The C-propeptide domain of procollagen can be replaced with a transmembrane domain without affecting trimer formation or collagen triple helix folding during biosynthesis.

The folding and assembly of procollagen occurs within the cell through a series of discrete steps leading to the formation of a stable trimer consisting of three distinct domains: the N-propeptide, the C-propeptide and the collagen triple helix flanked at either end by short telopeptides. We have established a semi-permeabilized cell system which allows us to study the initial stages in the folding and assembly of procollagen as they would occur in the intact cell. By studying the folding and assembly of the C-propeptide domain in isolation, and a procollagen molecule which lacks the C-propeptide, we have shown that this domain directs the initial association event and is required to allow triple helix formation. However, the essential function of this domain does not include triple helix nucleation or alignment, since this can occur when the C-propeptide is substituted with a single transmembrane domain. Also the telopeptide region is not involved in triple helix nucleation; however, a minimum of two hydroxyproline-containing Gly-X-Y triplets at the C-terminal end of the triple helix are required for nucleation to occur. Thus, the C-propeptide is required solely to ensure association of the monomeric chains; once these are brought together, the triple helix is able to nucleate and fold to form a correctly aligned triple helix.     

Photoperiodic time measurement in the aphid Megoura viciae

Megoura produces parthenogenetic virginoparae in long day conditions, gamic oviparae in short days. The nature of this photoperiodic response has been analysed by rearing parent apterae in a wide range of circadian and non-circadian light cycles. By varying the light and dark components independently in a two-component cycle it has been established that the time measuring function is associated primarily with the dark period. There is no evidence that an endogenous circadian oscillation is implicated: thus (a) the ‘short day’ response is abolished by ‘night interruptions’ positioned in the early or late night. But this bimodal response pattern remains unchanged when the duration of the ‘main’ photoperiod is varied from ca. 6 hr to at least 25·5 hr. The stability of the maxima within the scotophase is inconsistent with the ‘coincidence’ models of photoperiodic timing that have been proposed. It is suggested that the essential timing process operates on the hour-glass principle, beginning anew with the onset of each period of darkness; (b) night interruption experiments employing very long (up to 72 hr) scanned dark periods yielded response maxima explicable in terms of the hour-glass hypothesis but did not reveal any circadian relationship between the maxima.The ‘dark reaction’ comprises a sequence of four stages, definable by the effects of light. Stage 1, extending from dark hr 0 to ca. 2·5, is fully photoreversible: at the next dark period the entire timing sequence is repeated up to the 9·5 hr critical night length. Towards the end of stage 1 reversibility is gradually lost and after a light interruption the reaction is resumed from a later time equivalent than dark hr 0; the subsequent critical night length is therefore reduced. The extent of the photoreversal is related to light duration. The period of maximum light insensitivity (stage 2) is attained at the end of the fourth hour. From ca. dark hr 5 to just short of the critical night length light exerts an increasingly promotive action which favours the production of virginoparae. This dark process is not photoreversible. Stage 4, which begins at hr 9·5, marks the end of the timing sequence. Light will not then annul the non-promotive action of the previous long night.Light has three effects which are determined by its duration and position within the cycle. The two terminal effects, mentioned above, are associated with the interception of dark stages 1 and 3 by either short (1 hr) or longer photoperiods. Light also prepares or primes the dark period timer. Thus the critical length is increased, and timing accuracy lost, if the preceding photoperiod is less than ca. 6 hr. Light during stage 4 has a priming action but no terminal function. Repeated cycles are ‘read’ in various ways, depending on the cycle structure. For example, if light intercepts stage 3, a two-component cycle is interpreted as the overlapping sequence light/dark/light. One and the same photoperiod then acts terminally in respect of the preceding dark period and as a primer for the next dark period.There is also a mechanism for summing the promotive effects produced by repeated interruption of dark stage 3. With complex (four-component) cycles both halves of the same cycle may contribute. ‘Product accumulation’ falls below threshold if the frequency of presentation of a given promotive cycle is too low. This occurs if there are very long, relatively non-promotive dark components. Such cycles are accepted as ‘continuous darkness’.     

Characterization of <Emphasis Type="Italic">Brassica napus</Emphasis> L. genotypes utilizing sequence-related amplified polymorphism and genotyping by sequencing in association with cluster analysis

Identifying parental combinations that exhibit high heterosis is a constant target for commercial Brassica napus L. hybrid development programs. Finding high heterotic parental combinations can require hundreds of test crosses and years of yield evaluation. Heterotic pool development could be used to divide breeding material into specific breeding pools and focus the number of parental combinations created. Here, we report the genotypic characterization of 79 B. napus genotypes by calculating genetic distance based on sequence-related amplified polymorphism (SRAP) and genotyping by sequencing (GBS) in association with a neighbour-joining clustering algorithm. Despite the different genotypic analyses, neighbour-joining cluster analysis based on genetic distance of SRAP and GBS produced similar clusters. Homology between SRAP and GBS clusters was approximately 77 % when manually comparing clusters and 68 % when comparing clusters using Compare2Trees. This research demonstrates that SRAP can have similar efficacy when compared to next-generation sequencing technology for heterotic pool classification. This information may provide an important breeding scaffold for the development of hybrid cultivars based upon genetic distance and cluster analysis.     

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.     

Human fibroblasts transduced with CD80 or CD86 efficiently trans-costimulate CD4+ and CD8+ T lymphocytes in HLA-restricted reactions: implications for immune augmentation cancer therapy and autoimmunity.

Augmenting immunogenicity by genetically modifying tumor cells to express costimulatory molecules has proven to be a promising therapeutic strategy in murine tumor models and is currently under investigation in human clinical trials for metastatic cancer. However, there are significant technical and logistic problems associated with implementing strategies requiring direct gene modification of primary tumor cells. In an effort to circumvent these problems, we are developing a strategy in which the costimulatory signal required for tumor-specific T lymphocyte activation is provided by a genetically modified human fibroblast (trans-costimulation). We have evaluated the efficiency of CD80- and CD86-mediated trans-costimulation in the activation of human CD8+ and CD4+ T lymphocytes in MHC class I- and class II-restricted lymphoproliferation reactions. Our studies demonstrate that the efficiency of CD80- or CD86-mediated trans-costimulation of purified human CD8+ and CD4+ T lymphocytes is comparable to cis-costimulation under defined conditions. Moreover, a dose-response relationship consistent with the predicted two-hit kinetics of the reaction was evident in trans-costimulation reactions in which the ratio of target cells expressing either signal 1 or signal 2 was varied incrementally from 1:10 to 10:1. Importantly, the level of cell-surface CD86 required for trans-costimulation is equivalent to that constitutively expressed by human peripheral blood monocytes. These results may have significant implications for the clinical implementation of this type of cancer immunotherapy and also raise questions about the possibility of trans-costimulating autoreactive T lymphocytes in vivo.     

Effect of forward trunk inclination on joint power output in vertical jumping

It is commonly accepted that vertical jump performance is a good indicator of maximal joint power. Some studies, however, have indicated that knee joint power output in the vertical jump is limited due to forward trunk inclination early in the push-off. The aim of this experimental study was to investigate the effect of forward trunk inclination on joint power output in vertical jumping. A group of 20 male subjects performed maximal vertical countermovement jumps from stance while minimizing the contribution of arm swing by holding their hands on their hips (arms akimbo). They also performed maximal jumps while holding the trunk as upright as possible throughout the jump, still holding the arms akimbo. Jump height, joint kinematics (angles), and joint kinetics (torque, power) were calculated. Jump height of vertical jumps while holding the trunk upright was 10% less than in normal jumps. Hip joint power was decreased by 37% while knee joint power was increased by 13%. Ankle joint power did not change. These results demonstrated that maximal jump performance does not necessarily represent maximal power of each individual joint. The implication is that jump performance may well be a good representation of overall joint power; it is, however, not an accurate measure to evaluate maximal individual joint power as part of contemporary training and rehabilitation methods.     

X chromosome inactivation patterns in normal and X-linked hereditary nephropathy carrier dogs

Alport syndrome (AS) and hereditary nephropathy (HN) are glomerular nephropathies caused by mutations in the genes encoding the type IV collagens. In a mixed breed of dog, termed Navasota (NAV) dogs, X-linked hereditary nephropathy (XLHN) is caused by a 10-bp deletion in exon 9 of COL4A5. Males harboring this mutation succumb to end-stage renal disease before 18 months of age. In contrast, female carriers of this disease survive much longer, most have a normal life-span, and vary in disease progression as compared with XLHN-affected males. X chromosome inactivation (XCI) patterns have been studied in human X-linked AS carriers and some have been shown to have a high degree of skewed XCI. However, similar studies have never been reported in an animal model of this disease. Therefore, patterns of XCI were examined in XLHN-carrier NAV dogs. The variation in XCI among the 26 XLHN-carrier and seven normal female NAV dogs studied was low and only three were found to preferentially inactivate one X chromosome, all of which were XLHN-carriers. The average skewedness among all dogs was 59% and 57% among the XLHN-carriers. No significant difference in XCI was found between the two groups (P = 0.477). It is clear from these data that genotype does not seem to have an effect on inactivation; the majority of these dogs have random patterns of XCI. Highly skewed X chromosome inactivation also appears to be random, given that no difference was observed between the XLHN-carriers and normal females. Because of the apparent rarity of skewed XCI, these dogs may not be a suitable model for studying a potential correlation between this phenomenon and disease progression.