Time and Temperature Stability of TGF-ß1, EGF and IGF-1 in 20% and 100% Human Serum.

Autologous serum eye drops (ASEDs) are used as a treatment for severe dry eye disease. The concentration and stability of various growth factors in ASEDs is determinative for their efficiency. We therefore assessed the concentrations of transforming growth factor beta 1 (TGF-ß1), epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF-1) in ASEDs following storage at 4-8, -20, -80 and -156 °C. Twenty % and 100% sera from eight healthy volunteers were analysed by the sandwich enzyme immunoassay at different time intervals up to seven months. The mean levels of TGF-ß1 and EGF in undiluted and 20% serum did not differ significantly from the baseline levels in fresh serum for any storage conditions after 7 days at 4-8 °C, as well as after 4- and 7-month preservation at sub-zero temperatures. In 20% serum, no IGF-1 concentration decrease was found following 7 days of preservation at 4-8 °C. However, a decrease to 78 % and 81 % (P < 0.01) of baseline values was found in 20% serum after 4-month storage at -20 °C and 7-month storage at -156 °C, respectively. A more pronounced decrease in IGF-1 was observed in undiluted serum. All assessed growth factors present in 20% frozen serum remained stable for up to 7 months. The highest stability was achieved at -80 °C. At -20 and -156 °C, some decrease in IGF-1 occurred. Our results indicate that 20% ASEDs can be stored frozen up to 7 months under proper conditions.

Saccharomyces cerevisiae nuclear and nucleolar antigen preservation for immunoelectron microscopy.

Yeast cells in general are known to be difficult to prepare for electron microscopy investigations particularly when the preservation of antigenicity is required. In this work, we compare various protocols for preparation of Saccharomyces cerevisiae cells for immunoelectron microscopy, ranging from classical chemical fixation to high-pressure freezing followed by freeze-substitution in different kinds of substitution media. Our aim was to establish a protocol giving optimal, routinely reproducible results for simultaneous retention of fine ultrastructural details and antigen immunoreactivity, with particular focus on the preservation of nuclear and nucleolar architecture. This was demonstrated by ultrastructural immunolocalization of various nucleolar (Nop1 and Nsr1), nuclear (Nsp1) and alpha-tubulin antigens. The protocol which we found to yield the best preserved Saccharomyces cerevisiae cells for both morphological and immunological studies included cryo-fixation by high-pressure freezing followed by freeze-substitution in acetone with 0.1% uranyl acetate and embedding in Lowicryl HM20. In addition, immunofluorescence detection of the antigens was performed and correlated with immunolabelling at the electron microscopy level.

Chromatin conformation and salt-induced compaction: three-dimensional structural information from cryoelectron microscopy.

Cryoelectron microscopy has been used to examine the three-dimensional (3-D) conformation of small oligonucleosomes from chicken erythrocyte nuclei after vitrification in solutions of differing ionic strength. From tilt pairs of micrographs, the 3-D location and orientation of the nucleosomal disks, and the paths of segments of exposed linker can be obtained. In "low-salt" conditions (5 mM NaCl, 1 mM EDTA, pH 7.5), the average trinucleosome assumes the shape of an equilateral triangle, with nucleosomes at the vertices, and a length of exposed linker DNA between consecutive nucleosomes equivalent to approximately 46 bp. The two linker DNA segments converge at the central nucleosome. Removal of histones H1 and H5 results in a much more variable trinucleosome morphology, and the two linker DNA segments usually join the central nucleosome at different locations. Trinucleosomes vitrified in 20 mM NaCl, 1 mM EDTA, (the salt concentration producing the maximal increase in sedimentation), reveal that compaction occurs by a reduction in the included angle made by the linker DNA segments at the central nucleosome, and does not involve a reduction in the distance between consecutive nucleosomes. Frequently, there is also a change in morphology at the linker entry-exit site. At 40 mM NaCl, there is no further change in trinucleosome morphology, but polynucleosomes are appreciably more compact. Nevertheless, the 3-D zig-zag conformation observed in polynucleosomes at low salt is retained at 40 mM NaCl, and individual nucleosome disks remain separated from each other. There is no evidence for the formation of solenoidal arrangements within polynucleosomes. Comparison of the solution conformation of individual oligonucleosomes with data from physical measurements on bulk chromatin samples suggests that the latter should be reinterpreted. The new data support the concept of an irregular zig-zag chromatin conformation in solution over a range of ionic strengths, in agreement with other in situ (McDowall, A.W., J.M. Smith, and J. Dubochet. 1986, EMBO (Eur. Mol. Biol. Organ.) J.5: 1395-1402; Horowitz, R.A., D.A. Agard, J.W. Sedat, and C.L. Woodcock, 1994. J. Cell Biol. 125:1-10), and in vitro conclusions (van Holde, K., and J. Zlatanova. 1995. J. Biol. Chem. 270:8373-8376). Cryoelectron microscopy also provides a way to determine the 3-D conformation of naturally occurring chromatins in which precise nucleosome positioning plays a role in transcriptional regulation.

Postnatal appearance of uncoupling protein and formation of thermogenic mitochondria in hamster brown adipose tissue.

Brown adipose tissue of developing hamster was characterized by western blotting, enzyme activity measurements and immunoelectron microscopy. During the first postnatal week the tissue contained significant amounts of differentiating mitochondria and comparable quantities of active cytochrome oxidase and ATP synthase. The uncoupling protein appeared on the 7/8th day and its specific content increased 80-times between day 8 and day 17. In parallel, the specific content and activity of cytochrome oxidase increased 3-times but ATP synthase decreased 2-times. The total content of uncoupling protein and of cytochrome oxidase in interscapular brown adipose tissue increased 360- and 11-times, respectively. Analysis of isolated mitochondria showed that the observed differences result mainly from changes of the enzymic equipment of the mitochondrial membrane. During the same interval, propylthiouracil-insensitive "type II' thyroxine 5'-deiodinase activity in brown adipose tissue increased 10-times. It was concluded that the thermogenic function of the hamster brown adipose tissue develops after the first postnatal week due to highly differentiated synthesis of mitochondrial proteins leading to replacement of preexisting, uncoupling protein-lacking nonthermogenic mitochondria by thermogenic ones, similarly as shown in brown adipose tissue of the embryonic mouse and rat (Houstek, J., et al. (1988) Biochim. Biophys. Acta 935, 19-25).

Determination of DNA persistence length by cryo-electron microscopy. Separation of the static and dynamic contributions to the apparent persistence length of DNA.

Axial deflection of DNA molecules in solution results from thermal motion and intrinsic curvature related to the DNA sequence. In order to measure directly the contribution of thermal motion we constructed intrinsically straight DNA molecules and measured their persistence length by cryo-electron microscopy. The persistence length of such intrinsically straight DNA molecules suspended in thin layers of cryo-vitrified solutions is about 80 nm. In order to test our experimental approach, we measured the apparent persistence length of DNA molecules with natural "random" sequences. The result of about 45 nm is consistent with the generally accepted value of the apparent persistence length of natural DNA sequences. By comparing the apparent persistence length to intrinsically straight DNA with that of natural DNA, it is possible to determine both the dynamic and the static contributions to the apparent persistence length.

Opposite effect of counterions on the persistence length of nicked and non-nicked DNA.

Using cryo-electron microscopy we reconstructed the three-dimensional trajectories adopted in cryovitrified solutions by double-stranded DNA molecules in which the backbone of one strand lacked a phosphate at regular intervals of 20 nucleotides. The shape of such nicked DNA molecules was compared with that of DNA molecules with exactly the same sequence but without any single-stranded scissions. Upon changing the salt concentration we observed opposite effects of charge neutralization on nicked and non-nicked DNA. In low salt solutions (10 mM Tris-HCl, 10 mM NaCl) the applied dense nicking caused ca 3.5-fold reduction of the DNA persistence length as compared with non-nicked DNA. Upon increasing the salt concentration (to 150 mM NaCl and 10 mM MgCl2) the persistence length of non-nicked DNA appreciably decreased while that of nicked DNA molecules increased by a factor of 2.

The twist, writhe and overall shape of supercoiled DNA change during counterion-induced transition from a loosely to a tightly interwound superhelix. Possible implications for DNA structure in vivo.

A cryo-electron microscopy study of supercoiled DNA molecules freely suspended in cryo-vitrified buffer was combined with Monte Carlo simulations and gel electrophoretic analysis to investigate the role of intersegmental electrostatic repulsion in determining the shape of supercoiled DNA molecules. It is demonstrated here that a decrease of DNA-DNA repulsion by increasing concentrations of counterions causes a higher fraction of the linking number deficit to be partitioned into writhe. When counterions reach concentrations likely to be present under in vivo conditions, naturally supercoiled plasmids adopt a tightly interwound conformation. In these tightly supercoiled DNA molecules the opposing segments of interwound superhelix seem to directly contact each other. This form of supercoiling, where two DNA helices interact laterally, may represent an important functional state of DNA. In the particular case of supercoiled minicircles (178 bp) the delta Lk = -2 topoisomers undergo a sharp structural transition from almost planar circles in low salt buffers to strongly writhed "figure-eight" conformations in buffers containing neutralizing concentrations of counterions. Possible implications of this observed structural transition in DNA are discussed.

Microbiological characterization of winery effluents: an inventory of the sites for different treatment systems.

In a more and more regulated and socially pressured environment, the durable management of winery effluents must take into account their characteristics and their potential impact on their natural setting. The object of this exploratory study is to establish an inventory of the microbiological composition of winery effluents coming from different treatment systems. We have observed that winery effluents are charged with micro-organisms, by a factor that ranges from 10(5) to 10(8) UFC/ml, and that the level of "microbiological pollution" is independent of the type of system. The composition of the flora is closely tied to the time of year and therefore to winery activities, so certain micro-organisms will be favoured in certain periods and others will have a tendency to decrease. We have seen that from one year to another our observations remain identical; the flora equilibrium therefore occurs systematically and naturally. Faecal germs are found in very small quantities in winery effluent treatment systems. They represent minor sanitary risks. Good correlations were observed between some micro-organisms and some physical-chemical parameters (COD). It is, however, difficult to use these "easy-to-measure" parameters as reliable markers of certain microbial populations.

Ex-vivo and in-vivo platelet function in patients with severe hypercholesterolemia undergoing LDL-apheresis.

Patients with severe familial hypercholesterolemia (HC) show abnormal platelet function and shortened platelet survival. Atherosclerosis is associated with platelet hyperactivity. Low-density lipoporotein (LDL)-apheresis eliminates the most atherogenic lipid fraction and inhibits the progression of atherosclerosis inducing even regression. In order to assess the influence of LDL-apheresis on platelet function ex-vivo and in-vivo, 6 patients with severe heterozygous HC, all of them being pharmacologically treated with HMG-CoA reductase inhibitors and anion exchange resins were investigated. Ex-vivo platelet function was assessed by the aggregation response to ADP before starting apheresis treatment, as well as after 2 and 24 weeks, respectively. In-vivo platelet function was determined by measuring platelet survival after radiolabeling with 111In-oxine before starting LDL-apheresis and after 24 weeks of twice monthly treatment. LDL-apheresis therapy induced a significant (p < 0.01) drop in cholesterol by 64%, LDL-cholesterol by 77% and in triglycerides by 46% over a period of 24 weeks. ADP-induced platelet aggregation revealed a decreased aggregability of platelets with a decline in the maximal amplitude and the slope of the response curve. Changes in platelet sensitivity to prostaglandins (PG) were significantly for PGI2, but did not reach statistical significance for PGE1. The results revealed a significant (p < 0.001) increase in platelet survival of 111In-oxine-radiolabeled autologous platelets from a mean of 106.50 hours before to 137.50 hours (p < 0.01) after treatment, being accompanied by an increase in labeling efficiency (p < 0.001) and recovery (p < 0.001). These data provide evidence for improved hemostatic regulation in vivo as a result of maintainance of lipid-lowering achieved with LDL-apheresis.

The effects of demineralized bone matrix and direct current on an "in vivo" culture of bone marrow cells.

Bone marrow cells (BMCs) from rabbit femora and tibiae were grown in diffusion chambers implanted in rabbit muscle. At 42 days 80% of the BMC chambers exhibited cartilage formation within them. Demineralized bone matrix added to the marrow cell suspension in the chamber accelerated the appearance and increased the number of chambers with cartilage. Mineralization of the cartilage also occurred earlier in the chambers with bone matrix. In a second experiment, a 5-microA direct current cathode in the bone marrow chamber increased the number of chambers containing cartilage from 50 to 80% at day 25. Mineralization also occurred earlier in the chambers with direct current.

The contribution of secondary heavy particles to the absorbed dose from high-energy photon beams.

High-energy photon radiotherapy sources produce non-negligible numbers of secondary heavy particles (neutrons, protons and alphas) which contribute to the dose in a patient. They also present a radiation protection problem. The contribution of such particles to the absorbed dose in a tissue-like medium and to the equivalent dose is considered in this study experimentally. Track etch detectors were chosen as a basic type of measuring instrument: cellulose nitrate Kodak LR115 and polyallyldiglycol carbonate CR39 were used as detectors. Several methods of directly read datum (track density, track parameter, LET spectrum etc) interpretation have been analysed. It has been ascertained that the contribution of secondary heavy particles represents approximately 0.002 of the photon dose and 0.02 of the equivalent dose, both at 50 MeV as maximum photon energy. The contribution of a 20 MV bremsstrahlung beam is roughly one-fifth of that at 50 MV.

Serum thyroglobulin determinations in patients with differentiated thyroid carcinoma.

Serum thyroglobulin levels were measured in 636 patients with differentiated thyroid carcinoma, in whom altogether 1240 determinations were performed in different phases of disease and treatment. A modified sensitive radioimmunoassay was employed using an own high-specific thyroglobulin antibody. The results showed that both the majority of patients with functioning metastases accumulating radioiodine and with non-functioning metastases which could not be detected by scanning had higher serum TG levels, compared with a group of healthy subjects. However, "normal" TG values in patients with metastases, especially non-functioning, were found too. Thus, these findings decrease the diagnostic value of the TG determination. The highest TG values were found in patients with distant metastases of differentiated thyroid carcinoma (in lungs and bones); on the other hand, the proportion of patients with lymph-node(s) metastases and "normal" TG levels was relatively high. We suggest that the serum TG determination cannot generally replace scanning with 131I and cannot serve as the only test, while being a helpful indicator in the long-term follow-up of differentiated thyroid cancer patients.

Functional activity of a thyroid nodule under the influence of stable iodide administration in a hyperthyroid patient.

In a thyrotoxic patient with nodular goiter treated with stable iodide and Carbimazole, a series of 131I scans was performed at different intervals for proposed 131I therapy. Originally, total uptake was suppressed and the node was evaluated as active. The same type of scan was seen repeatedly with uptake increasing for about 6 months following stable iodide withdrawal. Only 9 months later, the effect of 127I disappeared and uptake rose considerably with a reversal of the scan to the picture of a hypofunctioning node. The same distribution was found after therapeutic 131I administration. 6 months following treatment, without any thyrostatic drugs, the patient was euthyroid and on the scan most of tracer was found in the node. It is believed that this change is best explained by the different sensitivity of the nodule and the paranodular tissue to the effect of stable iodide. It is believed that the hyperthyroidism originated in the paranodular tissue highly sensitive on the 127I, while the nodule (presumably an adenoma) was less sensitive and showed uptake only when the paranodular tissue was depressed by 127I or, later, injured by the effect of therapeutic 131I.

Epidermal growth factor (EGF) in serum of patients with differentiated carcinoma of thyroids.

Serum levels of epidermal growth factor (EGF) were investigated in 31 patients with differentiated carcinoma of thyroid. Patients with carcinoma had significantly decreased basal levels of serum EGF, however this decrease in serum EGF occurred only in the group of patients which had been evaluated at an interval of six weeks after ablation of residues of normal thyroid with radioiodine. In patients that had been treated with radioiodine for cancer this decrease was not observed. The change in serum EGF was not dependent on thyroidal function and did not correlate with the serum thyreoglobulin level. It is presumed that decreased serum EGF is a consequence of functional changes of platelets after whole body irradiation at ablative dose of radioiodine.

Sonography in the evaluation of treatment of differentiated thyroid cancer. First results in 158 patients.

Sonographic examination of soft tissues of the neck was used to search for recurrent thyroid cancer in 158 patients. Two hundred and thirty-six sonographic findings were evaluated. Specificity of the method, regardless of the type of recurrence, was 0.96, sensitivity 0.66, precision 0.81, predictive value of negative finding 0.72. This compares favorably with the results of examination by palpation (sensitivity 0.32 to 0.52, according to the type of recurrence), or with those of palpation combined with thyroglobulin estimation (sensitivity 0.50 to 0.60). Most important was the possibility of early detection of unpalpable recurrent lesions showing no radioiodine uptake, or relapses with low thyroglobulin production. Systematic evaluation of out-patients using sonography could lower the need for radioiodine scans that are necessarily connected with thyroid hormone therapy withdrawal and that are usually performed in in-patients.

Developmental changes in uncoupling protein 1 and F1-ATPase subunit levels in the golden hamster brown adipose tissue mitochondria as determined by electron microscopy in situ immunocytochemistry.

The postnatal developmental changes in mitochondrial uncoupling protein 1 (UCP 1) and F1-ATP synthase (ATPase) subunit levels in the interscapular brown adipose tissue (BAT) were studied in golden Syrian hamsters (Mesocricetus auratus) using electron microscopy in situ immunocytochemistry. The relatively low initial density of 5 nm gold conjugated anti-UCP 1 immunocomplexes gradually increased from 7- to 21-day-old animals and numerous immunocomplexes were found on the mitochondrial membranes of adult hamsters. At the age of 7-9 days, a positive reaction was also detected in the cytoplasm of BAT adipocytes. Immunolocalization of F1-ATPase subunit indicated its presence in BAT mitochondria and cytoplasm of 7- to 9-day-old animals. However, contrary to UCP 1, intensity of the immunostaining of F1-ATPase subunit rapidly decreased both in mitochondria and cytoplasm between the 10th and 21st postnatal day and it became stabilized in adult animals at a very low level restricted to mitochondria. These results confirm that profound changes in the enzymatic apparatus of BAT mitochondrial membranes, leading to formation of thermogenic mitochondria, occur not until the early postnatal period of hamster ontogenetic development.

Evaluation of selenium supply and status of inhabitants in three selected rural and urban regions of the Czech Republic.

Blood serum selenium of 65 men and hair selenium of 77 men from three regions of the Czech Republic (CR) were analyzed by neutron activation analysis, and 202 samples of urine from the same populations were analyzed for Se by the fluorimetric method to assess selenium status of these regions. Low status (53 micrograms Se/L of serum and 0.29 micrograms Se/g lyophilized hair as means) and very low urine selenium (8.7 micrograms/L urine) were detected. By these data, the CR is among the countries with the lowest Se intake. A comparison of studied regions is presented. Moreover, values of serum zinc were within the reference range, but mild to moderate deficiency in the supply of iodine was detected.

Dosimetry during the first IBIS facility flight.

The dosimetry of cosmic rays was performed during the first experimental flight of the IBIS facility. Different thermoluminescent detectors (TLD) have been used to measure the contribution of the low linear energy transfer component (LET < 10 keV/micrometer) and plastic nuclear track detectors (PNTD) for the high linear energy tranfer (LET) component. Several parameters of tracks have been measured to determine the LET spectra of primary and secondary charged particles. The total absorbed dose rate (TLD+PNTD) during the flight was 0.23 mGy/day and the dose equivalent rate using the ICRP 60 was 0.52 mSv/day. The corresponding mean quality factor was 2.4. These results are in agreement with those obtained aboard the MIR station with a tissue equivalent proportional counter.

Arthroscopic treatment of triangular fibrocartilage tears.

The triangular fibrocartilage complex is an intricate anatomic structure located at the ulnar aspect of the wrist. The triangular fibrocartilage is important to the stability and biomedical function of the ulnar carpus and distal radioulnar joint. This article reviews the anatomy and biomedical function of the triangular fibrocartilage. Diagnosis and treatment of traumatic injuries to the triangular fibrocartilage are also discussed.

The role of arthroscopy in the treatment of traumatic triangular fibrocartilage injuries.

The triangular fibrocartilage complex is a complex anatomic structure located at the ulnar aspect of the wrist and is important to the stability and biomechanical function of the ulnar carpus and distal radioulnar joint. This article will review the anatomy and biomechanical function of the TFCC and its relationship to the diagnosis and treatment of traumatic injuries to the TFCC.