New preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for fatty acids and derivatives

A preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for carboxylic acids is described. 9-Anthraldehyde hydrazone is oxidized with an organic oxidant, N-chlorosuccinimide, in an organic solvent such as ethyl acetate to give ADAM, and then the reaction mixture is directly used as the reagent solution for the derivatization of carboxylic acids. Both the oxidation and the derivatization reaction are carried out at room temperature, and an aliquot of the derivatization mixture is directly injected into a chromatograph. 9-Anthrylmethyl ester derivatives formed from ADAM and various carboxylic acids are sufficiently separated on a reversed-phase column and are sensitively detected fluorometrically. The present method was applied to the high-performance liquid chromatographic determination of long and short chain fatty acids, keto acids, and hydroxy acids.     

Measurement of specific radioactivity by high-performance liquid chromatograph with a synchronized accumulating radioisotope detector: determination of turnover rate and pool size of tryptophan in rat

A high-performance liquid chromatograph with a synchronized accumulating radioisotope detector was used to determine the turnover rate and pool size of tryptophan in rat. The specific radioactivity could be followed for three half-lives on the final slope of the specific radioactivity curve following intravenous administration of 15 muCi of carrier free tryptophan labeled with carbon-14. Remarkable individual differences were noted in turnover rate and in pool size among rats.     

Biliverdin IX delta and neobiliverdin IX delta, isolated from the ovaries of the marine snail, Turbo cornutus

The ovaries of the marine snail Turbo cornutus contain a number of pigments. So far, the presence of carotenoids and a chromoprotein with a bile pigment, called turboverdin (= 3(2)-hydroxy-mesobiliverdin IX alpha), as its prosthetic group are known. The present work describes the isolation and structure elucidation of two further bile pigments, biliverdin IX delta and neobiliverdin IX delta. This is the first report of naturally occurring bile pigments with IX delta structure.     

Immunogenic protein MPB57 from Mycobacterium bovis BCG: molecular cloning, nucleotide sequence and expression

Using a single-probe method, we have cloned the gene for an immunogenic MPB57 protein of Mycobacterium bovis BCG. The nucleotide sequence includes an ORF of 300 base pairs encoding a protein of 99 amino acids with an Mr of 10,818. This cloned gene was expressed in an Escherichia coli expression vector to give a mature protein which reacted with a polyclonal antibody raised against MPB57.     

Biochemical evidence for the local synthesis and retention of fibronectin in the tissue of human placenta

1. Human chorionic tissues were incubated with [14C]leucine and/or [3H]glucosamine, and fibronectin synthesis was examined. 2. Radio-labeled fibronectin was detected in the tissue fraction of the incubation mixture, but not in the medium fraction, indicating that fibronectin is synthesized and retained in the tissue. 3. The glycopeptides derived from 3H-labeled fibronectin showed the lectin-binding characteristics similar to those from unlabeled placenta fibronectin, but different from those of plasma fibronectin.     

To what extent does a minimal atherosclerotic plaque alter the arterial wall shear stress distribution? A model study by an electrochemical method

An electrochemical surface shear stress measurement was applied to a model of very thin unilateral arterial stenosis (height of 1/8 of the model pipe diameter with very smooth surface). Three dimensional wall shear stress distribution was measured under steady flow field from a relatively low Reynolds number, Re = 270, to a high Reynolds number, Re = 1200. There was a characteristic high and low wall shear distribution pattern around the stenosis. There were also remarkable high shear stress areas on the opposite wall and both side walls of the stenosis. It was clearly shown that three dimensional structure of the flow field, hence, the wall shear stress distribution, is affected by a minimal change on the arterial wall.     

Alpha-interferon treatment for adult T cell leukemia: low levels of circulating alpha-interferon and it's clinical effectiveness

We describe a patient with adult T cell Leukemia to whom alpha-interferon therapy was highly effective. Although a combination chemotherapy (ACVP) first introduced was effective in reducing total leukocyte counts, the percentage of leukemic cells relative to total leukocyte counts was decreased first after the institution of alpha-interferon therapy. The patient is now under complete remission for four years. It was noted in this patient that circulating alpha-interferon, measured by a sensitive radioimmunoassay, was consistently low as compared with the value found in the age-, sex-matched healthy control (p less than 0.001). Since adult T cell leukemia is pathogenetically related to the retrovirus infection, low levels of circulating alpha-interferon of the patient may be important from both pathogenetic and therapeutic standpoints. Alpha-interferon therapy may be an useful additive for the chemotherapy of adult T cell leukemia.     

Role of high-density lipoprotein in transport of circulating bilirubin in rats

The analbuminemic rat strain established by Nagase et al. (Nagase, S., Shimamune, K., and Shumiya, S. (1979) Science 205, 590-591) exhibits hereditary deficiency in albumin biosynthesis. Serum bilirubin concentration is rather lower in homozygous (aa) rats (0.009 +/- 0.002 mg/dl) as compared with heterozygous (Aa) rats (0.047 +/- 0.009 mg/dl) or wild-type Sprague-Dawley (AA) rats (0.034 +/- 0.006 mg/dl) as evidenced by high pressure liquid chromatography analysis of bilirubin. After intravenous administration of various amounts of [heme-3H]hemoglobin in rats, [3H]bilirubin derived from [3H]heme of hemoglobin in vivo is more efficiently excreted into bile in aa rats than in Aa or AA rats. [3H]Bilirubin is exclusively bound with high-density lipoprotein (HDL) in aa rats, and a significant amount of [3H]bilirubin is shown to bind with HDL in Aa or AA rats in vivo. Scatchard plots revealed that [3H]bilirubin is bound with HDL in three binding modes depending on the molar ratio of [3H]bilirubin to HDL: Kd = 0.8 X 10(-7) M (molar ratio, 0.02-0.06), Kd = 1.6 X 10(-6) M (molar ratio, 0.06-0.41), and Kd = 1.2 X 10(-4) M (molar ratio, 0.79-9.02). Even under extreme conditions of excess hemoglobin administration, the molar ratio remains under 0.041; and thus, expected the Kd value would remain around 0.8 X 10(-7) M. Binding of [3H]bilirubin to rat serum albumin revealed two distinct binding modes depending on the molar ratio of [3H]bilirubin to rat serum albumin: Kd = 3.6 X 10(-7) M (molar ratio, 0.03-0.21), and Kd = 5.0 X 10(-6) M (molar ratio, 0.21-2.46). Under physiological conditions in Aa or AA rats, the former mode would be more reliable than the latter. Thus, HDL could bind with approximately 4.5 times higher affinity than rat serum albumin in Aa or AA rats under physiological conditions in vivo.     

Single transient K channels in mammalian sensory neurons.

A single-channel recording of the transient outward current (A-current) was obtained from dorsal root ganglion cells in culture using patch-clamp techniques. Depolarization of the membrane patch elicited pulse like current of a uniform amplitude in an outward direction, of which the unitary conductance was 20 pS. Alteration of extracellular ionic compositions indicated that the charge carriers were K ions. A systematic study was made on the voltage-dependence of the ensemble average current; (a) the activation started at a potential around -60 mV; (b) the time course of the activation was relatively rapid; (c) the channel was completely inactivated at a potential positive to -40 mV. Two time constants (tau f = 100 ms and tau s = 4,000 ms) were detected in the decay of the current indicating that the channels had two different states of inactivation. A convulsant, 4-aminopyridine (4-AP), acted on the channel only from the intracellular side of the membrane. 4-AP (5 mM) reduced not only mean open time (by 50%) but also the single-channel conductance (by 20%). The properties of the channel were independent of Ca ions in the intracellular space.     

Distinction of G0 cells from senescent cells in cultures of non-cycling human fetal lung fibroblasts by anti-MAP-1 monoclonal antibody staining

On staining with a monoclonal antibody raised against microtubule-associated protein-1 (MAP-1), dot-like structures were seen in the nuclei of interphase cells, but not in those of non-cycling G0-arrested cells. Dots were also not seen in the nuclei of non-cycling senescent human cells (IMR-90). A SV40-DNA-transformed subline of IMR-90 with a limited growth potential showed progressive decrease of cells with nuclei containing dots in the final stage of their lifespan. The dots appeared in G0-arrested IMR-90 cells when these cells were incubated in medium of high osmotic pressure for 3 min. In contrast, no dots appeared in senescent cells or X-ray-irradiated young cells when they were incubated in medium of high osmotic pressure. Thus irreversibly non-cycling cells could be distinguished from G0-phase cells on the level of whole cultures. The results suggest that senescent cells lose their division potential by entering an irreversible cell-cycle stage differing from G0.