Mutations in cornea-specific keratin K3 or K12 genes cause Meesmann's corneal dystrophy.

The intermediate filament cytoskeleton of corneal epithelial cells is composed of cornea-specific keratins K3 and K12 (refs 1,2). Meesmann's corneal dystrophy (MCD) is an autosomal dominant disorder causing fragility of the anterior corneal epithelium, where K3 and K12 are specifically expressed. We postulated that dominant-negative mutations in these keratins might be the cause of MCD. K3 was mapped to the type-II keratin gene cluster on 12q; and K12 to the type-I keratin cluster on 17q using radiation hybrids. We obtained linkage to the K12 locus in Meesmann's original German kindred (Zmax = 7.53; theta = 0) and we also showed that the phenotype segregated with either the K12 or the K3 locus in two Northern Irish pedigrees. Heterozygous missense mutations in K3 (E509K) and in K12 (V143L; R135T) completely co-segregated with MCD in the families and were not found in 100 normal unrelated chromosomes. All mutations occur in the highly conserved keratin helix boundary motifs, where dominant mutations in other keratins have been found to severely compromise cytoskeletal function, leading to keratinocyte fragility phenotypes. Our results demonstrate for the first time the molecular basis of Meesmann's corneal dystrophy.

Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma). Genetic linkage to chromosome 12q in the region of the type II keratin gene cluster.

Epidermolytic hyperkeratosis (EHK) is an autosomal dominant genodermatosis characterized by hyperkeratosis and blistering of the skin. Histopathology demonstrates suprabasilar blister formation with aggregation of tonofilaments. In this study, we tested the hypothesis that the EHK phenotype is linked to one of the suprabasilar keratins (KRT10 or KRT1) present in the types I and II keratin gene clusters in chromosomes 17q and 12q, respectively. For this purpose, Southern hybridizations were performed with DNA from a large kindred with EHK, consisting of 11 affected individuals in three generations. Segregation analysis with markers flanking the keratin gene clusters demonstrated linkage (Z = 3.61 at theta = 0) to a locus on 12q, while markers on 17q were excluded. These data implicate KRT1, the type II keratin expressed in suprabasilar keratinocytes, as a candidate gene in this family with EHK.

Genetic linkage of type VII collagen (COL7A1) to dominant dystrophic epidermolysis bullosa in families with abnormal anchoring fibrils.

Epidermolysis bullosa (EB) in a group of genodermatoses characterized by the fragility of skin. Previous studies on the dystrophic (scarring) forms of EB have suggested abnormalities in anchoring fibrils, morphologically recognizable attachment structures that provide stability to the association of the cutaneous basement membrane to the underlying dermis. Since type VII collagen is the major component of the anchoring fibrils, we examined the genetic linkage of dominant dystrophic EB (EBDD) and the type VII collagen gene (COL7A1) locus, which we have recently mapped to chromosome 3p, in three large kindreds with abnormal anchoring fibrils. Strong genetic linkage of EBDD and COL7A1 loci was demonstrated with the maximum logarithm of odds (LOD) score of 8.77 at theta = 0. This linkage was further confirmed with two additional markers in this region of the short arm of chromosome 3, and these analyses allowed further refinement of the map locus of COL7A1. Since there were no recombinants between the COL7A1 and EBDD loci, our findings suggest that type VII collagen is the candidate gene that may harbor the mutations responsible for the EB phenotype in these three families.

Genetic linkage of recessive dystrophic epidermolysis bullosa to the type VII collagen gene.

Generalized mutilating recessive dystrophic epidermolysis bullosa (RDEB) is characterized by extreme skin fragility owing to loss of dermal-epidermal adherence. Immunohistochemical studies have implicated type VII collagen, the major component of anchoring fibrils, in the etiology of RDEB. In this study, we demonstrate genetic linkage of the type VII collagen gene and the generalized mutilating RDEB phenotype. We first identified a Pvull polymorphic site by digestion of an amplified product of the type VII collagen gene, which was shown to reside within the coding region. Genetic linkage analysis between this marker and the RDEB phenotype in 19 affected families which were informative for this polymorphism showed no recombination events, and gave a maximum lod score of 3.97 at a recombination fraction (theta) of 0, demonstrating that this DNA region is involved in this form of RDEB. These data provide strong evidence that the type VII collagen gene, which has also been linked with the dominant form of the disease, harbors the mutation(s) causing the generalized mutilating form of RDEB in these families, thus underscoring the major functional importance of type VII collagen in basement membrane zone stability.

Relationships of aerobic capacity and percent body fat with plasma free fatty acid following walking.

We investigated relationships of peak VO2 and percent body fat with postexercise plasma free fatty acid (FFA) and glycerol concentrations in 14 men using multiple-regression techniques. During 1 h of walking (36% of peak VO2), no significant differences in plasma-FFA response were attributed to either peak VO2 or percent body fat. However, individuals with higher peak VO2S tended to have greater elevations in plasma-glycerol concentration during exercise (p = 0.074). They also had greater peak FFA concentrations and FFA X glycerol(-1)-molar ratios immediately after exercise and faster subsequent clearing of excess FFA from the blood (p less than 0.05). Percent fat was not related to postexercise plasma glycerol, FFA, FFA X glycerol-1 responses. Differing postexercise FFA responses, as related to peak VO2, were due, not to varying rates of lipolysis but rather to different rates of FFA mobilization and utilization.

Use of DNA restriction fragment length polymorphisms to document marrow engraftment and mixed hematopoietic chimerism following bone marrow transplantation.

We have studied the feasibility of using DNA restriction fragment-length polymorphisms (RFLP) to study marrow engraftment in 27 patients after allogeneic bone marrow transplantation, and have compared these results with those obtained using red blood cell antigens, cytogenetics, and immunoglobulin allotypes. Using highly polymorphic DNA probes, we have documented stable chronic mixed hematopoietic chimerism, have identified transient mixed chimeras, have excluded mixed chimerism with high probability in retrospective studies even when a pretransplant DNA sample was not available, have documented marrow engraftment in the early posttransplant period, and have studied the origin of leukemic cells in patients with recurrent disease. We have evaluated the advantages and disadvantages of several genetic markers and have developed tentative statements concerning the prognosis of patients with mixed chimerism. We conclude that DNA RFLP are powerful and practical genetic markers in bone marrow transplantation studies and that further studies of mixed hematopoietic chimerism are warranted.

Exclusion of type II and type VI procollagen gene mutations in a five-generation family with multiple epiphyseal dysplasia.

We have studied a family with an autosomal dominant form of multiple epiphyseal dysplasia (MED) inherited through at least 5 generations. Bilateral deformity of the hips with subsequent degenerative arthritis was the most common and most severe change observed in the affected relatives. Abnormalities of the knees, ankles, and shoulders were also noted in some affected individuals. Radiological examination showed changes in affected joints consistent with epiphyseal dysplasia. In early stages, the articular surfaces appeared flattened or irregular in shape. In advanced stages, epiphyseal fragmentation, joint surface erosion, and extensive remodeling were observed. The abnormalities of the epiphyses suggested that the primary defect might be in a structural component of the epiphyseal cartilage matrix. The gene encoding type II collagen (COL2A1) was tested for genetic linkage to MED in this family by restriction fragment length polymorphism (RFLP) analysis. Recombination between COL2A1 and MED was observed, ruling out COL2A1 as the site of the mutation. The genes encoding the 3 chains of type VI collagen were also excluded on the basis of discordant inheritance. The disease in this family is therefore not the result of mutations in the genes encoding type II or type VI collagen.

Multiple epiphyseal dysplasia, ribbing type: a novel point mutation in the COMP gene in a South African family.

Multiple epiphyseal dysplasia is broadly categorised into the more severe Fairbank and the milder Ribbing types. In this paper we document mild MED in a South African kindred, and demonstrate that heterozygosity for a mutation in the cartilage oligomeric matrix protein (COMP) gene causes the condition. The mutation, C1594G, implies a N523K substitution, altering a residue at the carboxyl-terminal end of the calmodulin-like region of COMP. The identification of this mutation demonstrates that the spectrum of manifestations from mild MED through pseudoachondroplasia can all be produced by structural mutations in COMP.

Stickler syndrome. A mutation in the nonhelical 3' end of type II procollagen gene.

BACKGROUND: All of the mutations in the type II procollagen (COL2A1) gene that have been identified in families affected with Stickler syndrome have been located primarily in the triple helical region of the gene. We report what we believe is the first premature stop codon in the globular C-propeptide region encoded by the COL2A1 gene, in a family affected with Stickler syndrome. DESIGN: Genomic DNA from affected and unaffected family members of this three-generation family was amplified using the polymerase chain reaction. The polymerase chain reaction products were directly sequenced for DNA analysis. RESULTS: Direct sequencing showed a single base deletion in exon 50, resulting in a premature stop codon in exon 51 in the globular C-propeptide of COL2A1 gene in all affected members. CONCLUSIONS: These results implicate premature stop codons as a common cause of Stickler syndrome. The location of this premature stop codon in the far end of the nonhelical 3' end of the gene indicates that a truncated C-propeptide of at least 84 amino acid residues is inadequate for the functional gene product.

Effect of paraxanthine on FFA mobilization after intravenous caffeine administration in humans.

Because it has previously been shown that it takes much more caffeine to cause fat mobilization in vitro than in vivo, it has been suggested that there may be an active metabolite working with caffeine causing an increase in lipolysis in vivo. To determine the relationship between the appearance of paraxanthine (caffeine's major dimethylxanthine metabolite) and free fatty acid (FFA) mobilization after intravenous caffeine administration, 10 men were studied at rest after receiving a dose of 4 mg/kg lean body mass. Venous blood samples were obtained before dosing and at minutes 5, 10, 15, 30, 45, 60, 90, 120, 150, and 180. Serum levels of FFA, glycerol, caffeine, and paraxanthine were determined in duplicate. Concentrations of FFA and glycerol were corrected for plasma volume changes. A high negative correlation was seen between decreases in caffeine and increases in FFA (r = -0.90) and a high positive correlation was seen between the appearance of paraxanthine and FFA (r = 0.93). It was concluded that paraxanthine may play a role in increased lipolysis after caffeine administration in humans.

Accuracy of RPE from graded exercise to establish exercise training intensity.

The purpose of this investigation was to examine the accuracy of an exercise intensity prescription based upon perceptual responses obtained during a graded exercise test. Fifteen physically active men completed a graded exercise test (GXT) on a motor driven treadmill. Heart rate (HR), oxygen uptake (VO2), and RPE were measured each minute. An RPE intensity prescription was calculated as 75% of heart rate reserve from the GXT heart rate and RPE data. A minimum of 48 h later the subjects completed 10 min of exercise (EXT) on a level treadmill at the prescribed RPE. The subjects set the treadmill speed to elicit an exercise intensity equal to the prescribed perception of effort. There were significant mean differences (P < 0.05) in heart rate between the GXT (161.8 +/- 1.3) and EXT (154.9 +/- 4.5). However, by minute 6 the subjects were within four beats.min-1 of the target heart rate. There were no significant differences (P > 0.05) between GXT and EXT for VO2 (36.1 +/- 5.2, 33.1 +/- 6.4) and VE (64.1 +/- 10.8, 58.4 +/- 13.5) respectively. The present investigation demonstrates that a subject's perceptual response to a GXT can be used to accurately prescribe exercise intensity during level treadmill running. The intensity selected was within a typical range used for exercise prescription. The advantage of RPE as a method of exercise prescription is that an individual does not need to stop during exercise and measure a heart rate, but can make pace adjustments while exercising based solely upon the perception of effort.

Cardiovascular, metabolic, and ventilatory responses of women to equivalent cycle ergometer and treadmill exercise.

The relationships between age and cardiorespiratory and metabolic adjustments during cycle ergometer exercise and treadmill walking were investigated at 30%, 60%, 80%, and maximal aerobic power (VO2max). The subjects were 18 females between the ages of 17-40. Tr-admill walking resulted in an 8 percent greater maximal aerobic power than that observed during cycle ergometer exercise. Higher lactate concentrations, respiratory exchange ratios and lower heart rates during the cycle ergometer test supported the possibility of localized fatigue from anaerobic metabolism as a limitation to aerobic power potential. Contrary to previous observations in males, female subjects die not demonstrate a lower stroke volume or higher heart rate during submaximal cycling compared to treadmill walking. At exercise intensities above 30% Vi2max, plasma bicarbonate and pH were consistently lower following exercise on the cycle ergometer. Respiratory compensation as calculated from "excess ventilation," delta VE/delta HCO3-, was approximately 4 1 min-1/mEq 1-1. Compensation was found to be independent of the type of exercise but linearly related (r=0.99) to the resultant metabolic acidosis. It was concluded that cardiovascular responses to equivalent cycle ergometer and treadmill exercise are similar; however, occurrence of a greater metabolic acidosis during cycle ergometer exercise suggests that a smaller muscle mass is used.

Body temperature, respiration, and acid-base equilibrium during prolonged running.

The purpose of this investigation was to study respiratory responses related to acid-base equilibrium during two bouts of prolonged high intensity treadmill running, each at different body temperature levels. In an attempt to augment elevation in body temperature during prolonged running, incomplete rehydration procedures were employed. Rectal temperature was found to increase significantly during the course of each run. In addition, rectal temperature was found to be significantly higher during the second run at each respective time period. After the transition to exercise, plasma volume, arterial lactate concentration, arterial hydrogen ion concentration, arterial carbon dioxide partial pressure, arterial bicarbonate concentration, pulmonary ventilation, and ventilatory equivalent of carbon dioxide remained constant throughout each run. It was also found that arterial carbon dioxide partial pressure was lower and ventilatory equivalent of carbon dioxide was higher during the second run at each respective time period. For the studied population, these results indicate that after initial adjustments most of the examined variables remain fairly constant despite increasing rectal temperature during prolonged running.

Circulatory and thermal adjustments to prolonged exercise in paraplegic women.

The circulatory and thermal responses to 90 min of wheelchair ergometer exercise were examined in five wheelchair dependent (WD) women with low level spinal dysfunction and five able-bodied (AB) women who served as a comparison group. Metabolic rate during exercise was 221 W for WD and 255 W for AB (P greater than 0.05). Oral temperature (Tor), mean skin temperature (Tsk), oxygen uptake (VO2), heart rate (HR), and cardiac output (Qc) were assessed periodically throughout the exercise period. Ambient conditions were 24-25 degrees C and 38-52% relative humidity. A significant group X time interaction was found for Tor (P less than 0.001) and Tsk (P less than 0.001). Tor of the WD group steadily increased during the exercise, whereas the AB group showed a stable Tor. Tsk of WD increased rapidly during the first 5-10 min of exercise and continued to rise at a slower rate throughout the exercise. In contrast, Tsk of AB rose to a peak during the first 10 min and then showed a decreasing trend. VO2 and HR remained stable in both groups throughout the exercise period. Following an initial increase in Qc from minute 10 to minute 20 in both groups, values for WD continually decreased until Qc at 80 min was 14% lower than at 10 min. The findings suggest that the WD women had greater thermoregulatory strain than the AB women as indicated by a higher Tor and Tsk and by an inability to maintain Qc due to paralysis of the lower limbs and perhaps an increase in cutaneous blood volume.

Plasma volume changes and cardiovascular responses associated with weight lifting.

Ten experienced weight lifters were studied to determine the influence of strenuous, free-weight, squat lift exercise on changes in plasma volume, mean arterial pressure, and the rate pressure product. After a 40-min postural adjustment prior to upright exercise, each subject completed eight sets of eight repetitions of the squat exercise at 55% of the one-repetition maximum. Electrocardiogram heart rate was monitored throughout the test, and blood pressure was determined at the completion of each exercise set. Arterialized capillary blood was drawn during sitting and standing pre-exercise and at the end of the second, fourth, sixth, and eighth sets for the measurement of hemoglobin, hematocrit, and plasma protein concentration, and after the last set for blood lactate. Five additional subjects participated in a postural control comparison. The results of the ANOVA showed a significant loss in plasma volume over the trials which was 2 1/2 times greater than for the control subjects (17.9 vs 7.7% loss) over the same period of time. Exercise rate pressure product increased by 2.85 times resting while only 1.1 times resting for the control subjects. An elevation in mean arterial pressure was found to be correlated to changes in plasma volume, r = -0.98. This study found that plasma volume is reduced while the rate pressure product is increased during the squat lift exercise.

Physiological and performance characteristics of United States championship class orienteers.

The purpose of this investigation was to study the anaerobic and aerobic power of female and male United States championship orienteers and relate these data to competitive performance. In addition, it was considered valuable to obtain a general physical description of the athletes. Anthropometrically, neither the females (n=5) nor the males (n=13) conformed to a somatotype classification typical of endurance athletes. Although the mean anaerobic power values for the females (76.6 kg.m.sec-1) and the males (106.6 kg.m.sec-1) were consistent with what is known for distance runners, the respective aerobic powers of 46.1 and 61.6 ml.kg-1.min-1 were considerably below expectations for national class competitors. The explanation for this latter finding was attributed to their reported training programs. Partial residual plots and multiple regression procedures were used to determine the relationship between aerobic power and gender, respectively, with speed of competitive performance. The final equation to predict performance was P = -13.10 + 0.62(VO2max) + 0.26 (Experience, years) - 0.03 (Anaerobic power) + 1.21 (gender) - 0.01 (VO2max)2 with an R-square of 0.73 and P less than 0.01.

Effects of dehydration on isometric muscular strength and endurance.

To examine the effects of rapid dehydration on isometric muscular strength and endurance, seven men were tested at baseline (control) and after a dehydration (dHST) and a euhydration (eHST) heat stress trial. The dHST consisted of intermittent sauna exposure until 4% of body mass was lost, whereas the eHST consisted of intermittent sauna exposure (same duration as dHST) with water replacement. Peak torque was determined for the knee extensors and elbow flexors during three isometric maximal voluntary contractions. Time to fatigue was determined by holding a maximal voluntary contraction until torque dropped below 50% peak torque for 5 s. Strength and endurance were assessed 3.5 h after the HSTs (no food or water intake). Body mass was decreased 3.8+/-0.4% post dHST and 0.4+/-0.3% post eHST. Plasma volume was decreased 7.5+/-4.6% and 5.7+/-4.4%, 60 and 120 min post dHST, respectively. A small (1.6 mEq x L[-1]) but significant increase was found for serum Na+ concentration 60 min post dHST but had returned to predehydration level 120 min post dHST. Serum K+ and myoglobin concentrations were not affected by HSTs. Peak torque was not different (P > 0.05) among control, dHST, and eHST for the knee extensors (Mean (Nm)+/-SD, 285+/-79, 311+/-113, and 297+/-79) and elbow flexors (79+/-12, 83+/-15, and 80+/-12). Time to fatigue was not different (P > 0.05) among control, dHST and eHST for the knee extensors (Mean (s)+/-SD. 42.4+/-11.5, 45.3+/-7.6, and 41.8+/-6.0) and elbow flexors (48.2+/-8.9, 44.0+/-9.4, and 46.0+/-6.4). These results provide evidence that isometric strength and endurance are unaffected 3.5 h after dehydration of approximately 4% body mass.

Venous and fingertip blood to calculate plasma volume shift following exercise.

This study determined whether fingertip blood samples used to calculate percentage change in calculated plasma volume following exercise were in agreement with values obtained from venous blood samples. Twenty-five subjects engaged in two cycle ergometer exercises at 100 and 200 W, with percentage plasma volume shift (% PVS) determined after each from venous (VB) and fingertip (FT) blood. Values for % PVS were FT -6.25% compared with VB -8.04% (P less than 0.05), with the correlation between the two methods at r = 0.88. The following equation was established: corrected FT % PVS = (0.8662 * FT) - 2.625; SEE = 2.60%. In order to cross-validate this equation, fifteen additional subjects submitted to VB and FT. Corrected FT % PVS using the established equation resulted in a mean value of 9.53 compared with 10.53% for actual VB % PVS. Although these means were not significantly different, there was approximately a 25% chance that the corrected FT % PVS would be more than one standard error of estimate from the regression line. It was concluded that FT underestimates VB % PVS. However, when limited to group data, FT can be corrected to favorably represent VB % PVS following moderate to heavy cycle ergometer exercise.

Effect of physostigmine on plasma lactate and pyruvate in untrained/trained rats.

Physostigmine (Phy) is metabolized to eseroline, a phenolic compound that appears to alter mitochondrial functions. The effect of Phy on recovery from exercise and on time course of plasma lactate and pyruvate levels following an acute bout of exercise (AE) was examined in untrained and trained (ET) rats. Phy alone elicited significantly higher plasma lactate and pyruvate levels than sedentary control. AE + Phy had a significantly higher plasma lactate and pyruvate levels compared to AE 2 min postadministration. From 5-30 min postexercise, lactate and pyruvate levels did not differ between these two acutely exercised groups. ET + Phy exhibited significantly lower levels of plasma lactate and pyruvate from 5-60 min postexercise compared to ET. The data show that the "additive" effect of Phy on postexercise plasma lactate and pyruvate levels can be attenuated by an enhanced fitness level in these rats.

Effect of concurrent exercise and physostigmine on lactate and pyruvate in plasma, muscle, and brain tissue of rats.

The purpose of this investigation was to determine the effect of physostigmine (Phy) and/or concurrent exercise on lactate, pyruvate, and L/P ratio in plasma, skeletal muscle, and brain tissue in male Sprague-Dawley rats. The Phy-dosed (Phy-D) and Phy-dosed + concurrent acute exercise (Phy-D + CAE) groups elicited significantly higher L/P ratios in plasma compared to the acutely exercised (AE) group at 30 min postexercise. Physostigmine dosing, with or without exercise, resulted in significantly lower muscle pyruvate levels, from 30 to 50 min postdrug administration, in Phy-D and Phy-D + CAE groups compared to the AE group. In the brain, lactate values were significantly elevated in the acutely exercised groups at 5 min postexercise with or without Phy dosing. However, at 15 to 30 min postexercise, lactate values were significantly elevated in the Phy-D + CAE compared to the AE group. These data suggest that when Phy is administered prior to a 20-min moderately intensive exercise bout, there is an accumulation of lactate for a prolonged period of time in recovery.