Tetracycline-Inducible System for Regulation of Skeletal Muscle-Specific Gene Expression in Transgenic Mice

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.     

A phase-variable capsule is involved in virulence of Campylobacter jejuni 81-176

Campylobacter jejuni strain 81-176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K-digested whole-cell preparations. A site-specific insertional mutant in the kpsM gene results in loss of expression of a high-molecular-weight (HMW) glycan (apparent Mr 26 kDa to > 85 kDa) and increased resolution of a second ladder-like glycan (apparent Mr 26-50 kDa). The kpsM mutant of 81-176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM-dependent HMW glycan and the kpsM-independent ladder-like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81-176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an alpha2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81-176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM-dependent capsule undergoes phase variation at a high frequency.     

Direct innervation of GnRH neurons by encephalic photoreceptors in birds

In nonmammalian vertebrates, photic cues that regulate the timing of seasonal reproductive cyclicity are detected by nonretinal, nonpineal deep brain photoreceptors. It has long been assumed that the underlying mechanism involves the transmission of photic information from the photoreceptor to a circadian system, and thence to the reproductive axis. An alternative hypothesis is that there is direct communication between the brain photoreceptor and the reproductive axis. In the present study, light and confocal microscopy reveal that gonadotropin releasing hormone (GnRH) neurons and processes are scattered among photoreceptor cells (identified by their opsin-immunoreactivity) in the lateral septum (SL). In the median eminence (ME), opsin and GnRH immunoreactive fibers overlap extensively. Single and double label ultrastructural immunocytochemistry indicate that in the SL and preoptic area (POA), opsin positive terminals form axo-dendritic synapses onto GnRH dendrites. In the ME, opsin and GnRH terminals lie adjacent to each other, make contact with tanycytes, or terminate on the hypophyseal portal capillaries. These results reveal thatbrain photoreceptors communicate directly with GnRH-neurons; this represents a means by which photoperiodic information reaches the reproductive axis.     

Supplementation with alkaline minerals reduces symptoms in patients with chronic low back pain

The cause of low back pain is heterogeneous, it has been hypothesised that a latent chronic acidosis might contribute to these symptoms. It was tested whether a supplementation with alkaline minerals would influence symptoms in patients with low back pain symptoms. In an open prospective study 82 patients with chronic low back pain received daily 30 g of a lactose based alkaline multimineral supplement (Basica) over a period of 4 weeks in addition to their usual medication. Pain symptoms were quantified with the "Arhus low back pain rating scale" (ARS). Mean ARS dropped highly significant by 49% from 41 to 21 points after 4 weeks supplemention. In 76 out of 82 patients a reduction in ARS was achieved by the supplementation. Total blood buffering capacity was significantly increased from 77.69 +/- 6.79 to 80.16 +/- 5.24 mmol/L (mean +/- SEM, n = 82, p < 0.001) and also blood pH rose from 7.456 +/- 0.007 to 7.470 +/- 0.007 (mean +/- SEM, n = 75, p < 0.05). Only intracellular magnesium increased by 11% while other intracellular minerals were not significantly changed in sublingual tissue as measured with the EXA-test. Plasma concentrations of potassium, calcium, iron, copper, and zinc were within the normal range and not significantly influenced by the supplementation. Plasma magnesium was slightly reduced after the supplemenation (-3%, p < 0.05). The results show that a disturbed acid-base balance may contribute to the symptoms of low back pain. The simple and safe addition of an alkaline multimineral preparate was able to reduce the pain symptoms in these patients with chronic low back pain.     

Integration of hypoxic dilation signaling pathways for skeletal muscle resistance arteries

Mediator contributions to hypoxic dilation of rat gracilis muscle resistance arteries were determined by measuring dilation, vascular smooth muscle hyperpolarization, and metabolite production after incremental hypoxia. Nitric oxide (NO) synthase inhibition abolished responses to mild hypoxia, whereas COX inhibition impaired responses to more severe hypoxia by 77%. Blocking 20-hydroxyeicosatetraenoic acid (20-HETE) impaired responses to moderate hypoxia. With only NO systems intact, responses were maintained with mild hypoxia (88% normal) mediated via K(Ca) channels. When only COX pathways were intact, responses to moderate-severe hypoxia were largely retained (79% of normal) mediated via K(ATP) channels. Vessel responses to moderate hypoxia were retained with only 20-HETE systems intact mediated via K(Ca) channels. NO production increased 5.6-fold with mild hypoxia; greater hypoxia was without further effect. With increased hypoxia, 20-HETE levels fell to 40% of control values. 6-keto-PGF(1alpha) levels were not altered with mild hypoxia, but increased 4.6-fold with severe hypoxia. These results suggest vascular reactivity to progressive hypoxia represents an integration of NO production (mild hypoxia), PGI(2) production (severe hypoxia), and reduced 20-HETE levels (moderate hypoxia).     

Antihypertensive effect of mechanism-based inhibition of renal arachidonic acid omega-hydroxylase activity

The cytochrome P-450 eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor that is implicated in the regulation of blood pressure. The identification of selective inhibitors of renal 20-HETE formation for use in vivo would facilitate studies to determine the systemic effects of this eicosanoid. We characterized the acetylenic fatty acid sodium 10-undecynyl sulfate (10-SUYS) as a potent and selective mechanism-based inhibitor of renal 20-HETE formation. A single dose of 10-SUYS caused an acute reduction in mean arterial blood pressure in 8-wk-old spontaneously hypertensive rats. The decrease in mean arterial pressure was maximal 6 h after 10-SUYS treatment (17.9 +/- 3.2 mmHg; P < 0.05), and blood pressure returned to baseline levels within 24 h after treatment. Treatment with 10-SUYS was associated with a decrease in urinary 20-HETE formation in vivo and attenuation of the vasoconstrictor response of renal interlobar arteries to ANG II in vitro. These results provide further evidence that 20-HETE plays an important role in the regulation of blood pressure in the spontaneously hypertensive rat.     

Mechanobiology of cartilage: how do internal and external stresses affect mechanochemical transduction and elastic energy storage?

 Articular cartilage is a multilayered structure that lines the surfaces of all articulating joints. It contains cells, collagen fibrils, and proteoglycans with compositions that vary from the surface layer to the layer in contact with bone. It is composed of several zones that vary in structure, composition, and mechanical properties. In this paper we analyze the structure of the extracellular matrix found in articular cartilage in an effort to relate it to the ability of cartilage to store, transmit, and dissipate mechanical energy during locomotion. Energy storage and dissipation is related to possible mechanisms of mechanochemical transduction and to changes in cartilage structure and function that occur in osteoarthritis. In addition, we analyze how passive and active internal stresses affect mechanochemical transduction in cartilage, and how this may affect cartilage behavior in health and disease. Received: 8 February 2002 / Accepted: 9 July 2002