An azurocidin-like protein is induced in Trichoplusia ni larval gut cells after bacterial challenge

Trichoplusia ni immune genes up-regulated in response to bacterial infection have been isolated using differential display polymerase chain reaction. Here we report the cloning and characterisation of a gut-specific immune gene encoding an azurocidin-like protein. The deduced protein is 317 amino acid residues long with a hydrophobic C-terminus and a predicted 17-residue signal peptide. The mature T. ni protein shows 30% identity to human azurocidin, an antibacterial protein. Like azurocidin, the T. ni protein contains two amino acid substitutions in the active site triad normally present in serine proteases. The T. ni protein was synthesised with a six-histidine C-terminal extension using the baculovirus expression system. Sequencing of the recombinant azurocidin-like protein confirmed the predicted cleavage of the signal peptide. Northern blots show that T. ni azurocidin-like protein is expressed solely in the larval gut and that expression is up-regulated by injecting or feeding bacteria. Expression reaches its highest level at 10 h after bacteria injection.     

The gel test: use in the identification of unexpected antibodies to blood group antigens

The IgG GEL test was compared with the LISS tube test (L?w and Messeter's low-ionic-strength saline) for antibody identification. The suitability of red blood cells (RBCs) pretreated with ficin, dithiothreitol (DTT), or chloroquine diphosphate (CDP) also was assessed for use in the GEL test. In addition, time-in-motion studies were performed comparing GEL (12 panels per batch) with polyethylene glycol (PEG) tube tests (3 panels per batch). In 57 antibody identification studies, there were 63 GEL+ LISS+, 2 GEL+ LISS-, and 6 GEL-LISS+ antibodies. Among the GEL+ LISS+ antibodies were 19 that yielded stronger reactions in GEL than in LISS; by virtue of their specificity, 14 of these are considered potentially significant: D, 5 E, 2 e, 2 Jka, 2 S, K, and Fya. There were 38 antibodies that yielded equivalent results by both methods, including 31 that are considered potentially significant. Of six antibodies with significantly greater reactivity in LISS, there were three anti-Rh and three that are considered harmless with respect to transfusion management. The two GEL+ LISS- antibodies (anti-Jkb) were potentially significant. GEL- LISS+ reactions involved only harmless antibodies. Of the 50 antibodies of potential significance, GEL yielded equivalent or superior results in 47 (94%) instances. Additionally, GEL failed to detect 6 of 21 harmless antibodies. Expected results were obtained with normal serum or plasma and antibodies of known specificity in tests with RBCs treated with ficin, DTT, or CDP. Hands-on-time required for each GEL panel was 2 to 21/2 minutes compared with 12 minutes for PEG. These data document the suitability of GEL for use in antibody identification studies.     

Carrier frequency of the common mutation IVS8-1G>C in DHCR7 and estimate of the expected incidence of Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is a multiple congenital anomaly/mental retardation syndrome of variable severity with an incidence previously estimated at 1 in 20,000-60,000 based on case frequency surveys. Identification of the gene defect in SLOS has made it possible to calculate the carrier frequency and estimate disease incidence using molecular methods to identify carriers. Using a previously described PCR-RFLP assay we screened 1503 anonymous blood samples from random newborn screening blood spot cards for the presence of the common SLOS mutation IVS8-1G>C in order to determine the carrier frequency. Sixteen carriers were identified in the 1503 samples. Since the frequency of the IVS8-1G>C mutation among all SLOS gene mutations is known, the overall carrier frequency for all mutations can be calculated. The calculated carrier frequency for all mutations based on this result is 1 in 30, predicting an SLOS incidence of 1 in 1590 to 1 in 13,500. The current incidence estimate may, therefore, significantly underestimate the true incidence of SLOS. This discrepancy between calculated and observed incidence could be due to undiagnosed mild cases, misdiagnosed severe cases, death prior to diagnosis, or fetal loss. More comprehensive incidence studies are needed to determine if SLOS is as common as predicted by the very high (1 in 30) carrier frequency determined in this study.     

The non-immunosuppressive immunophilin ligand GPI-1046 potently stimulates regenerating axon growth from adult mouse dorsal root ganglia cultured in Matrigel

We used explant cultures of adult mouse dorsal root ganglia with spinal nerve attached growing in Matrigel to assess the effects of the non-immunosuppressive immunophilin ligand GPI-1046 [Snyder et al. (1998) TIPS 19, 21-26] on the growth rate of regenerating sensory axons and found a potent stimulation of axon growth. In these explant cultures, naked, unfasciculated axons emerge from the cut end of the spinal nerve and continue to grow in the Matrigel for up to eight days [Tonge et al. (1996) Neuroscience 73, 541-551]. Some axons are entirely smooth whilst others show prominent varicosities. Some of the former express the phosphorylated neurofilament epitope recognised by monoclonal antibody RT97, a marker for large calibre, myelinated axons, whilst the latter express calcitonin gene-related peptide, predominantly a marker for unmyelinated, and small diameter myelinated sensory axons. Many of the axons in these cultures also express the low-affinity neurotrophin receptor p75. GPI-1046 has been shown to have striking stimulatory effects on embryonic primary sensory axons growing in vitro and it was therefore of interest to see whether it could also enhance regenerating sensory axon growth from the adult ganglia in our cultures. GPI-1046 potently stimulated axon growth in our cultures in a dose-dependent manner. The stimulatory effect was not dependent on the class of sensory axon. These observations show that GPI-1046 is a potent stimulator of regenerating axons from adult, primary sensory neurones. The cellular site of action of GPI-1046 is unknown. To distinguish between a direct effect of the drug on neurones and an indirect effect we compared the effects of GPI-1046 on explant and dissociated cultures. In confirmation of previous results, we found that GPI-1046 potently stimulated axon outgrowth from explants of embryonic chick dorsal root ganglia. However, the drug was without effect on dissociated embryonic dorsal root ganglion neurones, suggesting that non-neuronal cells are important for axon growth stimulation.     

Genomic characterization of KIR2DL4 in families and unrelated individuals reveals extensive diversity in exon and intron sequences including a common frameshift variation occurring in several alleles

The KIR2DL4 gene including a portion of exon 1 through exon 9 was sequenced from two families and eight cell lines from the International Histocompatibility Workshop (IHWS). Two known alleles and eight variants were detected. Overall, there were five synonymous and three non-synonymous changes when the variants were compared to the coding sequences of the most closely related known alleles plus a common frameshift change in five of the variant alleles. Alignment of the new variants with all known alleles showed that the regions encoding the extracellular region and the cytoplasmic tail were the most polymorphic. Two non-synonymous changes, P146H and L161V, occurred in an extracellular immunoglobulin-like domain. Five of the eight variants had a single adenine deletion in the exon encoding the transmembrane region, potentially resulting in a truncated protein lacking the cytoplasmic tail. The distribution of the deletion variant among many KIR2DL4 alleles may explain the high frequency of this variation in the population. Four of the eight consanguineous IHWS cell lines were found to be heterozygous for KIR2DL4 carrying two alleles that differed from one another by a few nucleotide substitutions. Analysis of intron sequences in the families revealed the nature and distribution of interspersed repeat elements which comprise 46% of the KIR2DL4 nucleotide sequence and consist of 12 elements including six SINEs (13.73% of the total length), one LINE (12.41%), and five LTR elements (19.51%). The results revealed the presence of extensive diversity in the KIR2DL4 gene. This is the first extensive report providing both exon and intron data in related individuals.     

Experimental genital mycoplasmosis: time of infection influences pregnancy outcome.

Genital infection of rats with Mycoplasma pulmonis causes adverse pregnancy outcome and can result in in utero spread of infection to the fetus. The current study was designed to determine whether the stage of pregnancy when infection occurs influences pregnancy outcome. Rats were inoculated with 3 X 10(7) CFU of M. pulmonis at 10 days prior to breeding (-10) or at gestational day (gd) 11 or 14 and were necropsied at gd 11, 14, or 18 or within 24 h of parturition (term). Control rats received sterile broth. M. pulmonis was isolated from the placenta, amniotic fluid, or fetal tissues only from rats infected prior to breeding (P < 0.001). All infected rats had significantly more loss of pups than did control rats (P < 0.006), but rats infected prior to breeding or at the beginning of the third trimester (gd 14) were much more likely to have fetal losses. Rats infected in the early second trimester after implantation (gd 11) did not experience severe losses. Litter sizes, total litter weight, and individual pup weight from all infected rats, regardless of gestational stage when infected, were significantly smaller than those of control rats (P < 0.001). On the basis of the results of this study, we conclude that the time of infection plays a major role in determination of pregnancy outcome and spread of infection from the genital tract to the respiratory tract.     

Twelve novel HLA-B*15 alleles carrying previously observed sequence motifs are placed into B*15 subgroups

Twelve new B*15 alleles are described. All of the known B*15 alleles are divided into subgroups based on serologic assignments and/or nucleotide sequence polymorphisms. These groups might be used as a reference for DNA-based testing at an intermediate (i.e. "serologic") level of resolution.     

Nucleation of calcium phosphate by surface-bound extracellular matrix

The native extracellular matrix (ECM) laid down on silicon and titanium surfaces by osteoblast-like SAOS-2 cells was exposed by selective removal of cells. This type of material surface ECM-Si, ECM-Ti was shown to promote the nucleation of calcium phosphate from a simulated body fluid (SBF). Microscopic and spectroscopic results revealed the effect was associated with a collagen fiber-free extracellular matrix.