Evidence for targeted vesicular glutamate exocytosis in osteoblasts.

Regulated intercellular signaling is essential for the maintenance of bone mass. In recent work we described how osteoblasts and osteoclasts express functional receptors for the excitatory amino acid, glutamate, indicating that a signaling pathway analogous to synaptic neurotransmission exists in bone. Here, we show that osteoblasts also express the essential molecular framework for regulated glutamate exocytosis to occur as is present in presynaptic neurons. A combination of reverse transcription-polymerase chain reaction (RT-PCR) and northern and western blotting is used to show expression of the target membrane-SNARE (soluble NSF attachment protein receptor), proteins SNAP-25 and syntaxin 4 and the vesicular-SNARE protein VAMP (synaptobrevin), the minimum molecular requirements for core exocytotic complex formation. Immunofluorescent localizations reveal peripheral SNAP-25 expression on osteoblastic cells, particularly at intercellular contact sites, colocalizing with immunoreactive glutamate and the synaptic vesicle-specific protein, synapsin I. We also identify multiple accessory proteins associated with vesicle trafficking, including munc18, rSec8, DOC2, syntaxin 6, and synaptophysin, which have varied roles in regulated glutamate exocytosis. mRNA for the putative Ca(2+)-dependent regulators of vesicle recycling activity, synaptotagmin I (specialized for fast Ca(2+)-dependent exocytosis as seen in synaptic neurotransmission), and the GTP-binding protein Rab3A are also identified by northern blot analysis. Finally, we demonstrate that osteoblastic cells actively release glutamate in a differentiation-dependent manner. These data provide compelling evidence that osteoblasts are able to direct glutamate release by regulated vesicular exocytosis, mimicking presynaptic glutamatergic neurons, showing that a process with striking similarity to synaptic neurotransmission occurs in bone.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli.

A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59-241 has been cloned and expressed in Escherichia coli. A 1.0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a beta-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The beta-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-amino-benzyl-1-thio-beta-D-galactopyranoside agarose.

Inheritance of resistance to covered smut in barley and development of a tightly linked SCAR marker.

Inheritance of resistance to covered smut in the barley line Q21861 was studied using a doubled-haploid population produced by crossing Q21861 with the line SM89010. Based on 3 years of screening in the field and two seasons in the greenhouse, segregation for resistance/susceptibility fits a one-gene ratio, indicating a single major gene for resistance in Q21861. Of 440 random 10-mer primers tested using bulked segregant analysis, one primer (OPJ10) resulted in a reproducible polymorphic band. RAPD marker OPJ10(450) co-segregated in repulsion with the covered smut resistance. This marker was converted to a sequence-characterized amplified region (SCAR) marker linked in coupling (5.5 cM) with the covered smut resistant gene in Q21861. The SCAR marker was amplified in the line TR640 which is also resistant to covered smut, but not in the other resistant lines. The SCAR marker will be useful for marker-assisted selection for covered smut in barley breeding programs.

Effect of administration of a novel recombinant bovine interferon on length of oestrous cycle in cattle.

In ruminants, extensive reproductive loss occurs during the process of maternal recognition of pregnancy and it has been suggested that trophoblast interferons may be potential therapeutic agents. This paper reports results from a trial using eight first lactation Holstein-Friesian heifers to test the efficacy of a novel recombinant bovine interferon produced in bacteria in extending the life of the corpus luteum. Oestrus was synchronised in these animals and 0.1 mg of this non-glycosylated interferon was infused into the uterus twice daily for 13 days starting approximately 12 days after oestrus. This treatment resulted in an extension of the lifespan of the corpus luteum by 5.5 days (P=0.028) compared with untreated controls. In these animals the interovulatory period was extended by 6.4 days (P=0.009). Administration of this protein did not have any adverse effects either on body temperature or on daily milk yields. The results indicate that this novel interferon may have potential therapeutic application for reducing embryo mortality.

Effect of a novel recombinant bovine interferon and trophoblast secretory products on protein metabolism by endometrial explants from cattle and sheep.

The effect of a novel recombinant bovine interferon on the rate of protein synthesis, as measured by the rate of leucine incorporation, in endometrial explants from cows and ewes was investigated and compared with the effect of trophoblast secretory proteins and of a recombinant bovine interferon-alpha1. Exposure of cultured bovine endometrial tissue to the novel recombinant bovine interferon mimicked the effect of pregnancy in reducing the rate of protein synthesis in both caruncular and intercaruncular endometrium. This effect was similar to that seen with naturally produced trophoblast secretory proteins and it was more pronounced than that seen when endometrial tissue was treated with a related recombinant bovine interferon-alpha1. Ovine endometrial cultures were unaffected by exposure to this novel recombinant bovine interferon. This novel recombinant interferon exhibited potent antiviral properties (an activity of 1.6 x 10(10) international reference units/mg protein) and may prove to be a valuable reagent in studying the maternal recognition of pregnancy in the cow and reducing embryo mortality.

Mapping quantitative trait loci associated with barley net blotch resistance.

Net blotch of barley, caused by Pyrenophora teres Drechs., is an important foliar disease worldwide. Deployment of resistant cultivars is the most economic and eco-friendly control method. This report describes mapping of quantitative trait loci (QTL) associated with net blotch resistance in a doubled-haploid (DH) barley population using diversity arrays technology (DArT) markers. One hundred and fifty DH lines from the cross CDC Dolly (susceptible)/TR251 (resistant) were screened as seedlings in controlled environments with net-form net blotch (NFNB) isolates WRS858 and WRS1607 and spot-form net blotch (SFNB) isolate WRS857. The population was also screened at the adult-plant stage for NFNB resistance in the field in 2005 and 2006. A high-density genetic linkage map of 90 DH lines was constructed using 457 DArT and 11 SSR markers. A major NFNB seedling resistance QTL, designated QRpt6, was mapped to chromosome 6H for isolates WRS858 and WRS1607. QRpt6 was associated with adult-plant resistance in the 2005 and 2006 field trials. Additional QTL for NFNB seedling resistance to the more virulent isolate WRS858 were identified on chromosomes 2H, 4H, and 5H. A seedling resistance QTL (QRpts4) for the SFNB isolate WRS857 was detected on chromosome 4H as was a significant QTL (QRpt7) on chromosome 7H. Three QTL (QRpt6, QRpts4, QRpt7) were associated with resistance to both net blotch forms and lines with one or more of these demonstrated improved resistance. Simple sequence repeat (SSR) markers tightly linked to QRpt6 and QRpts4 were identified and validated in an unrelated barley population. The major 6H QTL, QRpt6, may provide adequate NFNB field resistance in western Canada and could be routinely selected for using molecular markers in a practical breeding program.

Best5: a novel interferon-inducible gene expressed during bone formation.

Regulation of bone formation is important in the pathogenesis of many conditions such as osteoporosis, fracture healing, and loosening of orthopedic implants. We have recently identified a novel rat cDNA (best5) by differential display PCR that is regulated during osteoblast differentiation and bone formation in vitro and in vivo. Expression of best5 mRNA is induced in cultures of osteoblasts by both interferon-alpha (IFN-alpha) or IFN-gamma. Whereas IFN-alpha induced a rapid, transient induction of best5 expression peaking at 4-6 h poststimulation, IFN-gamma elicited a more prolonged induction of best5 expression, which remained elevated 48 h poststimulation. A polyclonal antibody generated to a peptide derived from the best5 coding region recognized a 27 kDa protein on Western blot analysis of osteoblast lysates. We localized BEST5 protein in osteoblast progenitor cells and mature osteoblasts in sections of rat tibiae and in sections of bones loaded in vivo to induce adaptive bone formation. Best5 may therefore be a fundamental intermediate in the response of osteoblasts to stimuli that modulate proliferation/differentiation, such as interferons or mechanical loading. These findings highlight the close interactions between the immune system and bone cells and may open new therapeutic avenues in modulating bone mass.

Mapping of a resistance gene effective against Karnal bunt pathogen of wheat.

A set of 130 wheat recombinant inbred lines (RILs) developed from a cross between parents susceptible (WL711) and resistant (HD29) to Karnal bunt (caused by Tilletia indica), were screened for 3 years with the pathogen populations prevalent in northern India. When 90 simple sequence repeats (SSRs) and 81 amplified fragment length polymorphism (AFLP) loci were mapped on the RILs, markers on chromosomes 2A, 4B and 7B accounted collectively for about one-third of the variation in the disease reaction. The genomic region of largest effect, identified on the long arm of chromosome 4B, reduced Karnal bunt disease by half in three different experiments and accounted for up to 25% of the phenotypic variation for KB reaction. A closely linked SSR marker, GWM538, may be useful in marker-assisted selection for Karnal bunt resistance in wheat.