Gitelman's syndrome as a cause of poorly controlled hypokalemia.

Gitelman's syndrome is a rare autosomal recessive tubulopathy caused by a defect of the thiazide-sensitive sodium chloride co-transporter at the distal tubule, leading to hypokalemia, metabolic alkalosis, hypomagnesemia, hypocalciuria and low-to-normal blood pressure. Clinical features include transient periods of muscle weakness and tetany, dizziness, abdominal pains and constipation. Patients can also present with convulsions due to severe metabolic alkalosis or hypomagnesemia. Therefore, early recognition and treatment are important. Diagnosis of Gitelman's syndrome is usually made incidentally during adolescence or early adulthood based on clinical and biochemical findings. In this paper we present the case of a 23-year-old female patient referred to our nephrology department for severe hypokalemia. Complementary evaluation revealed hypokalemia, hypomagnesemia, metabolic alkalosis, increased chloride and sodium urinary excretion and reduced urinary calcium excretion with normal renal function. A diagnosis of Gitelman syndrome was established. Treatment included magnesium and potassium salts and potassium saving diuretics. In general, the long-term prognosis of Gitelman's syndrome is good if the patient adhere with the treatment.

Chondrogenic potential of human synovial mesenchymal stem cells in alginate.

OBJECTIVE: In a recent study, we demonstrated that mesenchymal stem cells (MSCs) derived from the synovial membranes of bovine shoulder joints could differentiate into chondrocytes when cultured in alginate. The purpose of the present study was to establish the conditions under which synovial MSCs derived from aging human donors can be induced to undergo chondrogenic differentiation using the same alginate system. METHODS: MSCs were obtained by digesting the knee-joint synovial membranes of osteoarthritic human donors (aged 59-76 years), and expanded in monolayer cultures. The cells were then seeded at a numerical density of 4x10(6)/ml within discs of 2% alginate, which were cultured in serum-containing or serum-free medium (the latter being supplemented with 1% insulin, transferrin, selenium (ITS). The chondrogenic differentiation capacity of the cells was tested by exposing them to the morphogens transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, insulin-like growth factor-1 (IGF-1), bone morphogenetic protein-2 (BMP-2) and BMP-7, as well as to the synthetic glucocorticoid dexamethasone. The relative mRNA levels of collagen types I and II, of aggrecan and of Sox9 were determined quantitatively by the real-time polymerase chain reaction (PCR). The extracellular deposition of proteoglycans was evaluated histologically after staining with Toluidine Blue, and that of type-II collagen by immunohistochemistry. RESULTS: BMP-2 induced the chondrogenic differentiation of human synovial MSCs in a dose-dependent manner. The response elicited by BMP-7 was comparable. Both of these agents were more potent than TGF-beta1. A higher level of BMP-2-induced chondrogenic differentiation was achieved in the absence than in the presence of serum. In the presence of dexamethasone, the BMP-2-induced expression of mRNAs for aggrecan and type-II collagen was suppressed; the weaker TGF-beta1-induced expression of these chondrogenic markers was not obviously affected. CONCLUSIONS: We have demonstrated that synovial MSCs derived from the knee joints of aging human donors possess chondrogenic potential. Under serum-free culturing conditions and in the absence of dexamethasone, BMP-2 and BMP-7 were the most potent inducers of this transformation process.

Osteogenic protein-1 (BMP-7) accelerates healing of scaphoid non-union with proximal pole sclerosis.

We randomly assigned 17 patients with scaphoid non-union at the proximal pole to three treatment groups: (1) autologous iliac graft (n=6), (2) autologous iliac graft + osteogenic protein-1 (OP-1; n=6), and (3) allogenic iliac graft + OP-1 (n=5). Radiographic, scintigraphic, and clinical assessments were performed throughout the follow-up period of 24 months. OP-1 improved the performance of both autologous and allogenic bone implants and reduced radiographic healing time to 4 weeks compared with 9 weeks in group 1. Helical CT scans and scintigraphy showed that in OP-1-treated patients sclerotic bone was replaced by well-vascularised bone. The addition of OP-1 to allogenic bone implant equalised the clinical outcome with the autologous graft procedure. Consequently the harvesting of autologous graft can be avoided.

Regulation of BMP-7 expression by retinoic acid and prostaglandin E(2).

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) gene superfamily of growth and differentiation factors. Members of the BMP family were originally cloned and characterized by their ability to induce ectopic bone formation. Of the various BMPs cloned, the bone inductive ability of BMP-7 (OP-1) and BMP-2 has been well characterized. Both BMP-7 and -2 have been shown to have clinical utility in the healing of non-union fractures. However, in spite of the various advances in BMP research, the physiological regulation of BMPs is not well understood. Here we studied the expression of BMP-7 by cloning a 4.6-kB fragment of the human BMP-7 promoter (hBMP-7p) and placing it upstream of a luciferase reporter. The promoter reporter construct was stably transfected into different cell backgrounds and its regulation by various factors was investigated. We show that retinoic acid (RA) treatment results in an upregulation of the hBMP-7p reporter activity. This regulation of the hBMP-7p was further confirmed by Northern blot, PCR, and Western blot analyses, which showed an increase in both BMP-7 mRNA and protein expression upon treatment with RA. We further show that RA specifically upregulates expression of osteocalcin via activation of BMP-7 mRNA and protein in vitro. Similarly, prostaglandin E(2) (PGE(2)) treatment increases BMP-7 mRNA and protein levels, but does not transcriptionally activate the hBMP-7p. Additionally, in vivo expression of BMP-7 in bone was increased upon PGE(2) treatment. In conclusion, RA and PGE(2) upregulate BMP-7 protein expression both in vitro and in vivo.

Separation and detection of intact noncovalent protein complexes from mixtures by on-line capillary isoelectric focusing-mass spectrometry.

Separation and mass spectrometric analysis of intact noncovalent protein-protein complexes from mixtures is described. Protein complexes were separated using isoelectric focusing in a capillary under native conditions. During the mobilization, molecular masses of the intact complexes were measured on-line (as they emerged from the capillary) using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. An FTICR "in-trap" ion cleanup procedure was necessary for some complexes to reduce high levels of adduction and to obtain accurate molecular mass measurements. Optimization of the conditions for analysis of different intact complexes is discussed. We have shown that either the intact noncovalent complexes or their constituent protein subunits can be detected by variation of sheath liquid (i.e., NH4OAc vs HOAc) added at the electrospray-mass spectrometer interface. Thus, two successive experiments permit a fast and efficient characterization of intact complex stoichiometry, the individual complex subunits and the possible presence of metal or other adducted species.

Characterization of human alcohol dehydrogenase isoenzymes by capillary isoelectric focusing-mass spectrometry.

The human liver alcohol dehydrogenase (ADH) isoenzymes are currently believed to play a major role in ethanol metabolism, accounting for most of the ethanol oxidized in the liver. They have similar molecular masses and similar isoelectric point (pI) values (the 13 possible isoenzymes having pIs in the range of 8.26-8.87), making their characterization a significant analytical challenge. Capillary isoelectric focusing (CIEF) coupled on-line with electrospray ionization - Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry was applied to separate and characterize mixtures of alphaalpha, beta1beta1 and beta3beta3 ADH isoenzymes. Seven different species were resolved by the separation in the pI 8.26-8.67 range. ESI-FTICR analysis of native ADHs revealed that each noncovalent ADH complex contains two monomeric protein units and four zinc atoms. The combination of CIEF separations with mass spectrometry appears well-suited for detailed characterization of ADH isozymes, and the attomole level sensitivity of FTICR should allow very small samples to be addressed.

Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures.

Capillary isoelectric focusing (CIEF) can provide high-resolution separations of complex protein mixtures, but until recently it has primarily been used with conventional UV detection. This technique would be greatly enhanced by much more information-rich detection methods that can aid in protein characterization. We describe progress in the development of the combination of CIEF with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and its application to proteome characterization. Studies have revealed 400-1000 putative proteins in the mass range of 2-100 kDa from total injections of approximately 300 ng protein in single CIEF-FTICR analyses of cell lysates for both Escherichia coli (E. coli) and Deinococcus radiodurans (D. radiodurans). We also demonstrate the use of isotope labeling of the cell growth media to improve mass measurement accuracy and provide a means for quantitative proteome-wide measurements of protein expression. The ability to make such comprehensive and precise measurements of differences in protein expression in response to cellular perturbations should provide new insights into complex cellular processes.

Biological repair of thyroid cartilage defects by osteogenic protein-1 (bone morphogenetic protein-7) in dog.

The efficacy of human recombinant osteogenic protein-1 (OP-1; bone morphogenetic protein-7) in regeneration of dog larynx was examined by treating thyroid cartilage defects (1.5 cm2) in dogs with thyroid allografts covered with host perichondrium or fascia. Prior to implantation allografts were frozen, thawed and demineralized. The treatment groups were as follows: I--Allograft control implant (n = 3); II--Implants coated with 500 micrograms OP-1 (n = 4); III--Implants coated with 100 micrograms OP-1 (n = 3); IV--Implants coated with 500 micrograms OP-1 and covered with neck fascia (n = 3); and V--Implants extracted with 1 M NaCl and guanidine hydrochloride, and coated with 500 micrograms OP-1 (n = 4). Dogs were sacrificed four months following surgery. Each larynx was removed, carefully dissected and a three-dimensional reconstruction of the defect area was performed on serial sections. The results revealed that the implants of control dogs remained intact with no apparent reduction in size and new tissue formation. OP-1 enriched thyroid allografts, dose dependently induced bone, cartilage and ligament-like structures comprising up to 80% of the total regenerated defect area. Boundaries of the defects healed by formation of new bone when bone resided within the old thyroid cartilage layers. Old cartilage not containing bone within its layers healed by complete integration with newly formed cartilage. Both new bone and cartilage were embedded into layers of new ligament-like tissue which expressed specific morphologic and molecular markers. The three newly formed tissues were tightly connected into a "bone-cartilage-ligament continuum" of tissues, suggesting that OP-1 served as a multiple tissue morphogen in this specific microenvironment.

Proteome analysis using selective incorporation of isotopically labeled amino acids.

A method is described for identifying intact proteins from genomic databases using a combination of accurate molecular mass measurements and partial amino acid content. An initial demonstration was conducted for proteins isolated from Escherichia coli (E. coli) using a multiple auxotrophic strain of K12. Proteins extracted from the organism grown in natural isotopic abundance minimal medium and also minimal medium containing isotopically labeled leucine (Leu-D10), were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled Leu residue has no effect on the CIEF separation of the protein, therefore both versions of the protein are observed within the same FTICR spectrum. The difference in the molecular mass of the natural isotopic abundance and Leu-D10 isotopically labeled proteins is used to determine the number of Leu residues present in that particular protein. Knowledge of the molecular mass and number of Leu residues present can be used to unambiguously identify the intact protein. Preliminary results show the efficacy of this method for unambiguously identifying proteins isolated from E. coli.

[Treatment of emergency conditions in the aged--suggestions for solutions]. / Lijecenje urgentnih stanja kod starije dobne skupine--prijedlozi rjesenja.

If it is true that we are living in the era of sure surgery and dangerous medications, then this fact is particularly relevant in the treatment of elderly patients from third age-group. The uncontrollable growth of the number of such people (demographic signs point to an increase of population by 100%), demands complete changes in the medical approach to patients from the third age-group, especially considering the peculiarities of the geriatric medicine: intermingling of involute molecular ageing changes; multimorbidity; especially chronic development of decease; changes in the selection of selection of appropriate medications and medical treatment. Problem of chronic illness is the most outstanding problem of old age, and therefore, within the total health-care system nothing can be right, nor in its place, until major questions of the health protection for elderly in the third age-group are resolved. One of the most important phenomenon associated with these problems is in hospitalisation where, because of a limited hospital capacity, there is usually not enough space for patients from middle age-group, and even more so for elderly from the third age-group. According to presented data, the fact that third-age group constitutes 23.11% of examined patients, points to an obvious urgency and the actuality of these matters. The health problems of patients from third age-group demand special training of physicians and their assistants of all profiles, and a special scientific research in this area.

Osteogenic protein-1 is produced by human fetal trophoblasts in vivo and regulates the synthesis of chorionic gonadotropin and progesterone by trophoblasts in vitro.

Osteogenic protein-1 (OP-1/BMP-7), a member of the transforming growth factor (TGF-beta) superfamily of proteins, is shown to be expressed at sites of epithelial-mesenchymal tissue interaction during human fetal development. In the present study, we examined the expression of OP-1 in human placentas (5-11 weeks of gestation and full term) using in situ hybridization and immunohistochemistry methods. The results show that OP-1 was expressed in cytotrophoblasts (Langhans layer) of the chorionic villi in early and full term placentas. Employing highly purified cytotrophoblast cultures which fused to form functional syncytiotrophoblasts, we showed that exogenously added recombinant OP-1, in the presence of low serum concentration, reduced the secretion of chorionic gonadotropin and progesterone by 60% and 36%, respectively, compared with control cultures. The results suggest that osteogenic protein-1 is synthesized locally by cytotrophoblasts and may be involved in the regulation of human pregnancy by controlling reproductive hormonal secretion.

Effect of pulse Doppler ultrasound on placental hormone excretion in vitro.

There is no literature data on the effects of ultrasound on the metabolism of placental tissue. In the present study, the authors have investigated the possible influence of pulsed field Doppler ultrasound on hormonal excretion of human placental trophoblasts in vitro. For the detection of placental hormones, specific immunoassays for chorionic gonadotropin (hCG), human placental lactogen (hPL), estrogen and progesterone were used. Compared to control trophoblast hormone excretion, pulsed field ultrasound exposure induced no changes in the excretion of the hormones in vitro. The authors were unable to demonstrate any alterations caused by pulsed field Doppler ultrasound in this in vitro model. Additional studies are required to deepen our understanding of the effect of pulsed Doppler ultrasound on other biomolecules and their metabolism.

[Angiotensin-converting enzyme activity in dehydrated rats]. / Aktivnost enzima konverzije angiotenzina kod dehidriranih stakora.

The serum activity of angiotensin converting enzyme in 10 rats before and after a five day dehydration was examined. It was measured spectrophotometrically and expressed in units corresponding to 1 mnol of hipuric acid liberated from Hip-Gly-Gly supstrate. The result show that serum activity of angiotensin converting enzyme was significantly increased in rats after the period of dehydration.

[N-acetyl-beta-D-glucosaminidase as an early marker and indicator of the extent of kidney and liver lesions in endotoxemia and endotoxic shock]. / N-acetil-beta-D-glukozaminidaza kao rani marker i indikator stupnja lezije bubrega i jetre u endotoksemiji i endotoksinskom soku.

The aim of the study was to answer the question whether the serum level and the urine level of N-acetyl-beta-D-glucosaminidase (NAG) could be the indicator of liver and kidney lesions, and the indicator of the stage of the lesions, in cases of endotoxemia and endotoxin shock. The authors performed the experiment using 60 rabbits as experimental animals. The animals were divided into six groups (10 rabbits in each group); control animals received an equal volume (1.0 ml/kg of body weight) of physiological saline solution. Endotoxin (Lipopolysaccharide E. coli 055 B-5) in doses of 20, 40, 60, 80, and 100 micrograms/kg of body weight were administered in a single intravenous injection to each animal of I-V experimental groups. Using endotoxin in that dose of application, the authors provoked different stages of endotoxemia on the rabbits in the first four experimental groups, and the endotoxin shock on the rabbits of the fifth experimental group. After 18 hours, the rabbits were decapitated, and the levels of NAG in serum, urine, kidney and liver tissues determined. The results showed that the serum increase of NAG activity is caused not only by the lesions of kidney parenchyma, but by the lesions of liver parenchyma too, in the cases of endotoxemia and endotoxin-produced shock; the increase is statistically significant. Urine increase of NAG activity is significant even in cases in which the authors administered very low doses of endotoxin (20 micrograms/kg of body weight), so it can be said that urine NAG activity is a very good indicator of early kidney parenchyma lesions. But, the urine increase of NAG activity is (absolutely) in correlation with the dose of administered endotoxin, so it can be said that urine activity of NAG is the indicator of stage of kidney lesions in cases of endotoxemia and endotoxin-provoked shock.

[Urinary polyamines before and after treatment of malignant tumors]. / Urinarni poliamini prije i nakon ljecenja malignih tumora.

The concentrations of polyamines (putrescine, spermidine and spermine) were determined in urine of twelve patients with malignant tumors before and after therapy and in twelve healthy patients. The results showed significant increase of urinary concentrations of putrescine, spermidine and spermine in urine of 12 malignant tumour patients. The concentration of urinary putrescine was higher approximately by 6.5 times; of spermidine by 5.8 times and of spermine by 9.8 times in comparison with the results in healthy patients. After the therapy the concentration of urinary putrescine was by 42.3% lower; of spermidine by 34.4% and of spermine by 52.2% lower in comparison with the results obtained before the therapy. The possibility of usage of concentration determination of urinary po-therapy efficiency are discussed.