Actin binding LIM protein 3 (abLIM3)

LIM domain proteins were demonstrated to play key roles in various biological processes such as embryonic development, cell lineage determination, and cancer differentiation. Actin binding LIM protein 1 (abLIM1) was reported to be localized in a genomic region often deleted in human cancers and suggested to be involved in axon guidance. Recently, existence of a second family member was reported, actin binding LIM protein 2. By means of computational biology and comparative genomics, we now characterized an additional, third member of the actin binding LIM protein subgroup, actin binding LIM protein 3 (abLIM3). The human mRNA sequence was previously annotated as differentially regulated in hepatoblastoma compared to normal livers. Conservation of key structural features of abLIM1 and abLIM2, four LIM domains and a VHD domain, suggested comparable biological function of abLIM3 as a linker between actin cytoskeleton and cell signaling pathways. AbLIM3 was found to be conserved in vertebrates, as orthologous sequences were characterized for mouse, fish, and frog. In addition, we report the existence of abLIM2 orthologs in fish and frog, suggesting a similar degree of evolutionary conservation. The intracellular localization of the abLIM3 protein was predicted to be nuclear by means of Reinhardt's neural network and the k-nearest neighbor algorithm. The corresponding abLIM3 gene was localized to chromosome 5q32 and spanned 119 kb, organized in 24 exons. An RT-PCR based expression profile available from the human unidentified gene-encoded (HUGE) database demonstrated highest expression for abLIM3 in heart, lung, liver, and brain/cerebellum accompanied by lower expression in multiple other tissues. Furthermore, abLIM3 was expressed in fetal liver, CNS, and spinal cord.     

Characterization of OEBT, a LIM protein

LIM Proteins have been demonstrated to play key roles in pattern formation during embryonic development, cell lineage determination, and cancer differentiation. These proteins are characterized by their conserved LIM domain, which functions as a specific protein-binding site. Recently, two new members of the LIM protein family, PRICKLE1 and PRICKLE2, were characterized in silico and demonstrated to be human orthologues of the Drosophila prickle proteins. We report on an additional member of this protein family, overexpressed breast tumor protein (OEBT). The corresponding gene was mapped to human chromosome 6p22.31. Orthologues in mouse and rat with 72 and 54% identities on a protein level were identified and the corresponding genes were mapped to mouse chromosome 17 and rat chromosome 9. The protein displays two LIM domains, as well as a PET domain, and was predicted to be localized in the nucleus. Expression of human OEBT was analyzed in silico, and the corresponding RNA was annotated to be highly expressed in a broad range of tissues. ESTs from several malignant tissue differentiations point towards a possible role of OEBT in cancer differentiation.     

Oxidative stress and the muscle reflex in heart failure

Muscle contraction stimulates thin fibre muscle afferents and evokes a reflex increase in blood pressure. In heart failure (HF) this reflex is accentuated. Of note, superoxide and other reactive oxygen species are increased in HF. In this report, we tested the hypothesis that excess superoxide contributes to the exaggerated muscle reflex in HF. HF was induced in rats by coronary artery ligation. Electrically induced 30 s hindlimb muscle contraction in decerebrate rats with myocardial infarction (MI) (left ventricular fractional shortening (FS) = 24 ± 1%; n = 15) evoked larger ( P < 0.05) increases in mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) as compared to control rats (FS = 47 ± 1%; n = 14). In the MI rats, the pressor and RSNA responses to contraction were reduced by intra-arterial injection into the hindlimb circulation of tempol (10 mg), a superoxide dismutase mimetic (ΔMAP: 22 ± 2 vs. 11 ± 1 mmHg; ∫ΔRSNA: 1032 ± 204 vs. 431 ± 73 arbitrary units (a.u.); before vs. after tempol; P < 0.05). Tempol also attenuated the RSNA response to 1 min intermittent (1–4 s stimulation to relaxation) bouts of static contraction in the MI rats (116 ± 17 vs. 72 ± 11 a.u.; P < 0.05; n = 16). In the control rats, tempol had no effect on these responses. These results suggest that excess superoxide in HF sensitizes mechanically sensitive muscle afferents engaged during contraction. We hypothesize that oxidative stress contributes to the exaggerated muscle reflex in HF.     

Immunoperoxidase demonstration of a new muscle protein (Z-protein) in myogenic tumors as a diagnostic aid.

Z-protein, a new muscle protein with a molecular weight of 55,000 daltons recently discovered in Japan, is an excellent marker for muscle differentiation in paraffin sections. Positive staining by a peroxidase-antiperoxidase technique for detection of intracellular Z-protein was observed in 2 of 2 cardiac rhabdomyomas, 5 of 5 leiomyomas, 9 of 10 leiomyosarcomas, and in 15 of 18 rhabdomyosarcomas including 5 of 5 of the pleomorphic type, 7 of 9 of the embryonal type, and 3 of 4 of the alveolar type. Other types of tumors (77 cases) that sometimes mimic myogenic tumors on histologic examination gave negative results. The immunoperoxidase method for detection of intracellular Z-protein is of use in distinguishing myogenic tumors from other types of tumors.     

Insulin-like growth factor-binding protein 7 (IGFBP 7) as a new biomarker in coronary heart disease

PurposeThe role of insulin-like growth factor-binding protein-7 (IGFBP-7) in atherosclerosis is still not well-known. The objective of this study was to find out the following: 1) whether IGFBP-7 may act as a biomarker of coronary artery disease (CAD) occurrence and extent; 2) whether IGFBP-7 is potentially related to the classical and new markers of cardiovascular risk (carotid intima-media thickness - cIMT); 3) whether IGFBP-7 may be a marker of mortality in the group of patients with myocardial infarction (MI).Materials/MethodsThe study group consisted of 212 patients with MI and 75 patients with stable CAD, the control group included 100 healthy volunteers. IGFBP-7 serum concentration was measured.ResultsIGFBP-7 value was considerably higher in the study group (MI and CAD patients - 35.1 ng/ml (P = 0.000001) and 32.7ng/ml (P = 0.0001), respectively), than in the controls – 25.2ng/ml. No statistically significant differences between IGFBP-7 concentrations in the MI and CAD group were found. No relationship between IGFBP-7 and the coronary lesions advancement in the study group was observed. No changes in IGFBP-7 concentration in the MI patients during hospitalization were observed. In the group of MI patients who died during follow-up, a considerably higher cIMT values were found whereas no statistically significant difference was observed in relation to IGFBP-7 (34.6 vs. 35.2 ng/ml).ConclusionsIGFBP-7 is a good biomarker of CAD occurrence but not of its advancement. We demonstrated the existence of the relation between higher IGFBP-7 concentration and the selected classical risk factors of cardiovascular events as well as cIMT values. IGFBP-7 cannot serve as a marker of acute ischemia. Also, IGFBP-7 was not confirmed as a predictor of mortality in the MI patients.     

DNA alkylation damage as a sensor of nitrosative stress in Mycobacterium tuberculosis

One of the cellular consequences of nitrosative stress is alkylation damage to DNA. To assess whether nitrosative stress is registered on the genome of Mycobacterium tuberculosis, mutants lacking an alkylation damage repair and reversal operon were constructed. Although hypersensitive to the genotoxic effects of N-methyl-N'-nitro-N-nitrosoguanidine in vitro, the mutants displayed no phenotype in vivo, suggesting that permeation of nitrosative stress to the level of cytotoxic DNA damage is restricted.     

Genotype-phenotype relationships involving hypertrophic cardiomyopathy-associated mutations in titin, muscle LIM protein, and telethonin

BACKGROUND: TTN-encoded titin, CSRP3-encoded muscle LIM protein, and TCAP-encoded telethonin are Z-disc proteins essential for the structural organization of the cardiac sarcomere and the cardiomyocyte's stretch sensor. All three genes have been established as cardiomyopathy-associated genes for both dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). Here, we sought to characterize the frequency, spectrum, and phenotype associated with HCM-associated mutations in these three genes in a large cohort of unrelated patients evaluated at a single tertiary outpatient center. METHODS: DNA was obtained from 389 patients with HCM (215 male, left ventricular wall thickness of 21.6+/-6 mm) and analyzed for mutations involving all translated exons of CSRP3 and TCAP and targeted HCM-associated exons (2, 3, 4, and 14) of TTN using polymerase chain reaction (PCR), denaturing high performance liquid chromatography (DHPLC), and direct DNA sequencing. Clinical data were extracted from patient records and maintained independent of the genotype. RESULTS: Overall, 16 patients (4.1%) harbored a Z-disc mutation: 12 had a MLP mutation and 4 patients a TCAP mutation. No TTN mutations were detected. Seven patients were also found to have a concomitant myofilament mutation. Seven patients with a MLP-mutation were found to harbor the DCM-associated, functionally characterized W4R mutation. W4R-MLP was also noted in a single white control subject. Patients with MLP/TCAP-associated HCM clinically mimicked myofilament-HCM. CONCLUSIONS: Approximately 4.1% of unrelated patients had HCM-associated MLP or TCAP mutations. MLP/TCAP-HCM phenotypically mirrors myofilament-HCM and is more severe than the subset of patients who still remain without a disease-causing mutation. The precise role of W4R-MLP in the pathogenesis of either DCM or HCM warrants further investigation.     

Excitatory membrane current in heart muscle (Purkinje fibers)

Summary In short Purkinje fibers current voltage relations were measured by a voltage clamp technique. The membrane was de- and repolarized at constant speeds of 0.3–30 V/s. The capacitive current flowing during such potential changes could be simulated by the current through a network, in which about two thirds of the total membrane capacity of 12 F/cm² were connected through a series resistor of 150 cm².When the membrane was depolarized with speeds above 1 V/s, sodium current was measured between about – 50 to 0 mV. Absence of sodium current at positive membrane potentials could result from temporal inactivation. The amplitude of the sodium current increased greatly with rising speed of depolarization. When the external Na⁺ concentration was varied between 0 and 145 mM/l, the direction of the sodium current was outward above, and inward below the respective sodium equilibrium potentials. The dependence of sodium permeability on membrane potential was not influenced by the external sodium concentration. The maximum Na⁺ permeabilities measured at a speed of depolarization of 30 V/s were in the range of 0.5 · 10^–3 cm/s.In addition to the excitatory negative Na⁺ current seen in Tyrode's solution a negative non-sodium current was observed in presence and absence of external Na⁺. The voltage dependence of the non-sodium current was similar to that of the Na⁺ current.

---

Zusammenfassung An verkürzten Purkinjefäden wurden bei geregelterSpannung Strom-Spannungs-Beziehungen gemessen. Dabei wurde die Membran mit jeweils konstanter Geschwindigkeit von 0,3–30 V/s de- und repolarisiert. Der kapazitive Strom, der während einer solchen Spannungsänderung floß, verhielt sich wie der Strom durch ein Netzwerk, in dem eine direkt gekoppelte Membrankapazität C _M (4 F/cm²) einer größeren über einen Serienwiderstand C _M (150 cm²) angeschlossenen Kapazität C _M (8 F/cm²) parallel liegt.Wenn die Membran mit Geschwindigkeiten über 1 V/s depolarisiert wurde, floß zwischen –50 und 0 mV ein Natriumstrom. Das Fehlen von Natriumstrom bei positiven Membranpotentialen könnte bei den besprochenen Depolarisationsgeschwindigkeiten auf zeitliche Inaktivierung zurückgeführt werden. Die Amplitude dieses Natriumstroms wuchs mit steigender Depolarisationsgeschwindigkeit stark an. Wurde die extracelluläre Na⁺-Konzentration zwischen 0 und 145 mM/l variiert, so flossen die Natriumionen oberhalb des entsprechenden Na⁺-Gleichgewichtspotentials aus der Faser, und unter diesem Potential in die Faser. Die Abhängigkeit der Na⁺-Permeabilität vom Membranpotential wurde von der extracellulären Na⁺-Konzentration nicht beeinflußt. Die bei einer Depolarisationsgeschwindigkeit von 30 V/s gemessene maximale Na⁺-Permeabilität lag bei 0,5 · 10^–3 cm/s.Zusätzlich zum erregenden negativen Na⁺-Strom in Tyrode-Lösung wurde ein negativer Nicht-Natrium-Strom beobachtet, der von der Anwesenheit extracellulärer Na⁺-Ionen unabhängig war. Der negative Nicht-Natrium-Strom hing in ähnlicher Weise wie der Na⁺-Strom vom Membranpotential ab. Die Ladungsträger für diesen Strom sind nicht bekannt.

---

---

This investigation was supported by the Deutsche Forschungsgemeinschaft.     

Glucose-regulated protein 78 (GRP78) is elevated in embryonic mouse heart and induced following hypoglycemic stress

This study investigates the distribution and heart levels of glucose regulated protein (GRP) 78 during normal development and in response to hypoglycemia in the mouse. Results demonstrate that GRP78 is strongly expressed with in the heart, neural tube, gut endoderm, somites, and surface ectoderm of mouse embryos during early organogenesis, and GRP78 staining remains prominent in the heart from gestational days 9.5 through 13.5. Cardiac myocytes are the primary site of GRP78 expression within the heart. GRP78 levels are highest in the heart during early organogenesis and levels decrease significantly by the fetal period. GRP78 expression is increased after 24 h of hypoglycemia in the early organogenesis-stage heart. Considering the tissue specific pattern of GRP expression and changes during development of the heart, GRPs may play significant roles in the normal differentiation and development of cardiac tissue. GRP induction may also be involved in hypoglycemia-induced cardiac dysmorphogenesis. Accepted: 25 January 2000     

Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress

Glycinergic interplexiform cells provide a feedback signal from the inner retina to the outer retina. To determine if cones receive such a signal, glycine was applied on cultured porcine cone photoreceptors recorded with the patch clamp technique. A minor population of cone photoreceptors was found to generate large currents in response to puff application of glycine. These currents reversed close to the calculated equilibrium potential for chloride ions. These glycine-elicited currents were sensitive to strychnine but not to picrotoxin consistent with the expression of α–β-heteromeric glycine receptors. Glycine receptors were also activated by taurine and β-alanine. The glycine receptor antibody mAb4a labelled a minority of the cone photoreceptors identified by an antibody specific for cone arrestin. Finally, expression of the β subunit of the glycine receptor was demonstrated by single cell RT-PCR in a similar proportion (∼13%) of cone photoreceptors freshly isolated by lectin-panning. The identity of cone photoreceptors was assessed by their specific expression of the cone arrestin mRNA. The population of cone photoreceptors expressing the glycine receptor was not correlated to a specific colour-sensitive subtype as demonstrated by single cell RT-PCR experiments using primers for S opsin, cone arrestin and glycine receptor β subunit. This glycine receptor expression in a minority of cones defines a new cone population suggesting an unexpected role for glycine in the visual information processing in the outer retina.     

Fabrication and characterizations of a novel drug delivery device liposomes-in-microsphere (LIM)

In the present work, we developed a novel drug delivery system, liposomes-in-microsphere (LIM) of biodegradable polymers, which is conceived from a combination of the polymer- and the lipid-based delivery systems and can thus integrate the advantages and avoid the drawbacks of the two systems. Liposomes were encapsulated into microspheres of biodegradable polymers by the solvent extraction/evaporation process to form LIMs. The integrity of the liposomes was preserved by modifying the microencapsulation process and coating the liposomes with chitosan. We demonstrated by scanning electron microscopy, laser light scattering and fluorescence spectroscopy that the particle size and surface morphology of the polymeric microspheres did not change significantly with the liposomes encapsulated, the liposomes remained intact within the polymeric matrix of the microspheres, and the encapsulated liposomes could be released from the microspheres in a controlled manner at a nearly constant release rate after an initial off-release period. Decreasing the particle size of liposomes and increasing the pore size of the polymeric matrix shortened the initial off-release period and increased the liposome release rate. In conclusion, a novel drug delivery system, liposomes-in-microsphere, was successfully developed and characterized. The liposome release kinetics could be controlled by the composition and fabrication parameters of the liposomes and polymeric microspheres. Such a novel controlled release system may have potential to be applied for drug delivery and gene therapy.     

心肌弹性的实验研究(二)

心脏的收缩和舒张功能与心肌弹性密切相关。为了研究心肌的弹性，探讨心衰的机理，作者对离体猪心的弹性模量进行了测试，实验结果表明，心肌的弹性优于骨骼肌、血管和腱。