Relationship between anisotropic diffusion properties and tissue morphology in porcine TMJ disc.

OBJECTIVE: To investigate the relationship between anisotropic solute diffusion properties and tissue morphology in porcine temporomandibular joint (TMJ) discs. DESIGN: TMJ discs from eleven pigs aged 6-8 months were divided into five regions: anterior, intermediate, posterior, lateral, and medial. The transport properties and tissue morphology were investigated in three orthogonal orientations: anteroposterior (AP), mediolateral (ML), and superoinferior (SI). The anisotropic diffusivity of fluorescein (332 Da) in the right discs was determined by the fluorescence recovery after photobleaching (FRAP) protocols. The tissue morphology in the left discs was quantified by scanning electron microscopy. RESULTS: The diffusivities of fluorescein in the TMJ disc were significantly anisotropic, except for the anterior region. In the medial, intermediate, and lateral regions, the diffusion along the fiber orientation (i.e., AP direction) was significantly faster than the diffusion in ML and SI directions. In the posterior region, the diffusion along the fiber orientation (i.e., ML direction) was significantly faster than the diffusion in AP and SI directions. The diffusion in the anterior region was mostly isotropic with the lowest degree of diffusion anisotropy, as well as collagen fiber alignment, likely due to the multi-directional fiber arrangement. The anterior region had the highest mean diffusivity [65.6 (49.3-81.8) µm(2)/s] in the disc, likely due to its high water content. The overall average diffusivity of fluorescein across the TMJ disc was 57.0 (43.0-71.0) µm(2)/s. CONCLUSIONS: The solute diffusion in porcine TMJ discs was strongly anisotropic and inhomogeneous, which associated with tissue structure (i.e., collagen fiber alignment) and composition (e.g., water content).

Hepatic transcription of the acute-phase alpha 1-inhibitor III gene is controlled by a novel combination of cis-acting regulatory elements.

mRNA coding for the abundant broad-range plasma proteinase inhibitor alpha 1-inhibitor III (alpha 1I3) was detected only in rat liver, while mRNA for the related proteins alpha 1-macroglobulin and alpha 2-macroglobulin was also found in a variety of nonhepatic tissues. cis-Acting control elements necessary for the hepatic transcription of alpha 1I3 were mapped by transfection and expression studies of control-region constructs in cultured hepatic and nonhepatic cells. The promoter-proximal 5'-flanking region contained four control elements, I to IV, located between -109 and -196 base pairs upstream of the transcriptional start site relevant for the hepatic transcription of this gene. Elements II and III were essential, and I and IV exerted strong modulatory effects. Elements I to III acted as positive regulators, and IV acted as a negative element. Element II contained the sequence TGGCA and is probably a binding site for a nuclear factor related to the known transcription factor NF1. The other three elements did not resemble consensus binding sites for known transcription factors that are involved in the hepatocyte-specific transcription of other well-characterized plasma protein genes, such as the prototype factor HNF-1. Thus, the alpha 1I3 gene achieves its highly hepatocyte-specific transcription through a novel combination of cis-acting control elements and trans-acting factors.

Primary mesenchymal cells isolated from SPARC-null mice exhibit altered morphology and rates of proliferation.

SPARC (secreted protein acidic and rich in cysteine)/BM 40/osteonectin is a matricellular protein shown to function as a counteradhesive factor that induces cell rounding and as an inhibitor of cell proliferation. These activities have been defined in cell culture, in which interpretation has been complicated by the presence of endogenous SPARC. We therefore sought to determine whether cell shape and proliferation would be affected by the absence of SPARC. Mesangial cells, fibroblasts, and aortic smooth muscle cells were isolated from SPARC-null and age-matched, wild-type mice. In contrast to wild-type cells, SPARC-null mesangial cells exhibited a flat morphology and an altered actin cytoskeleton. In addition, vinculin-containing focal adhesions were distributed over the center of SPARC-null cells, whereas in wild-type cells, the number of focal adhesions was reduced, and these structures were restricted largely to the cell periphery. Although the SPARC-null fibroblasts did not display overt differences in cell morphology, the cells responded to exogenous recombinant SPARC by rounding up in a manner similar to that of wild-type fibroblasts. Thus, the expression of endogenous SPARC is not required for the response of cells to SPARC. Additionally, SPARC-null mesangial cells, fibroblasts, and smooth muscle cells proliferated faster than their respective wild-type counterparts. Null cells also showed a greater sensitivity to the inhibition of cell cycle progression by the addition of recombinant SPARC. The increased proliferation rate of SPARC-null cells appeared to be mediated, at least in part, by an increase in the cell cycle regulatory protein cyclin A. We conclude that the expression of SPARC influences the cellular architecture of mesangial cells and that SPARC plays a role in the regulation of cell cycle in mesangial cells, fibroblasts, and smooth muscle cells.

The containment of toxic wastes: II. Metal movement in leachate and drainage at Parc lead-zinc mine, North Wales.

Nant Gwydyr, a tributary of the River Conwy in North Wales, has been affected by metal wastes, from a lead and zinc mine, Parc Mine, through contaminated mine drainage waters and episodal erosion of an unstable tailings heap. From 1954 when the mining operation was discontinued to 1978 when a reclamation programme aimed to stabilise the tailings was accomplished, 13 000 tonnes of metalliferous spoil, containing 43 tonne Pb, 104 tonne Zn, and 1 tonne Cd was eroded from the main tailings dam. Dispersal and redeposition during flood events caused extensive pollution of the agricultural land of the flood plain. Analysis of the present water quality of the Nant Gwydyr, 14 years after the stabilisation work, shows that although there has been a marked improvement and no particulate matter is released, the Nant Gwydyr is still a polluted stream. Under normal discharge conditions, it contributes approximately 1 tonne of Zn, 0.2 tonne of Pb and 0.05 tonne of Cd per year to the River Conwy. Most of this originates from water issuing from the mine adit which has been impossible to control. There is still, however, a major contribution by the leachate from the tailings heap, because the stabilisation method used does not prevent this. The control of pollution in mine drainage is discussed.

The containment of toxic wastes: I. Long term metal movement in soils over a covered metalliferous waste heap at Parc lead-zinc mine, North Wales.

In order to stabilise and contain a toxic metalliferous waste heap at Parc Mine, North Wales, it was covered with 30-40 cm layer of quarry waste in 1977-1978, and sown with a grass/clover seed mixture. This study has examined subsequent metal movement in the cover material and its effect on vegetation. The results, especially when compared with previous observations, give no evidence of upward migration of metals by capillarity in the cover material. Sideways movement of leachate, however, appears to be carrying the metals into the cover material on the sloping sides, giving rise to increasing concentrations of heavy metals in the vegetation and dieback in some places. Root growth on the flat top of the heap is greater than on the slope, but the roots have not penetrated the waste and the contents of Pb, Zn and Cd in surface vegetation remain low. Surface covering of toxic waste with coarse materials restricting capillary rise is therefore a valid reclamation technique so long as lateral movement of toxic leachate can be controlled.

LPS induces greater bone and PDL loss in SPARC-null mice.

Individuals with periodontal disease have increased risk of tooth loss, particularly in cases with associated loss of alveolar bone and periodontal ligament (PDL). Current treatments do not predictably regenerate damaged PDL. Collagen I is the primary component of bone and PDL extracellular matrix. SPARC/Osteonectin (SP/ON) is implicated in the regulation of collagen content in healthy PDL. In this study, periodontal disease was induced by injections of lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans in wild-type (WT) and SP/ON-null C57/Bl6 mice. A 20-µg quantity of LPS was injected between the first and second molars 3 times a week for 4 weeks, whereas PBS control was injected into the contralateral maxilla. LPS injection resulted in a significant decrease in bone volume fraction in both genotypes; however, significantly greater bone loss was detected in SP/ON-null maxilla. SP/ON-null PDL exhibited more extensive degradation of connective tissue in the gingival tissues. Although total cell numbers in the PDL of SP/ON-null were not different from those in WT, the inflammatory infiltrate was reduced in SP/ON-null PDL. Histology of collagen fibers revealed marked reductions in collagen volume fraction and in thick collagen volume fraction in the PDL of SP/ON-null mice. SP/ON protects collagen content in PDL and in alveolar bone in experimental periodontal disease.

Enhanced angiogenesis characteristic of SPARC-null mice disappears with age.

The impairment of angiogenesis in aging has been attributed, in part, to alterations in proteins associated with the extracellular matrix (ECM). SPARC (secreted protein acidic and rich in cysteine/osteonectin/BM-40) is a matricellular protein that regulates endothelial cell function as well as cell-ECM interactions. We have previously shown that angiogenesis, as reflected by fibrovascular invasion into subcutaneously implanted polyvinyl alcohol (PVA) sponges, is increased in SPARC-null mice (6-9 months of age) relative to their wild-type (WT) counterparts. In this study, we define the influence of aging on (a) the expression of SPARC and (b) fibrovascular invasion into sponge implants in SPARC-null and WT mice. The expression of SPARC in fibroblasts and endothelial cells derived from young donors (humans mean age less than 30 years and mice 4-6 months of age) and old donors (humans mean age over 65 years and mice 22-27 months of age) decreased 1.6 to 2.3-fold with age. Analysis of fibrovascular invasion into sponges implanted into old (22-27 months) SPARC-null and WT mice showed no differences in percent area of invasion or collagenous ECM. Moreover, sponges from old SPARC-null and WT mice contained similar levels of VEGF that were significantly lower than those from young (4-6 months) mice. In contrast to fibroblasts from young SPARC-null mice, dermal fibroblasts from old SPARC-null mice did not migrate farther, proliferate faster, or produce greater amounts of VEGF relative to their old WT counterparts. However, when stimulated with TGF-beta1, primary cells isolated from the sponge implants, and dermal fibroblasts from both old SPARC-null and WT mice, showed marked increases in VEGF secretion. These data indicate that aging results in a loss of enhanced angiogenesis in SPARC-null mice, as a result of the detrimental impact of age on cellular functions, collagen deposition, and VEGF synthesis. However, the influence of aging on these processes may be reversed, in part, by growth factor stimulation.

Adaptation of plants to soils containing toxic metals--a test for conceit.

Darwin, and many biologists afterwards, have seen few, if any, limits to the processes of adaptation by evolutionary change. Perhaps we have been conceited. A study of heavy-metal tolerance, and other conditions to which evolutionary adaptation has occurred, should overwhelm us with evidence for limits to the evolutionary process and limits to the adaptation it achieves. These limits clearly arise from restrictions in the supply of genetic variability. Nearly all species are in a condition of genostasis, in which there is a lack of appropriate variability for further evolutionary change. It is the molecular biologist who, by understanding the architecture of genes, will ultimately be able to explain what failures and limitations in genetic architecture at the molecular level cause the limits to adaptation itself.

The Croonian Lecture, 1991. Genostasis and the limits to evolution.

The Darwinian explanation for evolution is that it is the outcome of the interaction between genetic variation and natural selection. There is now good evidence for both the existence of genetic variation and the occurrence of natural selection, the latter potentially at high intensities. The outcome should be rapid evolutionary change; yet in practice very little change is found. Most species are very stable, and in situations where evolution is observed in one species often none is found in others despite equivalent opportunity. Evolutionary failure is commonplace. Despite the occurrence of high levels of protein polymorphism, there is good evidence that the supply of variation making a major contribution to fitness is very limited. As a result it is argued that lack of evolution in most species may be due more to lack of appropriate variability than to other causes: a condition for which the term 'genostasis' is proposed. In those situations where appropriate genetic variation is available for one reason or another, evolution is found to be very rapid. There are good theoretical and practical reasons for more attention being paid to the mechanisms of supply of new variation and to those situations where evolution appears not be taking place.