Lack of PPCA expression only partially coincides with lysosomal storage in galactosialidosis mice: indirect evidence for spatial requirement of the catalytic rather than the protective function of PPCA

Protective protein/cathepsin A (PPCA) is a pleiotropic lysosomal enzyme that complexes with beta-galactosidase and neuraminidase, and possesses serine carboxypeptidase activity. Its deficiency in man results in the neurodegenerative lysosomal storage disorder galactosialidosis (GS). The mouse model of this disease resembles the human early onset phenotype and results in severe nephropathy and ataxia. To understand better the pathophysiology of the disease, we compared the occurrence of lysosomal PPCA mRNA and protein in normal adult mouse tissues with the incidence of lysosomal storage in PPCA(-/-) mice. PPCA expression was markedly variable among different tissues. Most sites that produced both mRNA and protein at high levels in normal mice showed extensive and overt storage in the knockout mice. However, this correlation was not consistent as some cells that normally expressed high levels of PPCA were unaffected in their storage capability in the PPCA(-/-) mice. In addition, some normally low expressing cells accumulated large amounts of undegraded products in the GS mouse. This apparent discrepancy may reflect a requirement for the catalytic rather than the protective function of PPCA and/or the presence of cell-specific substrates in certain cell types. A detailed map showing the cellular distribution of PPCA in nomal mouse tissues as well as the sites of lysosomal storage in deficient mice is critical for accurate assessment of the effects of therapeutic interventions.     

Synthesis and triplex forming properties of an acyclic N7-glycosylated guanine nucleoside

A chiral acyclic nucleoside, one in which the ribose carbohydrate has been replaced with a glycerol-based linker, is prepared by glycosylating guanine at the N7-nitrogen. The stereochemically pure derivative is converted to a DMT-protected phosphoramidite for incorporation into DNA sequences. Sequence containing the acyclic N7-dG nucleoside are capable of forming DNA triplexes in which it is likely that the N1-H and N2-amino groups of the N7-dG are involved in recognition of the guanine base in G-C base pairs.     

Noninvasive determination of cardiac output using single breath CO2 analysis

OBJECTIVE: To examine the utility of single breath CO2 analysis as a noninvasive measure of cardiac output. SETTING: An animal laboratory in a university-affiliated medical center. DESIGN: A prospective, animal cohort study comparing 21 parameters derived from single breath CO2 analysis with cardiac output determined by an ultrasonic flow probe. SUBJECTS: Six healthy adult sheep. METHODS: The single breath CO2 analysis station consists of a mainstream capnometer, a variable orifice pneumotachometer, a signal processor, and computer software with capability for both on- and off-line data analysis. Twenty-one derived components of the CO2 expirogram were evaluated as predictors of cardiac output. Cardiac output was manipulated by successive injections of a hydraulic constrictor placed around the inferior vena cava. MEASUREMENTS AND MAIN RESULTS: Thirty-four measurements of cardiac output were available for comparison with derived variables from the CO2 expirogram. Stepwise linear regression identified two variables that were most predictive of cardiac output: a) the angle between the slope lines for phase II and III of the CO2 expirogram divided by the volume of CO2 per breath (angle/mL CO2); and b) the slope of phase II. The multivariate equation was highly statistically significant and explained 94% of the variance (adjusted r2 = .94, p < .0001). The bias and precision of the calculated cardiac output were .00 and .23, respectively. The mean percent difference for the cardiac output estimate derived from the single breath CO2 analysis station was 0.36%. CONCLUSIONS: Our data indicate that analysis of the CO2 expirogram can yield accurate information about the cardiovascular system. Specifically, two variables derived from a plot of expired CO2 concentration vs. expired volume predict changes in cardiac output in healthy adult sheep with an adjusted coefficient of determination of .94. Prospective application of this technology in the setting of lung injury and rapidly changing physiology will be essential in determining the clinical usefulness of the technique.     

Suppression of in vitro IgM rheumatoid factor production by diphtheria toxin interleukin 2 recombinant fusion protein (DAB 486IL-2) in patients with refractory rheumatoid arthritis

OBJECTIVE: To investigate the effect of diphtheria toxin interleukin 2 recombinant fusion protein (DAB 486IL-2) on in vitro synthesis of immunoglobulin and rheumatoid factor (RF) in patients with severe refractory rheumatoid arthritis (RA) enrolled in a phase II, double blind, placebo controlled study. METHODS: Anticoagulated venous blood samples were obtained before (Day 1) and after (Day 28) intravenous infusion of either DAB 486IL-2 at 0.075 mg/kg/day (12 patients) or saline placebo (10 patients) on Days 1-5. Peripheral blood leukocytes (PBL) were prepared by density gradient centrifugation, cultured in the presence and absence of pokeweed mitogen (PWM) for one week, and culture supernatants assayed for immunoglobulins and IgM RF by ELISA. RESULTS: Compared to placebo treated patients, PWM induced IgM RF synthesis by PBL decreased after treatment with DAB 486IL-2 (p = 0.043). However, there was no apparent correlation with clinical improvement. PWM induced IgM, IgA, and IgG synthesis also tended to decrease, although the changes did not attain statistical significance. In contrast, PWM induced IgM RF, IgM, IgA, and IgG synthesis by PBL from patients treated with placebo tended to increase during the observation period. Spontaneous immunoglobulin and IgM RF production by PBL from either the DAB 486IL-2 or placebo patients remained stable. CONCLUSION: These observations raise the possibility that DAB 486IL-2 may diminish B cell function either directly or indirectly through effects on T cell function, but the change may not correspond to clinical response.     

Characterising the energy deposition events produced by trapped protons in low earth orbit

Men and equipment in space vehicles in low earth orbit are exposed to a wide variety of radiations, but the majority of the dose is due to trapped protons, which have energies of the order of 100 MeV and are low LET particles. These high energy particles produce nuclear fragmentation with high LET secondaries that may be responsible for a significant fraction of dose equivalent. In order to understand better the biological effectiveness of this radiation environment, a portable tissue equivalent proportional counter spectrometer has been developed that automatically records the distribution of energy in a small tissue-like site as a function of time. This instrument weighs about 700 g and will be flown on a number of future space shuttle flights.     

Nutritional regulation of the activities of lipogenic enzymes of rat liver and brown adipose tissue

Nutrition-induced effects on the activity of enzymes of lipogenesis, fatty acid synthase (FAS: EC 2.3.1.85), ATP citrate lyase (ACL: EC 4.1.3.8), malic enzyme (ME; EC 1.1.1.40), glucose-6-phosphate dehydrogenase (G6PDH: EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (PGDH; EC 1.1.1.44) were investigated in liver and interscapular brown adipose tissue (BAT) of rats. The lipogenic enzymes could be grouped into two categories according to their response to dietary manipulations: FAS and ACL, both key enzymes of lipogenesis, responded fast and strongly to dietary manipulations. ME, G6PDH and PGDH, enzymes which also contribute to metabolic pathways other than lipogenesis, responded in a more sustained and less pronounced fashion. Feed deprivation caused the specific activities of lipogenic enzymes to decline several-fold. Refeeding of previously fasted (up to 3 days) animals increased the activities dramatically (10-to 25-fold) to far above pre-fasting levels ("overshoot"). Repetition of the fasting/refeeding regimen increasingly impaired the ability of both tissues to synthesize overshooting enzyme activities in the subsequent refeeding period. The fasting-induced decline of the activities was prevented when sugars were provided to the animals via drinking water. The sugars displayed different effectivities: sucrose = glucose > fructose > maltose > > lactose. Sugars as the sole nutrient after fasting were also able to induce overshooting enzyme activities. Again, activities of FAS and ACL responded in a more pronounced fashion than the other three enzymes. Transition from feeding one diet to feeding a new diet of different composition led to adaptation of the lipogenic enzyme activities to levels characteristic for the new diet. Replacing a low-carbohydrate with a high-carbohydrate diet proceeded with major alterations of enzyme activities. This process of attaining a new level took up to 20 days and involved pronounced oscillations of the specific activities. In contrast, when a high-carbohydrate diet was replaced with another diet. particular one high in fat, transition to new enzyme activities was completed within 2-3 days and proceeded without oscillations. All dietary manipulations caused more pronounced responses in young (35d-old) than in adult (180d-old) animals.     

Surgical salvage after radiation for laryngeal cancer

From January, 1962, through December, 1973, 1,084 patients with cancer of the intrinsic larynx were treated at the Mayo Clinic. Of the 1,084 patients, 136 were retreated after radiation therapy failed to cure their cancer (105 glottic, 30 supraglottic, and one subglottic cancers). Recurrence of glottic cancer was generally recognized later than recurrences in the supraglottic area; likewise, glottic cancers were more advanced at recurrence than supraglottic growths. When feasible, conservation surgery was carried out on both glottic and supraglottic growths. Unfortunately, many growths were too advanced for conservation surgery by the time recurrence was recognized. The concept of "radiate-and-watch" for early glottic and supraglottic cancers is designed to save larynges. The concept is not supported by this study.     

T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates

Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for gene-corrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.     

Novel bisquinoline antimalarials. Synthesis, antimalarial activity, and inhibition of haem polymerisation

We report the synthesis of two series of novel bisquinoline compounds that inhibit the growth of both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. To study the molecular basis of the action of these novel antimalarial drugs, we examined their ability to inhibit haem polymerisation in the presence and absence of parasite extracts. The level of antimalarial potency was correlated with the level of inhibition of haem polymerisation, suggesting that these bisquinolines exert their antimalarial activity by antagonising the sequestration of toxic haem moieties.     

Cyclophosphamide and 4-Hydroxycyclophosphamide/aldophosphamide kinetics in patients receiving high-dose cyclophosphamide chemotherapy

Using a recently developed gas chromatography and mass spectrometry method to determine whole-blood cyclophosphamide (CP) and 4-hydroxycyclophosphamide/aldophosphamide (4-HO-CP/AP) concentrations, we investigated their pharmacokinetics in women receiving CP therapy. Patients (n = 18) received one or two courses of CP: (a) a 90-min i.v. infusion (4 g/m2) followed by a 96-h i.v. infusion (6 g/m2) in combination with high-dose thiotepa; or (b) a 96-h i.v. infusion (6 g/m2) in combination with high-dose thiotepa. Whole-blood exposures to CP [area under the whole blood concentration versus time curve (AUCCP)] and 4-HO-CP/AP (AUC4HOCP) between courses 1 and 2 were compared after normalization to dose (g/m2). A nonproportional increase was observed for the AUCCP between the first course [1112 micrometer. h/g/m2 +/- 14% coefficient of variation (CV)] and the second course (1579 micrometer . h/g/m2 +/- 28% CV) (P < 0.001). In contrast, the AUC4HOCP (27 micrometer . h/g/m2 +/- 25% CV) determined for the first course was 29% higher than the AUC4HOCP (21 micrometer . h/g/m2 +/- 26% CV) for the second course (P < 0.01). The interpatient whole-blood exposures to both CP and 4-HO-CP/AP were remarkably consistent in this patient population with percent CVs ranging from 14 to 28%. Because thiotepa (800 mg/m2) was administered simultaneously with CP during the second course of treatment, possible inhibition of CP metabolism by thiotepa was investigated using human liver microsomes in vitro. IC50 values determined for inhibition of CP metabolism in three individual liver donors ranged from 1.0 to 40 micrometer. However, the clinical relevance of this observation has not been established.