High-performance liquid chromatography of type-III heptocarboxylic porphyrinogen isomers.

A reversed-phase h.p.l.c. system is described for the separation of the four type-III heptacarboxylic porphyrinogen isomers. The effects of buffer concentration, pH and type and proportion of organic modifier in the mobile phase on retention and resolution of isomers were studied. Optimum separation on an ODS-Hypersil column was by elution with a ternary mobile phase of acetonitrile, methanol and 1 M-ammonium acetate, pH 5.16 (7:3:90, by vol.). Isomer identification was based on a comparison of their retention times with those of authentic standards, and was further confirmed by h.p.l.c. analysis of the characteristic mixture of three pentacarboxylic porphyrins formed after partial decarboxylation of individual isomers in 0.3 M-HCl at 160 degrees C.     

Effect of egg cholesterol and dietary fats on plasma lipids, lipoproteins, and apoproteins of normal women consuming natural diets

Nine normal women, 22 to 37 years old, consumed controlled quantities of natural foods to test their responses to dietary cholesterol and saturated fat. All diets contained, as percentage of calories, 14% protein, 31% fat, and 55% carbohydrate. The main sources of polyunsaturated and saturated fats were corn oil and lard, respectively, and egg yolk was used for cholesterol supplementation. All subjects participated in four diet protocols of 15 days duration, and each diet period was separated by 3 weeks without diet control. The first diet (corn) was based on corn oil, had a polyunsaturated to saturated fat ratio (P/S) of 2.14, and contained 130 mg of cholesterol. The second diet (corn+) was identical to the first but contained a total of 875 mg of cholesterol. The third diet (lard) was based on lard, had a P/S ratio of 0.64, and contained 130 mg of cholesterol. The fourth diet (lard+) was identical to the third, but contained 875 mg of cholesterol per day. Changes of the plasma lipid, lipoprotein and apoprotein parameters relative to the corn diet were as follows: the corn+ diet significantly increased total plasma cholesterol, HDL-cholesterol, LDL-cholesterol, and apoB levels; the lard diet significantly increased total cholesterol, HDL-cholesterol, and apoB; and the lard+ diet significantly increased the total cholesterol, HDL-cholesterol, LDL-cholesterol, and apoA-I and apoB levels. There were no significant variations in VLDL-cholesterol, triglyceride, or apoE levels with these diets. The diets affected both the number of lipoprotein particles as well as the composition of LDL and HDL. Compared to the corn diet, cholesterol and saturated fat each increased the number of LDL particles by 17% and 9%, respectively, and the cholesterol per particle by 9%. The combination of saturated fat and cholesterol increased particle number by 18% and particle size by 24%. Switching from lard+ to lard, corn+, or corn diets reduced LDL-cholesterol of the group by 18%, 11%, and 28%, respectively, while a large inter-individual variability was noted. In summary, dietary fat and cholesterol affect lipid and lipoprotein levels as well as the particle number and chemical composition of both LDL and HDL. There is, however, considerable inter-individual heterogeneity in response to diet.     

Trifluoperazine Activates and Releases Latent ATP-Generating Enzymes Associated with the Synaptic Plasma Membrane

Neurone-specific enolase (NSE) and the brain form of creatine phosphokinase (CPK-BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two-dimensional gel (2-D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be "cryptic" unless the SPM were treated with Triton X-100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for "unmasking" the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK-BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2-D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70-kilodalton heat-shock cognate protein. Tubulin, synapsin I, and a 35-kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase on the basis of 2-D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.     

Rat corticotropin-releasing hormone gene: sequence and tissue-specific expression

The rat corticotropin releasing hormone (CRH) gene has been isolated and characterized by DNA sequence analysis. The gene exhibits a structural organization similar to that of the human CRH gene. The nucleotide sequence encoding the entire rat CRH precursor is located on the second exon, while exon I encodes the 5'-untranslated region of the mRNA. Analysis of the nucleotide sequence homology between the human and rat CRH genes reveals several highly conserved regions including the CRH peptide-encoding sequence and the 5'-flanking sequence. RNA blot analysis demonstrates that CRH mRNA can be observed in numerous regions of the rat brain as well as the spinal cord, adrenal gland, pituitary, and testis.     

Feedback optimization of fed-batch fermentation

The problem of feedback optimization of the feed rate for fed-batch fermentation processes is formulated in the framework of singular control theory and switching hypersurfaces. Using four differential balance equations that describe a general class of fedbatch processes and a general objective function to be minimized, it is shown that under certain restrictions the feedback optimization of the feed rate can be realized as a nonlinear function of the state variables, such as the concentrations of cell mass, substrate and product, and the fermentor volume. The restrictions on the initial conditions, the fermentation kinetics and the objective function, that are needed for realization of the feedback optimization, are provided. Fed-batch fermentation models of lysine and alcohol are used to construct switching curves and to illustrate the feedback optimization of the feed flow rates.     

A translocation associated, loss-of-function mutation in the nitrogen metabolite repression regulatory gene of Aspergillus nidulans can revert intracistronically

Summary The areA ^r -18 mutation is a loss-of-function mutation in areA, the positive acting regulatory gene mediating nitrogen metabolite repression in Aspergillus nidulans. It results from a reciprocal translocation which splits the coding region into 5 and 3 moieties. Surprisingly, we have selected rare intracistronic revertants of areA ^r -18. From crosses heterozygous for areA ^r -18 revertant alleles, duplication-deficiency progeny containing two copies of a substantial portion of chromosome IV but lacking part of chromosome III, including the 5 moiety of areA, have been obtained. For all four revertants analysed genetically, growth properties of these duplication-deficiency strains indicate that the reversion events involve the 3 portion of areA and that the 5 portion of areA is unnecessary for the revertant phenotype. This conclusion was directly confirmed for one revertant using Southern blotting. As all four reversion events involve additional chromosomal rearrangements, they probably fuse functional promoters, ribosome binding sites and in frame initiation codons to the 3 portion of the gene. In the course of characterisation of these mutations, new mapping data for a large region of chromosome IV have been generated, and a new reciprocal translocation activating the cryptic regulatory gene areB, whose product can substitute for that of areA, has been identified.     

Incorporation of measured photosynthetic rate in a mathematical model for calculation of non-structural saccharide concentration

A simple mathematical model for calculating the concentration of mobile carbon skeletons in the shoot of soya bean plants [Glycine max (L.) Merrill cv. Ransom] was built to examine the suitability of measured net photosynthetic rates (PN) for calculation of saccharide flux into the plant. The results suggest that either measurement of instantaneous PN overestimated saccharide influx or respiration rates utilized in the model were underestimated. If neither of these is the case, end-product inhibition of photosynthesis or waste respiration through the alternative pathway should be included in modelling of CH2O influx or efflux; and even if either of these is the case, the model output at a low coefficient of leaf activity indicates that PN still may be controlled by either end-product inhibition or alternative respiration.     

Isolation and characterization of cDNA clones encoding the 17.9 and 8.1 kDa subunits of Photosystem I from Chlamydomonas reinhardtii

cDNA clones encoding two Photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses of 18 and 11 kDa (thylakoid polypeptides 21 and 30; P21 and P30 respectively) were isolated using oligonucleotides, the sequences of which were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that both proteins are encoded by single-copy genes. The mRNA sizes of the two components are 1400 and 740 nucleotides, respectively. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the molecular masses of the mature proteins are 17.9 (P21) and 8.1 kDa (P30). Analysis of the deduced protein sequences predicts that both subunits are extrinsic membrane proteins with net positive charges. The amino acid sequences of the transit peptides suggest that P21 and P30 are routed towards the lumenal and stromal sides of the thylakoid membranes, respectively.Abbreviations OEE1, 2 and 3 oxygen evolution enhancer proteins 1, 2 and 3 - Rubisco ribulose bisphosphate carboxylase/oxygenase - PS photosystem - P21 and P30 C. reinhardtii thylakoid polypeptides 21 and 30