Vitamin B2-based blue-light photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting the circadian clock in mammals

In mammals the retina contains photoactive molecules responsible for both vision and circadian photoresponse systems. Opsins, which are located in rods and cones, are the pigments for vision but it is not known whether they play a role in circadian regulation. A subset of retinal ganglion cells with direct projections to the suprachiasmatic nucleus (SCN) are at the origin of the retinohypothalamic tract that transmits the light signal to the master circadian clock in the SCN. However, the ganglion cells are not known to contain rhodopsin or other opsins that may function as photoreceptors. We have found that the two blue-light photoreceptors, cryptochromes 1 and 2 (CRY1 and CRY2), recently discovered in mammals are specifically expressed in the ganglion cell and inner nuclear layers of the mouse retina. In addition, CRY1 is expressed at high level in the SCN and oscillates in this tissue in a circadian manner. These data, in conjunction with the established role of CRY2 in photoperiodism in plants, lead us to propose that mammals have a vitamin A-based photopigment (opsin) for vision and a vitamin B2-based pigment (cryptochrome) for entrainment of the circadian clock.     

pIRS1 and pTRS1 are present in human cytomegalovirus virions

The virus-coded proteins pIRS1 and pTRS1 were found associated with purified human cytomegalovirus virions. The proteins were not degraded when intact virions were treated with trypsin, which suggests that they are localized inside the viral particle. In transfection experiments pIRS1 and pTRS1 modestly activated expression from a reporter plasmid containing the viral major immediate-early promoter but did not influence the activity of a reporter carrying the irs1/trs1 immediate-early promoter. Both reporters were activated by the combination of pIRS1 or pTRS1 and pUL69, which is also present in virions.     

Putative environmental factors in Type 1 diabetes

Various environmental triggers, e.g. certain viruses and dietary factors, are thought to initiate the autoimmune process, leading to the destruction of pancreatic beta-cells and consequent Type 1 diabetes. A genetic predisposition is another prerequisite allowing the autoimmune process to progress. Twin studies, major geographical variations in incidence rates, temporal trends in the incidence and findings in migrant studies indicate that environmental factors play a crucial role in the development of Type 1 diabetes. In the present review the major focus is on dietary factors, and among them particularly the possible role of cow's milk proteins. The cow's milk and Type 1 diabetes hypothesis was developed more than 10 years ago, and the issue is still not settled. Among viral infections, enteroviruses are today the most interesting group of viruses in this respect, as recent prospective studies indicate that these viruses may trigger and potentiate existing beta-cell autoimmunity. Among toxins, particularly N-nitroso compounds are of potential interest, as they are probably involved in the aetiology of some cases. Finally, psychosocial factors and the interaction between genetic predisposition and environmental factors are briefly discussed.     

Putative nuclear cdk2 substrates in normal and transformed cells

The presence of putative substrates of cdk2 in a nuclear fraction obtained by DNase plus RNase extraction (S1 fraction) has been analyzed by immunoprecipitation using specific anti-cdk2 antibodies, followed by phosphorylation assays. S1 nuclear fractions from four different cellular types, two normal (rat hepatocytes and human T lymphocytes) and two transformed (HeLa and Namalwa cells), have been studied. Results indicate that the normal cells share three putative nuclear cdk2 substrates of 21, 37 and 57 kDa. On the other hand, only a substrate of 20 kDa is shared by the two transformed cell lines. On comparing the proliferating normal lymphocytes with the lymphoblastoid cell line Namalwa, it can be observed that they share two proteins of 40 and 70 kDa.     

The prostaglandin E2 and F2 alpha receptor genes are expressed in human myometrium and are down-regulated during pregnancy

Prostaglandin (PG) E2 and PGF2 alpha are believed to play important roles in the myometrial contraction and the initiation of labor. Myometrial contraction by these prostanoids is mediated mainly through EP3 and FP, which are specific receptors to PGE2 and PGF2 alpha, respectively. During normal pregnancy, uterine myometrium are relaxed until term. To explore the involvement of EP3 and FP in the myometrial relaxation during pregnancy, we examined the EP3 and FP gene expressions in nonpregnant and pregnant myometrium obtained by hysterectomy for gynecological diseases. In all samples examined, expressions of EP3 and FP genes were detected. During pregnancy, the expression of EP3 gene in human myometrium was significantly reduced, to 60% of that in nonpregnant myometrium. The expression of FP gene in human myometrium also decreased during pregnancy to 55% of that in nonpregnant myometrium. In the myometrium from the nonpregnant women taking combined oral contraceptives, the gene expressions of EP3 and FP were not significantly changed as compared to those in nonpregnant controls. The down-regulation of EP3 and FP during pregnancy may play a role in the relaxation of myometrium and thus in the maintenance of normal pregnancy in humans.     

BRCA1 and BRCA2 mRNA levels are coordinately elevated in human breast cancer cells in response to estrogen

The steady state levels of BRCA1 and BRCA2 mRNAs were shown to be coordinately elevated by the steroid hormone estrogen but not progesterone in the human breast cancer cell lines BT-483 and MCF-7. Two different antiestrogens, trans 4'-hydroxytamoxifen and ICI 182,780, blocked the elevation of BRCA1 and BRCA2 mRNA levels, confirming that the effect was mediated through the estrogen receptor. In BT-483 cells, BRCA1 and BRCA2 mRNA levels were both elevated 18 to 24 h after estrogen stimulation, suggesting that the effect of estrogen was indirect. Cycloheximide blocked the estrogen effect implying that estrogen induces synthesis of an unidentified estrogen-responsive protein(s) that then result in the elevation of BRCA1 and BRCA2 mRNAs.     

Laminin-1 and laminin-2 G-domain synthetic peptides bind syndecan-1 and are involved in acinar formation of a human submandibular gland cell line

The culture of human submandibular gland (HSG) cells on laminin-1 induces acinar differentiation. We identified a site on laminin involved in acinar differentiation using synthetic peptides derived from the C-terminal G-domain of the laminin alpha1 and alpha2 chains. The alpha1 chain peptide AG73 (RKRLQVQLSIRT) decreases the size of acini formed on laminin-1. Cells cultured with either AG73 or the homologous alpha2 chain peptide MG73 (KNRLTIELEVRT) form structures that appear acinar-like, but the cell nuclei are not polarized to the basal surface and no lumen formation occurs, indicating that additional sites on laminin are required for complete differentiation. The G-domain of laminin-1 contains both integrin and heparin binding sites, and anti-beta1-integrin antibodies disrupt acinar formation. Cell adhesion to the peptides and to E3, an elastase digest fragment of laminin-1 containing AG73, is specific, since other laminin peptides or EDTA do not compete the binding. Heparin and heparan sulfate decrease cell adhesion to AG73 and MG73 but anti-beta1-integrin antibodies have no effect. Treating the cell surface with heparitinase inhibits adhesion to both AG73 and MG73. We isolated cell surface ligands using both peptide affinity chromatography and laminin-1 affinity chromatography. Treating the material bound to the affinity columns with heparitinase and chondroitinase enriches for a core protein identified as syndecan-1 by Western blot analysis, thus identifying a syndecan-1 binding site in the globular domain of laminin-1 and laminin-2. In summary, multiple interactions between laminin and HSG cells contribute to acinar differentiation, involving both beta1-integrins and syndecan-1.     

Sialyl-Lewis x and Sialyl-Lewis a are associated with MUC1 in human endometrium

Endometrial epithelial cells express MUC1 with increased abundance in the secretory phase of the menstrual cycle, when embryo implantation occurs. MUC1 is associated with the apical surface of epithelial cells and is also secreted, being detectable in uterine fluid at elevated levels in the implantation phase. However, its physiological role is uncertain; it may either inhibit intercellular adhesion by steric hindrance or carry carbohydrate recognition structures capable of mediating cell-cell interaction. Here we show that endometrial epithelium expresses both Sialyl-Lewis x (SLex) and Sialyl-Lewis a (SLea), with a distribution and pattern of menstrual cycle regulation similar to that of MUC1. Using Western blotting and double determinant ELISA of uterine flushings, we demonstrate that SLex is associated with MUC1 core protein. The endometrial carcinoma cell lines HEC1A and HEC1B are shown to express MUC1 in a mosaic pattern, while three other cell lines express much lower amounts. HEC1A expresses both SLex and SLea while HEC1B expresses SLea only. Immunoprecipitation has been used to demonstrate that SLea is associated with MUC1 in HEC1B cells, and both SLex and SLea are associated with MUC1 in HEC1A cells.     

Rabbit cells expressing human CD4 and human CCR5 are highly permissive for human immunodeficiency virus type 1 infection

To evaluate the feasibility of using transgenic rabbits expressing CCR5 and CD4 as a small-animal model of human immunodeficiency virus type 1 (HIV) disease, we examined whether the expression of the human chemokine receptor (CCR5) and human CD4 would render a rabbit cell line (SIRC) permissive to HIV replication. Histologically, SIRC cells expressing CD4 and CCR5 formed multinucleated cells (syncytia) upon exposure to BaL, a macrophagetropic strain of HIV that uses CCR5 for cell entry. Intracellular viral capsid p24 staining showed abundant viral gene expression in BaL-infected SIRC cells expressing CD4 and CCR5. In contrast, neither SIRC cells expressing CD4 alone nor murine 3T3 cells expressing CCR5 and CD4 exhibited significant expression of p24. These stably transfected rabbit cells were also highly permissive for the production of virions upon infection by two other CCR5-dependent strains (JR-CSF and YU-2) but not by a CXCR4-dependent strain (NL4-3). The functional integrity of these virions was demonstrated by the successful infection of human peripheral blood mononuclear cells (PBMC) with viral stocks prepared from these transfected rabbit cells. Furthermore, primary rabbit PBMC were found to be permissive for production of infectious virions after circumventing the cellular entry step. These results suggest that a transgenic rabbit model for the study of HIV disease may be feasible.     

Intracellular Ca2+ and adaptation of voltage responses to light in Hermissenda photoreceptors

Fluorescent imaging of Ca2+ and intracellular recordings were used to assess Ca2+ increases and voltage responses during light presentations in Hermissenda B photoreceptors. Ca2+ levels increased and were sustained during a relatively long exposure to light. Repeated presentations of a brief light induced an elevation of intracellular Ca2+ that persisted throughout short interlight intervals, but which dissipated during long interlight intervals. In all instances, the magnitude of the intracellular Ca2+ signal was inversely related to the amplitude of the light-induced generator potential. Blocking of voltage-dependent Ca2+ channels did not significantly affect the magnitude of the Ca2+ signal, suggesting that the intracellular Ca2+ response arises primarily from release from intracellular stores. These results indicate that Ca2+ plays an important role in the modulation of the voltage responses to light, acting to suppress the response during repetitive or prolonged stimulation.     

The heterochronic Teopod1 and Teopod2 mutations of maize are expressed non-cell-autonomously

Teopod1 and Teopod2 are dominant, unlinked mutations in maize that cause dramatic morphological abnormalities, including inappropriate expression of juvenile traits in adult vegetative phytomers and the transformation of reproductive structures into vegetative ones. These phenotypes are consistent with the constitutive expression of a juvenile phase of development throughout shoot growth. To investigate the basis of the Tp1 and Tp2 phenotypes we have analyzed their cell-autonomy in mosaic Teopod:wild-type plants. Mosaic plants were generated by three different mechanisms. Tp1 has previously been shown to be non-cell-autonomous; to verify and extend these results, large wild-type sectors were generated on Tp1 plants by the spontaneous loss of a B-A translocation chromosome containing the Tp1 gene. Analysis of Tp2 cell-autonomy was complicated by a lack of useful markers on chromosome 10L proximal to Tp2. To circumvent this problem two strategies were used. A reciprocal translocation was used to link Tp2 the wild-type allele of lw2. Sectors were induced in plants of this type by irradiation of imbibed seeds. Also, a chromosome-breaking Ds element located proximal to Tp2 was used to generate somatic sectors that uncovered w2, an albino mutation distal to Tp2. Our results demonstrate conclusively that both Tp1 and Tp2 are non-cell-autonomous. The general use of these techniques for clonal analysis in plants and the potential role of a diffusible factor in regulating the juvenile phase of development in maize are discussed.     

Effects of removing extracellular Ca2+ on excitation and adaptation in Limulus ventral photoreceptors

In Limulus ventral photoreceptors, removing extracellular calcium (Ca2+o) increases the median latency of light-evoked discrete waves. Removal greatly lengthens the time-to-peak of responses in the dark-adapted cell, but not in the light-adapted cell. Removal does not block light-adaptation or the light-induced rise in intracellular calcium (Ca2+i). These results are interpreted in terms of the hypothesis that both sensitivity and the kinetics of excitation are dependent on Ca2+i, and that Ca2+i is dependent on Ca2+o in the dark-adapted cell, but in the light is dependent largely on Ca2+ released from intracellular compartments.     

Heterologous processing of prosomatostatin in constitutive and regulated secretory pathways. Putative role of the endoproteases furin, PC1, and PC2

Mammalian prosomatostatin (PSS) is cleaved at a dibasic Arg-Lys site to produce somatostatin-14 (SS-14) and at monobasic Arg and Lys sites to yield SS-28 and PSS(1-10) (antrin), respectively. Furin, PC1, and PC2 are three recently discovered mammalian endoproteases localized either to the constitutive (furin) or regulated (PC1, PC2) secretory pathways. In this study we have compared the heterologous processing of PSS in transiently transfected endocrine (AtT-20 pituitary) and nonendocrine (COS-7 monkey kidney, PC12 pheochromocytoma) tumor cells. We have correlated the efficiency of processing of PSS to SS-14, SS-28, and PSS(1-10) with (i) secretion through the constitutive or regulated pathways; (ii) endogenous expression of mRNA for furin, PC1, and PC2; and (iii) exogenous expression of PC1 and PC2 in cells that do not contain these enzymes in order to delineate the putative role of these enzymes in mediating PSS cleavage at dibasic and monobasic sites and to localize the proteolytic events to specific compartments of the secretory pathways. COS-7 and PC12 cells expressed only furin, secreted constitutively, and processed PSS preferentially at monobasic sites to SS-28 (40-43%) and antrin (27-29%). Processing, however, was inefficient as suggested by large amounts of unprocessed PSS. In contrast, AtT-20 cells showed regulated secretion, expressed all three endoproteases (with high levels of PC1), and processed PSS efficiently to mainly SS-14. PC1, but not PC2, exogenously coexpressed with PSS in COS-7 cells produced significant conversion to SS-14 but not SS-28. This study shows that PSS is capable of monobasic cleavage in the constitutive secretory pathway. Such processing could be mediated by a furin-like enzyme but is relatively inefficient. PC1 can effect dibasic cleavage of PSS whereas PC2 is without influence on PSS processing at least within the constitutive secretory pathway. Although monobasic and dibasic processing of PSS in COS-7 cells correlates with furin-like and PC1 activity, respectively, the relative inefficiency of such processing suggests that compartmentalization of proteolytic events in secretory vesicles or other more specific endoproteases may be required.     

Regulation of the TRP Ca2+ channel by INAD in Drosophila photoreceptors

Drosophila vision involves a G protein-coupled phospholipase C-mediated signaling pathway that leads to membrane depolarization through activation of Na+ and Ca2+ channels. InaD mutant flies have a M442K point mutation and display a slow recovery of the Ca2+ dependent current. We report that anti-INAD antibodies coimmunoprecipitate TRP, identified by its electrophoretic mobility, cross reactivity with anti-TRP antibody, and absence in a null allele trp mutant. This interaction is abolished by the InaD point mutation in vitro and in vivo. Interaction was localized to the 19 amino acid C-terminus of TRP by overlay assays, and to the PDZ domain of INAD, encompassing the point mutation. Given the impaired electrophysiology of the InaD mutant, this novel interaction suggests that INAD functions as a regulatory subunit of the TRP Ca2+ channel.     

Ca2+ induces an increase in cGMP-phosphodiesterase activity in squid retinal photoreceptors

In this paper we describe a rapid, isocratic high performance liquid chromatography (HPLC) method for the study of radioactive fatty acid incorporation into complex lipids of human erythrocytes, which allows the simultaneous separation of the major phospholipid classes and long-chain acylcarnitines. The lipid extract of erythrocytes pulsed with radioactive fatty acids was injected into an HPLC system equipped with a silica column. The individual components eluted were monitored by ultraviolet absorption and radioactive emission. With respect to the UV profile, the radioactive profile showed an additional peak between phosphatidyl-choline and phosphatidylethanolamine, which was identified as long-chain acylcarnitine by different experimental approaches. The radioactivity recovered in the long-chain acylcarnitines contains essential information enabling definition of acyl trafficking in red cells.     

Light-dependent changes in outer segment free-Ca2+ concentration in salamander cone photoreceptors

Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurrent from the inner segment, which was drawn into a suction pipette, a laser spot confocal technique was employed to evoke fluorescence from the outer segment of a cone loaded with the Ca2+ indicator fluo-3. When a dark-adapted cone was exposed to the intense illumination of the laser, the circulating current was completely suppressed and fluo-3 fluorescence rapidly declined. In the more numerous red-sensitive cones this light-induced decay in fluo-3 fluorescence was best fitted as the sum of two decaying exponentials with time constants of 43 +/- 2.4 and 640 +/- 55 ms (mean +/- SEM, n = 25) and unequal amplitudes: the faster component was 1.7-fold larger than the slower. In blue-sensitive cones, the decay in fluorescence was slower, with time constants of 140 +/- 30 and 1,400 +/- 300 ms, and nearly equal amplitudes. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allowed the calculation of the free-Ca2+ concentration, yielding values of 410 +/- 37 nM in the dark-adapted outer segment and 5.5 +/- 2.4 nM after saturating illumination (mean +/- SEM, n = 8). Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration. When the photopigment was regenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities. These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.     

Both T-helper-1- and T-helper-2-type lymphokines are depressed in posttrauma anergy

BACKGROUND: We have previously shown that an intrinsic postinjury T-cell dysfunction defined as lack of proliferative response to direct stimulation through the T-cell receptor, referred to here as "anergy," occurs in a subgroup of patients with severe trauma and is associated with organ failure. It has been suggested recently that a dominance of T-helper-2 (Th2) lymphokine production might be responsible for immunosuppression and associated with poor patient outcome. Here, we hypothesize that anergy is associated with global failure of T lymphokine (T LK) production, suggesting that poor outcome is not the result of an excess of immunosuppressive T LK (i.e., interleukin (IL)-10) but rather results from lost T-cell regulatory networking. METHODS: Purified T cells from 37 severely injured trauma patients were cultured and stimulated with alphaCD3/alphaCD4, and proliferation was assessed at 72 hours. Anergy is defined as occurring when the patient's T-cell proliferation to alphaCD3/alphaCD4 is less than 50% of the simultaneously run normal proliferation. Culture supernatants were assessed for T LK production by enzyme-linked immunosorbent assay. Clinical severity was measured by the multiple organ dysfunction syndrome (MODS) and Acute Physiology and Chronic Health Evaluation III scores. RESULTS: Anergy occurred in 20 of 37 patients, and it usually appeared at greater than 5 to 7 days after injury. There was a global reduction of T LK production during T-cell anergy (IL-2, 2.5%; interferon (IFN)gamma, 30.5%; IL-4, 11.8%; and IL-10, 16.9%) compared with increased or unchanged T LK production during the nonanergic state (IL-2, 83%; IFNgamma, 230%; IL-4, 110%; and IL-10, 307.9%; p < 0.01). There was a significant direct correlation between depressed IL-4 and depressed IFNgamma (r = 0.620, p < 0.001), indicating a diminished LK production of both types of T-helper cells (Th1 and Th2). Decreased IL-2 and IL-10 levels were also specifically correlated to each other during the anergic state (r = 0.91, p < 0.001). The average MODS score for patients during anergy was significantly higher (7.6) than their MODS score in the absence of anergy (4.0, p = 0.01). When IL-2 and IL-10 were measured simultaneously, a predominance of Th2 LK (IL-10) production would result in an IL-10/IL-2 ratio greater than 1. We found, however, that this ratio was not greater than 1 in 80% of assays in which T cells were anergic (p = 0.01). CONCLUSION: During T-cell anergy there is not a predominance of Th2 lymphokine production but rather a global depression of the T-cell lymphokine profile. Both depressed T-cell proliferation and depressed LK production correlate to poor clinical outcome.