Promotion of motoneuron survival and branching in rapsyn-deficient mice

Inhibition of programmed cell death of motoneurons during embryonic development requires the presence of their target muscle and coincides with the initial stages of synaptogenesis. To evaluate the role of synapse formation on motoneuron survival during embryonic development, we counted the number of motoneurons in rapsyn-deficient mice. RaDsyn is a 43 kDa protein needed for the formation of postsynaptic specialisations at vertebrate neuromuscular synapses. Here we show that the rapsyn-deficient mice have a significant increase in the number of motoneurons in the brachial lateral motor column during the period of naturally occurring programmed cell death compared to their wild-type littermates. In addition, we observed an increase in intramuscular axonal branching in the rapsyn-deficient diaphragms compared to their wild-type littermates at embryonic day 18.5. These results suggest that deficits in the formation of the postsynaptic specialisation at the neuromuscular synapse, brought about by the absence of rapsyn, are sufficient to induce increases in both axonal branching and the survival of the innervating motoneuron. Moreover, these results support the idea that skeletal muscle activity through effective synaptic transmission and intramuscular axonal branching are major mechanisms that regulate motoneuron survival during development. (C) 2001 Wiley-Liss, Inc.

MUC13, a novel human cell surface mucin expressed by epithelial and hemopoietic cells

Transmembrane mucins are glycoproteins involved in barrier function in epithelial tissues. To identify novel transmembrane mucin genes, we performed a tblastn search of the GenBank(TM) EST data bases with a serine/ threonine-rich search string, and a rodent gene expressed in bone marrow was identified. We determined the cDNA sequence of the human orthologue of this gene, MUC13, which localizes to chromosome band 3q13.3 and generates 3.2-kilobase pair transcripts encoding a 512-amino acid protein comprised of an N-terminal mucin repeat domain, three epidermal growth factor-like sequences, a SEA module, a transmembrane domain, and a cytoplasmic tail (GenBank(TM) accession no. AF286113), MUC13 mRNA is expressed most highly in the large intestine and trachea, and at moderate levels in the kidney, small intestine, appendix, and stomach, In situ hybridization in murine tissues revealed expression in intestinal epithelial and lymphoid cells. Immunohistochemistry demonstrated the human MUC13 protein on the apical membrane of both columnar and goblet cells in the gastrointestinal tract, as well as within goblet cell thecae, indicative of secretion in addition to presence on the cell surface. MUC13 is cleaved, and the beta -subunit containing the cytoplasmic tail undergoes homodimerization, Including MUC13, there are at least five cell surface mucins expressed in the gastrointestinal tract.

Sonographic estimation of fetal weight in macrosomic fetuses: diabetic versus non-diabetic pregnancies

The objective of this study is to compare the accuracy of sonographic estimation of fetal weight of macrosomic babies in diabetic vs non-diabetic pregnancies. Ali babies weighing 4000 g or more at birth, and who had ultrasound scans performed within one week of delivery were included in this retrospective study. Pregnancies with diabetes mellitus were compared to those without diabetes mellitus. The mean simple error (actual birthweight - estimated fetal weight); mean standardised absolute error (absolute value of simple error (g)/actual birthweight (kg)); and the percentage of estimated birthweight falling within 15% of the actual birthweight between the two groups were compared. There were 9516 deliveries during the study period. Of this total 1211 (12.7 %) babies weighed 4000 g or more. A total of 56 non-diabetic pregnancies and 19 diabetic pregnancies were compared. The average sonographic estimation of fetal weight in diabetic pregnancies was 8 % less than the actual birthweight, compared to 0.2 % in the non-diabetic group (p < 0.01). The estimated fetal weight was within 15% of the birthweight in 74 % of the diabetic pregnancies, compared to 93 % of the non-diabetic pregnancies (p < 0.05). In the diabetic group, 26.3 % of the birthweights were underestimated by more than 15 %, compared to 5.4 % in the non-diabetic group (p < 0.05). In conclusion, the prediction accuracy of fetal weight estimation using standard formulae in macrosomic fetuses is significantly worse in diabetic pregnancies compared to non-diabetic pregnancies. When sonographic fetal weight estimation is used to influence the mode of delivery for diabetic women, a more conservative cut-off needs to be considered.

Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice

Type I diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta -cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta -cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in A-PNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta -cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(-/-) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

Kinase-dependent and kinase-independent functions of EphA4 receptors in major axon tract formation in vivo

The EphA4 receptor tyrosine kinase regulates the formation of the corticospinal tract (CST), a pathway controlling voluntary movements, and of the anterior commissure (AC), connecting the neocortical temporal robes. To study EphA4 kinase signaling in these processes, we generated mice expressing mutant EphA4 receptors either lacking kinase activity or with severely downregulated kinase activity. We demonstrate that EphA4 is required for CST formation as a receptor for which it requires an active kinase domain. In contrast, the formation of the AC is rescued by kinase-dead EphA4, suggesting that in this structure EphA4 acts as a ligand for which its kinase activity is not required. Unexpectedly, the cytoplasmic sterile-alpha motif (SAM) domain is not required for EphA4 functions. Our findings establish both kinase-dependent and kinase-independent functions of EphA4 in the formation of major axon tracts.

Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copperATPase

Wilson disease is an autosomal recessive copper transport disorder resulting from defective biliary excretion of copper and subsequent hepatic copper accumulation and liver failure if not treated. The disease is caused by mutations in the ATP7B (WND) gene, which is expressed predominantly in the liver and encodes a copper-transporting P-type ATPase that is structurally and functionally similar to the Menkes protein (MNK), which is defective in the X-linked copper transport disorder Menkes disease. The toxic milk (tx) mouse has a clinical phenotype similar to Wilson disease patients and, recently, the tx mutation within the murine WND homologue (Wnd) of this mouse was identified, establishing it as an animal model for Wilson disease. In this study, cDNA constructs encoding the wild-type (Wnd-wt) and mutant (Wnd-tx) Wilson proteins (Wnd) were generated and expressed in Chinese hamster ovary (CHO) cells. The fx mutation disrupted the copper-induced relocalization of Wnd in CHO cells and abrogated Wnd-mediated copper resistance of transfected CHO cells. In addition, co-localization experiments demonstrated that while Wnd and MNK are located in the trans-Golgi network in basal copper conditions, with elevated copper, these proteins are sorted to different destinations within the same cell, Ultrastructural studies showed that with elevated copper levels, Wnd accumulated in large multivesicular structures resembling late endosomes that may represent a novel compartment for copper transport. The data presented provide further support for a relationship between copper transport activity and the copper-induced relocalization response of mammalian copper ATPases, and an explanation at a molecular level for the observed phenotype of fx mice.

Mouse Sox8 is located between, not within, the t-complex deletions t(w18) and t(h20) on chromosome 17

The SOX family of developmental transcription factors is known to play critical roles in cell lineage specification, fate determination and differentiation during development in diverse phyla. Their importance is underscored by their involvement in a number of human diseases and mouse mutants, and by targeted mutation in mice. SOX8 is broadly expressed during development and is located on human chromosome 16p and within the t-complex on mouse chromosome 17, in the vicinity of two mutations t(w18) and t(h20). Here we analyse mutant genomic DNA to show that the Sox8 gene locus lies outside the deletion regions of both t(w18) and t(h20) and between these deletions. These data exclude Sox8 from contributing to the t(w18) and t(h20) phenotypes, and provide an additional marker for structural characterization of this complex genomic region. Copyright (C) 2001 S. Karger AG, Basel.

Rapid diagnosis in pediatric infectious diseases: The past, the present and the future

The focus of rapid diagnosis of infectious diseases of children in the last decade has shifted from variations of the conventional laboratory techniques of antigen detection, microscopy and culture to that of molecular diagnosis of infectious agents. Pediatricians will need to be able to interpret the use, limitations and results of molecular diagnostic techniques as they are increasingly integrated into routine clinical microbiology laboratory protocols. PCR is the best known and most successfully implemented diagnostic molecular technology to date. It can detect specific infectious agents and determine their virulence and antimicrobial genotypes with greater speed, sensitivity and specificity than conventional microbiology methods. Inherent technical limitations of PCR are present, although they are reduced in laboratories that follow suitable validation and quality control procedures. Variations of PCR together with advances in nucleic acid amplification technology have broadened its diagnostic capabilities in clinical infectious disease to now rival and even surpass traditional methods in some situations. Automation of all components of PCR is now possible. The completion of the genome sequencing projects for significant microbial pathogens, in combination with PCR and DNA chip technology, will revolutionize the diagnosis and management of infectious diseases.

Oral disease in animals: The Australian perspective. Isolation and characterisation of black-pigmented bacteria from the oral cavity of marsupials

A wide range of animals suffer from periodontal disease. However, there is very little reported on disease and oral micro-biota of Australian animals. Therefore, the oral cavity of 90 marsupials was examined for oral health status. Plaque samples were collected from the subgingival margins using curettes; or swabs. Plaque samples were plated onto. non-selective trypticase soy agar plates, selective trypticase soy agar, non-selective and selective Wilkens Chalgrens, Agar. Plates were incubated in an anaerobic atmosphere and examined after 7-14 days for the presence of black-brown-pigmented colonies. A combination of morphological and biochemical tests were used (colonial morphology, pigmentation, aerobic growth, Gram reaction, fluorescence under long-wave UV light (360 nm), production of catalase, enzymatic activity with fluorogenic substrates and haemagglutination of sheep red cells) to identify these organisms. Black-pigmented bacteria were cultivated from the plaque of 32 animals including six eastern grey kangaroos, a musky rat kangaroo, a whiptail and a red-necked wallaby, 18 koalas, a bandicoot and five brushtail possums. No black-pigmented colonies were cultivated from squirrel or sugar gliders or quokkas or from marsupial mice. The majority of isolates were identified as Porphyromonas gingivalis-like species with the higher prevalence of isolation from the oral cavity of macropods (the kangaroos and wallabies). Oral diseases, such as gingivitis can be found in native Australian animals with older koalas having an increase in disease indicators and black-pigmented bacteria. Non-selective Wilkens Chalgren Agar was the medium of choice for the isolation of black-pigmented bacteria. (C) 2002 Elsevier Science Ltd. All rights reserved.

The role of CD4(+) cells in vivo on the induction of the immune response to Porphyromonas gingivalis in mice

Background: It has previously been suggested that CD4(+) T cells play a pivotal role in regulating the immune response to periodontal pathogens. The aim of the present study therefore was to determine delayed type hypersensitivity (DTH), spleen cell proliferation, serum and splenic anti-Porphyromonas gingivalis antibody levels, and lesion sizes following challenge with viable P. gingiualis in CD4-depleted BALB/c mice immunized with P. gingiualis outer membrane proteins (OMP). Methods: Four groups of BALB/c mice were used. Groups 1 and 2 were injected intraperitoneally (ip) with saline for 3 consecutive days and then weekly throughout the experiment. Groups 3 and 4 were injected ip with rat immunoglobulin and a monoclonal rat anti-mouse CD4 antibody, respectively. Two days later, group 1 mice were injected ip with saline only, while all the other groups were immunized ip with P. gingiualis OMP weekly for 3 weeks. One week later following the last immunization of OMP, 3 separate experiments were conducted to determine: 1) the DTH response to P. gingiualis OMP by measuring footpad swelling; 2) the levels of antibodies to P. gingiualis in serum samples and spleen cell cultures using an enzyme-linked immunosorbent assay, as well as spleen cell proliferation after stimulation with OMP; and 3) the lesion sizes after a subcutaneous challenge with viable P. gingiualis cells. Results: In CD4(+) T-cell-depleted mice (group 4), the DTH response and antigen-stimulated cell proliferation were significantly suppressed when compared to groups 2 and 3. Similarly, the levels of serum and splenic IgM, IgG, and all IgG subclass antibodies to P. gingiualis OMP were depressed. Delayed healing of P. gingivalis-induced lesions was also observed in the CD4(+) T-cell-depleted group. Conclusions: This study has shown that depletion of CD4(+) T cells prior to immunization with P. gingiualis OMP led to the suppression of both the humoral and cell-mediated immune response to this microorganism and that this was associated with delayed healing. These results suggest that the induction of the immune response to P. gingiualis is a CD4(+) T-cell-dependent mechanism and that CD4(+) T cells are important in the healing process.

Expression of a truncated Brca1 protein delays lactational mammary development in transgenic mice

To address the hypothesis that certain disease-associated mutants of the breast-ovarian cancer susceptibility gene BRCA1 have biological activity in vivo, we have expressed a truncated Brca1 protein (trBrca1) in cell-lines and in the mammary gland of transgenic mice. Immunofluorescent analysis of transfected cell-lines indicates that trBRCA1 is a stable protein and that it is localized in the cell cytoplasm. Functional analysis of these cell-lines indicates that expression of trBRCA1 confers an increased radiosensitivity phenotype on mammary epithelial cells, consistent with abrogation of the BRCA1 pathway. MMTV-trBrca1 transgenic mice from two independent lines displayed a delay in lactational mammary gland development, as demonstrated by altered histological profiles of lobuloalveolar structures. Cellular and molecular analyses indicate that this phenotype results from a defect in differentiation, rather than altered rates of proliferation or apoptosis. The results presented in this paper are consistent with trBrca1 possessing dominant-negative activity and playing an important role in regulating normal mammary development. They may also have implications for germline carriers of BRCA1 mutations.

Body mass index-related human adipocyte agouti expression is sex-specific but not depot-specific

Objective: To determine if human adipocyte agouti signal protein (ASIP) mRNA expression is associated with obesity and is gender and/or depot specific. Research Methods and Procedures: Subjects included 8 men (64 +/- 3 years) and 14 women (56 +/- 15 years) undergoing elective abdominal surgery. ASIP mRNA levels in isolated omental and subcutaneous abdominal adipocytes were measured by quantitative reverse transcription polymerase chain reaction. Results: No significant depot difference was observed between genders; ASIP mRNA levels of omental and subcutaneous abdominal adipocytes were pooled for this analysis. BMI and ASIP gene expression were negatively correlated in men (p = -0.70; p < 0.05), whereas a positive relationship was observed in women (p = 0.48; p < 0.05). No significant difference was observed in age, body weight, body mass index (BMI), and waist circumference between groups. Hip circumference was significantly higher in women than in men (p < 0.05). Also, no significant difference in ASIP mRNA expression was observed between men and women, regardless of the fat depot. Discussion: These results show that men and women of similar age and BMI present similar ASIP mRNA levels in omental and subcutaneous abdominal adipocytes. However, a sexual dimorphism exists in the relationship between ASIP expression and BMI. If ASIP is involved in appetite regulation or energy homeostasis in humans, this observation may contribute to the recognized differences in these parameters between men and women.

Effect of periparturient diseases accompanied by excessive vulval discharge and weaning to mating interval on sow reproductive performance

Objective To evaluate the effect of periparturient disease accompanied by vulval discharge, and weaning-to-mating intervals, on sow fertility and litter size. Design Reproductive data were collected and analysed from 19 Hungarian swine herds over a 4 year period. Conception rates, farrowing rates and litter sizes of sows with periparturient disease accompanied by vulval discharge were used to evaluate the relationship between duration of vulval discharge and subsequent fertility and litter size. The possibility of interactions between weaning-to-mating intervals and duration of vulval discharges was investigated to determine if there was any effect on subsequent fertility and litter size. Results and conclusions Both parity 1 and parity 2 to 8 sows having had periparturient disease accompanied by vulval discharge in excess of 6 days duration had significantly (P < 0.001) lower subsequent fertility (conception, farrowing and adjusted farrowing rates) compared with sows of similar parity where the duration of vulval discharge was < 4 or 4 to 6 days. There was no difference in fertility rates between sows, in both parity categories, with vulval discharge for < 4 days compared with 4 to 6 days. A duration of vulval discharge in excess of 6 days in parity 1 sows significantly reduced litter size (total born and live-born) in subsequent farrowings, but not in parity 2 to 8 sows. There was no interaction between the duration of vulval discharge and post-weaning to mating intervals. However sows with weaning to mating intervals between 7 and 10 days had smaller (P < 0.001) subsequent litter sizes compared with 3 to 6 or 11 to 14 day intervals. It was concluded that the duration of vulval discharge in excess of 6 days was an indication of a severe persistent endometritis adversely affecting fertility of sows.

Hepatic covalent adduct formation with zomepirac in the CD26-deficient mouse

Background and Aims: Zomepirac (ZP), a non-steroidal anti-inflammatory drug (NSAID), has been reported to cause immune-mediated liver injury. In vivo, ZP is metabolized to a chemically reactive acyl glucuronide conjugate (ZAG) which can undergo covalent adduct formation with proteins. Such acyl glucuronide-derived drug-protein adducts may be important in the development of immune and toxic responses caused by NSAID. We have shown using immunoabsorptions that the 110 kDa CD26 (dipeptidyl peptidase IV) is one of the hepatic target proteins for covalent modification by ZAG. In the present study, a CD26-deficient mouse strain was used to examine protein targets for covalent modification by ZP/metabolites in the liver. Methods and Results: The CD26-deficient phenotype was confirmed by immunohistochemistry, flow cytometry analysis, RT-PCR, enzyme assay and immunoblotting. Moreover, by using monoclonal antibody immunoblots, CD26 was not detected in the livers of ZP-treated CD26-deficient mice. Immunoblots using a polyclonal antiserum to ZP on liver from ZP-treated mice showed three major sizes of protein bands, in the 70, 110 and 140 kDa regions. Most, but not all, of the anti-ZP immunoreactivity in the 110 kDa region was absent from ZP-treated CD26-deficient mice. Conclusion: These data definitively showed that CD26 was a component of ZP-modified proteins in vivo. In addition, the data suggested that at least one other protein of approximately 110 kDa was modified by covalent adduct formation with ZAG. (C) 2002 Blackwell Science Asia Pty Ltd.