Polarisation-sensitive optical coherence tomography measurement of retardance in fibrosis, a non-invasive biomarker in patients with systemic sclerosis.

Polarisation-sensitive optical coherence tomography (PS-OCT) offers a novel, non-invasive method of assessing skin fibrosis in the multisystem disease systemic sclerosis (SSc) by measuring collagen retardance. This study aimed to assess retardance as a biomarker in SSc. Thirty-one patients with SSc and 27 healthy controls (HC) underwent PS-OCT imaging. 'Skin score' was assessed by clinical palpation (0-3 scale). A subset of ten patients and ten age/sex-matched HC had a biopsy and longitudinal imaging. Histological assessment included quantification of epidermal thickness, collagen content (to assess fibrosis) and matrix metalloproteinase (MMP) activity (in situ zymography). PS-OCT images were assessed for epidermal thickness (structure) and fibrosis (retardance). Positive correlation was observed between epidermal thickness as measured by histology and structural PS-OCT (r = 0.79; p < 0.001). Retardance was: HC mean 0.21 (SD 0.21) radian/pixel; SSc skin score 0, 0.30 (0.19); skin score 1, 0.11 (0.16); skin score 2, 0.06 (0.12); skin score 3, 0.36 (0.35). Longitudinal retardance decreased at one-week across groups, increasing at one-month for HC/skin score 0-1; HC biopsy site retardance suggests scarring is akin to fibrosis. Relationships identified between retardance with both biopsy and skin score data indicate that retardance warrants further investigation as a suitable biomarker for SSc-related fibrosis.

Simulating nailfold capillaroscopy sequences to evaluate algorithms for blood flow estimation.

The effects of systemic sclerosis (SSc)--a disease of the connective tissue causing blood flow problems that can require amputation of the fingers--can be observed indirectly by imaging the capillaries at the nailfold, though taking quantitative measures such as blood flow to diagnose the disease and monitor its progression is not easy. Optical flow algorithms may be applied, though without ground truth (i.e. known blood flow) it is hard to evaluate their accuracy. We propose an image model that generates realistic capillaroscopy videos with known flow, and use this model to quantify the effect of flow rate, cell density and contrast (among others) on estimated flow. This resource will help researchers to design systems that are robust under real-world conditions.

Comparative sequence analysis of the human and pufferfish Huntington's disease genes.

The Huntington's disease (HD) gene encodes a novel protein with as yet no known function. In order to identify the functionally important domains of this protein, we have cloned and sequenced the homologue of the HD gene in the pufferfish, Fugu rubripes. The Fugu HD gene spans only 23 kb of genomic DNA, compared to the 170 kb human gene, and yet all 67 exons are conserved. The first coding exon, the site of the disease-causing triplet repeat, is highly conserved. However, the glutamine repeat in Fugu consists of just four residues. We also show that gene order may be conserved over longer stretches of the two genomes. Our work describes a detailed example of sequence comparison between human and Fugu, and illustrates the power of the pufferfish genome as a model system in the analysis of human genes.

2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans.

As part of our effort to sequence the 100-megabase (Mb) genome of the nematode Caenorhabditis elegans, we have completed the nucleotide sequence of a contiguous 2,181,032 base pairs in the central gene cluster of chromosome III. Analysis of the finished sequence has indicated an average density of about one gene per five kilobases; comparison with the public sequence databases reveals similarities to previously known genes for about one gene in three. In addition, the genomic sequence contains several intriguing features, including putative gene duplications and a variety of other repeats with potential evolutionary implications.

Diffusible signal molecules controlling cell differentiation and patterning in Dictyostelium.

Slime moulds, such as Dictyostelium discoideum, have biochemical, physiological and probably developmental features in common with both plants and animals. During development separate Dictyostelium amoebae first aggregate into collecting centers to form small multicellular organisms which, in their slug form, can migrate over the substratum toward light. Eventually a slug culminates to form a fruiting body consisting of a cellular stalk supporting a mass of spores. Development is highly regulative, indicating that it is controlled by signalling between the cells. A number of diffusible signal molecules have been discovered, including cyclic AMP, the chemoattractant in aggregation, and DIF-1, a novel chlorinated phenyl alkanone, which acts as a specific inducer of stalk cell differentiation. The migrating slug contains three types of precursor cell: prespore, prestalk A and prestalk B cells. Differentiation of these cells from uncommitted amoebae can be brought about in cell cultures by cyclic AMP and DIF-1 acting in combination: cyclic AMP alone favours prespore, DIF-1 alone favours prestalk B, cyclic AMP and DIF-1 together favour prestalk A cell differentiation. There is evidence suggesting that these signals act in the same way in the intact aggregate. A cytoplasmic DIF-1 binding protein has been discovered, whose level increases as cells become sensitive to DIF-1 and which binds DIF-1 with an affinity and specificity suggestive of a receptor. At the same time, cells are able to inactivate DIF-1 by a metabolic pathway involving at least 12 metabolites. Metabolism may also serve to produce gradients of DIF-1 in the aggregate or other signal molecules from DIF-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Combinatorial control of cell differentiation by cAMP and DIF-1 during development of Dictyostelium discoideum.

At least three distinct types of cell arise from a population of similar amoebae during Dictyostelium development: prespore, prestalk A and prestalk B cells. We report evidence suggesting that this cellular diversification can be brought about by the combinatorial action of two diffusible signals, cAMP and DIF-1. Cells at different stages of normal development were transferred to shaken suspension, challenged with various combinations of signal molecules and the expression of cell-type-specific mRNA markers measured 1-2 h later. pDd63, pDd56 and D19 mRNAs were used for prestalk A, prestalk B and prespore cells respectively. We find the following results. (1) Cells first become responsive to DIF-1 for prestalk A differentiation and to cAMP for prespore differentiation at the end of aggregation, about 2 h before these cell types normally appear. (2) At the first finger stage of development, when the rate of accumulation of the markers is maximal, the expression of each is favoured by a unique combination of effectors: prespore differentiation is stimulated by cAMP and inhibited by DIF-1; prestalk A differentiation is stimulated by both cAMP and DIF-1 and prestalk B differentiation is stimulated by DIF-1 and inhibited by cAMP. (3) Half-maximal effects are produced by 10-70 nM DIF-1, which is in the physiological range. (4) Ammonia and adenosine, which can affect cell differentiation in other circumstances, have no significant pathway-specific effect in our conditions. These results suggest that cell differentiation could be brought about in normal development by the localized action of cAMP and DIF-1.

Morphogen hunting in Dictyostelium.

A highly regulative pattern of prestalk and prespore tissue is formed during Dictyostelium development, starting from separate amoebae. Potential morphogens controlling this process have been hunted biochemically, using bioassays to monitor activity. All those discovered to date are low MW diffusible compounds: cAMP, adenosine, NH3 and DIFs 1-3. The DIFs are assayed by their ability to induce isolated amoebae to differentiate into stalk cells and have been identified as a family of chlorinated phenyl alkanones. The diversification of amoebae into prestalk and prespore cells seems to be brought about by cAMP and DIF-1. cAMP is necessary for both pathways of differentiation but DIF-1 specifically induces the differentiation of prestalk cells while suppressing that of prespores. When DIF-1 is added to intact slugs, it causes a substantial enlargement of the prestalk tissue at physiological concentrations in the time previously shown to be required for pattern regulation. DIF-1 is a dynamic molecule and we have found that it is metabolized along a pathway involving at least 8 compounds. Metabolism is developmentally regulated and may be important in producing DIF gradients or other effector molecules from DIF. Although we almost certainly have some of the central actors, it is difficult to formulate a satisfactory theory of pattern formation in Dictyostelium at the moment. We suspect that at least one important actor is missing.

Cyclic AMP is an inhibitor of stalk cell differentiation in Dictyostelium discoideum.

Cyclic AMP and DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) together induce stalk cell differentiation in vitro in Dictyostelium discoideum strain V12M2. The induction can proceed in two stages: in the first, cyclic AMP brings cells to a DIF-responsive state; in the second, DIF-1 alone can induce stalk cell formation. We report here that during the DIF-1-dependent stage, cyclic AMP is a potent inhibitor of stalk cell differentiation. Addition of cyclic AMP at this stage to V12M2 cells appreciably delays, but does not prevent, stalk cell formation. In contrast, stalk cell differentiation in the more common strain NC4 is completely suppressed by the continued presence of cyclic AMP. This fact explains earlier failures to induce stalk cells in vitro in NC4. We now consistently obtain efficient stalk cell induction in NC4 by removing cyclic AMP in the DIF-1-dependent stage. Cyclic AMP also inhibits the production of a stalk-specific protein (ST310) in both NC4 and a V12M2 derivative. Adenosine, a known antagonist of cyclic AMP action, does not relieve this inhibition by cyclic AMP and does not itself promote stalk cell formation. Finally, stalk cell differentiation of NC4 cells at low density appears to require factors in addition to cyclic AMP and DIF-1, but their nature is not yet known. The inhibition of stalk cell differentiation by cyclic AMP may be important in establishing the prestalk/prespore pattern during normal development, and in preventing the maturation of prestalk into stalk cells until culmination.

Signals controlling cell differentiation and pattern formation in Dictyostelium.

The major inducers of cell differentiation in Dictyostelium appear to be cyclic AMP and DIF-1. Recently we have chemically identified DIF-1, together with the closely related DIF-2 and -3. They represent a new chemical class of potent effector molecules, based on a phenyl alkanone with chloro, hydroxy, and methoxy substitution of the benzene ring. Previous work has shown that DIF-1 can induce prestalk-specific gene expression within 15 min, whereas it suppresses prespore differentiation. Hence, DIF-1 can control the choice of pathway of cell differentiation in Dictyostelium and is therefore likely to be involved in establishing the prestalk/prespore pattern in the aggregate. In support of this, we show that DIF treatment of slugs results in an enlarged prestalk zone. Cyclic AMP seems less likely to have such a pathway-specific role, but later in development it becomes inhibitory to stalk cell differentiation. This inhibition may be important in suppressing terminal stalk cell differentiation until culmination. Spore differentiation can be induced efficiently by high levels of Br-cyclic AMP, a permeant analogue of cyclic AMP. In this, it phenocopies certain spore-maturation mutants, and we propose that during normal development spore differentiation is triggered by an elevation in intracellular cyclic AMP levels. How this elevation in cyclic AMP levels is brought about is not known. The experiments with Br-cyclic AMP also provide the first direct evidence that elevated levels of intracellular cyclic AMP induce differentiation in Dictyostelium.

Two distinct classes of prestalk-enriched mRNA sequences in Dictyostelium discoideum.

We have isolated cDNA clones derived from three mRNA sequences which are inducible by DIF, the putative stalk-specific morphogen of Dictyostelium. The three mRNA sequences are selectively expressed in cells on the stalk cell pathway of differentiation and we have compared them with previously characterized prestalk-enriched mRNA sequences. We find these latter sequences are expressed without a dependence on DIF, are much less highly enriched in prestalk over prespore cells and are expressed earlier during development than the DIF-inducible mRNA sequences. We propose two distinct mechanisms whereby a mRNA may become enriched in prestalk cells. An apparently small number of genes, represented by those we have isolated, is inducible by DIF and accumulates only in prestalk cells. We suggest that a second class of prestalk-enriched mRNA sequences are induced by cAMP to accumulate in all cells during aggregation and then become enriched in prestalk cells by selective loss from prespore cells.

Direct induction of Dictyostelium prestalk gene expression by DIF provides evidence that DIF is a morphogen.

We have isolated a gene that is very rapidly induced at the transcriptional level by DIF--a low molecular weight, diffusible factor necessary for stalk cell differentiation in Dictyostelium cells developing in vitro. The gene encodes a protein containing an N-terminal signal peptide preceding approximately 70 tandem repeats of a highly conserved 24 amino acid sequence with a high cysteine content. These features suggest it is an extracellular structural protein. During normal development, the gene is maximally expressed in the slug, in which the mRNA is very highly enriched in prestalk over prespore cells. The gene is not detectably expressed until the tipped aggregate stage, several hours later than prespore genes, suggesting that prespore cell differentiation precedes prestalk cell differentiation. The demonstration that DIF induces a gene normally only expressed in the prestalk zone of the slug provides strong evidence that DIF is a Dictyostelium morphogen.

Isolation and properties of an inducible and a constitutive beta-lactamase from Pseudomonas aeruginosa.

The inducible beta-lactamase from Pseudomonas aeruginosa NCTC 8203 and the constitutive beta-lactamase from strain 1822 S/H have been isolated and compared. These two enzymes are apparently periplasmic since they are released by freezing and thawing. They resemble each other closely in their molecular weights, amino acid composition, isoelectric points and electrophoretic mobility as well as in their catalytic properties, and they may be identical. Neither enzyme contains a free thiol group.

The mosaic organization of the apocytochrome b gene of Aspergillus nidulans revealed by DNA sequencing.

The coding section of the apocytochrome b gene (cobA) of the mitochondrial DNA of Aspergillus nidulans has been completely sequenced. The gene comprises two exons of 507 and 654 bp separated by one intron of approximately 1.1 kb. The derived amino acid sequence shows 61% homology with that of Saccharomyces cerevisiae and 51% homology with the cognate human sequence. Comparison of these sequences indicates that UGA codes for tryptophan in the A. nidulans mitochondrial system. The intron is in exactly the same place as intron 13 of the apocytochrome b gene of "long" strains of S. cerevisiae. Other introns present in "long" strains (I1 and I2) are absent in A. nidulans. Introns 14 and 15 present both "long" and "short" strains of yeast are also absent. This is particularly significant in view of the involvement of 14 in the splicing of both cob and oxi3 (coding for cytochrome oxidase subunit 1) mRNA precursors in S. cerevisiae. The partial sequence of the A. nidulans intron shows an open reading frame for at least the first 200 bp, and shows the intron to be closed in all frames at the carboxyl terminus.

Conservation and rearrangement of mitochondrial structural gene sequences.

Mitochondria contain the simplest DNA molecules that are present in eukaryotes. Mitochondrial DNA (mtDNA) is easily purified, and is an important model system for studying eukaryote gene structure and basic molecular processes. The protein sequences of mitochondrial gene products have been shown to be conserved from yeast to man, and there are definite similarities at the DNA sequence level. In contrast, the overall organization of the mitochondrial genome is drastically different in these organisms. To understand this, we need to extend work on mtDNA to a wider range of species. We have chosen to study the mtDNA of Aspergillus nidulans because a particularly comprehensive analysis of this system can be achieved using genetics as well as biochemistry, and like most eukaryotes it is an obligate aerobe, whereas Saccharomyces cerevisiae is not. We have investigated whether defined pieces of particular yeast mitochondrial genes show enough homology to Aspergillus mtDNA fragments to enable the corresponding Aspergillus genes to be located on the physical map. The results reported here show that this is the case for all five genes tested, and present the first data on the physical organization of the structural genes in the mitochondrial genome of A. nidulans.