Identification of cDNA clones encoding different domains of the basement membrane heparan sulfate proteoglycan

We have used antibodies to the basement membrane proteoglycan to screen lambda gt11 expression vector libraries and have isolated two cDNA clones, termed BPG 5 and BPG 7, which encode different portions of the core protein of the heparan sulfate basement membrane proteoglycan. These clones hybridize to a single mRNA species of approximately 12 kilobases. Amino acid sequences obtained on peptides derived from protease digests of the core protein were found in the deduced sequence, confirming the identity of these clones. BPG 5 spanned 1986 base pairs and has an open reading frame of 662 amino acids. The amino acid sequence deduced from BPG 5 contains two cysteine-rich domains and two internally homologous domains lacking cysteine. The cysteine-rich domains show homology to the cysteine-rich domains of the laminin chains. A globule-rod structure, similar to that of the short arms of the laminin chains, is proposed for this region of the proteoglycan. The other clone, BPG 7, is 2193 base pairs long and has an open reading frame of 731 amino acids. The deduced sequence contains eight internal repeats with 2 cysteine residues in each repeat. These repeats show homology to the neural-cell adhesion molecule N-CAM and the plasma alpha 1B-glycoprotein. Looping structures similar to these proteins and to other proteins of the immunoglobulin gene superfamily are proposed for this region of the proteoglycan. The sequence DSGEY was found four times in this domain and could be heparan sulfate attachment sites.     

Activation of phosphorylase kinase through autophosphorylation by membrane component phospholipids

Phosphatidic acid (PtdOH) has been shown not only to stimulate autophosphorylation and autoactivation of phosphorylase kinase of rabbit skeletal muscle but also to decrease the apparent Ka for Ca2+ on autophosphorylation sharply [Negami et al. (1985) Biochem. Biophys. Res. Commun. 131, 712-719]. In this study we investigated the interaction between PtdOH and other phospholipids on autophosphorylation and autoactivation of this enzyme. Acidic phospholipids, such as phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns) and PtdOH, stimulated this reaction about 2-4-fold, and the approximate Ka values of this reaction were 10 micrograms/ml, 6.3 micrograms/ml and 30 micrograms/ml respectively. The molar ratio of PtdIns and PtdSer with maximal effect on autophosphorylation was about 1:1. Under these conditions PtdOH stimulated the initial velocity of autophosphorylation about 5.2-fold. When fully autophosphorylated, about 12-13 mol phosphate per tetramer (alpha beta gamma delta) were incorporated in the presence of mixed acidic phospholipids (PtdOH:PtdIns:PtdSer = 2:1:1), which was about twice as much as values observed without effectors. In the presence of mixed acidic phospholipids there was a concomitant enhancement of kinase activity, about 30-40-fold at pH 6.8 and 2.5-3-fold at pH 8.2. Mixed acidic phospholipids sharply decreased an apparent Ka for Ca2+ from 4 X 10(-5) M to 8 X 10(-7) M. With mixed acidic phospholipids as effectors this autophosphorylation occurred through an intramolecular mechanism. Based on these results, autophosphorylation and autoactivation of phosphorylase kinase in the presence of acidic phospholipids may account for an important regulatory mechanism of glycogenolysis in muscle contraction.     

Amidase-like activity of calpain I and calpain II on substance P and its related peptides

Porcine calpains (Ca2+-dependent cysteine proteinases) I and II, which had been purified each to a homogeneous state, were found to hydrolyze specifically carboxyl-terminal amide of substance P and several other biologically active peptidyl amides. This amidase-like activity was demonstrated both by determining released ammonia and by separating products on high-performance liquid chromatography followed by amino acid analysis. The calpain-catalyzed deamidation of substance P occurred exclusively at the carboxyl-terminal amide, leaving the side-chain glutamine intact. Enkepharinamide and MSH-release inhibiting factor were scarcely deamidated. Calpains I and II showed similar specificities for these amide substances and similar profiles of inhibitions by various protease inhibitors, but distinctly different Ca2+ requirements. The specificity constants, kcat/Km, for substance P were found to be three to four orders of magnitude higher than those for the synthetic substrates.     

Modulation of postinhibitory increase in plasma prolactin by domperidone in patients with prolactin secreting adenoma

To evaluate the PRL secretory mechanism in patients with PRL-secreting adenoma (PRL-oma), plasma PRL responses to dopamine (DA) were studied in these cases and in normal subjects. Plasma PRL values showed clear decreases during the infusion of DA (5 micrograms/kg/min for 90 min) in both 6 normal and 7 PRL-oma subjects (%decrease: 43.8 +/- 3.9% vs. 53.9 +/- 5.6%; NS) and postinhibitory increases after the termination. However, the postinhibitory increase occurred more promptly and markedly in PRL-oma patients than in normal subjects, i.e. the postinhibitory increase exceeded the basal level 45 min after the termination of DA infusion in PRL-oma patients, whereas the increase in normal subjects did not exceed the basal level even 90 min after the infusion. When domperidone was injected at the termination of DA infusion, the postinhibitory increases were significantly enhanced in either PRL-oma or normal subjects. The maximal increments in plasma PRL in the combination test of DA plus domperidone were significantly larger in PRL-oma patients, but were almost the same in normal controls, compared to the single domperidone test. In contrast, TRH did not modify the postinhibitory rises in 9 PRL-oma patients. These results indicate that the secretory properties and the sensitivities of lactotrophs to decreasing action of DA might be different between PRL-oma patients and normal controls. Further, the postinhibitory rebound phenomenon in PRL-oma patients is possibly determined by an overshoot of PRL storage concomitantly with a decreasing DA action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of L-propargylglycine on metabolism of sulfur-containing amino acids in pregnant rats and their fetuses

Cystathionine accumulated in several tissues of dams and fetuses by a single intraperitoneal administration of L-proparglyglycine to pregnant rats. Cystathionine in the liver of dams reached its maximal level at about 15 hrs after L-proparglyglycine injection (10 mg/300g), while that in the kidney and brain of dams, and in the liver, kidney, and brain of fetuses reached a maximum at about 21 hrs. The content of cystine in the liver of fetuses decreased gradually in proportion to the amount of L-proparglyglycine administered. Cystathionine gamma-lyase activity in the liver of dams and fetuses decreased to about 2-4% of that of control rats at 15 hrs after L-proparglyglycine injection, and that in the kidney and pancreas of dams to about 10-20% of that of control rats. On the other hand, cystathionine beta-synthase activity did not show significant changes from that of control rats.     

Correlation between X-chromosome inactivation and cell differentiation in female preimplantation mouse embryos

By means of a cytological method involving BrdU incorporation and acridine orange fluorescence staining in combination with embryo manipulation, we studied X-chromosome activity in female preimplantation mouse embryos with special reference to the correlation between X-chromosome inactivation and cell differentiation. There was no sign of asynchronous replication between the two X chromosomes from the one-cell to intermediate blastocyst stage. The allocyclic X chromosome, first detected in late blastocysts, was paternal in origin, mostly replicating early in the S phase and limited to the trophectoderm. Subsequent X-chromosome inactivation occurring in the primary endoderm was also characterized by the involvement of the paternal X and early replication. Both X chromosomes continued to replicate synchronously in the embryonic ectoderm or epiblast at this stage. It was evident that overt cell differentiation preceded the appearance of the asynchronously replicating X chromosome in the trophectoderm and primary endoderm. This finding seems to support the view that cell differentiation is an important correlate of X-chromosome inactivation.     

Effect of ozone on breathing in dogs: vagal and nonvagal mechanisms

We exposed two awake dogs with a chronic tracheostomy and the cervical vagus nerves exteriorized in skin loops to 1.0 ppm of ozone (O3) for 2 h at intervals of 4 wk. We measured ventilatory variables before and after O3 exposure during rest and exercise before and after vagal block. We compared the effects of vagal blockade, exercise, and O3 on the primary determinants of breathing pattern (VT/TI, VT/TE, TI, and TE) in each of three conditions: base line (steady state), during hypercapnia, and after inhalation of 1% histamine. Under base-line conditions, O3 increased respiratory rate and decreased tidal volume (VT) by shortening time of expiration (TE) and time of inspiration (TI) without affecting VT/TI, an indicator of the neural drive to breathing. During progressive hypercapnia, O3 shortened TE and TI by effects both on tonic (nonvolume-related) and on phasic (volume-related) vagal inputs, and only the latter were prevented completely by cooling of the vagus nerves. Histamine-induced tachypnea was increased by O3 and was totally blocked by cooling the vagus nerves. We conclude that O3 shortens the timing of respiration without increasing ventilatory drive, shortens TI and TE through vagal and nonvagal pathways, increases tonic nonvagal and phasic vagal inputs, and stimulates more than one vagal fiber type.     

Hemodynamics and vascular sensitivity to circulating norepinephrine in normal skin and delayed and acute random skin flaps in the pig

Cutaneous circulation in 4 X 10 cm skin samples and delayed and acute random skin flaps constructed on the flanks of castrated Yorkshire pigs (13.3 +/- 0.7 kg; n = 12) were studied during intravenous infusion (0.5 ml per minute) of 5% dextrose solution (vehicle) and 5% dextrose containing norepinephrine (1 microgram/kg per minute). Total and capillary blood flow and A-V shunt flow were measured by the radioactive microsphere technique 6 hours after the raising of 4 X 10 cm single-pedicle acute and delayed random skin flaps using the technique and calculations published previously. Fluorescein dye test was also performed to assess vascular perfusion. It was observed that the capillary blood flow in the single-pedicle delayed skin flaps was similar to that in the normal skin, and the maintenance of this normal skin blood flow was not due to the closing of A-V shunt flow in the delayed skin flaps. Similarly, the significant (p less than 0.01) decrease in capillary blood flow and distal perfusion in the acute skin flaps compared with the delayed skin flaps was not due to the opening of A-V shunts in the acute skin flaps. There was no evidence to indicate that A-V shunt flow per se was the primary factor for the regulation of capillary blood flow in the acute and delayed skin flaps in the pig. Our data seemed to indicate that tissue ischemia in the distal portion of acute skin flaps was likely the result of vasoconstriction of the small random arteries which supplied blood to arterioles and A-V shunts, and locally released neurohumoral substances may play an important role in the pathogenesis of vascular resistance and ischemia in the acute skin flaps.     

Effect of 5-azacytidine on the differentiation of human leukemia K-562 cells

The treatment of K-562 cells with 10(-5) M to 10(-7) M 5-azacytidine induced a marked increase in benzidine-positive cells. Similarly, the exposure of K-562 cells to 2 X 10(-3) M butyric acid or 5 X 10(-7) M 1-beta-arabinofuranosylcytosine or 1 X 10(-3) M hydroxyurea induced an erythroid differentiation of K-562 cells. The activity of DNA-methyltransferase and the level of methylcytosine in newly synthesized DNA were significantly decreased when the cells were treated with 5-azacytidine or butyric acid, while 1-beta-arabinofuranosylcytosine or hydroxyurea had no inhibitory effect on DNA-methylation of K-562 cells. These results suggest that the inhibition of DNA-methylation is not necessarily a specific phenomenon for erythroid differentiation of K-562 cells.