Functional interaction of human neutrophil peptide-1 with the cell wall precursor lipid II

Defensins constitute a major class of cationic antimicrobial peptides in mammals and vertebrates, acting as effectors of innate immunity against infectious microorganisms. It is generally accepted that defensins are bactericidal by disrupting the anionic microbial membrane. Here, we provide evidence that membrane activity of human α-defensins does not correlate with antibacterial killing. We further show that the α-defensin human neutrophil peptide-1 (HNP1) binds to the cell wall precursor lipid II and that reduction of lipid II levels in the bacterial membrane significantly reduces bacterial killing. The interaction between defensins and lipid II suggests the inhibition of cell wall synthesis as a novel antibacterial mechanism of this important class of host defense peptides.     

Functional and structural remodeling of the myocardial microvasculature in early experimental hypertension

Advanced hypertension (HT), associated with left ventricular hypertrophy (LVH), impairs myocardial microvascular function and structure and leads to increased myocardial hypoxia and growth factor activation. However, the effect of HT on microvascular architecture and its relation to microvascular function, before the development of LVH (early HT), remains unclear. By way of method, pigs were studied after 12 wk of renovascular HT (n = 7) or control (n = 7) animals. Myocardial microvascular function (blood volume and blood flow at baseline and in response to adenosine) was assessed by using electron beam computed tomography (CT). Microvascular architecture was subsequently studied ex vivo using micro-CT, and microvessels (diameter, <500 microm) were counted in situ in three-dimensional images (40-microm on-a-side cubic voxels). Myocardial expression of vascular endothelial growth factor, basic fibroblast growth factor, and hypoxia-inducible factor-1alpha were also measured. By way of results, left ventricular muscle mass was similar between the groups. The blood volume response to intravenous adenosine was attenuated in HT animals compared with normal animals (+7.4 +/- 17.0 vs. +46.2 +/- 12.3% compared with baseline, P = 0.48 and P = 0.01, respectively). Microvascular spatial density in HT animals was significantly elevated compared with normal animals (246 +/- 26 vs. 125 +/- 20 vessels/cm2, P < 0.05) and correlated inversely with the blood volume response to adenosine. Growth factors expression was increased in HT animals compared with control animals. In conclusion, early HT elicits changes in myocardial microvascular architecture, which are associated with microvascular dysfunction and precede changes in muscle mass. These observations underscore the direct and early effects of HT on the myocardial vasculature.     

Store-operated Ca2+ entry modulates sarcoplasmic reticulum Ca2+ loading in neonatal rabbit cardiac ventricular myocytes

Store-operated Ca²⁺ entry (SOCE), which is Ca²⁺ entry triggered by the depletion of intracellular Ca²⁺ stores, has been observed in many cell types, but only recently has it been suggested to occur in cardiomyocytes. In the present study, we have demonstrated SOCE-dependent sarcoplasmic reticulum (SR) Ca²⁺ loading (load_SR) that was not altered by inhibition of L-type Ca²⁺ channels, reverse mode Na⁺/Ca²⁺ exchange (NCX), or nonselective cation channels. In contrast, lowering the extracellular [Ca²⁺] to 0 mM or adding either 0.5 mM Zn²⁺ or the putative store-operated channel (SOC) inhibitor SKF-96365 (100 µM) inhibited load_SR at rest. Interestingly, inhibition of forward mode NCX with 30 µM KB-R7943 stimulated SOCE significantly and resulted in enhanced load_SR. In addition, manipulation of the extracellular and intracellular Na⁺ concentrations further demonstrated the modulatory role of NCX in SOCE-mediated SR Ca²⁺ loading. Although there is little knowledge of SOCE in cardiomyocytes, the present results suggest that this mechanism, together with NCX, may play an important role in SR Ca²⁺ homeostasis. The data reported herein also imply the presence of microdomains unique to the neonatal cardiomyocyte. These findings may be of particular importance during open heart surgery in neonates, in which uncontrolled SOCE could lead to SR Ca²⁺ overload and arrhythmogenesis. cardiac ontogeny; cardiac excitation-contraction coupling; calcium homeostasis     

Binding characteristics of the Lactobacillus brevis ATCC 8287 surface layer to extracellular matrix proteins

Self-assembling proteins that form crystalline surface layers on many microorganisms can be involved in bacterial-host adhesion via specific interactions with components of the extracellular matrix. Here, we describe the interaction of the Lactobacillus brevis ATCC 8287 surface-layer protein SlpA with fibronectin, laminin, fibrinogen and collagen using surface plasmon resonance. SlpA was found to interact with high affinity to fibronectin and laminin, with a respective binding constant of 89.8 and 26.7 nM. The interaction of SlpA with collagen and fibrinogen was found to be of much lower affinity, with respective binding constants of 31.8 and 26.1 microM. The serine protease inhibitor benzamidine greatly reduced the affinity of SlpA for fibronectin, whereas the affinity for laminin remained unaffected. No protease activity of the purified SlpA protein could be detected. These data suggest that L. brevis may interact with host cells directly through high affinity interactions with laminin and fibronectin predominantly, involving distinct regions of the SlpA protein.     

Mechanism of graded persistent cellular activity of entorhinal cortex layer v neurons

Working memory is an emergent property of neuronal networks, but its cellular basis remains elusive. Recent data show that principal neurons of the entorhinal cortex display persistent firing at graded firing rates that can be shifted up or down in response to brief excitatory or inhibitory stimuli. Here, we present a model of a potential mechanism for graded firing. Our multicompartmental model provides stable plateau firing generated by a nonspecific calcium-sensitive cationic (CAN) current. Sustained firing is insensitive to small variations in Ca2+ concentration in a neutral zone. However, both high and low Ca2+ levels alter firing rates. Specifically, increases in persistent firing rate are triggered only during high levels of calcium, while decreases in rate occur in the presence of low levels of calcium. The model is consistent with detailed experimental observations and provides a mechanism for maintenance of memory-related activity in individual neurons.     

Random amplification of polymorphic DNA (RAPD) typing of carnobacteria isolated from hindgut chamber and large intestine of Atlantic cod (Gadus morhua l.)

Autochthonous and allochthonous bacteria were isolated from hindgut chamber and large intestine of fed and starved Atlantic cod (Gadus morhua L.). All bacterial strains isolated from hindgut chamber belong to carnobacteria. However, only 10.2% of the bacteria strains from the large intestine belong to carnobacteria. Random amplification of polymorphic DNA (RAPD) analysis using three selective primers, was performed to further identify the carnobacteria strains. Nine of these were isolated from hindgut chamber contents, ten associated with epithelial cells of the hindgut chamber, and six isolated from the large intestines of fed and starved fish. The 25 isolates segregated into eight clusters. The major cluster comprised nine strains isolated from the hindgut chamber of both fed and starved fish showing low similarity with the reference strains. The other strains isolated from the hindgut were located in clusters showing high similarity with Carnobacterium gallinarum or Carnobacterium piscicola. Strains isolated from large intestine appeared more divergent and were located in five different clusters. Autochthonous (indigenous) bacteria were clearly demonstrated in the hindgut chamber as transmission electron microscopy revealed rod-shaped bacteria between adjacent microvilli. Endocytosis of bacteria by epithelial cells was observed in the hindgut chamber.     

HIF-1alpha has an anti-apoptotic effect in human airway epithelium that is mediated via Mcl-1 gene expression

Hypoxia-inducible factor-1alpha (HIF-1alpha) and myeloid cell leukemia-1 (Mcl-1) proteins have been shown to regulate apoptosis in some cell systems but have not been studied in this context in airway epithelium. Using a model of anoxia/reoxygenation (A/R), the present study employed RNA interference (RNAi) targeting HIF-1alpha and Mcl-1 to evaluate their possible anti-apoptotic effects on HBE1 cells, an immortalized human bronchial epithelial cell line. The cells were either cultured under normoxic conditions or were transfected with small interfering RNA (siRNA) duplexes targeting HIF-1alpha or Mcl-1 mRNA and then immediately exposed to A/R. As controls, non-transfected HBE1 cells and cells transfected with scrambled RNA duplexes were subjected to A/R. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and RNAi was assessed by knockdown of HIF-1alpha and Mcl-1 mRNA and protein expression using real-time quantitative RT-PCR (Q-PCR), immunohistochemistry, and Western blots. HBE1 cells transfected with siRNA duplexes targeting either HIF-1alpha or Mcl-1 and subjected to A/R manifested considerable apoptosis, a finding not observed in either non-transfected cells or cells transfected with scrambled RNA duplexes. Specific knockdown of mRNA and protein expression by RNAi in HBE1 cells after A/R was shown for siRNA duplexes targeting either HIF-1alpha or Mcl-1. Unexpectedly, knockdown of HIF-1alpha induced parallel knockdown of Mcl-1 mRNA and protein expression, whereas Mcl-1 knockdown had no noticeable effect on HIF-1alpha expression. Thus, although both of these proteins were shown to be anti-apoptotic, the action of HIF-1alpha appeared to be mediated in part via Mcl-1.     

Exercise training and the antioxidant alpha-lipoic acid in the treatment of insulin resistance and type 2 diabetes

One hallmark of the insulin-resistant state of prediabetes and overt type 2 diabetes is an impaired ability of insulin to activate glucose transport in skeletal muscle, due to defects in IRS-1-dependent signaling. An emerging body of evidence indicates that one potential factor in the multifactorial etiology of skeletal muscle insulin resistance is oxidative stress, an imbalance between the cellular exposure to an oxidant stress and the cellular antioxidant defenses. Exposure of skeletal muscle to an oxidant stress leads to impaired insulin signaling and subsequently to reduced glucose transport activity. Numerous studies have demonstrated that treatment of insulin-resistant animals and type 2 diabetic humans with antioxidants, including alpha-lipoic acid (ALA), is associated with improvements in skeletal muscle glucose transport activity and whole-body glucose tolerance. An additional intervention that is effective in ameliorating the skeletal muscle insulin resistance of prediabetes and type 2 diabetes is endurance exercise training. Recent investigations have demonstrated that the combination of exercise training and antioxidant treatment using ALA in an animal model of obesity-associated insulin resistance provides a unique interactive effect resulting in a greater improvement in insulin action on skeletal muscle glucose transport than either intervention individually. Moreover, this interactive effect of exercise training and ALA is due in part to improvements in IRS-1-dependent insulin signaling. These studies highlight the effectiveness of combining endurance exercise training and antioxidants in beneficially modulating the molecular defects in insulin action observed in insulin-resistant skeletal muscle.     

Effects of Mesolithic hunter-gatherers on local vegetation in a non-uniform glacio-isostatic land uplift area, northern Sweden

The oldest early Mesolithic settlements found so far (i.e. 8600 b.p.) in the interior of northern Sweden, in the province of Norrbotten, have been discovered through the application of a model simulating glacio-isostatic land uplift. The objective of this study was to investigate, through pollen and charred particle analyses, vegetation composition and the influence of man on vegetation and fire pattern in the vicinity of two of these early settlements. Early Holocene vegetation was characterised by Betula, Hippophaë and Salix-species, but no initial impact by hunter-gatherers on vegetation was detected. Subsequently Betula and Pinus became dominant but abrupt changes in the tree layer followed, Poaceae, Hippophaë and Salix-species increased, and Humulus occurred. These changes, which were synchronous with dates of archaeological findings at the settlements, indicated local vegetation changes caused by man. The relationship between these changes and fire was, however, subtle. Eventually Pinus forests became dominant and human impact on vegetation became less apparent. It is concluded that the impact of early Mesolithic hunter-gatherers on vegetation was detectable, but that their effect on fire pattern was difficult to evaluate. Further, we show that the glacio-isostatic land uplift must be considered in the search for Mesolithic settlements in areas previously covered by the Weichselian Ice, and that the process has influenced the biological archives through re-deposition of material.     

Mornings with Art, lessons learned: feedback regulation, restriction threshold biology, and redundancy govern molecular stress responses

Work from the laboratory of Dr. Arthur B. Pardee has highlighted basic principles that govern cellular and molecular biological processes in living cells. Among the most important governing principles in cellular and molecular responses are: (i) threshold "restriction" responses, wherein a level of response is reached and a "point of no return" is achieved; (ii) feedback regulation; and (iii) redundancy. Lessons learned from the molecular biology of cellular stress responses in mammalian cancer versus normal cells after ionizing radiation (IR) or chemotherapeutic agent exposures reveal similar instances of these guiding principles in mammalian cells. Among these are the: (i) induction of cell death responses by beta-lapachone (beta-lap), a naphthoquinone anti-tumor agent that kills cancer cells via an NQO1 (i.e., X-ray-inducible protein-3, xip3)-dependent mechanism; (ii) induction of secretory clusterin (sCLU) in response to TGF-beta1 exposure, and the ability of induced sCLU protein to down-regulate TGF-beta1 signaling; and (iii) induction of DNA mismatch repair-dependent G(2) cell cycle checkpoint responses after exposure to alkylating agents. We have learned these lessons and now adopted strategies to exploit them for improved therapy. These examples will be discussed and compared to the pioneering findings of researchers in the Pardee laboratory over the years.