Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

We have investigated the expression and function of the isoforms of laminin bearing the alpha(5) chain, i.e. laminin-10/11 in neonatal and adult human skin. By immunostaining human skin derived from a variety of anatomic sites, we found that the laminin-alpha(5) chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well-studied laminin-5 isoform did not change significantly with age, laminin-10/11 (a5 chain) appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-10/11 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. Importantly, in vitro cell adhesion assays demonstrated that laminin-10/11 is a potent adhesive substrate for both neonatal and adult keratinocytes and that this adhesion is mediated by the alpha(3)beta(1), and alpha(6)beta(4) integrins. Adhesion assays performed with fractionated basal keratinocytes showed that stem cells, transit amplifying cells and early differentiating cells all adhere to purified laminin-10/11 via these receptors. Further, laminin-10/11 provided a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type-18 transformed tumorigenic keratinocyte cell line in vitro. Finally, laminin-10/11 was shown to stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin-10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.

Clinicopathological significance of the 'keloid-like' collagen and myxoid stroma in advanced rectal cancer

Aim: To establish the histological categorization of fibrotic stroma which reflects the biological behaviour of advanced rectal cancer. Methods and results: Six hundred and twenty-seven surgically resected cases of advanced rectal carcinoma were examined. We histologically categorized fibrotic stroma in the invasive frontal region into three groups: type A, multiple fine and mature fibres were stratified into layers: type B, broad bands of eosinophilic hyalinized collagen ('keloid-like' collagen) were intermingled: type C, myxoid stroma. Type A stroma was observed in 63% of patients, type B stroma in 25%, type C stroma in 12%.. The incidence of type A stroma decreased in accordance with Dukes stage (98% in Dukes A: 73% in B: 41%, in C1: 29% in C2) and conversely, there was an increase of C type (0%, in Dukes A; 4%, in B: 20% in C1: 54% in C2). Stroma type had a significant correlation with long-term survival (80% of 5-year survival in type A stroma: 54% in type B: 26% in type C). Based on multivariate analysis. it was found that the stromal pattern had independent prognostic value, together with nodal involvement. growth pattern. and lymphocyte infiltration. Conclusions: Tumour fibrotic stroma may play an important role as a regulator of neoplastic behaviour. Pathological categorization of the fibrotic stroma is helpful for predicting the prognostic outcome of patients with rectal carcinoma.

A sponge allelochemical induces ascidian settlement but inhibits metamorphosis

Secondary metabolites synthesised by sessile invertebrates appear to play a role in creating and maintaining space on hard substrata by repelling competitors. In this study, we investigated the responses of the larvae of the ascidian Herdmania curvata to haliclonacyclamine A (HA), the major component of a suite of cytotoxic alkaloids extracted from the sponge Haliclona sp. 628. Both Haliclona sp. 628 and Herdmania curvata inhabit the crest and slope of Heron Island Reef. High rates of settlement were induced in competent H. curvata larvae by a range of concentrations of HA, all lower than that naturally occurring in the sponge. HA did not induce precompetent larvae to settle. Although early metamorphosis of HA-induced larvae was normal, larvae exposed to all but the lowest concentration of HA were developmentally arrested after completion of tail resorption, at about 4 h after the initiation of metamorphosis. These postlarvae underwent extensive cellular necrosis within 24 h. We also demonstrate that the addition of a transcriptional inhibitor, actinomycin D, to larvae also causes inhibition of metamorphosis after tail resorption is completed. Analyses of incorporation of radiolabelled nucleotides to measure levels of transcription during normal development and after the addition of the transcriptional inhibitor indicate that there is a significant burst of transcriptional activity just after tail resorption is completed. Despite inhibiting metamorphosis at the same stage as actinomycin D, HA increases initial rates of RNA synthesis after induction of metamorphosis in a manner similar to that observed in normal postlarvae until the onset of cellular necrosis. We conclude that HA initially induces H. curvata larvae to settle and progress through early metamorphosis possibly by engaging the same pathway as other artificial and environmental cues but subsequently inhibits completion of metamorphosis, resulting in death of the postlarvae. Since HA does not affect overall transcription rates, it appears to disrupt another important developmental process during early metamorphosis.

Excited to death: different ways to lose your neurones

The selective loss of neurones in a range of neurodegenerative diseases is widely thought to involve the process of excitotoxicity, in which glutamate-mediated neuronal killing is elaborated through the excessive stimulation of cell-surface receptors. Every such disease exhibits a distinct regional and subregional pattern of neuronal loss. so processes must be locally triggered to different extents to account for this. We have studied several mechanisms which could lead to excitotoxic glutamate pathophysiology and compared them in different diseases. Our data suggest that glutamate can reach toxic extracellular levels in Alzheimer disease by malfunctions in cellular transporters, and that the toxicity may be exacerbated by continued glutamate release from presynaptic neurones acting on hypersensitive postsynaptic receptors. Thus the excitotoxicity is direct. In contrast, alcoholic brain damage arises in regions where GABA-mediated inhibition is deficient, and fails properly to dampen trans-synaptic excitation, Thus the excitotoxicity is indirect. A variety of such mechanisms is possible, which may combine in different ways.

A new technique for the quantification of peripheral edema with application in both unilateral and bilateral cases

Current noninvasive techniques for the routine and frequent quantification of peripheral lymphedema in patients are total limb volume measurement (by water immersion or by circumferential measurements) and bioelectrical impedance analysis (BIA). However both of these techniques require standardizing the measurement using a contralateral measurement from the unaffected limb, Hence these techniques are essentially restricted to unilateral lymphedema. This paper describes the results from a preliminary study to investigate an alternative approach to the analysis of the data from multiple frequency BIA to produce an index of lymphedema without the need for normalization to another body segment. Twenty patients receiving surgical treatment for breast cancer were monitored prior to surgery and again after diagnosis with unilateral lymphedema. The data recorded were total limb volume, by circumferential measurements; and BIA measurements of both limbs. From these measurements total limb volumes and extracellular fluid volumes were calculated and expressed as ratios of the affected limb to that of the unaffected limb. An index of the ratio of the extracellular fluid volume to the intracellular fluid volume was determined. This ECW/ICW index was calculated for both the affected and unaffected limbs at both measurement times. Results confirmed that the established techniques of total limb volume and extracellular fluid volume normalized to the unaffected contralateral limb were accurate in the detection of lymphedema (p < 10(-6)). Comparison of the ECW/ICW index from the affected limb after diagnosis with that from the pre-surgery measurement revealed a significant (p< 10(-6)) and considerable (75%) increase. The results of this pilot study suggest that by using multiple frequency bioelectrical impedance analysis, an index of the ECW/ICW ratio can be obtained and this index appears to have an equal, or better, sensitivity, than the other techniques in detecting lymphedema. More importantly, this index does not require normalization to another body segment and can be used to detect all types of peripheral edema including both unilateral and bilateral lymphedema.

The mitochondrial 12S gene is a suitable marker of populations of Sarcoptes scabiei from wombats, dogs and humans in Australia

We sequenced part of the mitochondrial 12S ribosomal RNA gene of 23 specimens of Sarcoptes scabiei from eight wombats, one dog and three humans. Twelve of the 326 nucleotide positions varied among these mites and there were nine haplotypes (sequences) that differed by 1-8 nucleotides. Phylogenetic analyses indicated that these mites were from two lineages: (1) mites from wombats from Victoria, Australia, and mites from the humans and dog from the Northern Territory, Australia (haplotypes 1-4, 9); and (2) mites from the humans and dog from the Northern Territory (haplotypes 5-8). Mites from the three different hosts (wombats, a dog and humans) had not diverged phylogenetically; rather, these mites had similar 12S sequences. Thus, we conclude that these mites from wombats, humans and a dog are closely related, and that they diverged from a common ancestor relatively recently. This conclusion is consistent with the argument that people and/or their dogs introduced to Australia the S. scabiei mites that infect wombats Australia. So, S. scabiei, which has been blamed for the extinction of populations of wombats in Australia, may be a parasitic mite that was introduced to Australia with people and/or their dogs. These data show that the mitochondrial 12S rRNA gene may be a suitable population marker of S. scabiei from wombats, dogs and humans in Australia.

Copper transfer from the Cu(I) chaperone, CopZ, to the repressor, Zn(II)CopY: Metal coordination environments and protein interactions

Extracellular copper regulates the DNA binding activity of the CopY repressor of Enterococcus hirae and thereby controls expression of the copper homeostatic genes encoded by the cop operon. CopY has a CxCxxxxCxC metal binding motif. CopZ, a copper chaperone belonging to a family of metallochaperones characterized by a MxCxxC metal binding motif, transfers copper to CopY. The copper binding stoichiometries of CopZ and CopY were determined by in vitro metal reconstitutions. The stoichiometries were found to be one copper(l) per CopZ and two copper(l) per CopY monomer. X-ray absorption studies suggested a mixture of two- and three-coordinate copper in Cu(1)CopZ, but a purely three-coordinate copper coordination with a Cu-Cu interaction for Cu(1)(2)CopY. The latter coordination is consistent with the formation of a compact binuclear Cu(l)-thiolate core in the CxCxxxxCxC binding motif of CopY. Displacement of zinc, by copper. from CopY was monitored with 2,4-pyridylazoresorcinol. Two copper(l) ions were required to release the single zinc(II) ion bound per CopY monomer. The specificity of copper transfer between CopZ and CopY was dependent on electrostatic interactions. Relative copper binding affinities of the proteins were investigated using the chelator, diethyldithiocarbamic acid (DDC). These data suggest that CopY has a higher affinity for copper than CopZ. However, this affinity difference is not the sole factor in the copper exchange: a charge-based interaction between the two proteins is required for the transfer reaction to proceed. Gain-of-function mutation of a CopZ homologue demonstrated the necessity of four lysine residues on the chaperone for the interaction with CopY. Taken together, these results suggest a mechanism for copper exchange between CopZ and CopY.

Axon navigation in the mammalian primary olfactory pathway: Where to next?

The process of establishing long-range neuronal connections can be divided into at least three discrete steps. First, axons need to be stimulated to grow and this growth must be towards appropriate targets. Second, after arriving at their target, axons need to be directed to their topographically appropriate position and in some cases, such as in cortical structures, they must grow radially to reach the correct laminar layer Third, axons then arborize and form synaptic connections with only a defined subpopulation of potential post-synaptic partners. Attempts to understand these mechanisms in the visual system have been ongoing since pioneer studies in the 1940s highlighted the specificity of neuronal connections in the retino-tectal pathway. These classical systems-based approaches culminated in the 1990s with the discovery that Eph-ephrin repulsive interactions were involved in topographical mapping. In marked contrast, it was the cloning of the odorant receptor family that quickly led to a better understanding of axon targeting in the olfactory system. The last 10 years have seen the olfactory pathway rise in prominence as a model system for axon guidance. Once considered to be experimentally intractable, it is now providing a wealth of information on all aspects of axon guidance and targeting with implications not only for our understanding of these mechanisms in the olfactory system but also in other regions of the nervous system.

Macrophages, myofibroblasts and neointimal hyperplasia after coronary artery injury and repair

Macrophages participate in the restenosis process through the release of cytokines, metalloproteinases and growth factors. Studies of peritoneal granulation tissue suggest that macrophages may be precursors of myofibroblasts. This study examined the contribution of monocyte/macrophage lineage cells to neointimal cellular mass in a porcine model of thermal vascular injury. Thermal coronary artery injury caused medial smooth muscle cell necrosis and transformation of the media into an extracellular matrix barrier. The neointimal hyperplasia that developed over the injury sites was evaluated by light microscopy, electron microscopy and immunohistochemistry. At day 3, blood monocytes were adhered to the vessel wall and infiltrated the fibrotic media. At day 14, 42 +/- 3.9% of neointimal cells had a monocytic nuclear morphology and expressed macrophage-specific antigen SWC3 (identified by monoclonal antibody DH59B). Moreover, 9.2+/-1.8% of neointimal cells co-expressed SWC3 and alpha-smooth muscle actin and had ultrastructural characteristics intermediate between macrophages and myofibroblasts. At day 28, 10.5 +/- 3.5%, of cells expressed SWC3 and 5.2+/-1.8% of cells co-expressed SWC3 and alpha-smooth muscle actin. This study indicates that hematopoietic cells of monocyte/macrophage lineage abundantly populate the neointima in the process of lesion formation and may be precursors of neointimal myofibroblasts after thermal vascular injury. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Bracken (Pteridium aquilinum)-induced DNA adducts in mouse tissues are different from the adduct induced by the activated form of the bracken carcinogen ptaquiloside

Following treatment with bracken fern (Pteridium aquilinum) extract and bracken spores a number of DNA adducts were detected by P-32-postlabeling. Three of these adducts have been described previously (Povey et al., Br. J. Cancer (1996) 74, 1342-1348) and in this study, using a slightly different protocol, four new adducts, with higher chromatographic mobility, were detected at levels ranging from 50 to 230% of those previously described, When DNA was treated in vitro with activated ptaquiloside (APT) and analysed by butanol extraction or nuclease P1 treatment, only one adduct was detected by P-32-postlabeling, This adduct was not present in the DNA from mice treated with bracken fern or spores, suggesting either that bracken contains genotoxins other than ptaquiloside or that the metabolism of ptaquiloside produces genotoxins not reflected by activated ptaquiloside. However, as the ATP-derived adduct has been detected previously in ileal DNA of bracken-fed calves, species-specific differences in the metabolism of bracken genotoxins may exist, thereby leading to differences in their biological outcomes. (C) 2001 Academic Press.

Neural control of the heart: developmental changes in ionic conductances in mammalian intrinsic cardiac neurons

The expression and properties of ionic channels were investigated in dissociated neurons from neonatal and adult rat intracardiac ganglia. Changes in the hyperpolarization-activated and ATP-sensitive K+ conductances during postnatal development and their role in neuronal excitability were examined. The hyperpolarization-activated nonselective cation current, I-h, was observed in all neurons studied and displayed slow time-dependent rectification. An inwardly rectifying K+ current, I-K(I), was present in a population of neurons from adult but not neonatal rats and was sensitive to block by extracellular Ba2+. Using the perforated-patch recording configuration, an ATP-sensitive K+ (K-ATP) conductance was identified in greater than or equal to 50% of intracardiac neurons from adult rats. Levcromakalim evoked membrane hyperpolarization, which was inhibited by the sulphonylurea drugs. glibenclamide and tolbutamide. Exposure to hypoxic conditions also activated a membrane current similar to that induced by levcromakalim and was inhibited by glibenclamide. Changes in the complement of ion channels during postnatal development may underlie observed differences in the function of intracardiac ganglion neurons during maturation. Furthermore, activation of hyperpolarization-activated and KATP channels in mammalian intracardiac neurons may play a role in neural regulation of the mature heart and cardiac function during ischaemia-reperfusion. (C) 2002 Elsevier Science B.V All rights reserved.

Basal nonselective cation permeability in rat cardiac microvascular endothelial cells

The presence of a basal nonselective cation permeability was mainly investigated in primary cultures of rat cardiac microvascular endothelial cells (CMEC) by applying both the patch-clamp technique and Fura-2 microfluorimetry. With low EGTA in the pipette solution, the resting membrane potential of CMEC was -21.2 +/- 1.1 mV, and a Ca2+-activated Cl- conductance was present. When the intracellular Ca2+ was buffered with high EGTA, the membrane potential decreased to 5.5 +/- 1.2 mV. In this condition, full or partial substitution of external Na+ by NMDG(+) proportionally reduced the inward component of the basal I-V relationship. This current was dependent on extracellular monovalent cations with a permeability sequence of K+ > Cs+ > Na+ > Li+ and was inhibited by Ca2+, La3+, Gd3+, and amiloride. The K+/Na+ permeability ratio, determined using the Goldman-Hodgkin-Katz equation, was 2.01. The outward component of the basal I-V relationship was reduced when intracellular K+ was replaced by NMDG(+), but was not sensitive to substitution by Cs+. Finally, microfluorimetric experiments indicated the existence of a basal Ca2+ entry pathway, inhibited by La3+ and Gd3+. The basal nonselective cation permeability in CMEC could be involved both in the control of myocardial ionic homeostasis, according to the model of the blood-heart barrier, and in the modulation of Ca2+ -dependent processes. (C) 2002 Elsevier Science (USA).

Mitochondrial genomes of parasitic flatworms

Complete or near-complete mitochondrial genomes are now available for 11 species or strains of parasitic flatworms belonging to the Trematoda and the Cestoda. The organization of these genomes is not strikingly different from those of other eumetazoans, although one gene (atp8) commonly found in other phyla is absent from flatworms. The gene order in most flatworms has similarities to those seen in higher protostomes such as annelids. However, the gene order has been drastically altered in Schistosoma mansoni, which obscures this possible relationship. Among the sequenced taxa, base composition varies considerably, creating potential difficulties for phylogeny reconstruction. Long non-coding regions are present in all taxa, but these vary in length from only a few hundred to similar to10 000 nucleotides. Among Schistosoma spp., the long non-coding regions are rich in repeats and length variation among individuals is known. Data from mitochondrial genomes are valuable for studies on species identification, phylogenies and biogeography.

Conserved modularity and potential for alternate splicing in mouse and human Slit genes

The vertebrate Slit gene family currently consists of three members;Slit1,Slit2 and Slit3. Each gene encodes a protein containing multiple epidermal growth factor and leucine rich repeat motifs, which are likely to have importance in cell-cell interactions. In this study, we sought to fully define and characterise the vertebrate Slit gene family. Using long distance PCR coupled with in silico mapping, we determined the genomic structure of all three Slit genes in mouse and man. Analysis of EST and genomic databases revealed no evidence of further Slit family members in either organism. All three Slit genes were encoded by 36 (Slit3) or 37 (Slit1 and Slit2) exons covering at least 143 kb or 183 kb of mouse or human genomic DNA respectively. Two additional potential leucine-rich repeat encoding exons were identified within intron 12 of Slit2. These could be inserted in frame, suggesting that alternate splicing may occur in Slit2 A search for STS sequences within human Slit3 anchored this gene to D5S2075 at the 5' end (exon 4) and SGC32449 within the 3' UTR, suggesting that Slit3 may cover greater than 693 kb. The genomic structure of all Slit genes demonstrated considerable modularity in the placement of exon-intron boundaries such that individual leucine-rich repeat motifs were encoded by individual 72 by exons. This further implies the potential generation of multiple Slit protein isoforms varying in their number of repeat units. cDNA library screening and EST database searching verified that such alternate splicing does occur.

Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ

Neuronal and glial high-affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non-metabolisable glutamate transporter substrate, D-aspartate. In the rat retina, pan-isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Muller cells. This effect was mimicked by rottlerin but not by Go6976, suggesting the involvement of the PKCdelta isoform, but not PKCalpha, beta or gamma. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCdelta selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform-specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.