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.

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.

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.

Different mechanisms of handling ingested polycyclic aromatic hydrocarbons in mammalian species: organ-specific response patterns of CYP1A1-induction after oral intake of PAH-contaminated soils.

Potential effects of xenobiotics on humans are largely derived from studies with animal models. However, due to species-specific processing of xenobiotics, susceptibilities to xenobiotic-dependent adverse effects are known to differ between species. We analysed the basal expression of cytochrome P450 (CYP) enzymes in several organs of minipigs and rats, and their inducibility upon oral intake of soils containing polycyclic aromatic hydrocarbons (PAH). CYP-specific enzymatic activities were determined in duodenum, liver and kidney microsomes. Upon ingestion of PAH-contaminated soils, CYP1A1 is differentially induced in a tissue-specific and dose-dependent manner in duodenum, liver and kidney of minipigs and rats. In the duodenum, the induction response is low in rats (about 4-fold) but it is high in minipigs (8-230-fold). By contrast, induced hepatic CYP1A1-dependent EROD-activity is higher in rats than in minipigs. The dose-response profile for renal CYP1A1 parallels that in the liver of either species but EROD-activities are 10-20 times lower than in the liver. Liver microsomal CYP2E1 is only slightly modulated in its expression by ingestion of PAH-contaminated soils in both species, whereas CYP3A-dependent testosterone 2beta- and 6beta-hydroxylation is increased in liver of rats but not in minipigs. The hepatic capacity for catechol oestrogen formation, i.e., the 2-hydroxylation of 17beta-oestradiol, is markedly increased in rats but not in minipigs by ingested PAH. It is concluded that different metabolic and transport pathways are used by minipigs and rats to process ingested PAH. Whereas in minipigs the duodenum appears as the first efficient barrier, rats respond by efficient metabolism in the liver. What is not known is which response profile is operative in man.

[Prediction of mortality, mobility and admission to long-term care after hip fractures]. / Prädiktion von Mortalität und soziofunktionellen Einschränkungen nach proximalen Femurfrakturen bei nicht institutionalisierten Senioren.

INTRODUCTION: Surgical treatment, discharge planning and rehabilitation procedures are rarely based upon defined assessment procedures. It might therefore be useful to develop simple and reliable screening tools to identify patients for early discharge, intensified rehabilitation and limited treatment. PATIENTS AND METHODS: 234 patients were initially contacted. From these 217 gave informed consent. The reported study included the 134 home dwelling elderly. All patients could be contacted or died after six month. Thus,data from more than 90% could be used for the analysis. The six month mortality was 10%. A population based cohort of elderly patients referred to five local hospitals was tested. Mortality, institutionalisation and mobility were defined as major outcome criteria. Only information that was available during the first week of treatment was used in the model. Predictors expressed as odd ratios (OR) were calculated using logistic regression with variable selection. RESULTS: The most important predictors for institutionalisation were age, inability to eat without assistance, postoperative night time confusion, stroke history, cognitive deficit and fear of falling. Outdoor mobility was strongly associated to the preoperative ADL performance measured as the Barthel-Index, history of malignancy and fear of falling. Six month mortality was associated with male sex, the Barthel-Index and fear of falling. DISCUSSION: It seems feasible to improve postoperative resource allocation by predictor led stratification. This need to be tested in intervention trials under the specific condition of the German health care system.

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.

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.

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.

Rescue of skeletal muscles of gamma-sarcoglycan-deficient mice with adeno-associated virus-mediated gene transfer.

In humans, a subset of cases of Limb-girdle muscular dystrophy (LGMD) arise from mutations in the genes encoding one of the sarcoglycan (alpha, beta, gamma, or delta) subunits of the dystrophin-glycoprotein complex. While adeno-associated virus (AAV) is a potential gene therapy vector for these dystrophies, it is unclear if AAV can be used if a diseased muscle is undergoing rapid degeneration and necrosis. The skeletal muscles of mice lacking gamma-sarcoglycan (gsg-/- mice) differ from the animal models that have been evaluated to date in that the severity of the skeletal muscle pathology is much greater and more representative of that of humans with muscular dystrophy. Following direct muscle injection of a recombinant AAV [in which human gamma-sarcoglycan expression is driven by a truncated muscle creatine kinase (MCK) promoter/enhancer], we observed significant numbers of muscle fibers expressing gamma-sarcoglycan and an overall improvement of the histologic pattern of dystrophy. However, these results could be achieved only if injections into the muscle were prior to the development of significant fibrosis in the muscle. The results presented in this report show promise for AAV gene therapy for LGMD, but underscore the need for intervention early in the time course of the disease process.

Differential requirement for individual sarcoglycans and dystrophin in the assembly and function of the dystrophin-glycoprotein complex.

Sarcoglycan is a multimeric, integral membrane glycoprotein complex that associates with dystrophin. Mutations in individual sarcoglycan subunits have been identified in inherited forms of muscular dystrophy. To evaluate the contributions of sarcoglycan and dystrophin to muscle membrane stability and muscular dystrophy, we compared muscle lacking specific sarcoglycans or dystrophin. Here we report that mice lacking (delta)-sarcoglycan developed muscular dystrophy and cardiomyopathy similar to mice lacking (gamma)-sarcoglycan. However, unlike muscle lacking (gamma)-sarcoglycan, (delta)-sarcoglycan-deficient muscle was sensitive to eccentric contraction-induced disruption of the plasma membrane. In the absence of (delta)-sarcoglycan, (alpha)-, (beta)- and (gamma)-sarcoglycan were undetectable, while dystrophin was expressed at normal levels. In contrast, without (gamma)-sarcoglycan, reduced levels of (alpha)-, (beta)- and (delta)-sarcoglycan were expressed, glycosylated and formed a complex with each other. Thus, the elimination of (gamma)- and (delta)-sarcoglycan had different molecular consequences for the assembly and function of the dystrophin-glycoprotein complex. Furthermore, these molecular differences were associated with different mechanical consequences for the muscle plasma membrane. Through this in vivo analysis, a model for sarcoglycan assembly is proposed.

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.

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.

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.

[Disabilities and handicaps due to hip fractures in the elderly]. / Unfallfolgen nach Sturz: Funktionelle Defizite und soziale Beeinträchtigungen nach proximalen Femurfrakturen Alterer.

OBJECTIVE: to examine mortality, mobility, pain, institutionalization rates six month after hip fractures. DESIGN: observational study, non-selected consecutive patients, time-set protocol. SETTING: urban area (population n = 116,5000), five hospitals. PARTICIPANTS: hip fracture patients (n = 214), age 65 + years (mean 82.4 years). MEASUREMENTS: age, gender, prefracture status, operative procedures, complications, comorbidity, cognition (MMSE), depression (GDS), fear of falling, ADL (Barthel) and mobility and pain status (SAHFE protocol). RESULTS: the incidence for this age group was 636.9/100,000, 31.8% were institutionalized elderly. 84% of the patients were female. Pre-existing comorbidity showed a high prevalence of neurodegenerative (cognitive deficits 53. 6%, Parkinson's disease/syndrome 11.2%) and cerebrovascular diseases (16.8%). Six month postfracture, the mortality was 17.6%. From those surviving 76.2% did walk indoors, 58.5% did also walk outdoors. Independent to dress were 54.6%. Severe pain was reported by 10.2%, whereas 36.9% described no pain. The institutionalization rate at six month was 19.0%. CONCLUSIONS: the study showed considerable mortality, a significant loss in function and social disintegration. Considerable differences were observed for subgroups of patients. Future treatment should focus on risk stratification and include postdischarge training programs. Moreover, preventive strategies should be implemented for high risk groups, such as ambulating patients with a history of stroke. Parkinson's disease and syndrome, dementia and nursing home residents.

Muscle degeneration without mechanical injury in sarcoglycan deficiency.

In humans, mutations in the genes encoding components of the dystrophin-glycoprotein complex cause muscular dystrophy. Specifically, primary mutations in the genes encoding alpha-, beta-, gamma-, and delta-sarcoglycan have been identified in humans with limb-girdle muscular dystrophy. Mice lacking gamma-sarcoglycan develop progressive muscular dystrophy similar to human muscular dystrophy. Without gamma-sarcoglycan, beta- and delta-sarcoglycan are unstable at the muscle membrane and alpha-sarcoglycan is severely reduced. The expression and localization of dystrophin, dystroglycan, and laminin-alpha2, a mechanical link between the actin cytoskeleton and the extracellular matrix, appears unaffected by the loss of sarcoglycan. We assessed the functional integrity of this mechanical link and found that isolated muscles lacking gamma-sarcoglycan showed normal resistance to mechanical strain induced by eccentric muscle contraction. Sarcoglycan-deficient muscles also showed normal peak isometric and tetanic force generation. Furthermore, there was no evidence for contraction-induced injury in mice lacking gamma-sarcoglycan that were subjected to an extended, rigorous exercise regimen. These data demonstrate that mechanical weakness and contraction-induced muscle injury are not required for muscle degeneration and the dystrophic process. Thus, a nonmechanical mechanism, perhaps involving some unknown signaling function, likely is responsible for muscular dystrophy where sarcoglycan is deficient.

Analysis of functional elements in the human Egr-1 gene promoter.

The early growth response (Egr)-1 gene encoding a zinc-finger transcription factor is transiently induced in many different cell types upon various differentiation signals. However, in synovial fibroblasts of rheumatoid arthritis patients, Egr-1 is constitutively expressed at high levels, and several genes with Egr-1 binding sites in their promoter regions have been associated with disease progression of RA. We analyzed the control of Egr-1 transcription by characterizing those regulatory elements in the Egr-1 promoter that induce Egr-1 expression in fibroblasts. Using reporter gene assays and deletion mutants of the Egr-1 promoter we could demonstrate that Egr-1 transcription is mainly activated by a single serum response element, whereas other transcription factor binding sites, including binding sites for AP-1 or Egr-1, were found to play a minor role. Furthermore, we identified a novel regulatory element in the human Egr-1 promoter similar to a NF kappa-B binding site. Deletion of this element enhanced Egr-1 promoter activity in 3T3 but not in L929 fibroblasts. Stimulation by phorbolester induced only transient Egr-1 expression in 3T3 fibroblasts but a extended expression of Egr-1 in L929 cells. These data suggest that in fibroblasts the most proximal serum response element in the Egr-1 promoter represents the major activation site, whereas binding of the NFkB-like factor may serve as negative regulatory signal for Egr-1 transcription in fibroblasts.

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.