Expression of human growth hormone by the eukaryotic alga, Chlorella.

A method to use Chlorella to express a recombinant heterologous protein that can be recovered from the extracellular medium has been developed. Plasmids are constructed with an extracellular secretion signal sequence inserted between a promoter region and a gene for human growth hormone (hGH). The plasmids also contain a Kanr region which confers resistance to the antibiotic G418. Protoplasts are prepared by enzymatic treatment, and the plasmid is introduced by incubation of the protoplasts with polyethylene glycol and dimethyl sulfoxide. Cells are then grown in the presence of G418, and the medium is collected from 6 days after transfection. hGH is measured by immunoassay, and values for expressed hGH of about 200-600 ng/ml are obtained.

Utilization of xylose for growth by the eukaryotic alga, Chlorella.

A green alga, Chlorella, was found to be capable of utilizing xylose or other pentose sugars (xylitol, arabinose) for enhanced growth rates when grown in the light, but not when grown heterotrophically in the dark. With selection for growth in xylose-containing medium, it was possible to improve dramatically the ability of selected Chlorella strains to grow on xylose mixotrophically. Growth on arabinose or xylitol was not changed in the xylose-selected strains.

Activin antiserum infused into the lateral hypothalamic area affects operant behavior of rats fed lysine-deficient diet.

Rats were trained to maintain a high rate of bar pressing to receive 50-mg pellets of a complete diet when given a lysine-deficient (Lys-def) diet ad libitum. This bar-pressing behavior was significantly inhibited when rats were also allowed ad libitum access to 0.4 M Lys to drink. A brain activin system may modulate motivation to engage in bar-pressing behavior, since previous work has established that antagonism of activin by infusion of inhibin or follistatin, but not activin, into the lateral hypothalamic area (LHA) also inhibits bar-pressing behavior. The present study sought to clarify whether the effect of inhibin or follistatin might be mediated by antagonism of endogenous activin or by a separate direct effect of inhibin or follistatin. Thus, we infused an antiserum, which specifically inhibits activin A activity, into the LHA. Infusion of antiserum greatly inhibited bar-pressing behavior of rats fed a Lys-def diet and was additive with Lys consumption further to decrease bar pressing. Ad libitum Lys consumption was unchanged from control levels, indicating that it is likely that an endogenous activin system in the LHA mediates behavioral responsiveness when rats are fed a Lys-def diet but does not appear specifically to affect appetite for Lys.

Recognition of deficient nutrient intake in the brain of rat withL-lysine deficiency monitored by functional magnetic resonance imaging, electrophysiologically and behaviorally.

EachL-amino acid (AA) in plasma and brain remains unchanged all day long while normal diet is available. But once restriction ofL-lysine (Lys) was introduced, strong anorexia happened. When Lys deficient diet was offered to rats, their growth were decreased depending upon dietary Lys intake, and they ingested Lys solution in choice quantitatively and both appetite and growth normalized. The recognition site for the deficit in rat's brain was identified by brain oxygenation using a functional MRI that higher signals in the ventromedial hypothalamus and lateral hypothalamic area (LHA) appeared, at 30-50 minutes after Lys injection i.p. and then recovered. Degree of Lys hunger, assayed by bar-pressing (50mg pellet of normal diet/30 presses), was suppressed by Lys micro-injection into the LHA, similar to free Lys ingestion but any other AA never did, suggesting the LHA as recognition site for Lys deficit in rats with Lys deficiency due to AA homeostasis.

Effect of inhibin, follistatin, or activin infusion into the lateral hypothalamus on operant behavior of rats fed lysine deficient diet.

To identify brain mechanisms which mediate hunger for amino acid (e.g. L-lysine; Lys) deficiency, rats were trained to bar press (FR30 schedule) to receive 50 mg pellets of a complete diet. Rats given a lysine deficient (Lys-def) diet ad libitum maintained a high rate of bar pressing but when allowed ad libitum access to 0.4 M Lys to drink had a significant decrease in pressing. Also, Lys continuously infused by minipump into the lateral hypothalamic area (LHA) inhibits pressing by rats given a Lys-def diet. The threshold maximal dose is between 0.1-0.5 nmol Lys/h. Therefore, animals lacking dietary Lys will work to receive complete diet, but replacement of Lys by voluntary consumption or by direct infusion into the LHA inhibits bar pressing for complete diet. The ratio of brain activin and inhibin may modulate motivation to work for a complete diet, since continuous inhibin or follistatin, but not activin, infusion into the LHA was found to inhibit bar pressing, which is normally quite strong in rats maintained on Lys-def diet. The inhibitory effect of LHA inhibin infusion was replicated, and concurrent availability of Lys solution ad libitum was additive with LHA inhibin infusion to depress responding further. This inhibitory effect of inhibin or follistatin did not result from altered ad lib. consumption of Lys-def diet. Although LHA Lys infusion did decrease consumption of a concurrently available Lys solution, inhibin did not change ad libitum Lys consumption. This indicates that inhibin may work in the LHA to inhibit bar pressing for complete diet via other mechanisms than sensation of Lys deficiency.

Proline, ascorbic acid, or thioredoxin affect jaundice and mortality in Long Evans cinnamon rats.

The Long Evans Cinnamon (LEC) rat spontaneously develops fulminant hepatitis, which is usually lethal due to excess copper accumulation in the liver and is considered an animal model of Wilson's disease. LEC rats show a strong appetite for proline solution. Daily oral (p.o.) administration of proline resulted in significant delay of mortality. Feeding a copper-deficient diet greatly delayed the onset of jaundice and mortality and voluntary consumption or p.o. administration of proline further delayed jaundice and prevented mortality. LEC rats also consume ascorbic acid solutions, and p.o. administration of ascorbate also results in a significant delay in the appearance of jaundice and mortality. Combined treatment with ascorbic acid and proline is additive to delay further jaundice and mortality. An endogenous antioxidant protein, thioredoxin, when infused by minipump IP, could also inhibit the incidence of jaundice. These results indicate that antioxidant treatment combined with proline may be of benefit in Wilson's disease and possibly other forms of hepatic dysfunction.

Effect of continuous infusion of lysine via different routes and hepatic vagotomy on dietary choice in rats.

The effect of continuous L-lysine (Lys) infusion on dietary choice between Lys deficient and protein-free diets in Sprague-Dawley rats was studied to determine the sensing site of Lys deficiency. After daily intake of each diet became constant, Lys was continuously infused for 11 days via intraperitoneal (IP), intragastric (IG) or intracerebroventricular (ICV) route, with an osmotic pump. Daily intake of each diet was measured. Intake of the Lys deficient diet compared with protein-free diet in either IP or IG Lys-infused group increased significantly (p < 0.001) vs. the intake in the baseline period. The selection of the Lys deficient diet was quite comparable between IP and IG groups. But the intake of the ICV group was unchanged. Hepatic vagotomy during IP infusion transiently delayed selection of the Lys deficient diet. These results imply the roles of postabsorptive mechanisms in sensing an amino acid deficiency, and possible involvement of the hepatic branch of the vagus in the sensing. However, sensing in the brain or indeed in the intestine was not excluded.

Gastrulation in the sea urchin, Strongylocentrotus purpuratus, is disrupted by the small laminin peptides YIGSR and IKVAV.

Laminin is present on the apical and basolateral sides of epithelial cells of very early sea urchin blastulae. We investigated whether small laminin-peptides, known to have cell binding activities, alter the development of sea urchin embryos. The peptide YIGSR-NH2 (850 microM) and the peptide PA22-2 (5 microM), which contains the peptide sequence IKVAV (Tashiro et al., J. Biol. Chem. 264, 16174, 1989), typically blocked archenteron formation when added to the sea water soon after fertilization. At lower doses, the YIGSR peptide allowed invagination of the archenteron but blocked archenteron extension and differentiation and evagination of the feeding arms. The effect of YIGSR and PA22-2 peptides declined when added to progressively older stages until no effect was seen when added at the mesenchyme blastula stage (24 hours after fertilization). Control peptides GRGDS, YIGSE, and SHA22, a dodeca-peptide with a scrambled IKVAV sequence, had no effect on development. The YIGSK peptide containing a conserved amino acid modification had only a small effect on gastrulation. The results suggest that YIGSR and IKVAV peptides specifically disrupt cell/extracellular matrix interactions required for normal development of the archenteron and feeding arms. Our recent finding that YTGIR is at the cell binding site of the B1 chain of S. purpuratus laminin supports this conclusion. Evidently, laminin or other laminin-like molecules are among the many extracellular matrix components needed for the invagination and extension of the archenteron during the gastrulation movements of these embryos.

Lysine deficient diet and lysine replacement affect food directed operant behavior.

As a test for specific hunger for amino acids with the goal of identifying brain regions which might mediate motivation to alleviate L-lysine (Lys) deficiency, rats were trained to lever press to receive complete diet. Rats were given Lys deficient (Lys-def) diet ad lib. Intraperitoneal (IP) injection of Lys, 2 h before the test session, inhibited lever pressing. Lys injected animals pressed at nearly the same low rate as did animals fed ad lib complete diet, although the response of saline injected animals was also partially decreased. Next, osmotic minipumps were implanted IP and animals given Lys-def diet ad lib. Chronic Lys infusion also strongly inhibited lever pressing. Rats allowed ad lib access to Lys to drink drank significantly more Lys than when given complete diet. Lys chronically infused by minipump into the lateral hypothalamus, also inhibited pressing by rats given Lys-def diet. Therefore, animals lacking Lys in the diet will work to receive complete diet, but replacement of Lys by drinking, chronic IP infusion, or directly into the lateral hypothalamic area inhibits bar pressing behavior.

Autonomic dysreflexia resulting from prolapsed hemorrhoids. Report of a case.

PURPOSE: This article reports a case of autonomic dysreflexia associated with hemorrhoidal disease in a patient with high spinal cord lesions and successful treatment by surgical hemorrhoidectomy. METHODS: Following an unsuccessful attempt at conservative treatment which included bulk agents and warm compresses, the patient subsequently underwent three-column, closed surgical hemorrhoidectomy. RESULTS: The patient was symptom free and had normal bowel activity six weeks postoperatively, and five-year follow-up showed no recurrence of the hemorrhoidal prolapse or dysreflexia. CONCLUSION: Carefully controlled hemorrhoidectomy, when conservative measures fail, may be effective in managing autonomic dysreflexia in high spinal cord transection patients when prolapse serves as the stimulus.

Measurements of collisional linewidths in the nu(2) band of H(2)O from Fourier-transformed flame spectra.

The collisional widths of more than 200 transitions belonging to the v(2) band of the H(2)O molecule were measured by using the Fourier-transformed spectra of an air-methane flame at 2000 K. A nonlinear least-squares method was used to determine the line widths for a wide range of J (up to 28) and K(a) (up to 14) quantum number values. Finally, an analysis of the results as functions of J and K(a) is presented.

Effects of clonidine on breathing movements and electrocortical activity in the fetal lamb.

Clonidine is a recommended antihypertensive for use during pregnancy, although little is known of its fetal effects. This study examines the effects of clonidine on breathing and sleep-state cycling in fetal lambs. Clonidine was infused into a fetal lateral ventricle for up to 24 hours at 128 to 135 days' gestation. Control infusions of artificial cerebrospinal fluid had no effect. Clonidine infusion significantly reduced the incidence and episode duration of fetal breathing for the duration of the infusion period. Cycling of electrocortical activity became irregular and rapid, and the incidence of high-voltage electrocortical activity (equivalent to quiet sleep) was reduced. Fetal heart rate decreased but arterial pressure was unaffected. After infusion the breathing incidence and episode duration both increased significantly compared with control, with continuous high-amplitude breathing for several hours, whereas the incidence of high-voltage electrocortical activity remained low. Because lung development is promoted by fetal breathing, long-term use of clonidine during pregnancy could slow lung development by reducing fetal breathing activity.

Central effects of an alpha 2-adrenergic antagonist on fetal lambs: a possible mechanism for hypoxic apnea.

In 12 chronically-prepared fetal lambs at 128-135 days gestation, a specific alpha 2-antagonist, L-657,743 (Merck) was infused into a lateral cerebral ventricle for periods of up to 24 h. Control infusions of artificial CSF had no effect. L-657,743 at a dose rate sufficient to block the actions of injected clonidine did not affect breathing incidence or episode duration, electrocortical activity, heart rate, arterial pressure or blood gases during normoxic conditions. However, 5 out of 6 preparations receiving L-657,743 while hypoxic continued to breathe during hypoxia instead of becoming apneic as normal. The central effects of alpha 2-adrenergic blockade appear to be specific to hypoxia. It is concluded that fetal hypoxic apnea may be mediated by inhibitory alpha 2-adrenergic receptors which can be blocked by the antagonist L-657,743.

The effects of brain-stem section on the breathing and behavioural response to morphine in the fetal sheep.

In the unanesthetized fetal sheep the administration of morphine causes initial apnoea followed by hyperpnoea. We thought that a section of the brain at midcollicular level might separate these two effects. Therefore we sectioned the brain stem of five fetuses at 132 +/- 1 (SEM) days of gestation and compared their responses to morphine (17 experiments) with that observed in seven intact fetuses at similar gestational ages (15 experiments). Brain stem sections were confirmed morphologically and histologically. Morphine, 1 mg/kg was injected in the fetal jugular vein during low-voltage electrocortical activity (ECoG). We measured ECoG, eye movements, diaphragmatic activity, blood pressure and amniotic pressure. Sectioned fetuses before the administration of morphine had a complete dissociation between ECoG and breathing activity. With the administration of morphine we found: (i) the length of the apnoea was 139.8 +/- 15.5 min in sectioned fetuses and 17.0 +/- 5.8 min in intact fetuses (P less than 0.01); and (ii) there was no hyperpneic response in the sectioned fetus whereas the length of hyperpnoea in the intact group was 99.1 +/- 11.8 min (P less than 0.001). The results support the idea of two central distinct areas of action of morphine in the fetal brain. The absence of hyperpnoea in the sectioned fetuses suggests that neurons inhibiting the 'respiratory neurons' are located rostrally to the mid-collicular line.

Protease inhibitors influence the direction of neurite outgrowth.

Addition of protease inhibitors to the culture medium has been shown to enhance neurite outgrowth by cultured mouse dorsal root ganglia (DRG). Those results are now extended to show that a diffusible source of soybean trypsin inhibitor (STI) or zones of immobilized STI can orient the direction of outgrowth towards the region of STI. However, a high concentration of diffusible STI promotes outgrowth in the opposite direction from the STI source. Immobilized leupeptin, L-lysine, or D-Phe-Pro-Arg-chloromethyl ketone can also direct outgrowth towards their immobilized areas, as do zones of laminin or fibronectin. However, derivatized zones containing urokinase or thrombin preferentially direct outgrowth away from those zones. These data support the hypothesis that a balance between extracellular protease and inhibitor is important in mediating interactions between neurite growth cone and extracellular matrix.