Comment on "An early Miocene extinction in pelagic sharks".

Sibert and Rubin (Reports, 4 June 2021, p. 1105) claim to have identified a previously unidentified, major extinction event of open-ocean sharks in the early Miocene. We argue that their interpretations are based on an experimental design that does not account for a considerable rise in the sedimentation rate coinciding with the proposed event, nor for intraspecific variation in denticle morphology.

Protease-activated receptor 2 mediates interleukin-8 and intercellular adhesion molecule-1 expression in response to Aggregatibacter actinomycetemcomitans.

INTRODUCTION: We investigated the mechanisms by which extracts of Aggregatibacter actinomycetemcomitans affect the inflammatory response in gingival epithelial cells. METHODS: Human gingival cells (Ca9-22) were cultured in bacterial extracts prepared from A. actinomycetemcomitans ATCC 29522. The cells were pretreated with protease inhibitors or transfected with small interfering RNA (siRNA) specific for protease-activated receptor 2 (PAR-2). RESULTS: The pretreatment of cells with serine protease inhibitors significantly inhibited A. actinomycetemcomitans extract-induced expression of interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1) at both the messenger RNA and protein levels. In addition, A. actinomycetemcomitans extract-induced IL-8 and ICAM-1 expression was significantly decreased in PAR-2/siRNA-transfected cells. CONCLUSIONS: A. actinomycetemcomitans extract-induced IL-8 and ICAM-1 expression in gingival epithelial cells is mediated by PAR-2.

Biomechanical testing of the fractured distal radius treated with a new bone cement--is it strong enough?

This study evaluates the in vitro ability of CAP, a bone graft substitute with osteointegration and osteoconductive properties, to restore the anatomy and strength of fractured distal radii. Ten pairs of cadaveric radii were imaged and tested to failure, simulating a fracture. The radii were reconstructed using CAP and were re-fractured and sequentially imaged. The deformities of the bones were determined through computerised evaluation of the radiographs. Radiographic analysis showed that CAP is capable of restoring the anatomy of the distal radius. The load and work required to fracture intact radii were compared to those required to fracture the reconstructed radii. The load to fracture was similar in the two situations. The work to fracture, however, was higher with reconstructed radii, suggesting that these are stronger than the original bones. Our study supports the hypothesis that CAP is capable of restoring the original anatomy and dimensions of the distal radius and re-establishing its mechanical strength.

Chemical modification by trinitrobenzenesulfonate of a lipid and proteins of intracytoplasmic membranes isolated from Chromatium vinosum and Azotobacter vinelandii.

1. The structure of intracytoplasmic membranes of a photosynthetic bacterium chromatium vinosum and a nitrogen-fixing bacterium Azotobacter vinelandii was studied by chemical modification of amino groups of phosphatidylethanolamine and proteins with trinitrobenzenesulfonate. 2. Almost all the constituents of intracytoplasmic membranes of C. vinosum were solubilized in a mixture of chloroform, methanol and trichloroacetic acid. One-third of proteins in the intracytoplasmic membranes of C. vinosum was found solubilized in a mixture of chloroform and methanol. By using a column chromatography with Sephadex LH-20 in organic solvents, the unmodified as well as the trinitrophenylated proteins and also the trinitrophenylated phosphatidylethanolamine were separated from the other colored substances. 3. In the chemical modification of the intracytoplasmic membrane preparations, 30% of phosphatidylethanolamine and 15% of protein amino groups in C. vinosum and 45% of phosphatidylethanolamine and 20% of protein amino groups in A. vinelandii were estimated to be exposed to the aqueous phase. In the single-layered liposomes composed of phosphatidylethanolamine and phosphatidylglycerol with a ratio of 2:1, 40% of phosphatidylethanolamine were estimated to be exposed to the aqueous phase.

Effect of sterical stabilization on macrophage uptake in vitro and on thickness of the fixed aqueous layer of liposomes made from alkylphosphocholines.

A serious problem using liposomes for therapeutic purposes is the fast removal from blood circulation by components of the reticuloendothelial system (RES) most likely after opsonization of the vesicles. This study was performed to quantify the reduction in macrophage uptake in vitro of sterically stabilized liposomes (PEG-liposomes) prepared from hexadecylphosphocholine, cholesterol and poly(ethylene glycol2000) distearoylphosphoethanolamine (PEG2000DSPE) for the first time. The uptake was determined using HPC-liposomes of different defined size (125, 250 and 1000 nm) without and with sterical stabilization by incorporating 5 mol% of PEG2000DSPE. HPTS was used as fluorescence marker allowing the discrimination between general uptake and the part of liposomes internalized into the low pH-compartment (Daleke, L.D., Hong, K. and Papahadjopoulos. D. (1990) Biochim. Biophys. Acta 1024, 352-366). Liposomal uptake by J774 mouse macrophage-like cells was time-dependent. Both the uptake and internalization were clearly reduced for PEG-liposomes compared to plain liposomes. Sterical stabilization reduced the general uptake of liposomes in vitro by more than 50% and the internalization by about 50-60%. PEG-liposomes additionally showed a delay in internalization into the macrophages during the first 6 h. Size of used liposomes had only a minor influence on liposomal uptake but highest concentration of lipid was found for large multilammelar vesicles (MLV). The fixed aqueous layer thickness (FALT) was determined by zeta potential measurements of plain and sterically stabilised HPC-liposomes (100 nm) in solutions of different ion concentrations. The calculation of the thickness was based on the linear correlation between ln zeta (zeta-potential) and kappa (Debye Hückel-Parameter). FALT was calculated and found to be for plain HPC-liposomes 0.83 +/- 0.17 nm and for PEG-HPC-liposomes 3.57 +/- 0.17 nm. Exchange of the HPC by an alkylphospholipid with different head group has no or only minor effect (PEG-OPP-liposomes 3.44 +/- 0.31 nm). Thus the reduced uptake of HPC-LUVET correlates with an increased thickness of the fixed aqueous layer around these liposomes and could support the hypothesis that the thickness is an important property responsible for preventing opsonization and resulting finally in a reduced macrophage uptake.

Association of bacteriochlorophyll a with the CsmA protein in chlorosomes of the photosynthetic green filamentous bacterium Chloroflexus aurantiacus.

The protein assumed to be associated with bacteriochlorophyll (BChl) a in chlorosomes from the photosynthetic green filamentous bacterium Chloroflexus aurantiacus was investigated by alkaline treatment, proteolytic digestion and a new treatment using 1-hexanol, sodium cholate and Triton X-100. Upon alkaline treatment, only the 5.7 kDa CsmA protein was removed from the chlorosomes among six proteins detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, concomitantly with the disappearance of BChl a absorption at 795 nm. Trypsin treatment removed two proteins with molecular masses of 11 and 18 kDa (CsmN and CmsM), whereas the spectral properties of BChl a and BChl c were not changed. By the new hexanol-detergent (HD) treatment, most BChl c and all of the detected proteins except CsmA were removed from the chlorosomes without changing the BChl a spectral properties. Subsequent proteinase K treatment of these HD-treated chlorosomes caused digestion of CsmA and a simultaneous decrease of the BChl a absorption band. Based on these results, we suggest that CsmA is associated with BChl a in the chlorosomes. This suggestion was supported by the measured stoichiometric ratio of BChl a to CsmA in isolated chlorosomes, which was estimated to be between 1.2 and 2.7 by amino acid analysis of the SDS-PAGE-resolved protein bands.

Biodistribution of liposomes containing synthetic galactose-terminated diacylglyceryl-poly(ethyleneglycol)s.

We describe the synthesis of biodegradable poly(ethyleneglycol)-coupled galactolipids in which the galactose moiety is separated from a diacylglyceride lipid anchor by poly(ethylene glycol) chains of 10, 20 or 40 oxyethylene residues (PEG10/20/40). These Gal-PEG lipids (Gal-PEG-Lip) were incorporated in the bilayer of liposomes. The surface exposure of the galactose was investigated by aggregation experiments with ricinus communis agglutinin 120. Only the liposomes containing the PEG10 galactolipid aggregated with the lectin. Therefore liposomes were prepared containing Gal-PEG10-Lip and a trace amount of [3H]cholesteryl oleyl ether with an average diameter of approximately 100 nm and injected intravenously into rats. The Gal-PEG10-Lip liposomes were cleared from plasma with a T1/2 of 0.3 h. Identically sized and composed control liposomes without the Gal-PEG10-Lip had a T1/2 of approximately 12 h. The rapid plasma elimination of the Gal-PEG10-Lip liposomes could be attributed entirely to increased uptake by the liver amounting to more than 90% of injected dose. Uptake by the spleen was decreased to less than 1% of injected dose. A single injection of N-acetylgalactosamine 1 min prior to Gal-PEG-Lip liposome administration reduced the initial rate of plasma clearance to control levels. The increased liver uptake was almost entirely attributable to increased uptake by the Kupffer cells. Incorporation of PEG-DSPE in the Gal-PEG10-Lip liposomes only partially reversed the effect of the galactolipid with respect to liver and spleen uptake as well as intrahepatic distribution. These experiments demonstrate that liposome surface-exposed galactose residues, even if attached at the distal end of a poly(ethyleneglycol) chain anchored in the liposomal bilayer are effectively recognized by the galactose particle receptor on the Kupffer cells but fail to achieve significant targeting to the asialoglycoprotein receptor on the hepatocytes.

Transfection of wild-type TP53 induces differentiation in human gingival carcinoma cells.

We investigated the effects of transfection of wild-type TP53 on the growth properties of a human gingival carcinoma cell line, KOSC-3, in which the TP53 gene is mutated at codon 248 and overexpressed. The wild-type TP53 expression plasmid, pCDM8-p53/neo and the control plasmid, pCDM8/neo, were each stably transfected into KOSC-3 cells by using the calcium phosphate method. The number of G418-resistant colonies from wild-type TP53-transfected cells was approximately half that from plasmid controls. Exogenous wild-type TP53 transcripts were identified in four of the 20 G418-resistant clones analysed by reverse transcription PCR. Although the growth rates of the wild-type TP53+ clones did not drastically change during log phase, their saturation density was significantly reduced. The wild-type TP53+ cells were morphologically flat and enlarged when cultured in vitro, and were less able to form colonies in soft agar. In nude mice, the wild-type TP53+ clones formed subcutaneous tumours with conspicuous keratinisation and notable cell death that was not manifested in the parental and plasmid control cells. These findings indicate that the wild-type TP53 gene, even when it coexists with a mutated form, may function as a growth suppressor and differentiation inducer under restricted conditions in gingival squamous cell carcinoma.

Iodine-impregnated drapes enable recording of precordial electrocardiogram.

Although we believed that the use of a polyester film drape for primary elective open sternum with delayed sternal closure made it impossible to record precordial electrocardiograms on the drape, we confirmed that it is possible to record electrocardiograms on an iodine-impregnated drape due to the iodine anion electrical conductibility.

Differential controls of small and large motor unit activity in the masseter muscle with incisal stimulation in humans.

The activity of masseter motor units with different spike amplitudes was recorded and reflex responses by force stimulation of an incisor were analyzed in humans. The effects of reflex responses varied depending on the level of prestimulus firing frequency (background activity, BGA). When small amplitude motor units and large amplitude motor units were tested at the same level of BGA, the former tended to exhibit excitatory reflexes and the latter inhibitory ones.

A comparison of the bactericidal effects and cytotoxic activity of three types of oxidizing water, prepared by electrolysis, as chemical dental plaque control agents.

Acid oxidizing water (AOW), neutral oxidizing water (NtOW) and acid oxidizing water with a low available chlorine concentration (AOW-LC) may be obtained by electrolyzing a solution of tap water containing various quantities of NaCl and HCl. This study compared the bactericidal effects of these waters on cariogenic and periodontopathogenic bacteria and their cytotoxicities against epithelial cells. AOW, NtOW and AOW-LC showed considerable bactericidal effects. The cytotoxicity of AOW-LC was significantly lower than the other solutions tested (P<0.0001). The results indicated that the three types of oxidizing water had similar activity in inhibiting bacterial plaque formation as conventional chemical plaque-control agents.

Heterogeneity of primary malignant melanomas in oral mucosa: an analysis of 43 cases in Japan.

In contrast to Caucasians, Japanese people have a relatively high incidence of malignant melanoma in the oral cavity and volar-subungual skin. The classification of oral melanoma has not been established. We report 7 new cases of oral melanoma, with a review of 36 cases, published in Japan, between 1970 and 1984. Most of the cases reviewed have radial growth phases, and their gross and microscopic features seem to correspond to those of acral lentiginous melanoma of the skin. However, 1 of our cases and 3 previously reported cases of oral nodular melanoma suggest the presence of melanoma heterogeneity in the oral cavity.

Determination of the thickness of the fixed aqueous layer around polyethyleneglycol-coated liposomes.

The zeta potentials of adriamycin-encapsulating liposomes containing 1-(monomethoxy polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) were measured in an isotonic solution of 10mM lactate buffer (pH 4.0) with sodium chloride and sucrose. The negativity of the zeta potentials of adriamycin-encapsulating liposomes containing PEG-DMG decreased with increases in NaCl concentration more steeply than that of adriamycin-encapsulating liposomes without PEG coating. From this observation, the electrical potential distributions near the membrane surfaces were shown to be different between adriamycin-encapsulating liposomes with and without PEG coating. Based on these zeta potential data, the thickness of the fixed aqueous layer around PEG-DMG-containing adriamycin liposomes was determined from the slope of 1n zeta potential versus Debye-Hückel parameter plot. As a result, a correlation was indicated to exist between the circulation time of liposomes and the thickness of the aqueous layer around the liposomes.

Short and long latency jaw-opening reflex responses elicited by mechanical stimulation in man.

Jaw-opening reflex responses elicited by tapping the chin during maximum clenching in incisal edge-to-edge contact position were studied in 10 healthy subjects. Stimuli were also delivered during weak clenching on a rubber stamp, separating the incisors by approx. 10 mm and protruding the mandible to the edge-to-edge incisor relationship. All four central incisors were stimulated simultaneously. With weak stimuli, there was a short-latency (9.5 ms) digastric response which may have had a disynaptic pathway. Taps of moderate strength produced long-latency (20 ms) responses, and sometimes a short-latency (9.5 ms) component as well. Strong (non-painful) taps produced an even longer-latency digastric response, 30 ms or more following the stimulus with less synchronization than earlier responses. Jaw-jerk reflexes occurred 8.5 ms following the tap, independently of the magnitude of the stimulus. Local anaesthesia of the upper and lower incisors abolished the digastric muscle response. Thus large periodontal afferents may be responsible for the early digastric reflex activity and smaller fibres for later effects. Temporal summation of the reflex response probably occurred when all incisors were stimulated simultaneously.

Determination of incorporated amounts of poly(ethylene glycol)-derivatized lipids in liposomes for the physicochemical characterization of stealth liposomes.

We describe a method for determining incorporated amounts of poly(ethylene glycol) (PEG)-derivatized lipids in liposomes for the physicochemical characterization of PEG-coated liposomes. This method is based on the spectrophotometric determination of complexes of polyethers with sodium ions after their extraction as picrates into 1,2-dichloroethane, developed by Favretto for measuring levels of polyoxyethylene alkylphenyl-ether non-ionic surfactants in waste water. The same assay was applied to the estimation of PEG-derivatized lipids in liposomes and percent incorporation of PEG-derivatized lipids into liposomes was successfully determined. To prevent the interference from liposomal lipids other than PEG-derivatized lipids in this assay, liposomal samples were diluted at least to a concentration of less than 0.2 mM. The percent incorporation of PEG-lipids varied, depending on the molecular weight of PEG and anchor acyl chain length in PEG-lipids and it was suggested that the percent incorporation of PEG-lipids into liposomes would be a good parameter of quality control of PEG-liposomes in manufacturing facility and the picrate method used in the present study allows for the determination of this parameter without the need for hazardous radioisotopes.

Excitatory and inhibitory controls of the masseter and temporal muscles elicited from teeth in the rat.

The periodontal mechanism that controls the jaw reflexes was examined in lightly anesthetized rats. Motor-unit activity in the masseter and temporal muscles was recorded electromyographically and pressure stimulation was applied to either an upper incisor or an upper molar. Reflex effects of dental stimulation varied depending on the level of ongoing activity (background activity, BGA) in each motor unit. Incisal or molar stimulation elicited excitatory reflexes in both the masseter and temporal motor units at a low BGA, but inhibitory reflexes in both types of motor unit at a higher BGA. In contrast to these synergistic reflex actions, the reciprocal reflex actions of the two motor units that belonged to the respective muscles occurred when the BGA was intermediate. The results suggest that different patterns of periodontal jaw reflexes may be elicited, depending on the different levels of BGAs. Furthermore, the present reflexes were modified with the site of a stimulated tooth within the dentition. Incisal stimulation produced greater excitation in the masseter motor unit than in the temporal one, and the opposite type of response occurred during molar stimulation. Moreover, smaller-amplitude motor units with a low reflex threshold and larger-amplitude motor units with a higher reflex threshold tended to exhibit excitatory and inhibitory reflexes, respectively.

Patterns of jaw reflexes induced by incisal and molar pressure stimulation in relation to background levels of jaw-clenching force in humans.

Patterns of jaw reflexes induced by periodontal stimulation were examined in ten adults. Surface electromyograms (EMGs) from the masseter and anterior temporal muscles were recorded when pressure stimulation was applied to either an incisor or a molar. Reflex responses to periodontal pressure stimulation varied, depending on the background levels of jaw-clenching force that preceded stimulation (background clenching force, BCF). At low BCF, excitatory reflexes were elicited from the jaw-closing muscles and jaw-clenching force. However, the magnitude of excitatory reflexes varied with the location of the stimulated tooth along the dentition. While excitatory reflexes were induced equally in the masseter and temporal muscles during incisal stimulation, stronger excitatory reflexes were induced in the temporal muscle than in the masseter muscle during molar stimulation. At high BCF, inhibitory reflexes in the jaw-closing muscles and jaw-clenching force were elicited in eight subjects (group A) during periodontal stimulation. However, excitatory reflexes in the muscles and force were elicited in the remaining two subjects (group B). In the subjects of group A, stronger inhibitory reflexes were elicited in the temporal muscle than in the masseter muscle, and jaw-clenching force also decreased during both incisal and molar stimulation. In the subjects of group B, the magnitude of excitatory reflexes decreased with increases in BCF.

Demonstration of the nasal septal branches of the sphenopalatine artery by use of a new intravascular injection method.

We injected a new injection material into the external carotid artery using a new method, which led to the successful demonstration of the nasal septal branches of the sphenopalatine artery in human cadavers. The result shows the trunk of the artery divided into three main branches, the upper two of which run toward Little's area. We believe that the knowledge of septal branches, shown in a photograph, is very useful, not only for nasal treatment, but also for anatomic demonstration to students.

Morphology of the lateral ligament in the human temporomandibular joint.

The morphology of the lateral ligament of the human temporomandibular joint is of two types: ligamentous and without distinct structure. Under the scanning electron microscope, a sheath-like structure that contained bundles of collagen was mainly found in the posterior region of the lateral ligament. Analysis of macromolecular components revealed that type III collagen was mainly present on the collagenous framework of the sheath-like structure. Type I collagen, laminin, and tenascin were found in the framework of the sheath-like structure. Supported collagenous bundles and the distribution of macromolecular components might be related to the stability of the temporomandibular joint. The sheath-like structure and other components of the lateral ligaments store energy and protect the capsule from stress and tension during movements of the jaw.

Relationship between levels of aspartate aminotransferase in gingival crevicular fluid and conventional measures of periodontal status assessed using PocketWatch: a cross-sectional study.

The aim of this cross-sectional study was to determine, using PocketWatch, the relationship between the level of aspartate aminotransferase (AST) in gingival crevicular fluid (GCF) and conventional measures of periodontal status, such as probing depth, attachment level, bleeding on probing and gingival index, in patients with untreated chronic periodontitis. A total of 15 patients with chronic periodontitis were enrolled. Their periodontal status and AST levels in their GCF were measured (n = 93) and statistically analyzed. There was a statistically significant difference in AST levels between diseased periodontal sites and healthy sites (p < 0.0001). The coefficients of correlation between AST levels and probing depth, attachment level and gingival index at all sites were 0.436, 0.266 and 0.468 (Spearman rank correlation). The correlation coefficients were too small to show a definite relationship between AST levels and individual measures of clinical periodontal status. However, AST levels may help to confirm clinical observations in patients with chronic periodontitis before therapy, since AST levels differentiate active and inactive periodontal diseased sites.