Characterization of a polymorphism in the 3′ part of the chicken vitellogenin gene

Summary An allele giving rise to a polymorphism within the 3 part of the chicken vitellogenin gene was cloned, sequenced, and compared to the previously cloned allele. The polymorphism is formed by a perfect copy of 343 bp from intron 32 in tandem array with a perfect copy of 244 bp from intron 33; this 587-bp element is inserted in a head-to-tail arrangement in intron 33. We propose a mechanism in which an unequal crossing-over resulted in a vitellogenin gene with two exons 33, one of which was subsequently deleted. Thus, intron 33 was enlarged by the tandem repeats without affecting the protein-encoding sequence of the gene. At the boundaries of the repeated elements, two short direct repeats are found that resemble the recombination signals of immunoglobulin genes. They may have had a key role in the formation of the new allele.     

Immunochemical study of transforming growth factor-β in the kidney of the rat and chicken

Transforming growth factor- (TGF-) is a homodimeric polypeptide of 25 kDa, which regulates cell growth and differentiation and influences extracellular matrix metabolism. Using immunochemical techniques, we identified TGF- in the loops of Henle and the collecting and Bellini ducts of rat kidney and in the loops of Henle of chicken kidney. Furthermore, we detected two TGF--immunoreactive proteins on kidney blots of the rat of 12.5 and 47 kDa, and three on chicken kidney blots of 12.5, 34, and 47 kDa. We suggest that the precursor forms of rat and chicken TGF-2 or 3, chicken TGF-4, and the mature form of all of them are expressed in the collecting and Bellini ducts of rat kidney and the loops of-Henle of rat and chicken kidney.     

Integrin-mediated regulation of TGFβ in fibrosis

Fibrosis is a major cause of morbidity and mortality worldwide. Currently, therapeutic options for tissue fibrosis are severely limited, and organ transplantation is the only effective treatment for end-stage fibrotic disease. However, demand for donor organs greatly outstrips supply, and so effective anti-fibrotic treatments are urgently required. In recent years, the integrin family of cell adhesion receptors has gained prominence as key regulators of chronic inflammation and fibrosis. Fibrosis models in multiple organs have demonstrated that integrins have profound effects on the fibrotic process. There is now abundant in vivo data demonstrating critical regulatory roles for integrins expressed on different cell types during tissue fibrogenesis. In this review, we will examine the ways in which integrins regulate these processes and discuss how the manipulation of integrins using function blocking antibodies and small molecule inhibitors may have clinical utility in the treatment of patients with a broad range of fibrotic diseases. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

Stability of recombinant bovine interferon-γ antiviral activity in the absence of stabilizing additives

The stability of recombinant bovine interferon-γ (rbIFN-γ) produced by a baculovirus expression system was investigated under different storage conditions: freezing-thawing and storage for 30 days at temperatures of -80, 4, 25, and 37°C. Antiviral activity was not significantly decreased by freeze-thawing at least five times. Furthermore, although not statistically different, antiviral activity gradually decreased as temperature increased. These findings suggest that rbIFN-γ possesses high thermal and freeze-thaw stability.     

Negative regulation of TGF-β signaling in development

The TGF-β superfamily members have important roles in controlling patterning and tissue formation in both invertebrates and vertebrates. Two types of signal transducers, receptors and Smads, mediate the signaling to regulate expression of their target genes. Despite of the relatively simple signal transduction pathway, many modulators have been found to contribute to a tight regulation of this pathway in a variety of mechanisms. This article reviews the negative regulation of TGF-β signaling with focus on its roles in vertebrate development.     

Characterization of early stage intermediates in the nucleation phase of Aβ aggregation

Alzheimer's disease (AD) is a common form of dementia, which is characterized by the presence of extracellular amyloid plaques comprising the amyloid β peptide (Aβ). Although the mechanism underlying AD pathogenesis remains elusive, accumulating evidence suggests that the process of amyloid fibril formation is a surface-mediated event, which plays an important role in AD onset and progression. In this study, the mechanism of Aβ aggregation on hydrophobic surfaces was investigated with dual polarization interferometry (DPI), which provides real-time information on early stages of the aggregation process. Aggregation was monitored on a hydrophobic C18 surface and a polar silicon oxynitride surface. The DPI results showed a characteristic Aβ aggregation pattern involving a decrease in the density of Aβ at the surface followed by an increase in the thickness on the hydrophobic C18 chip. Most importantly, the DPI measurements provided unique information on the early stages of Aβ aggregation, which is characterized by the presence of initially slow nucleus formation process followed by exponential fibril elongation. The dimensions of the putative nucleus corresponded to a thickness of ～5 nm for both Aβ40 and Aβ42, which may represent about 10-15 molecules. The results thus support the nucleation-dependent polymerization model as indicated by the presence of a nucleation phase followed by an exponential growth phase. These results are the first reported measurements of the real-time changes in Aβ molecular structure during the early stages of amyloid formation at the nanometer level.     

Characterization of Negative Feedback Network Motifs in the TGF-β Signaling Pathway

{Chung, 2009 #1}The transforming growth factor-β (TGF-β) superfamily of cytokines plays a fundamental role in a wide variety of cellular processes, including growth, differentiation, apoptosis, and tissue homeostasis. Its relevance is emphasized by the mutations of its core components that are associated with diverse human diseases, such as cancer and cardiovascular pathologies. A prominent regulator of the pathway is Smad7, which attenuates the signal and controls its duration in a cell-type-dependent manner through a negative feedback loop. Here, we characterize all the potential Smad7-mediated negative feedback network motifs and investigate their effects on the signaling dynamics upon stimulation with TGF-β and bone morphogenetic protein (BMP) ligands. The results show that the specific negative feedback implementation is a key determinant of both the response of the system to single and multiple ligands of the TGF-β superfamily and its robustness and sensitivity to parameter perturbations.     

Biochemical evidence that regulation of Ero1β activity in human cells does not involve the isoform-specific cysteine 262

In the ER (endoplasmic reticulum) of human cells, disulfide bonds are predominantly generated by the two isoforms of Ero1 (ER oxidoreductin-1): Ero1α and Ero1β. The activity of Ero1α is tightly regulated through the formation of intramolecular disulfide bonds to help ensure balanced ER redox conditions. Ero1β is less tightly regulated, but the molecular details underlying control of activity are not as well characterized as for Ero1α. Ero1β contains an additional cysteine residue (Cys²⁶²), which has been suggested to engage in an isoform-specific regulatory disulfide bond with Cys¹⁰⁰. However, we show that the two regulatory disulfide bonds in Ero1α are likely conserved in Ero1β (Cys⁹⁰–Cys¹³⁰ and Cys⁹⁵–Cys¹⁰⁰). Molecular modelling of the Ero1β structure predicted that the side chain of Cys²⁶² is completely buried. Indeed, we found this cysteine to be reduced and partially protected from alkylation in the ER of living cells. Furthermore, mutation of Cys¹⁰⁰–but not of Cys²⁶²–rendered Ero1β hyperactive in cells, as did mutation of Cys¹³⁰. Ero1β hyperactivity induced the UPR (unfolded protein response) and resulted in oxidative perturbation of the ER redox state. We propose that features other than a distinct pattern of regulatory disulfide bonds determine the loose redox regulation of Ero1β relative to Ero1α.     

Strain-specific differences in the regulation of the hepatic cytoplasmic 17β-hydroxysteroid dehydrogenase activity of the rat

• 1.1. The effect of prepuberal gonadectomy of Sprague-Dawley/NIH/HAN rats on cytoplasmic 17β-hydroxysteroid dehydrogenase was examined on day 30, 45, 60, 75, 90 and 105 of life.
• 2.2. The activity in male rats was not significantly affected by gonadectomy, whereas the activity in females showed an age-dependent oestrogen dependency.
• 3.3. This age-dependent oestrogen dependency could also be demonstrated in 5α-dihydrotestosterone treated intact females.
• 4.4. Cytoplasmic 17β-hydroxysteroid dehydrogenase activity of female Chbb:THOM rats also showed an age-dependent oestrogen dependency, whereas the enzyme activity of male rats of this strain showed a distinct androgen dependency absent in Sprague-Dawley/NIH/HAN rats.
• 5.5. On the basis of previous investigations it is concluded that the androgen dependency of the enzyme activity of male Chbb:THOM rats has been bred into this strain in the period 1974–1977.

     

5′-Nucleotidase activity in the pineal organ of the pike

Summary To date, it is still unknown whether the metabolism of purine nucleotides and nucleosides plays an important role in the pineal organ of lower vertebrates. We have therefore investigated the sites of 5-nucleotidase activity in the pineal organ of the pike (Esox lucius L.). Various ultracytochemical procedures were used. An intense ecto-5-nucleotidase activity was characteristic of the entire plasma membrane of the phototransducers (cone-like and modified photoreceptor elements) and the interstitial cells, with exception of the portions facing the basal lamina of the pericapillary spaces. Additionally, intracellular sites of activity were also visualized in the inner segment and the pedicle of the phototransducers. Most of the intracellular deposits were apparently cytosolic and only few seemed to be associated with the membrane of the clear synaptic vesicles of the pedicle. Phagocytotic cells in the pineal lumen also showed a strong enzymatic activity on the outer surface of their plasmalemma (in ectoposition). This was apparently not the case for the cell types of the tissues surrounding the pineal vesicle. The present study emphasizes the importance of the occurrence and metabolism of purine nucleotides and nucleosides in a photoreceptive pineal organ.     

Does the nucleus raphes participate in the regulation of resting and stress-induced hypothalamic-pituitary-adrenocortical activity in the pigeon?

Extensive multiple electrolytic lesions were placed into the nucleus raphes of the brain stem in the pigeon. Diurnal pituitary-adrenocortical rhythmicity appeared not to be altered and basal plasma corticosterone level remained quite normal in raphe lesioned birds. Electrical stimulations through permanently implanted electrode were delivered in various central nervous structures in unanaesthetized, freely moving pigeons. Stimulations of nucleus raphes and of various parts of formatio reticularis led to a significant rise in plasma corticosterone within 16 to 19 min after the beginning of the stimulating session. Then, plasma B came again to initial level within 15 minutes. Stimulations of the corticotropic area of the hypothalamus (n. posterior medialis hypothalami) and of archistriatum dorsalis induced an early plasma corticosterone increase occurring immediately after the stimulating burst (10 min). Stimulating the n. septum medialis also had an immediate, but reverse (decrease) effect on plasma corticosterone level. Stress-induced pituitary-adrenal cortical activation exhibited a temporal pattern quite similar to that observed after brain stem (n. raphes or formatio reticularis) stimulation. It is suggested that these various limbic and brain stem areas might be involved in some "limbic system-midbrain circuit" with two components : The forebrain component might be involved in the regulation and diurnal modulation of basal hypothalamic-pituitary-adrenocortical function, the brain-stem component interferring with stress-induced responses.     

Is the Rsp5 ubiquitin ligase involved in the regulation of ribophagy?

Under nutrient limiting conditions, cytoplasmic components are randomly sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for degradation and recycling. In the last few years, however, it has been observed that several cytoplasmic components such as organelles, pathogens or specific protein complexes can also be selectively targeted for degradation by autophagy-related pathways (reviewed in reference 1). We have recently shown that in S. cerevisiae, mature ribosomes are subject to such selective degradation by autophagy under starvation conditions, in a process that we termed ‘ribophagy’.² By genetic screening, we found that selective degradation of 60S large ribosomal subunits depends on the ubiquitin protease Ubp3 and its cofactor Bre5, implying that ribophagy is regulated by ubiquitin-dependent steps. Interestingly, several ubiquitinated proteins accumulate in ribosome fractions isolated from ubp3? cells, suggesting that the regulation of ribophagy by ubiquitin may be direct. Here we present data on a potential role of the ubiquitin ligase Rsp5 as a positive regulator of ribophagy, and discuss the possible involvement of ubiquitin as a signaling molecule in this process.

Addendum to: Kraft C, Deplazes A, Sohrmann M, Peter M. Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease. Nat Cell Biol 2008; 10:602-10.     

Is the Rsp5 ubiquitin ligase involved in the regulation of ribophagy?

Under nutrient limiting conditions, cytoplasmic components are randomly sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for degradation and recycling. In the last few years, however, it has been observed that several cytoplasmic components such as organelles, pathogens or specific protein complexes can also be selectively targeted for degradation by autophagy-related pathways (reviewed in ref. 1). We have recently shown that in S. cerevisiae, mature ribosomes are subject to such selective degradation by autophagy under starvation conditions, in a process that we termed 'ribophagy.'(2) By genetic screening, we found that selective degradation of 60S large ribosomal subunits depends on the ubiquitin protease Ubp3 and its cofactor Bre5, implying that ribophagy is regulated by ubiquitin-dependent steps. Interestingly, several ubiquitinated proteins accumulate in ribosome fractions isolated from ubp3Delta cells, suggesting that the regulation of ribophagy by ubiquitin may be direct. Here we present data on a potential role of the ubiquitin ligase Rsp5 as a positive regulator of ribophagy, and discuss the possible involvement of ubiquitin as a signaling molecule in this process.     

Evidence of absence is not proof of absence: the case of the New Brighton katipō

ABSTRACT

The katipō is an endemic New Zealand spider that was previously common in the sand dunes at New Brighton. At sites on Banks Peninsula, katipō were detected under dried seaweed on the strandline 70% of the time. However, we detected no katipō among strandlines at New Brighton after 382 sampling visits. Incorporating these results into binomial and iterative Bayesian sampling models, it appeared highly unlikely that katipō still existed at New Brighton given so many non-detection events. However, when re-visiting the site, katipō were observed in the dunes at two locations, although they were still not found on the strandline. This specific habitat may be avoided at New Brighton due to high exposure to the prevalent strong easterly winds that occur at this site. The results emphasise that sampling models that use non-detection to indicate the likelihood of species absence can be highly specific to the sampling method used.