A phylogenetically conserved APETALA2/ETHYLENE RESPONSE FACTOR, ERF12, regulates Arabidopsis floral development.

KEY MESSAGE: Arabidopsis ETHYLENE RESPONSE FACTOR12 (ERF12), the rice MULTIFLORET SPIKELET1 orthologue pleiotropically affects meristem identity, floral phyllotaxy and organ initiation and is conserved among angiosperms. Reproductive development necessitates the coordinated regulation of meristem identity and maturation and lateral organ initiation via positive and negative regulators and network integrators. We have identified ETHYLENE RESPONSE FACTOR12 (ERF12) as the Arabidopsis orthologue of MULTIFLORET SPIKELET1 (MFS1) in rice. Loss of ERF12 function pleiotropically affects reproductive development, including defective floral phyllotaxy and increased floral organ merosity, especially supernumerary sepals, at incomplete penetrance in the first-formed flowers. Wildtype floral organ number in early formed flowers is labile, demonstrating that floral meristem maturation involves the stabilisation of positional information for organogenesis, as well as appropriate identity. A subset of erf12 phenotypes partly defines a narrow developmental time window, suggesting that ERF12 functions heterochronically to fine-tune stochastic variation in wild type floral number and similar to MFS1, promotes meristem identity. ERF12 expression encircles incipient floral primordia in the inflorescence meristem periphery and is strong throughout the floral meristem and intersepal regions. ERF12 is a putative transcriptional repressor and genetically opposes the function of its relatives DORNRÖSCHEN, DORNRÖSCHEN-LIKE and PUCHI and converges with the APETALA2 pathway. Phylogenetic analysis suggests that ERF12 is conserved among all eudicots and appeared in angiosperm evolution concomitant with the generation of floral diversity.

DORNRÖSCHEN, DORNRÖSCHEN-LIKE, and PUCHI redundantly control floral meristem identity and organ initiation in Arabidopsis.

The biphasic floral transition in Arabidopsis thaliana involves many redundant intersecting regulatory networks. The related AP2 transcription factors DORNRÖSCHEN (DRN), DORNRÖSCHEN-LIKE (DRNL), and PUCHI individually execute well-characterized functions in diverse developmental contexts, including floral development. Here, we show that their combined loss of function leads to synergistic floral phenotypes, including reduced floral merosity in all whorls, which reflects redundant functions of all three genes in organ initiation rather than outgrowth. Additional loss of BLADE-ON-PETIOLE1 (BOP1) and BOP2 functions results in the complete conversion of floral meristems into secondary inflorescence shoots, demonstrating that all five genes define an essential regulatory network for establishing floral meristem identity, and we show that their functions converge to regulate LEAFY expression. Thus, despite their largely discrete spatiotemporal expression domains in the inflorescence meristem and early floral meristem, PUCHI, DRN, and DRNL interdependently contribute to cellular fate decisions. Auxin might represent one potential non-cell-autonomous mediator of their gene functions, because PUCHI, DRN, and DRNL all interact with auxin transport and biosynthesis pathways.

Arabidopsis floral phytomer development: auxin response relative to biphasic modes of organ initiation.

In the Arabidopsis inflorescence meristem (IM), auxin is considered a prepatterning signal for floral primordia, whereas a centripetal mode of positional information for floral organ identity is inherent to the ABCE model. However, spatio-temporal patterns of organ initiation in each whorl at the earliest initiation stages are largely unknown. Evidence suggests that initial flower development occurs along an abaxial/adaxial axis and conforms to phytomer theory. Use of the founder cell marker DORNRÖSCHEN-LIKE (DRNL) as a tool in leafy, puchi, and apetala 1 cauliflower mutant backgrounds suggests that bract founder cells are marked at the IM periphery. The DRNL transcription domain in the wild-type IM is spatially discrete from DR5 expression, suggesting that bract initiation is independent of canonical auxin response. When bracts develop in lfy and puchi mutant floral primordia the initiation of lateral sepals precedes the specification of medial sepals compared with wild type, showing an interplay between bract and abaxial sepal founder cell recruitment. In the perianthia (pan) mutant background, DRNL expression indicates that a radial outer whorl arrangement derives from splitting of sepal founder cell populations at abaxial and adaxial positions. This splitting of incipient sepal primordia is partially dependent on PRESSED FLOWER (PRS) activity and implies that sepal specification is independent of WUSCHEL and CLAVATA3 expression, as both marker genes only regain activity in stage-2 flowers, when patterning of inner floral organs switches to a centripetal mode. The transition from an initially abaxial/adaxial into a centripetal patterning programme, and its timing represent an adaptive trait that possibly contributes to variation in floral morphology, especially unidirectional organ initiation.

Live imaging of DORNRÖSCHEN and DORNRÖSCHEN-LIKE promoter activity reveals dynamic changes in cell identity at the microcallus surface of Arabidopsis embryonic suspensions.

KEY MESSAGE : Transgenic DRN::erGFP and DRNL::erGFP reporters access the window from explanting Arabidopsis embryos to callus formation and provide evidence for the acquisition of shoot meristem cell fates at the microcalli surface. The DORNRÖSCHEN (DRN) and DORNRÖSCHEN-LIKE (DRNL) genes encode AP2-type transcription factors, which are activated shortly after fertilisation in the zygotic Arabidopsis embryo. We have monitored established transgenic DRN::erGFP and DRNL::erGFP reporter lines using live imaging, for expression in embryonic suspension cultures and our data show that transgenic fluorophore markers are suitable to resolve dynamic changes of cellular identity at the surface of microcalli and enable fluorescence-activated cell sorting. Although DRN::erGFP and DRNL::erGFP are both activated in surface cells, their promoter activity marks different cell identities based on real-time PCR experiments and whole transcriptome microarray data. These transcriptome analyses provide no evidence for the maintenance of embryogenic identity under callus-inducing high-auxin tissue culture conditions but are compatible with the acquisition of shoot meristem cell fates at the surface of suspension calli.

Floral meristem initiation and emergence in plants.

Plant development and architecture is regulated by meristems that initiate lateral organs on their flanks. The gene regulatory networks that govern the transition of a vegetative shoot apical meristem into an inflorescence meristem (IM), together with those necessary to specify floral meristem (FM) identity have been elucidated in Arabidopsis thaliana and are highly complex and redundant. FMs are initiated in the axils of cryptic bracts and evidence suggests that FMs emerge and differentiate along an abaxial/adaxial axis, in contrast to existing models of centroradial positional information within FMs. Real-time imaging has revealed dynamic cell division and gene expression patterns associated with incipient primordia in the IM. This review, however, outlines how little is known concerning the identity of these primordia, the timing of FM specification and commitment in relation to the establishment of FM identity, and the interplay between bract and FM founder cell recruitment and development.

Rejection of murine heterotopic corneal transplants.

A murine heterotopic corneal transplant model has been developed using s.c. abdominal pouches as recipient sites. Donor-recipient genetic disparities involving H-2 antigens alone or H-2 plus non-H-2 antigens result in high rates of rejection. In addition, donor-recipient disparities involving non-H-2 antigens alone or H-Y antigen also result in significant, although lower, rates of rejection. In comparison, pretreatment of the donor grafts by hyperbaric oxygen, removal of the epithelial layer, or soaking in anti-Ia antibody plus complement results in statistically significant reductions in the rejection rates as compared with fresh, untreated donor tissue. These observations suggest that cells bearing Ia antigens (i.e., Langerhans cells) in the epithelial layer of donor corneas play a major role in host sensitization and subsequent rejection following corneal transplantation in this model. However, other antigens may play a role when Ia antigens are depleted from donor corneas.

Reduction in the incidence of rejection of heterotopic murine corneal transplants by pretreatment with ultraviolet radiation.

A reduction in the incidence of rejection of mouse heterotopic corneal allografts was achieved by in vitro pretreatment of the graft with ultraviolet light. By day 21, 68% of untreated BALB/c corneas transplanted into s.c. abdominal pouches of C57BL/6 recipients demonstrated rejection. In contrast, corneas pretreated with UV-A, -B, or -C at 150 mJ/cm2 showed an incidence of rejection at day 21 of 30%, 33%, and 18%, respectively. Experiments indicate a significant reduction in rejection with doses of UV-B radiation above 75 mJ/cm2. The mechanism of this reduction is unknown, but may be due to a depletion or alteration of ocular surface Langerhans cells residing in the corneal epithelium.

Lack of T6 induction on human corneal Langerhans cells in vitro.

Previous studies have shown that Langerhans cells (LC) in normal human corneas differ from their counterparts in other epithelia (eg, skin, gingival, cervical) by their lack of the thymocyte antigen T6 on their membranes. In those studies only three out of four very young infant corneas (newborn, 3-day-, 8-day-old) have displayed positive T6 staining to date. Corneas from older infants and adults have demonstrated no such staining. This study tested the capacity of corneal LC to express T6 by in vitro induction with several immunomodulating agents. Human gamma interferon (IFN gamma was tested at 1000 U/ml, 500 U/ml and 100 U/ml. Interleukin-1 (IL-1) was tested at 100 U/ml, 50 U/ml and 20 U/ml. Thymopoietin pentapeptide (TP-5) was tested at 10 micrograms/ml, 1 micrograms/ml and 0.1 microgram/ml. Combinations of these agents were also tested in a similar fashion. None of these immunomodulating agents or combinations of them were able to induce T6 expression on normal corneal LC. This may reflect an innate incapacity of these cells to express this antigen or dosage requirements in excess of those tested.

Proteoglycans of rabbit corneas with nonperforating wounds.

Rabbit corneal proteoglycans were labeled by intrastromal injection of 3H-glucosamine and 35S-sulfate 1 and 2 weeks after partial-thickness radial scalpel incisions. Proteoglycans were extracted with guanidine-HCl and purified by ion exchange chromatography. Wounding caused a marked decrease in the total incorporation of labeled precursors into proteoglycans. The labeled proteoglycans were more readily extracted with guanidine-HCl after wounding. Labeled proteoglycans from wounded corneas had a larger molecular size on gel filtration chromatography than did proteoglycans from control corneas, a result of an increased amount of keratan sulfate in the large molecular size fractions. Analysis of labeled glycosaminoglycan (GAG) from guanidine-extracted proteoglycans and from the corneal tissue after guanidine-HCl extraction showed an increase in the relative amount of heparan sulfate and keratan sulfate after wounding, and a decrease in relative amount of dermatan sulfate. The 35S:3H ratio of heparan and dermatan sulfates increased after wounding, and that of keratan sulfate decreased, suggesting changes in sulfation. Degradation of labeled dermatan sulfate with hyaluronidase and with periodate revealed a 2-fold increase in iduronic acid content and 2-4-fold increase in hyaluronidase-resistant dermatan sulfate in the wounded corneas. Reduction in proteoglycan content, reduced sulfation of keratan sulfate, and accumulation of a high-sulfate, high-iduronic acid dermatan sulfate are previously reported properties of proteoglycan in scar tissue from perforating corneal wounds. Demonstration of these properties in proteoglycan after wounds similar to radial keratotomy incisions suggests that deposition of scar tissue can result from wounds which do not damage Descemet's membrane.

Presence of Langerhans cells in the central corneas of normal human infants.

Nine normal adult and seven normal infant human corneas were studied for the presence of dendritic epithelial Langerhans cells in a masked fashion. Epithelial flatmounts were separated from the underlying corneal stroma using EDTA. The epithelial Langerhans cell densities were determined in the limbus as well as the peripheral, pericentral, and central corneal regions following staining with ATPase. Segments of the flatmounts were also studied by immunofluorescence to confirm that the dendritic cells contained class II histocompatibility antigens. The limbus, peripheral, and pericentral zones of adult and infant flatmounts contained similar densities of Langerhans cells. However, the central corneal Langerhans cell densities in infants were significantly elevated as compared with those in adults. These results suggest that Langerhans cells are a constant constituent of the human central corneal epithelium during late gestation and early infancy. They further suggest that perturbations of the corneal epithelium are not required for the presence of Langerhans cells in the corneal epithelium.

HLA-DR+/T6- Langerhans cells of the human cornea.

We have investigated normal human corneas for the presence of T6-marker on Langerhans cells. With the exception of one pair of newborn corneas and two pairs of very young infant corneas, all HLA-DR-positive cells in central and peripheral corneal epithelium were T6-negative by double-labeled immunofluorescence. In contrast, epidermal sheets from normal human eyelid skin displayed positive staining for T6 on most of the HLA-DR-positive Langerhans cells. Since T6 antigen is considered to be a specific Langerhans cell differentiation marker, we interpret this finding to indicate a nonactivated or undifferentiated state of Langerhans cells in normal human corneas.

Thymidine kinase activity of ocular herpes simplex isolates resistant to IUDR therapy.

Thymidine kinase (TK) activity was examined in extracts of TK-deficient cells infected with low passage isolates of herpes simplex virus type 1 (HSV-1) obtained from patients with herpetic keratitis. TK activity induced by the virus, relative phosphorylation rates of thymidine and iododeoxyuridine (IUDR), and Km values for thymidine and for IUDR were compared for TK induced by the different viral isolates. Four of the five isolates showing IUDR resistance in vitro and in vivo also exhibited alterations in properties of the viral TK. Two induced very low levels of viral TK. Three (including one with reduced TK) had increased Km values for IUDR. A fifth IUDR-resistant isolate induced TK similar to the IUDR-sensitive isolates. The results indicate that modification of viral gene for TK may be responsible for development of clinical resistance to IUDR in many occurrences of herpetic keratitis.

In vivo induction of Ia expression in murine cornea after intravitreal injection of interferon-gamma.

Intravitreal injection of interferon gamma (IFN-gamma) induces increased expression of Class II major histocompatibility complex (Ia) antigen expression on corneal endothelial cells and stromal fibroblasts. In contrast, IFN-gamma has no detectable effect on Ia antigen expression in epithelium. Induction of Ia antigen expression was rapid with increases detectable as early as 6-12 hours after a single injection of 1 x 10(5) units. Expression peaked at 24-48 hours and decreased to background levels by 120 hours. The Ia antigen expression increased in a dose-dependent manner, and IFN-gamma treatment also induced the synthesis of increased amounts of a 65-kilodalton (kD) protein in the cornea. Increased levels of this 65-kd protein are seen as early as 12 hours after treatment and can be induced with as little as 1 x 10(2) units of IFN-gamma. The function of the 65-kd protein is unknown. This model should be useful in studies on in vivo modulation of Ia antigen expression.

Prophylactic effects of silver nitrate and erythromycin on Chlamydia psittaci conjunctivitis.

An animal model has been developed to study the effects of various prophylaxis agents on acquisition of chlamydial conjunctivitis. When Hartley strain newborn guinea pigs received ocular inoculations of Chlamydia psittaci followed by the instillation of various agents, 1% AgNO3 significantly lowered the risk of developing chlamydial conjunctivitis if it was administered within 15 min after the inoculation with C. psittaci. However, if the AgNO3 was administered at either 1 hr or 2 hr following inoculation, it did not have any prophylactic effect on the development of chlamydial conjunctivitis. Erythromycin ointment, 0.5%, was also found to prevent chlamydial conjunctivitis. The prophylactic effect was similar to placebo when the drug was given at 15 min; however, erythromycin ointment prophylaxis 1 hr or 2 hr after inoculation with C. psittaci was statistically superior to placebo.

Analysis of viral DNA in isolates from patients with recurrent herpetic keratitis.

Isolates were obtained from 10 patients with recurrent herpes simplex type 1 infection of the eye, lids, or mouth. The viral DNA of successive isolates from each patients was analyzed by restriction endonuclease fingerprinting. Evaluation of the DNA banding patterns of all isolates by means of two different enzymes, performed in a masked fashion, demonstrated that all of the isolates from any one patient had the same genetic makeup. These results indicate that recurrent herpes infections of the eye and face in humans are caused not by unrelated, serial infections but rather by reactivation of the same latent virus that remains in the ganglion over a period of years.

Heparin and infarct coronary artery patency after streptokinase in acute myocardial infarction.

Anticoagulant therapy is frequently used after thrombolytic agents in the treatment of acute myocardial infarction (AMI) although it is unclear that such therapy will prevent subsequent infarct vessel reocclusion. The role of duration of heparin therapy in maintaining infarct artery patency was studied retrospectively in 53 consecutive AMI patients who received streptokinase therapy and underwent coronary angiography acutely and at 14 +/- 1 days. Of the 39 patients with initial infarct vessel patency, patency at follow-up angiography was observed in 100% (22 of 22) of those who received greater than or equal to 4 days of intravenous heparin but in only 59% (10 of 17) of those patients who received less than 4 days of heparin (p less than 0.05). Of the 14 patients not initially recanalized after streptokinase, patent infarct-related arteries at follow-up angiography were found in 3 of 8 (38%) treated with greater than or equal to 4 days of heparin therapy but in none of the 6 patients treated for less than 4 days (difference not significant). No significant difference in hemorrhagic complications was noted between the short- and long-term heparin treatment groups. Thus, greater than or equal to 4 days of intravenous heparin therapy after successful streptokinase therapy in AMI is more effective in maintaining short-term infarct vessel patency than a shorter duration of therapy and it may maintain the short-term patency of the infarct vessel in those patients who later spontaneously recanalize.

Photosynthesis and nutrient supply in needles of Sitka spruce [Picea sitchensis (Bong.) Carr.].

Growth and photosynthetic development were measured for currently developing needles of young trees of Sitka spruce [Picea sitchensis (Bong.) Carr.] throughout their fourth growth season. Treatments included trees that were fully fertilized (control), trees deficient in phosphorus (-P), or nitrogen (-N), and trees initially deficient but then supplied with phosphorus (-PR) or nitrogen (-NR). Growth was measured in terms of needle projected area, and the photosynthetic components measured were pigment concentration, net photosynthetic rate, (PN ), activity of ribulose-1,5-bisphosphate carboxylase, (RuBPC), stomatal conductance to CO2 , (Gs ), and the intercellular partial pressure of CO2 , (Ci ). Needle growth was rapid, beginning in early May and being complete by the end of the month or early in June. Free growth occurred in the -NR treatment. Photosynthesis increased throughout the season, reaching a peak in August, with some variables subsequently showing a decrease in value. PN increased more rapidly during needle expansion than either chlorophyll concentration or RuBPC activity. Phosphorus deficiency led to a reduction in RuBPC activity, which was restored to the control value following Refertilization with P. Nitrogen deficiency severely reduced values of all variables studied, except Ci , which was higher than for the controls. Refertilization of -N trees caused a very rapid increase in values of all variables, with an increase in Ci , representing a larger increase in mesophyll conductance to CO2 , (GM ), than GS , PN and RuBPC activity were significantly correlated with total chlorophyll concentration for all treatments, but PN was not correlated with GS or RuBPC activity.

Diphtheria corneal ulcers.

An adult with cutaneous diphtheria was admitted with bilateral purulent conjunctivitis and a perforated eye with most of the cornea absent due to Corynebacterium diphtheriae. At the time of admission of grayish patch of corneal epithelium was noted in the other eye, and in the next 24 hours there developed a large corneal perforation with dissolution of much of the cornea. Involvement of the external eye in diphtheria is rare but it is usually associated with cutaneous forms of the disease. Cutaneous diphtheria has been prominent in several recent outbreaks in the United States. Prompt recognition, early antibiotic treatment, and neutralization of the toxin with antitoxin are required for successful therapy.

Epithelial ingrowth.

A 74-year-old woman had a radical anterior segment resection for epithelial ingrowth following cataract extraction. Light microscopy demonstrated the presence of a sheet of cells morphologically similar to conjunctival epithelium covering the superior half of the inner surface of the cornea, iris, and ciliary processes. Electron microscopy revealed that frequent hemidesmosomes and a well-developed basal lamina were consistently present along the base of the ingrowing epithelium. The tissue immediately beneath the ingrowing epithelium, lining the cornea, trabecular meshwork and iris, morphologically resembled the subepithelial zone that has been described along the human skin epidermal-dermal junction. Additionally, focal areas of necrosis were noted in the trabecular meshwork. The major structural alterations induced on the surfaces of intraocular structures by the invading epithelium and the associated necrosis probably are responsible for the glaucoma which occurs with epithelial ingrowth.

Therapeutic uses of contact lenses.

Therapeutic use of contact lenses is an essential element in ophthalmic care. Materials currently in use include polymethyl methacrylate (PMMA), cellulose acetate butyrate, siloxane-containing polymethacrylates, silicones, and hydrogels. Suitability of a material for therapeutic contact lens use is determined by the physical, chemical, and mechanical properties (notably gas permeability and hydrophilicity, but also lipid absorption and lens movement, among others) and the condition to be treated; fabrication techniques are likewise important, affecting lens diameter and base curve. Selection and fitting of therapeutic contact lenses requires knowledge of how different contact lenses affect corneal physiology, as well as an understanding of the mechanisms whereby a contact lens can be therapeutic. In addition to these topics, general fitting guidelines are discussed, and results of therapeutic lens use in selected clinical situations (including recurrent erosion, metaherpetic ulcers and other epithelial defects, and keratitis sicca, and other dry eye states). Common therapeutic contact lens complications and their treatment are also discussed.