Structural mechanism for lipid activation of the Rac-specific GAP, beta2-chimaerin

The lipid second messenger diacylglycerol acts by binding to the C1 domains of target proteins, which translocate to cell membranes and are allosterically activated. Here we report the crystal structure at 3.2 A resolution of one such protein, beta2-chimaerin, a GTPase-activating protein for the small GTPase Rac, in its inactive conformation. The structure shows that in the inactive state, the N terminus of beta2-chimaerin protrudes into the active site of the RacGAP domain, sterically blocking Rac binding. The diacylglycerol and phospholipid membrane binding site on the C1 domain is buried by contacts with the four different regions of beta2-chimaerin: the N terminus, SH2 domain, RacGAP domain, and the linker between the SH2 and C1 domains. Phospholipid binding to the C1 domain triggers the cooperative dissociation of these interactions, allowing the N terminus to move out of the active site and thereby activating the enzyme.     

Novel identification of the different types of cones in the retina of the chicken

1. Although single cones and double cones in the chicken retina had been documented for more than 30 years, the exact morphology of these cells had never been studied by the scanning electronmicroscopy. In this brief report, we present the evidence for the first time the existence of two types of single cones and three types of double cones (termed as types A, B, and C), each with distinct morphology.2. The proportion of type A:type B:type C double cones, as estimated from the midperipheral and central regions of our scanning specimens, was about 30%:50%:20%.3. Based on the literature data that red oil droplets reside in single cones and yellow oil droplets in chief cones of the double cones, the proportion of single to double cones was deciphered and the relative proportion was estimated to be 1:0.91 in the central region, 1:0.92 in the midperiphery, and 1:0.84 in the periphery.     

C-fos antisense oligodeoxynucleotide decreases subcutaneous bee venom injection-induced nociceptive behavior and fos expression in the rat

Oligodeoxynucleotide complementary to c-fos mRNA was applied to characterize its effect on the spinal cord Fos expression and relevant nociceptive behaviors challenged by subcutaneous injection of bee venom to the rat hind paw. Nociceptive behavioral responses (spontaneous pain and hyperalgesia) following bee venom (0.2 mg/50 microl) injection were assessed in adult male Sprague-Dawley rats receiving intrathecal administration of c-fos antisense oligodeoxynucleotide (ASO, 50 microg/10 microl), sense oligodeoxynucleotide (SO, 50 microg/10 microl) and saline (10 microl) 4 h prior to bee venom injection. The lumbar spinal cord expression of Fos protein 2 h after bee venom injection in the ASO-, SO- and saline-treated animals was observed by immunohistochemistry. The results showed that pretreatment of c-fos ASO markedly reduced the flinching response and primary thermal hyperalgesia, but without significant effects on mechanical hyperalgesia and secondary thermal hyperalgesia. At the same time, ASO treatment also significantly decreased the expression of Fos protein within the lumbar region of the spinal cord ipsilateral to the injection. The results provide further evidence that Fos protein contributes to the activation of the spinal dorsal horn neurons and the generation and/or maintenance of spontaneous pain and primary thermal hyperalgesia induced by subcutaneous injection of bee venom.     

Proliferation and apoptosis in the epithelium of the developing human cornea and conjunctiva.

To determine the distribution of proliferating and apoptotic cells in the human cornea during prenatal and early postnatal development, we examined sections of the bulbar conjunctiva, the limbus as well as the central and peripheral cornea between 11 weeks of gestation and 6 months after birth. The objective was to localize dividing cells by proliferating cell nuclear antigen-like immunoreactivity (PCNA-LI) and apoptotic cells by terminal transferase-mediated nick-end labeling (TUNEL). Before the 17th gestational week, PCNA-LI was absent in all 4 regions examined, indicating negligible cell proliferation during early development. After 20 weeks, strong PCNA-labeling was observed in all regions examined suggestive of high proliferative activity not only in the limbus and the bulbar conjunctiva, but also in the central and peripheral cornea. This rise in proliferative activity was followed by a steady decline: after 28 weeks, anti-PCNA staining gradually disappeared in the central and peripheral cornea, so that, at 6 months after birth, it was confined to the limbus and the bulbar conjunctiva, resembling the picture described for the adult cornea. TUNEL-positive cells were virtually absent in all 4 regions examined before the 38th gestational week. Apoptotic cells only started to appear at 38 weeks; at this stage, they were confined to the bulbar conjunctival epithelium. At 6 months after birth, TUNEL-positive cells were observed in the bulbar conjunctival epithelium and the entire cornea; the limbus, however remained devoid of apoptotic cells throughout the entire prenatal and early postnatal period. The present study for the first time localizes proliferating and apoptotic cells in the epithelium of the developing human cornea. Three stages of development can be distinguished: Minimal proliferation (until 17th week), vigorous proliferation over the entire cornea including the limbus and the bulbar conjunctiva (until 28th week) and gradual decrease in proliferative activity (after 28th week) accompanied by the appearance of apoptotic cells.     

Specific glycanforms of type IX collagen accumulate in embryonic chick sterna after 17 days of development

Type IX collagen is a key component of the extracellular matrix of cartilage where it occurs at the surfaces of type II collagen fibrils as a glycanated molecule. The function of the glycosaminoglycan (GAG) side chain of the molecule is, however, unknown. We have shown that type IX collagen in chicken sternal cartilage is synthesized with a unimodal distribution of GAG chain size, but at post 17 days of development three predominant glycanforms of type IX collagen accumulate. Such accumulation did not occur in sterna from day 15 embryos. In day 17 embryos predominant glycanforms were found in the caudal region of the sternum. By day 19 of development the three predominant glycanforms are widespread throughout the caudal and cephalic regions. The results indicate that developmental and anatomical changes occur to type IX collagen that depend on the size of the GAG chain attached to the alpha2(IX) chain of the molecule.      

Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro.

Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.     

Ultraviolet-induced DNA excision repair in human B and T lymphocytes. II. Effect of inhibitors and DNA precursors.

Despite their great sensitivity to ultraviolet light purified human B and T lymphocytes are capable of complete repair provided that the ultraviolet dose does not exceed 0.5 Jm-2. Their capacity to repair, as measured by the restoration of DNA supercoiling in preparations of nucleoids, and their survival are significantly increased in the presence of deoxyribonucleosides. Certain agents which inhibit semi-conservative DNA synthesis (hydroxyurea, 1-beta-D-arabinofuranosylcytosine (arafCyt) either stop or delay the repair process in lymphocytes. The effect of hydroxyurea is eventually overcome spontaneously, but changes in the sedimentation behaviour of ultraviolet-irradiated nucleoids caused by arafCyt can only be neutralized by addition of deoxycytidine. The effective inhibition of repair by arafCyt permits the detection of extremely small amounts of ultraviolet damage and also the estimation of when repair is complete.     

Human B and T lymphocytes differ in UV-induced repair capacity.

Unstimulated human T lymphocytes are more readily killed by ultraviolet light (UV) than are B lymphocytes. The greater UV sensitivity of T cells can be explained by a less efficient process of excision repair; this was measured by following the restitution of DNA supercoiling in preparations of nucleoids obtained from purified and irradiated B and T lymphocytes after various periods of incubation. Differences in the sedimentation behaviour of irradiated B and T nucleoids in sucrose gradients are not attributable to differences in the degree of DNA supercoiling. The return to normal supercoiling for both B and T nucleoids is inhibited by hydroxyurea.     

Postnatal changes of vascular endothelial growth factor (VEGF) expression in the retinae of normal and hypertensive rats

Vascular endothelial growth factor (VEGF) has been shown to have potent mitotic activity specific to vascular endothelial cells and has been related to vascular permeability, angiogenesis and cell proliferation in both normal and pathological situations. The present study aimed at elucidating the spatio-temporal changes in the postnatal expression pattern of VEGF in the retinae of both normal and hypertensive rats. In situ hybridization with a riboprobe showed that in the pre-hypertensive stage (2 weeks postnatal, prior to the increase of the blood pressure of the hypertensive rat), VEGF expressed strongly in the retinal pigment epithelium (RPE) and inner nuclear layer (INL) but weakly in the ganglion cell layer and nerve fiber layer in both the normal and hypertensive rats. During the early hypertensive stage (6 weeks postnatal, initial increase of the blood pressure of the hypertensive rat), similar expression pattern was maintained but the INL of the hypertensive rat was found to have more positive cells in clusters than that of the normal rat. When a sustained high blood pressure was developed (12 weeks postnatal, sustained hypertensive stage) in the hypertensive rat, the VEGF expression was much reduced in all layers of the retina although weak expression was still observed in the RPE of the normal rat and RPE and INL of the hypertensive rat. Western blot analysis however showed that VEGF protein expression in the retina was much stronger in the hypertensive rat than in the normal rat at 2 and 6 weeks postnatal. At 12 weeks, the VEGF protein returned to a level comparable to that found in the normal rat. It is speculated that the change of the VEGF protein expression pattern during the early phase of the development of hypertension may be related to the subsequent changes in the retinal vasculature of the hypertensive rat.     

Changes in leucine uptake in the retina of the hamster after traumatic detachment

Protein metabolism was investigated in detached hamster retinas. By sucking off 0.2 ml of aqueous humor from the anterior chamber through limbic insertion of a 27-gauge needle, a tractional force pulled off the neural retina from the retinal pigment epithelium and created a simple detachment without retinal breaks in the right eyes of the hamsters. The left eyes were left untouched as normal controls and sham controls were induced by simple limbic insertion without suction. The animals were sacrificed at selected intervals of 1, 3, 6, 9, 16, 24, 32 days after the operation. Subsequently, scintillation counting and autoradiography were employed to study retinal protein metabolism using leucine uptake as an index. After tritiated leucine uptake, scintillation counting of radioactive substance indicated that detached retinas had taken in less tritiated leucine than normal controls from day 1 to 6 after the operation, but this change had normalized by day 9. For autoradiography, the change in leucine uptake rate was shown to be different in different layers. All the retinal cells seemed to show a decreased leucine uptake with the exception of the outer nuclear layer, in which leucine appeared to be significantly upregulated. This paper illustrates the patterns of protein metabolism and their change after traumatic detachment as well as their possible recovery.