The isolation and properties of phenylalanine hydroxylase from human liver

Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The K(m) values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria.     

Sporulation, Heat Resistance, and Biological Properties of Clostridium perfringens

A sporulation medium for 134 Clostridium perfringens strains, including types A, B, C, D, E, and F, was devised according to Grelet's observation that sporulation occurred when cultural environment became limited in any nutritional requirement indispensable for the growth of the organism. Sporulation took place most prominently when 10% cooked-meat broth (pH 7.2) containing 3% Proteose Peptone and 1% glucose was used for the preculture and 2% Poli Peptone medium (pH 7.8) was used for the subculture medium. Sometimes, terminal spores could be observed. A correlation between sporulation and heat resistance was examined by use of C. perfringens strains isolated from samples heated at different temperatures. Almost all strains isolated from unheated samples and from those heated at lower temperatures gave rise to spores in our sporulation medium, but the spores were weakly heat-resistant, whereas strains isolated from samples heated at 100 C for 60 min were highly heat-resistant but sporulated poorly. A majority of these heat-resistant strains were non-gelatinolytic and definitely salicin-fermenting.     

Vasoactive properties of dihomo-gamma-linolenic acid and series one prostaglandins in a freshwater teleost, Channa maculata

1. Prostaglandins A1, B1, E1 and F1 alpha (2-120 micrograms/kg), arachidonic acid and dihomo-gamma-linolenic acid (0.1-2 mg/kg) were injected intravenously into Channa maculata and changes in arterial blood pressure were recorded. 2. Injection of PGF1 alpha had no significant effect on arterial blood pressure. Injection of PGA1 and PGE1 was followed by dose-dependent hypotension whereas injection of PGB1 elicited significant dose-dependent increase in arterial blood pressure. 3. Both dihomo-gamma-linolenic acid and arachidonic acid were also depressor agents but dihomo-gamma-linolenic acid was more potent. 4. A single bolus intravenous injection of indomethacin (5 mg/kg) or 4 daily intraperitoneal injections (4 x 10 mg/kg) significantly lowered arterial blood pressure. One hour after pre-treatment of indomethacin, the vascular effects of both prostaglandin precursors were abolished. 5. It appears that the vascular effects of prostaglandins in Channa maculata are qualitatively different from those reported for mammals.     

Properties,synthesis, and accumulation of storage proteins in Pieris rapae L.

Two kinds of storage proteins (SP-1, SP-2) were confirmed in hemolymph and fat body of Pieris rapae during metamorphosis. Both proteins were present in high concentrations in the hemolymph during the last larval instar. Hemolymph concentrations of SP-1 and SP-2 dropped after pupation as the proteins were being deposited in fat bodies. SP-2 is present in a larger amount than SP-1. Detailed studies on storage proteins determined their properties, mode of synthesis, and accumulation in the fat body. SP-1 has a molecular weight of 500,000 and consists of one type of subunit (M_r 77,000), while SP-2 has a molecular weight of 460,000 and is composed of two types of subunits (M_r 80,000 and 69,000). The pl values of SP-1 and SP-2 were determined to be 6.97 and 7.06, respectively. Fat body cells from 1-day-old fifth instar larvae synthesized storage proteins in large amounts, whereas those from late prepupae exhibited high protein sequestration. Proteins taken up in fat body accumulated in dense granules during the pupal stage but sharply decreased at the adult stage. Morphological changes in the fat body tissues were observed during the larval-pupal transformation; the nuclei of fat body cells became irregularly shaped, and the boundaries between cells seemed to be obscure. Synthesis, storage, or degradation of storage proteins in fat body during development is closely associated with morphological changes in the tissues.     

Synteny on mouse chromosome 5 of homologs for human DNA loci linked to the Huntington disease gene

Comparative mapping in man and mouse has revealed frequent conservation of chromosomal segments, offering a potential approach to human disease genes via their murine homologs. Using DNA markers near the Huntington disease gene on the short arm of chromosome 4, we defined a conserved linkage group on mouse chromosome 5. Linkage analyses using recombinant inbred strains, a standard outcross, and an interspecific backcross were used to assign homologs for five human loci, D4S43, D4S62, QDPR, D4S76, and D4S80, to chromosome 5 and to determine their relationships with previously mapped markers for this autosome. The relative order of the conserved loci was preserved in a linkage group that spanned 13% recombination in the interspecific backcross analysis. The most proximal of the conserved markers on the mouse map, D4S43h, showed no recombination with Emv-1, an endogenous ecotropic virus, in 84 outcross progeny and 19 recombinant inbred strains. Hx, a dominant mutation that causes deformities in limb development, maps approximately 2 cM proximal to Emv-1. Since the human D4S43 locus is less than 1 cM proximal to HD near the telomere of chromosome 4, the murine counterpart of the HD gene might lie between Hx and Emv-1 or D4S43h. Cloning of the region between these markers could generate new probes for conserved human sequences in the vicinity of the HD gene or possibly candidates for the murine counterpart of this human disease locus.     

Expression of low density lipoprotein receptor gene in human placenta during pregnancy

Mammalian cells require cholesterol as a structural component of plasma membranes. It is also required for placental steroid synthesis. De novo synthesis of cholesterol is limited in human placenta and cholesterol is obtained mainly from plasma low density lipoprotein (LDL). Cholesterol delivery from LDL is mediated by receptor-mediated uptake and the receptor amount is the most important factor for cellular delivery. Thus, the regulation of receptor synthesis is important for placental development and function. Since the regulation of LDL receptor gene expression has not been studied in human placenta, LDL receptor mRNA was measured in placentae of 5-40 weeks of gestation by hybridization of RNA with 32P-labeled cDNA for human LDL receptor. Two mRNA species for LDL receptor were demonstrated by Northern blot analysis. The longer mRNA [5.3 kilobases (kb)] was much more abundant than the shorter mRNA (3.7 kb). The amount of 5.3 kb mRNA was highest early in gestation and decreased during pregnancy. However, the amount of 3.7 kb mRNA did not change appreciably during gestation. Dot blot analysis of 26 placental mRNAs obtained from various stages of gestation revealed a negative correlation between LDL receptor mRNA and gestation (r = -0.76, P less than 0.001). Considering the rapid growth of the trophoblast during gestation, especially in the first and the second trimester, increased expression of the LDL receptor gene and subsequent translation are expected for efficient cholesterol uptake to provide a sufficient substrate for cell growth. Possible mechanisms for the appearance of two mRNA species for LDL receptor are also discussed.     

A method for fractionation of cloned protein in recombinantSaccharomyces cerevisiae

In a spheroplasting method which allows the fractionation and quantification of cloned invertase activity in recombinantSaccharomyces cerevisiae cells, the yeast cell is selectively degraded with the enzyme Zymolyase for 60 minutes at 45°C to separate periplasmic proteins from cytoplasmic proteins. Most of the glucose-6-phosphate dehydrogenase (a cytoplasmic marker protein) was found in the cytoplasmic fraction.     

Human serum amyloid A protein. The assignment of the six major isoforms to three published gene sequences and evidence for two genetic loci

Serum amyloid A protein (apo-SAA) is an acute-phase reactant and an apolipoprotein of high density lipoproteins (HDL). Six major isoforms of apo-SAA occur in humans (pI 6.0, 6.4, 7.0, 7.4, 7.5, 8.0). In this report we have rationalized the phenotypic expression of apo-SAA isoforms with published apo-SAA structures predicted from apo-SAA cDNA's pA1 and pSAA82 and the genomic DNA SAAg9. The six apo-SAA isoforms fall into three pairs, pI 6.0/6.4, 7.0/7.5, and 7.4/8.0, which are products of cDNA pA1, cDNA pSAA82, and genomic DNA SAAg9, respectively. The second of each isoform pair (i.e. pI 6.4, 7.5, and 8.0) is the "primary" product: a 104-residue peptide with the NH2-terminal sequence Arg-Ser-Phe-Phe. Each primary product is processed either to a major 103-residue peptide with the NH2-terminal sequence Ser-Phe-Phe or processed to a minor 102-residue product which results from the loss of both an Arg and a Ser residue from the NH2 termini. These "secondary" products have the lower pI values of 6.0, 7.0, and 7.4, respectively. The isoelectric points of the SAAg9 products were confirmed by expression of SAAg9 in transfected mouse L-cells. Both the pI 8.0 and 7.4 isoforms were present in cellular extracts, suggesting that post-translational modification of apo-SAA may occur intracellularly. However, the greater relative abundance of the pI 7.4 isoform extracellularly suggests that the major conversion may occur after secretion. Whereas the gene corresponding to the pA1 cDNA sequence does not show allelic variation, the segregation characteristics of the pI 7.0/7.5 and 7.4/8.0 isoform pairs amongst individuals suggests that these isoforms are the products of genes (with sequences corresponding to pSAA82 and SAAg9, respectively) which are allelic variants at a single locus distinct from that for the pI 6.0/6.4 isoform pair.     

Long-acting nifedipine versus metoprolol as monotherapy for essential hypertension. A randomized,controlled crossover study.

We assessed the efficacy of long-acting nifedipine as monotherapy in 52 patients with mild to moderate essential hypertension in a randomized, controlled crossover study. Good blood pressure control was achieved in 34 of 40 patients (85%) receiving nifedipine (mean daily dose, 52 mg in 2 divided doses) compared with 23 of 40 patients (58%) receiving metoprolol (mean daily dose, 155 mg in 2 divided doses). After treatment for 4 weeks, the mean blood pressures with nifedipine (149.7 +/- 16.6/88.7 +/- 11.1 mm of mercury) and metoprolol administration (163.9 +/- 23.3/94.2 +/- 10.2 mm of mercury) were significantly lower than with placebo (176.7 +/- 17.3/100.9 +/- 7.1 mm of mercury) (P less than .05). The mean systolic pressure during nifedipine treatment was 14.2 mm of mercury lower (95% confidence interval [CI], 3.9 to 24.5 mm of mercury) and mean diastolic pressure 5.5 mm of mercury (95% CI, 0.3 to 10.7 mm of mercury) lower than with metoprolol therapy. Both drugs were reasonably well tolerated, and intolerance requiring withdrawal was encountered in 3 of 45 (7%) patients receiving nifedipine, compared with 1 of 45 (2%) of those taking metoprolol and placebo, respectively. Adverse effects of nifedipine, most of which were transient, included palpitations, headache, facial flushing, and ankle edema. Long-acting nifedipine is a promising agent when given alone for mild to moderate hypertension and can be safely administered in clinical practice.     

Transforming growth factor alpha and a PC12-derived growth factor induce neurites in PC12 cells and enhance the survival of embryonic brain neurons.

We have identified and characterized a 5000-Da protein that induces neurite outgrowth from PC12 pheochromocytoma cells, enhances the survival of embryonic rat brain neurons in primary culture, and induces the multiplication of embryonic rat brain astrocytes in primary culture. The factor is produced by a flat cell PC12 variant that expresses the activated ras oncogene after transfection of the gene. The factor resembles transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) in that it induces anchorage-independent colony formation of normal rat kidney cells in soft agar and competes with EGF for binding to the EGF receptor. Rat TGF alpha and human TGF alpha also induce neurite outgrowth from PC12 and enhance the survival of embryonic brain neurons. The PC12 variant-derived factor can be distinguished from TGF alpha and EGF immunologically and by migration rates on reversed-phase high-performance liquid chromatography.