Non-secretory solitary plasmacytoma of the spleen

A case of primary nonsecretory plasmacytoma of the spleen is reported. On laparotomy and splenectomy a 920 g spleen was removed, measuring 16×14×6 cm. The cut surface of the entire spleen showed that the tumour occupied most of the splenic tissue. A bone marrow aspirate and trephine, skeletal survey showed no signs of myeloma. Biopsy of the liver and regional lymph nodes was normal. Immunocytochemistry of the splenic tumour showed positivity for pan-B and plasma cell markers. After splenectomy the patient was treated with chemotherapy according to protocol VBCMP (M2).     

Biosynthesis of squalene and other triterpenes in Mentha piperita from mevalonate-2-14C

Unlike mono- and sesqui-terpenes, squalene and other triterpenes in peppermint readily incorporate mevalonate-2-¹⁴C label (greater than 30% incorporation of R-mevalonate in 4 hr). The labelled squalene produced turns over rapidly. Squalene derived from mevalonate-2-¹⁴C in incorporation times of 1, 4 and 7 hr was degraded chemically and shown to be equivalently labelled, according to theory, in the isopentenyl pyrophosphate-derived and dimethylallyl pyrophosphate-derived portions of the molecule. This contrasts with earlier studies on the biosynthesis of mono- and sesqui-terpenes in peppermint from ¹⁴C-precursors, in which the isopentenyl pyrophosphate-derived portions of the terpene molecules were found to be preferentially labelled, suggesting the presence of endogenous dimethylallyl pyrophosphate pools. The kinetics of squalene biosynthesis, and the labelling pattern of squalene, suggest that sites of triterpene biosynthesis are readily accessible to exogenous mevalonate and that endogenous dimethylallyl pyrophosphate pools do not participate in triterpene biosynthesis to any appreciable extent. The triterpene biosynthetic sites in peppermint thus appear to differ significantly from the monoterpene and sesquiterpene biosynthetic sites.     

Limonene Synthase, the Enzyme Responsible for Monoterpene Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland Secretory Cells

Circumstantial evidence based on ultrastructural correlation, specific labeling, and subcellular fractionation studies indicates that at least the early steps of monoterpene biosynthesis occur in plastids. (4S)-Limonene synthase, which is responsible for the first dedicated step of monoterpene biosynthesis in mint species, appears to be translated as a preprotein bearing a long plastidial transit peptide. Immunogold labeling using polyclonal antibodies raised to the native enzyme demonstrated the specific localization of limonene synthase to the leucoplasts of peppermint (Mentha × piperita) oil gland secretory cells during the period of essential oil production. Labeling was shown to be absent from all other plastid types examined, including the basal and stalk cell plastids of the secretory phase glandular trichomes. Furthermore, in vitro translation of the preprotein and import experiments with isolated pea chloroplasts were consistent in demonstrating import of the nascent protein to the plastid stroma and proteolytic processing to the mature enzyme at this site. These experiments confirm that the leucoplastidome of the oil gland secretory cells is the exclusive location of limonene synthase, and almost certainly the preceding steps of monoterpene biosynthesis, in peppermint leaves. However, succeeding steps of monoterpene metabolism in mint appear to occur outside the leucoplasts of oil gland cells.     

Regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha) species: cDNA isolation, characterization, and functional expression of (-)-4S-limonene-3-hydroxylase and (-)-4S-limonene-6-hydroxylase.

The oxygenation pattern of the cyclic monoterpenoids of commercial mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-4S-limonene. In peppermint (Mentha x piperita), C3-allylic hydroxylation leads to (-)-trans-isopiperitenol, whereas in spearmint, C6-allylic hydroxylation leads to (-)-trans-carveol. The microsomal limonene-6-hydroxylase was purified from the oil glands of spearmint, and amino acid sequences from the homogeneous enzyme were used to design PCR primers with which a 500-bp amplicon was prepared. This nondegenerate probe was employed to screen a spearmint oil gland cDNA library from which the corresponding full-length cDNA was isolated and subsequently confirmed as the C6-hydroxylase by functional expression using the baculovirus-Spodoptera system. The probe was also utilized to isolate two closely related full-length cDNA species from a peppermint oil gland cDNA library which were confirmed as the limonene-3-hydroxylase by functional expression as before. Deduced sequence analysis of these regiospecific cytochrome P450 monooxygenases indicates that both enzymes bear a typical amino-terminal membrane anchor, consistent with the microsomal location of the native forms, exhibit calculated molecular weights of 56,149 (spearmint) and about 56,560 (peppermint), and are very similar in primary sequence (70% identity and 85% similarity). The availability of these regiochemically distinct, yet very closely related, recombinant hydroxylases and their corresponding genes provides a unique model system for understanding structure-function relationships in cytochrome P450 substrate binding and catalysis, and a means for transgenic manipulation of monoterpene biosynthetic pathways in plants.     

Crystal Structure of the FeS Cluster–Containing Nucleotide Excision Repair Helicase XPD

DNA damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an α-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster–containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.     

Taxol biosynthesis: Identification and characterization of two acetyl CoA:taxoid-O-acetyl transferases that divert pathway flux away from Taxol production

Two cDNAs encoding taxoid-O-acetyl transferases (TAX 9 and TAX 14) were obtained from a previously isolated family of Taxus acyl/aroyl transferase cDNA clones. The recombinant enzymes catalyze the acetylation of taxadien-5α,13α-diacetoxy-9α,10β-diol to generate taxadien-5α,10β,13α-tri-acetoxy-9α-ol and taxadien-5α,9α,13α-triacetoxy-10β-ol, respectively, both of which then serve as substrates for a final acetylation step to yield taxusin, a prominent side-route metabolite of Taxus. Neither enzyme acetylate the 5α- or the 13α-hydroxyls of taxoid polyols, indicating that prior acylations is required for efficient peracetylation to taxusin. Both enzymes were kinetically characterized, and the regioselectivity of acetylation was shown to vary with pH. Sequence comparison with other taxoid acyl transferases confirmed that primary structure of this enzyme type reveals little about function in taxoid metabolism. Unlike previously identified acetyl transferases involved in Taxol production, these two enzymes appear to act exclusively on partially acetylated taxoid polyols to divert the Taxol pathway to side-route metabolites.     

Association of polyaminergic loci with anxiety, mood disorders, and attempted suicide

Background

The polyamine system has been implicated in a number of psychiatric conditions, which display both alterations in polyamine levels and altered expression of genes related to polyamine metabolism. Studies have identified associations between genetic variants in spermidine/spermine N1-acetyltransferase (SAT1) and both anxiety and suicide, and several polymorphisms appear to play important roles in determining gene expression.

Methodology/Principal Findings

We genotyped 63 polymorphisms, spread across four polyaminergic genes (SAT1, spermine synthase (SMS), spermine oxidase (SMOX), and ornithine aminotransferase like-1 (OATL1)), in 1255 French-Canadian individuals who have been followed longitudinally for 22 years. We assessed univariate associations with anxiety, mood disorders, and attempted suicide, as assessed during early adulthood. We also investigated the involvement of gene-environment interactions in terms of childhood abuse, and assessed internalizing and externalizing symptoms as endophenotypes mediating these interactions. Overall, each gene was associated with at least one main outcome: anxiety (SAT1, SMS), mood disorders (SAT1, SMOX), and suicide attempts (SAT1, OATL1). Several SAT1 polymorphisms displayed disease-specific risk alleles, and polymorphisms in this gene were involved in gene-gene interactions with SMS to confer risk for anxiety disorders, as well as gene-environment interactions between childhood physical abuse and mood disorders. Externalizing behaviors demonstrated significant mediation with regards to the association between OATL1 and attempted suicide, however there was no evidence that externalizing or internalizing behaviors were appropriate endophenotypes to explain the associations with mood or anxiety disorders. Finally, childhood sexual abuse did not demonstrate mediating influences on any of our outcomes.

Conclusions/Significance

These results demonstrate that genetic variants in polyaminergic genes are associated with psychiatric conditions, each of which involves a set of separate and distinct risk alleles. As several of these polymorphisms are associated with gene expression, these findings may provide mechanisms to explain the alterations in polyamine metabolism which have been observed in psychiatric disorders.