ONTOGENETIC CHANGES OF PROTEINS OF ENDOPLASMIC RETICULUM

The proteins of the smooth and rough endoplasmic reticulum from fetal, immature, and adult male rats were compared after incorporation of two radioactively labeled precursors, ¹⁴C-labeled amino acids and δ-aminolevulinic acid-³H by means of gel electrophoresis. The labeling patterns indicated that protein components present in two major electrophoretic bands underwent significant synthesis in fetal tissue while three actively incorporating protein bands were noted in adult tissue. Although the uptake of the amino acids-¹⁴C decreased for the smooth and rough elements of the endoplasmic reticulum as a whole during liver development, the qualitative patterns were not significantly different in adult and fetal livers. The over-all incorporation (disintegrations per minute per milligram protein) of the heme precursor into the smooth and rough elements also did not change with development. However, a change was noted in the distributional electrophoretic patterns with development. The estimation of molecular weight (by disc electrophoresis) and the incorporation of the heme precursor suggested the similarity of the two major protein bands to cytochrome P-450 and cytochrome b₅, components of the endoplasmic reticulum, thought to be involved in the mixed-function oxidase system. The evidence indicated that in fetal liver, at a time when the oxidase capability was low, the amino acid incorporation into these two protein groups was the same as in the adult. The incorporation of the heme moiety, however, was different, decreasing in the cytochrome b₅ region and increasing in the cytochrome P-450 region during development. These results correlate with the increase in oxidase activity associated with liver development.     

Connectivity of Caribbean coral populations: complementary insights from empirical and modelled gene flow

Understanding patterns of connectivity among populations of marine organisms is essential for the development of realistic, spatially explicit models of population dynamics. Two approaches, empirical genetic patterns and oceanographic dispersal modelling, have been used to estimate levels of evolutionary connectivity among marine populations but rarely have their potentially complementary insights been combined. Here, a spatially realistic Lagrangian model of larval dispersal and a theoretical genetic model are integrated with the most extensive study of gene flow in a Caribbean marine organism. The 871 genets collected from 26 sites spread over the wider Caribbean subsampled 45.8% of the 1900 potential unique genets in the model. At a coarse scale, significant consensus between modelled estimates of genetic structure and empirical genetic data for populations of the reef-building coral Montastraea annularis is observed. However, modelled and empirical data differ in their estimates of connectivity among northern Mesoamerican reefs indicating that processes other than dispersal may dominate here. Further, the geographic location and porosity of the previously described east-west barrier to gene flow in the Caribbean is refined. A multi-prong approach, integrating genetic data and spatially realistic models of larval dispersal and genetic projection, provides complementary insights into the processes underpinning population connectivity in marine invertebrates on evolutionary timescales.     

Exocyclic DNA adducts as oxidative stress markers in colon carcinogenesis: potential role of lipid peroxidation,dietary fat and antioxidants

Molecular pathways to colorectal cancer involve multiple genetic changes, whereby extensive oxyradical damage causes mutations in cancer-related genes and leads to a cycle of cell death and regeneration. Besides direct oxidative DNA-damage, reactive oxygen and nitrogen species can induce etheno (epsilon)-DNA adducts mainly via trans-4-hydroxy-2-nonenal, generated as the major aldehyde by lipid peroxidation (LPO) of omega-6 PUFAs. Patients with familial adenomatous polyposis (FAP) develop multiple colorectal adenomas. In affected tissues increased LPO could be triggered due to increased arachidonic acid metabolism as a result of elevated cyclooxygenases. Our studies demonstrated an increased epsilon-DNA adduct level in affected colon epithelia of FAP patients. Epsilon-DNA adducts are promutagenic and can cause genomic instability that drives colorectal adenoma to malignancy. We have further investigated the potential chemopreventive properties of olive oil and its polyphenolic components. 'Mediterranean diet', of which olive oil is a major fatty acid source, has protective effects against human breast and colorectal cancers. Olive oil extracts and the newly identified lignan fractions showed high antioxidant capacity in vitro. As epsilon-DNA adducts are biomarkers for oxidative stress and LPO induced DNA damage, they can verify the efficacy of newly identified antioxidants, e.g. from olive oil, as chemopreventive agents against colon carcinogenesis.     

beta-Hydroxyaspartic acid or beta-hydroxyasparagine in bovine low density lipoprotein receptor and in bovine thrombomodulin

All of the vitamin K-dependent plasma proteins with domains that are homologous to the epidermal growth factor (EGF) precursor have 1 hydroxylated aspartic acid residue in the NH2-terminal EGF-homology region. In addition, protein S has 1 hydroxylated asparagine residue in each of the three COOH-terminal EGF-homology regions. All of these proteins have been found to have the amino acid sequence, CX(D or N)XXXX(F or Y)XCXC (corresponding to residues 20 to 33 in EGF), where the Asp or Asn residue is hydroxylated. This sequence also appears in two of the three EGF-homology regions of the human low density lipoprotein receptor and in two of the six EGF-homology regions of bovine thrombomodulin so far identified, suggesting that they may have the modified amino acid. We have now identified beta-hydroxyaspartic acid in acid hydrolysates of both these proteins.     

Effect of methylmalonate on ketone body metabolism in developing rat brain

The oxidation of 3-hydroxy[3-¹⁴C]butyrate to CO₂ and its incorporation into cerebral lipids by cortex slices from one-week old rats were markedly inhibited by methylmalonate. However, methylmalonate had no effect on the metabolism of labelled aceto- acetate, glucose and acetate by brain slices. Addition of propionate in the incubation medium reduced cerebral lipogenesis from labelled 3-hydroxybutyrate and acetate. Acute methylmalonic acidemia induced in one-week old pups by injecting 3% methylmalonate solution caused a reduction in the incorporation of labelled 3-hydroxybutyrate into cerebral lipids. However, acute methylmalonic acidemia had no effect on cerebral lipogensis $\text{in vivo}$ from labelled acetate. These findings show (i) the conversion of 3-hydroxybutyrate to acetoacetate by 3-hydroxybutyrate dehydrogenase in the brain is inhibited by methylmalonate, and (ii) an inhibition of cerebral lipid synthesis by propionate, which also accumulates in patients with methylmalonic aciduria.     

First Report of a Deletion Encompassing an Entire Exon in the Homogentisate 1,2-Dioxygenase Gene Causing Alkaptonuria

Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5–16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband’s phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin.     

Medicinal history of North American Veratrum

Plants belonging to the genus Veratrum have been used throughout history for their medicinal properties. During the nineteenth and twentieth centuries, phytochemical investigations revealed a host of steroidal alkaloids in Veratrum species, some of which are potent bioactives. This review discusses Veratrum species that grow in North America with a focus on the medicinal history of these plants and the steroidal alkaloids they contain. While significant reviews have been devoted to singularly describing the plant species within the genus Veratrum (botany), the staggering breadth of alkaloids isolated from these and related plants (phytochemistry), and the intricacies of how the various alkaloids act on their biological targets (physiology and biochemistry), this review will straddle the margins of the aforementioned disciplines in an attempt to provide a unified, coherent picture of the Veratrum plants of North America and the medicinal uses of their bioactive steroidal alkaloids.     

Route-dependent effect of nutritional support on liver glucose uptake

The liver is a major site of glucose disposal during chronic (5 day) total parenteral (TPN) and enteral (TEN) nutrition. Net hepatic glucose uptake (NHGU) is dependent on the route of delivery when only glucose is delivered acutely; however, the hepatic response to chronic TPN and TEN is very similar. We aimed to determine whether the route of nutrient delivery altered the acute (first 8 h) response of the liver and whether chronic enteral delivery of glucose alone could augment the adaptive response to TPN. Chronically catheterized conscious dogs received either TPN or TEN containing glucose, Intralipid, and Travasol for either 8 h or 5 days. Another group received TPN for 5 days, but approximately 50% of the glucose in the nutrition was given via the enteral route (TPN+EG). Hepatic metabolism was assessed with tracer and arteriovenous difference techniques. In the presence of similar arterial plasma glucose levels (approximately 6 mM), NHGU and net hepatic lactate release increased approximately twofold between 8 h and 5 days in TPN and TEN. NHGU (26 +/- 1 vs. 23 +/- 3 micromol.kg(-1).min(-1)) and net hepatic lactate release (44 +/- 1 vs. 34 +/- 6 micromol.kg(-1).min(-1)) in TPN+EG were similar to results for TPN, despite lower insulin levels (96 +/- 6 vs. 58 +/- 16 pM, TPN vs. TPN+EG). TEN does not acutely enhance NHGU or disposition above that seen with TPN. However, partial delivery of enteral glucose is effective in decreasing the insulin requirement during chronic TPN.     

Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein

During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and 1H NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.