The hydrophobic effect and hydrogen bonding interactions have long been considered to be the dominant forces in protein folding. However, the contribution of hydrogen bonds to stabilizing proteins has been difficult to clarify. As the intramolecular hydrogen bonds are formed in place of hydrogen bonds with solvent during folding, measures of stability fail to give a significant change in free energy. Previous studies on hydrogen bonding interactions have shown that they are only marginally important. Three long-range side chain-main chain hydrogen bonds have been found in the alpha subunit of tryptophan synthase (αTS), a (βα)8TIM barrel protein. These long-range noncovalent interactions connect either the N-terminus of one β-strand with the C-terminus of the succeeding and anti-parallel α-helix (F19-D46 and I97-D124) or the N-terminus of an α-helix with the C-terminus of the succeeding β-strand (A103-D130). By analogy, these interactions are designated as βα- or αβ-hairpin clamps. Surprisingly, the removal of any one of these clamp interactions, by replacement of the aspartic acid with alanine, results in significantly decreased thermodynamic stability for the native state and a substantial loss of secondary structure. When compared to several other side chain-side chain and short-range side chain-main chain interactions in αTS, these hairpin clamps clearly play a unique role in the structure and stability of αTS. The generality of these observations for βα-hairpin clamps in TIM barrel proteins was tested by experimental analysis of the clamps in a pair of homologous indole-3-glycerol phosphate synthase (IGPS) TIM barrels of low sequence identity. The results suggest that only the subset of conserved βα-hairpin clamps with hydrogen bond length less than 2.80 Å make substantive contributions to stability and/or structure. Those clamps with longer hydrogen bonds make modest contributions to stability and structure, similar to other types of side chain-main chain or side chain-side chain hydrogen bonds. The role of these clamps in defining the structures of the super-family of TIM barrel proteins was examined by a survey of 71 TIM barrel proteins from the structural database. Conserved features of βα-hairpin clamps are consistent with a 4-fold symmetry, with a predominance of main chain amide hydrogen bond donors near the N-terminus of the odd-number β-strands and side chain hydrogen bond acceptors in the loops between the subsequent α-helices and even-numbered β-strands. In this configuration, the clamps provide an N-terminal cap to odd-number β- strands in the β-barrel. Taken together, these findings suggest that βα-hairpin clamps are a vestigial signature of the fundamental βαβ building block for the (βα)8 motif and an integral part of the basic TIM barrel architecture. The relative paucity of βα-hairpin clamps remaining in TIM barrel structures and their variable contributions to stability imply that other determinants for structure and stability of the barrel have evolved to render a subset of the clamp interactions redundant. Distinct sequence preferences for the partners in the βα-hairpin clamps and the neighboring segments may be useful in enhancing algorithms for structure prediction and for engineering stability in TIM barrel proteins.

Calmodulin is a prevalent calcium sensing protein found in all cells. Three genes exist for calmodulin and all three of these genes encode for the exact same protein sequence. Recently mutations in the amino acid sequence of calmodulin have been identified in living human patients. Thus far, patients harboring these mutations in the calmodulin sequence have only displayed an altered cardiac related phenotype. Calcineurin is involved in many key physiological processes and its activity is regulated by calcium and calmodulin. In order to assess whether or not calcineurin contributes to calmodulinopathy (a pathological state arising from dysfunctional calmodulin), a comprehensive search of relevant literature has been performed. Herein, the physiological roles of calcineurin and consequences of dysfunction have been reviewed for literature focused on the heart.

Several lipopeptidyl nucleoside antibiotics that inhibit bacterial translocase I (MraY) involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. A-90289 and muraminomicin are the two representative antibiotics that belong to this family. Bioinformatic analysis of the biosynthetic A-90289 gene clusters revealed that five enzymes are likely involved in the assembly and attachment of the aminoribosyl unit. These enzymes of A-90289 are functionally assigned by in vitro characterization. The results reveal a unique ribosylation pathway that highlighted by uridine-5′-monophosphate as the source of the sugar, a phosphorylase strategy to generate a sugar-1-phosphate, and a primary amine-requiring nucleotidylyltransferase that generates the NDP-sugar donor. Muraminomicin, which has a structure similar to A-90289, holds the distinction in that both ribose units are 2-deoxy sugars. The biosynthetic gene cluster of muraminomicin has been identified, cloned and sequenced, and bioinformatic analysis revealed a minimum of 24 open reading frames putatively involved in the biosynthesis, resistance, and regulation of muraminomicin. Similar to the A-90289 pathway, fives enzymes are still likely involved in the assembly of the 2,5-dideoxy-5-aminoribose saccharide unit, and two are now functionally assigned and characterized: Mra20, a 5′-amino-2′,5′-dideoxyuridine phosphorylase and Mra23, a UTP:5-amino-2,5-dideoxy-α-D-ribose-1-phosphate uridylyltransferase. The cumulative results are consistent with the incorporation of the ribosyl appendage of muraminomicin via the archetypical sugar biosynthetic pathway that parallels A-90289 biosynthesis

Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center where it interacts with both A-site and P-site tRNAs as well as with 23S rRNA. We observed that L27 in S. aureus and other Firmicutes is encoded with a short N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. The extension contains a conserved cleavage motif; nine N-terminal amino acids are post-translationally removed from L27 by a site-specific protease so that conserved residues important for tRNA stabilization at the peptidyl transferase center are exposed. We have identified a novel cysteine protease in S. aureus that performs this cleavage. This protease, which we have named Prp, is conserved in all bacteria containing the L27 N-terminal extension. L27 cleavage was shown to be essential in S. aureus; un-cleavable L27 did not complement an L27 deletion. Cleavage appears to play an essential regulatory role, as a variant of L27 lacking the cleavage motif could not complement. Ribosomal biology in eubacteria has largely been studied in E. coli; our findings indicate that there are aspects of the basic biology of the ribosome in S. aureus and other related bacteria that differ substantially from that of E. coli. This research lays the foundation for the development of new therapeutic approaches that target this novel, essential pathway.

A newly discovered cysteine protease, Prp, has been shown to perform an essential, site-specific cleavage of ribosomal protein L27 in Staphylococcus aureus. In Firmicutes and related bacteria, ribosomal protein L27 is encoded with a conserved N-terminal extension that must be removed to expose residues critical for ribosome function. Uncleavable and pre-cleaved variants were unable to complement an L27 deletion in S. aureus, indicating that this N-terminal processing event is essential and likely plays an important regulatory role. The gene encoding the responsible protease (prp) has been shown to be essential, and is found in all organisms encoding the N-terminal extension of L27. Cleavage of L27 by Prp represents a new target for potential antibiotic therapy. In order to characterize this protease, Prp has been overexpressed and purified. Using an assay we have developed, based on cleavage of a fluorogenic peptide derived from the conserved L27 cleavage sequence, we have undertaken an analysis of the enzyme kinetics and substrate specificity for Prp cleavage and tested predictions made based on a structural model using active-site mutants.

This dissertation addresses the role for Janus Kinase 3 (Jak3) in CD4+ T cell homeostasis and function. Jak3 is a protein tyrosine kinase whose activity is essential for signals mediated by the γc dependent cytokines IL-2, -4, -7, -9, -15, and -21. Previous data have demonstrated that peripheral CD4+ T cells from Jak3-deficient mice have a memory phenotype and are functionally impaired in both proliferative and IL-2 responses in vitro. Interestingly, Jak3/γc activity has been previously shown to play a role in the prevention of T cell anergy. These studies were initiated to more precisely define the role for Jak3/γc cytokines in the prevention of T cell anergy and the maintenance of functional CD4+ T cell responses. We began to address this question by assessing global gene expression changes between wild type and Jak3-/- CD4+ T cells. These data indicate that Jak3-/- CD4+ T cells have an increase in gene expression levels of inhibitory surface receptors as well as immunosuppressive cytokines. Further analyses confirmed that Jak3-deficient T cells express high levels of PD-1, secrete a Trl-type cytokine profile following direct ex vivo activation, and suppress the proliferation of wild type T cells in vitro. These characteristics indicate that CD4+ Jak3-/- T cells share properties with regulatory T cell subsets that have an important role in peripheral tolerance and the prevention of autoimmunity. We next addressed whether these regulatory characteristics were T cell intrinsic or rather the result of expanding in a Jak3-deficient microenvironment characterized by a number of immune abnormalities and a disrupted splenic architecture. Jak3-/- CD4+ T cells proliferate in vivoin a lymphopenic environment and selectively acquire regulatory T cell characteristics in the absence of any additional activation signals. While the precise mechanism by which Jak3-deficient T cells acquire these characteristics remains unclear, our data indicate that one important component is a T cell-intrinsic requirement for Jak3 signaling. These findings indicate several interesting aspects of T cell biology. First, these studies, demonstrate that the homeostatic proliferation of CD4+ T cells is not dependent on signaling via γc-dependent cytokine receptors. And, second, that the weak activation signals normally associated with homeostatic expansion are sufficient to drive Jak3-/- T cells into a non-conventional differentiation program. Previous data indicate that, for wild type T cells, signaling through both the TCR as well as γc-dependent cytokine receptors promote the homeostatic proliferation of T cells in lymphopenic hosts. Since Jak3-/- T cells are unable to receive these cytokine signals, their proliferation is likely to be wholly dependent on TCR signaling. As a consequence of this TCR signaling, Jak3-/- T cells proliferate, but in addition, are induced to up regulate PD-1 and to selectively activate the IL-10 locus while shutting off the production of IL-2. Since this fate does not occur for wild type T cells in a comparable environment, it is likely that the unique differentiation pathway taken by Jak3-/- T cells reflects the effects of TCR signaling in the absence of γc-dependent cytokine signaling. Interestingly, wild type T cells undergoing homeostatic expansion in lymphopenic hosts show many common patterns of gene expression to freshly-purified unmanipulated Jak3-/- T cells. For instance, micro array analysis of gene expression in wild type CD4+ T cells after lymphopenia induced homeostatic expansion show a similar pattern of upregulation in surface markers (PD-1 and LAG-3), and cytokine signaling molecules (IL-10 and IFN-γ cytokine, receptors, and inducible gene targets) to that of Jak3-/- CD4+ T cells immediately ex vivo. These data suggest that the process of homeostatic proliferation normally induces immune attenuation and peripheral tolerance mechanisms, but that full differentiation into a regulatory T cell phenotype is prevented by γc-dependent cytokine signals. Taken together these data suggest that Jak3 plays an important role in tempering typical immune attenuation mechanisms employed to maintain T cell homeostasis and peripheral tolerance.

DNA damage can cause genome instability, which may lead to human cancer. The most common form of DNA damage is DNA base damage, which is efficiently repaired by DNA base excision repair (BER). Thus BER is the major DNA repair pathway that maintains the stability of the genome. On the other hand, BER mediates DNA demethylation that can occur on the promoter region of important tumor suppressor genes such as Breast Cancer 1 (BRCA1) gene that is also involved in prevention and development of cancer. In this study, employing cell-based and in vitro biochemical approaches along with bisulfite DNA sequencing, we initially discovered that an oxidized nucleotide, 5’,2-cyclo-2-deoxyadenosine in DNA duplex can either cause misinsertion by DNA polymerase β (pol β) during pol β-mediated BER or inhibit lesion bypass of pol β resulting in DNA strand breaks. We then explored how a T/G mismatch resulting from active DNA demethylation can affect genome integrity during BER and found that pol β can extend the mismatched T to cause mutation. We found that AP endonuclease 1 (APE1) can use its 3'-5' exonuclease to remove the mismatched T before pol β can extend the nucleotide preventing a C to T mutation. The results demonstrate that the 3'-5' exonuclease activity of APE1 can serve as a proofreader for pol β to prevent mutation. We further explored the effects of exposure of environmental toxicants, bromate and chromate on the DNA methylation pattern on the promoter region of BRCA1 gene with bisulfite DNA sequencing. We found that bromate and chromate induced demethylation of 5-methylcytosines (5mC) at the CpG sites as well as created additional methylation at several unmethylated CpG sites at BRCA1 gene in human embryonic kidney (HEK) 293 cells. We further demonstrated that the demethylation was mediated by pol β nucleotide misinsertion and an interaction between pol β and DNA methyltransferase 1 (DNMT1) suggesting a cross-talk between BER and DNA methyltransferases. We suggest that DNA base damage and BER govern the interactions among the environment, the genome and epigenome, modulating the stability of the genome and epigenome and disease development.

The thesis titled “Stereoselective synthesis of bio-active styryllactones” comprises an introduction about styryllactones and three sections delineating the results and discussion about the synthesis of styryllactones and experimental section. Trees of the genus Goniothalamus of the plant family Annonaceae in South East Asia has been known for a long time for their proven use in folk medicine. The research group of McLaughlin isolated and characterized a series of styryllactones, possessing significant to marginal cytotoxic activity against human tumor cell lines. The structures and relative configurations of these compounds were determined either by X-ray crystallography or by extensive NMR spectral analysis. Classification of these styryllactones is based on the structural characteristics of the six different skeletons as shown in Figure 1. It was proposed by Shing et al. that the bio-synthesis of styryllactones 1-7 occur via the shikimic acid pathway. This proceeds through the formation of cinnamic acid from phenylalanine, followed by the incorporation of two acetate–malonate units activated as co-enzyme A, generating the styryl-pyrone, goniothalamin 9 a key styryllactone, which on further hydroxylation/oxidation leads to the formation of other styryllactones Section 1: Stereoselective synthesis of styryllactones containing furanofurone, pyrano-pyrone and styryl-pyrone structural units. In this section of the thesis, stereoselective total synthesis of furano-furone, pyrano-pyrone and styryl-pyrone type styryllactones (+)-7-epi-goniofufurone 1, (+)-goniofufurone 2, (+)goniopypyrone 3, (+)-goniotriol 4, (+)-9-deoxygoniopypyrone 5 and (+)-goniodiol 6 is discussed. It is anticipated that the masked tetrol 13, comprising an alkene tether and four contiguous hydroxy groups installed with definite configuration would serve as the intermediate for the synthesis of styryllactones 1-6. It is relied on exploiting the hydroxy directed lactonization via the oxidation of alkene in 13, and subsequent elaboration to styryllactones 1-6. Bis-dimethylamide 10, derived from D-(−)-tartaric acid was identified as the suitable precursor for the synthesis of 13. Synthesis of masked tetrol 13 is accomplished from 10 involving a combination of selective Grignard additions and a stereoselective reduction (Scheme 2). Section 2: Stereoselective synthesis of styryllactones containing tetrahydrofuran and furano-pyrone structural units This section deals with stereoselective synthesis of natural antitumor tetrahydrofuran containing natural product (+)-goniothalesdiol 8. Key features of the synthesis include a FeCl3 mediated formation of THF 15 with very high selectivity (Scheme 3). THF 15 is further elaborated into the furano-pyrone type styryllactones (+)-altholactone 7 and (−)-etharvensin 16 in good yields (Scheme 3). Section 3: Stereoselective total synthesis of (+)-cardiobutanolide Recently, a new styryllactone cardiobutanolide 20 was isolated from the stem bark of Goniothalamus cardiopetalus, together with four known styryllactones by Hisham et al. Stereoselective total synthesis of this natural product from D-(−)-tartaric acid is described in this section. Key features of the synthesis include the elaboration of the γ-hydroxy butyramide 17 obtained from the bis-dimethylamide 10, involving a combination of the addition of 1,3-dithian-2-yllithium and stereoselective reduction (Scheme 4). (For structural formula pl see the pdf file)

Vitamin B₁₂ deficiency has been difficult to diagnose, mainly due to the vitamin's lack of biological significance in serum in which it is usually assayed. This research has investigated the marker of vitamin B₁₂/cobalt (Co) deficiency in sheep, methylmalonic acid (MMA), in comparison with serum and liver vitamin B₁₂ concentrations in farm situations where vitamin B₁₂ deficiency is expected in order to establish more accurate reference ranges for serum and liver vitamin B₁₂, and MMA. In addition, an attempt was made to ascertain the vitamin B₁₂ requirements of preruminant (PR) lambs, and to determine whether metabolic demand for vitamin B₁₂ influences tissue concentrations. Furthermore, since the vitamin is active in biological tissues in form of its coenzymes, 5’ -deoxyadenosylcobalamin and methylcobalamin, a preliminary assessment of variation in the distribution of these coenzymes in liver in different situations has been sought. The first trial was set up to find out if the addition of propionate to the PR lamb's diet stimulated the uptake and/or storage of vitamin B₁₂ in the liver as a reflection of the need to deal with the incoming propionate. Sixteen ten day old lambs (Dorset Down/Coopworth cross-bred) were housed indoors soon after birth and fed on milk replacer. For half of the lambs 7.5 % (w:w) of the milk powder was replaced by propionate. Within each group, four lambs were treated with 250 µg vitamin B₁₂ twice weekly. Supplementation with vitamin B₁₂ increased liver concentrations from ~250 to ~900 nmol/kg fresh tissue, but there was no effect of propionate. Propionate addition did, however, result in increased plasma vitamin B₁₂ concentrations in vitamin B₁₂ supplemented groups, values being 3323 and 2355 pmol/l in propionate supplemented and control groups, respectively. This suggested that diet could influence plasma vitamin B₁₂ concentrations. An attempt was made to quantify the PR lamb's ability to absorb vitamin B₁₂ from the alimentary tract by comparing the ability of intra-muscular (IM) and oral vitamin B₁₂ to raise plasma and liver vitamin B₁₂ concentrations. Twenty-seven three to four day old lambs from a farm with marginal Co status were housed indoors and fed on milk replacer. They were divided into three groups: control (n=3), IM treatment (n=12) and oral treatment (n=12). The two treatment groups were further subdivided into five sub-groups. These received, respectively, 0.2 (n=3), 0.4 (n=2), 0.8 (n=2), 1.6 (n=2) and 3.2 µg OH-cbl/d (n=3). The oral groups received tenfold the amount of the comparable IM groups, on the assumption that if oral absorption of the vitamin is about 10 % both groups would show similar increases in plasma and liver vitamin B₁₂ concentration. None of the IM groups showed any significant change in plasma or liver vitamin B₁₂. In the oral groups only the group on the highest dose of vitamin B₁₂, viz 32 µg/d, showed increases in plasma and liver concentrations. It was concluded that either absorption of vitamin B₁₂ was greater than 10 % or that the vitamin was retained better when administered orally. The amount retained in the livers of the lambs in the highest oral group was calculated to represent ~ 7.5 % of the dose. In a follow-up 24 h trial, 14 of the above lambs were divided into three groups: Control (n=3), oral (n=6) and IM (n=5) treatment. The IM group received 3.2 µg OH-cbl and the oral group tenfold the amount as single doses at 0800 h. Blood samples were taken at regular intervals throughout the 24 h period and assayed for vitamin B₁₂, Vitamin B₁₂ concentrations in the IM group rose steeply within the first hour after injection to a concentration that was calculated to reflect 100 % uptake of the vitamin. It rose more slowly over about 8 h in the oral group. From the area under the curve absorption of the oral dose was estimated to be ~ 7 %. The next experiment involved a farm where Co deficiency had been reported previously. In the first year, 50 pregnant two-tooth Half-bred ewes were divided randomly into two groups of 25. One group received a Co bullet plus 1000 µg OH-cb1 IM, the other group remained unsupplemented. In the following year the trial was repeated. Ewes from the previous year's trial (by then four-tooths) were augmented by a new cohort of pregnant two-tooths to make up numbers to 75. After lambing the lambs were divided into four groups: first by their dams' vitamin B₁₂ treatment, then half of each group received injections of vitamin B₁₂ at approximately three weekly intervals while the other half remained untreated. The trials lasted about five months, from mid-pregnancy until weaning. Pasture Co was at its lowest at lambing in both years, 0.09 and 0.10µg/g DM, respectively. In the first year, vitamin B₁₂ concentrations in the untreated ewes rose from 340 to 950 pmol/l in plasma and decreased in liver from 330 to 170 nmol/kg fresh tissue. In the Co treated group, vitamin B₁₂ concentrations in plasma rose from 500 to 1550 pmol/l and in liver from 310 to 560 nmol/kg fresh tissue. In the second year, vitamin B₁₂ concentrations in serum in the unsupplemented groups fell from 500 to 260 pmol/l around lambing before rising again to starting values at weaning, and liver vitamin B₁₂ concentrations fell from 450 at the start to 230 nmol/kg fresh tissue at the end of the trial. Serum vitamin B₁₂ concentrations in the two-tooth supplemented group rose from < 500 to > 3000 pmol/l whereas in the four-tooth supplemented group serum vitamin B₁₂ levels started at ~2800 and rose to nearly 5000 pmol/l. The supplemented four-tooths maintained higher liver vitamin B₁₂ concentrations throughout compared to the supplemented two-tooths, viz 680 compared to below 400 at the start and 900 versus 650 nmol/kg fresh tissue at weaning, respectively. MMA in the untreated groups rose to 15 and to 8 µmol/l during early lactation in the first and second years, respectively, whereas MMA in the treated groups stayed below 3 µmol/l in the first season and below 1.5 µmol/l in the second season. There was a live weight response to treatment in the ewes as the unsupplemented groups showed a significantly lower weight gain during the trials than the supplemented groups, viz 10.0 versus 13.6 kg in the first year, and 10.6 versus 13.3 kg in the four-tooths and 9.9 versus 12.1 kg in the two-tooths in the second year. There was also a significant difference in faecal egg count (FEC) in the first year. FEC in the untreated group was higher during lactation than in the treated group, viz 590 versus 170 eggs per gram wet faeces (epg), respectively. In the second year, the two-tooths had a higher FEC than the four-tooths, viz 120 versus 40 epg during the same time span, respectively. While there was a trend for treatment having an effect on FEC similar to that in the first year it was not significant. Supplementation of ewes in the first year increased mean milk vitamin B₁₂ concentrations at lambing from 800 to 1400 pmol/l and at weaning from 1750 to 4000 pmol/l. In the second year, Co bullet treatment increased milk vitamin B₁₂ concentrations in the four-tooths and two-tooths from 1500 and 2300 to 4000 and 2900 pmol/l at lambing, and from 1800 and 1400 to 6200 and 4500 pmol/l at weaning, respectively. Treatment of ewes increased vitamin B₁₂ concentrations in the lambs which were not themselves supplemented. Plasma values in the first year increased from 160 to 325 pmol/l soon after birth and from 650 to 900 pmol/l at weaning, and liver values from 75 to 140 nmol/kg fresh tissue soon after birth and from 150 to 240 nmol/kg fresh tissue at weaning. In the second year, plasma vitamin B₁₂ concentrations increased from 160 to 380 pmol/l soon after birth and from 500 to 700 pmol/l at weaning, and in liver from 130 to 260 nmol/kg fresh tissue soon after birth and from 220 to 340 nmol/kg fresh tissue at weaning. There was also a significant effect of ewe supplementation on lamb MMA in 1997/1998 when values decreased from 19 to 8 µmol/l around the time of rumen development. MMA in the second year stayed below 3 µmol/l throughout in all groups of lambs. There was no difference in LWG between any groups of lambs. FEC was lowest in the group where both ewes and lambs were supplemented and highest in the group where neither ewes nor lambs were treated. Further investigations were conducted on farms in Southland with lambs post-weaning in order to compare changes in serum and liver vitamin B₁₂ with serum MMA and LWG to determine the critical time and level of deficiency. In the first year, three farms with 50 lambs each participated. Lambs from each farm were allocated to five groups of 10 animals each. The first group received a Co bullet at weaning, and each month another group was treated with a Co bullet. The lambs were weighed monthly, and blood and liver samples were taken prior to treatment and each subsequent month from five lambs of the first supplemented group. The trial lasted about four months. Serum vitamin B₁₂ concentrations in lambs at weaning were between 500 and 1000 pmol/l. Although supplementation increased serum levels for the first month this was followed by a drop to near or below starting concentrations. An exception was Farm 3 where serum vitamin B₁₂ concentrations rose again at the end of the trial. Liver vitamin B₁₂ concentrations also showed an overall decline from starting levels (200 to 300 nmol/kg fresh tissue) to the end of the trial (100 to 200 nmol/kg fresh tissue). MMA started around 2 µmol/l and reached between 6 and 7 µmol/l in the untreated lambs on Farms 1 and 3 two months after weaning before decreasing to around 3 µmol/l at the end of the trial, whereas the treated lambs maintained MMA concentrations around 2 µmol/l. On Farm 2 MMA started just below 5 µmol/l, decreased to around 1 µmol/l for treated and untreated lambs one month later and rose again to between 2.5 and 4 µmol/l, respectively, at the end of the trial. LWG was below average for all lambs (between 0.20 and 0.04 kg/d except for Farm I in the first month after weaning) but no significant differences were noted between treated and untreated lambs on any of the farms. Another trial was conducted on one of these farms in the following year. One hundred lambs were divided into two groups of 50 each at weaning and sampled monthly for about six months. One group was treated with two Co bullets, the other group remained untreated. Pasture Co was between 0.04 and 0.07 µg/g DM, yet serum levels for the untreated group stayed ~500 pmol/l throughout the trial. Serum vitamin B₁₂ concentrations for the treated group started at ~500 pmol/l, rose to ~2500 pmol/l before falling back to ~2000 pmol/l. Liver vitamin B₁₂ concentrations for the untreated and treated groups were 529 and 427 nmol/kg fresh tissue at weaning, respectively. This decreased for both groups to ~350 nmol/kg fresh tissue one month after weaning. In the untreated lambs liver values decreased further to ~290 nmol/kg fresh tissue whereas they increased to ~450 nmol/kg fresh tissue for the treated group at the end of the trial. MMA concentrations started between 2 and 3 µmol/l for both groups and increased to 4.5 µmol/l for the untreated group one month later before falling back to 3.2 µmol/l. In the treated group MMA decreased to ~1µmol/l and stayed at that level throughout the trial. There was no difference in weight gain. In order to obtain an understanding of the distribution of corrinoids in biological tissues a High Performance Liquid Chromatography method was developed. The sensitivity of the analytical method meant that it was only practical to assay mainly liver samples because of the higher vitamin B₁₂ concentrations than in other tissues. The general finding was that the coenzyme 5’ –deoxyadenosylcobalamin (ado-cbl) constituted the highest proportion of corrinoids in liver (45 %), followed by analogues (28 %), OH-cbl (24 %) and lastly methy1cobalamin (3 %). Ado-cbl did tend to be proportionately higher in supplemented than in unsupplemented animals (56 and 42 %, respectively), whereas biologically non-active analogues tended to be higher in untreated than in treated sheep (29 and 21 %, respectively). It was concluded that in the farm trials Co deficiency was only mild or not present although deficiency would have been predicted from the low vitamin B₁₂ concentrations in serum and liver and from raised MMA values. Therefore, currently used thresholds in New Zealand appear to be too high for vitamin B₁₂, and overseas values for MMA do not seem to be appropriate. Revised marginal ranges of 100 to 200 pmol/l for serum, 100 to 200 nmol/kg fresh tissue for liver and 10 to 20 µmol/l for MMA are suggested. Further, this work shows that Co bullets were effective in elevating blood and liver vitamin B₁₂concentrations for longer than one year. In the trials with preruminant lambs it was found that maintenance requirements were met by the vitamin B₁₂ content of milk replacer. There is evidence from indoor and farm trials that vitamin B₁₂ from milk was much more readily absorbed than vitamin B₁₂ from supplements. It was estimated that suckling lambs probably require between 1200 and 4000 pmol vitamin B₁₂/d, depending on age.

Very long chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD deficient mice and patients’ clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD deficient mice were treated systemically with 1x10 12 vector genomes of rAAV9-VLCAD. Expression was detected in the liver, heart and muscle. Also substantial expression of VLCAD was noted in the brain, where it was expressed across different sections of the brain and in different cell types with different morphologies. Biochemical correction was observed in vector-treated mice beginning two weeks post-injection, as characterized by a significant drop in long chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks post injection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD-/- mice dropped below 20°C and the mice became lethargic, requiring euthanasia. In contrast all rAAV9-treated VLCAD-/- mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD-/- mice maintained euglycemia, whereas untreated VLCAD-/- mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.