A starting place for the road to function

Published in 2003.     

A microfabricated array bioreactor for perfused 3D liver culture

We describe the design, fabrication, and performance of a bioreactor that enables both morphogenesis of 3D tissue structures under continuous perfusion and repeated in situ observation by light microscopy. Three-dimensional scaffolds were created by deep reactive ion etching of silicon wafers to create an array of channels (through-holes) with cell-adhesive walls. Scaffolds were combined with a cell-retaining filter and support in a reactor housing designed to deliver a continuous perfusate across the top of the array and through the 3D tissue mass in each channel. Reactor dimensions were constructed so that perfusate flow rates meet estimated values of cellular oxygen demands while providing fluid shear stress at or below a physiological range (<2 dyne cm(2)), as determined by comparison of numerical models of reactor fluid flow patterns to literature values of physiological shear stresses. We studied the behavior of primary rat hepatocytes seeded into the reactors and cultured for up to 2 weeks, and found that cells seeded into the channels rearranged extensively to form tissue like structures and remained viable throughout the culture period. We further observed that preaggregation of the cells into spheroidal structures prior to seeding improved the morphogenesis of tissue structure and maintenance of viability. We also demonstrate repeated in situ imaging of tissue structure and function using two-photon microscopy.     

The p11 subunit of annexin II heterotetramer is regulated by basic carboxypeptidase

The Ca(2+)-dependent phospholipid-binding protein annexin II heterotetramer (AIIt) is composed of two copies of annexin II and a p11 dimer. The interaction of the carboxyl-terminal lysine residues of the p11 subunit of AIIt with the lysine-binding kringle domains of plasminogen is believed to play a key role in plasminogen binding and stimulation of the tPA-catalyzed cleavage of plasminogen to plasmin. In the current report, we show that AIIt-stimulated plasminogen activation is regulated by basic carboxypeptidases, in vitro. The incubation of AIIt with a 1/400 molar ratio of carboxypeptidase B for periods as short as 2 min resulted in a significant loss in AIIt-stimulated plasminogen activation. Carboxypeptidase B (CpB) as well as thrombin-activated fibrinolysis inhibitor (TAFIa) and carboxypeptidase N (CpN) rapidly reduced AIIt-stimulated plasminogen activation by 80%. The molar ratio of carboxypeptidase/AIIt for half-maximal inhibition of AIIt was 1/4700, 1/700, and 1/500 for CpB, TAFIa, and CpN, respectively. Treatment of AIIt with carboxypeptidase resulted in loss of both carboxyl-terminal lysine residues from the p11 subunit, which correlated with a decrease in the k(cat) and an increase in the K(m) for plasminogen activation. The data reveal a novel mechanism for the regulation of AIIt-stimulated plasminogen activation.     

A mathematical approach towards nitrogen cycling in arable land inCajanus cajan field

Summary A balance sheet of nitrogen has been presented for an arable land in aCajanus cajan field. Quantitative inventory and transfer rates are suggested for several compartments. On the basis of data, it has been concluded that due to microbial activities appreciable nitrogen was returned to the available nitrogen pool by means of root and litter disappearance. Twenty-five percent of the total soil nitrogen has been taken up by fine roots, out of which 63.39 percent nitrogen locked up in the form of organic nitrogen and the rest of nitrogen (36.61%) was being added to the soil which governed the soil fertility. Transfer matrix of process model for the nitrogen transformation explaining component transferred from one compartment to another compartment and groups responsible for this transformation are dealt with. By means of several proposed equations the amount of nitrogen in soil at different intervals of time can be predicted. The quantifications of microbial transformations and their role in nitrogen turnover has been exploited. A high nitrogen content in the original material was found to promote decomposition.     

Role of an indigenous drug geriforte on blood levels of biogenic amines and its significance in the treatment of anxiety neurosis

In the recent past, several herbal drugs have proved their efficacy in decreasing anxiety and tension. Until recently, limited scientific study has been done to prove the clinical importance of these herbal drugs. Geriforte is an indigenous compound commonly advocated for arresting the aging process. This remedy has also been found beneficial in reducing anxiety and stress.-34 diagnosed cases of anxiety neurosis and 24 apparently normal individuals were selected for the present study. After 12 weeks of drug therapy a significant reduction in 5-HT levels was observed. Circulating MAO and GABA showed an increasing trend along with decreased glutamic acid levels after treatment, in the neurotic anxiety cases. Psychological complaints considerably decreased after 12 weeks of therapy. Since Geriforte regulates the altered circulating biogenic amines, this remedy can be advocated as an adjunctive therapy in the management of stress disorders.     

Actomyosin dynamics modulate microtubule deformation and growth during T-cell activation

Activation of T-cells leads to the formation of immune synapses (ISs) with antigen-presenting cells. This requires T-cell polarization and coordination between the actomyosin and microtubule cytoskeletons. The interactions between these two cytoskeletal components during T-cell activation are not well understood. Here, we elucidate the interactions between microtubules and actin at the IS with high-resolution fluorescence microscopy. We show that microtubule growth dynamics in the peripheral actin-rich region is distinct from that in the central actin-free region. We further demonstrate that these differences arise from differential involvement of Arp2/3- and formin-nucleated actin structures. Formin inhibition results in a moderate decrease in microtubule growth rates, which is amplified in the presence of integrin engagement. In contrast, Arp2/3 inhibition leads to an increase in microtubule growth rates. We find that microtubule filaments are more deformed and exhibit greater shape fluctuations in the periphery of the IS than at the center. Using small molecule inhibitors, we show that actin dynamics and actomyosin contractility play key roles in defining microtubule deformations and shape fluctuations. Our results indicate a mechanical coupling between the actomyosin and microtubule systems during T-cell activation, whereby different actin structures influence microtubule dynamics in distinct ways.     

Quantification of arbuscular mycorrhizal fungi activity by the glomalin concentration on hyphal traps

 Strips of horticultural film (16–32 cm²) were used to trap extraradical hyphae emanating from roots of sudangrass [Sorghum sudanense (Piper) Staph] enclosed in 40-μm mesh bags and colonized by Gigaspora rosea FL 224-1, Glomus intraradices EY 113/114, or Glomus caledonium UK 301-1. Strips of film were placed at opposite sides of 17–21 replicate sand culture pots for each isolate and were removed after 12–14 weeks of plant growth. To extract glomalin, a strip was cut into small pieces and submerged in 2 ml of 20 mM citrate, pH 7.0 and then autoclaved for 60 min. A quantitative enzyme-linked immunosorbent assay (ELISA) detected 0.005–0.04 μg glomalin in the volume of extract tested. The Bradford protein assay detected 1.25–5 μg of protein in the volume of extract tested. Both assays gave results ranging from 5–40 μg glomalin/cm² of film. Protein assay values were correlated with ELISA values (r=0.6091, P≤0.001, n=118). Analysis of variance indicated that isolates differed in Bradford protein values (P=0.001), but not ELISA values (P=0.154). Spatial variability of glomalin deposition ca. 7 cm from roots on opposite sides of pots was indicated by significant paired T tests (P<0.05) for protein values for each of the three isolates and ELISA for two isolates. These results indicate that hyphal traps, Bradford protein assay and ELISA are useful to assess hyphal activity over a growing season. Accepted: 11 October 1998     

Comparative analysis of the extracellular proteomes of two Clostridium sordellii strains exhibiting contrasting virulence

Clostridium sordellii is a toxin-producing anaerobic bacillus that causes severe infections in humans and livestock. C. sordellii infections can be accompanied by a highly lethal toxic shock syndrome (TSS). Lethal toxin (TcsL) is an important mediator of TSS. We recently obtained a clinical strain of C. sordellii (DA-108) lacking the TcsL-encoding tcsL gene, which was not fatal in rodent models of infection, in contrast to a tcsL⁺ reference strain (ATCC9714). Protein preparations derived from cell-free, stationary phase cultures obtained from ATCC9714 were lethal when injected into mice, while those obtained from DA-108 were not, a difference that was attributed to the unique presence of TcsL in the ATCC9714-derived proteins. We questioned whether there were other major differences between the extracellular proteomes of these two strains, apart from TcsL. Two-dimensional gel electrophoresis was conducted using crude cell-free supernatants from these strains and 14 differentially expressed proteins were subjected to mass spectrometric analysis. Nine of these 14 proteins were more highly expressed by DA-108 and 5 by ATCC9714. Twelve of the 14 proteins isolated from the 2-D gels were putatively identified by mass spectrometry. Several of these proteins were identical, possibly reflecting enzymatic cleavage, degradation, and/or post-translational modifications. Excluding identical sequences, only 5 unique proteins were identified. Four proteins (ferredoxin–nitrite reductase; formate acetyltransferase; Translation Elongation Factor G; and purine nucleoside phosphorylase) were over-expressed by DA-108 and 1 (N-acetylmuramoyl-l-alanine amidase) by ATCC9714. These results support the concept that TcsL is the major determinant of C. sordellii TSS during infection.     

Aquaculture planning for suspended bivalve farming systems: The integration of physiological response of green mussel with environmental variability in site selection

Bivalve Condition Index (CI) is widely employed in environmental monitoring programmes as it integrates physiological responses to stress with changes in somatic growth. Besides indicating, the commercial quality of a bivalve population it may be also be used to compare the relative health of animals in nearby populations. In this study, CI of green mussels, Perna viridis was evaluated as the intrinsic response to the variations in the environment in two potential mussel mariculture sites in the tropical monsoon region with diverse remoteness to riverine outflow. Condition index of mussels from site with relatively higher riverine influence (RI) was compared with mussels from area of reduced riverine (RR) influence along Karnataka coast (Eastern Arabian Sea). The dominant patterns of spatial trends in 12 environmental variables (temperature, salinity, pH, dissolved oxygen, rainfall, chlorophyll-a (chl-a), suspended particulate matter (SPM) particulate organic matter (POM), particulate inorganic matter (PIM), POM/SPM, chla/POM and PIM%) of the sites were resolved by Principal Component Analysis (PCA). The stepwise multiple regression analysis related the spatial variability in CI to variations in water temperature and organic content of the seston (chlorophyll-a and particulate organic matter). The discriminant analysis performed with monthly mean water temperature, chl-a levels and CI ratio [high CI ratio (CI_high) and low CI ratio (CI_low)] indicated that 83.9% of the CI_high was associated with high chl-a and high water temperature group whereas, 72.4% of CI_low were associated with low chl-a and low water temperature regime. The RR site presented less variation in environmental parameters offering a more conducive environment for the growth of mussels, characterised by better CI with low seasonal variations. Whereas, in the site proximate to riverine discharge, reserves were alternatively channelled into energy-consuming processes and hence unfavourable environmental conditions showed poor tissue condition due to utilization of energy reserves which were potentially destined for growth. Thus, average CI, besides its representation of the general health condition of the bivalves can potentially be used in selecting suitable sites for bivalve mariculture in comparable hydrological environment.