REVIEW ARTICLE: Cyanogenesis in cassava (Manihot esculenta Crantz)

Cassava is the most agronomically important of the cyanogeniccrops. Linamarin, the predominant cyanogenic glycoside in cassava,can accumulate to concentrations as high as 500 mg kg^–1fresh weight in roots and to higher levels in leaves. Recently,the pathway of linamarin synthesis and the cellular site oflinamarin storage have been determined. In addition, the cyanogenicenzymes, linamarase and hydroxynitrile lyase, have been characterizedand their genes cloned. These results, as well as studies onthe organ- and tissue-specific localization of linamarase andhydroxy-nitrile lyase, allow us to propose models for the regulationof cyanogenesis in cassava. There remain, however, many unansweredquestions regarding the tissue-specific synthesis, transport,and accumulation of cyanogenic glycosides. The resolution ofthe sequestions will facilitate the development of food processing,biochemical and transgenic plant approaches to reducing thecyanogen content of cassava foods. Key words: Cyanide, cyanogenic glycosides, linamarin, cyanogens     

Comparative chromosome painting between two marsupials: origins of an XX/XY1Y2 sex chromosome system

Cross-species chromosome painting was used to investigate genome rearrangements between tammar wallaby Macropus eugenii (2n = 16) and the swamp wallaby Wallabia bicolor (2n = 10♀/11♂), which diverged about 6 million years ago. The swamp wallaby has an XX female:XY₁Y₂ male sex chromosome system thought to have resulted from a fusion between an autosome and the small original X, not involving the Y. Thus, the small Y₁ should represent the original Y and the large Y₂ the original autosome. DNA paints were prepared from flow-sorted and microdissected chromosomes from the tammar wallaby. Painting swamp wallaby spreads with each tammar chromosome-specific probe gave extremely strong and clear signals in single-, two-, and three-color FISH. These showed that two tammar wallaby autosomes are represented unchanged in the swamp wallaby, two are represented by different centric fusions, and one by a tandem fusion to make the very long arms of swamp wallaby Chromosome (Chr) 1. The large swamp wallaby X comprises the tammar X as its short arm, and a tandemly fused 7 and 2 as the long arm. The acrocentric swamp wallaby Y₂ is a 2/7 fusion, homologous with the long arm of the X. The small swamp wallaby Y₁ is confirmed as the original Y by its painting with the tammar Y. However, the presence of sequences shared between the microdissected tammar Xp and Y on the swamp wallaby Y₂ implies that the formation of the compound sex chromosomes involved addition of autosome(s) to both the original X and Y. We propose that this involved fusion with an ancient pseudoautosomal region followed by fission proximal to this shared region. Received: 16 October 1996/Accepted: 30 January 1997     

The Mouse Spam1 maps to proximal Chromosome 6 and is a candidate for the sperm dysfunction in Rb(6.16)24Lub and Rb(6.15)lAld heterozygotes

We have determined the chromosomal localization of the murine gene encoding the 68-kDa sperm adhesion molecule 1, Spam1 or Ph-20. Using two independent approaches, fluorescence in situ hybridization (FISH) and interspecific backcross analysis, we show that Spam1 maps to proximal mouse Chromosome (Chr) 6. This map position is within the conserved linkage group corresponding to human Chr 7q, where the human homolog, SPAM 1, has been shown to map previously. Genetic mapping shows the gene to be very closely linked to Met, one of the most proximal loci on MMU 6. It thus places the gene near the centromere and the junction of the Rb(6.16)24Lub and Rb(6.15)1Ald translocations. The essential role of the Spam1 sperm antigen in mouse sperm-egg interactions and its gene location provide strong support for its candidacy as the gene involved in the dysfunction of mouse sperm bearing the Rb(6.16)24Lub or Rb(6.15)1Ald translocation. Received: 16 July 1996 / Accepted: 23 September 1996     

Role for a Glycan Phosphoinositol Anchor in Fcγ Receptor Synergy

While many cell types express receptors for the Fc domain of IgG (FcγR), only primate polymorphonuclear neutrophils (PMN) express an FcγR linked to the membrane via a glycan phosphoinositol (GPI) anchor. Previous studies have demonstrated that this GPI-linked FcγR (FcγRIIIB) cooperates with the transmembrane FcγR (FcγRIIA) to mediate many of the functional effects of immune complex binding. To determine the role of the GPI anchor in Fcγ receptor synergy, we have developed a model system in Jurkat T cells, which lack endogenously expressed Fcγ receptors. Jurkat T cells were stably transfected with cDNA encoding FcγRIIA and/or FcγRIIIB. Cocrosslinking the two receptors produced a synergistic rise in intracytoplasmic calcium ([Ca²⁺]_i) to levels not reached by stimulation of either FcγRIIA or FcγRIIIB alone. Synergy was achieved by prolonged entry of extracellular Ca²⁺. Cocrosslinking FcγRIIA with CD59 or CD48, two other GPI-linked proteins on Jurkat T cells also led to a synergistic [Ca²⁺]_i rise, as did crosslinking CD59 with FcγRIIA on PMN, suggesting that interactions between the extracellular domains of the two Fcγ receptors are not required for synergy. Replacement of the GPI anchor of FcγRIIIB with a transmembrane anchor abolished synergy. In addition, tyrosine to phenylalanine substitutions in the immunoreceptor tyrosine-based activation motif (ITAM) of the FcγRIIA cytoplasmic tail abolished synergy. While the ITAM of FcγRIIA was required for the increase in [Ca²⁺]_i, tyrosine phosphorylation of crosslinked FcγRIIA was diminished when cocrosslinked with FcγRIIIB. These data demonstrate that FcγRIIA association with GPI-linked proteins facilitates FcγR signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored FcγR of human PMN.     

Increase in percutaneous muscle biopsy yield with a suction-enhancement technique

Hennessey, James V., Joseph A. Chromiak, ShirleyDellaVentura, Jennifer Guertin, and David B. MacLean. Increasein percutaneous muscle biopsy yield with a suction-enhancementtechnique. J. Appl. Physiol. 82(6):1739-1742, 1997.The percutaneous muscle biopsy technique is usedin clinical practice and biomedical research. We developed a newenhanced-suction technique [suction-enhancing nipples(SEN)] and compared it with techniques currently in practice byassessing biopsy yields on anesthetized pigs. We applied the enhanced-suction technique to human subjects participating in aclinical trial. In the pig, there was a mean 91% (1.9-fold) increasein the size of the samples obtained with the 4-mm needle when SEN wasused and a mean 507% (fivefold) increase in sample size when the SENwas applied to the 6-mm needles. Nine passes of the 6-mm needle withSEN obtained from five consecutive human subjects yielded a meanindividual sample size of 109.4 mg or 219.4 mg per needle pass whenusing the double-sample technique. Adequate tissue samples forhistomorphometric and other analyses were obtained in all samplesobtained. The percutaneous muscle biopsy performed with enhancedsuction using inexpensive, readily available nipples enhances tissueyield two- to fivefold.

     

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the and subunits of the simpler proteasome isolated fromThermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that -type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these -type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulatedin vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.     

Regulation of neuronal nitric oxide synthase by histone,protamine, and myelin basic protein

We examined the effects of endogenous basic proteins rich in the amino acidL-arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca²⁺ gradient by a Ca²⁺ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influex of extracellular Ca²⁺ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca²⁺ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism ofL-arginine intoL-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively.     

Solvent polarity-dependent structural refolding: A CD and NMR study of a 15 residue peptide

A close association between the HIV surface protein gp120 and the CD4 T cell receptor initiates the viral multiplication cycle. A 15 amino acid peptide (LAV) within the CD4 binding domain of gp 120 has been shown to retain receptor binding ability. The structural behavior of the LAV peptide has been studied by CD and NMR methods in aqueous solution and upon addition of trifluoroethanol (TFE) to emulate the relatively apolar conditions at the membrane bound receptor. Previous work has shown that the LAV peptide folds into a β-pleated structure in more polar buffer/TFE mixtures, while a concerted structural change can be observed at a concentration of 60% TFE (v/v). This abrupt, cooperative refolding from a regular β-sheet to a helical secondary structure is known as “switch” behavior. Former CD experiments with LAV sequence variants have supported the assumption that four amino acids at the N-terminus (LPCR) are indispensable for the “switch.” The tetrad has a strong β-turn forming potential. The suggestion has been formulated that the tetrad can act as a nucleation site governing the refolding. The present NMR study of the LAV peptide in TFE gives evidence for a 3₁₀-helix suggesting that the tetrad adopts a type III β-turn and promotes the formation of a similar bend in the next overlapping tetrad until the sequence is restructured into a 3₁₀-helix at a critical polarity favoring intrachain hydrogen bonds. © 1995 Wiley-Liss, Inc.