Maleic acid is a biomarker for maleylacetoacetate isomerase deficiency; implications for newborn screening of tyrosinemia type 1.

Dried blood spot succinylacetone (SA) is often used as a biomarker for newborn screening (NBS) for tyrosinemia type 1 (TT1). However, false-positive SA results are often observed. Elevated SA may also be due to maleylacetoacetate isomerase deficiency (MAAI-D), which appears to be clinically insignificant. This study investigated whether urine organic acid (uOA) and quantitative urine maleic acid (Q-uMA) analyses can distinguish between TT1 and MAAI-D. We reevaluated/measured uOA (GC-MS) and/or Q-uMA (LC-MS/MS) in available urine samples of nine referred newborns (2 TT1, 7 false-positive), eight genetically confirmed MAAI-D children, and 66 controls. Maleic acid was elevated in uOA of 5/7 false-positive newborns and in the three available samples of confirmed MAAI-D children, but not in TT1 patients. Q-uMA ranged from not detectable to 1.16 mmol/mol creatinine in controls (n = 66) and from 0.95 to 192.06 mmol/mol creatinine in false-positive newborns and MAAI-D children (n = 10). MAAI-D was genetically confirmed in 4/7 false-positive newborns, all with elevated Q-uMA, and rejected in the two newborns with normal Q-uMA. No sample was available for genetic analysis of the last false-positive infant with elevated Q-uMA. Our study shows that MAAI-D is a recognizable cause of false-positive TT1 NBS results. Elevated urine maleic acid excretion seems highly effective in discriminating MAAI-D from TT1.

[Low muscular mass and ketoacidosis]. / Lage spiermassa en een ketoacidose.

Fasting ketoacidosis is especially an underdiagnosed problem in patients with neuro-muscular disease with severely depleted muscular mass. During periods of prolonged fasting, low levels of insulin and high levels of glucagon induce lipolysis in the peripheral fat tissue. This will result in elevated free fatty acids levels in de blood and increased ketogenesis in the liver. These ketones pass into the blood, leading to a ketoacidosis. Patients with low muscular mass are more susceptible to develop ketoacidosis due to lower energy reserves and reduced glycogen stores on the one hand, and a reduced uptake of ketones by their low muscular mass on the other hand. During periods of increased metabolism and in the absence of adequate caloric intake, this can easily lead to severe ketoacidosis. An adequate oral caloric intake is essential in the prevention and treatment of fasting ketoacidosis.

Measurement of the helicity asymmetry E for the reaction γ p → π 0 p : The CBELSA/TAPS Collaboration.

A measurement of the double-polarization observable E for the reaction γ p → π 0 p is reported. The data were taken with the CBELSA/TAPS experiment at the ELSA facility in Bonn using the Bonn frozen-spin butanol (C 4 H 9 OH) target, which provided longitudinally-polarized protons. Circularly-polarized photons were produced via bremsstrahlung of longitudinally-polarized electrons. The data cover the photon energy range from E γ = 600 to 2310 MeV and nearly the complete angular range. The results are compared to and have been included in recent partial wave analyses.

Observation of the pη

Data on the beam asymmetry Σ in the photoproduction of η mesons off protons are reported for tagged photon energies from 1130 to 1790 MeV (mass range from W=1748 MeV to W=2045 MeV). The data cover the full solid angle that allows for a precise moment analysis. For the first time, a strong cusp effect in a polarization observable has been observed that is an effect of a branch-point singularity at the pη^{'} threshold [E_{γ}=1447 MeV (W=1896 MeV)]. The latest BnGa partial wave analysis includes the new beam asymmetry data and yields a strong indication for the N(1895)1/2^{-} nucleon resonance, demonstrating the importance of including all singularities for a correct determination of partial waves and resonance parameters.

N(1520) 3/2(-) helicity amplitudes from an energy-independent multipole analysis based on new polarization data on photoproduction of neutral pions.

New data on the polarization observables T, P, and H for the reaction γp→pπ(0) are reported. The results are extracted from azimuthal asymmetries when a transversely polarized butanol target and a linearly polarized photon beam are used. The data were taken at the Bonn electron stretcher accelerator ELSA using the CBELSA/TAPS detector. These and earlier data are used to perform a truncated energy-independent partial wave analysis in sliced-energy bins. This energy-independent analysis is compared to the results from energy-dependent partial wave analyses.

First measurement of the helicity asymmetry for γp → pπ0 in the resonance region.

The first measurement of the helicity dependence of the photoproduction cross section of single neutral pions off protons is reported for photon energies from 600 to 2300 MeV, covering nearly the full solid angle. The data are compared to predictions from the SAID, MAID, and BnGa partial wave analyses. Strikingly large differences between data and predictions are observed, which are traced to differences in the helicity amplitudes of well-known and established resonances. Precise values for the helicity amplitudes of several resonances are reported.

Successful treatment of metastatic retinoblastoma with high-dose chemotherapy and autologous stem cell rescue in South America.

High-dose chemotherapy (HDC) followed by autologous stem cell rescue (ASCR) is the only curative treatment for metastatic retinoblastoma, but its feasibility in developing countries is unknown. We report 11 consecutive children (six unilateral) treated in three South-American middle-income countries with HDC-ASCR. One patient had metastatic retinoblastoma at diagnosis and the remaining ones had a metastatic relapse. Metastatic sites included BM=6, bone=4, orbit=5 and central nervous system (CNS)=4. All patients received induction with conventional chemotherapy achieving CR at a median of 5.7 months from the diagnosis of metastasis. Conditioning regimens included carboplatin and etoposide with thiotepa in six or with CY in four or melphalan in one patient. All patients engrafted after G-CSF-mobilized peripheral blood ASCR and no toxic deaths occurred. Two children received post-ASCR CNS radiotherapy. Seven children have disease-free survival (median follow-up 39 months). CNS relapse, isolated (n=3) or with systemic relapse (n=1), occurring at a median of 7 months after ASCT was the most common event. In the same period, five children with metastatic retinoblastoma did not qualify for HDC-ASCR and died. We conclude that HDC-ASCR is a feasible and effective treatment for children with metastatic retinoblastoma in middle-income countries.

Glomerular hypertrophy precedes albuminuria and segmental loss of podoplanin in podocytes in Munich-Wistar-Frömter rats.

Focal segmental glomerulosclerosis (FSGS) is a common cause of end-stage renal disease. Albuminuria is a risk factor for FSGS and is influenced by environmental, genetic, and sex-specific factors. Podocytes play a central role in the development of albuminuria, but the precise relationship between early glomerular and podocyte-associated damage and albuminuria is unclear. Furthermore, experimental findings demonstrate a sex difference in development of albuminuria and FSGS. We investigated the early glomerular changes in male Munich-Wistar-Frömter (MWF) rats, which spontaneously develop albuminuria, and male albuminuria-resistant spontaneously hypertensive rats (SHR). In addition, since female MWF rats are protected from overt proteinuria and progressive renal disease, we compared the phenotypic changes in podocytes during early development of albuminuria in male and female MWF rats. In male MWF rats, glomerular hypertrophy preceded the onset of albuminuria and was greater than in male SHR. Albuminuria developed starting at 6 wk of age and coincided with focal and segmental loss of podoplanin, increased expression of desmin, entrapment of albumin in affected podocytes, and focal and segmental foot process effacement at the ultrastructural level. Other podocyte-associated molecules, such as nephrin and zonula occludens 1, were unaffected. Early glomerular hypertrophy and podocyte damage did not differ between male and female MWF rats. Our data show for the first time that albuminuria in male and female MWF rats is preceded by glomerular hypertrophy and accompanied by focal and segmental loss of podoplanin when FSGS was not yet present.

[An adult with mechanical ileus in association with non-rotation of the intestine]. / Een volwassene met een strengileus samenhangend met non-rotatie van de darm.

A mechanical ileus was considered in the differential diagnosis of a 28-year-old man who presented to the Emergency Clinic with acute, severe, painful cramps in the lower abdomen of 2 hours' duration, without radiation and with an urge to move constantly. An emergency laparotomy was then performed, revealing non-rotation of the intestine; the last segment ofthe small intestine was pinched off by a strangulation. Several strangulations were cleaved, after which the symptoms disappeared. Non-rotation, a form of malrotation, is a congenital anomaly of intestinal rotation. In adults, non-rotation is a rare diagnosis with a variable presentation. Surgical intervention is necessary in both the acute and the more chronic presentation. The chronic presentation is usually discovered by chance in patients who have had aspecific recurrent abdominal complaints for a long time; if malrotation is suspected, additional investigation, for example by means of a gastrointestinal contrast study, is necessary before resorting to surgery. In the acute situation, immediate surgery is the only proper decision. Surgical intervention comprises reduction of the volvulus, inspection of the mesenteric bands (Ladd's bands) that run from the coecum to the lateral peritoneum and compress the duodenum, and an appendectomy: the Ladd procedure.

Use of a telemetered dispensing system for controlling nutrient additions to experimental patch reefs in the ENCORE study at One Tree Island, Great Barrier Reef, Australia.

A telemetrically controlled system was developed to add nutrients automatically to experimental patch reefs in a remote marine environment. The experiment, called ENCORE, was done in the lagoon of One Tree Island, a remote research station at the southern end of the Great Barrier Reef. Nutrient dispensing units (NDUs), moored adjacent to patch reefs in the lagoon, were telemetrically linked to a base station on the island. The base station, about 3 km away from the furthest NDU, consisted of a dedicated computer, controller and radio transmitter, which relayed coded signals to a radio receiver mounted on each NDU. This activated a solenoid valve to discharge a measured quantity of concentrated nutrient solution from a measuring chamber using compressed air from a SCUBA tank. The solution was discharged through 4-8 PVC outlets into the basins of the patch reefs to allow thorough mixing. The base station interrogated each NDU to find out if the operation had been successful and stored the information on disk to provide a daily log of operations. Nutrient samples taken within the patch reefs demonstrated that calculated initial mean concentrations of 2 micrograms-at PO4-P l-1 and 10 micrograms-at NH4-N l-1 were achieved. The system we have developed can be used in many situations where regular perturbations need to be introduced to aquatic ecosystems. It uses state-of-the-art technology, yet all components are commercially readily available and relatively inexpensive. Detailed specifications and drawings are available from the Great Barrier Reef Marine Park Authority.

ENCORE: the effect of nutrient enrichment on coral reefs. Synthesis of results and conclusions.

Coral reef degradation resulting from nutrient enrichment of coastal waters is of increasing global concern. Although effects of nutrients on coral reef organisms have been demonstrated in the laboratory, there is little direct evidence of nutrient effects on coral reef biota in situ. The ENCORE experiment investigated responses of coral reef organisms and processes to controlled additions of dissolved inorganic nitrogen (N) and/or phosphorus (P) on an offshore reef (One Tree Island) at the southern end of the Great Barrier Reef, Australia. A multi-disciplinary team assessed a variety of factors focusing on nutrient dynamics and biotic responses. A controlled and replicated experiment was conducted over two years using twelve small patch reefs ponded at low tide by a coral rim. Treatments included three control reefs (no nutrient addition) and three + N reefs (NH4Cl added), three + P reefs (KH2PO4 added), and three + N + P reefs. Nutrients were added as pulses at each low tide (ca twice per day) by remotely operated units. There were two phases of nutrient additions. During the initial, low-loading phase of the experiment nutrient pulses (mean dose = 11.5 microM NH4+; 2.3 microM PO4(-3)) rapidly declined, reaching near-background levels (mean = 0.9 microM NH4+; 0.5 microM PO4(-3)) within 2-3 h. A variety of biotic processes, assessed over a year during this initial nutrient loading phase, were not significantly affected, with the exception of coral reproduction, which was affected in all nutrient treatments. In Acropora longicyathus and A. aspera, fewer successfully developed embryos were formed, and in A. longicyathus fertilization rates and lipid levels decreased. In the second, high-loading, phase of ENCORE an increased nutrient dosage (mean dose = 36.2 microM NH4+; 5.1 microM PO4(-3)) declining to means of 11.3 microM NH4+ and 2.4 microM PO4(-3) at the end of low tide) was used for a further year, and a variety of significant biotic responses occurred. Encrusting algae incorporated virtually none of the added nutrients. Organisms containing endosymbiotic zooxanthellae (corals and giant clams) assimilated dissolved nutrients rapidly and were responsive to added nutrients. Coral mortality, not detected during the initial low-loading phase, became evident with increased nutrient dosage, particularly in Pocillopora damicornis. Nitrogen additions stunted coral growth, and phosphorus additions had a variable effect. Coral calcification rate and linear extension increased in the presence of added phosphorus but skeletal density was reduced, making corals more susceptible to breakage. Settlement of all coral larvae was reduced in nitrogen treatments, yet settlement of larvae from brooded species was enhanced in phosphorus treatments. Recruitment of stomatopods, benthic crustaceans living in coral rubble, was reduced in nitrogen and nitrogen plus phosphorus treatments. Grazing rates and reproductive effort of various fish species were not affected by the nutrient treatments. Microbial nitrogen transformations in sediments were responsive to nutrient loading with nitrogen fixation significantly increased in phosphorus treatments and denitrification increased in all treatments to which nitrogen had been added. Rates of bioerosion and grazing showed no significant effects of added nutrients. ENCORE has shown that reef organisms and processes investigated in situ were impacted by elevated nutrients. Impacts were dependent on dose level, whether nitrogen and/or phosphorus were elevated and were often species-specific. The impacts were generally sub-lethal and subtle and the treated reefs at the end of the experiment were visually similar to control reefs. Rapid nutrient uptake indicates that nutrient concentrations alone are not adequate to assess nutrient condition of reefs. Sensitive and quantifiable biological indicators need to be developed for coral reef ecosystems. The potential bioindicators identified in ENCORE should be tested in future research on coral reef/nutrient interactions. Synergistic and cumulative effects of elevated nutrients and other environmental parameters, comparative studies of intact vs. disturbed reefs, offshore vs. inshore reefs, or the ability of a nutrient-stressed reef to respond to natural disturbances require elucidation. An expanded understanding of coral reef responses to anthropogenic impacts is necessary, particularly regarding the subtle, sub-lethal effects detected in the ENCORE studies.

Z/AP, a double reporter for cre-mediated recombination.

The Cre/loxP site-specific recombination system combined with embryonic stem cell-mediated technologies has greatly expanded our capability to address normal and disease development in mammals using genetic approaches. The success of this emerging technology hinges on the production of Cre-expressing transgenic lines that provide cell type-, tissue-, or developmental stage-specific recombination between loxP sites placed in the genome. Here we describe and characterize the production of a double-reporter mouse line that provides a convenient and reliable readout of Cre recombinase activity. Throughout all embryonic and adult stages, the transgenic animal expresses the lacZ reporter gene before Cre-mediated excision occurs. Cre excision, however, removes the lacZ gene, allowing expression of the second reporter, the human alkaline phosphatase gene. This double-reporter transgenic line is able to indicate the occurrence of Cre excision in an extremely widespread manner from early embryonic to adult lineages. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site-specific recombinase system.

Transcripts of Grg4, a murine groucho-related gene, are detected in adjacent tissues to other murine neurogenic gene homologues during embryonic development.

The groucho-related genes (Grg) of the mouse comprise at least four family members. In Drosophila, groucho is one of the neurogenic genes that participates in the Notch signalling pathway. The Groucho protein interacts with Hairy-related transcription factors to regulate segmentation, neurogenesis and sex determination. Thus, by analogy to the Drosophila proteins, murine Grg proteins may interact with mammalian Hairy and E(spl) homologues (Hes proteins) and take part in a signalling pathway downstream of murine Notch. We have isolated murine Grg4 cDNAs and examined Grg4 expression during embryogenesis. Transcripts of Grg4 were detected in proliferating epithelial tissues undergoing mesenchymal induction, overlapping with Grg3, Notch1 and Hes1 expression. Grg4 was also expressed in the central nervous system and somites, but in cells adjacent to Grg3-, Notch1-, and Hes1-expressing cells. This distinct pattern of expression suggests a role for Grg4 in later stages of cell differentiation than for the other mouse neurogenic gene homologues.

Herpes simplex virus VP16 rescues viral mRNA from destruction by the virion host shutoff function.

Herpes simplex virus (HSV) virions contain two regulatory proteins that facilitate the onset of the lytic cycle: VP16 activates transcription of the viral immediate-early genes, and vhs triggers shutoff of host protein synthesis and accelerated turnover of cellular and viral mRNAs. VP16 and vhs form a complex in infected cells, raising the possibility of a regulatory link between them. Here we show that viral protein synthesis and mRNA levels undergo a severe decline at intermediate times after infection with a VP16 null mutant, culminating in virtually complete translational arrest. This phenotype was rescued by a transcriptionally incompetent derivative of VP16 that retains vhs binding activity, and was eliminated by inactivating the vhs gene. These results indicate that VP16 dampens vhs activity, allowing HSV mRNAs to persist in infected cells. Further evidence supporting this hypothesis came from the demonstration that a stably transfected cell line expressing VP16 was resistant to host shutoff induced by superinfecting HSV virions. Thus, in addition to its well known function as a transcriptional activator, VP16 stimulates viral gene expression at a post-transcriptional level, by sparing viral mRNAs from degradation by one of the virus-induced host shutoff mechanisms.

Binding sites for the herpes simplex virus immediate-early protein ICP4 impose an increased dependence on viral DNA replication on simple model promoters located in the viral genome.

We examined the ability of binding sites for the herpes simplex virus immediate-early protein ICP4 to alter the regulation of closely linked promoters by placing strong ICP4 binding sites upstream or downstream of simple TATA promoters in the intact viral genome. We found that binding sites strongly reduced the levels of expression at early times postinfection and that this effect was partially overcome after the onset of viral DNA replication. These data confirm that DNA-bound ICP4 can inhibit the activity of a closely linked promoter and raise the possibility that ICP4 binding sites contribute to temporal regulation during infection.

Role of aldehyde dehydrogenase in the biological activity of spermine dialdehyde, a novel immunosuppressive/purging agent.

The antitumour and immunosuppressive activities of spermine dialdehyde (SDA), a synthetic, oxidized form of spermine, were examined using L1210 cell lines and murine bone marrow cells. SDA acted as a high affinity substrate for aldehyde dehydrogenase (ADH) derived from different sources, with kinetic profiles similar to other aldehyde substrates. The murine leukaemic, cyclophosphamide-resistant L1210/CPA cells, having high levels of intracellular ADH activity, were less sensitive to SDA compared to ADH deficient L1210/O cells as measured by [3H]-thymidine incorporation in proliferation studies. Furthermore, pretreatment of L1210/CPA cells with the ADH inhibitor, diethyl aminobenzaldehyde (DEAB), resulted in potentiation of the SDA response. Murine bone marrow cells were more resistant to SDA than splenic T cells. However, addition of DEAB to bone marrow cultures potentiated the sensitivity of progenitor cells to SDA, as measured by colony formation. The results indicate that levels of ADH in the target tissues would determine the potency of SDA and subsequently offer selectivity and specificity to the therapeutic potentials of this putative purging agent.