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.     

Effect of megestrol acetate on milk production in sows and piglet growth

A group of 16 German Landrace sows, nursing 155 piglets, was used for the demonstration of the effect of 20 mg of the progestogen megestrol acetate, administered orally from Day 8 to 21 post-partum, on milk production, milk composition and piglet weight. Eight sows remained untreated. Megestrol acetate treatment increased significantly milk yield by 35% and piglet weight at weaning (Day 35) by 30% over controls. A carryover effect after cessation of drug treatment was dtected, which kept milk production at an increased level for 8 more days.     

Typing of Nontypable Staphylococci by Lysogeny

Strains of coagulase-positive staphylococci which were nontypable with the routine typing set of phages could be typed by lysogeny with phage-propagating strains as indicators and with ultraviolet induction. About 10% of the strains could be typed without induction. About 36% of them could be typed by this method when ultraviolet irradiation was used as an inducing agent. The phage groups from which the majority of the nontypable staphylococci originated were easily identified by this method of typing.     

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.     

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.     

IL-4 blocks the up-regulation of IL-2 receptors induced by IL-2 in normal human B cells

A negative influence of IL-4 on the IL-2-induced B cell proliferation and differentiation has recently been reported. In this study, we have further investigated a role of IL-4 on human tonsillar B cell proliferation and IL-2R expression. IL-4 enhanced Staphylococcus aureus Cowan 1 strain (SAC)-induced B cell proliferation, reaching the peak on day 3. However, from day 4, IL-4 inhibited IL-2-induced proliferation. In the cross-linking study, IL-4 enhanced the density of 125I-IL-2-binding protein at low affinity binding condition (2 nM of 125I-IL-2) in SAC-activated B cells. However, IL-4 blocked the enhancement in the density of 125I-IL-2-binding proteins induced by IL-2, from day 3, in both high (50 pM of 125I-IL-2) and low affinity binding conditions, suggesting that IL-4 is able to block IL-2-induced IL-2R up-regulation. This was confirmed by a binding study: B cells that cultured for 3 days with SAC plus IL-2 expressed an average of 180 +/- 20 high affinity receptors/cell with a Kd of 12 pM and 5800 +/- 500 low affinity receptors/cell with a Kd of 980 pM. By coculturing with IL-4, high affinity receptors were almost undetectable and the expression of low affinity receptors was reduced by more than 80%. IL-4-mediated inhibition of IL-2-induced IL-2R expression does not seem to be due to the direct interaction between IL-4 and cell surface receptors, inasmuch as preincubation of cells with IL-4 for 60 min at 37 degrees C did not alter the binding of 125I-IL-2 to cells previously cultured for 3 days with SAC plus IL-2. These data suggest that IL-4 has a capacity to block the up-regulation of the high as well as low affinity IL-2R-induced by IL-2 in normal human B cells, and could provide a possible explanation for the decreased responsiveness of B cells to IL-2 in the presence of IL-4.     

Tight junctional inhibition of entry of Toxoplasma gondii into MDCK cells

Various conditions of cultures were performed to investigate the role of tight junctions formed between adjacent MDCK cells on the entry of Toxoplasma. When MDCK cells were cocultured with excess number of Toxoplasma at the seeding density of 1 x 10(5), 3 x 10(5), and 5 x 10(5) cells/ml for 4 days, the number of intracellular parasites decreased rapidly as the host cells reached saturation density, i.e., the formation of tight junctions. When the concentration of calcium in the media (1.8 mM in general) was shifted to 5 microM that resulted in the elimination of tight junction, the penetration of Toxoplasma increased about 2-fold (p less than 0.05) in the saturated culture, while that of non-saturated culture decreased by half. Trypsin-EDTA which was treated to conquer the tight junctions of saturated culture favored the entry of Toxoplasma about 2.5-fold (p less than 0.05) compared to the non-treated, while that of non-saturated culture decreased to about one fifth. It was suggested that the tight junctions of epithelial cells play a role as a barrier for the entry of Toxoplasma and Toxoplasma penetrate into host cells through membrane structure-specific, i.e., certain kind of receptors present on the basolateral rather than apical surface of MDCK cells.     

The fate of spargana inoculated into the cat brain and sequential changes of anti-sparganum IgG antibody levels in the cerebrospinal fluid

To establish an animal model of intracranial sparganosis, the fate and behavior of the experimentally inoculated spargana were observed. A total of 102 scolices of spargana were injected into 22 cat brains, and the cats were sacrificed at 2 weeks, 1 month, 3 months and 6 months after the inoculation. Neurosparganosis was established in 77% of the cats. Of 43 recovered worms, 19 (44%) were located in the subdural or subarachnoid space, 16 (37%) in the brain parenchyme, and 2 (5%) in the lateral ventricle. One was detected at the diploic space of the skull and 5 were outside the cranial cavity. All but one were alive, and had grown tails. They were distributed in the brain parenchyme randomly. There was no place which they could not invade. No adult was found in the intestine. Cerebrospinal fluid (CSF) was collected before inoculation, 1 week, 2 weeks, 1 month, 3 months and 6 months after inoculation. The level of anti-sparganum IgG antibody in CSF measured by ELISA began to increase above the criteria of positivity 1 month after inoculation. Three months after inoculation, the values markedly increased. The present findings reveal that intracranial inoculation of spargana into the brains of cats would be a good animal model of experimental neurosparganosis.     

Regulation of apolipoprotein E synthesis and mRNA by diet and hormones

Rats were fasted or fasted and refed simple purified diets so the effects of individual carbohydrates or fats could be studied. Freshly isolated hepatocytes from these animals were used to measure both apoE synthesis and mRNA levels so any changes in apoE synthesis that might occur without changes in its mRNA could be detected. Some of these experiments were done with both sexes. Both fasting and fasting and refeeding a 60% glucose fat-free diet significantly increased spoE synthesis. However, cyclic AMP is not likely to rapidly mediate the effect of fasting since dibutyryl cAMP slightly lowered (rather than increased) apoE synthesis and mRNA when injected into rats for 4.5 h. Dietary fat had no effect either in the absence of carbohydrate or when consumption of carbohydrate was constant in pair-fed rats. ApoE mRNA levels remained normal for 4 days in primary hepatocytes cultured in medium that had only amino acids as an energy source. Added hormones or fructose had no significant effect. Thus, only fasting and fasting and refeeding glucose were able to significantly change apoE synthesis or mRNA levels. Synthesis of apoE may be regulated to increase when apoE is secreted with very low density lipoprotein or when apoE in secreted high density lipoprotein is needed to acquire cholesteryl esters for the synthesis of bile salts and acids by liver.