Effect of thyroid hormone treatment on responses of the isolated working rat heart

Hearts from rats pretreated either with L-triiodothyronine (T3) or with L-thyroxine (T4) exhibited changed function curve characteristics on the working heart apparatus compared with hearts from vehicle-treated rats. There was no supersensitivity of the hyperthyroid myocardium to the inotropic effect of isoproterenol as estimated by pD2 values. There were significant increases in +dP/dt and -dP/dt in hyperthyroid working hearts over the entire range of the function curve. T3 hearts showed much shorter relaxation times and total contraction times throughout the function curve. T4 hearts showed significantly reduced relaxation times and total contraction times as compared with control at all left atrial filling pressures under 15 cm of water. At high filling pressures T4 heart relaxation times and total contraction times were not different from control, but were however, significantly increased from those of T3 hearts. Area under the left ventricular pressure curve was unchanged by thyroid hormone pretreatment. Heart weight increased about 15% following either T3 or T4 treatment while the increases in (+) or (-) dP/dt and the left ventricular developed pressure (LVDP) were about 20-30%. The increase in cardiac mass certainly played a role in the increased cardiac function. Potency of isoproterenol in hyperthyroid working heart preparations was unchanged from control. The pD2 values, as determined from +dP/dt data, were 8.8 +/- 0.15 for T3-treated hearts, 8.25 +/- 0.40 for T4-treated hearts, and 8.18 +/- 0.12 for euthyroid hearts. While the mechanism(s) for the altered performance of the hyperthyroid working heart are not absolutely known, possible biochemical and physiological changes which may be implicated are discussed.     

Effect of Biotin on Fatty Acids and Phospholipids of Biotin-Sensitive Strains of Rhizobium japonicum

The effect of biotin on fatty acids and intact lipids was studied by comparing a biotin-requiring, a biotin-inhibited, and a biotin-indifferent strain of Rhizobium japonicum. These organisms were grown in a defined medium with added levels of 0, 0.3, and 0.5 μg of biotin per liter, and were analyzed for fatty acids and lipid components. Myristic, palmitic, and octadecenoic acids were found to be the major fatty acids in these strains. The indifferent strain also contained large amounts of C₁₉ cyclopropane acid and small amounts of a C₁₇ cyclopropane acid. Several unidentified acids were present in the other two strains. The percentages of fatty acids showed statistically significant changes corresponding with changes in level of biotin in the medium. When biotin concentration was increased in the medium, the C₁₈ monoenoic acids of the biotin-requiring strain increased significantly, and those of the biotin-inhibited and biotin-indifferent strains decreased significantly. Palmitic acid showed a statistically significant increase in the indifferent strain with increasing biotin concentration. The principal intact lipid components in these strains are phospholipids. The major phospholipids are phosphatidylserine, phosphatidylcholine, phosphatitidylethanolamine, and cardiolipin. These phospholipids were not affected by biotin level and were independent of medium composition.     

Sucrose Catabolism in Clostridium pasteurianum and Its Relation to N2 Fixation

The growth constant and Y (sucrose) (grams of cells per mole of sucrose) for NH(3)-grown cultures of Clostridium pasteurianum were 1.7 times those of N(2)-grown cultures, whereas the rate of sucrose utilized per gram of cells per hour was similar for both conditions. The Y (sucrose) of chemostat cultures grown on limiting NH(3) under argon at generation times equal to those of N(2)-fixing cultures was less than that of cultures grown on excess NH(3), but cells of NH(3)-limited cultures contained the N(2)-fixing system in high concentration. The concentration of the N(2)-fixing system in whole cells, when measured with adenosine triphosphate (ATP) nonlimiting, was more than twofold greater than the amount needed for the N(2) actually fixed. Thus, energy production from sucrose, and not the concentration of the N(2)-fixing system nor the maximal rate at which N(2) could be fixed, was the limiting factor for growth of N(2)-fixing cells. Either NH(3) or some product of NH(3) metabolism partially regulated the rate of sucrose metabolism since, when cultures fixing N(2), growing on NH(3), or growing on limiting NH(3) in the absence of N(2) were deprived of their nitrogen source, the rate of sucrose catabplism decreased. Calculations showed that the rate of ATP production was the growth rate-limiting factor in cells grown on N(2), and that the increased sucrose requirement of N(2)-fixing cultures in part reflected the energy demand of N(2) fixation. Calculations indicated that whole cells require about 20 moles of ATP for the fixation of 1 mole of N(2) to 2 moles of NH(3).     

Immobilized flounder muscle glyceraldehyde 3-phosphate dehydrogenase

Summary Partially purified flounder muscle (Pseudopleuronectus americanus) glyceraldehyde 3-phosphate dehydrogenase was immobilized on cyanogen bromide-activated Sepharose. The catalytic properties of the immobilized preparation were studied to determine if immobilization alters the kinetic properties of the native holoenzyme. The results indicate that the pH activity profile of immobilized glyceraldehyde 3-phosphate dehydrogenase did not differ from that of the native enzyme. The Michaelis constants (Km) for NAD and glyceraldehyde 3-phosphate were somewhat altered. The enzyme stability toward various inactivation treatments in the presence and absence of NAD was characterized and compared to that of he native enzyme. When either form of the enzyme was incubated with urea at concentrations greater than 2m, inactivation occurred very rapidly. Incubation in 0.1% trypsin for 60 minutes decreased the activity of immobilized glyceraldehyde 3-phosphate dehydrogenase by 45% and of the native soluble enzyme by 70%. The immobilized enzyme also exhibited considerably more stability than the native soluble enzyme when exposed to a temperature of 50° or to 20 mm ATP. In all cases NAD either greatly reduced the rate of inactivation or completely protected the enzyme from inactivation.     

Sequencing along DNA fragments cloned in bacteriophage M13 by using internal primers. The sequence analysis of a yeast DNA fragment containing a replication origin

In the `shotgun' procedure for sequencing DNA, DNA fragments are cloned into a phage M13 vector and sequenced by using a flanking primer. In a variation of this procedure a longer DNA sequence is cloned into M13, the two single-stranded recombinants identified and sequenced by using a set of internal primers prepared by exonuclease III digestion of restriction fragments.     

Keratin protein domains within the human epidermis

Three species of human keratins are shown to have specific localizations within the epidermis. Using an immunohistochemical technique with rabbit antisera prepared against purified human keratins, two distinct epidermal domains were defined. The 45K and 46K MW keratins occur predominantly in the basal epidermal layer, whereas 55K keratin protein occurs chiefly in the suprabasal, differentiated squamous cells. Commitment of proliferating basal cells to terminal differentiation is accompanied by changes in the proportions of keratin species.     

Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry: I. Localization of catecholamines and gonadotropin-releasing hormone in the rat median eminence

Adjacent tissue sections through the rat median eminence were examined for the distribution of gonadotropin-releasing hormone (GnRH) and catecholamines (CA). A simultaneous visualization technique was employed for this correlative neuroanatomical analysis. At rostral and mid-central levels of the median eminence the majority of GnRH terminals do not appear in coexistence with CA terminals; the latter were confined to the outer-most 10 μm of the median eminence while the densest concentration of GnRH terminals was located internal to this layer. However, individual GnRH fibers appeared to penetrate the outer CA zone wherein they were found in juxtaposition to portal capillaries. At caudal levels of the median eminence, there was an extensive overlap of CA and GnRH varicosities adjacent to the tubero-infundibular sulcus. In addition, numerous GnRH terminals were seen adjacent to portal vessels. The differences in the positions of CA and GnRH terminals between rostral and caudal median eminence may provide a morphological basis for the hypothesis of separate regulatory mechanisms for CA upon GnRH secretion at these two levels of the median eminence.     

Phenolsulfonphthalein (phenol red) metabolism in primary monolayer cultures of adult rat hepatocytes

Summary The sulfonic acid dye, phenolsulfonphthalein (PSP or phenol red), has been incorporated as a pH indicator in many tissue culture media formulations since the emergence of tissue culture methodologies. The present study was designed to examine the pathway, time course, and degree of metabolism of this anionic dye in monolayer cultures of adult rat hepatocytes. Thin layer chromatographic studies coupled with β-glucuronidase studies show that glucuronidation is the major metabolic pathway for PSP in vitro. About 20% of the dye is metabolized in the first 24 h, but this functional activity is decreased by approximately half at 48 h, and even further at 72 h of culture. This metabolic activity was not affected by continuous exposure to the dye. The effect of PSP concentration on its rate of metabolism by the adult rat hepatocyte in culture seemed to be biphasic, and at concentrations of less than 100μM there was indication of a saturable process. Although PSP seemed not to be toxic to hepatocyte cultures, it is partially metabolized by these cells (as opposed to no observed metabolism in human fibroblasts or HeLa cells). Therefore, its incorporation into tissue culture media formulations for use in hepatocyte cultures should be avoided, especially when studying the mechanism(s) of glucuronidation or metabolic pathways thought to be affected by this anionic dye. This study was supported in part by NIH Grants HL-11945-11 and 1 R01 AM 26520-01A1.