Copper Centers in the Cryo-EM Structure of Particulate Methane Monooxygenase Reveal the Catalytic Machinery of Methane Oxidation.

The particulate methane monooxygenase (pMMO) is the first enzyme in the C1 metabolic pathway in methanotrophic bacteria. As this enzyme converts methane into methanol efficiently near room temperature, it has become the paradigm for developing an understanding of this difficult C1 chemistry. pMMO is a membrane-bound protein with three subunits (PmoB, PmoA, and PmoC) and 12-14 coppers distributed among different sites. X-ray crystal structures that have revealed only three mononuclear coppers at three sites have neither disclosed the location of the active site nor the catalytic mechanism of the enzyme. Here we report a cyro-EM structure of holo-pMMO from Methylococcus capsulatus (Bath) at 2.5 Å, and develop quantitative electrostatic-potential profiling to scrutinize the nonprotein densities for signatures of the copper cofactors. Our results confirm a mononuclear CuI at the A site, resolve two CuIs at the B site, and uncover additional CuI clusters at the PmoA/PmoC interface within the membrane (D site) and in the water-exposed C-terminal subdomain of the PmoB (E clusters). These findings complete the minimal set of copper factors required for catalytic turnover of pMMO, offering a glimpse of the catalytic machinery for methane oxidation according to the chemical principles underlying the mechanism proposed earlier.

Plasma and saliva levels of nerve growth factor and neuropeptides in chronic migraine patients.

OBJECTIVES: To investigate the level and interrelationship of nerve growth factor (NGF) and sensory neuropeptides [substance P (SP), calcitonin gene-related peptide (CGRP)] in plasma and saliva of chronic migraine patients, and to analyze the association between pain intensity and their concentration. MATERIALS AND METHODS: Plasma and resting whole saliva were collected from 33 chronic migraine patients and 36 control subjects. NGF, SP, and CGRP concentrations were measured by enzyme immunoassay and pain intensity of each subject was measured using the Graded Chronic Pain Scale. RESULTS: Chronic migraine patients showed higher NGF and neuropeptide levels in both plasma and saliva compared to the control subjects. Plasma NGF, and plasma and saliva levels of SP and CGRP were highly associated with pain intensity. There was a significant positive correlation between NGF and both neuropeptide levels in plasma, and between the neuropeptide levels in both plasma and saliva. Plasma levels of SP and CGRP were significantly correlated with their saliva level. CONCLUSIONS: The increased production of NGF and sensory neuropeptides may play an important role in the maintenance of pain in chronic migraine and analysis results of human saliva could act as an index of disease state and therapeutic outcome.

Comparison of the composition of oral mucosal residual saliva with whole saliva.

OBJECTIVE: Compared with whole saliva, residual saliva comprising the oral mucosal film shows a high protein concentration. The purpose of this study was to compare the composition of residual saliva with unstimulated and stimulated whole saliva in normosalivators. MATERIALS AND METHODS: The composition of oral mucosal residual saliva in 30 healthy individuals was investigated and compared with that of whole saliva. The concentrations of total protein, secretory immunoglobin A (sIgA), lactoferrin, total carbohydrate, and sialic acid were examined. The activities of peroxidase, lysozyme and alpha-amylase were determined. RESULTS: Residual saliva had higher levels of total protein and carbohydrate than whole saliva, with a higher carbohydrate to protein ratio in the residual saliva suggesting that salivary glycoproteins are concentrated on the oral mucosal surface. sIgA, lactoferrin and sialic acid were present as highly concentrated forms in residual saliva. The enzymatic activity of peroxidase in residual saliva was higher than that of whole saliva. CONCLUSIONS: These concentrated carbohydrate and antimicrobials on the oral mucosal surface work for mucosal defence and could be used for targeting sites for the delivery of therapeutic agents.

Viscosity and wettability of animal mucin solutions and human saliva.

OBJECTIVE: The purpose of this study was to compare viscosity and wettability between animal mucin solutions and human saliva. MATERIALS AND METHODS: Human whole and glandular saliva, porcine gastric mucin, bovine submaxillary mucin, and a mucin-based saliva substitute were used. Viscosity was measured with a cone-and-plate digital viscometer, while wettability on acrylic resin and Co-Cr alloy was determined by the contact angle. RESULTS: The viscosity of animal mucin solutions was proportional to mucin concentration, with the animal mucin solution of concentration 5.0 mg ml(-1) displaying similar viscosity to stimulated whole saliva. A decrease in contact angle was found with increasing animal mucin concentration. For the saliva samples tested, viscosity increased in the following order: stimulated parotid saliva, stimulated whole saliva, unstimulated whole saliva, stimulated submandibular-sublingual saliva. Contact angles of human saliva on the tested solid phases were inversely correlated with viscosity. Contact angles of human saliva on acrylic resin were much lower than those of animal mucin solutions and of those on Co-Cr alloy (P < 0.01). CONCLUSIONS: The effectiveness of animal mucin solutions in terms of their rheological properties was objectively confirmed, indicating a vital role for mucin in proper oral function as well as the development of effective salivary substitutes.

Nerve growth factor concentration in human saliva.

OBJECTIVE: The aims of this study were to measure the normal concentration of nerve growth factor (NGF) in healthy human saliva and to investigate the effects of age and gender differences on saliva NGF level. MATERIALS AND METHODS: Resting whole, stimulated parotid, and stimulated submandibular/sublingual saliva were collected from 127 healthy volunteers with ages ranging from 20 to 81 years. The saliva NGF concentration was measured by enzyme immunoassay. RESULTS AND CONCLUSIONS: The mean concentrations of NGF were 901.4 +/- 75.6 pg ml(-1) in resting whole saliva, 885.9 +/- 79.9 pg ml(-1) in stimulated parotid saliva, and 1066.1 +/- 88.1 pg ml(-1) in stimulated submandibular/sublingual saliva. The stimulated submandibular saliva showed lower NGF concentrations with increasing age (rho = -0.296, P = 0.001). The NGF concentrations of resting whole saliva (P = 0.025) and stimulated parotid saliva (P = 0.005) were significantly higher in women than men. The NGF concentration of stimulated submandibular saliva was significantly higher than stimulated parotid saliva (P = 0.005) and significantly correlated with stimulated parotid saliva NGF level (rho = -0.244, P = 0.008). We found measurable concentrations of NGF in all three sources of saliva; the concentration was affected by the source for the stimulated parotid and submandibular saliva, age for stimulated submandibular saliva, and gender difference for resting whole saliva and stimulated parotid saliva.

Influence of topical capsaicin on facial sensitivity in response to experimental pain.

Capsaicin, the pungent component of the red pepper, has been used as an analgesic in a variety of pain conditions, but sensory impairment after long-term treatment has been concerned. This study investigated the influence of topical capsaicin on various types of sensations including pain in the facial areas innervated by the mental nerve, and also evaluated whether the measurement of cutaneous current perception threshold (CPT) is reliable for the quantification of sensory change following capsaicin application. Twenty healthy subjects were given topical capsaicin cream (0.075%), which was applied to the mental area unilaterally, four times daily for 2 weeks. Burning sensation after capsaicin application gradually decreased with repeated applications. Repeated topical capsaicin resulted in reduced sensation to mechanical, heat and cold pain without changing non-painful tactile sensation. It also resulted in increased CPTs at 5 Hz and 250 Hz stimuli but no change in the CPTs at 2000 Hz from the first evaluation after capsaicin treatment and throughout the treatment period. This study demonstrated that topical capsaicin treatment for the management of chronic localized pain can be safely applied to the face without affecting non-painful normal sensations, and that CPT testing is a clinically useful tool for the quantification of sensory changes following capsaicin application.

Regulation of osteoclast differentiation by the redox-dependent modulation of nuclear import of transcription factors.

This study sought to characterize the reduced glutathione (GSH)/oxidized GSSG ratio during osteoclast differentiation and determine whether changes in the intracellular redox status regulate its differentiation through a RANKL-dependent signaling pathway. A progressive decrease of the GSH/GSSG ratio was observed during osteoclast differentiation, and the phenomenon was dependent on a decrease in total glutathione via downregulation of expression of the gamma-glutamylcysteinyl synthetase modifier gene. Glutathione depletion by L-buthionine-(S,R)-sulfoximine (BSO) was found to inhibit osteoclastogenesis by blocking nuclear import of NF-kappaB and AP-1 in RANKL-propagated signaling and bone pit formation by increasing BSO concentrations in mature osteoclasts. Furthermore, intraperitoneal injection of BSO in mice resulted in an increase in bone density and a decrease of the number of osteoclasts in bone. Conversely, glutathione repletion with either N-acetylcysteine or GSH enhanced osteoclastogenesis. These findings indicate that redox status decreases during osteoclast differentiation and that this modification directly regulates RANKL-induced osteoclastogenesis.

Experimental salivary pellicles formed on the surface of self-curing resin.

The aim of this study was to identify the salivary components present in the pellicles formed on self-curing resin and to investigate the qualitative variations in adsorbed salivary pellicle compositions according to different exposure time to saliva. Experimental pellicles were formed by the incubation of polymerized resin particles with fresh human parotid or submandibular-sublingual saliva for either 20 min or 2 h. Pellicles were extracted using formic acid and lyophilized, they were then subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting to identify the adsorbed salivary components. The amino acid profiles of the 2 h-pellicles were analysed and compared with those of fresh glandular salivas. There was a difference in the 2 h-pellicle components on the self-curing resin compared with those of other dental materials as well as tooth enamel. The amino acid profiles of the 2 h-pellicles were also different from those of fresh glandular salivas. In the case of submandibular-sublingual saliva, the components of the 2 h-pellicle showed a different pattern compared with those of the 20 min-pellicle. However, there was no significant difference between the components of the 2 h- and 20 min-pellicles in the case of parotid saliva. A distinct difference was found in the surface binding affinities of immunoglobulin (IgA) from different glandular salivas. The findings of this study provide information concerning the initial bacterial adhesion on the surfaces of self-curing resin.

Facial and glossal distribution of anaesthesia after inferior alveolar nerve block.

The aim of this study was to subjectively determine the distribution of anaesthesia by mapping areas of sensory loss following inferior alveolar nerve block. Fifty healthy dental students were the subjects of this study (men 32, women 18). They were asked to draw the anaesthetized area on a diagram of the face and tongue 20 min after inferior alveolar nerve block. They evaluated the degree of anaesthesia by touching their faces and moving their tongues. All of the 50 subjects reported anaesthesia in the facial area. Of these, 21 (42%) reported the cutaneous distribution of anaesthesia on mental nerve territory only. Seventeen subjects (34%) reported anaesthesia on mental and buccal nerve territory. Nine subjects (18%) reported anaesthesia on mental, buccal, and auriculotemporal nerve territory. Two subjects (4%) reported anaesthesia on mental and auriculotemporal nerve territory and one subject (2%) on mental, buccal and infra-orbital nerve territory. Forty-seven of the 50 subjects (94%) reported anaesthesia of the tongue with the various degree of anaesthesia according to the area. Of these, 17 subjects (34%) reported strong anaesthesia on the anterior area and weak anaesthesia on the middle part of the tongue. Nineteen subjects (38%) reported strong anaesthesia of the lateral area and weak anaesthesia on the medial area, and 11 subjects (22%) reported anaesthesia on only the lateral side of the tongue. Three subjects (6%) reported no anaesthesia of the tongue. The distribution of anaesthesia of the facial and glossal regions determined subjectively after inferior alveolar nerve block, varies significantly between individuals.

Laparoscopic Repair of Perforated Peptic Ulcer.

The recent introduction of laparoscopy into the armamentarium of the general surgeon has revolutionized many aspects of surgical practice. The repair of perforated peptic ulceration is ideally suited to a laparoscopic approach. An accurate diagnosis is obtained, and closure of the perforation with thorough peritoneal toilet safely can be undertaken. The main advantages include the avoidance of a major incision and the reduction of postoperative pain. This may benefit the patient with associated respiratory disease in particular. Although mobilization may be improved, early discharge from the hospital is unlikely because the patient must recover from the peritonitis and associated ileus. The main drawback is a longer operating time caused by technical difficulty with laparoscopic suturing. Many innovative techniques have been described to simplify the procedure. The reported experience with laparoscopic repair of perforated peptic ulcers is encouraging, and randomized trials comparing it with open surgery are eagerly awaited.