Occurrence and distribution of atrial natriuretic peptide-containing cells in the left ventricle of hypertensive rats

In the ventricles of adult mammalian hearts, production of atrial natriuretic peptide (ANP) is negligible, restricted to the impulse-conducting cells, the papillary muscles, and a minority of subendocardial myocytes. ANP expression is reinduced in the ventricles of pressure-overloaded and failing hearts and is frequently used as a marker for myocyte hypertrophy. Using an immunohistochemical approach, we have characterized the size distribution of ANP-containing myocytes in the left ventricle of the spontaneously hypertensive rat (SHR) before and after chronic antihypertensive therapy and compared the results to age-matched normotensive Wistar rats (WR). Our findings show that in SHR the frequency of cells presenting ANP granularity is positively correlated with myocyte size (r=0.746, P<0.02). The highest proportion of ANP-positive myocytes (55-57%) was measured among cells of diameters 30-34 microm. In any corresponding cell size, the proportion of ANP-presenting myocytes was five- to tenfold higher in SHR than in the normotensive WR. We studied the effects of the antihypertensive drugs captopril, hydralazine, and nifedipine and found that, regardless of their effect on blood pressure or hypertrophy, all three eliminated ANP immunoproducts from the majority of the left ventricular myocytes and reduced the level of ANP mRNA, captopril being the most effective. The positive correlation between myocyte size and ANP expression was not maintained in the hearts of drug-treated SHR. Myocytes on the border of fibrotic areas or in regions of ANP presentation within the normal heart resisted the suppressive effect of the antihypertensive therapy, indicating that blood pressure or hypertrophy are not the sole correlates for ANP expression.     

Transforming growth factor-β1 downregulates beating frequency and remodeling of cultured rat adult cardiomyocytes

We have observed increased levels of transforming growth factor-₁ (TGF-₁) in human hibernating myocardium (HM). Impaired ventricular function in HM is known to be restored to normal following revascularization implying that myocardial structure in HM is to a certain degree preserved. We have therefore tested whether TGF-₁ can imitate features of HM by reducing the number and frequency of beating cells (chronotropism) and structural remodeling of cultured adult rat cardiomyocytes (ARC), thus saving substrate, energy, and oxygen. Parameters measured were cell size, protein synthesis, protein degradation, protein content, myofibrillogenesis, and chronotropism. ARC were stimulated for 6 days with sera from patients with coronary heart disease, as this period led to a maximum response of cells. An increase of 90% in cell surface area following such treatment was reduced to a 20% increase of the original size by TGF-₁. Concomitantly, the rate of protein synthesis dropped from 3.6-fold to 2.4-fold, and myofibrillogenesis was reduced. TGF-₁ downregulated both the number of contracting cells from 81% to 10% and the frequency from 52 to nine beats per minute. However, TGF-₁ treatment did not reduce the augmentation of protein content (1.28-fold versus 1.25-fold) indicating that protein degradation was also inhibited. Similar results were obtained with serum from healthy volunters. The effects of TGF-₁ were reversible. We conclude that TGF-₁ constrains protein turnover and beating activity in underperfused myocardium, thus mediating protection by adapting myocytes to shortages in blood supply.T. Kubin and M. Tomars contributed equally to this work.     

Activity and diversity of methanogens in a petroleum hydrocarbon-contaminated aquifer

Methanogenic activity was investigated in a petroleum hydrocarbon-contaminated aquifer by using a series of four push-pull tests with acetate, formate, H(2) plus CO(2), or methanol to target different groups of methanogenic Archaea. Furthermore, the community composition of methanogens in water and aquifer material was explored by molecular analyses, i.e., fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes amplified with the Archaea-specific primer set ARCH915 and UNI-b-rev, and sequencing of DNA from dominant DGGE bands. Molecular analyses were subsequently compared with push-pull test data. Methane was produced in all tests except for a separate test where 2-bromoethanesulfonate, a specific inhibitor of methanogens, was added. Substrate consumption rates were 0.11 mM day(-1) for methanol, 0.38 mM day(-1) for acetate, 0.90 mM day(-1) for H(2), and 1.85 mM day(-1) for formate. Substrate consumption and CH(4) production during all tests suggested that at least three different physiologic types of methanogens were present: H(2) plus CO(2) or formate, acetate, and methanol utilizers. The presence of 15 to 20 bands in DGGE profiles indicated a diverse archaeal population. High H(2) and formate consumption rates agreed with a high diversity of methanogenic Archaea consuming these substrates (16S rRNA gene sequences related to several members of the Methanomicrobiaceae) and the detection of Methanomicrobiaceae by using FISH (1.4% of total DAPI [4',6-diamidino-2-phenylindole]-stained microorganisms in one water sample; probe MG1200). Considerable acetate consumption agreed with the presence of sequences related to the obligate acetate degrader Methanosaeata concilii and the detection of this species by FISH (5 to 22% of total microorganisms; probe Rotcl1). The results suggest that both aceticlastic and CO(2)-type substrate-consuming methanogens are likely involved in the terminal step of hydrocarbon degradation, while methanogenesis from methanol plays a minor role. DGGE profiles further indicate similar archaeal community compositions in water and aquifer material. The combination of hydrogeological and molecular methods employed in this study provide improved information on the community and the potential activity of methanogens in a petroleum hydrocarbon-contaminated aquifer.     

Regional variation in canopy transpiration of Central European beech forests

Forest hydrologists have hypothesised that canopy transpiration (E_c) of European temperate forests occurs at rather similar rates in stands with different tree species and hydrologic regimes. We tested this hypothesis by synchronously measuring xylem sap flow in four mature stands of Fagus sylvatica along a precipitation gradient with the aim (1) of exploring the regional variability of annual canopy transpiration (E_c(t)) in this species, and (2) of analysing the relationship between precipitation (P) and E_c(t). E_c(t) rates of 216, 225, 272 and 303 mm year^–1 corresponded to precipitation averages of 520, 710, 801 and 1,040 mm year^–1 in the four stands. We explored the regional variability of E_c(t) in Central European colline to sub-montane beech stands in two meta-analyses based on (1) existing sap flow data on beech (n=5 observations), or (2) all canopy transpiration data on beech obtained by different techniques (sap flow, micrometeorological or soil water budget approaches, n=25). With a coefficient of variation (CV) of 20%, the regional variability of E_c(t) (213–421 mm year^–1) was smaller than the variation in corresponding precipitation (550–1,480 mm year^–1). The mean E_c(t) for beech was 289 (±58) mm year^–1 (n=25). A humped-shaped relationship between E_c(t) and P, with a broad transpiration maximum in the precipitation range from ca. 700 to 1,000 mm year^–1, was found which may indicate soil moisture limitation of transpiration for P –1, and reduced transpiration by increased cloudiness or leaf wetness for P>1,000 mm year^–1. Thus, the precipitation level significantly influences canopy transpiration of humid temperate forests; however, the size of the P influence on E_c(t) and, in part, the direction of its effect differ from forests in semi-arid or arid climates. European beech has the capacity to maintain high E_c rates in both humid and partly dry summer climates (P<550 mm year^–1).     

Translocation of group 1 capsular polysaccharide in Escherichia coli serotype K30. Structural and functional analysis of the outer membrane lipoprotein Wza

The late steps in assembly of capsular polysaccharides (CPS) and their translocation to the bacterial cell surface are not well understood. The Wza protein was shown previously to be required for the formation of the prototype group 1 capsule structure on the surface of Escherichia coli serotype K30 (Drummelsmith, J., and Whitfield, C. (2000) EMBO J. 19, 57-66). Wza is a conserved outer membrane lipoprotein that forms multimers adopting a ringlike structure, and collective evidence suggests a role for these structures in the export of capsular polymer across the outer membrane. Wza was purified in the native form and with a C-terminal hexahistidine tag. WzaHis6 was acylated and functional in capsule assembly, although its efficiency was slightly reduced in comparison to the native Wza protein. Ordered two-dimensional crystals of WzaHis6 were obtained after reconstitution of purified multimers into lipids. Electron microscopy of negatively stained crystals and Fourier filtering revealed ringlike multimers with an average outer diameter of 8.84 nm and an average central cavity diameter of 2.28 nm. Single particle analysis yielded projection structures at an estimated resolution of 3 nm, favoring a structure for the WzaHis6 containing eight identical subunits. A derivative of Wza (Wza*) in which the original signal sequence was replaced with that from OmpF showed that the native acylated N terminus of Wza is critical for formation of normal multimeric structures and for their competence for CPS assembly, but not for targeting Wza to the outer membrane. In the presence of Wza*, CPS accumulated in the periplasm but was not detected on the cell surface. Chemical cross-linking of intact cells suggested formation of a transmembrane complex minimally containing Wza and the inner membrane tyrosine autokinase Wzc.     

Trichoderma harzianum metabolites pre-adapt mushrooms to Trichoderma aggressivum antagonism

Trichoderma spp. is the cause of green mold, a disorder that affects cultivated mushrooms. The aims of the study were to establish whether improvement of mushroom resistance to Trichoderma aggressivum could be obtained by inducing reaction mechanisms before contact with the pathogen and whether this ability was species or strain dependent. Twenty nine isolates of Agaricus bisporus, 29 isolates of Lentinula edodes and 18 isolates of Pleurotus spp. were studied. The effect of T. harzianum metabolites on mycelial growth of these isolates was evaluated on YMEA (yeast, malt extract and agar), supplemented or not with Lysing Enzymes from T. harzianum (Sigma?, L1412). Mycelial growth generally was affected by Lysing Enzymes, but some L. edodes and Pleurotus spp. adapted to Lysing Enzymes. When mycelium was taken from a first culture with Lysing Enzymes and placed on YMEA with Lysing Enzymes for a second culture, their growth rate was not different from those of the controls. In the case of A. bisporus, only partial adaptation was obtained with a few isolates. The effect of adaptation to Lysing Enzymes on resistance to T. aggressivum was assayed for one strain of each group. Trichoderma aggressivum was exposed to the margin of 5- to 9-day-old mushroom colonies. Agaricus bisporus produced brown droplets, and T. aggressivum overgrew its mycelium. Lentinula edodes and P. ostreatus produced brown lines blocking the progression of T. harzianum, both on YMEA and YMEA plus Lysing Enzymes. The line was visible after 3 d on YMEA and after only 2 d on YMEA plus Lysing Enzymes. Improvement in the resistance to antagonists by introduction of some of their metabolites to the culture medium is a method for mushroom protection.     

Metabolic control analysis of anaerobic glycolysis in human hibernating myocardium replaces traditional concepts of flux control

Myocardial hibernation represents an adaptation to sustained ischemia to maintain tissue vitality during severe supply-demand imbalance which is characterized by an increased glucose uptake. To elucidate this adaptive protective mechanism, the regulation of anaerobic glycolysis was investigated using human biopsies. In hibernating myocardium showing an increase in anaerobic glycolytic flux metabolizing exogenous glucose, the adjustment of flux through this pathway was analyzed by flux:metabolite co-responses. By this means, a previously unknown pattern of regulation using multisite modulation was found which largely differs from traditional concepts of metabolic control of the Embden-Meyerhof pathway in normal and diseased myocardium.     

Two different targeting signals direct human peroxisomal membrane protein 22 to peroxisomes

The 22-kDa peroxisomal membrane protein (PMP22) is a major component of peroxisomal membranes in mammals. Although its precise role in peroxisome function is poorly understood, it seems to be involved in pore forming activity and may contribute to the unspecific permeability of the organelle membrane. PMP22 is synthesized on free cytosolic ribosomes and then directed to the peroxisome membrane by specific targeting information. Previous studies in rats revealed that PMP22 contains one distinct peroxisomal membrane targeting signal in the amino-terminal cytoplasmic tail. We cloned and characterized the targeting signal of human PMP22 and compared it with the already described characteristics of the corresponding rat protein. Amino acid sequence alignment of rat and human protein revealed 77% identity including a high conservation of several protein motifs. We expressed various deletion constructs of PMP22 in fusion with the green fluorescent protein in COS-7 cells and determined their intracellular localization. In contrast to previous studies on rat PMP22 and most other peroxisomal membrane proteins, we showed that human as well as rat PMP22 contains two distinct and nonoverlapping peroxisomal membrane targeting signals, one in the amino-terminal and the other in the carboxyl-terminal protein region. They consist of two transmembrane domains and adjacent protein loops with almost identical basic clusters. Both of these peroxisomal targeting regions interact with PEX19, a factor required for peroxisome membrane synthesis. In addition, we observed that fusing the green fluorescent protein immediately adjacent to the targeting region completely abolishes targeting function and mislocalizes PMP22 to the cytosol.     

Clustering of CD40 ligand is required to form a functional contact with CD40

Receptor clustering is a key event in the initiation of signaling by many types of receptor molecules. Here, we provide evidence for the novel concept that clustering of a ligand is a prerequisite for clustering of the cognate receptor. We show that clustering of the CD40 receptor depends on reciprocal clustering of the CD40 ligand (gp39, CD154). Clustering of the CD40 ligand is mediated by an association of the ligand with p53, a translocation of acid sphingomyelinase (ASM) to the cell membrane, an activation of the ASM, and a formation of ceramide. Ceramide appears to modify preexisting sphingolipid-rich membrane microdomains to fuse and form ceramide-enriched signaling platforms that serve to cluster CD40 ligand. Genetic deficiency of p53 or ASM or disruption of ceramide-enriched membrane domains prevents clustering of CD40 ligand. The functional significance of CD40 ligand clustering is indicated by the finding that clustering of CD40 on B lymphocytes upon co-incubation with CD40 ligand-expressing T cells depends on clustering of the CD40 ligand and is abrogated by inhibition of CD40 ligand clustering.