The Mouse GalR2 Galanin Receptor: Genomic Organization, cDNA Cloning, and Functional Characterization

Abstract: The diverse physiological actions of galanin are thought to be mediated through activation of galanin receptors (GalRs). We report the genomic and cDNA cloning of a mouse GalR that possesses a genomic structure distinct from that of GalR1 and encodes a functional galanin receptor. The mouse GalR gene consists of two exons separated by a single intron within the protein-coding region. The splicing site for the intron is located at the junction between the third transmembrane domain and the second intracellular loop. The cDNA encodes a 370-amino acid putative G protein-coupled receptor that is markedly different from human GalR1 and rat GalR3 (38 and 57%) but shares high homology with rat GalR2 (94%). In binding studies utilizing membranes from COS-7 cells transfected with mouse GalR2 cDNA, the receptor displayed high affinity ( K _D = 0.47 n M ) and saturable binding with ¹²⁵I-galanin ( B _max = 670 fmol/mg). The radioligand binding can be displaced by galanin and its analogues in a rank order: galanin ⋍ M40 ⋍ M15 ⋍ M35 ⋍ C7 ⋍ galanin (2–29) ⋍ galanin (1–16) ≫ galanin (10–29) ⋍ galanin (3–29), which resembles the pharmacological profile of the rat GalR2. Receptor activation by galanin in COS-7 cells stimulated phosphoinositide metabolism, which was not reversed by pertussis toxin. Thus, the galanin receptor encoded in the cloned mouse GalR gene is the type 2 galanin receptor and is active in both ligand binding and signaling assays.     

In Vivo Volatile Organic Compound Signatures of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of a chronic enteric disease of ruminants. Available diagnostic tests are complex and slow. In vitro, volatile organic compound (VOC) patterns emitted from MAP cultures mirrored bacterial growth and enabled distinction of different strains. This study was intended to determine VOCs in vivo in the controlled setting of an animal model. VOCs were pre-concentrated from breath and feces of 42 goats (16 controls and 26 MAP-inoculated animals) by means of needle trap microextraction (breath) and solid phase microextraction (feces) and analyzed by gas chromatography/ mass spectrometry. Analyses were performed 18, 29, 33, 41 and 48 weeks after inoculation. MAP-specific antibodies and MAP-specific interferon-γ-response were determined from blood. Identities of all marker-VOCs were confirmed through analysis of pure reference substances. Based on detection limits in the high pptV and linear ranges of two orders of magnitude more than 100 VOCs could be detected in breath and in headspace over feces. Twenty eight substances differed between inoculated and non-inoculated animals. Although patterns of most prominent substances such as furans, oxygenated substances and hydrocarbons changed in the course of infection, differences between inoculated and non-inoculated animals remained detectable at any time for 16 substances in feces and 3 VOCs in breath. Differences of VOC concentrations over feces reflected presence of MAP bacteria. Differences in VOC profiles from breath were linked to the host response in terms of interferon-γ-response. In a perspective in vivo analysis of VOCs may help to overcome limitations of established tests.     

Effect of preservation period on the viscoelastic material properties of soft tissues with implications for liver transplantation

The liver harvested from a donor must be preserved and transported to a suitable recipient immediately for a successful liver transplantation. In this process, the preservation period is the most critical, since it is the longest and most tissue damage occurs during this period due to the reduced blood supply to the harvested liver and the change in its temperature. We investigate the effect of preservation period on the dynamic material properties of bovine liver using a viscoelastic model derived from both impact and ramp and hold experiments. First, we measure the storage and loss moduli of bovine liver as a function of excitation frequency using an impact hammer. Second, its time-dependent relaxation modulus is measured separately through ramp and hold experiments performed by a compression device. Third, a Maxwell solid model that successfully imitates the frequency- and time-dependent dynamic responses of bovine liver is developed to estimate the optimum viscoelastic material coefficients by minimizing the error between the experimental data and the corresponding values generated by the model. Finally, the variation in the viscoelastic material coefficients of bovine liver are investigated as a function of preservation period for the liver samples tested 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h after harvesting. The results of our experiments performed with three animals show that the liver tissue becomes stiffer and more viscous as it spends more time in the preservation cycle.     

Regulation of DMBT1 via NOD2 and TLR4 in intestinal epithelial cells modulates bacterial recognition and invasion

Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes the regulation and function of DMBT1 in intestinal epithelial cells, which form the primary immunological barrier for invading pathogens. We report that intestinal epithelial cells up-regulate DMBT1 upon proinflammatory stimuli (e.g., TNF-alpha, LPS). We demonstrate that DMBT1 is a target gene for the intracellular pathogen receptor NOD2 via NF-kappaB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn's disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF-kappaB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology of barrier dysfunction in Crohn's disease.     

Sequence and length recognition of the C-terminal turnover element of LpxC, a soluble substrate of the membrane-bound FtsH protease

The membrane-anchored FtsH protease is essential in Escherichia coli as it adjusts the cellular amount of LpxC, the key enzyme in lipopolysaccharide (LPS) biosynthesis. Both accumulation and depletion of LpxC are toxic to E. coli. By continuous proteolysis of LpxC, FtsH maintains a low concentration of LpxC and, hence, the proper equilibrium between LPS and phospholipids. The C terminus of LpxC is required for turnover. By adding this tail to glutathione-S-transferase (GST) we show that it is necessary but not sufficient for FtsH-mediated degradation. A detailed mutational analysis revealed six non-polar residues in the C terminus of LpxC that are critical for degradation. Alteration of the C-terminal AVLA motif towards the SsrA-like sequence ALAA directed LpxC to other cellular proteases reinforcing the importance of the C-terminal tail for targeting to FtsH. Short C-terminal truncations stabilized LpxC. Most mutations in the C terminus of LpxC left its enzymatic activity intact as was shown by growth assays, microscopy and 2-keto-3-deoxyoctonate (KDO) determination. The critical length of the turnover element was defined by internal deletions. A C-terminal tail of about 20 amino acids length is required for proteolysis of LpxC by FtsH.     

Effects of Stress Modifier Biostimulants on Vegetative Growth,Nutrients, and Antioxidants Contents of Garden Thyme (Thymus vulgaris L.) Under Water Deficit Conditions

The aim of this study is to investigate the effect of stress modulators on vegetative growth, antioxidants, and nutrient content of Thymus vulgaris L. under water deficit stress conditions. A factorial experiment was performed in the form of a randomized complete block design with 10 treatments and 3 replications in the 2019–2020 growing season. The factors were stress modulators at 5 levels (ZN: zinc nano-fertilizer, AA: amino acid, SW: seaweed, HA: humic acid and C: control) and irrigation regime at 2 levels [FIrr: full irrigation (100% field capacity) and DIrr: deficit irrigation (50% field capacity)]. The highest plant height, number of branches, and total dry weight of the garden thyme plant were observed in the foliar application of HA and SW under full irrigation conditions. Relative water content, chlorophyll a and b, and uptake of nutrients (N, P, and K) were reduced under water deficit stress, but the foliar application of stress modulators increased relative water content, chlorophyll content, and nutrient uptake of the garden thyme plant significantly compared with control. The water deficit increased proline content, total flavonoid, and phenol content in the garden thyme plant. So, the highest total flavonoid and phenol content was obtained from plants treated with HA, whereas proline content was higher in the control plants. Soluble sugars and essential oil increased significantly under water deficit stress conditions. The foliar application of HA compared to the control plant increased soluble sugars and essential oil in garden thymes. The activities of catalase, superoxide dismutase, and ascorbate peroxidase enzymes were improved in stress modulator treatments such as HA and SW compared to control plants under water deficit stress conditions. The plants of garden thymes showed a good response to stress modulator treatments under water stress conditions, and HA and SW treatments were found to be more effective.