High efficiency nano-focusing kinoform optics for synchrotron radiation.

Modern synchrotron sources have provided for decades intense beams of photons over a large energy spectrum. The availability of improved optics and detectors has opened up new opportunities for the study of matter at the micrometre and nanometre scale in many disciplines. Whilst exploitation of micro-focused beams occurs almost daily in many beamlines, the production of beams of 100 nm is achieved on few instruments which use specialised optics. Refractive lenses, zone plates, curved mirrors, multilayers, and multilayer Laue lenses, can all focus x-rays to less than 50 nm under strict beam stability conditions. Focusing the synchrotron radiation to beam sizes smaller than 10 nm is considered the ultimate goal for the current decade. Silicon micro-technology has so far provided some of the most advanced x-ray refractive lenses; we report on design and characterisation of a novel silicon kinoform lens that is capable of delivering nano-beams with high efficiency.

Evaluation of different cooling management strategies for lactating Holstein × Gir dairy cows.

Heat stress negatively impacts production, reproduction, and health of ruminants and strategies to alleviate these losses are warranted. Therefore, four experiments evaluated different cooling strategies on vaginal temperature (VT) of Holstein × Gir cows. Experiment 1 compared different amounts of water (2- or 4-L) over a 1-hour period from 1000 to 1100 h and 1600 to 1700 h. Experiment 2 evaluated the effects of sprinkling duration (in hours; 1- or 2-H), whereas Experiment 3 evaluated the effects of water amount (4- or 8-L) applied for 1- or 2-H. Lastly, the effects of a cooling strategy on specific hours of the day, starting at either 0700 (T-1) or 1100 h (T-2; Experiment 4), were evaluated. In all experiments, 12 Holstein × Gir cows were used in a 2 × 2 Latin Square Design containing two periods of 6 days each. Temperature and humidity index (THI) were recorded hourly and VT was recorded every 10-min throughout the experiments. As expected, an hour effect was observed for THI (P < 0.0001), which peaked early in the afternoon. In Experiment 1, a treatment × hour interaction was observed (P < 0.0001) for VT, as animals assigned to receive 4-L had a reduced VT at 1100, 1600, 1700, and 2300 h (P ≤ 0.03). During the cooling applications, cows receiving 4-L for 1 h had a reduced VT from 60 to 150 min (P ≤ 0.04). In Experiment 2, a treatment × hour interaction was observed (P < 0.0001) for VT, as animals assigned to receive 4-L of water for 2-H had a reduced VT at 1200 h (P = 0.05). Moreover, during the cooling process, VT was reduced for 2-H cows from 140 to 170 min after the beginning of the cooling process (P ≤ 0.05). In Experiment 3, animals assigned to receive 4-L + 2H had a reduced VT at 1200, 1700, 1800, and 1900 h (P < 0.001). A treatment × hour interaction was observed (P < 0.0001), as VT was reduced for 4-L + 2-H cows from 130 to 180 min after the beginning of the cooling process (P ≤ 0.05). In Experiment 4, by the time when the first cooling cycle of T-1 was applied (0700 h), T-1 cows consistently had (P ≤ 0.05) a reduced VT up to the hottest hours and greatest THI of the day (1400 and 1500 h). This pattern was maintained until the end of the last cooling cycle, whereas T-2 cows had a reduced VT. In summary, 4 L of water over a 5-min cycle for a period of 2 hours twice a day maintained VT of Holstein × Gir cows at lower levels. Moreover, the hour at which the first cooling cycle starts also should be considered when evaluating the efficacy of a cooling strategy for an entire day.

Genetic elements used for a murine lupus anti-DNA autoantibody are closely related to those for antibodies to exogenous antigens.

The mRNAs encoding heavy and light chains of a hybridoma-derived monoclonal IgM kappa anti-DNA autoantibody from lupus-prone MRL/Mp-lpr/lpr mice (Ighj) have been transcribed into cDNA copies and molecularly cloned, and their complete nucleotide sequences have been determined. The mRNA for the heavy chain variable region, including leader peptide and 5' untranslated region, is transcribed from a heavy chain variable region (VH) gene closely related (and possibly allelic) to VH genes of the C57BL/6 (Ighb) nitrophenyl antibody family. The deduced amino acid sequence corresponding to the light chain variable region of this autoantibody shows extensive similarities with non-autoantibody molecules of the V kappa 1 group, suggesting a common variable gene origin. The joining segments, constant regions, and 3' untranslated regions of both the heavy and light chain mRNAs are nearly identical to corresponding sequences of non-autoantibodies from normal mice. Our findings suggest that this anti-DNA autoantibody originated from the same germline repertoire as antibodies to exogenous antigens.

Characterization of germanium linear kinoform lenses at Diamond Light Source.

The unprecedented brilliance achieved by third-generation synchrotron sources and the availability of improved optics have opened up new opportunities for the study of materials at the micrometre and nanometre scale. Focusing the synchrotron radiation to smaller and smaller beams is having a huge impact on a wide research area at synchrotrons. The key to the exploitation of the improved sources is the development of novel optics that deliver narrow beams without loss of brilliance and coherence. Several types of synchrotron focusing optics are successfully fabricated using advanced miniaturization techniques. Kinoform refractive lenses are being developed for hard X-ray beamlines, and the first test results at Diamond are discussed in this paper.

The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations.

cDNA clones of a neuronal-specific mRNA encoding a novel 25-kD synaptosomal protein, SNAP-25, that is widely, but differentially expressed by diverse neuronal subpopulations of the mammalian nervous system have been isolated and characterized. The sequence of the SNAP-25 cDNA revealed a single open reading frame that encodes a primary translation product of 206 amino acids. Antisera elicited against a 12-amino acid peptide, corresponding to the carboxy-terminal residues of the predicted polypeptide sequence, recognized a single 25-kD protein that is associated with synaptosomal fractions of hippocampal preparations. The SNAP-25 polypeptide remains associated with synaptosomal membrane components after hypoosmotic lysis and is released by nonionic detergent but not high salt extraction. Although the SNAP-25 polypeptide lacks a hydrophobic stretch of residues compatible with a transmembrane region, the amino terminus may form an amphiphilic helix that may facilitate alignment with membranes. The predicted amino acid sequence also includes a cluster of four closely spaced cysteine residues, similar to the metal binding domains of some metalloproteins, suggesting that the SNAP-25 polypeptide may have the potential to coordinately bind metal ions. Consistent with the protein fractionation, light and electron microscopic immunocytochemistry indicated that SNAP-25 is located within the presynaptic terminals of hippocampal mossy fibers and the inner molecular layer of the dentate gyrus. The mRNA was found to be enriched within neurons of the neocortex, hippocampus, piriform cortex, anterior thalamic nuclei, pontine nuclei, and granule cells of the cerebellum. The distribution of the SNAP-25 mRNA and the association of the protein with presynaptic elements suggest that SNAP-25 may play an important role in the synaptic function of specific neuronal systems.

Localization of amyloid beta protein messenger RNA in brains from patients with Alzheimer's disease.

The distribution of cells containing messenger RNA that encodes amyloid beta protein was determined in hippocampi and in various cortical regions from cynomolgus monkeys, normal humans, and patients with Alzheimer's disease by in situ hybridization. Both 35S-labeled RNA antisense and sense probes to amyloid beta protein messenger RNA were used to ensure specific hybridization. Messenger RNA for amyloid beta protein was expressed in a subset of neurons in the prefrontal cortex from monkeys, normal humans, and patients with Alzheimer's disease. This messenger RNA was also present in the neurons of all the hippocampal fields from monkeys, normal humans and, although to a lesser extent in cornu ammonis 1, patients with Alzheimer's disease. The distribution of amyloid beta protein messenger RNA was similar to that of the neurofibrillary tangles of Alzheimer's disease in some regions, but the messenger RNA was also expressed in other neurons that are not usually involved in the pathology of Alzheimer's disease.

Equine amnionitis and fetal loss: the case definition for an unrecognised cause of abortion in mares.

A series of abortions occurred in mares in New South Wales during 2004 that involved similar and unusual findings on post mortem examination of aborted fetuses and fetal membranes. The term Equine Amnionitis and Fetal Loss (EAFL) was developed to describe the condition. This form of abortion had not been previously recognised in Australia. The pathology alone is not specific for EAFL and diagnosis requires demonstration of a combination of certain pathological and bacteriological features. The purpose of this paper is to describe patterns considered consistent with EAFL cases as a working case definition for use by veterinarians and veterinary pathologists in identifying future cases of EAFL. More detailed papers are in preparation to fully describe the epidemiological, histopathological, and microbiological aspects of EAFL.

Tottering and leaner mutations perturb transient developmental expression of tyrosine hydroxylase in embryologically distinct Purkinje cells.

The mouse mutants tottering and leaner exhibit neurologic disorders associated, in part, with global noradrenergic hyperinnervation. Therefore, the expression of tyrosine hydroxylase (TH) mRNA and protein was examined in mutant and control mice. TH expression was normal in the major catecholaminergic nuclei. However, TH was expressed in vermal Purkinje cells of adult mutant but not control mice. TH expression in the Purkinje cells of both mutants was first observed on P21 and persisted throughout adulthood; in contrast, Purkinje cells of normal mice expressed TH transiently during development from P21 to P35. Thus, tottering and leaner mice are deficient in suppressing the normal transient expression of TH in developing Purkinje cells, suggesting that the protein encoded by the tg locus may play a crucial role in neuronal development.

Chinese hamster polyadenylated messenger ribonucleic acid: relationship to non-polyadenylated sequences and relative conservation during messenger ribonucleic acid processing.

We have further analyzed the metabolism of specific messenger ribonucleic acid (mRNA) sequences within the cytoplasmic and nuclear RNA of Chinese hamster ovary (CHO) cells by using a set of previously constructed complementary deoxyribonucleic acid (DNA) clones (Harpold et al., Cell 17:1025-1035, 1979) as specific molecular probes in a variety of RNA:DNA hybridization experiments. The majority of the labeled mRNA complementary to each of the nine clones was found in the polyribosomes, with some variation between individual sequences. The great majority of each specific mRNA labeled for 3 h or less was in the polyadenylated [poly(A)+] fraction. However, the amount of each sequence increased in the non-poly(A)+ [poly(A)-] fraction after very long label times, suggesting the derivation of the poly(A)- RNA from the poly(A)+ RNA. Eight of the nine mRNA's have cytoplasmic half-lives ranging from 8 to 14 h, whereas one of the mRNA's, the scarcest in the group, has a somewhat shorter half-life of approximately 3 h. The proportion of each of the specific long-lived mRNA's within the total labeled mRNA increased as a function of labeling time, indicating that a large fraction, probably greater than 50%, of the initially labeled poly(A)+ mRNA in CHO cells has a half-life of less than 3 h. A quantitative analysis of the kinetics of labeling of specific nuclear and cytoplasmic sequences indicated that a significant fraction of the mRNA sequences transcribed from genes containing these nine CHO sequences were successfully processed into mRNA. However, two of the CHO mRNA sequences were only partially conserved during nuclear processing to yield mRNA. These studies demonstrated that events at two post-transcriptional levels, differential nuclear processing efficiency of different primary transcripts and cytoplasmic stability of different mRNA's, can be involved in the determination of the cytoplasmic concentrations of different mRNA's.

Large heterogeneous nuclear ribonucleic acid has three times as many 5' caps as polyadenylic acid segments, and most caps do not enter polyribosomes.

The rate of synthesis in Chinese hamster cells of 5' cap structures, m7 GpppNmp, in large (greater than 700 bases) heterogeneous nuclear ribonucleic acid (RNA) molecules is two to three times faster than the synthesis of 3'-terminal polyadenylic acid segments. As judged by presence of caps, newly synthesized polysomal messenger RNA, exclusive of messenger RNA the size of histone messenger RNA, is more than 90% in the polyadenylated category. It appears, therefore, that between half and two-thirds of the long capped heterogeneous nuclear RNA molecules do not contribute a capped polysomal derivative to the cytoplasm. There are capped, nonpolysomal, non-polyadenylated molecules with a rapid turnover rate that fractionate with the cytoplasm. These metabolically unstable molecules either could represent leakage into the cytoplasm during fractionation or could truly spend a brief time in the cytoplasm before decay.

T-cell receptor repertoire in matched MART-1 peptide-stimulated peripheral blood lymphocytes and tumor-infiltrating lymphocytes.

Characterization of tumor-associated antigens (TAAs) recognized by CTLs makes the consideration of therapeutic strategies based on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendritic cells, and direct vaccination with TAA-derived peptides. A critical component of any of these peptide-based strategies is the requirement that the patient's PBLs are able to react productively against the presented TAA. The purpose of this study, through the study of T-cell receptor (TCR) usage, was to evaluate the T-cell response in matched MART-1(27-35) peptide-stimulated PBLs and tumor-infiltrating lymphocytes (TILs). MART-1(27-35)-reactive PBL and TIL cultures were generated from three patients by in vitro stimulation with an immunodominant peptide of MART-1 (MART-1(27-35)). All cultures had a human leukocyte antigen A2-restricted, MART-1(27-35)-specific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR beta clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8-16 subfamilies were used) and a predominant usage of a different variable beta chain (BV) by each of these MART-reactive T cells. These predominant BV rearrangements were derived from multiple clonotypes because different variable, diversity, and junctional regions were observed. However, a similar pattern of expansion was present for both PBLs and TILs; the relative usage of each prevailing BV was more marked in TILs (36, 50, and 78% of TILs versus 26, 20, and 24% of PBLs, respectively), a broader TCR repertoire was used by PBLs (P > 0.05), and similar TCR subfamily usage was noted when TIL and PBL cultures from the same patient were compared (8 of 11, 7 of 9, and 7 of 8 for patients 1, 2, and 3, respectively). Furthermore, the exact same clonotypes derived from predominant TCR subfamilies in the PBLs and TILs were present in each patient, suggesting peptide-stimulated expansion in both biological compartments. These studies suggest that there will not be a limited and predictable TCR subfamily response to a specific TAA, although reproducible patterns of PBL and TIL expansion are present from patient to patient. Additionally, identical T-cell clonotypes having the same potential for antigen-driven expansion were present in a patient's PBLs and TILs. As such, our data support the conceptualization of approaches using adoptive transfer or vaccination based on TAA-derived peptide stimulation of PBLs.

Genomes of coral dinoflagellate symbionts highlight evolutionary adaptations conducive to a symbiotic lifestyle.

Despite half a century of research, the biology of dinoflagellates remains enigmatic: they defy many functional and genetic traits attributed to typical eukaryotic cells. Genomic approaches to study dinoflagellates are often stymied due to their large, multi-gigabase genomes. Members of the genus Symbiodinium are photosynthetic endosymbionts of stony corals that provide the foundation of coral reef ecosystems. Their smaller genome sizes provide an opportunity to interrogate evolution and functionality of dinoflagellate genomes and endosymbiosis. We sequenced the genome of the ancestral Symbiodinium microadriaticum and compared it to the genomes of the more derived Symbiodinium minutum and Symbiodinium kawagutii and eukaryote model systems as well as transcriptomes from other dinoflagellates. Comparative analyses of genome and transcriptome protein sets show that all dinoflagellates, not only Symbiodinium, possess significantly more transmembrane transporters involved in the exchange of amino acids, lipids, and glycerol than other eukaryotes. Importantly, we find that only Symbiodinium harbor an extensive transporter repertoire associated with the provisioning of carbon and nitrogen. Analyses of these transporters show species-specific expansions, which provides a genomic basis to explain differential compatibilities to an array of hosts and environments, and highlights the putative importance of gene duplications as an evolutionary mechanism in dinoflagellates and Symbiodinium.

Expression and distribution of lactate/monocarboxylate transporter isoforms in pancreatic islets and the exocrine pancreas.

Transport of lactate across the plasma membrane of pancreatic islet beta-cells is slow, as described by Sekine et al. (J Biol Chem 269:4895-4902, 1994), which is a feature that may be important for normal nutrient-induced insulin secretion. Although eight members of the monocarboxylate transporter (MCT) family have now been identified, the expression of these isoforms within the exocrine and endocrine pancreas has not been explored in detail. Using immunocytochemical analysis of pancreatic sections fixed in situ, we demonstrated three phenomena. First, immunoreactivity of the commonly expressed lactate transporter isoform MCT1 is near zero in both alpha- and beta-cells but is abundant in the pancreatic acinar cell plasma membrane. No MCT2 or MCT4 was detected in any pancreatic cell type. Second, Western analysis of purified beta- and non-beta-cell membranes revealed undetectable levels of MCT1 and MCT4. In derived beta-cell lines, MCT1 was absent from MIN6 cells and present in low amounts in INS-1 cell membranes and at high levels in RINm5F cells. MCT4 was weakly expressed in MIN6 beta-cells. Third, CD147, an MCT-associated chaperone protein, which is closely colocalized with MCT1 on acinar cell membranes, was absent from islet cell membranes. CD147 was also largely absent from MIN6 and INS-1 cells but abundant in RINm5F cells. Low expression of MCT1, MCT2, and MCT4 contributes to the enzymatic configuration of beta-cells, which is poised to ensure glucose oxidation and the generation of metabolic signals and may also be important for glucose sensing in islet non-beta-cells. MCT overexpression throughout the islet could contribute to deranged hormone secretion in some forms of type 2 diabetes.

Characterization of a sustained-release delivery system for combined cytokine/peptide vaccination using a poly-N-acetyl glucosamine-based polymer matrix.

Identification of tumor-associated antigens (TAAs) and their class I MHC-restricted epitopes now allows for the rational design of peptide-based cancer vaccines. A biocompatible system capable of sustained release of biologically relevant levels of cytokine and TAA peptide could provide a more effective microenvironment for antigen presentation. Our goal was to test a sustained-release cytokine/TAA peptide-based formulation using a highly purified polysaccharide [poly-N-acetyl glucosamine (p-GlcNAc)] polymer. Granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 microgram) and MART-1(27-35) peptide (128 microgram in DMSO) were formulated into p-GlcNAc. Peptide release was assayed in vitro using interleukin 2 production from previously characterized MART-1(27-35)-specific Jurkat T cells (JRT22). GM-CSF release was assayed via ELISA and proliferation of M-07e (GM-CSF-dependent) cells. Local bioavailability of MART-1(27-35) peptide for uptake and presentation by antigen-presenting cells was demonstrated for up to 6 days (>0.5 microgram/ml). More than 1.0 microgram/ml GM-CSF was concomitantly released over the same period. Biocompatibility and local tissue response to p-GlcNAc releasing murine GM-CSF was determined in C57BL/6 mice via s.c. injection using murine GM-CSF (0. 2 microgram/ml) in 200 microliter of a 2.5% polymer gel. Significant lymphocytic and eosinophilic infiltration was observed 2-7 days after injection with polymer containing murine GM-CSF. The results of our studies show that this biocompatible system is capable of a sustained concomitant release of biologically active peptide and cytokine into the local microenvironment. These findings support further studies to validate a p-GlcNAc delivery system vehicle for a cytokine/TAA peptide-based cancer vaccine.

Coloboma mouse mutant as an animal model of hyperkinesis and attention deficit hyperactivity disorder.

Hyperkinesis and developmental behavioral deficiencies are cardinal signs of attention-deficit hyperactivity disorder. In mice, the mutation coloboma (Cm) corresponds to a contiguous gene defect that results in phenotypic abnormalities including spontaneous hyperactivity, head-bobbing, and ocular dysmorphology. In addition, coloboma mutant mice exhibit delays in achieving complex neonatal motor abilities and deficits in hippocampal physiology, which may contribute to learning deficiencies. The hyperkinesis is ameliorated by low doses of the psychostimulant D-amphetamine and can be rescued genetically by a transgene encoding SNAP-25, located within the Cm deletion. Together with syntaxin and synaptobrevin/VAMP, SNAP-25 constitutes a core protein complex integral to synaptic vesicle fusion and neurotransmitter release. Despite the ubiquitous role of SNAP-25 in synaptic transmission, and uniformly decreased expression in the mutants, coloboma mice show marked deficits in Ca2+-dependent dopamine release selectively in dorsal but not ventral striatum. This suggests that haploinsufficiency of SNAP-25 reveals a specific vulnerability of the nigrostriatal pathway which regulates motor activity and may provide a model for impaired striatal input into executive functions encoded by the prefrontal cortex associated with ADHD.

Successful treatment of genetically hypophosphatemic mice by 1 alpha-hydroxyvitamin D3 but not 1,25-dihydroxyvitamin D3.

The X-linked hypophosphatemia (Hyp) mutation in the mouse, a model for X-linked familial hypophosphatemic rickets in man, is characterized by defective phosphate transport. The role of vitamin D3 in the defective phosphate transport was investigated in three experiments by treatment of mutant mice with the natural hormonal form of vitamin D3, 1,25-dihydroxyvitamin D3, or its potent synthetic analog, 1 alpha-hydroxyvitamin D3. The results showed that both compounds were able to increase urinary phosphate conservation and improve rachitic bone morphology. Only 1 alpha-hydroxyvitamin D3, however, repaired the critically important hypophosphatemia and significantly increased intestinal transport of phosphate. These results indicate that defective phosphate transport in genetic hypophosphatemia is amenable to effective treatment. We hypothesize that the intestinal phosphate transport system is not genetically deleted but, instead, is unable to respond to 1,25-dihydroxyvitamin D3. Elucidation of the mechanism whereby 1 alpha-hydroxyvitamin D3 is able to stimulate the defective phosphate transport may provide fresh insight into the metabolic basis of the disease.

Human cDNA clones encoding two different isoforms of the nerve terminal protein SNAP-25.

Two distinct cDNA sequences, corresponding to alternative isoforms of the human nerve terminal protein SNAP-25 (synaptosomal associated protein of 25 kDa), were cloned and characterized. Sequence analysis demonstrated that the two isoforms are generated by alternative splicing between two distinct but homologous exons 5, a and b each encoding 39 amino acids (aa). Although the two isoforms, SNAP-25a and SNAP-25b, differ by only 9 aa, this domain encodes the portion of the protein that is a substrate for post-translational fatty acylation, and therefore might be important for regulating subcellular localization and membrane targeting.

Botulinum neurotoxin types A and E require the SNARE motif in SNAP-25 for proteolysis.

Botulinum neurotoxins type A and E (BoNT/A and BoNT/E) are metalloproteases with a unique specificity for SNAP-25 (synaptosome-associated protein of 25 kDa), an essential protein component of the neuroexocytotic machinery. It has been suggested that this specificity is directed through the recognition of a nine residue sequence, termed SNARE motif, that is common to the other two SNARE proteins: VAMP (vesicle-associated membrane protein) and syntaxin, the only known substrates of the other six clostridial neurotoxins. Here we analyse the involvement of the four copies of the SNARE motif present in SNAP-25 in its interaction with BoNT/A and BoNT/E by following the kinetics of proteolysis of SNAP-25 mutants deleted of SNARE motifs. We show that a single copy of the motif is sufficient for BoNT/A and BoNT/E to recognise SNAP-25. While the copy of the motif proximal to the cleavage site is clearly involved in recognition, in its absence, other more distant copies of the motif are able to support proteolysis. Also, a non-neuronal isoform of SNAP-25, Syndet, is shown to be sensitive to BoNT/E, but not BoNT/A, whilst the SNAP-25 isoforms from Torpedo marmorata and Drosophila melanogaster were demonstrated not to be substrates of these metalloproteases.

Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds.

SNAP-25, a membrane-associated protein of the nerve terminal, is specifically cleaved by botulinum neurotoxins serotypes A and E, which cause human and animal botulism by blocking neurotransmitter release at the neuromuscular junction. Here we show that these two metallo-endopeptidase toxins cleave SNAP-25 at two distinct carboxyl-terminal sites. Serotype A catalyses the hydrolysis of the Gln197-Arg198 peptide bond, while serotype E cleaves the Arg180-Ile181 peptide lineage. These results indicate that the carboxyl-terminal region of SNAP-25 plays a crucial role in the multi-protein complex that mediates vesicle docking and fusion at the nerve terminal.

Characterization and ontogeny of synapse-associated proteins in the developing facial and hypoglossal motor nuclei of the Brazilian opossum.

The characterization and ontogeny of synapse-associated proteins in the developing facial and hypoglossal motor nuclei were examined in the Brazilian opossum (Monodelphis domestica). Immunohistochemical markers utilized in this study were the synaptic vesicle-associated proteins synaptophysin and synaptotagmin; a synaptic membrane protein, plasma membrane-associated protein of 25 kDa (SNAP-25); a growth cone protein, growth-associated phosphoprotein-43 (GAP-43); and the microtubule-associated proteins axonal marker tau and dendritic marker microtubule-associated protein-2 (MAP-2). In this study, we have found that, during the first 10 postnatal days (1-10 PN), the facial motor nucleus lacked immunoreactivity for synaptophysin, synaptotagmin, GAP-43, tau, and SNAP-25. After 10 PN, immunoreactivity increased in the facial motor nucleus for synaptophysin, synaptotagmin, GAP-43, and tau, whereas immunoreactivity for SNAP-25 was not evident until between 15 and 25 PN. Conversely, immunoreactivity for MAP-2, was present in the facial motor nucleus from the day of birth. In contrast, the hypoglossal motor nucleus displayed immunoreactivity from 1 PN for synaptophysin, synaptotagmin, SNAP-25, GAP-43, tau, and MAP-2. These results suggest that the facial motor nucleus of the opossum may not receive afferent innervation as defined by classical synaptic markers until 15 PN and, further, that characteristic mature synapses are not present until between 15 and 25 PN. These results indicate that there may be a delay in synaptogenesis in the facial motor nucleus compared to synaptogenetic events in the hypoglossal motor nucleus. Because the facial motor nucleus is active prior to completion of synaptogenesis, we suggest that the facial motoneurons are regulated in a novel or distinct manner during this time period.