Prevalence of honeybee viruses in different regions of China and Argentina.

Honeybees are threatened by various pathogens and parasites. More than 18 viruses have been described in honeybees and many of them have been detected in China and Argentina. In China, both Apis cerana and Apis mellifera are raised. In Argentina, beekeepers raise different ecotypes of A. mellifera: European honeybees (in both temperate and subtropical regions) and Africanised honeybees (in subtropical areas only). A thorough study was carried out in both China and Argentina to analyse the current virus presence and distribution in different climatic zones and gather information on different bee species/subspecies. Adult honeybees were collected from apiaries in temperate and subtropical regions of China (including areas with exclusive populations of A. mellifera, areas where A. mellifera and A. cerana co-exist, and areas with exclusive populations of A. cerana) and Argentina. Six viruses, namely, deformed wing virus (DWV), black queen cell virus (BQCV), sacbrood virus (SBV), chronic bee paralysis virus (CBPV), acute bee paralysis virus (ABPV) and Israeli acute paralysis virus (IAPV) were detected in China, both in A. cerana and in A. mellifera, while four viruses (DWV, BQCV, CBPV and ABPV) were present in Argentina. Interestingly, multiple infections were commonly found in China, with up to five different viruses co-circulating in some colonies without apparent abnormalities. In this study, no Chinese samples were positive for slow bee paralysis virus. The most prevalent viruses were BQCV (China) and DWV (Argentina). Kashmir bee virus was absent from samples analysed for both countries.

Les populations d'abeilles mellifères sont menacées par de nombreux agents pathogènes et parasites. Parmi eux, 18 virus ont été décrits, dont plusieurs ont été détectés en Chine et en Argentine. Les espèces d'abeilles mellifères élevées en Chine sont Apis cerana et Apis mellifera. En Argentine, les apiculteurs élèvent plusieurs écotypes d'A. mellifera : le type européen dans les régions tempérées et subtropicales et le type africanisé dans les zones subtropicales. Une étude approfondie a été réalisée en Chine et en Argentine dans le but d'identifier les virus présents, d'analyser leur distribution dans différentes zones climatiques et de réunir des informations sur les différentes espèces et sous-espèces d'abeilles présentes. Des abeilles mellifères adultes ont été collectées dans des ruchers des régions tempérées et subtropicales de Chine (zones peuplées exclusivement d'A. mellifera ou d'A. cerana et zones où A. mellifera et A. cerana coexistent) et d'Argentine (A. mellifera seulement). En Chine, six virus, à savoir le virus des ailes déformées, le virus des cellules royales noires, le virus du couvain sacciforme, le virus de la paralysie chronique de l'abeille, le virus de la paralysie aiguë de l'abeille et le virus israélien de la paralysie aiguë ont été détectés aussi bien chez A. cerana que chez A. mellifera ; en Argentine, quatre virus ont été détectés (virus des ailes déformées, virus des cellules royales noires, virus de la paralysie chronique de l'abeille et virus de la paralysie aiguë de l'abeille). Fait intéressant, les infections multiples étaient fréquentes en Chine, avec parfois jusqu'à cinq virus différents circulant dans certaines colonies sans provoquer de manifestations anormales apparentes. Aucun des échantillons analysés en Chine n'a été trouvé positif pour le virus de la paralysie lente de l'abeille. Les virus les plus fréquents étaient, en Chine, le virus des cellules royales noires et en Argentine, le virus des ailes déformées. Le virus du Cachemire n'a été trouvé dans aucun des échantillons analysés dans les deux pays.

Las abejas melíferas están amenazadas por diversos patógenos y parásitos. Se han descrito más de 18 virus que las afectan, muchos de los cuales se han detectado en China y la Argentina. En China se cultivan tanto Apis cerana como Apis mellifera, mientras que los apicultores argentinos crían diferentes ecotipos de A. mellifera: abejas europeas en las regiones templadas y subtropicales y abejas africanizadas en las zonas subtropicales. Los autores exponen un minucioso estudio realizado a la vez en China y la Argentina con el fin de analizar la actual presencia y distribución de virus en diferentes zonas climáticas y reunir información sobre distintas especies y subespecies de abeja. En primer lugar se recogieron abejas adultas de colmenares situados en regiones templadas y subtropicales de China (algunas donde hay exclusivamente poblaciones de A. mellifera, otras donde coexisten A. mellifera y A. cerana y otras zonas que albergan solo poblaciones de A. cerana) y la Argentina (solamente A. mellifera). En las poblaciones chinas tanto de A. cerana como de A. mellifera se detectaron seis virus: virus de las alas deformes (VAD); virus de las celdas reales negras (VCRN); virus de la cría ensacada (VCE); virus de la parálisis crónica de la abeja (VPCA); virus de la parálisis aguda de la abeja (VPAA); y virus de la variante israelí del virus de la parálisis aguda (VPAI), mientras que en la Argentina se observó la presencia de cuatro virus (VAD, VCRN, VPCA y VPAA). Un dato interesante es que en China se observaron con frecuencia infecciones múltiples, con hasta cinco virus diferentes circulando a la vez en algunas colonias sin que ello diera lugar a anormalidades aparentes. Ninguna de las muestras chinas analizadas en el estudio resultó positiva al virus de la parálisis lenta de la abeja. Los virus más prevalentes fueron el VCRN (China) y el VAD (Argentina). El virus Cachemira de las abejas estaba ausente de las muestras analizadas en ambos países.

No 1,25-dihydroxyvitamin D3 receptors on osteoclasts of calcium-deficient chicken despite demonstrable receptors on circulating monocytes.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is known to stimulate osteoclastic bone resorption in vivo and whole organ bone culture systems in vitro. It has not been established whether 1,25(OH)2D3 acts directly on osteoclasts or whether its action on osteoclasts is mediated via other bone cells (e.g., osteoblasts) or recruitment of osteoclast precursor cells. Circulating monocytes have been characterized as osteoclast precursors. In the present study, vitamin D3-replete chicken on a calcium-deficient diet were studied. Circulating monocytes, whole bone cell preparations, and isolated osteoclasts (differential sedimentation) were examined for presence of 1,25(OH)2D3 receptors. Reversible, specific, and saturable binding of [3H]-1,25(OH)2D3 to a 3.5 S macromolecule was demonstrated in nuclear fractions of monocytes (maximal binding capacity, 48 fmol/mg protein; dissociation constant, 1.3 X 10(-10) M) and of whole bone cell preparations. 1,25(OH)2D3 receptors were not demonstrable in osteoclast preparations (70% pure; detection threshold, 2 fmol/mg protein). Data are consistent with indirect action of 1,25(OH)2D3 on osteoclastic bone resorption.

Identification and regulation of 1,25-dihydroxyvitamin D3 receptor activity and biosynthesis of 1,25-dihydroxyvitamin D3. Studies in cultured bovine aortic endothelial cells and human dermal capillaries.

Because 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to play roles in both proliferation and differentiation of novel target cells, the potential expression of 1,25(OH)2D3 receptor (VDR) activity was investigated in cultured bovine aortic endothelial cells (BAEC). Receptor binding assays performed on nuclear extracts of BAEC revealed a single class of specific, high-affinity VDR that displayed a 4.5-fold increase in maximal ligand binding (Nmax) in rapidly proliferating BAEC compared with confluent, density-arrested cells. When confluent BAEC were incubated with activators of protein kinase C (PKC), Nmax increased 2.5-fold within 6-24 h and this upregulation was prevented by sphingosine, an inhibitor of PKC, as well as by actinomycin D or cycloheximide. Immunohistochemical visualization using a specific MAb disclosed nuclear localized VDR in venular and capillary endothelial cells of human skin biopsies, documenting the expression of VDR, in vivo, and validating the BAEC model. Finally, additional experiments indicated that BAEC formed the 1,25(OH)2D3 hormonal metabolite from 25(OH)D3 substrate, in vitro, and growth curves of BAEC maintained in the presence of 10(-8) M 1,25(OH)2D3 showed a 36% decrease in saturation density. These data provide evidence for the presence of a vitamin D microendocrine system in endothelial cells, consisting of the VDR and a 1 alpha-hydroxylase enzyme capable of producing 1,25(OH)2D3. That both components of this system are coordinately regulated, and that BAEC respond to the 1,25(OH)2D3 hormone by modulating growth kinetics, suggests the existence of a vitamin D autocrine loop in endothelium that may play a role in the development and/or functions of this pathophysiologically significant cell population.

Environment or beekeeping management: What explains better the prevalence of honey bee colonies with high levels of Varroa destructor?

Varroa destructor is one of the major threats to honey bee colonies. The mite abundance in the colonies is affected by environmental conditions as well as by beekeeping management. The aim of this study was to recognize the main drivers associated with autumn V. destructor infestation in honey bee colonies when different regions from Argentina are compared. A total of 361 colonies distributed in five Argentinean eco-regions were examined to evaluate Varroa mite infestation rate during autumn and Nosema sp. presence. Regions were different regarding annual temperature, precipitation and especially vegetation landscape. In addition, beekeeping management practices were obtained from a checklist questionnaire answered by the beekeepers. The prevalence of colonies with high infestation level was lower in semi-arid Chaco followed by humid and transition Chaco regions. Also, colonies that were positive for Nosema sp. showed a higher Varroa infestation rate. The "environmental" effect was stronger compared with the influence of secondary drivers associated with beekeeping activities. As well, a significant association between V. destructor infestation rates and Nosema presence was identified. Under contrasting natural conditions, environment seems a predominant driver on Varroa destructor infestation level in honey bee colonies.

Hyperparathyroidism and abnormal calcitriol metabolism in the spontaneously hypertensive rat.

Abnormalities of calcium metabolism and of its two principal regulating hormones, parathyroid hormone and 1,25-dihydroxyvitamin D3 (calcitriol), have been reported in the spontaneously hypertensive rat (SHR). Reports of abnormal calcitriol metabolism in the SHR by several groups have not provided measurements of tissue calcitriol receptors. Similarly, few data are available as to the parathyroid status of the SHR. In the present study, circulating calcitriol levels and intestinal and parathyroid gland calcitriol receptor status were determined in male SHR and in Wistar-Kyoto (WKY) rats. Parathyroid status was investigated by determination of parathyroid gland mass together with tissue micromorphometry and by quantitative histology of bone as a measure of the biological action of parathyroid hormone. Circulating calcitriol levels were reduced in the 11-week-old SHR compared with the WKY rat (165 +/- 23 vs. 194 +/- 28 pmol/l, p less than 0.01, mean +/- SD). Calcitriol-free ratio was diminished and maximal specific binding capacity for calcitriol was increased in the SHR in parathyroid tissue (172 +/- 4.9 vs. 123 +/- 6.6 fmol/mg protein, p less than 0.01) and in intestinal mucosa with no change of receptor affinity. Plasma ionized calcium (1.29 +/- 0.05 vs. 1.45 +/- 0.35 mmol/l, p less than 0.05) and phosphate (1.5 +/- 0.26 vs. 2.4 +/- 0.03 mmol/l, p less than 0.05) were significantly lower in the SHR. Parathyroid gland mass was increased in the SHR (59 +/- 12 vs. 17 +/- 7 micrograms/100 g body wt, p less than 0.001) as a result of hyperplasia and not hypertrophy. Higher osteoclast numbers were observed in SHR bone (27.6 +/- 0.79 vs. 23.9 +/- 0.66 osteoclasts/mm2, p less than 0.01), suggesting increased parathyroid hormone activity. In summary, in the 11-week-old SHR we observed reduced circulating calcitriol levels together with increased tissue calcitriol receptor numbers, increased parathyroid gland mass, and histological evidence of hyperparathyroidism. It is possible that these abnormalities influence the development of hypertension in the SHR.

Immunohistochemical detection of 1,25-dihydroxyvitamin D3 receptors and estrogen receptors by monoclonal antibodies: comparison of four immunoperoxidase methods.

We developed an immunohistochemical method for visualization of vitamin D (VDR) and estrogen receptors (ER) in cryostat sections, using monoclonal antibodies (MAb) to the vitamin D receptor and estrogen receptor, respectively. This method is based on an avidin-biotin labeling technique (LAB). To establish a reliable and sensitive method which can be used easily as a routine diagnostic procedure, we systematically compared four different immunoenzymatic methods with respect to their efficiency in detecting vitamin D and estrogen receptors. Compared to the indirect bridged avidin-biotin (IBRAB), the peroxidase- anti-peroxidase (PAP), and the avidin-biotin complex (ABC) methods, the LAB method produced stronger staining intensities and had higher detection efficiency for both vitamin D and estrogen receptors. In addition, the LAB method had a higher spatial resolution compared to the ABC technique in detection of VDR in normal human skin biopsies. In the case of steroid receptors, i.e., nuclear antigens, immunohistochemistry must deal with a relatively low number of antigenic sites per cell, restricted accessibility of the antigens, and slight differences in antigen concentrations among cells. Under these particular conditions, the chemical properties of the conjugates used in the LAB method may explain why it is superior to the other methods. Consequently, the LAB method is recommended for visualization of ER and VDR.

Risk factors associated with the presence of Varroa destructor in honey bee colonies from east-central Argentina.

Varroa destructor is considered one of the major threats for worldwide apiculture. Damage caused by varroa mite includes body weight loss, malformation and weakening of the bees. It was also suggested as the main cause associated with colony winter mortality and as an important vector for several honey bee viruses. Little is known about multiple factors and their interaction affecting V. destructor prevalence in apiaries from South America. The aim of this study was to identify risk factors associated with V. destructor prevalence in east-central Argentina. Parasitic mite infestation level and colony strength measures were evaluated in 63 apiaries distributed in 4 different regions in east-central Argentina in a cross sectional study. Data regarding management practices in each apiary were collected by means of a questionnaire. A mixed-effects logistic regression model was constructed to associate management variables with the risk of achieving mite infestation higher than 3%. Colonies owned by beekeepers who indicated that they did not monitor colonies after mite treatment (OR=2.305; 95% CI: 0.944-5.629) nor disinfect hives woodenware material (OR=2.722; 95% CI: 1.380-5.565) were associated with an increased risk of presenting high intensity infestation with V. destructor (>3%). On the other hand, beekeepers who reported replacing more than 50% of the queens in their operation (OR=0.305; 95% CI: 0.107-0.872), feeding colonies protein substitute containing natural pollen (OR=0.348; 95% CI: 0.129-0.941) and feeding colonies High Fructose Corn Syrup (HFCS) (OR=0.108; 95% CI: 0.032-0.364), had colonies that were less likely to have V. destructor infestations above 3%, than beekeepers who did not report using these management practices. Further research should be conducted considering that certain management practices were associated to mite infestation level in order to improve the sanitary condition in the colonies. Epidemiological studies provide key information to design surveillance programs against one the major threat to worldwide beekeeping.

Demonstration and characterization of 1,25-dihydroxyvitamin D3 receptors in human mononuclear blood cells.

Circulating human mononuclear blood cells were studied for the presence of specific 1,25-dihydroxyvitamin D3 (calcitriol) binding macromolecules. Cells were isolated by density gradient centrifugation and characterized by surface markers. Specific reversible high affinity binding by a 3.5 S macromolecule was demonstrated in malignant B-cells and circulating monocytes. In monocytes specific calcitriol binding was found both in the presence and absence of vitamin D3 to saturate the vitamin D3 binding serum protein. No specific calcitriol binding was found in resting B or T lymphocytes. The data suggest a role of calcitriol in the control of mononuclear blood cell proliferation/differentiation.

Nuclear testicular 1,25-dihydroxyvitamin D3 receptors in Sertoli cells and seminiferous tubules of adult rodents.

1,25(OH)2D3 receptors were studied in whole testes, Sertoli cells, seminiferous tubules, Leydig cells and spermatogonia of adult NMRI mice and SD rats. Specific reversible high affinity binding (KD 1.4 x 10(-10)M; Nmax 72 fmol/mg protein) by a 3.5 S macromolecule was demonstrated in whole testes, Sertoli cells and seminiferous tubules. With identical techniques, no receptors were found in Leydig cells despite previous reports of 1,25(OH)2D3 actions on Leydig cell function.

Demonstration of 1,25(OH)2 vitamin D3 receptors and actions in vascular smooth muscle cells in vitro.

Binding of [3H] 1,25(OH)2D3 and effects of 1,25(OH)2D3 on cell ultrastructure were evaluated in vascular smooth muscle cells (VSMC) primary cultures (aortic media). Specific reversible binding of [3H] 1,25(OH)2D3 by a 3.5 S macromolecule with DNA binding, KD 6.2 X 10(-10) M and Nmax 16 fmol/mg protein was demonstrated. Incubation of VSMC with 10(-8) M 1,25(OH)2D3, but not 25(OH)D3, in the presence of 10% FCS for up to three weeks caused rapid reversible appearance in the cytoplasm of membrane-bounded electron-dense lysosomal particles which on electronspectroscopic imaging contained Ca and Pi. VSMC are targets for vitamin D.

Fluid volume distribution within superficial shell tissues along body axis during changes of body posture in man: the application of a new miniature plethysmographic method.

In up to six different sides along the body axis during tilting manoeuvres, volume shifts into or out off superficial tissues were followed with a newly developed miniature plethysmograph. It was possible to localize a region where no or only minor volume changes during the tilt table experiments occurred. This region is identical with the Hydrostatic Indifferent Point (HIP) being localized below the apex of the heart in the upper third of abdominal vena cava. Above the HIP fluid is drained out off the tissues during assumption of upright posture whereas below the HIP fluid volume is pooled. The volume changes occurred in two phases. Within the first 5 s in the cephalad parts of the body a rapid decrease occurred, thereafter the volume remained unchanged or even increased; below the HIP within the first 5 s a large volume increase was followed by a slow continuous volume increment. The functional peculiarities of the low pressure system as a whole were visible studying only superficial shell tissues of the body with the non invasive miniature plethysmographic technique.

No decrease of 1,25(OH)2D3 receptors and duodenal calbindin-D9k in uraemic rats.

In parathyroids of uraemic patients or animals, decreased specific binding of 1,25(OH)2D3 has been observed and implicated in the genesis of secondary hyperparathyroidism of renal failure. We re-examined binding of 1,25(OH)2D3 using chromatin preparations for receptor characterization which differed from previous studies (a) by inclusion of protease inhibitors (PMSF, aprotinin) and molybdate in the extraction buffer and (b) by omitting the K-extraction step. With this method, the Nmax in the intestinal mucosa and parathyroids of uraemic animals was significantly higher, while the receptor sedimentation constant (S), DNA affinity and KD were all unchanged. The ratio of occupied to total receptors was not significantly altered. The regulation of 1,25(OH)2D3 receptors in response to acute injection of 1,25(OH)2D3 was abnormal. Calbindin-D9k concentration in the intestines of uraemic and control rats was comparable both before and after administration of 1,25(OH)2D3. The present data demonstrate (a) increased 1,25(OH)2D3 receptors and (b) unchanged 1,25(OH)2D3-dependent synthesis of calcium binding protein (CaBP) in experimental uraemia.

Effects of 1,25(OH)2D3 on compensatory renal growth in the growing rat.

Renal compensatory growth after uninephrectomy (UNX) was examined in vitamin D replete male 100 g Sprague-Dawley rats. Five days after UNX, the contralateral kidney wet weight increased by 25% with the kidney weight/body weight ratio reaching a plateau by day 7 after UNX. The early weight increase was primarily due to an increased cell number, as evaluated by a stereological technique in perfusion-fixed kidneys. Twenty pmol 1,25(OH)2D3 by daily s.c. injection increased time-averaged 1,25(OH)2D3 concentrations 3.3-fold and reduced the increment in the kidney weight of UNX pairfed rats compared to solvent UNX controls. The number of mitoses (whole kidney and different nephron segments) were significantly reduced by giving 1,25(OH)2D3 to UNX animals at different levels of food intake. The effect was also demonstrable in PTX animals on a constant infusion of exogenous PTH (100 ng/kg/hr 1,34 bPTH by osmotic minipump). The data suggest that changes of 1,25(OH)2D3 concentration within a physiologically relevant range modulate compensatory (and possibly basal) growth of the kidney.

Diminished parathyroid 1,25(OH)2D3 receptors in experimental uremia.

In Sprague Dawley rats, six days after subtotal nephrectomy, serum 1,25(OH)2D3 concentration was diminished (59.8 +/- 17.5 pg/ml vs. 121 +/- 48; P less than 0.01). Despite low circulating 1,25(OH)2D3 levels, maximal specific binding capacity for 1,25(OH)2D3 in parathyroid glands was diminished (Nmax 87.5 fmol/mg protein and 3.52 fmol/mg DNA vs. 143 fmol/mg protein and 4.75 fmol/mg DNA, respectively). There was no change of KD, apparent molecular size (sucrose density gradient) and DNA binding affinity (DNA cellulose chromatography) pointing to intactness of the receptor. Since 1,25(OH)2D3 is a potent negative feedback signal for parathyroids, the data are potentially relevant for the genesis of secondary renal hyperparathyroidism.

Evidence for in vivo upregulation of 1,25(OH)2 vitamin D3 receptor in human monocytes.

Mammalian cells increase net expression of 1,25(OH) 2D3 receptors after exposure to physiological concentrations of 1,25(OH) 2D3 in vitro. We examined specific binding of 1,25(OH) 2D3 by human monocytes before and after daily administration of 1.5-2 micrograms 1,25(OH) 2D3 p.o. for 3 days in 5 healthy normal D-replete probands. Median specific binding (Nmax) at baseline was 793 molecules/cell and 2052 or 2828 at 24h and 72h of 1,25(OH) 2D3 treatment respectively. The results suggest (a) upregulation of 1,25(OH) 2D3 receptors occurs in man and (b) monocyte preparations can be used to assess receptor regulation in vivo.

1,25(OH)2 vitamin D3 inhibits parathyroid cell proliferation in experimental uremia.

Parathyroid cell proliferation and parathyroid hyperplasia are features of renal secondary hyperparathyroidism. Since parathyroids have recently been recognized as an important target for 1,25(OH)2D3, the effects of administration of variable doses of 1,25(OH)2D3 on ex vivo radiothymidine incorporation in the parathyroid glands, on parathyroid cell mitoses, on parathyroid weight, morphometric indices and on parathyroid protein/DNA ratio were examined in rats with uremia (subtotal nephrectomy; NX) or with calcium deficiency. 3H-thymidine incorporation (3 hr; 37 degrees C; PBS with 10 mmol glucose) was elevated in NX animals, that is, 204 +/- 51 dpm/micrograms DNA versus 96 +/- 28 in controls. In vivo pretreatment with 1,25(OH)2D3, either by intermittent i.p. injection or by osmotic minipump, dose-dependently decreased 3H-thymidine incorporation and parathyroid cell mitoses without affecting morphometric indices of parathyroid cells. Prophylactic administration (i.p.) of 1,25(OH)2D3, starting on the day of nephrectomy, prevented parathyroid hyperplasia (NX + 1,25(OH)2D3 0.84 micrograms tissue/g body wt vs. 1.25 micrograms in untreated NX and 0.54 in ad libitum fed controls), but 10 days of treatment beginning on the 21st day of uremia did not reverse existing hyperplasia (NX + 1,25(OH)2D3 1.5 micrograms/g body wt vs. 1.37 micrograms in untreated NX and 0.56 micrograms in ad libitum fed controls). The inhibitory effect was specific for 1,25(OH)2D3 and not imitated by Dexamethason. However, the effect was not specific for parathyroid hyperplasia of uremia, since similar inhibition of 3H-thymidine incorporation by 1,25(OH)2D3 was also observed in rats on low calcium diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue compliance in superficial tissues along body axis in man.

A previously described miniature plethysmograph which allowed the measurement of tissue volumes in superficial tissues was enclosed in a small plexiglass chamber and attached to the frontal area, sternum, dorsum and the tibia. The tissues interposed between bone and skin underneath the chamber were exposed to pressures between +/- 3 and +/- 15 mmHg in order to test tissue deformability. The pressure application induced within the first 5 s a fast component of tissue deformation comprising between 75-90% of the total deformation followed by a slow component which lasted till the end of the pressure application. The highest deformability was found in the tissues of the sternum and dorsum whereas the stiffest tissues were in the pretibial area. Assuming the tissue deformation is due to a translocation of fluid into or out of the pressurized tissue, the tissue compliance was calculated. This calculated tissue compliance was 19.2 ml . 1,000 ml-1 . mmHg-1 in the sternum and 6.4 ml . 1,000 ml-1 . mmHg-1 (P < 0.01) in the pretibial area applying a pressure of +/- 3 mmHg. The differences observed are due to the morphological arrangement of the tissue fibres which in turn have to counteract the gravity forces to which the tissues are usually exposed during upright standing.

A new miniature plethysmograph to measure volume changes in small circumscribed tissue areas.

With an ultrasonic method tissue layer thickness was measured in man in circumscribed superficial tissue areas where the underlaying bone provided good backwall echos. In a 5 mm tissue layer changes of +/- 0.2% could be reliably detected. Knowing the height of the tissue cylinder between the surface of the skin and the bone allowed to calculate the tissue volume. The ultrasonic probes could therefore serve as miniature plethysmograph. Several probes were attached in the frontal region, sternum, along the vertebral column and along the tibia simultaneously. Changes of the volume content of the superficial shell tissues were induced by orthostasis, water immersion and heat exposure. It was possible to assess quantitatively the volume shifts into or out of the superficial tissues. During orthostasis 166 ml of fluid left the superficial tissues of the cephalad parts of the body and 164 ml could be traced in the dependent parts. Heat exposure was followed by a pooling 140 ml in the tissues studied. The most pronounced tissue volume changes were observed in the forehead region during heat exposure.

Hyperparathyroidism and abnormal 1,25(OH)2 vitamin D3 metabolism in experimental lead intoxication.

Clinical evidence points to disturbed calcium metabolism in lead (Pb) intoxication. To further clarify the mechanisms involved, serum levels of 1,25(OH)2D3, receptors for 1,25(OH)2D3 as well as size and ultrastructure of parathyroid glands were examined in Wistar Kyoto rats exposed to 1% lead (Pb) acetate in drinking water for 10 weeks (short-term study) or 0.001-1% Pb acetate for 24 weeks (long-term study). After administration of Pb for 10 weeks, bone Pb was significantly increased (641 +/- 66.9 (SD) vs. 0.648 +/- 0.39 mg kg-1 ash in controls). Total serum calcium and ionized Ca2+ (1.15 +/- 0.031 vs. 1.25 +/- 0.03 mmol l-1) were significantly decreased. Renal function (Ccr) was unchanged, but urinary cAMP excretion and circulating 1,25(OH)2D3 (177 +/- 10.9 vs. 232 +/- 18.9 pmol l-1) were diminished. Specific binding of 1,25(OH)2D3 was increased in parathyroids (Bmax 128 +/- 4.7 vs. 108 +/- 0.6 fmol mg-1 protein) and intestinal muscosa; Bmax failed to adequately rise in response to pretreatment with 1,25(OH)2D3 (2 x 10 ng day-1 for 4 d) in Pb-exposed animals. Receptor characteristics (sedimentation constant, KD, DNA affinity) were unchanged. Parathyroid weight was significantly increased (178 +/- 25 vs. 96 +/- 34 micrograms) with no change of estimated nuclear volume, cell volume or cell ultrastructure. After 24 weeks of Pb exposure, a dose-dependent but non-linear increase of parathyroid weight was noted between 0.001% and 1% Pb in drinking fluid. The present study documents secondary hyperparathyroidism associated with, and presumably caused by, hypocalcaemia and low 1,25(OH)2D3 levels, in experimental Pb intoxication.

Demonstration and characterization of 1,25-dihydroxyvitamin D3 receptors in basal cells of epidermis of neonatal and adult mice.

Nuclear and cytosolic receptors for 1,25-dihydroxycholecalciferol [1,25(OH)2D3] were demonstrated in the epidermis of neonatal and adult mice. The macromolecular binding protein sedimented at 3.5 S (sucrose density gradient) and was distinct from the 6.0 S binding protein for 25-hydroxycholecalciferol [25(OH)D3]. Analysis at different ionic strengths suggested the presence of unoccupied nuclear receptors. Digestion with proteases or nucleases, respectively, and inactivation with alkylating agents demonstrated that the binding macromolecule is a protein with SH groups at the active site. Binding of 1,25(OH)2D3 was specific and reversible. In neonatal mice KD was 1.6 X 10(-10) M for both cytosolic and nuclear fractions, binding capacity was 54 fmol/mg protein in the cytosolic and 108 in the nuclear fractions, respectively. The phenotypic expression of the 1,25(OH)2D3 receptor (dissociation constant, binding capacity) was identical in neonatal and adult epidermis. Half maximal displacement of 1,25(OH)2D3 was achieved with an 80-fold and 200-fold molar excess of 25(OH)D3 and 1-alpha-hydroxycholecalciferol [1(OH)D3], respectively. Using Percoll density gradient centrifugation, 1,25(OH)2D3 receptors could be localized in the basal cell fraction. DNA cellulose chromatography with 1,25(OH)2D3 receptor elution from DNA at 0.25 M KCl (linear gradient) points to a possible role in gene transcription. In mouse primary epidermal cell cultures, 1,25(OH)2D3, but not 25(OH)D3, 24,25(OH)2D3, and 1(OH)D3 influenced [3H]thymidine incorporation (at physiological concentrations); the magnitude of change depending on the concentration of 1,25(OH)2D3 and the time of incubation. These data demonstrate that skin is a target organ for the active vitamin D secosterol.