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PubMed1997

Synthesis and turnover of beta2-microglobulin in Ad12-transformed cells defective in assembly and transport of class I major histocompatibility complex molecules.

In primary embryonal fibroblasts from transgenic mice expressing H-2 genes and a miniature swine class I transgene (PD1), transformation with the highly oncogenic Ad12 results in a reduction in peptide transporter and proteasome-associated (LMP2 and LMP7) gene expression, and suppression in transport and cell surface expression of all class I antigens. The selective suppression in transport of H-2 (but not of PD1) molecules in cells reconstituted for the expression of peptide transporter and LMP genes implied that an additional factor(s) is involved in the assembly of class I complexes. Here we show that the beta2m, H-2Db, and H-2Kb genes are transcribed and translated in Ad12-transformed cells. However, unlike normal and E1Ad5-transformed cells, in which beta2m is either secreted unbound or bound to class I heavy chains, in Ad12-transformed cells significant amounts of beta2m are retained in the cell bound to the membrane, but free of class I heavy chains. This abnormal turnover of beta2m in the Ad12-transformed cells suggests the existence of a novel beta2m-binding molecule(s) that sequesters beta2m, and this process may provide a mechanism by which transformation with Ad12 may subvert class I complex formation.

Adenoviruses, Human↗

PubMed1998

MHC class I heavy chain mRNA must exceed a threshold level for the reconstitution of cell surface expression of class I MHC complexes in cells transformed by the highly oncogenic adenovirus 12.

In primary embryonal fibroblasts from transgenic mice expressing H-2(b) genes and a miniature swine class I transgene (PD1), transformation with adenovirus 12 results in suppression of assembly and cell surface expression of all class I complexes. Cell surface expression of PD1 can be recovered by transfecting the cells with peptide transporter genes. However, reconstitution of the H-2Kb gene expression requires, in addition, a 2-fold increase in the steady state level of the H-2Kb mRNA that can be attained by treatment of the cells with interferons or by transfecting them with the H-2Kb gene. A detailed analyses of the biogenesis of class I molecules has revealed the steady state expression of free class I heavy chains that are not converted into conformed complexes even when peptide transporter genes are overexpressed. The fact that class I complex assembly seems to be highly inefficient in certain cell lines might be a major in vivo obstacle for the elimination of transformed or virus-infected cells by cytotoxic T lymphocytes, especially in view of the fact that the level of class I gene transcription is often down-regulated in cancer cells and/or that assembly of class I major histocompatibility complexes can be subverted by virus-encoded proteins.

Adenoviridae↗

PubMed2004

Evidence that D1 processing is required for manganese binding and extrinsic protein assembly into photosystem II.

Photosystem II (PSII) is a large membrane protein complex that catalyzes oxidation of water to molecular oxygen. During its normal function, PSII is damaged and frequently turned over. The maturation of the D1 protein, a key component in PSII, is a critical step in PSII biogenesis. The precursor form of D1 (pD1) contains a C-terminal extension, which is removed by the protease CtpA to yield PSII complexes with oxygen evolution activity. To determine the temporal position of D1 processing in the PSII assembly pathway, PSII complexes containing only pD1 were isolated from a CtpA-deficient strain of the cyanobacterium Synechocystis 6803. Although membranes from the mutant cell had nearly 50% manganese, no manganese was detected in isolated DeltactpAHT3 PSII, indicating a severely decreased manganese affinity. However, chlorophyll fluorescence decay kinetics after a single saturating flash suggested that the donor Y(Z) was accessible to exogenous Mn(2+) ions. Furthermore, the extrinsic proteins PsbO, PsbU, and PsbV were not present in PSII isolated from this mutant. However, PsbO and PsbV were present in mutant membranes, but the amount of PsbV protein was consistently less in the mutant membranes compared with the control membranes. We conclude that D1 processing precedes manganese binding and assembly of the extrinsic proteins into PSII. Interestingly, the Psb27 protein was found to be more abundant in DeltactpAHT3 PSII than in HT3 PSII, suggesting a possible role of Psb27 as an assembly factor during PSII biogenesis.

Manganese↗

PubMed1996

LMP-associated proteolytic activities and TAP-dependent peptide transport for class 1 MHC molecules are suppressed in cell lines transformed by the highly oncogenic adenovirus 12.

Expression of class I major histocompatibility complex antigens on the surface of cells transformed by adenovirus 12 (Ad12) is generally very low, and correlates with the in vivo oncogenicity of this virus. In primary embryonal fibroblasts (H-2b) that express transgenic swine class I antigen (PD1), Ad12-mediated transformation results in inhibition in transport of newly synthesized class I molecules, as well as significant reduction in transporter associated with antigen presentation (TAP) gene expression. In this report we show that reexpression of TAP molecules either by stable transfection of mouse TAP genes or by infection with recombinant vaccinia viruses expressing human TAP genes, only partially reconstitutes the expression and transport of the class I molecules. Further analysis of Ad12-transformed cells revealed that the expression of both LMP2 and LMP7, but not of other proteasome complex components, was downregulated, resulting in altered proteolytic activities of the 20S proteasomes. Reconstitution of both TAP and LMP expression resulted in complete restoration of PD1 cell surface expression and enhanced expression of the endogenous H-2D(b) molecules encoded by recombinant vaccinia viruses, in reconstituted Ad12-transformed cells, efficient transport of H-2 class I molecules could only be achieved by treatment of the cells with gamma-interferon. These data suggest that an additional factor(s) that is interferon-regulated plays a role in the biosynthetic pathway of the class I complex, and that its function is deficient in this cell system. Thus, Ad12 viral transformation appears to suppress the expression of multiple genes that are important for antigen processing and presentation, which allows such transformed cells to escape immune surveillance. This coordinate downregulation of immune response genes must likely occur through their use of common regulatory elements.

ATP-Binding Cassette Transporters↗

PubMed2000

A new genetic method for isolating functionally interacting genes: high plo1(+)-dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast.

We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product. The screen identifies mutants whose growth depends on high levels of expression of that gene. We apply this to the plo1(+) gene that encodes a fission yeast homologue of the polo-like kinases. plo1(+) regulates both spindle formation and septation. We have isolated 17 high plo1(+)-dependent (pld) mutants that show defects in mitosis or septation. Three mutants show a mitotic arrest phenotype. Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2. One of the pld mutants, cdc7-PD1, was selected for suppressor analysis. As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1(+), sce3(+), cdc8(+), and rho1(+), and two previously uncharacterized genes, mpd1(+) and mpd2(+). mpd1(+) exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2(+) resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes. As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division. cdc16(+), dma1(+), byr3(+), byr4(+) and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations. These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes.

Amino Acid Sequence↗

PubMed2006

Effects of oral contraceptive, synthetic progestogen or natural estrogen pre-treatments on the hormonal profile and the antral follicle cohort before GnRH antagonist protocol.

BACKGROUND: Steroid pre-treatments may be useful to program GnRH antagonist IVF/ICSI cycles. This prospective study assessed hormonal and ultrasound data collected during the free period after the discontinuation of three different pre-treatments to provide information on the optimal time interval required before starting stimulation. METHODS: Women were randomized to receive oral contraceptive pill (OCP) [ethinyl estradiol (E(2)) 30 microg + desogestrel 150 microg] (n = 21) or norethisterone 10 mg/day (n = 23) or 17-betaE(2) 4 mg/day (n = 25) or no pre-treatment (n = 24) for one cycle before IVF. Assessments were performed on post-treatment day (PD) 1, 3 and 5, or on spontaneous cycle day (CD) 1 and 3. RESULTS: After OCP and progestogen administration, FSH and LH concentrations shifted from strongly suppressed PD1 levels to PD5 values similar to those observed on CD1. Meanwhile, follicle sizes remained small up to PD5. In contrast, estrogen pre-treatment poorly reduced FSH levels on PD1 compared with OCP or progestogen. Consequently, follicle size was more heterogeneous. FSH rebound was maximal on PD3, whereas LH levels were slightly increased up to PD5. CONCLUSIONS: A 5-day free interval after OCP or progestogen offers the advantages of gonadotrophin recovery and homogeneous follicular cohort, whereas early FSH rebound occurring after estrogen pre-treatment argues for a short free period.

Adult↗

PubMed1996

Genetic engineering of the processing site of D1 precursor protein of photosystem II reaction center in Chlamydomonas reinhardtii.

The D1 protein (D1) of photosystem II (PSII) reaction center is synthesized as a precursor (pD1) and then processed at its carboxyl terminus to establish the function of water cleavage. The amino acid sequence of the carboxyl terminal extension excised by this process is poorly conserved except for a residue after the cleavage site at position of 345. We have constructed a vector for site-directed mutagenesis of the chloroplast psbA gene encoding D1 of the green alga, Chlamydomonas reinhardtii. The vector enables one to transform the chloroplasts of a psbA deletion mutant (Fud7) and directly select transformants for resistance to spectinomycin. Using this transforming vector, we have substituted Ser345 to Gly, Cys, Val and Phe in order to investigate effects of the amino acid side chain at this position on the processing rate. All of the resulting transformants exhibited the PSII activity as wild type and grew normally under photoautotrophic conditions even under strong light where rapid turnover of D1 protein is expected to occur. Western blotting analysis demonstrated that mature D1 accumulates in these transformants at wild type level. Pulse and chase labeling of chloroplast-encoded proteins using [35S] sulfate revealed that the processing of D1 precursor protein occurs in all four transformants as efficiently as in wild type, at least under the experimental conditions examined. The results suggest that either the amino acid side chain at position of 345 (+1 position) is not crucial to the enzymatic cleavage of pD1 in vivo or the apparent rate of processing in vivo is not limited by the enzymatic cleavage.

Animals↗

PubMed2006

Protophloem differentiation in early Arabidopsis thaliana development.

During Arabidopsis embryogenesis, procambial cells undergo coordinated, asymmetric cell divisions, giving rise to vascular precursor cells (protophloem and protoxylem precursors). After germination, these cells terminally differentiate into specialized conducting cells, referred to as protophloem and protoxylem cells. Few readily identifiable markers of the onset of specification and differentiation are available, hampering the molecular genetic analysis of protophloem development. Confocal microscopy was used to investigate the patterning and differentiation of phloem cells during early plant development. Longitudinal divisions of phloem initials allowed the identification of protophloem precursor cells and adjacent metaphloem initials along the length of the plant. During germination, protophloem differentiation was observed at two independent locations, in the cotyledons and the hypocotyl. In both locations, differentiation was concomitant with cell elongation. We identified five gene-trap lines (PD1-PD5) with marker gene expression in immature protophloem elements. The spatio-temporal marker expression pattern of the lines divides them into two groups. The early specification markers PD4 and PD5 were expressed in developing organs before procambium formation and then became restricted to phloem initial cells. The protophloem precursor markers PD1-PD3 were expressed in differentiating protophloem cells at different stages of their development. All markers were expressed transiently and iteratively during the differentiation of protophloem in newly formed organs. Flanking genes were identified for four out of five gene-trap insertion lines. The possible function of these genes with respect to phloem differentiation is discussed.

Arabidopsis↗

PubMed2004

Changes of dopamine turnover in the progression of Parkinson's disease as measured by positron emission tomography: their relation to disease-compensatory mechanisms.

An increase in dopamine turnover has been shown to occur early in Parkinson's disease (PD). This study investigated changes of dopamine turnover as a function of PD duration using the effective distribution volume (EDV) for dopamine, determined by positron emission tomography with 6-[18F]-fluoro-L-dopa, and compared them with changes in dopamine synthesis and storage ability, quantified with the fluorodopa uptake rate constant Ki. Six healthy subjects, 9 early PD patients (PD1), and 13 advanced PD patients (PD2) participated in the study. In the caudate, the Ki and EDV for PD1 were not significantly different from the normal values, whereas in the putamen Ki was 63% of normal and EDV was only 35%. Between PDI and PD2 the decline in EDV was higher than that for Ki (caudate 44% and putamen 46% for EDV vs. 21% and 34%, respectively, for Ki). Turnover was higher in the caudate than the putamen in controls, whereas the PD patients exhibited the reverse pattern. This comparison of changes in Ki and EDV as a function of disease progression indicates that a relatively slower decrease in dopamine synthesis and a relatively faster increase in turnover in early disease likely act as compensatory mechanisms, and that the clinical onset of PD reflects a global failure of dopaminergic compensatory mechanisms.

Brain↗

PubMed2005

ATG deserts define a novel core promoter subclass.

The MHC class I gene, PD1, has neither functional TATAA nor Initiator (Inr) elements in its core promoter and initiates transcription at multiple, dispersed sites over an extended region in vitro. Here, we define a novel core promoter feature that supports regulated transcription through selective transcription start site (TSS) usage. We demonstrate that TSS selection is actively regulated and context dependent. Basal and activated transcriptions initiate from largely nonoverlapping TSS regions. Transcripts derived from multiple TSS encode a single protein, due to the absence of any ATG triplets within approximately 430 bp upstream of the major transcription start site. Thus, the PD1 core promoter is embedded within an "ATG desert". Remarkably, extending this analysis genome-wide, we find that ATG deserts define a novel promoter subclass. They occur nonrandomly, are significantly associated with non-TATAA promoters that use multiple TSS, independent of the presence of CpG islands (CGI). We speculate that ATG deserts may provide a core promoter platform upon which complex upstream regulatory signals can be integrated, targeting multiple TSS whose products encode a single protein.

Animals↗

PubMed2005

Development of sensory innervation in rat tibia: co-localization of CGRP and substance P with growth-associated protein 43 (GAP-43).

The development of sensory innervation in long bones was investigated in rat tibia in fetuses on gestational days (GD) 16-21 and in neonates and juvenile individuals on postnatal days (PD) 1-28. A double immunostaining method was applied to study the co-localization of the neuronal growth marker growth-associated protein 43 (GAP-43) and the pan-neuronal marker protein gene product 9.5 (PGP 9.5) as well as that of two sensory fibre-associated neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP). The earliest, not yet chemically coded, nerve fibres were observed on GD17 in the perichondrium of the proximal epiphysis. Further development of the innervation was characterized by the successive appearance of nerve fibres in the perichondrium/periosteum of the shaft (GD19), the bone marrow cavity and intercondylar eminence (GD21), the metaphyses (PD1), the cartilage canals penetrating into the epiphyses (PD7), and finally in the secondary ossification centres (PD10) and epiphyseal bone marrow (PD14). Maturation of the fibres, manifested by their immunoreactivity for CGRP and SP, was visible on GD21 in the epiphyseal perichondrium, the periosteum of the shaft and the bone marrow, on PD1 in the intercondylar eminence and the metaphyses, on PD7 in the cartilage canals, on PD10 in the secondary ossification centres and on PD14 in the epiphyseal bone marrow. The temporal and topographic pattern of nerve fibre appearance corresponds with the development of regions characterized by active mineralization and bone remodelling, suggesting a possible involvement of the sensory innervation in these processes.

Animals↗

PubMed2005

Effect of a single dose of mifepristone on expression of pinopodes in endometrial surface of mice.

AIM: To investigate the effect of mifepristone (RU486) as a single dose on pinopodes expression in the endometrial surface of mice at the time of implantation. METHODS: Pregnant mice in the treated four groups received mifepristone subcutaneously (0.1 mg) between 07:00 and 08:00 AM on Pd (day of pregnancy)1, Pd2, Pd3, and Pd4. Pregnant mice in the non-treated group were used as controls. The uterine horns were collected randomly from two mice in each group between 21:30 and 22:00 PM on Pd4, and from another two mice of the same group between 09:30 and 10:00 AM on Pd5. The specimens were examined by scanning electron microscopy for the detection of pinopodes. RESULTS: When mifepristone was given on Pd1, developing and fully developed pinopodes were observed, but the expression was markedly reduced compared to the control group. When mifepristone was administered on Pd2, only a few developing pinopodes were present. When mifepristone was administered on Pd3, developing pinopodes were observed. When mifepristone was administered on Pd4, different development stage pinopodes were present in specimens collected between 21:30 and 22:00 PM on Pd4, but no pinopodes was observed in specimens taken between 09:30 and 10:00 AM on Pd5. CONCLUSION: These findings suggest that administration of a single dose of RU486 subcutaneously on Pd1, Pd2, Pd3, and Pd4 might play a role in inhibiting development and maturation of endometrium, hence affecting embryo implantation in mice.

Animals↗

PubMed1991

Molecular cloning of the extracellular endodextranase of Streptococcus salivarius.

We report the cloning in Escherichia coli of the gene encoding an extracellular endodextranase (alpha-1,6-glucanhydrolase, EC 3.2.1.11) from Streptococcus salivarius PC-1. Recombinants from a S. salivarius PC-1-Lambda ZAP II genomic library specifying dextranase activity were identified as plaques surrounded by zones of clearing on blue dextran agar. One such clone, PD1, had a 6.3-kb EcoRI fragment insert which encoded a 190-kDa protein with dextranase activity. The recombinant strain also produced two lower-molecular-mass polypeptides (90 and 70 kDa) that had dextranase activity. Native dextranase was recovered from concentrated culture fluids of S. salivarius as a single 110-kDa polypeptide. PD1 phage lysate and PC-1 culture supernatant fluid extract were used to measure substrate specificity of the recombinant and native forms of dextranase, respectively. Analysis of these reaction products by thin-layer chromatography revealed the expected isomaltosaccharide products yielded by the recombinant-specified enzyme but was unable to resolve the larger polysaccharide products of the native enzyme. Furthermore, S. salivarius utilized neither the substrates nor the products of dextran hydrolysis for growth.

Blotting, Southern↗

PubMed2002

Plasmid-encoded asp operon confers a proton motive metabolic cycle catalyzed by an aspartate-alanine exchange reaction.

Tetragenococcus halophila D10 catalyzes the decarboxylation of L-aspartate with nearly stoichiometric release of L-alanine and CO(2). This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an L-aspartate-beta-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter --> aspD --> aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known L-aspartate-beta-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of L-aspartate-beta-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.

Alanine↗

PubMed1999

Ventilatory function and personal breathing zone dust concentrations in Lancashire textile weavers.

BACKGROUND: To report findings on ventilatory function and estimations of concentrations of personal breathing zone dust in Lancashire textile weavers. Weaving room dust is considered to be less harmful than that encountered in the cardroom or spinning room and weavers are generally thought to have less respiratory disability than carders or spinners. However, this occupational group has not been extensively studied. METHODS: Each person was given a respiratory symptom questionnaire (modified Medical Research Council, UK, questionnaire on respiratory diseases). Ventilatory function tests, forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) were performed on each person. A representative sample of workers from each occupational group underwent dust sampling in their personal breathing zone. Dust concentrations and ventilatory tests were analysed statistically with the Student's t test, Pearson's correlation coefficient, and forward step regression for relations with symptoms and environmental factors. Significance was p > or = 0.05. RESULTS: The FEV1 and FVC were reduced in workers with respiratory symptoms (non-specific chest tightness, shortness of breath, persistent cough, and wheezing) as well as in preparation room workers, current and former smokers, Asians, those working with predominantly cotton fibre (> 50% cotton) and starch size. Mean total dust concentration (pd1) in the personal breathing zone was 1.98 mg/m3. The corresponding value for total dust with large fibres lifted off the filter paper (pd2) was 1.55 mg/m3. There was a strong correlation (r = 0.94, p < 0.0001) between pd1 and pd2. Non-specific chest tightness was predicted by low dust concentrations and persistent cough by high dust concentrations. On regression analysis, impairment of ventilatory function (FEV1, FVC) was predicted by smoking, male sex, age, not working in the weaving shed, not being white, and personal dust concentrations. CONCLUSIONS: The FEV1 and FVC were impaired in smokers and those exposed to high dust concentrations in the personal breathing zone. Symptoms were inconsistently related to dust concentrations in the personal breathing zone.

Dust↗

PubMed1994

Respiratory symptoms and dust exposure in Lancashire cotton and man-made fiber mill operatives.

A cross-sectional study of work-related symptoms and cotton dust exposure was made in 404 man-made fiber and 1,048 cotton operatives in Lancashire spinning mills; 39 cotton-exposed operatives (3.7%) had symptoms of byssinosis. This was associated on regression analysis with cumulative lifetime cotton dust exposure (p < 0.001), total years spent carding (p < 0.001), and currently working in the carding area (p = 0.0041). Smoking habit did not differ significantly between byssinotic and nonbyssinotic workers. Other work-related symptoms were common: chronic bronchitis (CB) and persistent cough. The prevalence of CB correlated positively with dust exposure (r = 0.59). Cotton dust sampling was performed in the work area (SDPRES) and personal breathing zone (PD1). A retrospective estimate of lifetime cotton dust exposure based on SDPRES correlated best with the prevalence of byssinosis (r = 0.797), although correlations with PD1 (r = 0.709) and SDPRES (r = 0.594) were also significant.

Adult↗

PubMed2006

The production of a diabetic mouse using constructs encoding porcine insulin promoter-driven mutant human hepatocyte nuclear factor-1alpha.

A diabetic mouse model was produced using a mutant human hepatocyte nuclear factor-1alpha gene (HNF1alphaP291fsinsC) regulated by the porcine insulin promoter. The functionality of two different constructs containing HNF1alphaP291fsinsC, termed PD1 and PD2 (cytomegalovirus enhancer minus and plus), were examined in transgenic mice. The blood glucose levels and body weights of the PD1 transgenic mice did not differ from their non-transgenic littermates over the period from 3 to 8 weeks of age. Conversely, the PD2 transgenic mice exhibited hyperglycemia and decreased body weight. Western blot analysis demonstrated that mutant HNF-1alpha protein (HNF1alphaP291), derived from the PD2 transgene, was expressed in the PD2 mice. Morphometric studies of the pancreas of a PD2 mouse revealed that the number of pancreatic islets present was less than that in the non-transgenic mice, indicating disturbed islet neogenesis. These results suggest that impaired insulin secretion in disrupted islets causes hyperglycemia. In addition, the phenotype of PD2 transgenic mice similar to that of the HNF-1alpha gene-deficient mouse, which displays growth retardation and impaired viability. These results indicate that HNF1alphaP291 expression driven by the porcine insulin promoter, together with the cytomegalovirus enhancer, induces a diabetic phenotype in transgenic mice.

Animals↗

PubMed1999

Anatomical and functional characterisation of a dopaminergic system in the suprachiasmatic nucleus of the neonatal Siberian hamster.

In altricial rodents, maternal influences entrain the developing circadian system in the perinatal period before the capacity to respond directly to photic cues develops. The aim of these studies was to investigate the potential role of dopamine in this process in the Siberian hamster. An initial study investigated the ontogeny of retinal innervation of the suprachiasmatic nuclei (SCN) by using cholera toxin B subunit as a tracer. This revealed that retinal fibres first innervate the SCN on postnatal day 3 (PD3), and ingrowth of fibres is extensive by PD6. In situ hybridisation studies revealed the presence of D1-dopamine receptor (D1-R) mRNA in the SCN on PD2, and levels of expression were similar in PD6 pups and adult hamsters. Immunocytochemical staining for tyrosine hydroxylase revealed abundant catecholaminergic fibres within the ventromedial zone of the SCN from the day of birth through PD20; however, in contrast, few fibres were present in adult SCN. Dopamine-beta-hydroxylase-immunoreactive fibres were absent from the neonatal and adult SCN, suggesting that the fibres in the SCN are dopaminergic. The function of this dopaminergic system was investigated by determining the effects of D1-R agonists on the expression of the immediate-early gene c-fos in the SCN. This was assessed in pups ages PD1- PD5 by in situ hybridisation and immunocytochemical localisation of its protein product. No induction was seen in the SCN, in marked contrast to studies in the developing rat. A final series of studies investigated dopaminergic function by determining whether a D1-agonist could induce phosphorylation of Ca2+/cyclic AMP response element-binding protein (CREB) on Ser133. Hypothalamic slices containing SCN taken from PD1 and PD2 hamsters were treated with D1-R agonists, and levels of phosphorylated CREB were assayed by Western blots. Phosphorylation of CREB was stimulated by D1-R agonists in both Syrian and Siberian hamster hypothalamus, but the response was far greater in Syrian hamster tissue (+138%+/-28%) than in Siberian hamster tissue (+43%+/-11%). Although the anatomical studies demonstrate the existence of a dopaminergic system in the SCN of the early postnatal Siberian hamster, the unresponsiveness of c-fos expression and the relative lack of phosphorylation of CREB after D1-R activation suggests a diminished role for dopamine in the regulation of circadian events during the postnatal period in this species.

Animals↗