Mechanical spectroscopy and imaging of skin components in vivo: Assignment of the observed moduli.

BACKGROUND: The need to quantitatively identify the composition and organization of the macromolecular components of skin, skin lesions, scars, tumors, extracellular matrices (ECMs), and wound tissue has been a goal of researchers for many decades. A variety of studies have been recently reported applying optical coherence tomography (OCT) to image skin and cutaneous lesions. MATERIALS AND METHODS: This article describes the use of vibrational OCT to image and noninvasively characterize the macromolecular components of the ECM of skin. RESULTS: We report that the major macromolecular components of skin and scar can be identified noninvasively by their characteristic moduli calculated from measurements of the resonant frequency and tissue thickness. Moduli for fat (0.03 MPa), elastic tissue (0.8 MPa), skin (2 MPa), and scar (7 MPa) can be differentiated using images and measurements of the resonant frequency and the sample thickness obtained from OCT. CONCLUSIONS: Using vibrational OCT, it is possible to identify and map the location of the macromolecular components in skin and skin lesions.

Fractal dimensions and branching characteristics of placental chorionic surface arteries.

We present a novel approach to characterize and compare the placental chorionic surface arteries (PCSA) of normal, preterm, diabetic and preeclamptic pregnancies using their fractal dimension (FD) and branch point number (BPN). Mean FD of PCSA of normal pregnancies was similar to those of diabetic and preeclamptic pregnancies, but significantly different from those of preterm pregnancies. In contrast, the BPN of PCSA of normal and preterm pregnancies was similar but significantly different from diabetic and preeclamptic pregnancies. The results suggest that branching properties of normal and pathological pregnancies are intrinsically different.

Morphomechanics of dermis-A method for non-destructive testing of collagenous tissues.

BACKGROUND: Collagenous tissues store, transmit and dissipate elastic energy during mechanical deformation. In skin, mechanical energy is stored during loading and then is dissipated, which protects skin from mechanical failure. Thus, energy storage (elastic properties) and dissipation (viscous properties) are important characteristics of extracellular matrices (ECMs) that support the cyclic loading of ECMs without tissue failure. METHODS: Uniaxial stress-strain measurements on decellularized human dermis have been made and compared to results of a non-destructive technique involving optical coherence tomography (OCT) combined with vibrational analysis. In addition, Poisson's ratio has been determined for tensile deformation of decellularized dermis. RESULTS: The modulus of decellularized dermis measured using standard tensile stress-strain tests and that determined from calculations derived from natural frequency measurements give similar results. It is also observed that Poisson's ratio for dermis is between 0.38 and 0.63 after correction for changes in volume that occur during tensile deformation. These results suggest that the assumption that dermis and other ECMs deform at constant volume is incorrect and will lead to differences in the calculated modulus by conventional tensile stress-strain measurements. CONCLUSIONS: It is proposed that OCT in conjunction with vibrational analysis is a convenient way to non-destructively measure the modulus of decellularized dermis, ECMs and other materials that have a positive curvature to their stress-strain curves. Tensile deformation of dermis and possibly other ECMs is associated with an increase in Poisson's ratio consistent with a model of fluid expulsion from collagen fibrils during stretching. The value of Poisson's ratio should be considered in analyzing the mechanical properties of ECMs since at least dermis appears to be compressible during tensile deformation. Fluid expression during tensile deformation may play a role in mechanotransduction in skin in a similar manner to cartilage and bone tissue.

Villus packing density and lacunarity: Markers of placental efficiency?

We evaluate, in routine H&E histology slides, villus quantity in a given area (villous packing density, VPD) and the pattern or "gappiness" of villous distribution (lacunarity), and test for correlations with a proxy for fetoplacental metabolic rate, ß calculated as (ln (placental weight)/ln (birthweight)) from Kleiber's law [1]. Three ∼4.3 mm2 images each were obtained from 88 term placentas. Ranges of VPD and lacunarity were each correlated with ß (r = 0.31, p = 0.003, r = 0.23, p = 0.03 and respectively). The relationship between ß and within-placenta variation in VPD and lacunarity highlights the need to study not merely the mean but the variance of villous geometries and spatial distributions.

Shape matching algorithm to validate the tracing protocol of placental chorionic surface vessel networks.

Variability in placental chorionic surface vessel networks (PCSVNs) may mark developmental and functional changes in fetal health. Here we report a protocol of manually tracing PCSVNs from digital 2D images of post-delivery placentas and its validation by a shape matching method to compare the similarity between paint-injected and unmanipulated (uninjected and deflated vessels) tracings of PCSVNs. We show that tracings of unmanipulated vessels produce networks that are very comparable to the networks obtained by tracing paint-injected PCSVNs. We suggest that manual tracings of unmanipulated PCSVNs can extract features of PCSVN growth and structure that may impact fetal wellbeing.

Comparative efficacy of different estrus synchronization protocols on estrus induction response, fertility and plasma progesterone and biochemical profile in crossbred anestrus cows.

AIM: To evaluate estrus induction response and fertility including plasma progesterone and biochemical profile following use of three standard hormonal protocols in anestrus crossbred cows. MATERIALS AND METHODS: The study was carried out on 40 true anestrus and 10 normal cyclic cows. 10 anestrus cows each were treated with standard intravaginal controlled internal drug release (CIDR) device, Ovsynch (GPG) protocol, and Norgestomet ear implant with fixed-time artificial insemination (FTAI). 10 anestrus cows were kept as untreated control while 10 cows exhibiting the first estrus within 90 days postpartum without any treatment served as normal cyclic control. Blood samples were obtained from treated cows on day 0, 7, 9 (AI) of treatment and day 21 post-AI, and from control groups on the day of AI and day 21 post-AI for estimation of plasma progesterone, protein, cholesterol, calcium, and inorganic phosphorus profile. RESULTS: The use of CIDR, Ovsynch, and Norgestomet ear implant protocols resulted in 100% estrus induction with conception rates at induced estrus of 60%, 50%, and 50%, and the overall of three cycles as 80%, 80%, and 70%. In untreated anestrus control (n=10), only three cows exhibited spontaneous estrus within 90 days of follow-up and conceived giving the first service and overall conception rates of 66.66% and 30.00%, respectively. In normal cyclic control (n=10), the conception rates at first and overall of three cycles were 50% and 80%. The overall mean plasma progesterone (P4) concentrations in anestrus cows studied on day 0 (initiation), 7 (prostaglandin injection and/or removal of implant), 9 (FTAI) of treatment and on day 21 post-AI revealed that the values on day 7 and 21 were significantly (p<0.01) higher than other two periods in all three groups. The concentrations were significantly (p<0.05) higher in conceived than non-conceived group on day 21 post-AI in CIDR (4.36±0.12 vs. 1.65±0.82 ng/ml) and Ovsynch (4.85±0.62 vs. 1.59±0.34 ng/ml), but not in Norgestomet ear implant (4.50±0.53 vs. 3.02±1.15 ng/ml) or normal cyclic group (5.39±0.67 vs. 3.13±0.37 ng/ml). The cholesterol and protein levels were significantly higher, but not the calcium and phosphorus, in normal cyclic control than in anestrus groups. The influence of treatment days and pregnancy status was not significant for any of the biochemical constituents in any of the groups. CONCLUSION: Ovsynch and/or CIDR synchronization protocol can be effectively used to improve fertility up to 80% in anestrus cows, as compared to 30% in anestrus control, combined with plasma progesterone to delineate the reproductive status before and after treatment.

Abrogation of DNA vector-based RNAi during apoptosis in mammalian cells due to caspase-mediated cleavage and inactivation of Dicer-1.

RNA interference (RNAi) is used as a reverse-genetic tool to examine functions of a gene in different cellular processes including apoptosis. As key cellular proteins are inactivated during apoptosis, and as RNAi requires cooperation of many cellular proteins, we examined whether DNA vector-based RNAi would continue to function during apoptosis. The short hairpin RNA transcribed from the DNA vector is processed by Dicer-1 to form small interfering RNA that is incorporated in the RNA-induced silencing complex (RISC) to guide a sequence-specific silencing of the target mRNA. We report here that DNA vector-based RNAi of three different genes, namely poly(ADP-ribose) polymerase-1, p14(ARF) and lamin A/C are abrogated during apoptosis. The failure of DNA vector-based RNAi was not at the level of Ago-2 or RISC-mediated step of RNAi but due to catalytic inactivation of Dicer-1 on specific cleavage at the STTD(1476) and CGVD(1538) sites within its RNase IIIa domain. Using multiple approaches, caspase-3 was identified as the major caspase responsible for the cleavage and inactivation of Dicer-1. As Dicer-1 is also the common endonuclease required for formation of microRNA (miRNA) in mammalian cells, we observed decreased levels of mature forms of miR-16, miR-21 and let-7a. Our results suggest a role for apoptotic cleavage and inactivation of Dicer-1 in controlling apoptotic events through altered availability of miRNA.

Analysis of ADP-ribose polymer sizes in intact cells.

Poly(ADP-ribose) is a polymer (pADPr) that is synthesized by poly (ADP-ribose) polymerases in response to DNA damaging agents. For instance, chemical alkylating agents such as MNNG or physical stimulation of cells by gamma-rays are well known to induce pADPr synthesis. PARPs are members of a growing family of enzymes which includes PARP-1, PARP-2, S-PARP-1, tankyrase and V-PARP. The association of PARP-1 and PARP-2 in DNA damage signaling pathways has been characterized, but tankyrase and V-PARP seem to be independent of DNA repair mechanisms. Poly(ADP-ribosyl)ation leads to heterogenous chain lengths of up to 200 units (mers) in vitro. While most of these will be covalently bound to proteins, they may be released under alkaline conditions for analysis. Previous immunological methods such as immunoblots showed that about 60-70% of the 6-8 mers pADPr were lost during fixation and that the very short pADPr (2-5 mers) were very weakly bound to the membrane. Furthermore, detection of cellular pADPr using enzyme-linked immunosorbent assay (ELISA) revealed that some molecules of pADPr are also lost during fixation and washings. This phenomenon leads to underestimation of the short pADPr population in cells. Thus, evaluating which pADPr sizes are present in cells and tissues becomes critical. We report here the development of a new highly sensitive immunological method to detect synthesized pADPr sizes distribution in intact cells.

Caspase-3-mediated processing of poly(ADP-ribose) glycohydrolase during apoptosis.

Poly(ADP-ribose) glycohydrolase (PARG) is responsible for the catabolism of poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerase (PARP-1) and other PARP-1-like enzymes. In this work, we report that PARG is cleaved during etoposide-, staurosporine-, and Fas-induced apoptosis in human cells. This cleavage is concomitant with PARP-1 processing and generates two C-terminal fragments of 85 and 74 kDa. In vitro cleavage assays using apoptotic cell extracts showed that a protease of the caspase family is responsible for PARG processing. A complete inhibition of this cleavage was achieved at nanomolar concentrations of the caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting the involvement of caspase-3-like proteases. Consistently, recombinant caspase-3 efficiently cleaved PARG in vitro, suggesting the involvement of this protease in PARG processing in vivo. Furthermore, caspase-3-deficient MCF-7 cells did not show any PARG cleavage in response to staurosporine treatment. The cleavage sites identified by site-directed mutagenesis are DEID(256) downward arrow V and the unconventional site MDVD(307) downward arrow N. Kinetic studies have shown similar maximal velocity (V(max)) and affinity (K(m)) for both full-length PARG and its apoptotic fragments, suggesting that caspase-3 may affect PARG function without altering its enzymatic activity. The early cleavage of both PARP-1 and PARG by caspases during apoptosis suggests an important function for poly(ADP-ribose) metabolism regulation during this cell death process.

Immunological determination and size characterization of poly(ADP-ribose) synthesized in vitro and in vivo.

Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized rapidly and transiently from beta-NAD in response to DNA damaging agents. In order to study the physiological significance of poly(ADP-ribose) metabolism, we have developed immunological methods which enable us to study endogenous poly(ADP-ribose) without interfering with cell metabolism and integrity. For this purpose, we produced a highly specific polyclonal anti-poly(ADP-ribose) antibody which immunoreacts with polymers and oligomers. In addition to the immunodot blot method recently described by us (Affar et al., Anal. Biochem. 259 (1998) 280-283), other applications were investigated in cells: (i) detection of poly(ADP-ribose) by ELISA; (ii) characterization of poly(ADP-ribose) size using high resolution gel electrophoresis of polymers, followed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody; (iii) immunocytochemistry and flow cytometry analyses allowing poly(ADP-ribose) study at the level of individual cells.

Poly(ADP-ribose) turnover in quail myoblast cells: relation between the polymer level and its catabolism by glycohydrolase.

The concerted action of poly(ADP-ribose) polymerase (PARP) which synthesizes the poly(ADP-ribose) (pADPr) in response to DNA strand breaks and the catabolic enzyme poly(ADP-ribose) glycohydrolase (PARG) determine the level of polymer and the rate of its turnover. In the present study, we have shown that the quail myoblast cells have high levels of basal polymer as compared to the murine C3H10T1/2 fibroblasts. We have conducted this study to investigate how such differences influence polymer synthesis and its catabolism in the cells in response to DNA damage by alkylating agent. In quail myoblast cells, the presence of high MNNG concentration such as 200 microM for 30 min induced a marginal decrease of 15% in the NAD content. For C3H10T1/2 cell line, 64 microM MNNG provoked a depletion of NAD content by approximately 50%. The induction of the polymer synthesis in response to MNNG treatment was 6-fold higher in C3H10T1/2 cells than in quail myoblast cells notwithstanding the fact that 3-fold higher MNNG concentration was used for quail cells. The polymer synthesis thus induced in quail myoblast cells had a 4-5 fold longer half life than those induced in C3H10T1/2 cells. To account for the slow turnover of the polymer in the quail myoblast cells, we compared the activities of the polymer catabolizing enzyme (PARG) in the two cell types. The quail myoblast cells had about 25% less activity of PARG than the murine cells. This difference in activity is not sufficient to explain the large difference of the rate of catabolism between the two cell types implicating other cellular mechanisms in the regulation of pADPr turnover.

Determination of genotoxicity of the metabolites of the pesticides Guthion, Sencor, Lorox, Reglone, Daconil and Admire by 32P-postlabeling.

Commercial formulations of the pesticides: Guthion (azinphos methyl), Sencor (metribuzin), Lorox (linuron), Reglone (diquat), Daconil (chlorothalonil) and Admire (imidacloprid) were studied for their genotoxicity by 32P-postlabeling. Metabolites of the pesticides were obtained enzymatically using arochlor induced rat liver S9 fraction, in an NADPH generating system. The resulting metabolites were reacted with calf thymus DNA and the DNA was analyzed for presence of adducts by either the nuclease P1 or butanol enrichment. Nuclease P1 enrichment resulted in adducts for all the pesticides. Compared to the level of adducts in control DNA, the levels in pesticide-treated DNA were higher for all the pesticides, except Daconil. The increase in adduct numbers for pesticide-treated DNAs ranged from 4.9-12.4 times the control-DNA indicating pesticide genotoxicity in this in vitro system. Enrichment using butanol extraction gave three adducts unique to Sencor-DNA. These adducts were different from those obtained with nuclease P1 enrichment of the same. B(alpha)P was the positive control for the in vitro metabolism, and two adduct enrichment procedures: nuclease P1 digestion and butanol extraction.

Different cleavage pattern for poly(ADP-ribose) polymerase during necrosis and apoptosis in HL-60 cells.

Human promyelomonocytic leukemia cells HL-60 were treated with etoposide or cytochalasin B to induce apoptosis or necrosis, respectively. We report here that during necrosis, the DNA-repair associated nuclear enzyme poly(ADP-ribose) polymerase (PARP) was degraded differently from that observed during apoptosis. While apoptotic HL-60 cells exhibit only the signature 89 kDa fragment of PARP, necrosis of these cells was accompanied by formation of major fragments at MWr approximately 89 and 50 kDa and minor fragments at approximately 40 and 35 kDa. The necrosis-specific degradation of PARP was coincident with other changes detected by flow cytometric analysis, but earlier than the extensive degradation of DNA. Therefore, the unique necrotic degradation of PARP could be used as a sensitive indicator for necrotic death of cells.

Episodic release of luteinizing hormone and testosterone in Surti and Marwari bucks younger than one year of age.

Studies on the episodic release of luteinizing hormone (LH) and testosterone in Surti (n=2) and Marwari (n=2) bucks younger than one year of age (6 to 8 months) were carried out by collecting blood plasma during the breeding season. The studies revealed that definite pulsatile releases of LH and testosterone occur in both breeds of bucks. The overall number of LH and testosterone pluses over a 24 hour period was 9.1+/-1.00 and 7.5+/-0.28, respectively. The peak, basal and mean LH and testosterone concentrations did not show significant differences between the two breeds. The duration and interval of LH and testosterone pulses differed during light and dark hours. The time interval between LH peak followed by the testosterone peak was significantly (P<0.05) longer during the night than the day hours for both the breeds. The physiological basis of the findings are discussed.

Evidence for the presence of an antitumor factor in serum of normal animals.

We have previously reported finding a factor with antitumor activity (TNF, tumor necrosis factor) in extracts of serum from normal mice. The possibility that TNF exists in the blood of normal animals of other species was explored. Horse, mouse, dog, human, sheep, calf, rat and shark serums were fractionated with (NH4)2SO4 and filtered through S-200 sephacryl gel. Proteins of molecular weight 90,000 to 180,000 were pooled, concentrated and dialyzed. TNF, determined by L-cell assay in vitro and Meth A assay in vivo found in fractions from mouse, dog and human serum. Agarose electrophoresis of the TNF from mouse and human serum indicated the principle components were alpha 1-alpha 2 globulins. Preparative PAGE indicated that mouse TNF migrated slowly and was made up of at least 4 components while human TNF was a faster moving, monomeric protein.