Characterization and sequence analysis of a developmentally regulated putative cell wall protein gene isolated from soybean

A cDNA clone, pTU04, which hybridizes to two different sizes of mRNA on Northern blots was isolated from soybean suspension culture cell poly(A) RNA. Northern analysis reveals that meristematic tissue produces a 1050-nucleotide mRNA while quiescent mature cells produce primarily a 1220-nucleotide mRNA homologous to pTU04. The cDNA and its corresponding genomic clone have been partially characterized. The nucleotide sequence of the gene predicts a proline-rich protein, designated SbPRP1, which contains a signal peptide sequence and 43 repeats of a sequence consisting primarily of Pro-Pro-Val-Tyr-Lys (CCA-CCA-GTT-TAC-AAG). From nuclease S1 and hybrid-select translation analyses, the cDNA clone pTU04 appears to represent the mRNA for the mature tissue 1220-nucleotide RNA observed on Northern blots. Although there is no direct proof that the encoded protein is a cell wall protein, it has the properties similar to previously isolated cell wall proteins: 1) it is very basic with a high content of Pro, Tyr, and Lys; 2) it has similar hydropathic properties; and 3) its repeating unit shares sequence homology with that of more highly characterized cell wall proteins, generally termed extensin (Chen, J., and Varner, J. E. (1985) EMBO J. 4, 2145-2151; Smith, J. J., Muldoon, E. P., Willard, J. J., and Lamport, D. T. A. (1986) Phytochemistry 25, 1021-1030.     

Absence of heat shock protein synthesis in isolated mitochondria and plastids from maize

Examination of the proteins synthesized by isolated mitochondria, chloroplasts, or proplastids from maize tissues showed that a heat treatment at 40 degrees C does not induce or enhance the synthesis of any protein when compared to preparations treated at the control temperature of 28 degrees C. These observations are consistent with the results obtained by labeling proteins in vivo under sterile conditions. In vivo labeling in the presence of cycloheximide during heat shock showed no heat shock protein synthesis. Labeling in the presence of chloramphenicol during heat shock showed a similar heat shock protein pattern as in the absence of the inhibitor. It is concluded that maize organelles do not synthesize heat shock proteins and that, if present, they may be due to bacterial contamination.     

Hepatic metabolism and secretion of a cholesterol-enriched lipoprotein fraction

A potentially important source of cholesterol secreted in bile is cholesterol-rich lipoproteins. However, the fate of the cholesterol carried in these lipoproteins after hepatic uptake has not been investigated. We harvested an apoE- and cholesterol-rich lipoprotein fraction (d 1.02-1.06 g/ml) from hypercholesterolemic rats and examined the acute effects of these lipoproteins on hepatic cholesterol metabolism, very low density lipoprotein (VLDL) secretion, and biliary lipid secretion. Administration of a lipoprotein bolus (20 mg of cholesterol) to rats resulted in a significant decrease in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and a significant increase in acyl-coenzyme A:cholesterol acyltransferase activity over controls at 1 hr. Hepatic cholesteryl ester content increased 400% with no change in hepatic free cholesterol content or biliary cholesterol secretion. These cholesterol-rich lipoproteins delivered in the isolated perfused liver effected a fivefold increase in hepatic VLDL secretion with no change in composition. Therefore, cholesterol-rich lipoproteins do not acutely alter biliary cholesterol secretion. Rather, the majority of the cholesterol delivered to the liver in these lipoproteins is either esterified and stored as cholesteryl ester or resecreted as free and esterified cholesterol in hepatic VLDL.     

Phosphatidylinositol Phosphodiesterase (Phospholipase C) Activity in the Pineal Gland: Characterization and Photoneural Regulation

Phosphatidylinositol phosphodiesterase (PL-C) appears to be a key element in the adrenergic regulation of pineal cyclic AMP levels. In the present study, the rat pineal enzyme was characterized using exogenous [3H]phosphatidylinositol (0.5 mM) as substrate. Half the enzyme activity was found in the cytosolic fraction, but the highest specific concentration was associated with the membrane fraction. Two pH optima (5.5 and 7.5) of enzyme activity were observed for the membrane fraction but only one in the cytosol fraction (pH 5.5). Enzyme activity in both fractions was Ca2+ dependent. In the case of the membrane protein in pH 7.5, the enzyme activity was sensitive to changes in Ca2+ in the 10-100 nM range. Addition of an equimolar concentration of phosphatidylinositol 4-phosphate nearly completely inhibited the hydrolysis of [3H]phosphatidylinositol; other phospholipids (1.0 mM) were less potent. This may reflect our present finding that [3H]phosphatidylinositol 4-phosphate is a better substrate than [3H]phosphatidylinositol for the enzyme. Stimulus deprivation (2 weeks of constant light or superior cervical ganglionectomy) reduced the cytosolic activity by 30% and had no effect on the membrane-associated enzyme.     

Ethanol inhibits dual receptor stimulation of pineal cAMP and cGMP by vasoactive intestinal peptide and phenylephrine

Concurrent activation of vasoactive intestinal peptide and alpha 1-adrenergic receptor resulted in greater than 20-fold increases in pineal cAMP and cGMP accumulation. We now find that an intoxicating level of ethanol (0.2%, 34 mM) inhibits greater than 50% the large increases in pineal cAMP and cGMP produced by concurrent treatment with vasoactive intestinal peptide and phenylephrine. The potency of the various alcohols tested was directly related to their chain length. This inhibition appears to be specific since a five-fold higher concentration of ethanol does not inhibit the stimulation of cAMP and cGMP accumulation produced by concurrent treatment with isoproterenol and phenylephrine. Accordingly, it seems that one mechanism of action of ethanol on neural function may be its ability to selectively inhibit ethanol-sensitive integrative mechanisms which regulate cyclic nucleotides.     

Cardiac glycosides stimulate phospholipase C activity in rat pinealocytes

Ouabain and related cardiac glycosides stimulate phospholipase C activity 5-fold in rat pinealocytes. The combined treatment of ouabain and norepinephrine, which also stimulates phospholipase C, produces an additive effect. The effects of either ouabain or norepinephrine are blocked by EGTA. However, there are notable differences. The stimulatory effect of ouabain is lost when extracellular Na+ is reduced to 20 mM and is not blocked by prazosin. In contrast, the stimulatory effect of norepinephrine is not blocked when extracellular Na+ is reduced to 20 mM but is blocked by prazosin. Ouabain appears to increase phospholipase C activity through a mechanism involving inhibition of Na+,K+-ATPase, and an accumulation of intracellular Na+ and Ca2+, not involving alpha 1-adrenoceptors. These findings raise the possibility that activation of phospholipase C might be a more general effect of cardiac glycosides.     

The induction of a specific form of cytochrome P-450 (P-450j) by fasting

In previous work we have demonstrated that liver microsomal N-nitrosodimethylamine demethylase (NDMAd) activity is increased in rats by fasting, and we have postulated that this is due to the induction of a specific form of cytochrome P-450. This communication provides evidence for such a hypothesis. Fasting for 24 and 48 h caused 59 and 116% increases, respectively, in NDMAd activity in male rats, and fasting for 48 h caused a 63% increase in female rats. These increases were accompanied by corresponding increases of cytochrome P-450j (P-450ac) determined by immunoblotting. Fasting for 24 and 48 h also increased the mRNA for P-450j by 153 to 250%, as determined by hybridization with a cDNA probe of this cytochrome. The results suggest that fasting affects the gene expression of P-450j.     

Enzymatic production of ethanol from cellulose using soluble cellulose acetate as an intermediate

A two-stage process for the enzymatic conversion of cellulose to ethanol is proposed as an alternative to currently incomplete and relatively slow enzymatic conversion processes employing natural insoluble cellulose. This alternative approach is designed to promote faster and more complete conversion of cellulose to fermentable sugars through the use of a homogeneous enzymatic hydrolysis reaction. Cellulose is chemically dissolved in the first stage to form water-soluble cellulose acetate (WSCA). The WSCA is then converted to ethanol in a simultaneous saccharification-fermentation with Pestal-otiopsis westerdijkii enzymes (containing cellulolytic and acetyl esterase components) and yeast.Water-soluble cellulose acetate was successfully prepared from purified wood cellulose (Solka Floe) and chemical reagents. Enzyme pretreatment of WSCAto form metabolizable sugars was a necessary step in achieving practical conversion of WSCA to ethanol using yeast. The results showed that WSCA has a low enzyme requirement and a high convertibility to reducing sugars with enzymes from P. westerdijkii fungus. Pestalotiopsis westerdijkii enzymes were found to be superior to enzymes from Trichoderma viride in producing metabolizable glucose from WSCA. The yeast utilized 55-70% of the hydrolyzate sugars that were produced by P. westerrlijkii enzymes on WSCA and produced ethanol. The acetate that was liberated into solution by the action of acetyl esterase enzymes on WSCA was found to have a stimulatory effect on ethanol production in yeast. This is an important feature that can be used to advantage in manipulating the conversion to maximize the production of ethanol. Hence, the simultaneous saccharification-fermentation of WSCA to ethanol using P. westerdijkii enzymes and yeast has features that are highly desirable for developing an economical cellulose conversion process.     

Unsteady-state operation of continuous fermentor for enhancement of cell mass production

The transient behavior of continuous fermentation is studied concentrating on the time scale intrinsic to the system. The time scale is the time required for the fermentorto reach a stable steady state after the disturbance of cell mass is introduced. When the cell concentration is disturbed from the steady-state value, in particular, at the dilution rate near washout, the transient period becomes extended significantly, and the steady state is resumed sluggishly. This sluggish transient behavior could be turned to an advantage for enhancing the cell mass output rate. The proposed transient operation is a continuous fermentation whereby a positive disturbance in the cell mass is introduced, so that the cell concentration is higher than the steady-state value for an extended transient period. It is shown that a significantly higher cell mass production than that from the steady-state continuous fermentation can be achieved. Simple experiments were performed to demonstrate the improvement of cell (Candida utilis) productivity.