Research Note: Relative bioavailability of zinc in zinc hydroxychloride for chicks.

A chick assay was conducted to determine the effects of Zn source on performance and to establish a Zn relative bioavailability value (RBV) for a new source of Zn hydroxychloride. In the assay, 8-day-old chicks were fed a Zn-deficient soy protein concentrate diet supplemented with 0, 7, and 15 mg Zn/kg from feed grade ZnSO4 monohydrate for 14 d to establish a standard response curve. The same basal diet was supplemented with 3, 7, and 10 mg Zn/kg from a new Zn hydroxychloride (SAMZn). A second source of Zn hydroxychloride (IBZn) was supplemented at 10 mg Zn/kg as a direct comparison to the highest level of SAMZn. Weight gain increased (P < 0.05) with increasing Zn level, regardless of source. Weight gain of chicks fed 7 mg Zn/kg from SAMZn was not different (P > 0.05) from chicks fed 15 mg Zn/kg from ZnSO4. Weight gain was not different (P > 0.05) when comparing the 2 sources of Zn hydroxychloride supplemented at 10 mg Zn/kg. Tibia ash Zn and total tibia Zn were increased (P < 0.05) by all Zn sources and responded linearly (P < 0.05) to Zn supplementation from ZnSO4 and SAMZn. Total tibia Zn concentration was not different (P > 0.05) for chicks fed 10 mg Zn/kg from either source of Zn hydroxychloride. Multiple linear regression of total tibia Zn on supplemental Zn intake (R2 = 0.95) resulted in a RBV of 115% for SAMZn compared with ZnSO4 (set at 100%). The RBV of SAMZn was higher (P < 0.05) than ZnSO4. In conclusion, relative bioavailability of Zn (based on tibia Zn) in Zn hydroxychloride from SAMZn was higher than feed grade ZnSO4 based on multiple regression slope-ratio analysis and was similar to that of IBZn Zn hydroxychloride based on tibia Zn responses to 10 mg/kg supplemental dietary Zn.

Contamination of the cold water distribution system of health care facilities by Legionella pneumophila: do we know the true dimension?

German water guidelines do not recommend routine assessment of cold water for Legionella in healthcare facilities, except if the water temperature at distal sites exceeds 25°C. This study evaluates Legionella contamination in cold and warm water supplies of healthcare facilities in Hesse, Germany, and analyses the relationship between cold water temperature and Legionella contamination. Samples were collected from four facilities, with cases of healthcare-associated Legionnaires' disease or notable contamination of their water supply. Fifty-nine samples were from central lines and 625 from distal sites, comprising 316 cold and 309 warm water samples. Legionella was isolated from central lines in two facilities and from distal sites in four facilities. 17% of all central and 32% of all distal samples were contaminated. At distal sites, cold water samples were more frequently contaminated with Legionella (40% vs 23%, p <0.001) and with higher concentrations of Legionella (≥1,000 colony-forming unit/100 ml) (16% vs 6%, p<0.001) than warm water samples. There was no clear correlation between the cold water temperature at sampling time and the contamination rate. 35% of cold water samples under 20 °C at collection were contaminated. Our data highlight the importance of assessing the cold water supply of healthcare facilities for Legionella in the context of an intensified analysis.

Gamma-sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin.

gamma-Sarcoglycan is a transmembrane, dystrophin-associated protein expressed in skeletal and cardiac muscle. The murine gamma-sarcoglycan gene was disrupted using homologous recombination. Mice lacking gamma-sarcoglycan showed pronounced dystrophic muscle changes in early life. By 20 wk of age, these mice developed cardiomyopathy and died prematurely. The loss of gamma-sarcoglycan produced secondary reduction of beta- and delta-sarcoglycan with partial retention of alpha- and epsilon-sarcoglycan, suggesting that beta-, gamma-, and delta-sarcoglycan function as a unit. Importantly, mice lacking gamma-sarco- glycan showed normal dystrophin content and local- ization, demonstrating that myofiber degeneration occurred independently of dystrophin alteration. Furthermore, beta-dystroglycan and laminin were left intact, implying that the dystrophin-dystroglycan-laminin mechanical link was unaffected by sarcoglycan deficiency. Apoptotic myonuclei were abundant in skeletal muscle lacking gamma-sarcoglycan, suggesting that programmed cell death contributes to myofiber degeneration. Vital staining with Evans blue dye revealed that muscle lacking gamma-sarcoglycan developed membrane disruptions like those seen in dystrophin-deficient muscle. Our data demonstrate that sarcoglycan loss was sufficient, and that dystrophin loss was not necessary to cause membrane defects and apoptosis. As a common molecular feature in a variety of muscular dystrophies, sarcoglycan loss is a likely mediator of pathology.

Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein.

Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin-glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin-glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a gamma- and delta-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.

Activation of the Lbc Rho exchange factor proto-oncogene by truncation of an extended C terminus that regulates transformation and targeting.

The human lbc oncogene product is a guanine nucleotide exchange factor that specifically activates the Rho small GTP binding protein, thus resulting in biologically active, GTP-bound Rho, which in turn mediates actin cytoskeletal reorganization, gene transcription, and entry into the mitotic S phase. In order to elucidate the mechanism of onco-Lbc transformation, here we report that while proto- and onco-lbc cDNAs encode identical N-terminal dbl oncogene homology (DH) and pleckstrin homology (PH) domains, proto-Lbc encodes a novel C terminus absent in the oncoprotein that includes a predicted alpha-helical region homologous to cyto-matrix proteins, followed by a proline-rich region. The lbc proto-oncogene maps to chromosome 15, and onco-lbc represents a fusion of the lbc proto-oncogene N terminus with a short, unrelated C-terminal sequence from chromosome 7. Both onco- and proto-Lbc can promote formation of GTP-bound Rho in vivo. Proto-Lbc transforming activity is much reduced compared to that of onco-Lbc, and a significant increase in transforming activity requires truncation of both the alpha-helical and proline-rich regions in the proto-Lbc C terminus. Deletion of the chromosome 7-derived C terminus of onco-Lbc does not destroy transforming activity, demonstrating that it is loss of the proto-Lbc C terminus, rather than gain of an unrelated C-terminus by onco-Lbc, that confers transforming activity. Mutations of onco-Lbc DH and PH domains demonstrate that both domains are necessary for full transforming activity. The proto-Lbc product localizes to the particulate (membrane) fraction, while the majority of the onco-Lbc product is cytosolic, and mutations of the PH domain do not affect this localization. The proto-Lbc C-terminus alone localizes predominantly to the particulate fraction, indicating that the C terminus may play a major role in the correct subcellular localization of proto-Lbc, thus providing a mechanism for regulating Lbc oncogenic potential.

Subcutaneous microdialysis for children - safe biochemical tissue monitoring based on a minimal traumatizing no touch insertion technique.

BACKGROUND: Microdialysis (MD) enables analysis of extracellular metabolites without performing blood tests. Changes in the concentration of various metabolites can be monitored frequently on almost every type of human tissue. Microdialysis of subcutaneous tissue (sc MD) is of particular significance in the case of pediatric patients because diurnal profiles can be generated without repeated blood sampling. There are only a few scientific articles that describe the application of sc MD on neonates, infants, or children. So far, side effects have not been investigated comprehensively. This prospective study scrutinizes side effects of sc MD in pediatric patients, focusing on a Minimal Traumatizing Insertion Technique of the MD catheter. PATIENTS AND METHODS: 42 pediatric patients within four age categories participated in the study which involved bedside monitoring using sc MD, including 5 extremely low birth weight (ELBW) infants with a body weight <1000g. A total of 48 sc MD catheters were inserted. Selection criteria were risk of hypoglycaemia (n = 29), elevated lactate levels (n = 16), or aminoacidopathies (n = 3). Duration of sc MD ranged from 1 to 16 days. We used a Minimal Traumatizing Insertion Technique to safely insert the MD catheter into the subcutaneous tissue, characterized by blunt dissection of the tissue and by the use of a plastic cannula guidance to avoid desterilisation of the catheter. Complications and side effects related to sc MD were documented in standardized forms. RESULTS: The MD probe was easily placed even in the scanty adipose tissue of ELBW infants. During insertion of sc MD catheters accidental venous puncture occurred to 8%, and minor bleeding to 27%. Even with local anaesthesia insertion was painful for 40%. During the course of sc MD complications were rare: disturbance of perfusion flow 4%, catheter dislocation 4%, local bleeding 4%. No signs of systemic or local infection were observed, there were no cases of local incompatibility. All catheters were withdrawn completely without leaving a scar. Repeated measurements allowed the generation of diurnal metabolic profiles. In some cases (respiratory chain complex I deficiency, PDH-deficiency) significant therapeutical effects on the patients' metabolism were demonstrated. CONCLUSIONS: The present study proves long-term sc MD to be suitable and safe for biochemical tissue monitoring. Using our insertion technique, it can be applied to children of all ages without causing discomfort or severe side effects. As it permits frequent sampling it allows evaluating and optimizing therapy and means a substantial progress for pediatric observation.

Muscle-specific promoters may be necessary for adeno-associated virus-mediated gene transfer in the treatment of muscular dystrophies.

Recombinant adeno-associated virus (rAAV) vectors allow efficient gene transfer and expression in the muscle; therefore, rAAVs represent a potential gene therapy vector for muscular dystrophies. For further investigations, we used a mouse muscular dystrophy model (gsg(-/-) mice) gamma-sarcoglycan, a subunit of the dystrophin-glycoprotein complex, is missing. gsg(-/-) mice develop progressive dystrophy representative of a severe human phenotype disease. We previously showed high levels and stable expression of gamma-sarcoglycan in myofibers after direct muscle injection into gsg(-/-) mice of a recombinant AAV vector (AAV.dMCK.gSG) carrying the gamma-sarcoglycan cDNA driven by a muscle-specific promoter (truncated version of muscle creatine kinase). Here, we show that when gamma-sarcoglycan expression is driven by the ubiquitous cytomegalovirus (CMV) promoter (AAV.CMV.gSG), lower levels of transgene expression are observed and are associated with a humoral response to gamma-sarcoglycan. When using an rAAV vector, expressing the highly immunogenic product gamma-galactosidase under the CMV promoter (AAV.CMV.LacZ), we measured a strong cellular and humoral immune response to the transgene after intramuscular injection into gsg(-/-) mice. This study suggests that restriction of transgene expression to the muscle is an important criterion for the treatment of muscular dystrophies and will aid in the design of protocols for gene therapy.

Extraocular muscle is spared despite the absence of an intact sarcoglycan complex in gamma- or delta-sarcoglycan-deficient mice.

Models of the dystrophin-glycoprotein complex do not reconcile the novel sparing of extraocular muscle in muscular dystrophy. Extraocular muscle sparing in Duchenne muscular dystrophy implies the existence of adaptive properties in these muscles that may extend protection to other neuromuscular diseases. We studied the extraocular muscle morphology and dystrophin-glycoprotein complex organization in murine targeted deletion of the gamma-sarcoglycan (gsg(-/-)) and delta-sarcoglycan (dsg(-/-)) genes, two models of autosomal recessive limb girdle muscular dystrophy. In contrast to limb and diaphragm, the principal extraocular muscles were intact in gsg(-/-) and dsg(-/-) mice. However, central nucleated, presumptive regenerative, fibers were seen in the accessory extraocular muscles (retractor bulbi, levator palpebrae superioris) of both strains. Skeletal muscles of gsg(-/-) mice exhibited in vivo Evans Blue dye permeability, while the principal extraocular muscles did not. Disruption of gamma-sarcoglycan produced secondary displacement of alpha- and beta-sarcoglycans in the extraocular muscles. The intensity of immunofluorescence for dystrophin and alpha- and beta-dystroglycan also appeared to be slightly reduced. Utrophin localization was unchanged. The finding that sarcoglycan disruption was insufficient to elicit alterations in extraocular muscle suggests that loss of mechanical stability and increased sarcolemmal permeability are not inevitable consequences of mutations that disrupt the dystrophin-glycoprotein complex organization and must be accounted for in models of muscular dystrophy.

Sarcoglycans in muscular dystrophy.

Muscular dystrophy is a heterogeneous genetic disease that affects skeletal and cardiac muscle. The genetic defects associated with muscular dystrophy include mutations in dystrophin and its associated glycoproteins, the sarcoglycans. Furthermore, defects in dystrophin have been shown to cause a disruption of the normal expression and localization of the sarcoglycan complex. Thus, abnormalities of sarcoglycan are a common molecular feature in a number of dystrophies. By combining biochemistry, molecular cell biology, and human and mouse genetics, a growing understanding of the sarcoglycan complex is emerging. Sarcoglycan appears to be an important, independent mediator of dystrophic pathology in both skeletal muscle and heart. The absence of sarcoglycan leads to alterations of membrane permeability and apoptosis, two shared features of a number of dystrophies. beta-sarcoglycan and delta-sarcoglycan may form the core of the sarcoglycan subcomplex with alpha- and gamma-sarcoglycan less tightly associated to this core. The relationship of epsilon-sarcoglycan to the dystrophin-glycoprotein complex remains unclear. Animals lacking alpha-, gamma- and delta-sarcoglycan have been described and provide excellent opportunities for further investigation of the function of sarcoglycan. Dystrophin with dystroglycan and laminin may be a mechanical link between the actin cytoskeleton and the extracellular matrix. By positioning itself in close proximity to dystrophin and dystroglycan, sarcoglycan may function to couple mechanical and chemical signals in striated muscle. Sarcoglycan may be an independent signaling or regulatory module whose position in the membrane is determined by dystrophin but whose function is carried out independent of the dystrophin-dystroglycan-laminin axis.

Mobilization of PAH and PCB from contaminated soil using a digestive tract model.

Environmental contaminants are mainly incorporated by ingestion. In general only those contaminants mobilized by the digestive juices are available for absorption in the digestive tract, while pollutants still fixed to indigestible particles leave the body without any effect. To evaluate the different health risks arising from the ingestion of individual types of polluted soil or other materials, we developed an in vitro test system which simulates the transition of pollutants from contaminated materials into digestive juices by means of a standardized artificial gastro-intestinal model. The test system simulates the influence of the acidic environment of the stomach (gastric model) followed by the neutral or slightly alkaline environment of the small intestine (gastro-intestinal model). Investigations on small amounts of polluted soil, sewage sludge, asphalt, metal scrap and blast sand showed that the mobilization of polycyclic aromatic hydrocarbons (PAH) and polychlorinated biphenyls (PCB) by artificial gastric juice reaches 3% up to 22% of the pollutant concentration introduced into the test system. Elutions of the contaminated materials under gastric and subsequently under intestinal conditions with bile concentrations of 3 g/l resulted in PAH- and PCB-mobilizations in the range of 5% up to 40%. The degree of mobilization depends considerably on supplementary food material added to the test system. Lyophilized milk increased the fraction of mobilized PAH and PCB to 40%-85%. Application of the test system on 22 different contaminated soils showed that the mobilization of PAH under gastro-intestinal conditions with the addition of lyophilized milk ranged from 7% up to 95%, and the mobilization of PCB ranged from 32% up to 83%. This indicates that the test system can be a useful tool for evaluating the individual health risks arising from polluted soil or other materials.

Effect of the topoisomerase-II inhibitor etoposide on meiotic recombination in male mice.

Unlike other chemicals that have been tested in mammalian germ cells, the type-II topoisomerase inhibitor etoposide exhibits significant mutagenicity in primary spermatocytes. Because this is the cell stage during which meiotic recombination normally occurs, and because topoisomerases play a role in recombination, we studied the effect of etoposide on crossing-over in male mice. Exposure to those meiotic prophase stages (probably early to mid-pachytene) during which specific-locus deletion mutations can be induced resulted in decreased crossing-over in the p-Tyr(c) interval of mouse chromosome 7. Accompanying cytological studies with fluorescent antibodies indicated that while there was no detectable effect on the number of recombination nodules (MLH1 foci), there were marked changes in the stage of appearance and localization of RAD51 and RPA proteins. These temporal and spatial protein patterns suggest the formation of multiple lesions in the DNA after MLH1 has already disappeared from spermatocytes. Since etoposide blocks religation of the cut made by type II topoisomerases, repair of DNA damage may result in rejoining of the original DNA strands, undoing the reciprocal exchange that had already occurred and resulting in reduced crossing-over despite a normal frequency of MLH1 foci. Crossing-over could conceivably be affected differentially in different chromosomal regions. If, however, the predominant action of etoposide is to decrease homologous meiotic recombination, the chemical could be expected to increase nondisjunction, an event associated with human genetic risk. Three periods in spermatogenesis respond to etoposide in different ways. Exposure of (a) late differentiating spermatogonia (and, possibly, preleptotene spermatocytes) results in cell death; (b) early- to mid-pachytene induces specific-locus deletions and crossover reduction; and, (c) late pachytene-through-diakinesis leads to genetically unbalanced conceptuses as a result of clastogenic damage.

Comparison of different digestive tract models for estimating bioaccessibility of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) from red slag 'Kieselrot'.

'Kieselrot' (red slag), a highly PCDD/F-contaminated leaching residue from a copper production process, has been used as surface layer for more than 1,000 sports fields, playgrounds and pavements in Germany and neighbouring countries. Children can ingest this material directly by hand-to-mouth activities or soil-pica behaviour. Furthermore secondary contamination of farm land or kitchen gardens by drift of red slag dust may lead to an enrichment of PCDD/F within the food-chain. PCDD/F can be mobilized from contaminated materials by digestive juices and thus become bioaccessible for intestinal absorption. Two different digestive tract models were used to estimate the bioaccessibility of PCDD/F from red slag and to study the influence of food material on the mobilization of the contaminants. The bioaccessibility of PCDD/F from red slag depends on the charge of red slag material used, the bile content of the intestinal juice and on the presence of lipophilic foodstuffs. A low bioaccessibility of less than 5% was found when using a digestive tract model with a low bile content and in absence of food material. The bioaccessibility was estimated to be more than 60% when using a model with a higher bile content and in the presence of whole milk powder. A low bioaccessibility of PCDD/F from red slag in general--as assumed until now and mentioned in legal provision--was not confirmed by our study. Considering observations for the different homologue groups it is obvious that bioaccessibility is the first of several important steps to estimate human health risks arising from contaminated materials. In case red slag contaminated with PCDD/F their absorption rate in the digestive tract and/or metabolism might be at least just like important.

Glucose-monitoring with continuous subcutaneous microdialysis in neonatal diabetes mellitus.

BACKGROUND: Neonatal diabetes mellitus can be extremely brittle. In this situation close glucose monitoring is essential for adequate insulin treatment. Continuous subcutaneous microdialysis is a promising approach for the babies to reduce the painful stress caused by diagnostic blood sampling. The goal of this study was to evaluate the feasibility of continuous subcutaneous microdialysis for glucose monitoring in a baby with neonatal diabetes and to assess the correlation between the blood and the subcutaneous glucose profile. PATIENT AND METHOD: During a period of seven days glucose monitoring was performed on a six month old infant with neonatal diabetes mellitus. In addition to frequent capillary blood glucose determinations, a continuous subcutaneous microdialysis device was used for the detection of the subcutaneous interstitial glucose concentration. RESULTS: Subcutaneous tissue glucose concentrations were measured in a wide range from 1.7 to 23.8 mM. Variations in the adipose tissue glucose concentration closely paralleled changes in the capillary blood glucose. Based on 104 reference pairs there was a high overall correlation (r = 0.89) between the subcutaneous interstitial tissue (X) and the blood (Y) glucose concentration (Y = 1.1 X + 0.29). However the glucose profiles demonstrated a considerable variation of the time lag, up to one hour, between blood and subcutaneous interstitial glucose concentration. CONCLUSIONS: Continuous subcutaneous microdialysis helps the glucose monitoring of infants with diabetes mellitus by providing additional informations about the rise and fall of the glucose concentration. Further studies should focus on how to get a tighter link between blood glucose and the subcutaneous interstitial glucose concentration in the area around the microdialysis probe. Thus monitoring the subcutaneous interstitial glucose concentration will become a reliable procedure for real-time glucose monitoring.

Detection of approximal caries with a new laser fluorescence device.

The laser device DIAGNOdent developed for the detection of occlusal caries has limited value on approximal surfaces. The aim of this study was to develop and to test a new laser fluorescence (LF) device for the detection of approximal caries. Light with a wavelength of 655 nm was transported to the approximal surface using two different sapphire fibre tips. Seventy-five teeth were selected from a pool of extracted permanent human molars, frozen at -20 degrees C until use. Before being measured, they were defrosted, cleaned and calculus was removed with a scaler. The molars were set in blocks simulating the contact area of adults. Bitewing radiographs were obtained using Kodak Insight films. After two independent assessments with the new LF device, the teeth were histologically prepared, and assessed for caries extension. Using the laser, specificity values for D1 threshold (outer half of enamel), D2 threshold (inner half of enamel), D3 threshold (dentine) ranged between 0.81 and 0.93, sensitivity between 0.84 and 0.92 with no difference between the two tips. Bitewing radiography showed an inferior performance compared to LF (p<0.05). Intraex aminer reproducibility was high (kappa>.74). The new LF system might be a useful additional tool in detecting approximal caries. Because of its good reproducibility, it could be used to monitor caries regression or progression on approximal surfaces.

Respirator protection factors: Part I -- Development of an anthropometric test panel.

Respirators are currently approved by testing them on a number of subjects without specifying facial sizes. Anthropometrically designed test panels were developed that represent the majority of the working population in terms of relevant facial measurements.

Automated bacteriuria screening using the Berthold LB 950 luminescence analyser.

The Berthold LB950 Automatic Luminescence Analyser was used to estimate bacterial adenosine triphosphate in urine. The system provided a rapid (15 min) and fully automated screening test for bacteriuria at the 10(5) CFU/ml level. Bioluminescence results for 1040 urines were compared with viable counts using two reference culture methods and frequency distributions of bacterial counts and adenosine triphosphate levels were calculated. With a specificity of 79% the automated method showed a sensitivity of 84% using a pour plate reference count and 91% using a standard loop reference count. When contaminated urines were excluded the sensitivity improved to 98%. The automated bioluminescence test, though expensive, was shown to work well with good quality specimens.

Respirator protection factors: Part II-protection factors of supplied-air respirators.

Protection Factors provided by 25 NIOSH approved supplied-air respirators were determined while the devices were worn by a panel of test subjects anthropometrically selected to represent adult facial sizes. Polydispersed DOP aerosol was used for respirator fit tests on continuous flow, demand, and pressure-demand respirators. Based on facepiece leakage measurements it appears that demand-type respirators should neither be used nor approved. The highest level of protection was provided by pressure-demand devices.

Simple method for identification of plasmid-coded proteins.

Proteins encoded by plasmid DNA are specifically labeled in UV-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA.