The exon junction complex in neural development and neurodevelopmental disease.

Post-transcriptional mRNA metabolism has emerged as a critical regulatory nexus in proper development and function of the nervous system. In particular, recent studies highlight roles for the exon junction complex (EJC) in neurodevelopment. The EJC is an RNA binding complex composed of 3 core proteins, EIF4A3 (DDX48), RBM8A (Y14), and MAGOH, and is a major hub of post-transcriptional regulation. Following deposition onto mRNA, the EJC serves as a platform for the binding of peripheral factors which together regulate splicing, nonsense mediated decay, translation, and RNA localization. While fundamental molecular roles of the EJC have been well established, the in vivo relevance in mammals has only recently been examined. New genetic models and cellular assays have revealed core and peripheral EJC components play critical roles in brain development, stem cell function, neuronal outgrowth, and neuronal activity. Moreover, human genetics studies increasingly implicate EJC components in the etiology of neurodevelopmental disorders. Collectively, these findings indicate that proper dosage of EJC components is necessary for diverse aspects of neuronal development and function. Going forward, genetic models of EJC components will provide valuable tools for further elucidating functions in the nervous system relevant for neurodevelopmental disease.

Oxygen effects on glucose meter measurements with glucose dehydrogenase- and oxidase-based test strips for point-of-care testing.

OBJECTIVES: To determine the effects of different oxygen tensions (Po2) on glucose measurements with glucose dehydrogenase (GD)-based and glucose oxidase (GO)-based test strips, to quantitate changes in glucose measurements observed with different Po2 levels, and to discuss the potential risks of oxygen-derived glucose errors in critical care. DESIGN: Venous blood from healthy volunteers was tonometered to create different oxygen tensions simulating patient arterial Po2 levels. Venous blood from diabetic patients was exposed to air to alter oxygen tensions simulating changes in Po2 during sample handling. Whole-blood glucose measurements obtained from these samples with six glucose meters were compared with reference analyzer plasma glucose measurements. Glucose differences were plotted vs. different Po2 levels to identify error trends. Error tolerances were as follows: a) within +/-15 mg/dL of the reference measurement for glucose levels 100 mg/dL. SETTING AND SUBJECTS: Five healthy volunteers in the bench study and 11 diabetic patients in the clinical study. RESULTS: In the bench study, increases in Po2 levels decreased glucose measured with GO-based amperometric test strips, mainly at Po2 levels >100 torr. At nearly constant glucose concentrations, glucose meter systems showed large variations at low (39 torr) vs. high (396 torr) Po2 levels. Glucose measured with GD-based amperometric and GO-based photometric test strips generally were within error tolerances. In the clinical study, 31.6% (Precision PCx), 20.2% (Precision QID), and 23.0% (Glucometer Elite) of glucose measurements with GO-based amperometric test strips, 14.3% (SureStep) of glucose measurements with GO-based photometric test strips, and 4.6% (Accu-Chek Advantage H) and 5.9% (Accu-Chek Comfort Curve) of glucose measurements with GD-based amperometric test strips were out of the error tolerances. CONCLUSIONS: Different oxygen tensions do not significantly affect glucose measured with the GD-based amperometric test strips, and have minimal effect on GO-based photometric test strips. Increases in oxygen tension lowered glucose measured with GO-based amperometric test strips. We recommend that the effects of different oxygen tensions in blood samples on glucose measurements be minimized by using oxygen-independent test strips for point-of-care glucose testing in critically ill and other patients with high or unpredictable blood Po2 levels.

Superantigen-induced CD4 T cell tolerance mediated by myeloid cells and IFN-gamma.

We have previously shown that systemic staphylococcal enterotoxin A (SEA) injections cause CD4 T cells in TCR-transgenic mice to become tolerant to subsequent ex vivo restimulation. An active IFN-gamma-dependent mechanism of suppression was responsible for the apparent unresponsiveness of the CD4 T cells. In this study, we analyze the response of CD4 T cells isolated throughout the first 10 days of the in vivo response to injected SEA. We show that CD4 T cells isolated at the peak of the in vivo response undergo very little activation-induced cell death after sterile FACS sorting or restimulation in the presence of neutralizing Abs to IFN-gamma. We also show that the IFN-gamma-dependent tolerance develops soon after SEA injection in the spleens of both normal and TCR-transgenic mice. This suppression is dependent upon myeloid cells from the SEA-treated mice and is optimal when inducible NO synthase activity and reactive oxygen intermediates are both present. The data indicate that IFN-gamma, myeloid cells, and a combination of NO and reactive oxygen intermediates all contribute to a common pathway of T cell death that targets activated or responding CD4 T cells. Sorted Gr-1(+) cells from SEA-treated mice also directly suppress the response of naive CD4 T cells in mixed cultures, indicating that this tolerance mechanism may play a role in down-regulating other vigorous immune responses.

Correlation between the light scattering and the mitochondrial content of normal tissues and transplantable rodent tumors.

The development of noninvasive optical studies necessitates an understanding of the biological parameters which affect light propagation in soft tissues. In the present report, we have measured the optical properties of various normal (i.e., perfused liver, brain, skeletal muscle, white adipose tissue) and neoplastic rodent tissues (i.e., glioma, hepatoma, mammary adenocarcinoma) by using time-resolved spectroscopy. The contribution of the hemoglobin (+ myoglobin in the case of muscle) to the total light absorption at 780 nm has been determined. This contribution varies from about 25% (brain, skeletal muscle) to about 100% (white adipose tissue, 13762A mammary adenocarcinoma, 9L glioma). These results are explained by different blood volume fractions in the tissues and by the existence at 780 nm of other chromophores, such as the mitochondrial cytochrome oxidase. Secondly, the dependence of the light scattering of the tissue on both the cell and the mitochondrial content has been analyzed. The results indicate that there is no correlation between the light scattering and the DNA content, measured as an indicator of the cell number in the tissue. The scattering coefficient is proportional to both the succinate dehydrogenase activity and the mitochondrial protein content of the tissue, which are indicators of the mitochondria content of the tissue when based upon estimates of tissue wet weight.

Inhibition of rate of tumor growth by creatine and cyclocreatine.

Growth rate inhibition of subcutaneously implanted tumors results from feeding rats and athymic nude mice diets containing 1% cyclocreatine or 1%, 2%, 5%, or 10% creatine. The tumors studied included rat mammary tumors (Ac33tc in Lewis female rats and 13762A in Fischer 344 female rats), rat sarcoma MCI in Lewis male rats, and tumors resulting from the injection of two human neuroblastoma cell lines, IMR-5 and CHP-134, in athymic nude mice. Inhibition was observed regardless of the time experimental diets were administered, either at the time of tumor implantation or after the appearance of palpable tumors. For mammary tumor Ac33tc, the growth inhibition during 24 days after the implantation was approximately 50% for both 1% cyclocreatine and 1% creatine, and inhibition increased as creatine was increased from 2% to 10% of the diet. For the other rat mammary tumor (13762A), there was approximately 35% inhibition by both 1% cyclocreatine and 2% creatine. In the case of the MCI sarcoma, the inhibitory effect appeared more pronounced at earlier periods of growth, ranging from 26% to 41% for 1% cyclocreatine and from 30% to 53% for 1% creatine; there was no significant difference in growth rate between the tumors in the rats fed 1% and 5% creatine. The growth rate of tumors in athymic nude mice, produced by implantation of the human neuroblastoma IMR-5 cell line, appeared somewhat more effectively inhibited by 1% cyclocreatine than by 1% creatine, and 5% creatine feeding was most effective. For the CHP-134 cell line, 33% inhibition was observed for the 1% cyclocreatine diet and 71% for the 5% creatine diet. In several experiments, a delay in appearance of tumors was observed in animals on the experimental diets. In occasional experiments, neither additive inhibited tumor growth rate for the rat tumors or the athymic mouse tumors.

Antigenic evidence for simultaneous expression of two different lipooligosaccharides by some strains of Haemophilus influenzae type b.

Isolates of Haemophilus influenzae type b (Hib) can be divided into three antigenic groups based on their reactivities with a set of two monoclonal antibodies (MAbs) directed against epitopes in the oligosaccharide region of Hib lipooligosaccharide (LOS) (P. A. Gulig, C. C. Patrick, L. Hermanstorfer, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 55:513-520, 1987). Approximately 24% of Hib strains react with both of these LOS-specific MAbs. Immunoprecipitation experiments involving several of these strains indicated that the epitopes recognized by these MAbs resided in two different LOS molecules, both of which were synthesized by these particular Hib strains. In addition, Western blot (immunoblot) analysis of proteinase K-treated cell extracts of these strains that had been subjected to sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis revealed two different LOS staining patterns when they were probed independently with the two MAbs. Colony blot radioimmunoassay of hundreds of colonies of one of these Hib strains showed that each colony bound both MAbs. Immune electron microscopy confirmed that individual cells of this same Hib strain expressed both types of LOS molecule at the same time. An antibody accessibility radioimmunoassay was used to show that different Hib strains of this type varied in the relative amounts of each of the two MAbs that they could bind to their cell surfaces. These findings indicate that some Hib strains can synthesize two antigenically distinct LOS molecules simultaneously.

Primary structure of the porin protein of Haemophilus influenzae type b determined by nucleotide sequence analysis.

Sequencing techniques for single- and double-stranded DNA were used to determine the nucleotide sequence of the gene encoding P2, the major outer membrane (porin) protein of Haemophilus influenzae type b (Hib). The open reading frame encoding the P2 protein comprised 361 amino acid codons. Comparison of the inferred amino acid sequence with data obtained by amino acid sequencing of the N terminus of the mature or fully processed P2 protein revealed that this protein has a signal peptide composed of 20 amino acids. N-terminal amino acid sequencing of tryptic peptides derived from purified P2 allowed direct identification of 158 of the 341 amino acids in the fully processed P2 protein; there was 100% correlation between these amino acid sequences and that inferred from the nucleotide sequence. The amino acid sequence of Hib P2 protein had 23 to 25% homology with the sequence of the OmpF porin of Escherichia coli and with that of the Neisseria gonorrhoeae porin P.IA. Codon usage in the Hib P2 gene was significantly different from that observed for a gene encoding a porin of E. coli. DNA hybridization studies indicated that there is a single copy of the P2 gene in the Hib chromosome. The availability of the nucleotide and amino acid sequences for the Hib P2 protein will facilitate investigation of the antigenic characteristics and structure-function relationship of this porin.

Cloning of the gene encoding the major outer membrane protein of Haemophilus influenzae type b.

The major outer membrane protein (P2) of Haemophilus influenzae type b (Hib) with an apparent molecular weight of 37,000 to 40,000 has been previously shown to function as a porin and also as a target for antibodies protective against experimental Hib disease. The gene encoding the Hib P2 protein was cloned by using a shuttle vector capable of replication in both Escherichia coli and H. influenzae. The amino acid sequence of the amino terminus of the Hib P2 protein was determined and used to design an oligonucleotide probe corresponding to the first 20 amino acids of this protein. This oligonucleotide probe was used to identify Hib chromosomal DNA fragments containing the Hib P2 gene. These DNA fragments were ligated into the plasmid vector pGJB103 and then used to transform a rec-1 mutant of H. influenzae Rd. Recombinant clones expressing the Hib P2 protein were identified in a colony blot-radioimmunoassay by using a monoclonal antibody specific for a surface epitope of the Hib P2 protein. The gene encoding this Hib protein was present on a 10-kilobase Hib DNA insert in the recombinant plasmid. Transformation experiments involving the recombinant plasmid suggested that unregulated synthesis of Hib P2 is a lethal event in E. coli. The recombinant Hib P2 protein was exposed on the surface of the recombinant H. influenzae strain. This recombinant strain was used to develop a system for detecting polyclonal serum antibodies directed against surface determinants of the Hib P2 protein. The availability of the gene encoding the Hib P2 protein should facilitate investigation of both the immunogenicity and the structure-function relationship(s) of this major outer membrane protein.

Effect of TPN on bone marrow kinetics--therapeutic implications.

Stimulation of tumor and host tissue proliferation occurs with nutritional support of the tumor-bearing host. This study was performed to determine the effect of parenteral nutrition on bone marrow cytokinetics in tumor-bearing animals. Forty-three Lewis/Wistar rats with subcutaneous mammary tumor implants (AC-33) were protein-depleted for 8 days, underwent superior vena cava cannulation and were randomized to receive intravenous saline or total parenteral nutrition (TPN). Animals receiving TPN were sacrificed after 2, 6, 12, 24, and 48 hr and bone marrow cells were harvested for cell cycle analysis by flow cytometry. Compared to control animals, a significant increase in bone marrow cells in S (DNA synthetic) phase was demonstrated after 24 hr of TPN. The ratio of sensitive/resistant bone marrow cells to chemotherapy specific for cells actively synthesizing DNA was increased after 24 hr of TPN. This alteration in bone marrow cytokinetics was transient and distinct from the previously documented stimulation of tumor growth kinetics. The potential therapeutic implications of differentially stimulating host tissue and tumor growth kinetics with TPN are discussed.

Reduction of methotrexate toxicity with improved nutritional status in tumor-bearing animals.

The administration of chemotherapy in clinical situations is limited frequently because of the associated toxicity to normal bone marrow cells, gastrointestinal epithelium, and other host tissues. Although nutritional support has been advocated to reduce chemotherapy-related toxicity in cancer patients, few studies substantiate this clinical impression. The current study was performed to determine the role of nutritional status and enteral nutrient intake as determinants of methotrexate (MTX) toxicity in a well-controlled, tumor-bearing animal model. After subcutaneous mammary tumor (AC-33) inoculation, 56 female Lewis/Wistar rats were assigned randomly to one of the following two nutritional regimens for 14 days: (1) protein-depleted chow (PC) (0.03% protein; 4.27 kcal/g) or (2) standard chow (RC) (22.0% protein; 3.50 kcal/g). After 7 days of dietary control, all animals received one of three weight-adjusted doses of MTX (5, 10, or 20 mg/kg intramuscularly [IM] ) or placebo. All animals received leucovorin rescue (0.6 mg IM) at 6 and 24 hours after MTX injection. Improved nutritional status was associated with a significant reduction in objective measures of MTX-related morbidity and mortality. At low doses of MTX (5 and 10 mg/kg), the mean duration of clinical signs of toxicity (i.e., hair loss, lethargy, and diarrhea) and severity of leukopenia were greater in protein-depleted (PD) animals. With high-dose MTX (20 mg/kg), mortality was increased significantly in PD animals (100%) compared with well-nourished animals (0%). Equivalent tumor response was observed in PD and well-nourished animals. Thus, improved nutritional status by enteral nutrition reduced the morbidity and mortality associated with MTX significantly in this tumor-bearing animal model.

Haemophilus influenzae type b lipooligosaccharide: stability of expression and association with virulence.

Spontaneous antigenic and phenotypic variations in the lipooligosaccharide (LOS) of two strains of Haemophilus influenzae type b (Hib) were previously shown to be associated with changes in virulence (A. Kimura and E.J. Hansen, Infect. Immun. 51:69-79, 1986). The goal of the present study was to define further the stability of LOS expression by this pathogen and the role of Hib LOS in virulence. Variation in LOS antigenic reactivity, as detected with LOS-specific monoclonal antibodies, was observed in 3 of 30 Hib strains after single-colony passage. When large numbers of individual colonies from seven other Hib strains were screened, however, spontaneous LOS antigenic variation was detected in all of the strains. Antigenic variation was not consistently associated with an altered LOS phenotype, as determined by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and silver staining of LOS preparations. Changes in the LOS antigenic phenotype were correlated with altered virulence potential in two strains. In these strains, acquisition of reactivity with certain LOS-directed monoclonal antibodies was associated with the synthesis of a higher-molecular-weight LOS, enhanced virulence, and increased resistance to serum killing involving the classical complement pathway.

Enhanced tumor response to cycle-specific chemotherapy by pulse total parenteral nutrition.

Exogenous nutrient administration has been shown to significantly stimulate tumor growth in numerous animal models. The present study was performed to determine if substrate-induced alterations in tumor metabolism could be exploited to potentiate tumor response to cycle-specific chemotherapy. Following subcutaneous mammary tumor (AC-33) implantation, 55 female Lewis/Wistar rats were randomly assigned to one of three nutritional regimens for 48 hr: (1) protein-depleted chow (0.03% protein) ad lib per os, (2) standard rat chow (22.0% protein) ad lib per os, or (3) total parenteral nutrition (TPN; 18.6% dextrose/2.8% amino acids). One-half of the animals in each group received a single dose of methotrexate (5 mg/kg im) while the remaining animals received placebo (saline) injections. At sacrifice, methotrexate-treated animals receiving TPN demonstrated a significantly smaller tumor volume (0.47 +/- 0.44 cm3) compared to animals given either protein depleted chow (1.30 +/- 0.76 cm3) or standard rat chow (1.34 +/- 0.83 cm3) (P less than 0.01). In this animal model, adjuvant TPN was found to significantly potentiate tumor response to cycle-specific chemotherapy with no detectable exacerbation of host toxicity.

Use of 111In-bleomycin for combining radiotherapy and chemotherapy on glioma-bearing mice.

Mice bearing transplanted glioma received 0.9% NaCl, 0.1 mg of BLM, or 200-250 microCi of 111In-BLM (0.1 mg BLM) daily for 5 days intraperitoneally. After therapy, tumor sizes were in the order NaCl greater than BLM greater than 111In-BLM. On the 11th day after the first injection, tumor size (mm3) in the 111In-BLM group was 1,220; in the BLM group, it was 2,310 (P less than .025). After intratumor injection of a total dose of 0.1 mg of BLM/gm tumor weight, or of 1 mCi/gm tumor weight of 111In-BLM (carried by 0.1 mg of BLM/gm tumor weight), the tumor size decreased in the 111In-BLM group more than in the BLM group. On the 5th day after the 2nd dose therapy, the tumor size in the 111In-BLM group was 2,020; in the BLM group it was 4,220 (P less than .05). Host weights for these two groups were similar. The necrotic area in the tumor was much greater in the 111In-BLM group than in the BLM group. These results suggest the use for radiotherapy and chemotherapy.

A new 111In-bleomycin complex for combined radiotherapy and chemotherapy.

Six days after tumor transplantation three daily intraperitoneal doses of 0.9% NaCl, bleomycin (BLM), or a new 111In-bleomycin complex (BLMC, 15 microCi/g body weight) were administered to glioma-bearing mice. After therapy, tumors in mice treated with 111In-BLMC were smaller than those treated with BLM. Sixteen days after the first injection tumor size for 111In-BLMC-treated mice was 560 (240-1,030) mm3, 1,980 (1,400-3,290) mm3 for BLM (P less than 0.025), and 4,830 (2,580-9,180) mm3 for NaCl (0.1 less than P less than 0.2). Thirteen days after tumor transplantation glioma-bearing mice received single intratumor injection of 0.9% NaCl, BLM, or 111In-BLMC (1.5 mCi, carried by 0.5 mg BLM/g tumor weight). The average tumor size for 111In-BLMC was smaller than that for BLM by a factor of 2.5-3.7. Host weights for these two groups were similar, and morphologic abnormalities were not found in kidney or liver.

A new tumor imaging agent--111In-bleomycin complex. Comparison with 67Ga-citrate and 57Co-bleomycin in tumor-bearing animals.

We have found a new 111In-bleomycin complex (BLMC), which has high affinity to tumor, does not bind to transferrin and is stable in vivo. Distribution in animals bearing glioma, hepatoma, or mammary adenocarcinoma at 48 hours showed: the ratios of tumor to blood, brain, heart, lung, liver, pancreas, stomach, and femur were 1.4-22.4 times as high for 111In-BLMC as for 67Ga-citrate. In mammary adenocarcinoma, 111In-BLMC bound more to viable and 57Co-Bleomycin (BLM) more to necrotic tumor. In viable tumor, the concentration of 111In-BLMC was similar to that of 57Co-BLM. The ratios of tumor to stomach and pancreas were higher, to blood, brain, muscle, heart, and femur were lower for 111In-BLMC than those for 57Co-BLM. The ratios of tumor to lung, liver, spleen, skin, and kidney were similar for the two compounds. Tumors were imaged more distinctly with the new 111In-BLMC and 57Co-BLM than with 67Ga-citrate. 111In-BLMC is promising for tumor imaging.

Sequential changes in the activities of lipoprotein lipase and lipogenic enzymes during tumor growth in rats.

The sequential changes in lipid metabolism during tumor growth were evaluated in inbred Lewis rats bearing a mammary adenocarcinoma (AC33). Serum lipids, insulin, glucagon, and liver and adipose tissue lipogenic enzymes were measured in tumor-bearing and control rats after 6, 12, 18, 24, and 32 days of tumor growth. Lipoprotein lipase (LPL) activity in heart, soleus muscle, and epididymal fat pads was also determined. On the sixth day, the activity of LPL was reduced in the adipose tissue and remained lower throughout the duration of the experiment. Serum triglycerides were elevated from the 12th day followed by an increase in free fatty acid levels from the 18th day of tumor growth. These changes were accompanied by a decrease in serum insulin levels in the tumor-bearing rats from Day 12. The presence of the tumor also decreased the activities of some of the lipogenic enzymes in liver and adipose tissue, but these changes occurred at the later time points. On the 24th day, a decrease in fat pad weights was found and characterized by a decrease in fat cell size but not in fat cell number. These results suggest that a defect in clearance, due to the decrease in the activity of adipose tissue LPL, may be responsible for the early development of hypertriglyceridemia during tumor growth. In this study, the alterations in the lipogenic enzymes and LPL cannot be attributed to reduced food intake but may be due to the direct or an indirect effect of the tumor on a hormone such as insulin.

A new 111In-bleomycin complex for tumor imaging: preparation, stability, and distribution in glioma-bearing mice.

A new 111In-bleomycin complex (111In-BLMC) is here reported. Its radiochemical purity was 99% by thin-layer chromatography (TLC) (Rf 0.65) and in 5% agarose gel electrophoresis in 0.02 M NaHCO3 it migrated toward the anode. Autoradiographs of TLC and gel electrophoresis plates showed no change on storage for 3 weeks. Urine and plasma from untreated or glioma-bearing mice after injection of 111In-BLMC were analyzed by TLC and gel electrophoresis. Results indicated stability in vivo, nonbinding to transferrin, affinity to viable tumor, and excretion faster than 111In-BLM-B2, 111In-BLM, or 57Co-BLM. Tissue distributions 24 hr after injection of radiopharmaceutical showed activity ratios of tumor to blood, muscle, and brain of 13.1, 12.4, and 81.6, respectively, which were significantly higher than those for previously prepared 111In-BLM-B2 or 111In-BLM (except for brain, 0.05 less than P less than 0.1). The new 111In-BLM complex may be useful in clinical imaging and for combining radionuclide radiotherapy and chemotherapy.