Are Geriatrics-Focused Primary Care Clinics Better at Diagnosing Dementia Than Traditional Clinics? A Matched Cohort Study.

BACKGROUND: Dementia and mild cognitive impairment (MCI) are prevalent but underdiagnosed. OBJECTIVE: To compare new dementia/MCI diagnosis rates in geriatrics-focused primary care clinics and traditional primary care clinics. DESIGN: Secondary analysis of a prospective matched cohort study that spanned 2017-2021. PARTICIPANTS: Community-dwelling Veterans over 65 receiving primary care in a geriatrics-focused medical home (GeriPACT) or traditional primary care home (PACT) at one of 57 Veterans Affairs sites. We excluded individuals with a documented diagnosis of dementia or MCI in the year prior to enrollment. MAIN MEASURES: Diagnoses obtained from EHR. Cognitive status was assessed using modified Telephone Interview for Cognitive Status (mTICS) tool. KEY RESULTS: The 470 participants included in this analysis were predominantly white, non-Hispanic males with an average age of 80.3 years. 9.4% of participants received a diagnosis of dementia/MCI after 24 months: 11.5% in GeriPACT and 7.2% in PACT. Adjusted OR for dementia/MCI diagnosis based on GeriPACT exposure was 1.47 (95% CI 0.65-3.29). Low mTICS score (≤ 27) (OR 4.89, 95% CI 2.36-10.13) and marital status (married/partnered) (OR 1.89, CI 0.99-3.59) were independent predictors of dementia/MCI diagnosis. When stratified by cognitive status: diagnosis rates were 20.8% in GeriPACT and 16.7% in PACT among those who scored lower on the cognitive assessment (mTICS ≤ 27); 7.4% in GeriPACT and 3.6% in PACT among those who scored higher (mTICS > 27). The OR for new dementia/MCI diagnosis in GeriPACT was 1.19 (95% CI 0.49-2.91) among those with a low mTICS score and 1.85 (95% CI 0.70-4.88) among those with a higher mTICS score. CONCLUSIONS: Observed rates of new dementia/MCI diagnosis were higher in GeriPACT, but with considerable uncertainty around estimates. Geriatrics-focused primary care clinics may be a promising avenue for improving the detection of dementia in older adults, but further larger studies are needed to confirm this relationship.

Hair nicotine concentration measurement in cats and its relationship to owner-reported environmental tobacco smoke exposure.

OBJECTIVES: To investigate the association between hair nicotine concentration in cats and owner-reported exposure to environmental tobacco smoke. MATERIALS AND METHODS: Owner questionnaires documented exposure. Nicotine was extracted from hair by sonification in methanol followed by hydrophilic interaction chromatography with mass spectrometry. Relationships between hair nicotine concentration and owner-reported exposure were examined using hypothesis-testing statistics and receiver operating characteristic curve analysis. RESULTS: The hair nicotine concentration of reportedly exposed cats was significantly higher than unexposed cats and groups of cats with different levels of exposure had significantly different median hair nicotine concentrations corresponding to exposure. A hair nicotine concentration of 0·1 ng/mg had a specificity of 98% (95% confidence interval: 83 to 100) and a sensitivity of 69% (95% confidence interval: 54 to 84) for detecting environmental tobacco smoke exposure. Outdoors access, coat colour, urban or rural environment and length of time living with the owner were not obviously associated with hair nicotine concentration. CLINICAL SIGNIFICANCE: Feline hair nicotine concentration appears strongly associated with owner-reported environmental tobacco smoke exposure. Feline hair nicotine concentration could therefore be used as a biomarker for tobacco smoke exposure, allowing future studies to assess whether exposed cats have an increased risk of specific diseases.

Chemotherapy-induced dynamic gene expression changes in vivo are prognostic in ovarian cancer.

BACKGROUND: The response of ovarian cancer patients to carboplatin and paclitaxel is variable, necessitating identification of biomarkers that can reliably predict drug sensitivity and resistance. In this study, we sought to identify dynamically controlled genes and pathways associated with drug response and its time dependence. METHODS: Gene expression was assessed for 14 days post-treatment with carboplatin or carboplatin-paclitaxel in xenografts from two ovarian cancer models: platinum-sensitive serous adenocarcinoma-derived OV1002 and a mixed clear cell/endometrioid carcinoma-derived HOX424 with reduced sensitivity to platinum. RESULTS: Tumour volume reduction was observed in both xenografts, but more dominantly in OV1002. Upregulated genes in OV1002 were involved in DNA repair, cell cycle and apoptosis, whereas downregulated genes were involved in oxygen-consuming metabolic processes and apoptosis control. Carboplatin-paclitaxel triggered a more comprehensive response than carboplatin only in both xenografts. In HOX424, apoptosis and cell cycle were upregulated, whereas Wnt signalling was inhibited. Genes downregulated after day 7 from both xenografts were predictive of overall survival. Overrepresented pathways were also predictive of outcome. CONCLUSIONS: Late expressed genes are prognostic in ovarian tumours in a dynamic manner. This longitudinal gene expression study further elucidates chemotherapy response in two models, stressing the importance of delayed biomarker detection and guiding optimal timing of biopsies.

Interactive molecular networks obtained by computer-aided conversion of microarray data from brains of alcohol-drinking rats.

Lists of differentially expressed genes in a disease have become increasingly more comprehensive with improvements on all technical levels. Despite statistical cutoffs of 99% or 95% confidence intervals, the number of genes can rise to several hundreds or even thousands, which is barely amenable to a researcher's understanding. This report describes some ways of processing those data by mathematical algorithms. Gene lists obtained from 53 microarrays (two brain regions (amygdala and caudate putamen), three rat strains drinking alcohol or being abstinent) have been used. They resulted from analyses on Affymetrix chips and encompassed approximately 6 000 genes that passed our quality filters. They have been subjected to four mathematical ways of processing: (a) basic statistics, (b) principal component analysis, (c) hierarchical clustering, and (d) introduction into Bayesian networks. It turns out, by using the p-values or the log-ratios, that they best subdivide into brain areas, followed by a fairly good discrimination into the rat strains and the least good discrimination into alcohol-drinking vs. abstinent. Nevertheless, despite the fact that the relation to alcohol-drinking was the weakest signal, attempts have been made to integrate the genes related to alcohol-drinking into Bayesian networks to learn more about their inter-relationships. The study shows, that the tools employed here are extremely useful for (a) quality control of datasets, (b) for constructing interactive (molecular) networks, but (c) have limitations in integration of larger numbers into the networks. The study also shows that it is often pivotal to balance out the number of experimental conditions with the number of animals.

Does orally administered doxycycline reach the tear film?

BACKGROUND/AIMS: Orally administered doxycycline, a broad-spectrum antibiotic, is an established treatment for ocular surface diseases, particularly rosacea, meibomian gland dysfunction and recurrent epithelial cell erosion. In recent times, its efficacy in treating these diseases has been ascribed to an ability to inhibit matrix metalloproteinase (MMP) activity and both MMP and interleukin-1 (IL-1) synthesis. Since these functions are concentration-dependent, the aim of this project was to determine whether sufficient doxycycline reached the tear film to fulfil these roles in vivo. METHODS: Doxycycline was extracted with 1-butanol from tear and blood plasma samples obtained from patients with ocular surface disease and healthy individuals and quantified spectrophotometrically. The MMPs present in the patients tear films before and during doxycycline treatment were analysed zymographically. RESULTS: The quantity of doxycycline detected in the blood plasma samples of patients undergoing treatment ranged from 1.83 to 13.18 microM. Although doxycycline was not detected in their tear samples, the treatment caused the disappearance of the MMPs symptomatic of disease progression. CONCLUSION: The inability to detect doxycycline in the tear film of patients undergoing treatment indicates that doxycycline does not directly inhibit MMP activity or the synthesis/secretion of these proteases and IL-1 from corneal epithelial cells.

Species-specific in vitro pharmacological effects of the cannabinoid receptor 2 (CB2) selective ligand AM1241 and its resolved enantiomers.

BACKGROUND AND PURPOSE: Racemic (R,S) AM1241 is a cannabinoid receptor 2 (CB(2))-selective aminoalkylindole with antinociceptive efficacy in animal pain models. The purpose of our studies was to provide a characterization of R,S-AM1241 and its resolved enantiomers in vitro and in vivo. EXPERIMENTAL APPROACH: Competition binding assays were performed using membranes from cell lines expressing recombinant human, rat, and mouse CB(2) receptors. Inhibition of cAMP was assayed using intact CB(2)-expressing cells. A mouse model of visceral pain (para-phenylquinone, PPQ) and a rat model of acute inflammatory pain (carrageenan) were employed to characterize the compounds in vivo. KEY RESULTS: In cAMP inhibition assays, R,S-AM1241 was found to be an agonist at human CB(2), but an inverse agonist at rat and mouse CB(2) receptors. R-AM1241 bound with more than 40-fold higher affinity than S-AM1241, to all three CB(2) receptors and displayed a functional profile similar to that of the racemate. In contrast, S-AM1241 was an agonist at all three CB(2) receptors. In pain models, S-AM1241 was more efficacious than either R-AM1241 or the racemate. Antagonist blockade demonstrated that the in vivo effects of S-AM1241 were mediated by CB(2) receptors. CONCLUSIONS AND IMPLICATIONS: These findings constitute the first in vitro functional assessment of R,S-AM1241 at rodent CB(2) receptors and the first characterization of the AM1241 enantiomers in recombinant cell systems and in vivo. The greater antinociceptive efficacy of S-AM1241, the functional CB(2) agonist enantiomer of AM1241, is consistent with previous observations that CB(2) agonists are effective in relief of pain.

Tissue inhibitor of metalloproteinase-3 differentially binds to components of Bruch's membrane.

BACKGROUND: Sorsby's fundus dystrophy (SFD) is caused by mutations in tissue inhibitor of metalloproteinase (TIMP)-3 and, with the exception of early onset, is similar to age-related macular degeneration. The pathological features of this condition relate to the accumulation of TIMP-3 in Bruch's membrane. AIMS: To compare the extracellular membrane-binding characteristics of wild-type and four SFD-mutant TIMP-3s. METHODS: COS-7 cells were transfected with wild-type, Ser-181, Gly-167, Ser-156 and Tyr-168 SFD-mutant TIMP-3 cDNA. The TIMP-3 proteins subsequently synthesised were harvested, analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, semiquantified by ELISA and used in binding assays on the basis of the retention of the wild-type and SFD-mutant TIMP-3 proteins by components of Bruch's membrane. RESULTS: SFD-mutant TIMP-3s could not be distinguished from wild-type TIMP-3 by the extents to which they aggregated or adhered to type-I collagen, type-IV collagen, fibronectin, laminin, elastin, chondroitin sulphates A, B and C, and heparin sulphate. Of these macromolecules, the wild-type and SFD-mutant TIMP-3s exhibited greatest affinity for elastin and laminin. CONCLUSION: The similarity in the physical and extracellular membrane-binding characteristics of wild-type and SFD-mutant TIMP-3s indicates that these properties are not responsible for the difference in timing of onset of SFD and age-related macular degeneration.

Keratoconus: matrix metalloproteinase-2 activation and TIMP modulation.

Keratoconus is an ocular condition that causes corneal thinning, cone formation and scarring. In view of a hypothesis that activated MMP-2 may initiate or facilitate disease progression, the MMP-2/TIMP systems of stromal cells derived from normal and keratoconic corneas have been compared. To achieve this, stromal cell cultures were established from normal, clear keratoconic (KCS-1) and scarred keratoconic (KCS-2) corneas. The secreted MMP-2 was assayed using [(3)H]Type IV collagen and analysed by zymography. Optimally maintained and nutrient deprived cells were subsequently incubated with [(3)H]lysine. The secreted radiolabelled macromolecules were separated and quantified. The results obtained indicated that optimally maintained KCS-1 stromal cells produced more MMP-2 than normal stromal cells but not TIMP. Nutrient deprivation induced MMP-2 activation and cell death. Surviving cells upregulated TIMP-1 synthesis and in this respect became similar to the KCS-2 stromal cells that did not excessively generate activated MMP-2 or die as a consequence of nutrient deprivation. From these results, it was concluded that KCS-1 stromal cells over-expressed MMP-2 without increasing TIMP production. This may facilitate MMP-2 activation in vivo and hence advance the keratoconic condition. KCS-2 cultures over-expressed both MMP-2 and TIMP-1. Because TIMP-1 inhibits MMP-2 activity and protects against cell death it may be of significance in initiating repair processes and curtailing keratoconus.

Doxycycline-a role in ocular surface repair.

BACKGROUND/AIMS: Doxycycline is a broad spectrum antibiotic that chelates metal ions and is frequently used as part of the treatment of ocular surface diseases. Its therapeutic value has been ascribed to an ability to inhibit matrix metalloproteinase (MMP) activity and both MMP and IL-1 synthesis. The aim of this study was to evaluate the role of doxycycline as an inhibitor of corneal MMPs and assess its contribution to ocular surface repair mechanisms. METHODS: Corneal epithelial cell and keratocyte cultures were grown to confluence and incubated with IL-1alpha, LPS, doxycycline, or doxycycline and LPS in serum free medium for 4 days. The cells were either harvested and assayed for caspase-3 activity or stained with either AE5 or antivimentin antibodies. Media samples were concentrated and assayed for MMP activity by zymography or using a fluorigenic substrate. ELISA was used to quantify IL-1alpha, MMPs -1,-2,-3,-9, and TIMPs -1 and -2. RESULTS: IL-1alpha and LPS had no effect on MMP/TIMP production by cultured corneal epithelial cells and keratocytes. Corneal MMP-2 inhibition by doxycycline was partially [Ca(2+)] dependent but irreversible. At the minimum inhibitory concentration, 100 micro m, doxycycline had no apparent effect on MMP and TIMP production, but ultimately caused the death of keratocytes and some of the epithelial cells that detached from their basement membrane. Caspase-3 activity was not detected in dead or dying keratocytes. The mechanism of cell death in cultured corneal epithelial cells was not caspase-3 related apoptosis as the activity of this enzyme, normally detectable, was lost. The epithelial cells that survived doxycycline treatment did not bind antivimentin antibody and compared with controls, reacted less with the AE5 antibody. They were probably transient amplifying cells. CONCLUSIONS: Doxycycline irreversibly inhibits corneal MMP-2 activity by chelating the metal ions that are catalytically and structurally essential. Corneal MMP/TIMP production in vitro is not modulated by IL-1alpha, LPS, or doxycycline. The therapeutic value of doxycycline may depend upon its effective concentration at the ocular surface and probably relates to its chelating properties.

A framework for integrating the songbird brain.

Biological systems by default involve complex components with complex relationships. To decipher how biological systems work, we assume that one needs to integrate information over multiple levels of complexity. The songbird vocal communication system is ideal for such integration due to many years of ethological investigation and a discreet dedicated brain network. Here we announce the beginnings of a songbird brain integrative project that involves high-throughput, molecular, anatomical, electrophysiological and behavioral levels of analysis. We first formed a rationale for inclusion of specific biological levels of analysis, then developed high-throughput molecular technologies on songbird brains, developed technologies for combined analysis of electrophysiological activity and gene regulation in awake behaving animals, and developed bioinformatic tools that predict causal interactions within and between biological levels of organization. This integrative brain project is fitting for the interdisciplinary approaches taken in the current songbird issue of the Journal of Comparative Physiology A and is expected to be conducive to deciphering how brains generate and perceive complex behaviors.

Femoral impaction bone grafting with the Elite Plus stem.

This study evaluates the results of impaction grafting of the femoral stem with the Elite Plus system (DePuy, Leeds, UK) in revision hip arthroplasty with irradiated allograft. Nineteen hips in 19 patients (12 men and 7 women), at an average age of 59 years were followed for an average of 33 months. Endo-Klinik grading of bone stock loss was 5 grade 1, 6 grade 2, 8 grade 3 and no grade 4. Radiographic analysis revealed evidence of graft incorporation in 32% but no evidence of trabecular remodelling or cortical repair. Four patients have undergone revision of the femoral stem, one patient died while awaiting revision and one patient is unfit for revision. Most complications occurred in patients classified as Endo-Klinik grade 3. We found that impaction grafting with the Elite Plus system was associated with a high failure rate, especially in those cases with more severe bone stock loss. (Hip International 2002; 1: 11-6).

Tear film MMP accumulation and corneal disease.

BACKGROUND/AIMS: Matrix metalloproteinases (MMPs) accumulate in the tears of patients with active peripheral ulcerative keratitis (PUK) but it is unknown whether these enzymes have a central role in disease progression. The aims of the present investigation were to determine the source of these enzymes and to ascertain whether their accumulation in tears is a phenomenon specific to PUK or a general feature of other anterior segment diseases. METHODS: The experimental samples were obtained from the culture media of conjunctival and corneal epithelial cells, from fractionated blood plasma and leucocytes of healthy subjects and patients with rheumatoid arthritis, and from the tears of healthy subjects and patients with a variety of anterior segment diseases. The MMPs of all samples were visualised by zymography and tear samples were assayed using nitrophenol acetate and an MMP-9 susceptible quenched fluorescent peptide as substrate. RESULTS: The major MMPs that accumulate in the tears of patients with rheumatoid arthritis with active ocular disease are MMP-9 and a species of M(r) 116,000. By comparing the zymographic activity profiles of the gelatinases present in the samples obtained, it was deduced that the main source of these MMPs was granulocytes. Their accumulation in tears was not unique to patients with PUK; detectable amounts of the enzymes also occurred in the tears of patients with keratoconus with associated atopic disease, patients undergoing treatment for herpetic eye disease, and patients with systemic and non-systemic dry eye disease. CONCLUSION: The MMPs that accumulate in tears are mainly derived from granulocytes. This may be effected by autoimmune diseases that involve ocular tissue or by ocular diseases that induce an inflammatory response.

Matrix metalloproteinase 2 activation in cultured corneas.

PURPOSE: Corneas that are maintained in tissue culture medium shed their epithelial cells and repopulation following graft surgery is an essential facet of the healing process. Failure to do so may be a result of structural damage to the epithelial basement membrane of a donor cornea. The purpose of the present investigation was to ascertain whether MMP-2, the matrix metalloproteinase produced by corneal keratocytes, may be activated during storage and hence cleave the type IV collagen component of the epithelial cell basement membrane. METHODS: Fresh and transplant rejected corneas that had been stored in culture medium for varying time periods and of known donor age were collected. The soluble protein fractions of these corneas were obtained. Their MMP-2 proteins were visualised by zymography on SDS gelatin polyacrylamide gels and assayed for activity against nitrophenyl acetate and denatured [(3)H]type I collagen. RESULTS: The stromal tissue of fresh, normal corneas produced inactive MMP-2 of M(r) 66,000. Although the cultured corneas did not up-regulate MMP-2 production, they contained additional MMP-2 activities of M(r) 62,000 and M(r) 43,000. The appearance of these additional MMP-2 activities correlated with corneal culture time but not donor age. The ability to cleave denatured [(3)H]type I collagen correlated with the appearance of the M(r) 43,000 activity but not the M(r) 62,000 activity. CONCLUSION: Activated MMP-2 is produced in cultured corneas. For this reason the corneas donated for all graft procedures should not be held in culture medium for periods exceeding 4 weeks.

Matrix metalloproteinase 2: involvement in keratoconus.

PURPOSE: The activation of matrix metalloproteinase-2 (MMP-2) is postulated to be a crucial pathogenic factor behind progressive and chronic diseases in which basement membranes are disrupted. An ocular example is keratoconus. The purpose of the present enquiry was therefore to investigate and compare the activities of the MMP-2 secreted by keratocytes of normal and keratoconic corneas. METHODS: The spectrum of MMP-2 activities secreted by cultures of keratocytes derived from normal and keratoconic corneas was analysed by zymography. Subsequently, selected preparations were assayed for peptidase activity, using Type I, Type III, Type IV and Type V collagen as substrate, under native conditions and after treatment with a variety of putative activating reagents. RESULTS: Although MMP-2 of Mr 65,000 on SDS gelatin polyacrylamide gels is the major protease secreted by keratocytes of normal corneas, the keratocytes of early-phase keratoconic corneas secrete an additional zymographic activity of Mr 61,000. From their N-terminal amino acid sequences, both these proteins were shown to be conformers of proMMP-2. Treatment with SDS followed by protein fractionation was required to achieve in vitro activation of the MMP-2 secreted by normal corneal keratocytes. Treatment with SDS alone partially activated the enzyme produced by early-phase keratoconic corneal keratocytes. This procedure and autocatalysis, yielded an enzyme of Mr 43,000 that selectively hydrolysed Type IV and denatured Type 1 collagen. CONCLUSIONS: The zymographic gelatinase activities of apparent Mr 65,000 and 61,000 are conformers of corneal proMMP-2. Activated enzyme, of Mr 43,000, is more readily generated from protein preparations of the culture media of early phase keratoconic corneal keratocytes than from protein preparations of the culture media of normal corneal keratocytes.

The control of matrix metalloproteinase-2 expression in normal and keratoconic corneal keratocyte cultures.

PURPOSE: Early phase keratoconic corneas and their cultured keratocytes abnormally produce the Mr 62,000 form of the matrix metalloproteinase-2 (MMP-2). It is known that platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are involved in the regulation of MMP activity and tissue inhibitor of metalloproteinase (TIMP) production in non-ocular tissues. The purpose of this enquiry was to determine whether these growth factors also play a role in the activity and/or production of corneal MMP-2 and TIMP, and whether their activity could account for the existence of the Mr 62,000 form of MMP-2 in keratoconic corneas. METHODS: Confluent cultures of normal and early-phase keratoconic corneal keratocytes were established and incubated in serum-free media in the presence or absence of PDGF and TGF-beta. The proteins secreted by these cells over periods of 7 days were harvested for analysis. The total protein produced was determined spectrophotometrically. MMP-2 was visualised by SDS-gelatin polyacrylamide gel electrophoresis and assayed using radiolabelled type IV collagen as substrate. The enzyme inhibitors, TIMP-1 and TIMP-2, were quantified by dot blot immunoassay. RESULTS: The addition of PDGF or TGF-beta to the culture medium of keratoconic corneal keratocytes had no significant effect on overall protein production, MMP-2 activity or on the amounts of TIMP- 1 and TIMP-2 secreted. These observations also applied to normal corneal keratocytes, with the exception that PDGF induced expression of the Mr 62,000 species of MMP-2. CONCLUSIONS: PDGF may be involved in the production of the Mr 62,000 species of MMP-2 that is abnormally produced by early-phase keratoconic corneal keratocytes.

Nonunion of the distal radius.

We report five cases of nonunion of the distal radius. There were three women and two men. The mean age was 44 years (range, 34-56). All five patients were heavy tobacco smokers and three had a history of alcohol abuse. In three patients, union of the distal radius was obtained. Two had a persistent nonunion which was salvaged with a total wrist fusion.

Role of ocular matrix metalloproteinases in peripheral ulcerative keratitis.

AIM: Peripheral ulcerative keratitis (PUK) is an ocular manifestation of rheumatoid arthritis and other similar systemic diseases. The purpose of this inquiry was to investigate the involvement of matrix metalloproteinases (MMPs) in the induction and/or maintenance of PUK. METHODS: Substrate gel electrophoresis was used to characterise the MMP activities secreted by primary cultures of keratocytes derived from normal and perforated pathological corneal specimens, and those present in tears of normal subjects and patients with PUK. Substrate specificity and the in vivo activity status of the secreted MMPs was assessed by SDS-polyacrylamide gel electrophoresis of standard collagens incubated in the presence or absence of the various enzyme preparations. RESULTS: In addition to MMP-2 of M(r) 66,000, cultured keratocytes derived from perforated corneas of patients with PUK abnormally produce the MMP-2 of apparent M(r) 62,000. Other MMPs and in particular MMP-9 of M(r) 92,000, also occur in the tears of these patients. Their visualisation on substrate polyacrylamide gels correlated with clinical manifestations of disease activity; during periods of disease quiescence they were barely detectable. The steroid prednisolone, frequently used in systemic therapy, had no effect on the in vitro activity of MMP-2, or on its production by cultured corneal keratocytes. Although the in vitro activity of MMP-2 was inhibited by both Cu(2+) and Zn(2+), Cu(2+) apparently induced the keratocytes to produce activated enzyme and Zn(2+) irreversibly inhibited their production of MMP-2. CONCLUSION: Overexpression of corneal MMP-2 and tear film MMP-9 are characteristic features of patients with PUK and their activation may be a crucial facet of disease initiation or progression. Although effective in systemic therapy for PUK, prednisolone had no direct control over corneal MMP-2 production or activity. Zn(2+) on the other hand inhibited both MMP-2 production and MMP-2 activity and may, therefore, be of therapeutic value if suitably formulated and used in conjunction with systemic steroid treatment.

Localisation of connective tissue and inhibition of autofluorescence in the human optic nerve and nerve head using a modified picrosirius red technique and confocal microscopy.

The use of picrosirius red to localise connective tissue in thin tissue sections viewed by bright-field microscopy is well documented. Its use on thin tissue sections imaged by fluorescence confocal microscopy has also been reported. Here we describe modifications to published procedures that allow picrosirius red staining of thick 60-microm sections and their subsequent analysis by confocal microscopy. The use of phosphomolybdic acid pre-treatment was found to be essential for confocal analysis; in addition to preventing non-specific staining, it also quenched tissue autofluorescence. By incubating sections free-floating, pre-treating them with phosphomolybdic acid for 30 min and imaging them using an argon ion laser we were able to use confocal microscopy to image the entire depth of 60-microm human optic nerve and nerve head sections stained with picrosirius red. The application of this modified picrosirius red and confocal microscopy technique should be useful for analysing the three-dimensional structure of the optic nerve and other tissues with a similarly complex arrangement of connective tissue.

Over-expression of a gelatinase A activity in keratoconus.

Keratoconus is an ocular disorder in which the central cornea becomes thin, conical and frequently scarred. We are exploring the possibility that this condition is induced and maintained by proteases that exist in the corneal matrix in an activated form. In this study, the activities of the proteases secreted in vitro and in vivo by keratocytes of normal, clear keratoconic, scarred keratoconic and traumatically scarred corneas have been compared and partially characterised. Data obtained by assaying acyl transferase activity showed that the matrix metalloproteinases account for a minimum of 95% of the total protease secreted by cultured keratocytes. Their summated specific activity was consistently and significantly higher in the culture medium of keratoconic keratocytes than in the medium of other keratocyte cultures. Analysis of the individual protease activities secreted by these corneal keratocytes in vitro and in vivo by SDS-gelatin polyacrylamide gel electrophoresis showed that a gelatinase of molecular weight 65,000 is the major protease secreted by normal keratocytes. Whereas clear keratoconic and traumatically scarred corneal keratocytes secrete an additional activity of molecular weight 61,000, scarred keratoconic corneal keratocytes generally produced little or none of this gelatinase activity. Both activities may be ascribed to gelatinase A, and although the 61,000 molecular weight form may be a significant feature of keratoconus, neither appears to be active as secreted.

The Distribution of Gibberellins in Vegetative Tissues of Pisum sativum L. : I. Biological and Biochemical Consequences of the le Mutation.

The concentrations of endogenous gibberellin (GA) 1, 5, 8, 19, 20, and 29 in the component tissues of maturing tall (Le) and dwarf (le) pea (Pisum sativum) plants have been determined. The following conclusions were drawn from the data obtained: (a) GA(20) and its metabolites accumulate only in the growing regions of Le and le plants; (b) the le mutation is biochemically expressed in all immature tissues of the dwarf plants; (c) the quantitative composition of the GA metabolites in the various immature tissues is variable; (d) the total GA concentration in apical buds, unexpanded leaves, and tendrils is considerably higher than in GA(1)-responsive stem tissue; and (e) there is very little GA accumulation of the inactive 2beta-hydroxylated GAs (GA(8) and GA(29)) in either the mature vegetative tissues or the roots of pea plants.