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Background: Prescreening of biopsies has the potential to improve pathologists' workflow. Tools that identify features and display results in a visually thoughtful manner can enhance efficiency, accuracy, and reproducibility. Machine learning for detection of glomeruli ensures comprehensive assessment and registration of four different stains allows for simultaneous navigation and viewing., Methods: Medical renal core biopsies (4 stains each) were digitized using a Leica SCN400 at ×40 and loaded into the Corista Quantum research platform. Glomeruli were manually annotated by pathologists. The tissue on the 4 stains was registered using a combination of keypoint- and intensity-based algorithms, and a 4-panel simultaneous viewing display was created. Using a training cohort, machine learning convolutional neural net (CNN) models were created to identify glomeruli in all stains, and merged into composite fields of views (FOVs). The sensitivity and specificity of glomerulus detection, and FOV area for each detection were calculated., Results: Forty-one biopsies were used for training (28) and same-batch evaluation (6). Seven additional biopsies from a temporally different batch were also evaluated. A variant of AlexNet CNN, used for object recognition, showed the best result for the detection of glomeruli with same-batch and different-batch evaluation: Same-batch sensitivity 92%, "modified" specificity 89%, average FOV size represented 0.8% of the total slide area; different-batch sensitivity 90%, "modified" specificity 98% and average FOV size 1.6% of the total slide area., Conclusions: Glomerulus detection in the best CNN model shows that machine learning algorithms may be accurate for this task. The added benefit of biopsy registration with simultaneous display and navigation allows reviewers to move from one machine-generated FOV to the next in all 4 stains. Together these features could increase both efficiency and accuracy in the review process., (Copyright: © 2020 Journal of Pathology Informatics.)

Telokin is identical in sequence to the C-terminal portion of myosin light chain kinase but is expressed independently. We have used monoclonal antibodies specific to the non-telokin portion of myosin light chain kinase and to telokin, immunofluorescence microscopy and image reconstruction to demonstrate the presence of telokin in cardiac myocytes and to study its subcellular distribution. Antibodies to telokin labeled the intercalated discs of adult cardiac myocytes and similar structures in isolated intercalated disc preparations. Antibodies specific to the non-telokin portion of myosin light chain kinase did not label intercalated discs in either of these preparations. Western blots of isolated intercalated discs with anti-telokin revealed a 23kDa protein that co-migrates with purified telokin on SDS-PAGE. Deconvolution, reconstruction and analysis of fluorescence images of isolated intercalated discs labeled with anti-telokin and anti-beta-catenin, anti-gamma-catenin or anti-connexin43 indicated that telokin is only partially co-localized with these proteins at the discs.

Caldesmon is a heat-stable protein found in both muscle and non-muscle tissue. It binds to a number of contractile and cytoskeletal proteins and may be involved in regulating acto-myosin interaction in smooth muscle cells and/or the assembly of microfilaments in muscle and non-muscle cells. We have shown previously that caldesmon is localized at the Z-lines in adult cardiac myocytes and that both the low- and high-molecular-weight forms (/-caldesmon and h-caldesmon, respectively) are present in atrial and ventricular myocytes. Here we examined the expression of caldesmon and its localization in freshly isolated cardiac myocytes during postnatal development and when these myocytes were grown in culture. We found that /-caldesmon is expressed in both neonatal and adult rat ventricular myocytes. The expression of h-caldesmon, however, was not detected in myocytes from newborn animals but increased during the first 2 weeks of postnatal development. Caldesmon was generally not co-localized with alpha-actinin at the Z-lines in neonatal myocytes but became increasingly more so during the first 2 weeks of postnatal development. When myocytes from 5- and 10-day-old rats were grown in primary culture, h-caldesmon expression decreased and caldesmon could not be detected at the Z-lines in the cultured cells. These results indicate that caldesmon plays a role at the Z-lines in adult cardiac myocytes; however, its localization at the Z-lines is not necessary for the prenatal development that occurs at these sites or for the establishment of a contractile phenotype in cultured cardiac myocytes.

A Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart can be eluted from a DEAE-cellulose column either in the free form by buffers containing 0.1 mM ethylene glycol bis(beta-aminoethyl ether)N-N,N'N'-tetraacetic acid (EGTA) or as a complex of the enzyme with its protein modulator by buffers containing 0.01 mM CaCl2. A purification procedure based primarily on the significantly different affinity of the two forms of the enzyme for DEAE-cellulose was developed for the purification of the enzyme from bovine heart. The procedure involves ammonium sulfate fractionation, three chromatographic steps on DEAE-cellulose, and gel filtration on Sephadex G-200 with a 5000-fold purification over the crude extract. The purified enzyme has a specific activity of 120 mumol of cAMP/mg/min, can be activated 5-fold by Ca2+, but is only 80% pure as judged by analytical disc gel electrophoresis. The purified enzyme is unstable but can be stabilized by addition of Ca2+ and the protein modulator; this is in contrast to the less pure preparations of Ca2+-activatable phosphodiesterase which are destabilized by the protein modulator in the presence of Ca2+.

Calmodulin-dependent cyclic nucleotide phosphodiesterase was purified from bovine brain to apparent homogeneity by a new procedure involving DEAE-cellulose, Affi-Gel blue, calmodulin-Sepharose 4B, and Sephadex G-200 column chromatographies. The enzyme was purified more than 3,000-fold from the brain extracts with greater than 12% yield. The purified phosphodiesterase could be activated 10- to 15-fold by calmodulin and Ca2+ to a specific enzyme activity of more than 300 mumol of cAMP hydrolyzed/min/mg of protein. Molecular weight of the enzyme was determined to be 115,800 by the sedimentation equilibirum method or 124,000 from the sedimentation constant and Stokes radius of the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight of 58,000. These results suggested that the calmodulin-dependent phosphodiesterase from bovine brain has a subunit structure of alpha2. Molecular weight of the complex of calmodulin and phosphodiesterase was the complex of calmodulin and phosphodiesterase was also calculated from the sedimentation constant and Stokes radius to be 159,000. Since calmodulin has a molecular weight of about 17,000, the result indicated that the stoichiometry of the complex is calmodulin2 alpha2. The catalytic subunit of cylic AMP-dependent protein kinase was found to catalyze the phosphorylation of the purified phosphodiesterase with the incorporation of 2 mol of phosphate/mol of the enzyme.

An inhibitor protein of cyclic nucleotide phosphodiesterase is demonstrated in bovine brain extract and separated from modulator binding protein, a recently discovered inhibitory factor of phosphodiesterase. The new inhibitor protein is similar to the cyclic AMP phosphodiesterase inhibitor from bovine retina (Dumler, I. L., and Etingof, F. N. 1976) Biochim. Biophys, Acta 429, 474-484) in its heat stability: it retains full activity upon heating in a boiling water bath for 2 min. The new inhibitor protein counteracts the activation of the Ca2+-activatable cyclic nucleotide phosphodiesterase by the Ca2+-dependent modulator protein without affecting the basal activity of the enzyme. The inhibition of phosphodiesterase by the inhibitor can be reversed by high concentrations of modulator protein but is not influenced by a 20-fold increase in Ca2+ concentration. In contrast, a Ca2+-independent form of cyclic nucleotide phosphodiesterase is not inhibited by the inhibitor protein. These results suggest that the heat-stable inhibitor protein is specific against the action of the Ca2+-dependent modulator protein. Gel filtration analyses on Sephadex G-75 and G-100 columns have shown that the inhibitor protein and the modulator protein may associate in the presence of Ca2+. The molecular weights determined by the gel filtration for the free inhibitor protein and the complex of the inhibitor and modulator protein are about 70,000 and 85,000, respectively.

Glycogen phosphorylase b modified by NaBH4 and aliphatic aldehydes of varying chain length: ranging from acetaldehyde to n-heptanaldehyde were purified by heat-treatment. Kinetic studies showed that the various purified enzyme derivatives were similar to native phosphorylase b in kinetic properties with respect to glucose-I-P. They, however, exhibited different affinities toward AMP. For acetaldehyde, propionaldehyde and butyraldehyde modified phosphorylases b, increase in chain length of the modifying aldehyde resulted in a decrease in AMP affinity of the modified enzyme. For the other aldehydes increase in chain length resulted in an increased AMP affinity of the modified enzyme. Consequently, the apparent values of butyraldehyde and heptanaldehyde modified phosphorylase b exhibited greatest deviation from that of native phosphorylase b. These two enzyme derivatives were studied in more detail. The activation of both enzyme derivatives by AMP could be greatly stimulated by spermine at suboptimal levels of AMP. Both derivatives could be activated by IMP, UMP or CMP in addition to AMP. The extents of activation of heptanaldehyde modified phosphorylase b by these nucleotides were higher than those found for native phosphorylase b. Thus, under certain conditions which included using suboptimal AMP or using IMP, UMP or GMP instead of AMP, heptanaldehyde modified phosphorylase b had much higher catalytic activity than native phosphorylase b. The interactions between burtyraldehyde or heptanaldehyde modified phosphorylases b with IMP were respectively weaker or stronger than that between native phosphorylase b and IMP. The interactions between butyraldehyde or heptanaldehyde modified phosphorylase b with glucose-6-P, an inhibitor of phosphorylase b partially competitive with respective to AMP, were also respectively weaker or stronger than that between the inhibitor and the native enzyme.