CYCLIC PEPTIDES: X. Bitter Taste and Chymotryptic Hydrolysis of Cyclic Depsidipeptides Containing a Tryptophan Residue*

Four stereoisomers (I-LL, I-LD, I-DL and I-DD) of a cyclic depsidipeptide (I) containing a tryptophan and a 2-hydroxy-4-methylpentanoic acid residue were synthesized, and their taste and chymotryptic susceptibility were examined. Compound I-LL is a depsipeptide analog of a bitter principle BP-II, cyclo(-L-Trp-L-Leu-), obtained from casein hydrolyzate. All of the four stereoisomers of I are strongly bitter to taste. Another depsipeptide analog, L-aspartyl-L-2-hydroxy-3-phenylpropanoic acid methyl ester, of the sweet H-L-Asp-L-Phe- OMe showed bitter taste instead of sweet. Chymotrypsin hydrolyzed I-LL and I-LD in moderate rates, and I-DL and I-DD very slowly.     

ENZYME-CATALYZED FORMATION OF SEMISYNTHETIC STAPHYLOCOCCAL NUCLEASE USING A NEW SYNTHETIC FRAGMENT, [48-GLYCINE] SYNTHETIC-(6–49)

While trypsin can catalyze resynthesis of the peptide bond between fragments in the noncovalent complex of nuclease-T-(6–48) and nuclease-T-(49–149), this reaction leads to excision of Lys 49 and formation of inactive [des Lys 49]-nuclease-(6–149). To provide a method for making active covalent semisynthetic nuclease, we chemically synthesized the fragment of residues 6 to 49 in which lysine 48 was replaced by glycine. This peptide was made using the recently described solid phase support, 4-(oxymethyl)phenylacetamidomethyl-polystyrene. The resultant crude polypeptide exhibited 30–50% of native nuclease-T enzymatic activity when added to native nuclease-T-(50–149). When the noncovalent complex formed by native nuclease-T-(50–149) and a 10-fold molar excess of [Gly 48]synthetic-(6–49) was equilibrated with trypsin in 90% glycerol, an increase in enzymatic activity from 8 to 32% (versus nuclease) was observed. Simultaneously, approximately 20% conversion of nuclease-T-(50–149) to nuclease-molecular weight material was observed by gel electrophoretic analysis. These data indicate that a covalent semisynthetic species is formed with activity about equal to that of native nuclease. The results confirm the importance of loop integrity on catalytic site organization. The Gly 48-containing fragment system defined above can allow preparation of semisynthetic nuclease sequence analogs.     

Electron Microscopic Study of Cell Lines Established from Adult T-Cell Leukemia--Coexistence of Adult T-Cell Leukemia Virus and Epstein-Barr Virus

Six cell lines established from five patients with adult T-cellleukemia (ATL) were studied by electron microscopy. From onepatient two cell lines were established, an interleukin 2-dependentline and a nondependent line. The interleukin 2-dependent T-cellline had only ATL virus (ATLV) particles. The interleukin 2-nondependentB-cell line had both ATLV particles and Epstein-Barr virus (EBV)particles. In two other B-cell lines and one undetermined cellline, both ATLV particles and EBV particies were seen. In oneB-cell line only a few EBV particles were seen. These findingssuggest that (I) interleukin 2 is necessary for the growth ofleukemic T-cells from ATL tissue samples, and (2) ATLV can infectnot only T-cells but also B-cells.     

Evidence for the in Vivo Localization of Heterologous Anti-Leukocyte and Anti-Bone Marrow Antibodies in the Rat Bone Marrow

1. The intravenous injection of I¹³¹-labeled heterologous anti-leukocyte and anti-bone marrow antibodies into rats resulted in a high specific in vivolocalization in the bone marrow, indicating the presence of localizing anti-body.

2. No in vivo localization in lungs was demonstrated, in possible contrastto the earlier concept of pulmonary emboli of clumped leukocytes sensitizedwith antibodies in experimental immunoleukopenia.

3. Heterologous anti-leukocyte and anti-bone marrow antibodies injectedintravenously disappeared from plasma and fixed to peripheral leukocytesand bone marrow cells within 1 hour.

Submitted on June 25, 1962 Accepted on December 24, 1962     

Transient Manifestation of Antegrade Conduction Over a Kent Bundle After Ventricular Pacing

Postventricular Pacing Preexcitation. Antegrade conduction over a Kent bundle was transiently manifested after the cessation of ventricular overdrive pacing in two patients; otherwise, the heart behaved as if it had a concealed accessory pathway. Conventional electrophysiologic study suggested either a longitudinal dissociation of an accessory pathway or closely located double accessory pathways. The mechanism of the transient manifestation of ventricular preexcitation was discussed. (J Cardiovasc Electrophysiol, Vol. 3, pp. 423–430, October 1992)     

Electrophysiological Abnormalities of the Atrial Muscle in Patients with Manifest Wolff-Parkinson-White Syndrome Associated with Paroxysmal Atrial Fibrillation

We investigated the electrophysiological properties of the atrial muscle in 33 patients with manifest Wolff-Parkinson-White syndrome. Group I consisted of 13 patients with paroxysmal atrial fibrillation and group II consisted of 20 patients without paroxysmal atrial fibrillation. The anterograde and retrograde effective refractory periods of the accessory pathway and the inducibility of atrioventricular reciprocating tachycardia were not significantly different between the two groups. Endocardial electrograms, obtained by right atrial catheter mapping, were recorded during sinus rhythm from 12 sites of the right atrium in 12 of the 13 group I patients and in all group II patients. An abnormal atrial electrogram was defined as 100 msec or longer in duration, and/or the occurrence of eight or more deflections. Ten (83%) of the 12 group I patients had abnormal atrial electrograms, while only two (10%) of the 20 group II patients had abnormal atrial electrograms, and the difference was significant (P less than 0.01). Thirty-six (26%) of the total 139 electrograms obtained from 12 group I patients and two (1%) of the total 199 electrograms obtained from 20 group II patients fulfilled the criteria for an abnormal atrial electrogram, and the difference was significant (P less than 0.01). The fragmented atrial activity zone, interatrial conduction delay zone, and repetitive atrial firing zone obtained by right atrial extrastimulation were significantly wider in group I than in group II, respectively. It was concluded that electrical abnormalities of the atrial muscle may play an important role in the occurrence of paroxysmal atrial fibrillation in patients with Wolff-Parkinson-White syndrome.     

A New Pacemaker for Monitoring Asystole and Prevention of Adams-Stokes Syndrome and Sudden Cardiac Death

Patients with suspected Adams-Stokes syndrome are examined by Holter monitoring. During the monitoring, there is the danger of syncopes occurring and there are even reports of sudden cardiac death. We therefore developed a pacemaker for cardiac arrest monitoring and the prevention of Adams-Stokes syndrome and sudden cardiac death, which has the following functions: (1) the longest escape interval of the pacemaker not exceeding the value at which syncope is induced is determined by the decline of the mean heart rate including the asystole to a certain threshold rate; (2) once the pacemaker escapes from the interval it continues pacing for a while at a physiological rate to allow recover from ischemias in organs or tissues; and (3) to prevent overdrive suppression to the heart, the pacing rate gradually declines and stops pacing until the next asystole. This pacemaker is useful not only in the diagnosis of Adams-Stokes syndrome but also in pharmacological and pathophysiological studies and in determining when pacing should cease.     

Intestinal Brush-border Membrane Transport of Monocarboxylic Acids Mediated by Proton-coupled Transport and Anion Antiport Mechanisms

Intestinal brush-border membrane transport of monocarboxylic acids was investigated by using rabbit intestinal brush-border membrane vesicles (BBMVs) and isolated intestinal tissues mounted on Ussing-type chambers. [³H]Mevalonic acid uptake by BBMVs showed an overshoot phenomenon in the presence of an inwardly directed proton gradient, but not in the presence of an inwardly directed sodium gradient or an outwardly directed HCO₃^? or chloride gradient. Initial uptake of mevalonic acid was saturable in the presence of a proton gradient. Uptake of [³H]mevalonic acid was inhibited by various monocarboxylic acids, including acetic acid, benzoic acid, lactic acid, nicotinic acid, pravastatin, salicylic acid and valproic acid, but not by dicarboxylic acid or amino acids. Acetic acid, which is transported by both anion antiport and proton-coupled transport systems, induced serosal bicarbonate-dependent alkalinization in the mucosal-side bathing solution of rabbit jejunal tissues, when examined in Ussing-type chambers. Pravastatin, which is a structural analogue of mevalonic acid and is absorbed via proton-coupled transport like mevalonic acid, did not. The result demonstrates that acetic acid is transported by the bicarbonate-dependent anion antiport system, whereas pravastatin is not. So, it is suggested that monocarboxylic acids are transported by at least two independent transporters, namely, a proton-coupled transporter for most monocarboxylic acids, including mevalonic acid, pravastatin and acetic acid, and an anion antiporter for acetic acid, but not for mevalonic acid or pravastatin. Activation of anion antiporter can induce HCO₃^? secretion in intact intestine.