Microstructured dental implants and palatal mucosal grafts in cleft patients: a retrospective analysis.

BACKGROUND: In cleft patients, implant dentistry has become an integral part of oral rehabilitation. However, a lack of keratinized mucosa is found in many cases which may have adverse effects on the long-term success of dental implants with microstructured surfaces. Therefore, the aim of this study was to evaluate whether mucogingival surgery is of value in the treatment of these patients. PATIENTS: Between 1991 and 2002, a total of 35 microstructured dental implants were inserted in 32 cleft patients. In 18 patients, vestibular scars extended to the rim of the marginal mucosa of the implants and the gingiva of the adjacent teeth. To enhance the soft tissue condition, mucogingival surgery was performed using palatal mucosal grafts. METHODS: In May 2002, 29 implants and 16 mucosal grafts were evaluated. Assessment included radiological and clinical parameters. RESULTS: Three implants were lost. Most mucosal grafts showed shrinkage of up to 30%. Clinical and radiological parameters, however, showed results that were very similar to those from non-cleft patients. CONCLUSION: These results support the hypothesis that keratinized mucosal grafts show long-term success in the cleft region as well. Moreover, it may be concluded that a combination of dental implants with a rough surface and palatal mucosal grafts can be recommended for oral rehabilitation of cleft patients.     

Production of nitric oxide and superoxide by activated macrophages and killing of Leishmania major

Murine macrophages can be activated to produce nitric oxide (NO) and superoxide and these two radicals can react to form peroxynitrite, a powerful oxidant which may be involved in parasite killing. We now show that murine macrophages activated with zymosan and interferon-γ (ZYM/IFN-γ) produced both superoxide (peaking 1–2 h after stimulation, then rapidly declining) and NO (barely detectable at 6 h, peaking by 24 h). Macrophages activated with ZYM alone produced only superoxide, while stimulation with lipopolysaccharide (LPS) and IFN-γ induced NO but not superoxide. Cells stimulated with ZYM/IFN-γ or LPS/IFN-γ killed Leishmania major to a similar degree, an effect that was completely blocked by the addition of N-iminoethyl-L -ornithine. However, macrophages stimulated with ZYM alone were unable to kill L. major. S-nitroso-acetyl-penicillamine, which release NO, was highly leishmanicidal when added directly to the parasites. 3-morpholino-sydnonimine hydrochloride which releases both NO and superoxide simultaneously, was also efficient at killing L. major and this cytotoxicity was greatly enhanced by the addition of superoxide dismutase. Finally, authentic peroxynitrite failed to induce any cytotoxic effect, even at a high concentration. Thus macrophages can produce either NO, superoxide or both, depending on the stimulus. However, the killing of L. major is dependent only on the production of NO.     

Migratory patterns and developmental potential of trunk neural crest cells in the axolotl embryo.

Using cell markers and grafting, we examined the timing of migration and developmental potential of trunk neural crest cells in axolotl. No obvious differences in pathway choice were noted for DiI-labeling at different lateral or medial positions of the trunk neural folds in neurulae, which contributed not only to neural crest but also to Rohon-Beard neurons. Labeling wild-type dorsal trunks at pre- and early-migratory stages revealed that individual neural crest cells migrate away from the neural tube along two main routes: first, dorsolaterally between the epidermis and somites and, later, ventromedially between the somites and neural tube/notochord. Dorsolaterally migrating crest primarily forms pigment cells, with those from anterior (but not mid or posterior) trunk neural folds also contributing glia and neurons to the lateral line. White mutants have impaired dorsolateral but normal ventromedial migration. At late migratory stages, most labeled cells move along the ventromedial pathway or into the dorsal fin. Contrasting with other anamniotes, axolotl has a minor neural crest contribution to the dorsal fin, most of which arises from the dermomyotome. Taken together, the results reveal stereotypic migration and differentiation of neural crest cells in axolotl that differ from other vertebrates in timing of entry onto the dorsolateral pathway and extent of contribution to some derivatives.     

Adenosine: A Sensitive Indicator of Cerebral Ischemia during Carotid Endarterectomy

Background: For the human brain, there are no data available concerning the significance of adenosine and its metabolites as biochemical indicators of cerebral ischemia. Since adenosine may counteract key pathogenetic mechanisms during cerebral ischemia, its sensitivity and specificity as a marker of cerebral ischemia was investigated in relation to hypoxanthine and lactate.

Methods: Arterial and jugular venous concentration changes of adenosine, hypoxanthine, and lactate were studied in 41 patients undergoing carotid endarterectomy. Cerebral tissue oxygenation was monitored continuously by somatosensory-evoked potentials. A carotid artery shunt (n = 6) was placed only after complete loss of somatosensory-evoked potentials.

Results: Before carotid artery clamping jugular venous concentrations of adenosine, hypoxanthine, and lactate in subsequently shunted patients were 229 +/- 88 nM, 1105 +/- 116 nM, and 0.85 +/- 0.52 mM, respectively (mean +/- SD). In patients who required shunting, carotid artery clamping induced a significant increase in jugular venous adenosine (389 +/- 114 nM) and jugular venous hypoxanthine (1444 +/- 168 nM). In contrast, the increase in jugular venous lactate (0.91 +/- 0.48 mM) did not reach statistical significance. Focal cerebral ischemia was indicated by jugular venous adenosine with a sensitivity and specificity of 0.83 and 0.71, respectively.     

Plastidial transporters KEA1, -2, and -3 are essential for chloroplast osmoregulation,integrity, and pH regulation in Arabidopsis

Multiple K⁺ transporters and channels and the corresponding mutants have been described and studied in the plasma membrane and organelle membranes of plant cells. However, knowledge about the molecular identity of chloroplast K⁺ transporters is limited. Potassium transport and a well-balanced K⁺ homeostasis were suggested to play important roles in chloroplast function. Because no loss-of-function mutants have been identified, the importance of K⁺ transporters for chloroplast function and photosynthesis remains to be determined. Here, we report single and higher-order loss-of-function mutants in members of the cation/proton antiporters-2 antiporter superfamily KEA1, KEA2, and KEA3. KEA1 and KEA2 proteins are targeted to the inner envelope membrane of chloroplasts, whereas KEA3 is targeted to the thylakoid membrane. Higher-order but not single mutants showed increasingly impaired photosynthesis along with pale green leaves and severely stunted growth. The pH component of the proton motive force across the thylakoid membrane was significantly decreased in the kea1kea2 mutants, but increased in the kea3 mutant, indicating an altered chloroplast pH homeostasis. Electron microscopy of kea1kea2 leaf cells revealed dramatically swollen chloroplasts with disrupted envelope membranes and reduced thylakoid membrane density. Unexpectedly, exogenous NaCl application reversed the observed phenotypes. Furthermore, the kea1kea2 background enables genetic analyses of the functional significance of other chloroplast transporters as exemplified here in kea1kea2Na⁺/H⁺ antiporter1 (nhd1) triple mutants. Taken together, the presented data demonstrate a fundamental role of inner envelope KEA1 and KEA2 and thylakoid KEA3 transporters in chloroplast osmoregulation, integrity, and ion and pH homeostasis.The regulation of ion and pH homeostasis is a vitally important feature of all living organisms. The existence of organelles in eukaryotic cells has added complexity to this circumstance. Proper function of chloroplasts in plant cells is not only crucial for the organism’s survival but affects all life forms as chloroplasts convert light into chemical energy and fix carbon from the atmosphere. With up to 10% of the dry weight, K⁺ is the most abundant cation found in plants; it fulfills numerous essential roles: for example, in osmoregulation, as a pH regulator, in motor cell movements, and in membrane polarization (1).Early studies on isolated chloroplasts suggested that K⁺ transport occurs in exchange for H⁺ (2, 3). Later studies on reconstituted envelope membranes supported the notion that K⁺ and H⁺ transport are functionally connected (4, 5). A K⁺ transport system across envelope membranes has been proposed to be crucial for chloroplast function because even small changes in the osmotic pressure or electrochemical potential led to a dramatic decrease in photosynthesis (6, 7). However, the molecular mechanisms that mediate K⁺ transport across chloroplast membranes and that regulate the osmotic pressure and pH and ion homeostasis of the chloroplast are poorly understood. Two possible mechanisms have been proposed for K⁺ transport across the inner envelope membrane: K⁺ channels (4, 5) and K⁺/H⁺ antiporters (7, 8). Furthermore, it remains unknown whether K⁺ transport across chloroplast membranes is rate-limiting for chloroplast function. These gaps in knowledge could be closed by genetic analyses to assess the proposed roles of K⁺ in chloroplast function and could also lead to a mechanistic understanding for transport models. In this study we sought to characterize K⁺ transporters of the chloroplast and to identify their biological functions in plants.A variety of different ion/H⁺ transporters, K⁺ channels, and H⁺ pumps are located in the plasma and endomembranes of plants. These transporters maintain organelle-specific ion contents and pH, which create gradients over membranes that not only energize secondary transport processes but also result in unique biochemical reaction compartments. The electro neutral cation/H⁺ antiporters in Arabidopsis thaliana build the superfamily of monovalent cation/proton antiporters (CPA) (44 predicted genes), which further subdivides into the CPA1 and CPA2 families (9, 10). CPA1 consists of the Na⁺(K⁺)(Li⁺)/H⁺ exchangers NHX1–8. Although NHX1–6 were identified in endomembranes (11–13), NHX7 and -8 localize to the plasma membrane (14, 15) and are more distantly related to the first six members, thus forming a subfamily (10, 16). SOS1/NHX7 has been studied in detail because loss-of-function of the gene leads to salt sensitivity (14).The second family CPA2 covers two larger subfamilies, which include Cation/H⁺ exchangers (CHX) and putative K⁺-efflux antiporters (KEA) (10). Twenty-eight different CHXs exist in A. thaliana, with members targeted to the plasma membrane, prevacuolar membrane, or the endoplasmic reticulum, where they exchange K⁺ against H⁺ (17–19). The significance of this transporter class was shown in chx20 loss-of-function mutants, in which endomembrane dynamics and osmoregulation needed for stomatal opening is affected (20). Therefore, K⁺/H⁺ antiporters represent major osmo- and pH-regulators for organelles (9). A CPA2 family member, CHX23, long thought to be a chloroplast K⁺/H⁺ antiporter, was recently found not to target to chloroplasts but to the endoplasmic reticulum (19, 21). In addition, CHX23 was found to be preferentially expressed in pollen (9), and the described phenotypes in A. thaliana CHX23 RNAi and chx23-1 tilling mutants (22) could not be confirmed in T-DNA mutants (19, 21); thus, the molecular nature and biological function of chloroplast K⁺ transporters remain unknown.Recently, strong evidence was presented that the KEA2 protein could fulfill the role of plastidial K⁺/H⁺ antiport. A half-sized C-terminal transmembrane domain containing an AtKEA2 fragment was shown to complement a yeast mutant deficient in the endosomal Na⁺(K⁺)/H⁺ exchanger NHX1p (23). In addition, in vitro measurements showed K⁺/H⁺ transport capacity for the half-sized protein fragment. A 100-aa N-terminal protein fragment of AtKEA2 suggested that the full-length AtKEA2 protein may be targeted to chloroplasts (23). However, no mutant phenotypes or chloroplast functions have yet been ascribed for KEA2 and investigation of the full-length gene was unsuccessful because of gene toxicity in Escherichia coli (23). Here, we have identified three members of the CPA2 family, KEA1, KEA2, and KEA3, as chloroplast K⁺/H⁺ antiporters that have critical function in the inner envelope (KEA1, KEA2) and in the thylakoid membrane (KEA3). Our findings reveal their essential role in plant chloroplast function, osmoregulation, and pH regulation.