Cell-free translation of mRNA encoding an arterial smooth muscle cell proteoglycan core protein

The size and immunological reactivity of the primary gene products of a small non-aggregating dermatan sulfate proteoglycan from bovine and monkey arterial smooth muscle cells were examined after cell-free translation of mRNA. Antisera against the dermatan sulfate proteoglycans from bovine articular cartilage, DSPG II [Rosenberg et al. J. Biol. Chem. 260, 6304 (1985)] and human skin fibroblasts [Glossl et al. J. Biol. Chem. 259, 14144 (1984)] were used to show that the unmodified smooth muscle precursor core protein was immunologically related to both the cartilage and fibroblast core proteins. The size of the precursor core proteins within each species was identical regardless of the tissue source. Comparison of the precursor core proteins synthesized by primate and bovine cells revealed that the bovine core proteins were approximately 1500 Da larger than the primate core proteins as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A similar size difference was observed when the mature core proteins of monkey smooth muscle cells and bovine articular chondrocytes were compared after removal of the glycosaminoglycan chains. These results indicate that arterial smooth muscle cells synthesize a dermatan sulfate proteoglycan whose core protein is similar to, if not the same as, the cartilage and fibroblast dermatan sulfate proteoglycan core proteins. These core proteins may be encoded by the same gene that has diverged in size during speciation.     

Localisation and activation of the neurokinin 1 receptor in the enteric nervous system of the mouse distal colon

The substance P neurokinin 1 receptor (NK₁R) regulates motility, secretion, inflammation and pain in the intestine. The distribution of the NK₁R is a key determinant of the functional effects of substance P in the gut. Information regarding the distribution of NK₁R in subtypes of mouse enteric neurons is lacking and is the focus of the present study. NK₁R immunoreactivity (NK₁R-IR) is examined in whole-mount preparations of the mouse distal colon by indirect immunofluorescence and confocal microscopy. The distribution of NK₁R-IR within key functional neuronal subclasses was determined by using established neurochemical markers. NK₁R-IR was expressed by a subpopulation of myenteric and submucosal neurons; it was mainly detected in large multipolar myenteric neurons and was colocalized with calcitonin gene-related peptide, neurofilament M, choline acetyltransferase and calretinin. The remaining NK₁R-immunoreactive neurons were positive for nitric oxide synthase. NK₁R was expressed by most of the submucosal neurons and was exclusively co-expressed with vasoactive intestinal peptide, with no overlap with choline acetyltransferase. Treatment with substance P resulted in the concentration-dependent internalisation of NK₁R from the cell surface into endosome-like structures. Myenteric NK₁R was mainly expressed by intrinsic primary afferent neurons, with minor expression by descending interneurons and inhibitory motor neurons. Submucosal NK₁R was restricted to non-cholinergic secretomotor neurons. These findings highlight key differences in the neuronal distribution of NK₁R-IR between the mouse, rat and guinea-pig, with important implications for the functional role of NK₁R in regulating intestinal motility and secretion.     

Morphology of the pericellular capsule in articular cartilage revealed by hyaluronidase digestion

To allow a more valid comparison between our previous ultrastructural data and the immunolocalization of type IX and other minor collagen species in cryosectioned cartilage, we examined both normal and testicular hyaluronidase-digested canine tibial cartilage by electron microscopy. Removal of matrix proteoglycans caused the pericellular capsule to collapse against the cell surface, suggesting that its normal anatomical position is mediated by pericellular matrix hydration. Detailed examination of the pericellular capsule and pericellular channel revealed fine, faintly banded fibrils and an amorphous component somewhat similar in structure to basement membrane collagens. Matrix vesicles and the electron-dense material of the interterritorial matrix were only partially digested by hyaluronidase. We propose that the pericellular capsule is composed of a "felt-like" network of minor collagen species which act synergistically to maintain both the composition of the pericellular matrix and the integrity of the chondrocyte/pericellular matrix complex during compressive loading.     

Mesenchymal stem cells in autoimmune diseases: hype or hope?

Intervention with mesenchymal stem cells (MSCs) represents a promising therapeutic tool in treatment-refractory autoimmune diseases. A new report by Schurgers and colleagues in a previous issue of Arthritis Research & Therapy sheds novel mechanistic insight into the pathways employed by MSCs to suppress T-cell proliferation in vitro, but, at the same time, indicates that MSCs do not influence T-cell reactivity and the disease course in an in vivo arthritis model. Such discrepancies between the in vitro and in vivo effects of potent cellular immune modulators should spark further research and should be interpreted as a sign of caution for the in vitro design of MSC-derived interventions in the setting of human autoimmune diseases.     

Dynamics of microvascular oxygen pressure during rest-contraction transition in skeletal muscle of diabetic rats

Type I diabetes reduces dramatically the capacity of skeletal muscle to receive oxygen (QO(2)). In control (C; n = 6) and streptozotocin-induced diabetic (D: n = 6, plasma glucose = 25.3 +/- 3.9 mmol/l and C: 8.3 +/- 0.5 mmol/l) rats, phosphorescence quenching was used to test the hypothesis that, in D rats, the decline in microvascular PO(2) [Pm(O(2)), which reflects the dynamic balance between O(2) utilization (VO(2)) and QO(2)] of the spinotrapezius muscle after the onset of electrical stimulation (1 Hz) would be faster compared with that of C rats. Pm(O(2)) data were fit with a one or two exponential process (contingent on the presence of an undershoot) with independent time delays using least-squares regression analysis. In D rats, Pm(O(2)) at rest was lower (C: 31.2 +/- 3.2 mmHg; D: 24.3 +/- 1.3 mmHg, P < 0.05) and at the onset of contractions decreased after a shorter delay (C: 13.5 +/- 1.8 s; D: 7.6 +/- 2.1 s, P < 0.05) and with a reduced mean response time (C: 31.4 +/- 3.3 s; D: 23.9 +/- 3.1 s, P < 0.05). Pm(O(2)) exhibited a marked undershoot of the end-stimulation response in D muscles (D: 3.3 +/- 1.1 mmHg, P < 0.05), which was absent in C muscles. These results indicate an altered VO(2)-to-QO(2) matching across the rest-exercise transition in muscles of D rats.     

Change of speciation of Cu(II) in growth medium due to uptake of ammonia byPseudomonas testosteroni during growth

Growth ofPseudomonas testosteroni in a medium containing 1mm Cu(II) causes a color change from blue to green. The spectrum of the supernatant solution from the blue culture shows an absorption at 660 nm, identical to that of 1mm [Cu(II)] in the medium. The green supernatant solution shows a UV absorption, which tails into the visible and so is responsible for the green color, and ad-d absorption at 720 nm. The absorption at 660 nm for the blue supernatant solution is probably due to [Cu(NH₃)₃(H₂O)₃]²⁺. Growth of the organism causes loss of ammonia and a speciation change to [Cu(NH₃)₂(H₂O)₄]²⁺, with a shift in absorption maximum from 660 to 720 nm. These conclusions are based upon the spectra of known aquaammine complexes of Cu(II) and calculations of the speciation of Cu(II) before and after growth. Change in metal speciation owing to nutrient uptake by an organism does not appear to have been recognized previously.     

Persistence of cartilage proteoglycan and link protein during matrix-induced endochondral bone development: An immunofluorescent study

Monospecific antibodies to cartilage proteoglycan monomer and link protein were employed with immunofluorescence microscopy to determine the tissue distribution of these constituents during matrix-induced endochondral bone development. Subcutaneous implantation of demineralized diaphyseal bone matrix resulted in new endochondral bone formation. On Day 3, the implant consisted of mesenchymal tissue which did not contain any demonstrable cartilage-related proteoglycan or link protein. With the onset of early chondrogenesis on Day 5, cartilage proteoglycan monomer and link protein were first localized together in the cartilage matrix, particularly around chondrocytes in territorial sites. Progressively more staining around cells was observed at Days 7 and 9. On Day 9, when mineralization was first observed, there was no evidence of a net loss of these molecules prior to mineralization of the cartilage matrix. On Day 11 and thereafter, bone formation was observed by appositional growth on calcified cartilage spicules. Whereas the osteoblasts and bone matrix were devoid of any staining for cartilage proteoglycan and link components, the residual, partly mineralized cartilage spicules still reacted with antibodies to cartilage proteoglycan monomer and link protein in territorial sites, but in reduced amounts, indicating a loss of these molecules associated with a loss of hypertrophic chondrocytes. Since mineral prevented the access of Fab' antibody subunits, demineralization after fixation was routinely employed. The results reveal that cartilage proteoglycan monomer and link protein are present around chondrocytes in hyaline cartilage during the early stages of endochondral bone formation and that there is no net loss of these molecules prior to mineralization of this cartilage matrix as was previously thought.     

Activity of one of two engineered heterodimers of AhpF, the NADH:peroxiredoxin oxidoreductase from Salmonella typhimurium, reveals intrasubunit electron transfer between domains

AhpF, the flavoprotein reductase component of the Salmonella typhimurium alkyl hydroperoxide reductase system, catalyzes the reduction of an intersubunit disulfide bond in the peroxidatic active site of the system's other component, AhpC, a member of the peroxiredoxin family. Previous studies have shown that AhpF can be dissected into two functional units, a thioredoxin reductase-like C-terminus (containing FAD and a redox-active disulfide, Cys345-Cys348) and an N-terminal domain containing a second redox-active disulfide center (Cys129-Cys132). The role of the N-terminal domain as the direct reductant of AhpC, mediating electron transfer from the C-terminal redox centers of AhpF, has been firmly established by several approaches. Not known, however, was whether the transfer of electrons between the C-terminal and N-terminal disulfide centers occurred as an inter- or intrasubunit process in dimeric AhpF. Two heterodimeric AhpF species were therefore created in which one of the two pathways was completely disrupted while the other was left partially intact in each construct. Only the heterodimer containing one monomer of wild type AhpF and a monomer of mutated (and truncated) AhpF exhibited peroxidase activity with AhpC indicating that electron transfer between domains of AhpF is an intrasubunit process.     

Extent of invasion of Tasmanian native vegetation by the exotic bumblebee Bombus terrestris (Apoidea: Apidae)

Abstract Observations of the large earth bumblebee, Bombus terrestris (L.), in native vegetation were collated to determine the extent to which this exotic species has invaded Tasmanian native vegetation during the first 9 years after its introduction. The range of B. terrestris now encompasses all of Tasmania's major vegetation types, altitudes from sea level to 1260m a.s.L, and the entire breadth of annual precipitation in the state from more than 3200 mm to less than 600 mm. Observations of workers carrying pollen, together with the presence of large numbers of bumblebees at many localities across this range indicate that colonies are frequently established in native vegetation. Evidence that colonies are often successful was obtained from repeated observations of the species during more than 1 year at particular sites. Unequivocal evidence of colonies was obtained from six National Parks, including four of the five in the Tasmanian Wilderness World Heritage Area (WHA). Indeed, the species has been present in the WHA for at least as long as it has in the city of Hobart, where it was first recorded. In southwestern Tasmania, evidence of colonies was obtained up to 40km from gardens, 61 km from small towns and 93 km from large towns. Hence, contrary to previous suggestions, the species is established in the most remote parts of Tasmania and is not dependent on introduced garden plants. Given their strong record of invasion, it is likely that B. terrestris will form feral populations on the mainland of Australia and in many other parts of the world if introduced. Because of their likely negative impacts on native animals and plants, and potential to enhance seed production in weeds, the spread of bumblebees should be avoided.