve function.