Maternal regulation of the hypothalamic-pituitary-adrenal axis in the 20-day-old rat: consequences of laboratory weaning.

There is a large body of evidence that the development of the hypothalamic-pituitary-adrenal (HPA) system in the rat is under maternal regulation. One method used to study the influence of the dam-pup interaction in neonates and weanlings is the separation of mother and litter for 24 h. Previous studies showed that, even at the time of weaning, maternal deprivation results in a dysregulation of the HPA axis at multiple levels. However, the maternal deprivation paradigm usually includes deprivation of food and water, and it was not clear to which extent the observed effects are due to either maternal cues or dehydration and fasting. The primary purpose of the present study was to determine the role of fasting and/or maternal separation on the HPA axis at the time of weaning. Pups at 20 days after parturition are capable of self-feeding and no longer require tactile stimulation to induce eliminative functions. The results indicated that 24 h of fasting led to increased basal levels and further increases in stress induced corticosterone secretion. Fasting also appeared to contribute to the down regulation of basal glucocorticoid receptor mRNA in the hippocampus. In contrast, abrupt weaning irrespective of fasting or dehydration resulted in a suppressed adrenocorticotropin hormone response to an injection of isotonic saline. Although there was an effect of maternal separation on corticotropin-releasing factor mRNA in the paraventricular nucleus, this effect was further exacerbated by the absence of food. Finally, all rats that were separated from their dams showed more efficient negative-feedback. Thus, different aspects of the HPA system appear to respond differentially to either the absence of food or the absence of the mother or both.

Distinct roles of tumor necrosis factor-alpha and nitric oxide in acute liver injury induced by carbon tetrachloride in mice.

Macrophages are known to release a number of different inflammatory mediators with cytotoxic potential. In the present studies we analyzed the role of two macrophage-derived mediators, tumor necrosis factor-alpha (TNF-alpha) and nitric oxide, in liver injury induced by carbon tetrachloride (CCl4). Treatment of mice with CCl4 resulted in a dose- and time-dependent induction of centrilobular hepatic necrosis. This was observed within 12 h with 0.3 ml/kg CCl4 and was correlated with increases in serum transaminase levels. CCl4 administration also caused increases in hepatic TNF-alpha mRNA expression and serum TNF-alpha levels, as well as inducible nitric oxide synthase (NOS II) protein expression in the liver. To study the role of TNF-alpha and nitric oxide in hepatotoxicity, we used knockout mice lacking the gene for the 55-kDa TNF-alpha receptor (TNFR1/p55), the TNF-alpha cytokine, or NOS II. We found that CCl4 was significantly less effective in inducing hepatotoxicity in mice lacking TNFR1/p55 or the TNF-alpha cytokine. In contrast, CCl4-induced liver injury was increased in knockout mice lacking the gene for NOS II. This was associated with an increase in hepatic TNF-alpha mRNA expression and serum TNF-alpha levels. These data suggest that the hepatoprotective effects of nitric oxide in this model may be due in part to inhibition of TNF-alpha.

alpha 1(Xx) collagen, a new member of the collagen subfamily, fibril-associated collagens with interrupted triple helices.

Chick cDNA clones for a new member of the FACIT (fibril-associated collagens with interrupted triple helices) subfamily have been isolated and sequenced. The collagen chain encoded by these cDNAs was assigned the next consecutive number, making it the alpha1(XX) collagen chain. Assignment of type XX collagen to the FACIT family was based on sequence similarities to types XII and XIV collagen. Type XX collagen mRNA is not abundant in the chick embryo. It is most prevalent in corneal epithelium. It is also detectable by reverse transcription polymerase chain reaction in embryonic skin, sternal cartilage, and tendon, but is barely detectable in calvaria, notochord, or neural retina at select stages of development, suggesting that it is not expressed in these tissues. The cDNA predicts that the alpha1(XX) collagen polypeptide is smaller than the short forms of collagen XII and XIV. A polyclonal antibody against a synthetic alpha1(XX) peptide reacts with polypeptide bands of 185, 170, and 135 kDa by Western blot analysis. From its similarity to types XII and XIV collagen, type XX is expected to bind to collagen fibrils, projecting the amino-terminal domains away from the fibrillar surface. The projecting NC 3 domains are predicted to be about half the length of those of collagen XIV.

Expression of type XIV collagen in developing chicken tendons: association with assembly and growth of collagen fibrils.

Collagen fibril assembly is a multistep process involving multiple macromolecular interactions. Type XIV collagen contains multiple domains and is capable of interacting with collagen fibrils and other extracellular matrix components. During tendon development, naturally changing expression of type XIV collagen and its variants may modulate such interactions. Type XIV collagen was studied using immunochemical and molecular approaches. Western analysis demonstrated that type XIV collagen content was high between days 14 and 19, decreasing sharply at hatching. Immunoelectron microscopy demonstrated that type XIV collagen was fibril-associated, with a periodicity of 67 nm, indicating specific interactions. Decreased fibril-associated reactivity for type XIV collagen was seen at hatching, indicating a removal of collagen XIV from the fibril surface. The expression of two NC1 splice variants was analyzed. Overall, type XIV collagen mRNA decreased significantly from day 14 to hatching. The long NC1 splice variant was the predominant species at 14 days; at 19 days the two variants were expressed in lower amounts at nearly a 1:1 ratio; at hatching, both variants were expressed minimally. Changes in splice variant expression, suggest that different functional forms of type XIV collagen are present, allowing modified interactions with fibrils during development. In conclusion, type XIV collagen is fibril-associated and developmentally regulated. Modulation of expression of the NC1 splice variants may mediate the fibril interactions that allow the transition from growing fibril intermediates to mature fibrils.

Complete primary structure of the chicken alpha1(V) collagen chain.

Chicken alpha1(V) collagen cDNAs have been cloned by a variety of methods and positively identified. We present here the entire translated sequence of the chick polypeptide and compare selected regions to other collagen chains in the type V/XI family.

Lasting effect of repeated cocaine administration on acoustic and fear-potentiated startle in rats.

RATIONALE: Following cocaine withdrawal, humans may experience an abstinence syndrome with high levels of anxiety. Studying anxious behavior in animals following repeated cocaine administration may help elucidate important variables that contribute to a withdrawal syndrome. OBJECTIVES: This study investigated whether repeated cocaine pre-exposure produced lasting increases in conditioned fear as measured by fear-potentiated startle responses in rats. METHODS: Startle was measured in response to 50 ms acoustic stimuli of 95, 105 and 115 dB. Cocaine (20 mg/kg, IP) or saline was administered for 7 days and after each injection rats were either placed in startle chambers for 30 min or returned to the home cage. After a 1-week cocaine-free period, most rats were given ten light-footshock pairings in the startle chamber. Fear-potentiated startle was tested by presenting acoustic startle-eliciting stimuli of 95, 105 and 115 dB in the presence or absence of the light. Rats that were not fear conditioned received acoustic stimuli one week after 7 days of 20 mg/kg cocaine. RESULTS: Startle responses, both in the presence and absence of the light CS, were greater in fear-conditioned rats that received cocaine pre-exposure in the startle chamber than in saline pre-exposed rats. Startle responses in the presence of the light CS were further augmented at 115 dB. In contrast, home-cage exposure to cocaine did not enhance startle responses. In rats that were not fear conditioned, cocaine pre-exposure reduced acoustic startle. CONCLUSIONS: Repeated cocaine pre-exposure can increase, decrease or not change acoustic startle depending on whether fear conditioning occurred and whether cocaine was given in the testing chamber. The data suggest that cocaine pre-exposure may act as a contextually conditioned occasion setting stimulus that can facilitate anxious behavior similar to the postulated human cocaine abstinence syndrome.

Characterization of the gene for human EMMPRIN, a tumor cell surface inducer of matrix metalloproteinases.

EMMPRIN (extracellular matrix metalloproteinase inducer) also known as CD147 and basigin, is a member of the immunoglobulin family that is present on the surface of tumor cells and stimulates nearby fibroblasts to synthesize matrix metalloproteinases. Using our EMMPRIN cDNA, we have isolated a cosmid clone that contains the human EMMPRIN gene. S1 analysis with a fragment of the gene clone and primer extension of the mRNA was performed to determine the transcription start site. PCR and sequence analysis have defined the exon/intron organization of the gene and show that it is highly conserved with the mouse EMMPRIN/basigin gene. About 950 bases of the 5'-flanking region were examined for transcription factor consensus binding sites, locating three SP1 sites and two AP2 sites. The transcription start site was found to be located in a CpG island. Elements in the proximal promoter region were conserved in the human and mouse genes.

Development and roles of collagenous matrices in the embryonic avian cornea.

Corneal development requires the production, assembly and sometimes replacement of a number of collagenous matrices. The embryonic chick cornea is well-characterized and offers certain advantages for studying the assembly and roles of these matrices. We will first describe the matrices to be examined. These include the corneal stroma proper, first formed as the primary stroma and subsequently as the secondary (mature) stroma; Bowman's Membrane; Descemet's Membrane; and the hemidesmosome of the epithelial cell attachment complex. We will then describe the characteristics of the collagen types involved, including: the fibrillar collagens (types I, II and V), the fibril-associated collagens (types IX, XII and XIV), and the transmembrane collagen of the hemidesmosome (type XVII). Then, in each subsequent section we will examine in detail the structure, assembly and development of each collagenous matrix, and how each specific collagen and/or combination of collagens are thought to provide the matrices with their unique properties. The work and views presented here are largely from our own laboratories. Thus, this article is not meant to be a comprehensive review of the literature. For pertinent references by others, when possible, we will cite recent reviews.

Factors regulating neurogenesis and programmed cell death in mouse olfactory epithelium.

To identify factors regulating neurogenesis and programmed cell death in mouse olfactory epithelium (OE), and to determine the mechanisms by which these factors act, we have studied mouse OE using two major experimental paradigms: tissue culture of embryonic OE and cell types isolated from it; and ablation of the olfactory bulb ('bulbectomy') of adult mice, a procedure that induces programmed cell death of olfactory receptor neurons (ORNS) and a subsequent surge of neurogenesis in the OE in vivo. Such experiments have been used to characterize the cellular stages in the ORN lineage, leading to the realization that there are at least two distinct stages of proliferating neuronal progenitor cells interposed between the ORN and the stem cell that ultimately gives rise to it. The identification of a number of different factors that act to regulate proliferation and survival of ORNs and progenitor cells suggests that these multiple cell stages may each serve as a control point at which neuron number in the OE is regulated. Our recent studies of neuronal colony-forming progenitors (putative stem cells) of the OE suggest that even these cells, at the earliest stage in the ORN lineage so far identified, are subject to such regulation: if colony-forming progenitors are cultured in the presence of a large excess of differentiated ORNs, then the production of new neurons by progenitors is dramatically inhibited. This result suggests that differentiated ORNs produce a signal that feeds back to inhibit neurogenesis by their own progenitors, and provides a possible explanation for the observation that ORN death, consequent to bulbectomy, results in increased neurogenesis in the OE in vivo: death of ORNs may release neuronal progenitor cells from this inhibitory signal, produced by the differentiated ORNs that lie near them in the OE. Our current experiments are directed toward identifying the molecular basis of this inhibitory signal, and the cellular mechanism(s) by which it acts.

Stimulation of matrix metalloproteinase production by recombinant extracellular matrix metalloproteinase inducer from transfected Chinese hamster ovary cells.

Many of the tumor-associated matrix metalloproteinases that are implicated in metastasis are produced by stromal fibroblasts within or surrounding the tumor in response to stimulation by factors produced by tumor cells. In this study we transfected Chinese hamster ovary cells with putative cDNA for human extracellular matrix metalloproteinase inducer (EMMPRIN), a transmembrane glycoprotein that is attached to the surface of many types of malignant human tumor cells and that has previously been implicated in stimulation of matrix metalloproteinase production in fibroblasts. We show that these transfected cells synthesize EMMPRIN that is extensively post-translationally processed; this recombinant EMMPRIN stimulates human fibroblast production of interstitial collagenase, stromelysin-1, and gelatinase A (72-kDa type IV collagenase). We propose that EMMPRIN regulates matrix metalloproteinase production during tumor invasion and other processes involving tissue remodeling.

Type XVII collagen (BP 180) in the developing avian cornea.

PURPOSE: Previous sequence analyses of hemidesmosomal BP 180/collagen XVII cDNA from human skin and of a similar chicken corneal cDNA showed some similarities, but major differences as well. The authors examined whether, in one species, the same mRNA is present in cornea and skin. They also studied the developmental expression of the molecule and compared it to the transmembrane hemidesmosome component, alpha 6 beta 4 integrin, and to the formation of hemidesmosomes themselves. METHODS: Cornea and skin BP 180/collagen XVII cDNAs were cloned by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. Developmental expression was evaluated by quantitative RT-PCR, immunoblotting, and immunofluorescence microscopy. alpha 6 beta 4 integrin was evaluated by immunofluorescence microscopy, and hemidesmosome formation was assessed by electron microscopy. RESULTS: The same alpha 1 (XVII) collagen/BP 180 mRNA is present in cornea and skin. The appearance of alpha 1 (XVII) collagen mRNA and protein shows similar temporal patterns of expression, with changes in the mRNA preceding those of the protein by approximately 2 days. The appearance of mature hemidesmosomes lags still further. Immunofluorescence histochemistry of alpha 1 (XVII) collagen and alpha 6 beta 4 integrin shows that their developmental appearance is regulated closely. CONCLUSIONS: The differences between human BP 180/collagen XVII and the chicken corneal molecule represent species divergence. The appearance of alpha 1 (XVII) collagen mRNA and protein is regulated closely, with the protein lagging. Mature hemidesmosomes, once present, have a low turnover rate. The developmental appearance of alpha 1 (XVII) collagen and alpha 6 beta 4 integrin are regulated closely. However, the component responsible for initiating hemidesmosome formation remains unknown.

Positive regulation of corneal type V collagen mRNA: analysis by chicken-human heterokaryon formation.

Our previous studies have suggested that type V collagen is at least one factor responsible for the characteristically small, uniform diameter of striated collagen fibrils of the corneal stroma. These fibrils, which are heterotypic combinations of collagen types I and V, contain four- to fivefold more type V collagen than those of tendon and sclera. The latter are much larger and more heterodisperse. This high content of type V collagen in cornea is reflected by an equally elevated content of alpha1(V) chain mRNA in corneal fibroblasts. Thus, the increased production of the molecule in cornea appears to be regulated at the level of transcription and/or mRNA stability. One possible explanation for this is that corneal fibroblasts contain positive regulatory factors that specifically upregulate transcription of the type V collagen genes and/or increase their mRNA stability. To test this possibility, we have produced transient heterokaryons by fusing chicken corneal fibroblasts with two human noncorneal cell lines selected as containing little if any alpha1(V) mRNA. If the chicken corneal cells contain positive regulators that can act across species, these regulators should result in increased levels of the human alpha1(V) transcript. The results were evaluated by reverse transcript-polymerase chain reaction employing a primer pair selected for its ability specifically to amplify part of the human alpha1(V) mRNA. In fusions between chicken corneal fibroblasts and the human cell lines, after a lag of 10-14 h the heterokaryon-containing cultures showed de novo appearance or upregulation of human alpha1(V) chain mRNA, compared with that of the parental cell lines. Cultures of the mixed cell types that had not been fused showed no such upregulation, so the effect was not mediated by diffusible substances acting between the cells. Chicken tendon fibroblasts, a low producer of type V collagen, when tested in the same assay, evoked no detectible increase in the human transcript. Thus, corneal cells do contain positive regulators for alpha1(V) chain mRNA, and this effect is at least somewhat cell specific.

A self-expanding endoluminal graft for treatment of aneurysms: results through the development phase.

BACKGROUND: The results of two and a half years' experience of endoluminal treatment of aneurysmal disease (from March 1993 to December 1995) are reported. METHODS: The endoluminal grafts were individually made at Royal Perth Hospital. They are based on Dacron-covered stainless steel self-expanding 'Z' stents with Gianturco barbed stents (Cook Pty, Australia) for proximal anchorage for grafts within the aorta. RESULTS: Fourteen straight tube grafts (nine for aortic aneurysm, four for iliac aneurysm and one for subclavian aneurysm) and 24 bifurcate grafts were deployed; all were in patients considered high-risk for conventional repair. Seventy-two per cent of the straight tube grafts successfully excluded the aneurysm. The bifurcate grafts, in use since July 1994, successfully excluded the aneurysm in 88%. There were two delayed deaths from rupture after the grafts failed to exclude the aneurysms; two patients required conversion to open repair and survived; three patients have persistent endoleaks; and three of the bifurcate grafts subsequently occluded a graft limb but did not require further intervention. Ninety per cent of these complications occurred in the first half of the series (prior to January 1995). CONCLUSIONS: A learning and development curve was clearly apparent. The results thereafter compare favourably to those for open repair in similar high-risk groups, suggesting that these techniques hold promise for all patients with aneurysms.

Characterization and developmental regulation of avian corneal beta-1,4-galactosyltransferase mRNA.

Corneal development involves the synthesis and assembly of a number of specialized extracellular matrices. These matrices have distinctive properties derived from a unique assembly of collagens, proteoglycans and glycoproteins. The synthesis of each of these requires a number of enzymes. By probing a corneal cDNA library for genes that appeared to be up-regulated in cornea we have isolated a cDNA that represents an mRNA encoding the enzyme beta-1,4-galactosyltransferase. In cornea, a major function for this enzyme is likely to be in the synthesis of the keratan sulfate proteoglycan, lumican. Employing quantitative reverse transcript-polymerase chain reaction, we have observed that the steady-state level of mRNA for the molecule is elevated during certain stages of corneal development. It is also elevated in corneal fibroblasts in culture that have a greatly decreased synthesis of the mature lumican molecule. These data are consistent with, and complement, studies by others that show a corresponding regulation of the lumican core protein during development and in corneal fibroblast cultures.

Human keratinocytes express EMMPRIN, an extracellular matrix metalloproteinase inducer.

Increased levels of matrix metalloproteinases are associated with tissue degradation and remodeling during tumor invasion and wound healing. In both processes, there is evidence that cell interactions between fibroblasts and tumor cells or keratinocytes lead to increases in metalloproteinase production. We have previously isolated and purified a tumor cell surface protein, EMMPRIN (extracellular matrix metalloproteinase inducer), which stimulates production of interstitial collagenase, gelatinase A, and stromelysin-1 by fibroblasts, and we have obtained cDNA clones that encode the EMMPRIN protein from LX-1 human lung carcinoma cells. In this study we report immunolocalization of EMMPRIN around the surface of human keratinocytes in vitro and in vivo, and isolation of cDNAs that encode the entire open reading frame for EMMPRIN from a human keratinocyte library. Comparison of the EMMPRIN cDNAs from normal human keratinocytes and LX-1 human tumor cells by nucleotide sequence analysis, expression of the recombinant proteins, and in vitro translation using the cDNAs from the two sources indicate that they express very similar forms of EMMPRIN. Native EMMPRIN isolated directly from extracts of keratinocytes, however, is slightly smaller in size and is present at a lower concentration compared with that from LX-1 tumor cells. These results establish the presence of EMMPRIN in the normal epidermis and raise the possibility of its involvement in regulation of matrix remodeling at the epidermal-dermal interface.

Temporal expression of types XII and XIV collagen mRNA and protein during avian corneal development.

Using immunohistochemistry and competitive PCR for collagen types XII and XIV, we have followed the expression of these fibril-associated molecules during development of the avian cornea. By immunofluorescence histochemistry, both molecules are found in the acellular primary stroma and are therefore presumably of epithelial origin. During formation and development of the secondary corneal stroma, which is populated by mesenchymal cells, the molecules generally appear to be spatially segregated from each other. Type XIV collagen is found throughout most of the stroma, and therefore is predominantly a product of stromal fibroblasts. During subsequent compaction of the cornea, an event necessary for corneal transparency, the collagen XIV mRNA level increases dramatically, suggesting that this molecule may play a role in this event. Type XII collagen is more localized, occurring mainly in regions of the secondary stroma where matrices interface, such as where Bowman's membrane and Descemet's membrane abut the orthogonally layered collagen fibrils of the stromal matrix. These interfacial regions are highly stable areas of the cornea as determined previously by protease digestion and thermal denaturation studies. Type XII collagen may be involved in this stabilization.

Dynamics of MASH1 expression in vitro and in vivo suggest a non-stem cell site of MASH1 action in the olfactory receptor neuron lineage.

Disruption of the mouse gene encoding the transcription factor MASH1 leads to loss of certain classes of neurons, including receptor neurons of the olfactory epithelium (OE). Here we investigate the nature of the cell type expressing MASH1 in mouse OE by manipulating olfactory receptor neuron (ORN) neurogenesis in vitro and in vivo to alter the dynamics of neuronal production. The results indicate that MASH1 is expressed in cells of the ORN lineage, but not in ORNs themselves nor in their immediate precursors. Data on how changes in the numbers and proliferative states of MASH+ cells correlate with induced changes in overall neurogenesis strongly suggest that MASH1-expressing cells give rise to the immediate precursors of ORNs, but are not the self-renewing stem cells of the OE. The results imply that multiple progenitor stages are employed in generating ORNs and suggest that the action of MASH1 occurs predominantly at an intermediate stage.

The management of splenic rupture in infectious mononucleosis.

Non-operative management of splenic trauma is now well established; however, the role of conservative management in spontaneous splenic rupture is undetermined. The leading cause of spontaneous splenic rupture is infectious mononucleosis. We report on the management of four patients with spontaneous rupture, in association with infectious mononucleosis. Three patients eventually required splenectomy, and one was successfully managed non-operatively. The comparative risks of operative and non-operative management are discussed. We believe that when splenic rupture complicates infectious mononucleosis, early splenectomy is the most appropriate management.

Analysis of transcriptional isoforms of collagen types IX, II, and I in the developing avian cornea by competitive polymerase chain reaction.

The genes for the alpha 1(IX), alpha 1(II), and alpha 2(I) collagen chains can give rise to different isoforms of mRNA, generated by alternative promotor usage [for alpha 1(IX) and alpha 2(I)] or alternative splicing [for alpha 1(II)]. In this study, we employed competitive reverse transcriptase PCR to quantitate the amounts of transcriptional isoforms for these genes in the embryonic avian cornea from its inception (about 3 1/2 days of development) to 11 days. In order to compare values at different time points, the results were normalized to those obtained for the "housekeeping" enzyme, glycerol-3-phosphate dehydrogenase (G3PDH). These values were compared to those obtained from other tissues (anterior optic cup and cartilage) that synthesize different combinations of the collagen isoforms. We found that, in the cornea, transcripts from the upstream promotor of alpha 1(IX) collagen (termed "long IX") were predominant at stage 18-20 (about 3 1/2 days), but then fell rapidly, and remained at a low level. By 5 days (just before stromal swelling) the major mRNA isoform of alpha 1(IX) was from the downstream promoter (termed "short IX"). The relative amount of transcript for the short form of type IX collagen rose to a peak at about 6 days of development, and then declined. Throughout this period, the predominant transcriptional isoform of the collagen type II gene was IIA (i.e., containing the alternatively spliced exon 2). This indicates that the molecules of type II collagen that are assembled into heterotypic fibrils with type I collagen possess, at least transiently, an amino-terminal globular domain similar to that found in collagen types I, III, and V. For type I, the "bone/tendon" mRNA isoform of the alpha 2(I) collagen gene was predominant; transcripts from the downstream promotor were at basal levels. In other tissues expressing collagen types IX and II, long IX was expressed predominantly with the IIA form in the anterior optic cup at stage 22/23; in 14 1/2 day cartilage, long IX was expressed predominantly along with the IIB form of alpha 1(II). The downstream transcript of the alpha 2(I) gene (Icart) was found at high levels only in cartilage.