Assessing intracellular pH regulation in H+-ATPase-rich ionocytes in zebrafish larvae using in vivo ratiometric imaging.

The H+-ATPase-rich (HR) cells of zebrafish larvae are a sub-type of ion-transporting cell located on the yolk sac epithelium that are responsible for Na+ uptake and H+ extrusion. Current models of HR cell ion transport mechanisms in zebrafish larvae are well established, but little is known about the involvement of the various ion transport pathways in regulating intracellular acid-base status. Here, a ratiometric imaging technique was developed and validated to monitor intracellular pH (pHi) continuously in larval zebrafish HR cells in vivo Gene knockdown or CRISPR/Cas9 knockout approaches were used to evaluate the roles of the two principal apical membrane acid excretory pathways, the Na+/H+ exchanger (NHE3b; slc9a3.2) and the H+-ATPase (atpv1aa). Additionally, the role of HR cell cytosolic carbonic anhydrase (CAc) was investigated because of its presumed role in providing H+ for Na+/H+ exchange and H+-ATPase. The temporal pattern and extent of intracellular acidification during exposure of fish to 1% CO2 and the extent of post-CO2 alkalisation were altered markedly in fish experiencing knockdown/knockout of CAc, NHE3b or H+-ATPase. Although there were slight differences among the three knockdown/knockout experiments, the typical response was a greater degree of intracellular acidification during CO2 exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. The metabolic alkalosis and subsequent acidification associated with 20 mmol l-1 NH4Cl exposure and its washout were largely unaffected by gene knockdown. Overall, the results suggest markedly different mechanisms of intracellular acid-base regulation in zebrafish HR cells depending on the nature of the acid-base disturbance.

Endothelial apoptosis in Braf-deficient mice.

Tyrosine kinase growth factor receptors and Ras/Raf/MEK/MAPK signalling have been implicated in the suppression as well as augmentation of programmed cell death. In addition, a Ras-independent role for Raf as a suppressor of programmed cell death has been suggested by the recent finding that Craf1 interacts with members of the Bcl-2 family at mitochondrial membranes. However, genetic studies of C. elegans and Drosophila, as well as the targeted mutagenesis of the murine Araf gene, have failed to support such a role. Here we show that mice with a targeted disruption in the Braf gene die of vascular defects during mid-gestation. Braf -/- embryos, unlike Araf -/- or Craf1 -/- embryos (L.W. et al., unpublished), show an increased number of endothelial precursor cells, dramatically enlarged blood vessels and apoptotic death of differentiated endothelial cells. These results establish Braf as a critical signalling factor in the formation of the vascular system and provide the first genetic evidence for an essential role of Raf gene in the regulation of programmed cell death.

Rhabdomyosarcomas and radiation hypersensitivity in a mouse model of Gorlin syndrome.

Gorlin (or nevoid basal cell carcinoma) syndrome is characterized by a variety of clinical problems including generalized overgrowth of the body, cysts, developmental abnormalities of the skeleton and a predisposition to benign and malignant tumors. The syndrome results from germline mutations of the human homolog of the drosophila segment polarity gene patched (ptc). Here we report that mice heterozygous for ptc develop many of the features characteristic of Gorlin syndrome and that they exhibit a high incidence of rhabdomyosarcomas (RMS), the most common soft-tissue sarcoma in children. The downstream signalling partner of ptc, gli1, was overexpressed in all RMSs analyzed, indicating that abnormal signalling of the ptc-gli1 pathway may be common for the various tumors associated with the syndrome. igf2, implicated in the formation of RMSs, was also overexpressed, suggesting cross-talk between the ptc and igf2 pathways in tumorigenesis. Developmental defects in Gorlin syndrome resemble those induced by ionizing radiation. We show that ptc heterozygous mice exhibit increased incidence of radiation-induced teratogenesis. This suggests a role for ptc in the response to ionizing radiation and provides a model for both the systemic (developmental) and stochastic (cancer) abnormalities observed in Gorlin syndrome.

Early-life stress influences ion balance in developing zebrafish (Danio rerio).

As a key endocrine axis involved in responding to stress, the hypothalamic-pituitary-interrenal axis plays dual roles in mobilizing energy and maintaining ionic/osmotic balance in fishes. Although these roles have been examined independently in detail in adult fishes, less attention has been paid to the effects of an endogenous stress response during early life, particularly with respect to its potential effects on ionic/osmotic balance. The present study tested the hypothesis that exposure of zebrafish to stress during early development would alter ion balance later in life. Zebrafish at three developmental stages (4, 7, or 15 days post-fertilization, dpf) were subjected to an air-exposure stressor twice a day for 2 days, causing elevation of whole-body cortisol levels. Individuals stressed early in life exhibited decreased survival and growth, altered cortisol responses to a subsequent air-exposure stressor, and increased whole-body Na+ and Ca2+ concentrations. Changes in whole-body Ca2+ concentrations were accompanied by increased ionocyte abundance at 7 dpf and increased rates of Ca2+ uptake from the environment. Differences in whole-body ion concentrations at 15 and 35 dpf were not accompanied by altered ion uptake rates. Across all ages examined, air-exposure stress experienced at 7 dpf was particularly effective at eliciting phenotypic changes, suggesting a critical window at this age for a stress response to influence development. These findings demonstrate that early-life stress in zebrafish triggers developmental plasticity, with age-dependent effects on both the cortisol stress axis and ion balance.

Regulation of RAR beta 2 mRNA expression: evidence for an inhibitory peptide encoded in the 5'-untranslated region.

Regulation of mRNA translation and stability plays an important role in the control of gene expression during embryonic development. We have recently shown that the tissue-specific expression of the RAR beta 2 gene in mouse embryos is regulated at the translational level by short upstream open reading frames (uORFs) In the 5'-untranslated region (Zimmer, A., A.M. Zimmer, and K. Reynolds. 1994. J. Cell Biol. 127:1111-1119). To gain insight into the molecular mechanism, we have performed a systematic mutational analysis of the uORFs. Two series of constructs were tested: in one series, each uORF was individually inactivated by introducing a point mutation in its start codon; in the second series, all but one ORF were inactivated. Our results indicate that individual uORFs may have different functions. uORF4 seems to inhibit translation of the major ORF in heart and brain, while uORFs 2 and 5 appear to be important for efficient translation in all tissues. To determine whether the polypeptide encoded by uORF4 or the act of translating it, is the significant event, we introduced point mutations to create silent mutations or amino acid substitutions in uORF4. Our results indicate that the uORF4 amino acid coding sequence is important for the inhibitory effect on translation of the downstream major ORF.

Tissue specific expression of the retinoic acid receptor-beta 2: regulation by short open reading frames in the 5'-noncoding region.

The 40-S subunit of eukaryotic ribosomes binds to the capped 5'-end of mRNA and scans for the first AUG in a favorable sequence context to initiate translation. Most eukaryotic mRNAs therefore have a short 5'-untranslated region (5'-UTR) and no AUGs upstream of the translational start site; features that seem to assure efficient translation. However, approximately 5-10% of all eukaryotic mRNAs, particularly those encoding for regulatory proteins, have complex leader sequences that seem to compromise translational initiation. The retinoic-acid-receptor-beta 2 (RAR beta 2) mRNA is such a transcript with a long (461 nucleotides) 5'-UTR that contains five, partially overlapping, upstream open reading frames (uORFs) that precede the major ORF. We have begun to investigate the function of this complex 5'-UTR in transgenic mice, by introducing mutations in the start/stop codons of the uORFs in RAR beta 2-lacZ reporter constructs. When we compared the expression patterns of mutant and wild-type constructs we found that these mutations affected expression of the downstream RAR beta 2-ORF, resulting in an altered regulation of RAR beta 2-lacZ expression in heart and brain. Other tissues were unaffected. RNA analysis of adult tissues demonstrated that the uORFs act at the level of translation; adult brains and hearts of transgenic mice carrying a construct with either the wild-type or a mutant UTR, had the same levels of mRNA, but only the mutant produced protein. Our study outlines an unexpected role for uORFs: control of tissue-specific and developmentally regulated gene expression.

Pharmacokinetics of 99mTc(Sn)- and 131I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments in nude mice.

The biodistribution, radioimmunoimaging, and high pressure liquid chromatography activity profiles of 99mTc(Sn) and 131I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments were compared. Nude mice, bearing specific (colon carcinoma, LS174T) and nonspecific (pancreatic carcinoma, MIA) xenografts were given injections of the respective radiolabeled antibody fragments and also of irrelevant 125I-labeled antibody fragments (MOPC-21). The animals were imaged at 24 h after being given injections, they were sacrificed, and biodistribution studies were performed. Results of the study showed high kidney uptake [48.6% injected dose (ID)/g +/- 8.1% (SD)] and low tumor uptake (1.5% ID/g +/- 0.6%) for 99mTc(Sn)-labeled fragments and higher uptake (4.4% ID/g +/- 0.6%) for 131I-labeled fragments, resulting in a higher localization index for the radioiodinated monoclonal antibody fragments. Imaging results showed good tumor visualization at 24 h after injection for the 131I-labeled fragments and poor tumor visualization with predominant kidney uptake for 99mTc(Sn)-labeled fragments. After radiolabeling, high pressure liquid chromatography analysis indicated that 131I was primarily associated with F(ab')2 fragments, whereas 99mTc was mostly associated with Fab' fragments.

Cannabinoid withdrawal syndrome is reduced in pre-proenkephalin knock-out mice.

The functional interactions between the endogenous cannabinoid and opioid systems were evaluated in pre-proenkephalin-deficient mice. Antinociception induced in the tail-immersion test by acute Delta9-tetrahydrocannabinol was reduced in mutant mice, whereas no difference between genotypes was observed in the effects induced on body temperature, locomotion, or ring catalepsy. During a chronic treatment with Delta9-tetrahydrocannabinol, the development of tolerance to the analgesic responses induced by this compound was slower in mice lacking enkephalin. In addition, cannabinoid withdrawal syndrome, precipitated in Delta9-tetrahydrocannabinol-dependent mice by the injection of SR141716A, was significantly attenuated in mutant mice. These results indicate that the endogenous enkephalinergic system is involved in the antinociceptive responses of Delta9-tetrahydrocannabinol and participates in the expression of cannabinoid abstinence.

Absence of delta -9-tetrahydrocannabinol dysphoric effects in dynorphin-deficient mice.

The involvement of dynorphin on Delta-9-tetrahydrocannabinol (THC) and morphine responses has been investigated by using mice with a targeted inactivation of the prodynorphin (Pdyn) gene. Dynorphin-deficient mice show specific changes in the behavioral effects of THC, including a reduction of spinal THC analgesia and the absence of THC-induced conditioned place aversion. In contrast, acute and chronic opioid effects were normal. The lack of negative motivational effects of THC in the absence of dynorphin demonstrates that this endogenous opioid peptide mediates the dysphoric effects of marijuana.

Radioimmunodetection and radioimmunotherapy of cutaneous T cell lymphomas using an 131I-labeled monoclonal antibody: an Illinois Cancer Council Study.

A radiolabeled murine monoclonal antibody (T101) was used for imaging and therapy of six patients with cutaneous T cell lymphoma (CTCL). Radioimmunodetection was performed with a 5.6 to 13.1 mCi 131I-T101 preparation (9.6 to 10.5 mg). A therapeutic dose of 100.5 to 150.1 mCi 131I on 9.9 to 16.9 mg of antibody was administered to five patients, with subsequent retreatment following plasmapheresis in three patients at the time of disease progression. All patients responded to their initial therapy and two patients responded to retreatment. Regression of skin lesions and peripheral adenopathy was witnessed. All patients reported resolution of their chronic pruritus. The duration of response ranged from 3 weeks to 3 months. Acute toxicity included fevers, pruritus, and mild dyspnea in two instances. Myelosuppression was seen in patients receiving the 144.7 mCi, 145.0 mCi, and 150.1 mCi 131I-T101 doses. Radioimmunodiagnostic and therapy studies included gamma scintigraphy, plasma, urinary, and wholebody antibody clearances, and biodistribution determined from skin, bone marrow, and liver biopsies. Immunologic studies included immunoperoxidase staining of target tissues, immunofluorescent flow cytometric analysis on peripheral blood and bone marrow, assays for serum blocking factors, determination of a human antimouse antibody (HAMA) response, and quantitation of circulating T101 levels. These preliminary data suggest that 131I-T101 has therapeutic potential in CTCL and that myelosuppression will be the limiting toxicity.

Craf-1 protein kinase is essential for mouse development.

The three mammalian Raf serine/threonine protein kinases mediate the transduction of proliferative and differentiative signals from a variety of cell surface receptors to the nucleus. We report here that Craf-1 is essential for mouse development, as its mutation results in embryonic lethality. Developmental defects are found in mutant placentas as well as in the skin and in the lungs of mutant embryos. Craf-1 mutants also display a generalized growth retardation which is consistent with the ubiquitous expression of Craf-1 and which could be due to the reduced proliferation of mutant cells. Interestingly, the time-point of embryonal death varies depending on the genetic background. This suggests that Craf-1-mediated signaling is affected by genetic background-specific alleles of other genes.

Long-term persistence of human anti-murine antibody responses following radioimmunodetection and radioimmunotherapy of cutaneous T-cell lymphoma patients using 131I-T101.

Five cutaneous T-cell lymphoma (CTCL) patients have been imaged and treated with radiolabeled murine monoclonal antibody 131I-T101 in our laboratory. All patients developed human anti-murine antibody responses (HAMA) 14 days after the primary antibody infusion. HAMA responses could still be detected more than 22 months after T101 treatment. A substantial proportion of HAMA was crossreactive with any IgG2a antibody tested, although there did exist a specific anti-idiotypic component to HAMA. HAMA were of both IgM and IgG isotype. We also analyzed the effects of plasmapheresis on the specific HAMA isotypes in three patients who were retreated at the time of disease progression.

Region selective up-regulation of micro-, delta- and kappa-opioid receptors but not opioid receptor-like 1 receptors in the brains of enkephalin and dynorphin knockout mice.

The role of endogenous opioid peptides and receptors has recently been investigated using knockout mice. Although the affinities of opioid peptides for opioid receptors has been known for many years there is still some uncertainty over which receptor is the endogenous target for each peptide. To address this issue we have studied using quantitative autoradiography the levels of all four opioid receptor subtypes (micro, delta, kappa and opioid receptor-like 1 [ORL1]) in brains sectioned from enkephalin and dynorphin knockouts, as well as from double knockouts. Because receptor up-regulation has been observed when its cognate ligand-peptide is genetically ablated, regional changes in receptor binding in knockout mice may reflect areas where the peptide ligand is tonically active at its receptor or played a role in receptor regulation. In addition, the study aimed to correlate previously observed behaviour in these animals with receptor modulation. Marked region-specific up-regulation of the micro, delta, and kappa opioid receptors but not ORL1 receptors was observed in proenkephalin and prodynorphin knockouts. In proenkephalin knockouts this was most pronounced for the micro- and delta-receptor and in prodynorphin knockouts for the kappa-receptor. Combinatorial double knockouts did not show any changes in addition to those observed in single knockouts. The largest changes were observed in limbic regions and our results suggest that proenkephalin peptides are tonically active at micro and delta-receptors predominantly in these areas. Prodynorphin peptides appear to regulate mostly the kappa-receptor but they are also modulators of micro- and delta-receptors.

The paper spot test: a rapid method for quantitating stannous concentrations in radiopharmaceutical kits.

We have developed a simplified, semiquantitative test for the determination of stannous tin in pyrophosphate and other tin-containing radiopharmaceuticals, excluding those stabilized with ascorbic acid and MAA preparations. The test involves the formation and disappearance of a positive red color complex in the presence of Sn(II) and in acidified porphyrin solution. With this technique, the time of spot disappearance is directly proportional to the Sn(II) concentration spotted. The procedure is easy to use, requiring only a high-intensity light source (30-watt light bulb) and a timing device. The test is accurate, reproducible, and sensitive to Sn(II) levels as low as 40 micrograms/ml. Because the procedure is rapid (requiring less than 5 min), it can easily be incorporated into the routine radiopharmaceutical quality-control program of any nuclear medicine facility.

Rapid miniaturized chromatographic quality-control procedures for Tc-99m radiopharmaceuticals.

Our laboratory has adopted a complete miniaturized charomatography system for Tc-99m radiopharmaceuticals in order to improve upon the commercial systems currently available. Three distinct, separate, chromatographic procedures are used to determine the labeling efficiencies of Tc-99m-labeled sulfur colloid, MAA, stannous chloride, phytate, DMSA, DTPA, pyrophosphate, diphosphonate, methylene diphosphonate, polyphosphate, and glucoheptonate. The chromatographic systems include Whatman 31 ET paper and acetone, Gelman ITLC-SG and 0.9% sodium chloride, and Gelman ITLC-SA and acetone. The chromatographic strips are miniaturized (1 X 6 cm), colored-coded, marked, and numbered. All the chromatographic quality-control procedures are simple, rapid, and can easily be incorporated into the routine quality-control program of any nuclear medicine facility.

Enzymatic inhibition of diphosphonate: a proposed mechanism of tissue uptake.

Enzymes have been proposed as tissue receptors that bine 99mTc-stannous diphosphonate and its analogs. Incubation of diphosphonate with several enzymes demonstrated inhibition of acid and alkaline phosphatase activity but showed no effect on glutamic oxalacetic transaminase and lactate dehydrogenase activity. Complete reversal of the diphosphonate-induced inhibition of alkaline phosphatase activity occurred when calcium ion was added to the reaction. The specificity of calcium to induce reversal was dispelled when magnesium ion gave identical results. Diphosphonate-induced inhibition of acid phosphatase, however, was not reversed by calcium or magnesium.

Assay of 32P-sodium phosphate using a commercial dose calibrator.

Dose calibrators are not usually used to measure the activity of pure beta-emitting radionuclides. In this work, the activity of 32P-sodium phosphate was accurately measured with a Capintec CRC-2 dose calibrator. Using a calibration knob setting of 012 on the 1-mCi range, the 32P dose (in mCi) could be calculated directly simply by multiplying the instrument readout by 10. The dose calibrator response was found to be linear at this knob setting and moderate alterations in geometry produced no significant changes.

Radioimmunotherapy of patients with cutaneous T-cell lymphoma using an iodine-131-labeled monoclonal antibody: analysis of retreatment following plasmapheresis.

Radioimmunotherapy retreatment of patients receiving radiolabeled murine monoclonal antibodies is difficult because of human antimurine antibody (HAMA) formation. Retreatment therapy was initiated in three patients at the time of disease progression using a radioiodinated monoclonal antibody (T101). The clinical protocol consisted of a two day plasma exchange (4-6 L) to reduce HAMA titers. Immunoimaging was performed with 5 mCi 131I-T101 (10 mg). Gamma scintillation images were obtained 18 hr postinfusion, and radiation dosimetry estimates were performed. At 24 hr postinfusion, each patient received a 100-mCi 131I-T101 (10 mg) therapy dose. Results obtained after plasmapheresis showed a significant reduction, ranging from 28%-61%, in HAMA titers. Blood clearances were markedly different between initial therapy and retreatment therapy for patient with high HAMA titers, reflecting immune complex formation. Two patients responded to retreatment therapy with responses lasting 1 to 2 mo. Minimal acute and no chronic toxicities were observed during the retreatment protocol.

Considerations for tomographic imaging of monoclonal antibodies.

Monoclonal antibodies have begun to assume a significant role in clinical research. The ability to label these agents has initiated research in the areas of radioimmunodetection and radioimmunotherapy. In the case of antibodies directed against tumor antigens, imaging has been employed to help assess location and extent of disease, and to provide information and extent of disease, and to provide information concerning biodistribution to be used in subsequent dosimetric calculations. Because of the low counting statistics characteristic of such images, the use of single photon emission computed tomography (SPECT) is suggested as a potential method of improving the diagnostic yield from image data. Careful attention to acquisition parameters and image processing options is needed if these goals are to be achieved.

Nuclear medicine imaging workstations based on personal computer technology.

The development of personal computer technology has resulted in extremely powerful, inexpensive computers available as consumer items. With the addition of suitable hardware for gamma camera interfacing and image display, such systems can be transformed into fully functional nuclear medicine computers capable of performing all of the acquisition and processing tasks required in a modern radioisotope imaging department. Such an approach to nuclear medicine computerization offers many advantages in terms of flexibility, speed, cost, and expandability.