Immunohistochemical identification of tubular segments in percutaneous renal biopsies

Summary To identify the renal cortical tubular segments involved in tubulo-interstitial disease in formalin-fixed, paraffin-embedded percutaneous kidney biopsies, we developed multiple immunolabeling protocols using segment-specific tubular markers. The present study of biopsies from patients with minimal change or thin basement membrane nephropathy provides a baseline for interpretation of histopathology. Proximal tubules were stained either by the PAS reaction or by the biotinylated Phaseolus vulgaris erythroagglutinin (PHA-E)-streptavidin-gold-silver system (brush borders black). The anti-Tamm-Horsfall (THP) antibody-immunoperoxidase (aminoethylcarbazole, AEC-IPO), and anti-epidermal cytokeratins (ECK) antibodies-immunoalkaline-Fast Blue BB methods marked the distal straight tubules and the cortical collecting system red-brown and blue, respectively. When these immunolabelings were combined, the coapplication of AEC-PO-labeled peanut agglutinin (PNA) or anti-epithelial membrane antigen antibody-AEC-IPO technique (both are markers for distal nephron) visualized the apical membranes of distal convoluted tubules. In the protocol PHA-E + PNA + THP + ECK, the tubular basement membranes were outlined by the anti-laminin antibody-AEC-IPO staining, carried out simultaneously. The protocol PNA + THP + ECK + PAS was found to be a quite appropriate multiple immunolabeling method for the tubules, and is recommended for use as a tool in the study of tubulo-interstitial diseases.Abbreviations PAS periodic acid-Schiff reaction - PHAE Phaseolus vulgaris erythroagglutinin - PNA Peanut agglutinin - EMA epithelial membrane antigen - THP Tamm-Horsfall glycoprotein - ECK epidermal cytokeratins - PO peroxidase - Biot-PHA-E biotinylated PHA-E - APAAP complexes of alkaline phosphatase and mouse monoclonal anti-alkaline phosphatase - SWARI swine anti-rabbit immunoglobulins - FCS fetal calf serum - TBS Tris-buffered saline - AEC aminoethylcarbazole - DAB diaminobenzidine - FBBB Fast Blue BB - IA immunoalkaline - GL glomerulus - PT proximal tubule - DST distal straight tubule - DCT distal convoluted tubule - CCS cortical collecting system - CT connecting tubule - CD collecting duct     

Inhibition of the restriction endonuclease BanII using modified DNA substrates. Determination of phosphate residues critical for the formation of an active enzyme-DNA complex

The restriction endonuclease BanII catalyzes the cleavage of double-stranded DNA and recognizes the degenerate sequence 5'-GPuGCPyC-3'. The poly-linker of M13mp18 contains one such sequence, 5'-GAGCTC-3'. The three other possible sites recognized by the enzyme were prepared by site-directed muta-genesis. The substitution of phosphate groups by phosphorothioate residues at some positions within the various recognition sites had relatively little effect on the rate of cleavage of the DNA. However, when the DNA contained a phosphorothioate group at the site of cleavage the rate of linearization of the DNA was decreased by a factor of 9. Interestingly, DNA which contained an additional phosphorothioate internucleotidic linkage immediately 3'-outside the recognition site could not be linearized by the enzyme. The results indicate that an important contact between enzyme and substrate is perturbed by the presence of the sulfur atom at this position.     

Wood excavations of three species of subterranean termites

We compared the feeding excavations on wood blocks of three species of subterranean termites, Coptotermes formosanus Shiraki, Reticulitermes flavipes (Kollar), and R. virginicus (Banks). Feeding rate followed the order C. formosanus > R. flavipes > R. virginicus. Wood surface area (mm²) exposed per unit feeding was higher for C. formosanus and R. flavipes than for R. virginicus. This was caused by the tendency of C. formosanus and R. flavipes to make internally penetrating tunnels, thereby increasing surface area, whereas R. virginicus made trough- and bowl-like depressions on the outside of blocks, sometimes decreasing the size of blocks outwardly without a corresponding high increase in surface area typical with the tunnels of the other species. Consequently, wood surface area was sometimes reduced, rather than increased as a result of feeding by R. virginicus. Different patterns of wood excavation suggest that these termites have divergent roles in wood decay processes.

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Résumé Les organismes pionniers qui modifient le bois et le rendent acceptable par les insectes qui le perforent sont généralement des champignons du bois pourri. Cependant, une fois que les termites ou autres insectes perforant le bois ont pénétré, leurs galeries favorisent les bactéries fixatrices d'azote, permettent l'invasion d'autres organismes décomposeurs, et de ce fait régularisent la décomposition du bois (Ausmus, 1977). L'exposition de la surface à l'intérieur des perforations jouant un rôle très important dans le processus de pourrissement, il est souhaitable de pouvoir quantifier la surface des galeries dues à l'alimentation des termites. Une courbe type permettant de prédire l'aire de la surface perforée a été construite en perçant 109 morceaux de bois de trous cylindriques de différents diamètres, en calculant l'aire de la surface des morceaux de bois, en appliquant et pesant une couche de vernis pour bois au polyuréthane, et en divisant la masse de polyuréthane par l'aire de la surface. Le modèle prédictif qui en découle est: Y=0,01443×-3,51825 (P=0,0001; r=0,68), y étant la masse de polyuréthane (en g) et x la surface (en mm²) du morceau de bois. En traitant de la même façon au polyuréthane les morceaux de bois perforés par les termites, nous pourrions déduire leur surface.Une expérience a été effectuée avec 3 espèces de rhinotermitides,- Coptotermes formosanus Shiraki, Reticulitermes flavipes (Kollar) et R. virginicus (Banks). Des groupes de chaque espèce se sont alimentés pendant 11 ou 12 jours sur des morceaux de bois non contaminés par des champignons. Nous avons déterminé la survie, la consommation, la modification de la surface du morceau de bois (par utilisation du modèle prédictif) et le changement de surface par terminte.La survie est la même, mais la consommation est dans l'ordre suivant: C. formosanus > R. flavipes > R. virginicus. L'aire de la surface exposée par unité d'alimentation était plus élevée pour C. formosanus et R. flavipes que pour R. virginicus (Tab. 1). Ceci est dû à la tendance de C. formosanus et R. flavipes de creuser des galeries vers l'intérieur, tandis que R. virginicus fait des cuvettes à la surface du bois. Les attaques superficielles de R. virginicus réduisent parfois le volume du morceau de bois sans accroître proportionnellement la surface comme le font les espèces creusant des galeries. Ainsi, avec R. virginicus la surface peut être réduite au lieu d'augmenter. Des différences entre colonies s'observent avec toutes les variables (Tab. 2).Nos résultats suggèrent que C. formosanus et R. flavipes contribuent plus que R. virginicus à exposer le bois aux autres organismes décomposeurs. Cependant, ces résultats peuvent être modifiés par un conditionnement préalable du bois par des champignons.

     

Carbon-13 NMR relaxation studies of pre-melt structural dynamics in [4-13C-uracil] labeled E. coli transfer RNAIVal.

We report 67.8 MHz carbon-13 spin-lattice relaxation studies on [4-13C-uracil] labeled tRNAIVal purified from E. coli SO-187. Following 13C-enriched C4 carbonyl resonances from modified and unsubstituted uridines scattered throughout the polymer backbone enables us to determine dynamical features in both loop and helical stem regions. The experimental results have been analyzed in terms of a model of isotropic overall molecular reorientation. "Anomalous" residues for which the experimental data cannot be accounted for in terms of the model provide an assessment of local and regional properties. Thus, "native" tRNAIVal under physiological conditions of magnesium (10 mM) and temperature (20 degrees - 40 degrees C), exhibits the following characteristics: 1) uridines held rigidly in helical stems and tertiary interactions display correlation times for rotational reorientation of 15-20 nsecs, typical for overall tRNA motion; 2) uridines in loops such as the wobble residue uridine-5-oxyacetic acid (V34) are quite accessible to solvent; moreover V34 and another loop residue, D17, exhibit local mobility; 3) the tertiary interactions involving 4-thio uridine (s4U8) and A14 and ribothymidine (rT54) and A58 are weakened as temperature increases.     

Specificity in the inactivation of enzymes by periodate-oxidized nucleotides

Periodate-oxidized ATP (oATP) inactivates the partial reaction of aminoacyl-tRNA synthetases in which amino acid is transferred to tRNA without altering the other partial reaction in which ATP is a substrate or a product. The inactivation has been shown to be nonspecific with regard to substituents on the dialdehyde and with regard to the enzymes susceptible to inactivation; oxidized GTP and oxidized uridine react as well as oATP with aminoacyl-tRNA synthetases and all three dialdehydes also inactivate rabbit muscle aldolase.     

A regression analysis of the summer population dynamics of Polyarthra vulgaris in a northern Michigan bog lake

The population dynamics of a planktonic rotifer (Polyarthra vulgaris) were examined in a brown water, acid lake in northern Michigan, U.S.A. Predation by Chaoborus punctipennis and low food (Navicula spp. and Cyclotella spp.) concentrations were the main factors limiting P. vulgaris populations of all factors examined. The data presented here support a hypothesis for zooplankton limitation by an invertebrate predator.     

Dynamic geometry of the intact left ventricle

Knowledge of left ventricular chamber dynamics is central to our understanding of cardiac physiology. The complicated changes in left ventricular geometry observed in the dog during various phases of the cardiac cycle can be represented as distinct linear relationships between chamber eccentricity and intracavitary volume during diastole and ejection, and probably represent structural properties of the ventricular wall. Chamber geometry of the left ventricle is a major determinant of overall myocardial function. The slope of the radius of curvature (r) to wall thickness (h) relationship is a geometric constant that determines the mural force at any given transmural pressure. Chronic pressure and volume overload produce changes in this geometric relationship as a result of increased mural force resisting ejection. The adaptive mechanism of ventricular hypertrophy in this setting alters the r/h ratio and returns systolic mural force toward normal. Coronary occlusion induces acute changes in regional geometry characterized by holosystolic wall bulging and systolic wall thinning, which shift the r/h relationship upward and to the left. The geometric alteration during ischemia probably increases systolic mural force and could adversely affect myocardial function. Recent studies with patients have shown the r/h ratio to be of value in distinguishing between reversible and irreversible impairment of myocardial performance. Because most myocardial diseases produce major alterations in the structure of the ventricular wall, analysis of dynamic chamber geometry may prove of prognostic value in assessing patients with cardiac disorders.