Characterization of a heat-stable NADP-dependent isocitrate dehydrogenase from the obligate thermophile Thermoleophilum minutum YS-4

Summary A thermostable NADP-dependent isocitrite dehydrogenase (IDH; EC. 1.1.1.42) was purified from the obligately thermophilic hydrocarbonoclastic bacterium Thermoleophilum minutum YS-4 (ATCC 35265). This was accomplished by affinity chromatography and electroelution from a nondenaturing polyacrylamide gel. The enzyme has an M _r of 60 000 and is composed of two identical subunits of M _r 30 500. The amino acid composition has an Arg/Lys ratio of 4:1 and very high levels of glycine. Under nondenaturing conditions, the enzyme has a distinct difference in electrophoretic mobility relative to IDHs obtained from other genera including the genus Thermus. The secondary strcuture consists of 16% -helix, 20% -sheet, 25% -turn and 37% random coil as determined by circular dichroism spectroscopy. The optimum pH and temperature for activity were 7.2 and 75° C respectively and the apparent K _mvalues for DL-isocitrate adn NADP⁺ were 33 M, and 48 M, respectively. The enzyme requires divalent cations, such as Mn²⁺ or Mg²⁺ for activity. NAD⁺ cannot substitute for NADP⁺. Oxaloacetate plus glyoxylate exert considerable inhibition on IDH activity while other glycolytic and tricarboxylic acid cycle intermediates have a lesser effect. p-Chloromercuribenzoic acid was inhibitory to the IDH although isocitrate and Mn²⁺ offered some protection from this inactivation. The enzyme is thermostable, retaining 84% and 57% of initial activity after incubation for 1 h at 60° and 70° C, respectively. Isocitrate provided protection from thermal inactivation allowing the IDH to maintain 21% activity after 1 h at 80° C. Offprint requests to: J. J. Perry     

The interaction of troponin-I with the N-terminal region of actin

The interaction between troponin-I and actin that underlies thin-filament regulation in striated muscle has been studied using proton magnetic resonance spectroscopy. A restricted portion of skeletal muscle troponin-I (residues 96-116) has previously been shown to be capable of inhibiting the MgATPase activity of actomyosin in a manner enhanced by tropomyosin [Syska et al. (1976) Biochem. J. 153, 375-387]. On the basis of homologous spectral effects for signals of specific groups observed in different complexes formed using the native proteins and a variety of defined peptides, it is concluded that the segment of troponin-I which has inhibitory activity interacts with the N-terminal region of actin. The surface of contact of the inhibitory segment of troponin-I with actin involves two regions of the N-terminal of actin. These are located between residues 1-7 and 19-44. The data are discussed in the context of a structural mechanism for the inhibition of myosin ATPase activation.     

Fine needle aspiration biopsy of metastatic melanoma. A morphologic analysis of 174 cases

The prognostic and therapeutic decisions in cases of metastatic melanoma depend upon the morphologic documentation of metastatic disease, which may rapidly and accurately be done by fine needle aspiration (FNA) biopsy of clinically suspicious lesions. The tumor cells derived from malignant melanomas demonstrate a wide range of appearances, however, and other neoplasms may be mimicked. Furthermore, additional neoplasms of other types are more frequent in melanoma patients: the possibility of a new primary tumor must be considered if the morphology of the tumor cells is uncharacteristic. Therefore, a study was undertaken to analyze the morphologic changes seen in FNA biopsy specimens from metastatic malignant melanoma and to determine which features could be the most useful in establishing a definitive diagnosis. A total of 174 consecutive cases, comprising 151 malignant aspirates and 23 inconclusive aspirates, were reviewed. The most significant features for identification of melanoma over other tumor types were the cell shape and nuclear position, the presence of numerous isolated neoplastic cells and occasional binucleated or multinucleated cells. Intracellular melanin in neoplastic cells was diagnostic when present, but it was absent in 60% of the cases. Macronucleoli and/or intranuclear cytoplasmic invaginations were characteristic but variable features. Morphology was also found to vary by site and cell type. Lung aspirates were less cellular and more likely to contain melanin. Aspirates of subcutaneous nodules were more often composed of spindle-shaped cells or of other variant cell types. Lymph node aspirates more often yielded epithelioid cells with macronucleoli and/or intranuclear invaginations. Spindle-cell melanomas usually demonstrated inconspicuous nuclei and rarely showed enlarged nucleoli. Epithelioid-cell tumors contained multinucleated cells and areas of cell wrapping more frequently than did spindle-cell tumors. The findings in this study emphasize that a full awareness of the spectrum of morphologic presentations of metastatic melanoma as well as of the clinical history are needed for greater precision in its diagnosis and for avoidance of the pitfall of misdiagnosing nonmelanomas with similar appearances.     

Stimulation by epinephrine of the membrane transport of long chain fatty acid in the adipocyte

In isolated rat adipocytes, epinephrine rapidly stimulates the transport of long chain fatty acid across the plasma membrane. At a concentration of unbound oleate of 0.1 microM ([fatty acid]/[albumin] = 1) and 5 min exposure to the hormone, the minimal effective concentration of epinephrine is 0.03 and the optimal concentration 0.3 microM (0.01 and 0.1 microgram/ml). The stimulated rates are 5-10-fold the basal rate of influx or efflux. The hormone effect is on the transport process specifically as shown by isolation of the product of transport in either direction as unesterified fatty acid and inhibition by the transport inhibitors phloretin and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. This effect of epinephrine on transport coordinates physiologically with lipase activation to bring about fatty acid release from adipose tissue.     

Changes in Protein Content of Goldfish Optic Nerve During Degeneration and Regeneration Following Nerve Crush

Abstract: After the goldfish optic nerve was crushed, the total amount of protein in the nerve decreased by about 45% within 1 week as the axons degenerated, began to recover between 2 and 5 weeks as axonal regeneration occurred, and had returned to nearly normal by 12 weeks. Corresponding changes in the relative amounts of some individual proteins were investigated by separating the proteins by two-dimensional gel electrophoresis and performing a quantitative analysis of the Coomassie Brilliant Blue staining patterns of the gels. In addition, labelling patterns showing incorporation of [³H]proline into individual proteins were examined to differentiate between locally synthesized proteins (presumably produced mainly by the glial cells) and axonal proteins carried by fast or slow axonal transport. Some prominent nerve proteins, ON1 and ON2 (50–55 kD, pI ～6), decreased to almost undetectable levels and then reappeared with a time course corresponding to the changes in total protein content of the nerve. Similar changes were seen in a protein we have designated NF (～130 kD, pI ～5.2). These three proteins, which were labelled in association with slow axonal transport, may be neurofilament constituents. Large decreases following optic nerve crush were also seen in the relative amounts of α- and β-tubulin, which suggests that they are localized mainly in the optic axons rather than the glial cells. Another group of proteins, W2, W3, and W4 (35–45 kD, pI 6.5–7.0), which showed a somewhat slower time course of disappearance and were intensely labelled in the local synthesis pattern, may be associated with myelin. A small number of proteins increased in relative amount following nerve crush. These included some, P1 and P2 (35–40 kD, pIs 6.1–6.2) and NT (～50 kD, pI ～5.5), that appeared to be synthesized by the glial cells. Increases were also seen in one axonal protein, B (～45 kD, pI ～4.5), that is carried by fast axonal transport, as well as in two axonal proteins, HA1 and HA2 (～60 and 65 kD respectively, pIs 4.5–5.0), that are carried mainly by slow axonal transport. Other proteins, including actin, that showed no net changes in relative amount (but presumably changed in absolute amount in direct proportion to the changes in total protein content of the nerve), are apparently distributed in both the neuronal and nonneuronal compartments of the nerve.     

Establishment of a statistical base for use of ELISA in diagnostic serology for infectious bovine rhinotracheitis

The critical statistical parameters of an enzyme-linked immunosorbent assay were determined to enable quantitation of antibody responses in cattle affected with infectious bovine rhinotracheitis. A system of controlling well-to-well variations in optical density reading across a microtitre plate was evolved and dose--response assays were carried out to determine the dilution of serum which gave the greatest discrimination between acute and convalescent sera from an infected animal. Use of a standard serum was studied in further assays. An increase in optical density value of 0.15 was set as a diagnostic criterion for a significantly rising antibody response. This compared well with the conventional criterion of a fourfold rise in virus neutralizing antibody titre.     

Mating behavior,egg deposition,incubation period,and hatching in the clearnose skate,Raja eglanteria

Synopsis Adult clearnose skates, Raja eglanteria, were captured during the winters of 1981 and 1983, and observed to mate in captivity. Mating and egg depositions take place on the central west coast of Florida from December through mid-May. During copulation the male holds the trailing edge of the female's right or left pectoral fin firmly in his mouth, swings his tail beneath hers and inserts one clasper into the distal end of her reproductive tract. Copulation may last one to four hours during which time sperm pass from the urogenital papilla of the male along the clasper groove to the female. Sperm move cranially to the upper portion of the shell gland where they are stored and remain viable for at least three months. The ovum is fertilized in the shell gland. The egg case bears a prominent projection or horn at each corner. The two posterior ones are shorter and bear tendrils which are covered with a sticky substance that insures attachment to the substrate when the egg is deposited. Fertilized eggs are laid in pairs at intervals ranging from 1 to 13 days (mean of 4.5 ± 2.2 days). As development proceeds within the egg case a plugged slit on the lateral side of each horn opens and permits seawater to wash the developing embryo. Incubation periods for eggs maintained between 20–22°C decrease in duration throughout the egg laying season, ranging from 94 days initially to 77 days for eggs laid later in the spring. At hatching, the anterior end of the egg case ruptures, and the skate emerges abruptly with its pectoral fins rolled dorsally.     

Exclusion of plasma lipoproteins of intestinal origin from avian egg yolk because of their size

Increasing the fat content of the diet increases the proportion of large triglyceride-rich (TGR) lipoproteins (portomicrons) in laying-hen plasma, but has no effect on the size distribution of yolk TGR-lipoproteins. Electromicrographs of the ovarian follicle walls of hens fed a high-fat diet show the presence of numerous portomicron-like particles in the lumen of the thecal capillaries, in the pericapillary spaces and in the theca interna, but portomicrons were absent from the basal lamina, between the granulosa cells and in newly deposited yolk. Most of the lipoprotein lipase activity in the ovarian follicles is associated with the granulosa cells, but total activity in the follicle is very small compared to heart or adipose tissue. The results indicate that the ovarian follicle of the laying-hen specifically excludes lipoproteins of intestinal origin from yolk, most probably because they are too large to pass through the connective tissue matrix of the basal lamina. The low lipoprotein lipase activity of the ovarian follicle, together with its distribution within the follicle wall, indicates that the ovarian follicles make little contribution to catabolism of circulating portomicrons.     

Cytogenetic analysis of chorionic villi: a technical assessment

Summary Eighty-five samples of chorionic villi from women undergoing prenatal diagnosis at 8 to 12 weeks' gestation were subjected to cytogenetic analysis. Samples were prepared by a direct technique that permits limited analysis within two hours and by a short-term culture technique that permits detailed structural analysis within one week. An adequate number of cell divisions for cytogenetic analysis was obtained from 96% of living fetuses. Using both the direct technique and short-term culture, satisfactory banded chromosomal preparations were made in 93% of cases. Eleven of 12 pregnancies (92%) shown by ultrasound to be dead shortly before sampling, had cytogenetic abnormalities. Further studies are needed to develop banding definition equivalent to that available on cultured amniocytes.