Acceptance of oral health promotion programmes by dental hygienists and dental nurses in public dental service.

OBJECTIVES: The aim of this study was to analyse how dental hygienists and in-service trained dental nurses accepted new health promotion programmes, how did they experience them in practice, and how did these programmes affect their attitudes to work. METHODS: The subjects were all the dental hygienists and in-service trained dental nurses (n = 28) involved in health promotion of small children. Education and written instructions on two new programmes had been given to the professionals in two areas of Vantaa and those in the third area used the routine programme. The transtheoretical model (TTM) was selected as the theoretical framework for counselling. A structured questionnaire of 31-35 items was sent to all subjects. Independent samples Mann-Whitney U and Fisher's exact tests were used as statistical methods. RESULTS: The response rate was 89%. All respondents felt that the work they had carried out had always been important during their working career. Twenty-one of 25 respondents reported that the instructions and education were suitable for oral health promotion. The respondents within the new programmes felt they had advanced more as health professionals (P = 0.020) and acquired more confidence from the education (P = 0.018) compared with the routine programme. CONCLUSIONS: The new programmes for small children were well accepted by the dental hygienists and the in-service trained dental nurses, and the majority of them gained some new practices for their work.

Arthroscopic anterior cruciate ligament reconstruction using a hamstring graft with interference screw fixation.

BACKGROUND AND AIMS: This study assessed the clinical and radiographic results 3 to 8 years after an arthroscopic ACL reconstruction using a hamstring graft with interference screw fixation on femoral and tibial sites. MATERIAL AND METHODS: Seventy-six of 100 patients, who underwent an arthroscopic ACL reconstruction, could be re-examined with a mean follow-up of 5 years. RESULTS: The mean Tegner activity score was 4 (scale: 0 to 10) before surgery, and 5 at the follow-up. Fifty-four patients (78%) achieved a satisfactory result in the IKDC rating. The mean Lysholm score was 85 (scale: 0 to 100), the score being better in patients without meniscal resection than those with (P = 0.04). The mean side-to-side difference in anterior-posterior knee laxity was 1.8 mm, and 62 patients (90%) had good stability. Sixty-four patients (93%) had normal range of motion of the knee. Twelve patients (46%) with meniscal resection had osteoarthritic changes, compared with only 8 of those (20%) without meniscal resection (P = 0.03). CONCLUSIONS: This medium-term follow-up study showed that good stability and function and normal range of motion of the knee can be achieved in majority of the patients who have undergone an ACL reconstruction with a hamstring graft and interference screw fixation.

Lake eutrophication and brownification downgrade availability and transfer of essential fatty acids for human consumption.

Fish are an important source of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for birds, mammals and humans. In aquatic food webs, these highly unsaturated fatty acids (HUFA) are essential for many physiological processes and mainly synthetized by distinct phytoplankton taxa. Consumers at different trophic levels obtain essential fatty acids from their diet because they cannot produce these sufficiently de novo. Here, we evaluated how the increase in phosphorus concentration (eutrophication) or terrestrial organic matter inputs (brownification) change EPA and DHA content in the phytoplankton. Then, we evaluated whether these changes can be seen in the EPA and DHA content of piscivorous European perch (Perca fluviatilis), which is a widely distributed species and commonly consumed by humans. Data from 713 lakes showed statistically significant differences in the abundance of EPA- and DHA-synthesizing phytoplankton as well as in the concentrations and content of these essential fatty acids among oligo-mesotrophic, eutrophic and dystrophic lakes. The EPA and DHA content of phytoplankton biomass (mgHUFAg-1) was significantly lower in the eutrophic lakes than in the oligo-mesotrophic or dystrophic lakes. We found a strong significant correlation between the DHA content in the muscle of piscivorous perch and phytoplankton DHA content (r=0.85) as well with the contribution of DHA-synthesizing phytoplankton taxa (r=0.83). Among all DHA-synthesizing phytoplankton this correlation was the strongest with the dinoflagellates (r=0.74) and chrysophytes (r=0.70). Accordingly, the EPA+DHA content of perch muscle decreased with increasing total phosphorus (r2=0.80) and dissolved organic carbon concentration (r2=0.83) in the lakes. Our results suggest that although eutrophication generally increase biomass production across different trophic levels, the high proportion of low-quality primary producers reduce EPA and DHA content in the food web up to predatory fish. Ultimately, it seems that lake eutrophication and brownification decrease the nutritional quality of fish for human consumers.

Human spleen cysteineproteinase inhibitor. Purification, fractionation into isoelectric variants and some properties of the variants.

The papain inhibitor from human spleen was purified by extraction in isotonic sucrose, acetone fractionation, papain-Sepharose affinity chromatography and gel filtration on Sephadex G-50. The purified inhibitor was fractionated by electrofocusing into four major isoelectric variants with pI values of 4.7, 5.0, 6.0 and 6.5. These variants can be classified into two groups: the acidic type, comprising the variants with pI 4.7 and 5.0, and the neutral type, comprising the variants with pI 6.0 and 6.5. The following properties distinguish the two types: 1. Immunological properties: antibodies raised against either of the neutral variants precipitated both of these, but not the acidic variants. The antiserum against the human epidermal cysteineproteinase inhibitor precipitated the acidic variants, but not the neutral variants. 2. Molecular size: two-dimensional electrophoresis of the purified inhibitor gave molecular weights of 11400 for the acidic variants and 12000 for the neutral variants. The pI 6.0 variant contained two compounds with molecular weights of 12000 and 12800. 3. Enzyme spectrum: human cathepsin B was inhibited by the acidic type, while the neutral type was a poor inhibitor. Both types inhibited cathepsin H, papain, ficin and bromelain, although the inhibition of bromelain did not exceed 70%. Human cathepsin D, bovine trypsin and chymotrypsin and porcine elastase were not inhibited by either type.

Precision of measurement by dual-energy X-ray absorptiometry of bone mineral density and content in rat hindlimb in vitro.

Dual-energy x-ray absorptiometry (DXA) is a precise and effective method for measuring bone mineral density (BMD) and content (BMC) at various skeletal sites in the human, but its precision for measuring low BMD and BMC in tiny rat bones has scarcely been evaluated. The short-term precision of DXA (Norland XR-26) for eight regions of interest (ROI) was determined by measuring dissected, not totally defleshed hindlimbs from four rats of differently weights 10 times consecutively. The accuracy of the BMD (area) measurement was also evaluated. The following average precision values (coefficients of variation) with negligible reanalysis error were obtained for BMD and BMC measurements: 0.7 and 1.2% (total); 0.9 and 1.2% (femur); 1.3 and 2.8% (tibia); 1.3 and 2.1% (foot); 2.1 and 4.7% (proximal femur); 1.4 and 2.9% (femoral shaft); 1.8 and 5.6% (distal femur); and 2.7 and 7.6% (proximal tibia). As for the accuracy, BMD measured in tiny bones is likely to be markedly underestimated. We conclude that the standard DXA technique without additional modification for small animal measurements is a precise method for BMD and BMC measurements at multiple sites in rat hindlimb in vitro.

Osteoporosis in men with a history of tibial fracture.

The long-term effects of bone fractures on bone mineral density (BMD) at various skeletal sites are poorly established, although a serious fracture, such as a tibia fracture, followed by long immobility and disuse may lead to permanently decreased BMD and, through this mechanism, may be a risk factor for osteoporotic fractures in later life. To determine whether such an injury leads to osteoporosis, we measured the areal BMD (g/cm2) from the lumbar spine (L2-4), right distal radius and ulna, and the femoral neck, distal femur, patella, proximal tibia, distal tibia, and calcaneus of both extremities in 14 men with a history of primarily nonunited (finally bone-grafted) shaft fracture of the tibia and 20 men with a history of primary union. For evaluation of the patients' BMD in the spine and distal forearm, 22 age-, weight-, and height-matched normal men were also measured. The average time of immobilization in a long plaster cast was 27 weeks in the former group of patients and 16 weeks in the latter. The measurements were performed an average of 9 years after the fracture using a dual-energy x-ray absorptiometric scanner. Compared with normal men (mean +/- SD = 1.116 +/- 0.160), the spinal BMDs were significantly lower in men with a history of a primary nonunion (0.979 +/- 0.100, -12.3%) and union (1.010 +/- 0.124, -9.5%). In distal radius and distal ulna, there were no significant differences between the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

A tibial shaft fracture sustained in childhood or adolescence does not seem to interfere with attainment of peak bone density.

High peak bone mass or density in early adulthood is an important protective factor against osteoporotic fractures in later life, but it is not known whether injuries on growing bones affect the attainment of peak bone mass and density. The purpose of this study was therefore to examine with dual-energy X-ray absorptiometry the areal bone mineral density (BMD) of the injured and uninjured extremity (the femoral neck, trochanter area of the femur, distal femur, patella, proximal tibia, and distal tibia), lumbar spine, and distal radius of young adults with a history of early life tibial shaft fracture and to find out whether the fracture had affected the attainment of peak bone density of these patients. The second objective was to clarify whether any background or clinical follow-up variable would predict the BMD difference between the affected and unaffected extremity. Thus, the BMD and clinical status of 45 patients (34 men and 11 women), who had sustained a tibial shaft fracture in childhood or in adolescence (between 7 and 15 years of age) an average 11 years before the study, were examined. The results showed that the fracture had created a small but statistically significant injured-to-uninjured side BMD difference (proximal tibia -1.7%; p = 0.011, and distal tibia 2.6%; p = 0.014), while the other sites showed no significant side-to-side differences. There were neither significant differences in the spinal or radial BMDs between the patients and their age-, height-, and weight-matched healthy controls. A further analysis of the data showed that the better the muscle strength in the injured lower limb, the lower the side-to-side BMD deficit in the proximal tibia of the same limb (r = 0.51; p < 0.001). Smoking had a significant association with the relative BMD in the injured distal tibia (mean injured-to-uninjured side BMD difference: smokers 6.1% vs. nonsmokers -0.6%, p = 0.016). Also patient's age at the time of the injury showed an association: the younger the patient at the time of the injury, the lower the side-to-side BMD deficit in the injured distal tibia (r = -0.35; p = 0.048). In conclusion, this study indicates that early life tibial fracture leads to a small long-term BMD deficit in the fractured bone while the other sites of the skeleton seem not to be affected. Thus, a tibial shaft fracture sustained in childhood or adolescence seems to only marginally interfere the attainment of peak bone density, the important predictor of the osteoporotic fractures in later life.

A rotator cuff rupture produces permanent osteoporosis in the affected extremity, but not in those with whom shoulder function has returned to normal.

Areal bone mineral density (BMD) and clinical status of 34 men treated surgically 9 years earlier for a rotator cuff rupture of the dominant side shoulder were determined. The BMD was measured at the lumbar spine (L2-L4) and the proximal humerus, humeral shaft, radial shaft, ulnar shaft, distal forearm, and hand of both extremities using a dual-energy X-ray absorptiometric (DXA) scanner. Thirty-four age-, height-, weight-, and profession-matched normal men (controls) were also measured. The patients' mean side-to-side BMD difference (dominant minus nondominant/nondominant x 100%) was significantly lower in the proximal humerus (patients -3.5% vs. controls +2.4%, p = 0.0002), humeral shaft (-2.6% vs. +1.6%, p = 0.0005), radial shaft (-0.4% vs. +1.9%, p = 0.0311), distal forearm (-0.2% vs. +2.4%, p = 0.0158), and hand (+2.3% vs. +4.0%, p = 0.0047). In the ulnar shaft, this difference was almost the same in the patients (-0.2%) and controls (+0.2%) (NS). Also, the lumbar spine BMD did not differ significantly between these groups (mean +/- SD = 1.098 +/- 0.148 g/cm2 in patients vs. 1.066 +/- 0.156 g/cm2 in controls). In patients, the relative BMDs of the injured extremity did not significantly associate with the size of the rupture; time delay between the injury and the surgery; type of surgery and postoperative treatment; postoperative immobilization time; follow-up time; patient's age, muscle strength or pain assessment; and subjective assessment of shoulder function. However, they strongly associated with the objective assessment of the shoulder function: the better the observed function of the shoulder, the less bone loss caused by the injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced bone mineral density in men with a previous femur fracture.

This study determined the areal bone mineral density (BMD) from the lumbar spine (L2-4), right distal radius and ulna, and the femoral neck, trochanter area of the femur, distal femur, patella, proximal tibia, and calcaneus of both extremities in 29 men who had a femur shaft fracture 10 years earlier. For evaluation of the patients' BMDs in the spine and distal forearm, 29 age-, weight-, and height-matched normal men were also measured. Compared with normal men (mean +/- SD = 1.123 +/- 0.153), the spinal BMDs of the patients were significantly (p = 0.0054) lower (1.018 +/- 0.119, -9.3%). Distal radius and distal ulna showed no significant group differences. In patients, the mean BMD of the injured extremity (compared with the uninjured side) was significantly lower in the distal femur (-6.8%; p = 0.0000), patella (-5.4%; p = 0.0000), proximal tibia (-4.7%; p = 0.0000), and calcaneus (-2.2%; p = 0.0259). In the proximal femur, this value was at the same level (femoral neck 1.3%, NS) or higher (trochanter area 6.3%, p = 0.0002) than that in the uninjured extremity. The relative BMDs of the injured extremity did not associate with the fracture type, fracture location, age, muscle strength, follow-up time, or non-weight-bearing time but showed significant (r = 0.33-0.64) positive correlation with low pain assessment and high functional scores of the injured extremity.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of osteocalcin in the patella of experimentally immobilized and remobilized rats.

The exact mechanisms by which mechanical loading-unloading affects bone tissue are mostly unknown. Recently, osteocalcin, a direct product of osteoblasts, has been shown to reflect the activity of the mineralization phase of the newly formed bone matrix, and therefore, the in situ detection of osteocalcin could be used for studying the effects of physical activity-inactivity on the osteoblast function or bone formation in the target bone. In this study, the effect of various loading states (immobilization and three forms of subsequent remobilization) on the in situ expression of osteocalcin in the rat patellas and their osteotendinous junctions was studied immunohistochemically using a polyclonal rat antiosteocalcin as the primary antibody. Following immobilization for 3 weeks, the immunoreactivity of osteocalcin was markedly decreased or was completely absent in all the patellar areas which normally show intense reaction as a sign of mineralization of the newly formed bone, that is, in the subperiosteal and subchondral regions, in the osteoid tissue that lies on the surface of the trabecular bone, and around the cortical lacunae. The same was true in the mineralized fibrocartilage zone of the osteotendinous junction of the quadriceps tendon. Free remobilization for 8 weeks (free cage activity) could not improve the situation, but after intensified remobilization of the same duration (low and especially high intensity treadmill running) high osteocalcin expression was observed in the above-mentioned anatomic sites. These findings indicate that formation of new bone tissue is rapidly regulated by the loading states of the bone. Higher than normal activity seems to be needed to restore the bone formation from the disuseinduced depression to normal.

Randomized controlled study of effects of sudden impact loading on rat femur.

Physical loading creating high peak strains on the skeleton at high strain rates is suggested to be the most effective type of activity in terms of bone mineral acquisition. This study assessed the effects of sudden impact loading on mineral and mechanical bone properties in 13-week-old Sprague-Dawley rats. The rats were randomly assigned as sedentary controls (SED, n = 10), control animals receiving low-intensity exercise (EX, n = 15), and experimental animals receiving low-intensity exercise combined with sudden impact-loading (EX + IMP, n = 15). In the EX group, the rats walked in a walking mill at a speed of 10 cm/s for 20 minutes/day, 5 days/week for 9 weeks. In the EX + IMP group, the program was identical to the EX group except for the additional sudden impacts administered to their skeleton during the walking exercise. At the start, there were 50 impacts per session, after which their number was gradually increased to 200 impacts per session by week 6 and then kept constant until the end of the experiment, week 9. These horizontally and vertically directed body impacts were produced by a custom-made walking mill equipped with computer-controlled high-pressure air cylinders. After sacrifice, both femora of each rat were removed and their dimensions, bone mineral content (BMC) by dual-energy X-ray absorptiometry, and mechanical properties by femoral shaft three-point bending and femoral neck compression were determined. The cortical wall thickness increased significantly in the EX and EX + IMP groups as compared with SEDs (+7.6%, p = 0.049 and +10%, p = 0.020, respectively). The EX + IMP group showed +9.0% (p = 0.046) higher cross-sectional moment of inertia values than the EX group. No significant intergroup differences were seen in the BMC values, while the breaking load of the femoral shaft (EX + IMP vs. SED +8.8%,p = 0.047) and femoral neck (EX + IMP vs. SED +14.1%, p = 0.013) was significantly enhanced by the impact loading. In conclusion, this study indicates that mechanical loading can substantially improve the mechanical characteristics of a rat femur without simultaneous gain in its mineral mass. If this is true in humans too, our finding gives an interesting perspective to the numerous longitudinal exercise studies (of women) in which the exercise-induced gains in bone mass and density have remained mild to moderate only.

A cruciate ligament injury produces considerable, permanent osteoporosis in the affected knee.

Bone mineral density (BMD) and clinical status of 42 patients treated surgically 10-11 years earlier for an acute knee ligament injury were determined. The BMD was measured at the spine (L2-4) and the femoral neck, distal femur, patella, proximal tibia, and calcaneus of both lower extremities using dual-energy x-ray absorptiometric (DXA) scanner. The relative BMD results of the injured knee were correlated with sex, age, activity level, knee stability, and functional scores of the patient. In the 11 patients with moderate injury (isolated rupture of the medial collateral ligament), the BMDs of the injured and uninjured extremities were equal. In the 31 patients with severe injury (cruciate ligament rupture), the BMDs were significantly lower in the injured knee: distal femur, -6.0% (p = 0.0000); patella, -9.0% (p = 0.0000); and proximal tibia, -3.3% (p = 0.0012). Neither the femoral neck nor the calcaneus showed any differences. There were no significant differences either between men and women or between patients with different activity levels. The relative BMDs of the injured knee did not correlate with age or static knee stability but correlated significantly (r = 0.42-0.78, p < 0.01-0.001) with the functional scores of the same knee: the better the knee function in comparison with the healthy knee, the higher the relative BMD. The spinal BMDs corresponded with the age-adjusted reference values of the used densitometry. In conclusion, a severe knee ligament injury results in permanently decreased BMD in the injured knee. Other parts of the same extremity and lumbar spine are not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of free mobilization and low- to high-intensity treadmill running on the immobilization-induced bone loss in rats.

After an immobilization period of 3 weeks, the effects of free remobilization (8 weeks) as well as low- and high-intensity treadmill running on the bone mineral content (BMC) and density (BMD) of the hindlimbs of Sprague-Dawley rats (n = 70) were studied using a dual-energy x-ray absorptiometric scanner. In the low-intensity running program, the rats were allowed to move freely in the cage for 1 week, after which they started to run on a treadmill twice a day for 7 weeks. The speed of the treadmill was 20 cm/s, with an uphill inclination of 10 degrees. The running time was gradually increased from 20 minutes per session to 45 minutes per session. In the high-intensity group, the program was similar, with the exception that the speed of the treadmill was 30 cm/s, with an uphill inclination of 30 degrees. Immobilization for 3 weeks produced a significant BMC and BMD loss in the immobilized left femur and tibia (mean loss 9.6%, p < 0.001) but did not affect the right free limbs. Both low- and high-intensity running restored mineral content in the immobilized limb; however, an average 5% difference (p < 0.05) in mineral content of the right and left limb bones persisted. In the running groups, the values for the immobilized left limbs were at the same level or exceeded (range 3.8-11.6%, p < 0.05-0.01) and those of the free right limbs exclusively exceeded (range 5.3-15.9%, p < 0.05-0.01) the corresponding values of the age-matched control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of immobilization, three forms of remobilization, and subsequent deconditioning on bone mineral content and density in rat femora.

Disuse is associated with bone loss, which may not be recoverable. It is not known whether intensified remobilization is beneficial in restoring disuse-related bone loss nor if any such benefit would depend upon continuing mobilization for its maintenance. After an immobilization period of 3 weeks, the effects of free remobilization (11 weeks), and low-and high-intensity treadmill running (11 weeks) with and without subsequent deconditioning (18 weeks) on the bone mineral content (BMC) and density (BMD) of the hindlimb femora of Sprague-Dawley rats (n = 98) were studied using a dual-energy X-ray absorptiometric (DXA) scanner. Our hypothesis was that intensified remobilization is beneficial in restoring the BMC and BMD from disuse to normal while subsequent deconditioning is deleterious to these parameters. Immobilization for 3 weeks produced a significant BMC and BMD loss in the immobilized left femur (range -4.4 to -12.8%; p < 0.05-0.001). In the groups with free remobilization (free cage activity), the body weight-adjusted BMCs and BMDs always remained below those in the controls (range -2.3 to -12.1%; p values ranging from NS to < 0.01). Both low- and high-intensity running restored BMC and BMD in the immobilized limb, the effect being better in the latter group. In both of these groups, the values of the immobilized left limbs and those of the free right limbs exclusively exceeded the corresponding values of the age-matched control rats (left limb values 3.0-21.1% higher with p values ranging from NS to < 0.01; right limb values 7.9-21.4% higher with p < 0.05-0.01). However, after the deconditioning period of 18 weeks, the above described beneficial effects of low- and high-intensity running were lost, the left and right limb BMC and BMD values being lower than those in the age-matched controls (range -3.8 to -8.7%; p values ranging from NS to < 0.05). In conclusion, this study clearly indicates the need for greater than normal activity to restore the BMC and BMD after disuse to normal levels. However, the benefits of intensified remobilization are lost if the activity is terminated, and therefore, after immobilization and disuse, bone loading activities should be continued, perhaps indefinitely.

Tumor promoter and fibronectin induce actin stress fibers and focal adhesion sites in spreading human erythroleukemia (HEL) cells.

The effects of plasma fibronectin (pFn) and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on adhesion and cytoskeletal organization of human erythroleukemia (HEL) cells were studied. HEL cells, that normally grow in suspension, attached rapidly on pFn-coated growth substratum and some cells showed spreading. Upon exposure to TPA most of the cells adhered and showed some degree of spreading also when plated on plastic. The spread cells showed mostly peripheral accumulations of F-actin in addition to actin fibers seen in some of the cells. When the cells were plated in the presence of TPA on pFn or on pFn-fragments, containing the cell binding site, all the cells adhered rapidly, spread extensively, organized prominent F-actin stress fibers and typical ventral plaques of vinculin and alpha-actinin. Both proteins were revealed also in the suspended cells by Western blot analysis. When plated on substratum coated with other pFn-fragments or laminin, the HEL cells did not adhere or spread. Both adhesion on pFn as well as formation of stress fibers in the presence of TPA could be prevented by the synthetic peptide Arg-Gly-Asp-Ser (RGDS). HEL cells were also able to organize typical ventral fibrillar arrays of Fn. Immunostaining and metabolic labeling experiments showed that the cells did not contain or synthesize Fn, indicating that the plaques were formed from exogenous pFn by the cells. The results suggest that Fn and TPA synergistically induce the organization of the actomyosin system in HEL cells by promoting the formation of prominent actin stress fibers and focal adhesion sites.

Purification and some characteristics of the human epidermal SH-protease inhibitor.

An inhibitor of papain and other SH-proteases was purified 520-fold from human epidermis extracts by acetone fractionation, heat treatment, papain-Sepharose affinity chromatography, and Sephadex G-50 chromatography. The purified inhibitor had a molecular weight of 12,600 and contained no hexose, as tested by the anthrone reaction. The inhibitor survived in a boiling water bath, in 5% trichloroacetic acid, 20 mM Na3PO4 (pH 12.1) and 4 M NH4OH (pH 11.9). By isoelectric focusing 2 major activity peaks with pI's of 4.6 and 4.8, and a minor peak with a pI of 4.9 was fractioned, and 3 corresponding protein bands were seen after analytical isoelectric focusing. Immunization of rabbits with the purified inhibitor yielded a highly specific anti-inhibitor serum. The purified inhibitor inhibited papain, ficin, human cathepsins B and C, and slightly inhibited bromelain. No inhibition of serine proteases (bovine trypsin and chymotrypsin A, porcine elastase) or an acid protease (human cathepsin D) was observed. Evidence was obtained that the inhibitor formed a complex with both dithiothreitol-activated papain and enzymatically inactive mercuripapain.

The low-molecular-weight SH-protease inhibitor in rat skin is epidermal.

The epidermis and dermis of rat skin were separated and the presence of the high-molecular-weight SH-protease inhibitor I1 and the low-molecular-weight inhibitor I2 in both was studied. Gel filtrations of the extracts revealed that 97% of the epidermal inhibitor activity was due to I2 and 89% of the dermal activity to I1. The presence of I2 mainly in the epidermis extract was confirmed by immunodiffusion of specific rabbit anti-I2 serum against purified I2, epidermis and dermis extracts, and rat serum. Most of the immunoreactive protein was seen in the epidermis extract, traces in the dermis extract and none in the rat serum. I2 was localized in rat skin by indirect immunofluorescence using rabbit anti-I2 serum and fluorescein isothiocyanate conjugate of goat anti-rabbit immunoglobulins. Intense fluorescence, much brighter than in the controls treated with rabbit non-immune serum, was seen in the epidermis, being most pronounced in the cytoplasms of cells in the granular layer. The weak fluorescence of the hair follicles, sebaceous glands, connective tissue cells and fibres was unspecific and was also seen in the controls. In view of its epidermal location, the name epidermal SH-protease inhibitor is suggested for I2.

A rat skin acid esteroprotease hydrolyzing benzoylarginine ethylester: purification and properties.

A benzoylarginine ethylester hydrolyzing enzyme from rat skin has been purified 54-fold by chromatography on arginine methylester-CH-Sepharose and Sephadex G-200 prior to isoelectrofocusing. The molecular weight of the enzyme is approximately 125,000. The enzyme hydrolyzes bensoylarginine ethylester, acetyltyrosine ethylester, benzoylarginine methylester, benzoyllysine methylester, and benzoylalanine methylester. Casein is slightly hydrolyzed at an optimum pH of 6.5. The enzyme is inhibited by diisopropylfluorophosphate, and p-chloromercuribenzoic acid. KC1 enhances the extraction of the enzyme, increased its activity, and is essential for its stability.

Purification and biochemical characterization of rat skin cathepsin D.

The hemoglobin-hydrolyzing, acidic proteinase activity of rat skin was purified by using ammonium sulfate precipitation. Sephadex G-100 gel column chromatography, acid treatment, and DEAE-cellulose column chromatography, giving a purification coefficient of 182. The pH optimum, molecular size, substrate specificity, as well as inhibitor and activator sensitivity of the enzyme preparation, corresponded closely to those of cathepsin D. The enzyme activity was separated from cathepsin B1. The present status of the knowledge of skin cathespins is reviewed.

Partial purification and some properties of a new papain inhibitor from psoriatic scales.

A new papain inhibitor was purified from psoriatic epidermal scales using gel chromatography and anion exchange chromatography. The purified protein inhibited papain and ficin but not cathepsin B, cathepsin H, trypsin, or chymotrypsin. Isoelectric focusing revealed 3 major inhibitor variants with pI's of 7.3, 6.9, and 6.5. A Mr of 38,000 was obtained by a gel chromatographic method for the crude inhibitor. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr values of the isoelectric variants were: 43,000 for the variant pI 7.3, 43,000 and 35,000 for the variant pI 6.9, and 34,000-35,000 for the variant pI 6.5. An antiserum of the inhibitor was used to locate the inhibitor in the psoriatic and normal epidermis. In psoriatic epidermis, the inhibitor was found in the peripheral cytoplasm of spinous cells and in the scale. In normal epidermis, the staining was seen only in orifices of hair follicles. An inhibitor with similar size and antigenic properties to that isolated from the psoriatic scales was demonstrated in extracts made from the whole-thickness epidermis but not in extracts from the healthy epidermal scales, the dermis, the liver, the spleen, or the blood serum.