Ageing of the human corneal stroma: structural and biochemical changes.

High and low angle X-ray diffraction patterns from the corneal stroma give information about the mean intermolecular spacing of the collagen molecules and the mean interfibrillar spacing of the collagen fibrils, respectively. X-ray data were collected, using a high intensity synchrotron source, from human corneas and sclera at approximately physiological hydration. The spacings were measured as a function of tissue age. Between birth and 90 years there is an increase in the cross-sectional area associated with each molecule in corneal collagen from approx. 3.04 nm2 to 3.46 nm2, and an increase in scleral collagen from approx. 2.65 nm2 to 3.19 nm2. These changes may be due to an increase in the extent of non-enzymic cross-linking between collagen molecules over the age range. We have investigated this possibility by measuring collagen glycation using the thiobarbituric acid assay and the subsequent advanced glycation end-products (AGEs) using fluorescence emission. The results obtained have shown an age-related increase in glycation and AGEs in both tissues. We have also demonstrated a decrease in the interfibrillar spacing of corneal collagen with increasing age which may be related to changes in the proteoglycan composition of the interfibrillar matrix.     

OCCURRENCE AND LOCALIZATION OF BRAIN PHENOLSULPHOTRANSFERASE

—Rat brain contains the enzyme which forms sulphate conjugates of phenols, phenolsulphotransferase (EC 2.8.2.1), but the physiological role of the enzyme is unclear. The enzyme is unevenly distributed in rat brain, with the activity 13 times higher in the hypothalamus than in the cerebellum. Phenolsulphotransferase does not seem to be primarily located in glial cells. Cultured cells (type C6 astrocytoma) derived from rat glia had less than 1 per cent of the phenolsulphotransferase activity of whole rat brain. Sulphate conjugation of neutral compounds may be important in their removal from brain. The pineal and pituitary glands, areas outside the blood-brain barrier had very low phenolsulphotransferase activity. The activity of the enzyme in brain varied widely among different species: rabbit and rat had much higher levels of activity than mouse or frog; the activity in human brain was intermediate. Phenolsulphotransferase also occurred in other organs, including liver, heart, testes, lung, spleen, salivary glands, and intact or decentralized superior cervical ganglion. There was no correlation of enzyme activity with adrenergic or cholinergic innervation, or with the known roles of various tissues in drug metabolism or detoxification. The enzyme activity does not seem to be under neuronal control since ganglionectomy did not affect the phenolsulphotransferase activity of salivary glands. The precise localization of phenolsulphotransferase remains to be established, as well as the physiological importance of sulphate conjugation of phenols in brain and other organs.     

Quantitative Assay of Paravaccinia Virus Based on Enumeration of Inclusion-Containing Cells

Bovine paravaccinia virus produces cytoplasmic inclusion bodies on infection of bovine embryonic kidney cells; these were easily recognized when stained with acridine orange or May-Grünwald-Giemsa stain. The inclusions could be shown to contain newly synthesized deoxyribonucleic acid by autoradiography. Counts of inclusion-containing cells decreased when virus suspensions were treated with immune serum before being used to inoculate cell cultures. At 24 hr after infection, the number of cells containing inclusions was directly proportional to the concentration of infectious virus inoculated. These observations provide the basis for a virus assay which is simpler, faster, and more sensitive than the plaque assay.     

Decreased synthesis of nucleolar RNA in aging human cells in vitro

Normal human fibroblasts aged during serial passage in cell culture are known to display a reduction in total RNA synthetic activity. We have used “ghost monolayers”, which have been stripped of cytoplasm, in order to determine by autoradiographic techniques whether this decreased synthetic activity in aged cells could be localized to particular structures within the nuclei. Results indicated that the age-related decline in RNA synthesis was entirely due to decreased synthesis of nucleolar RNA, while nucleoplasmic synthesis was unchanged.     

HDM2 negatively affects the Chk2-mediated phosphorylation of p53

By GST pull downs and co-immunoprecipitation analyses we found that recombinant Chk2 and HDM2 can form stable complexes in vitro. Chk2/HDM2 complexes were also detected in transfected Cos-1 cells over-expressing both proteins. Furthermore, we show that HDM2, as would be expected, severely affects the Chk2-catalyzed phosphorylation of p53. HDM2 itself is only slightly phosphorylated by Chk2. However, whereas HDM2 inhibits the Chk2-catalyzed p53 phosphorylation, HDM2 phosphorylation by Chk2 doubles in the presence of p53. The significance of the HDM2 phosphorylation is unknown, but it is possible that it might influence the stability of the HDM2/p53 complex.     

Phage-peptide display identifies the interferon-responsive, death-activated protein kinase family as a novel modifier of MDM2 and p21WAF1

Phage-peptide display is a versatile tool for identifying novel protein-protein interfaces. Our previous work highlighted the selection of phage-peptides that bind to specific isoforms of MDM2 protein and in this work we subjected the putative MDM2-binding proteins to phage-peptide display to expand further on putative protein interaction maps. One peptide that bound MDM2 had significant homology to members of the death-activated protein kinase (DAPK) family, an enzyme family of no known direct link to the p53 pathway. We examined whether a nuclear member of the DAPK family named DAPK3 or ZIP kinase had direct links to the p53 pathway. ZIP kinase was cloned, purified, and the enzyme was able to phosphorylate MDM2 at Ser166, a site previously reported to be modified by Akt kinase, thus demonstrating that ZIP kinase is a bona fide MDM2-binding protein. Native ZIP kinase fractions were then subjected to phage-peptide display and one ZIP kinase consensus peptide motif was identified in p21(WAF1). ZIP kinase phosphorylates p21(WAF1) at Thr145 and alanine-substituted mutations in the p21(WAF1) phosphorylation site alter its ability to be phosphorylated by ZIP kinase. Thus, although ZIP kinase consensus sites were then defined as containing a minimal RKKx(T/S) consensus motif, alternate contacts in ZIP kinase binding are implicated, since amino acid residues surrounding the phospho-acceptor site can effect the specific activity of the kinase. Transfected ZIPK can promote the phosphorylation of p21(WAF1) at Thr145 in vivo and can increase the half-life of p21(WAF1), while the half-life of p21(WAF1[T145A]) is not effected by ZIP kinase. Thus, phage-peptide display identified an interferon-responsive protein kinase family as a novel modifier of two components of the p53 pathway, MDM2 and p21(WAF1), and underscores the utility of phage-peptide display for gaining novel insights into biochemical pathways.     

Functional variation among frugivorous birds: implications for rainforest seed dispersal in a fragmented subtropical landscape

Seed dispersal plays a critical role in rainforest regeneration patterns, hence loss of avian seed dispersers in fragmented landscapes may disrupt forest regeneration dynamics. To predict whether or not a plant will be dispersed in fragmented forests, it is necessary to have information about frugivorous bird distribution and dietary composition. However, specific dietary information for frugivorous birds is often limited. In such cases, information on the seed-crushing behaviour, gape width and relative dietary dominance by fruit may be used to describe functional groups of bird species with respect to their potential to disperse similar seeds. We used this information to assess differences in the seed dispersal potential of frugivorous bird assemblages in a fragmented rainforest landscape of southeast Queensland, Australia. The relative abundance of frugivorous birds was surveyed in extensive, remnant and regrowth rainforest sites (16 replicates of each). Large-gaped birds with mixed diets and medium-gaped birds with fruit-dominated diets were usually less abundant in remnants and regrowth than in continuous forest. Small-gaped birds with mixed diets and birds with fruit as a minor dietary component were most abundant in regrowth. We recorded a similar number of seed-crushing birds and large-gaped birds with fruit-dominated diets across site types. Bird species that may have the greatest potential to disperse a large volume and wide variety of plants, including large-seeded plants, tended to be less abundant outside of extensive forests, although one species, the figbird Sphecotheres viridis, was much more abundant in these areas. The results suggest that the dispersal of certain plant taxa would be limited in this fragmented landscape, although the potential for the dispersal of large-seeded plants may remain, despite the loss of several large-gaped disperser species.     

Selective inhibition of the DNA-dependent protein kinase (DNA-PK) by the radiosensitizing agent caffeine

Caffeine inhibits cell cycle checkpoints, sensitizes cells to ionizing radiation-induced cell killing and inhibits the protein kinase activity of two cell cycle checkpoint regulators, Ataxia-Telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). In contrast, caffeine has been reported to have little effect on the protein kinase activity of the DNA-dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks. Previously, we reported that DNA-PK phosphorylates Thr²¹ of the 32 kDa subunit of replication protein A (RPA32) in response to camptothecin. In this report we demonstrate that the camptothecin-induced phosphorylation of RPA32 on Thr²¹ is inhibited by 2 mM caffeine. In addition, we show that caffeine inhibits immunoprecipitated and purified DNA-PK, as well as DNA-PK in cell extracts, with an IC₅₀ of 0.2–0.6 mM. Caffeine inhibited DNA-PK activity through a mixed non-competitive mechanism with respect to ATP. In contrast, 10-fold higher concentrations of caffeine were required to inhibit DNA-PK autophosphorylation in vitro and caffeine failed to inhibit DNA-PKcs dependent double-strand break repair in vivo. These data suggest that while DNA-PK does not appear to be the target of caffeine-induced radiosensitization, caffeine cannot be used to differentiate between ATM, ATR and DNA- PK-dependent substrate phosphorylation in vivo.     

A recent insertion of an alu element on the Y chromosome is a useful marker for human population studies

A member of the Alu family of repeated DNA elements has been identified on the long arm of the human Y chromosome, Yq11. This element, referred to as the Y Alu polymorphic (YAP) element, is present at a specific site on the Y chromosome in some humans and is absent in others. Phylogenetic comparisons with other Alu sequences reveal that the YAP element is a member of the polymorphic subfamily-3 (PSF-3), a previously undefined subfamily of Alu elements. The evolutionary relationships of PSF-3 to other Alu subfamilies support the hypothesis that recently inserted elements result from multiple source genes. The frequency of the YAP element is described in 340 individuals from 14 populations, and the data are combined with those from other populations. There is both significant heterogeneity among populations and a clear pattern in the frequencies of the insertion: sub-Saharan Africans have the highest frequencies, followed by northern Africans, Europeans, Oceanians, and Asians. An interesting exception is the relatively high frequency of the YAP element in Japanese. The greatest genetic distance is observed between the African and non-African populations. The YAP is especially useful for studying human population history from the perspective of male lineages.