Immunohistochemical demonstration of keratins in the epidermal layers of the Malayan pangolin (Manis javanica), with remarks on the evolution of the integumental scale armour.

Using immunohistochemistry, the study demonstrates the distribution of keratins (pan-keratin with CK1-8, 10, 14-16, 19; keratins CK1, 5, 6, 9, 10; hair keratins AE13, AE14) in the epidermis of the Malayan pangolin (Manis javanica). A varying reaction spectrum was observed for pan-keratin, with body region-dependent negative to very strong reaction intensities. The dorsolateral epidermis exhibited positive reactions only in its vital layers, whereas the abdominal epidermis showed strong positive reactions in the soft two outer strata. The single acidic and basic-to-neutral (cyto)keratins produced clear variations compared to the pan-keratin tinging. E.g., CK1 appeared in all epidermal layers of both body regions, except for the ventral stratum corneum, whereas CK5, 6, 9, 10 were restricted to the soft ventral epidermis. Here, distinctly positive reactions were confined to the stratum granulosum, except for CK6 that appeared in the soft stratum corneum. A different staining pattern was obvious for the hair keratins, i.e., positive reactions of AE13 concentrated only in the granular layer of the dorsal epidermis. In the abdominal epidermis, remarkable tinging for AE14 was visible in the stratum basale, decreasing toward the corneal layer, but was also found in the outer root sheath cells of the hair follicles in the ventral body part. Our findings are discussed related to the evolution of the horny dorsal scales of the pangolin, which may have started from the tail root, projecting forward to the head.

Detection of myocardial ischaemia by epicardial accelerometers in the pig.

BACKGROUND: We describe a novel technique for continuous real-time assessment of myocardial ischaemia using a three-axis accelerometer. METHODS: In 14 anaesthetized open-chest pigs, two accelerometers were sutured on the left ventricle (LV) surface in the perfusion areas of the left anterior descending (LAD) and circumflex (CX) arteries. Acceleration was measured in the longitudinal, circumferential, and radial directions, and the corresponding epicardial velocities were calculated. Regional LV dysfunction was induced by LAD occlusion for 60 s. Global LV function was altered by nitroprusside, epinephrine, esmolol, and fluid loading. Epicardial velocities were compared with strain by echocardiography during LAD occlusion and with aortic flow and LV dP/dt(max) during interventions on global LV function. RESULTS: LAD occlusion induced ischaemia, shown by lengthening in systolic strain in the LV apical anterior region (P<0.01) and concurrent changes in LAD accelerometer circumferential velocities during systole (P<0.01) and during the isovolumic relaxation phase (P<0.01). The changes in accelerometer circumferential velocities during LAD occlusion were greater compared with the changes during the interventions on global function (P<0.01). For the LAD accelerometer circumferential velocities, sensitivity was 94-100% and specificity was 92-94% in detecting ischaemia. CONCLUSIONS: Myocardial ischaemia can be detected with epicardial three-axis accelerometers. The accelerometer had the ability to distinguish ischaemia from interventions altering global myocardial function. This novel technique may be used for continuous real-time monitoring of myocardial ischaemia during and after cardiac surgery.

A covalently cross-linked gel derived from the epidermis of the pilot whale Globicephala melas.

The rheological properties of the stratum corneum of the pilot whale (Globicephala melas) were investigated with emphasis on their significance to the self-cleaning abilities of the skin surface smoothed by a jelly material enriched with various hydrolytic enzymes. The gel formation of the collected fluid was monitored by applying periodic-harmonic oscillating loads using a stress-controlled rheometer. In the mechanical spectrum of the gel, the plateau region of the storage modulus G' (<1200 Pa) and the loss modulus G" (>120 Pa) were independent of frequency (omega = 43.98 to 0.13 rad x s(-1), tau = 15 Pa, T = 20 degrees C), indicating high elastic performance of a covalently cross-linked viscoelastic solid. In addition, multi-angle laser light scattering experiments (MALLS) were performed to analyse the potential time-dependent changes in the weight-average molar mass of the samples. The observed increase showed that the gel formation is based on the assembly of covalently cross-linked aggregates. The viscoelastic properties and the shear resistance of the gel assure that the enzyme-containing jelly material smoothing the skin surface is not removed from the stratum corneum by shear regimes during dolphin jumping. The even skin surface is considered to be most important for the self-cleaning abilities of the dolphin skin against biofouling.

A zymogel enhances the self-cleaning abilities of the skin of the pilot whale (Globicephala melas).

Enzyme activity in the stratum corneum of the pilot whale Globicephala melas was investigated employing colorimetric enzyme screening assays combined with NATIVE PAGE, size exclusion chromatography (SEC) and histochemical staining procedures. Applying different substrates, several enzymes were detected. The histochemical demonstration of some selected hydrolytic enzymes enriched in the stratum corneum showed high extracellular accumulation. As demonstrated by size exclusion chromatography, high molar mass aggregates were built up from a glycoprotein-rich 20-30-kD fraction. Using NATIVE PAGE experiments under non-reducing conditions, a selection of five degrading enzymes was recovered within the above-reported aggregates. Activity of extracellular aggregate-attached enzymes in the superficial layer of the stratum corneum exhibited no remarkable decrease potentially resulting from self-degradation. We thus conclude that due to their enclosure within the microenvironment of aggregates, a zymogel is formed and autolysis of the stratum corneum is reduced. With respect to the skin surface, the zymogel with hydrolytic activities covering major parts of it enhances the self-cleaning abilities of the skin of the pilot whale based on physical pre-requisites by hydrolyzing adhesive glycoconjugates of settling biofouling organisms considered as primary steps in fouling.

Genetics and phenotypes of RPE65 mutations in inherited retinal degeneration.

PURPOSE: To characterize the spectrum of RPE65 mutations present in 453 patients with retinal dystrophy with an interest in understanding the range of functional deficits attributable to sequence variants in this gene. METHODS: The 14 exons of RPE65 were amplified by polymerase chain reaction (PCR) from patients' DNA and analyzed for sequence changes by single-strand conformation polymorphism (SSCP) and direct sequencing. Haplotype analysis was performed using RPE65 intragenic polymorphisms. Patients were examined clinically and with visual function tests. RESULTS: Twenty-one different disease-associated DNA sequence changes predicting missense or nonsense point mutations, insertions, deletions, and splice site defects in RPE65 were identified in 20 patients in homozygous or compound heterozygous form. In one patient, paternal uniparental isodisomy (UPD) of chromosome 1 resulted in homozygosity for a probable functional null allele. Eight of the disease-associated mutations (Y79H, E95Q, E102X, D167Y, 669delCA, IVS7+4a-->g, G436V, and G528V) and one mutation likely to be associated with disease (IVS6+5g-->a) have not been reported previously. The most commonly occurring sequence variant identified in the patients studied was the IVS1+5g-->a mutation, accounting for 9 of 40 (22.5%) total disease alleles. This splice site mutation, as well as R91W, the most common missense mutation, exists on at least two different genetic backgrounds. The phenotype resulting from RPE65 mutations appears to be relatively uniform and independent of mutation class, suggesting that most missense mutations (15 of 40 disease alleles [37.5%]) result in loss of function. At young ages, this group of patients has somewhat better subjective visual capacity than is typically associated with Leber congenital amaurosis (LCA) type I, with a number of patients retaining some useful visual function beyond the second decade of life. CONCLUSIONS: RPE65 mutations account for a significant percentage (11.4%) of disease alleles in patients with early-onset retinal degeneration. The identification and characterization of patients with RPE65 mutations is likely to represent an important resource for future trials of rational therapies for retinal degeneration.

Blood cells and plasma proteins of chickens fed a diet supplemented with (1-->3),(1-->6)-beta-D-glucan and enrofloxacin.

The effects of (1-->3),(1-->6)-beta-D-glucan from Saccharomyces cerevisiae and of the fluochinolone enrofloxacin were studied on red and white blood cells and plasma proteins of growing chickens up to the 35th day of life. The prominent findings within the leukocyte population on a per cent scale are: (i) increase of leukocyte count; increase of neutrophils and decrease of lymphocytes in the control and in the antibiotic group from day 17 to day 35; (ii) a minor decrease of neutrophils and no change of lymphocytes in the glucan group; (iii) the monocytes increase from 2.5 +/- 1.8% to 6.5 +/- 7.6% in the glucan group; (iv) the basophils increase in the control group and scale down in the other groups from day 17 to day 35. The total count of leukocytes increases in the controls and in the glucan group. The total protein content of blood plasma, beta-globulin and gamma-globulin increase and the albumin-globulin-ratio and alpha-globulin decline during chickens growth. These changes are most prominent in the glucan group. The haemoglobin concentration shows in all three dietary groups a highly significant increase from day 17 to day 35 by about 17 to 27 per cent; no changes are seen in packed cell volume and number of erythrocytes per litre blood.

The relationship of oxidized lipids to coronary artery stenosis.

A total of 1200 patients with angina were cardiac catheterized establishing that 63% had 70-100% stenosis, 12% had 10-69% stenosis of one or more of their coronary arteries and 25% had microvascular angina listed as 0% stenosis. Prior to catheterization 10 ml of blood was drawn and the plasma subjected to analysis for the concentration of cholesterol, lipid peroxides (LPX), total antioxidant capacity (TAOC), fibrinogen (FB), ceruloplasmin (CP) and activation of polymorphonuclear leukocytes (PMNLs). Comparisons were made to non-smoking controls without angina. Significant differences in LPX were found between the patients with 0 and 10-69% stenosis (P<0.001), with 10-69 and 70-100% stenosis (P<0.001), and with 0 and 70-100% stenosis (P<0.001). Under 70 years of age there was a significant difference in LPX between patients with all levels of stenosis and age and sex matched controls (P<0.001). Differences in the mean plasma cholesterol concentration for different levels in the degree of stenosis were not significant, indicating that LPX provided consistent data on the severity of stenosis while the plasma cholesterol concentration did not. Compared with controls an increase in activation of PMNLs (P<0.01), an increase in concentration of both FB and CP (P<0.01) and a decrease in total antioxidant capacity were noted in the plasma of catheterized patients. In summary the concentration of oxidation products rather than the concentration of cholesterol in the plasma identified stenosis in cardiac catheterized patients.

An excess concentration of oxysterols in the plasma is cytotoxic to cultured endothelial cells.

To test if there is an excess concentration of oxysterols in the plasma of the patients with cardiovascular disease, we analyzed the oxysterol content in the plasma from 105 cardiac catheterized patients with angina and 80+/-8% stenosis in their coronary arteries. The result showed that the plasma contained a significantly higher concentration of oxysterols than did plasma from 105 age- and sex-matched, non-catheterized and angina-free controls (P<0.05). We used endothelial cells (ECs) cultured in medium containing either [3H]thymidine, [3H]mevalonolactone or 45Ca(2+) to determine how the plasma from the patients influences cell growth and function. We found that less [3H]thymidine (P<0.05), less [3H]mevalonolactone (P<0.05) and more 45Ca(2+) (P<0.001) was incorporated into ECs cultured in the plasma from 36 patients with 83+/-4% stenosis than from the 36 controls. When synthetic 7beta-hydroxycholesterol, cholesterol 5beta,6beta-epoxide, cholesterol 5alpha,6alpha-epoxide and 7-ketocholesterol were added to the plasma from the controls, the influx of 45Ca(2+) into ECs then equaled that in the plasma of patients. The enhanced incorporation of 45Ca(2+) into the ECs cultured in the plasma both from the patients and from controls with added synthetic oxysterols substantiates in vitro the hypothesis that oxysterols increase the influx of calcium into cells. These data indicated that an excess of oxysterols in the plasma of the patients was cytotoxic to the cultured cells.

The hemolytic activity of homocysteine is increased by the activated polymorphonuclear leukocytes.

Homocysteine is an accepted risk factor when its plasma level exceeds the physiological upper limit of 12 mumol/L. We found in vitro that homocysteine is able to lyse the erythrocytes (RBCs) at higher concentrations than 15 mumol/L only when activated polymorphonuclear leukocytes (PMNL) are present. The hemolytic effect of homocysteine was higher in RBCs obtained from cardiac catheterized patients with 100% stenosis of the coronary arteries. Homocysteine was also able to increase the activation in vitro of PMNLs triggered by opsonized zymosan. The hemolytic action of homocysteine was found to be dependent on the ratio of PMNLs to RBCs. This relationship may help to explain the great individual variations in the hemolytic activity noticed in blood obtained from cardiac catheterized patients, and also may explain the mild anemia in some patients suffering from cardiovascular disease.

Relationship of transcranial Doppler flow velocities and arteriovenous malformation feeding artery pressures.

BACKGROUND AND PURPOSE: Feeding mean arterial pressure immediately proximal to the nidus of arteriovenous malformations may influence the frequency of spontaneous intracerebral hemorrhage. This study assessed the usefulness of transcranial Doppler ultrasound velocities as a noninvasive estimate of feeding mean arterial pressure. METHODS: We studied 41 patients undergoing 73 staged treatments of arteriovenous malformations with endovascular embolization, surgery, or both. Before treatment during the awake state, transcranial Doppler mean and peak velocities were recorded in proximal Willisian vessels. During superselective angiography with the patient under conscious sedation or during surgery with the patient under general anesthesia, feeding mean arterial pressure was measured through a 1.5F transfemoral intracranial microcatheter or a 26-g needle by direct puncture. Measurement of insonated artery diameter was possible in 41 embolizations, and a flow velocity index (mL/min) and Reynolds' number were estimated. RESULTS: Mean +/- SEM feeding mean arterial pressure was 38 +/- 2 mm Hg at a systemic mean arterial pressure of 77 +/- 2 mm Hg; mean velocity was 102 +/- 4 cm/s. There was an inverse correlation between feeding mean arterial pressure and parent artery mean velocity (y = -0.74x + 130, r = .35, P = .0025). The best correlation was for the first treatment in each patient (n = 27) using the highest peak velocity obtainable in a Willisian vessel ipsilateral to the arteriovenous malformation (y = -1.61x + 221, r = .62, P = .0005). Flow velocity index (775 +/- 106 mL/min) did not correlate with feeding mean arterial pressure, but there was a weak correlation with Reynolds' number (y = -12x + 1616, r = .27, P = .1283). Mean Reynolds' number was 1257 +/- 119. CONCLUSIONS: Transcranial Doppler mean velocity is correlated with feeding mean arterial pressure but only weakly predictive. Considerations influencing the relation of distal feeding mean arterial pressure to proximal mean velocity might include the influence of other fistulae in the circuit between major inflow and outflow channels as well as turbulent flow at vascular branch points between point of insonation and the nidus, as suggested by Reynolds' number values of more than 400.

Desflurane and isoflurane have similar effects on cerebral blood flow in patients with intracranial mass lesions.

BACKGROUND: Before desflurane is advocated for patients undergoing neurosurgical procedures, it is necessary to determine the effect of desflurane on cerebral blood flow (CBF). In this study, CBF values are compared between desflurane and isoflurane at two doses. In addition, CBF reactivity to CO2 and the effect of prolonged exposure were compared between the two agents. METHODS: Cerebral blood flow measurements with intravenous 133Xe were performed in 24 patients undergoing craniotomy for mass lesions, randomized to receive either isoflurane or desflurane in oxygen and air. Cerebral blood flow was determined at 1 and 1.5 MAC concentrations at PaCO2 of 25 mmHg in the absence of surgical stimulation. Intraoperatively, with 1.25 MAC anesthesia, CBF was determined at target PaCO2 of 25 and 35 mmHg. In 15 patients, an additional measurement at 1.25 MAC was made before closure. RESULTS: At 1.0 MAC, mean +/- SD CBF values for the desflurane and isoflurane groups were 18 +/- 2 and 20 +/- 3 ml x 100 g-1 x min-1, respectively. At 1.5 MAC, CBF values were the same for the two anesthetics; 17 +/- 3 ml x 100 g-1 x min-1 for isoflurane and 19 +/- 4 ml.100 g-1 x min-1 for desflurane. During 1.25 MAC anesthesia, there were no differences between groups, with CO2 reactivity 1.3 +/- 1.2 ml x 100 g-1 x min-1 x mmHg-1 for desflurane and 1.6 +/- 0.6 ml.100 g-1 x min-1 x mmHg-1 for isoflurane. There was no demonstrable decrease in CBF with prolonged exposure to either agent. CONCLUSIONS: Desflurane and isoflurane are similar in terms of absolute CBF, the response to increasing doses, and the preservation of CO2 reactivity.

Pressure autoregulation is intact after arteriovenous malformation resection.

The loss of autoregulatory control of cerebral perfusion to changes in perfusion pressure in tissue remote from an arteriovenous malformation (AVM) has been proposed as the mechanism underlying "normal perfusion pressure breakthrough." This study is the first direct test of this mechanism. Studies were performed during the resection of moderate to large AVMs in 25 patients undergoing 28 procedures under isoflurane anesthesia. Cerebral blood flow (CBF) was measured (xenon-133 method) in the hemisphere adjacent to the nidus before resection after dural exposure (pre), after AVM removal before dural closure at spontaneous systemic blood pressure (post), and, finally, with the mean arterial pressure increased by 20 mm Hg, using phenylephrine (post-BP). AVM resection resulted in a significant enhancement of perfusion in the adjacent hemisphere (30 +/- 2 vs. 25 +/- 1 ml/100g/min, P < 0.01), but no further increase of CBF occurred during increased perfusion pressure (30 +/- 2 ml/100g/min). One patient suffered a postoperative hemorrhage and another developed intraoperative brain swelling during the course of the resection that necessitated staging the procedure. These two patients had the highest increases in CBF, but intact pressure autoregulation. Preserved autoregulation to increased mean arterial pressure after resection does not support a hemodynamic mechanism for the observed increase in CBF from before the resection to after the resection. Pathological events, however, do appear to be related to increases in hemispheric perfusion.

Role of nutrient HCO3(-) in protection of amphibian gastric mucosa.

In in vitro bullfrog fundic mucosa inhibited with 10(-3) M metiamide and exposed to a luminal pH of 2 a progressive slow decline in potential difference (PD) and short-circuit current (Isc) and a rise in resistance (R) were observed when the nutrient solution (N) contained 18 mM HCO3(-), but these changes were restored by an N containing 50 mM HCO3(-). Substitution of PO4(3-) or N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid for NHO3(-) in N caused a rapid drop in PD and Isc in inhibited tissues, changes that could be prevented by 10(-4) M histamine. Ulceration occurred more frequently in metiamide-inhibited gastric sacs exposed to artificial gastric juice with an N of 18 mMHCO3(-) than with 50 mM HCO3(-), but histamine prevented ulceration in the 18 mM HCO3(-) solution. JnetCl approximated Isc under most experimental conditions in inhibited mucosa and was reduced dramatically as were both Jn leads to sCl and Js leads to nCl when HCO3(-) was removed from N. In histamine-stimulated tissues, removal of nutrient HCO3(-) did not influence Cl- transport. Our results are consistent with the proposal that HCO3(-) in N supports normal Cl- flux and that the alkaline tide of actively secreting oxyntic cells can do the same in the absence of ambient HCO3(-).

Effect of 16-16-dimethyl-prostaglandin E2 on ulceration of isolated amphibian gastric mucosa.

In order to investigate the cytoprotective action of 16-16-dimethyl-prostaglandin E2 (16-16D), we studied its effect on sacs of isolated amphibian gastric mucosa known to ulcerate with high frequency in the absence of nutrient HCO3-. The actions of 16-16D, 2.85 X 10(-6) M, were also studied in an in vitro chamber. The incidence of ulceration in HCO3- -free nutrient media containing 10(-4) M histamine was significantly reduced by 16-16D. When artificial gastric juice (AGJ) was instilled into the lumen of the sac, protection occurred only after a pretreatment period of 60 min. Cytoprotection was not observed when active secretion of H+ was inhibited with 10(-3) M metiamide, and AGJ was placed in the lumen of the sac. In open sheets of fundus stimulated with 10(-4) M histamine, 16-16D did not influence H+ secretion. Isc was significantly higher than in control tissues. We conclude that 16-16D is cytoprotective in amphibian gastric mucosa by an action independent of changes in acid secretion or in blood flow. In addition, our studies suggest that 16-16D may increase the availability to the surface cells of nutrient HCO3- produced by actively secreting oxyntic cells.