[Diurnal fluctuations of protein contents and the pH dependence of beta2-microglobulin stability in urine].

Diurnal fluctuations of protein excretion into urine and the effect of urinary pH on the urinary protein concentrations were studied in patients with various kidney diseases. The diurnal kinetics of gamma-immunoglobulin, transferrin, albumin, alpha1-microglobulin, gamma-immunoglobulin light chains, and the retinol-binding protein proved to positively correlate with the diurnal fluctuations of proteinuria and to negatively correlate with urinary pH. Diurnal changes in urinary beta2-microglobulin content did not correlate with those of any other protein. Oral bicarbonate intake alkalinized the urine, increased the urinary beta2-microglobulin content, and led to a direct correlation between beta2-microglobulin excretion and excretion of other low-molecular proteins. Thus, proteinuria, single protein excretion, and urinary pH displayed diurnal rhythmicity in the patients; beta2-microglobulin was unstable in acid urine and its urinary level depended on the urinary pH.

[Treatment of gastrointestinal hemorrhage manifestation induced by nonsteroidal anti-inflammatory agents]].

INTRODUCTION: The risk of upper gastrointestinal tract bleeding in the patients taking NSAID estimates 1% per year. Bleeding stops spontaneously in approximately 80% of all cases. Persistent and repeated bleeding (expecialy during the initial hospitalization) still represent the serious clinical problem. In this group of patients, mortality rate is between 6-10%, which in the USA counts 10-20000 patients per year. AIM OF THE STUDY: The purpose of this review is to update the current knowledge of the use of different therapeutic strategies in patients with NSAID induced upper gastrointestinal bleeding. RESULTS: Proton pump inhibitors (PPI) therapy is effective as a prevention of NSAID induced acidopeptic lesions, and also represents the first and best therapeutic option for the treatment of complications, such as upper gastrointestinal bleeding. In the last three decades use of early flexibile (diagnostic and therapeutic) endoscopy, agressive acidosupression (PPIs), and surgical treatment in restrictive indications, resulted in decreasing of the mortality rates from 25-35% to 6-10%. When PPIs and flexible endoscopy are not sufficient in the control of upper gastrointestinal bleeding, use of systemic hemostatic drugs could be taken into consideration. CONCLUSION: Multidisciplinary approach, precise diagnostic and therapeutic critearia would probably result in better outcome of patients with active upper gastrointestinal bleeding.

[Iron deficiency anemia and its importance in gastroenterology clinical practise].

Iron deficiency anemia (IDA) is a universal problem involving individuals of all ages and both sexes and is a common cause of referral to medical departments. This anemia is one of the most common types of anemia. IDA impairs growth and intellectual development in children and adolescent. In women IDA is most common in reproductive period because of menstrual and pregnancy iron losses. IDA affects roughly 10-30% of all pregnancies and, among others morbidities, may contribute of developing postpartum depression. Among other adult patient, chronic occult gastrointestinal bleeding is the leading cause of IDA. Approximately, one third of patients with anemia have iron deficiency and up to two thirds of patients with IDA have serious gastrointestinal lesions detected with esophagogastroduodenoscopy and colonoscopy, including 10-15% with malignancy. However, in practice not all anemic patients undergo appropriate diagnostic tests to detect iron deficiency. Furthermore, a substantial proportion of patients with IDA do not undergo endoscopic evaluation. The approach to its investigation and subsequent therapy depends upon a comprehensive understanding of iron metabolism and heme synthesis. Once diagnosis of iron deficiency or IDA is established, evaluation for the cause of anemia must be appropriate performed and treatment must include corrective replenishment of body stores.

[Systemic hemostatic drugs].

Understanding the haemostatic changes is crucial in developing strategies for the management of haemorrhage syndroma. In recent years, the revised model of coagulation ("cell based" model) provided a much more authentic description of the coagulation process. Pharmacological intervention, especially desmopresin, antifibrinolytics (synthetics and nature) and increasingly recombinant activated factor VII are being used in prevention and therapeutically to control bleeding of variety etiologies. Skillfull surgery combined with blood saving methods and careful management of blood coagulation will all help in sucessfull haemorrhage prevention and treatment, and reduce unnecessary blood loss and transfusion requirements and its attendant risks. Among the all avalaible tests, the use of thromboelastography has allowed for more detailed dynamic assessment of the various steps of hemostasis.

Delayed hemolytic transfusion reaction due to anti-Jk(a).

BACKGROUND: Kidd antibodies are very heterogeneous and difficult to detect. They have been frequently implicated in delayed hemolytic transfusion reactions (DHTRs). CASE REPORT: A 64 year old female (6 pregnancies, 2 deliveries, 4 abortions) with none red cell (RBC) transfusions in the history was admitted to hospital due to pneumonia and severe anemia. On admittance hemoglobin (Hb) level was 63g/L and hematocrit (Ht) 0.21 L/L. The blood sample of the patient was sent to laboratory for serologic testing since RBC transfusions were required. Patient appeared to beO Rh(D)+ with negative both direct antiglobulin (DAT) and routine antibody screen (ID-DiaCell I+II+III-P). Three units of packed RBCs with negative crossmatch (tube method) were prepared. Patient received two units on Day 2 and one more on Day 3 without any discomfort. Hematological values after the third unit were: Hb 116g/L and Ht 0.37 L/L. On Day 6 she started to feel week, tired, with nausea and mild jaundice. Her Hb and Ht had dropped to 99 g/L and 0.33 L/L respectively, with tendency of dropping further (Day 7: Hb 83 g/L, Ht 0.26 L/L). Total serum bilirubin was 58.9 umol/L (normal range 20.5 umol/L) and direct fraction was 14.9 umol/L (normal range 7 umol/L). DTHR was suspected. Antibody identification performed by ID-DiaMed Gel Techique (GT) showed the presence of anti-Jk(a) with dosage phenomenon. All three previously transfused units were typed Jk(a) and the patient s RBCs were Jk(a-b+). She received two units of Jk(a) negative packed RBCs and was well enough to be discharged on Day 14. CONCLUSION: It is important to monitor clinical effect of transfusion regularly and to provide good team work between specialists of transfusion medicine and related medical staff. The policy of transfusion practice is to keep pretransfusion sample for three weeks and to perform cross-match tests on the samples no older then 24h and 48h respectively.

[Acute hemolytic transfusion reaction]. / Akutna transfuziona hemolizna reakcija.

Alloogenous blood and/or corresponding haemoproduct transfusion is an efficient and relatively safe supportive treatment. Despite the fact that pre transfusion investigation of both patients and donors ensure high degree of safety of this type of treatment, occurrence of adverse haemotherapy effects is possible and often unpredictable. Acute haemolytic transfusion reaction occur as a consequence of immune conflict between red blood cell membrane agents and specific antibodies present in plasma. Since it is impossible to completely avoid the occurrence of transfusion reactions, wherein acute transfusion haemolytic reaction present a serious, possibly life threatening complication, it is an imperative to continue to improve the knowledge on pathogenesis mechanisms leading to complications associated with these reaction and to define the most efficient therapeutical modalities.

[Acute transfusion-related lung injury]. / Akutno ostecenje pluca izazvano transfuzijom (transfusion related acute lung injury--TRALI).

Transfusion Related Acute Lung Injury (TRALI) is the most serious, potentially lethal, transfusion reaction caused by anti leukocyte antibodies carried passively into recipient's circulation by transfused haemoproduct. It is manifested most frequently within first four hours after transfusion of allogenous haemoproduct containing anti leukocyte antibodies. It is characterized with symptoms and signs of acute respiratory distress syndrome. This rare, but severe transfusion reaction with mortality rate of around 10% is often misdiagnosed, since TRALI signs are assigned to other clinical conditions. Thus, an education for timely recognition and urgent care of TRALI should be initiated.

[Comparative and quantitative analysis of proteins in the urine peritoneal fluid, separated by gel electrophoresis and stained with silver]. / Sravnitel'nyi i kolichestvennyi analiz belkov mochi i peritoneal'noi zhidkosti, razdelennykh metodom gel'-élektroforeza i okrashennykh serebrom.

The method for the silver staining of proteins fractionated with SDS electrophoresis is described as a new approach for the complex analysis of biological fluids allowing to obtain the valuable diagnostic information and to determine quantitatively individual proteins in urine and peritoneal fluid, in particular in nephropathologies.

Discontinuities in the temperature function of transmembrane water transport in Chara: relation to ion transport.

The NMR (nuclear magnetic resonance) method of Conlon and Outhred (1972) was used to measure diffusional water permeability of the nodal cells of the green alga Chara gymnophylla. Two local minima at 15 and 30 degreesC of diffusional water permeability (Pd) were observed delimiting a region of low activation energy (Ea around 20 kJ/mol) indicative of an optimal temperature region for membrane transport processes. Above and below this region water transport was of a different type with high Ea (about 70 kJ/mol). The triphasic temperature dependence of the water transport suggested a channel-mediated transport at 15-30 degreesC and lipid matrix-mediated transport beyond this region. The K+ channel inhibitor, tetraethylammonium as well as the Cl- channel inhibitor, ethacrynic acid, diminished Pd in the intermediate temperature region by 54 and 40%, respectively. The sulfhydryl agent p-(chloromercuri-benzensulfonate) the water transport inhibitor in erythrocytes also known to affect K+ transport in Chara, only increased Pd below 15 degreesC. In high external potassium ('K-state') water transport minima were pronounced. The role of K+ channels as sensors of the optimal temperature limits was further emphasized by showing a similar triphasic temperature dependence of the conductance of a single K+ channel also known to cotransport water, which originated from cytoplasmic droplets (putatively tonoplast) of C. gymnophylla. The minimum of K+ single channel conductance at around 15 degreesC, unlike the one at 30 degreesC, was sensitive to changes of growth temperature underlining membrane lipid involvement. The additional role of intracellular (membrane?) water in the generation of discontinuities in the above thermal functions was suggested by an Arrhenius plot of the cellular water relaxation rate which showed breaks at 13 and 29 degreesC.

Polarographic investigation of beta 2-microglobulin and ferritin thermal denaturation.

A temperature dependence of the corresponding signals, obtained by differential pulse (d.p.) and alternating current (a.c.) polarography, from a buffered aqueous solution of ferritin and beta 2-microglobulin is used for the characterization of a protein thermal denaturation process. The method is based on the significant differences in the interaction of folded and unfolded protein forms with a dropping mercury electrode due to a different accessibility, for the redox process, of protein electroactive groups. From the analysis of the resulting current, or capacitance, signals in function of temperature the thermal transition reversibility of different protein forms in the solution, protein melting points, and the apparent activation energies of the corresponding processes were determined.

Isolation and characterization of isoforms of retinol binding protein by isoelectrofocusing.

Retinol binding protein prepared from human urine was fractionated by chromatofocusing into four isoforms: two retinol-containing (holo-) and two retinol-free (apo-) species. The pl values of the isoforms ascertained by isoelectrofocusing with an immobiline pH gradient were: holo(I) 4.79-4.77; apo(II) 4.61-4.56; holo(III) 4.63 and apo(IV) 4.46-4.41. In vitro aging experiments with apo(II) under conditions favoring deamidation (37 degrees C, pH 7-10, 3-28 days) resulted in formation of the more acidic apo(IV)-isoform. The aging rate was consistent with pH increase. It appears that the urinary RBP mixture is composed of two apo-holo pairs: a native form with genuine protein structure and an acidic form generated upon aging.

[Silver staining of proteins separated by polyacrylamide gel electrophoresis: mathematical model]. / Okrashivanie serebrom belkov, razdelennykh élektroforeticheski v poliakrilamidnom gele: matematicheskaia model'.

Silver staining of proteins separated by electrophoresis in the thin layer of polyacrylamide gel can be quantitatively measured when the results are statistically treated. In the mathematical model, the reduction of protein-bound silver ions (crucial silver staining step) is considered to be autocatalytic. According to the model, the reaction rate strongly depends on the stability constant of silver complexes with functional groups of protein amino acids; and the shape of the calibration curve is determined by the ratio of concentration of silver-binding side chain groups to the reciprocal value of the stability constant. The complex shape of the curves can be explained the combination of autocatalysis and saturation. High sensitivity of this reaction to experimental conditions, contribution of complexes with different stability constants to the intensity of protein band staining, and optical effects are analyzed.

D2O-induced ion channel activation in Characeae at low ionic strength.

Effects of D2O were studied on internodal cells of the freshwater alga Nitellopsis obtusa under plasmalemma perfusion (tonoplast-free cells) with voltage clamp, and on Ca2+ channels isolated from the alga and reconstituted in bilayer lipid membranes (BLM). External application of artificial pond water (APW) with D2O as the solvent to the perfused plasmalemma preparation led to an abrupt drop of membrane resistance (Rm = 0.12 +/- 0.03 k omega.cm2), thus preventing further voltage clamping. APW with 25% D2O caused a two-step reduction of Rm: first, down to 2.0 +/- 0.8 k omega.cm2, and then further to 200 omega.cm2, in 2 min. It was shown that in the first stage, Ca2+ channels are activated, and then, Ca2+ ions entering through them activate the Cl- channels. The Ca2+ channels are activated irreversibly. If 100 mM CsCl was substituted for 200 mM sucrose (introduced for iso-osmoticity), no effect of D2O on Rm was observed. Intracellular H2O/D2O substitution also did not change Rm. In experiments on single Ca2+ channels in BLM H2O/D2O substitution in a solution containing 100 mM KCl (trans side) produced no effect on channel activity, while in 10 mM KCl, at negative voltage, the open channel probability sharply increased. This effect was irreversible. The single channel conductance was not altered after the H2O/D2O substitution. The discussion of the possible mechanism of D2O action on Ca2+ and Cl- channels was based on an osmotic-like stress effect and the phenomenon of higher D-bond energy compared to the H-bond.

Effects of D2O on permeation and gating in the Ca(2+)-activated potassium channel from Chara.

We studied the effects of H2O/D2O substitution on the permeation and gating of the large conductance Ca(2+)-activated K+ channels in Chara gymnophylla droplet membrane using the patch-clamp technique. The selectivity sequence of the channel was: K+ > Rb+ >> Li+, Na+, Cs+ and Cl-. The conductance of this channel in symmetric 100 mM KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D2O as compared to H2O. The blockade of channel conductance by cytosolic Ca2+ weakened in D2O as a result of a decrease in zero voltage Ca2+ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D2O primarily due to the change in Ca2+ binding to the channel during the activation step. The Hill coefficient for Ca2+ binding was 3 in D2O and around 1 in H2O. The values of the Ca2+ binding constant in the open channel conformation were 0.6 and 6 microM in H2O and D2O, respectively, while the binding in the closed conformation was much less affected by D2O. The H2O/D2O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1 mM internal Ca2+.

1H NMR assignments and secondary structure of human beta 2-microglobulin in solution.

Sequence-specific resonance assignments of human beta 2-microglobulin (M(r) 12,000) and its secondary structure are determined by 2D NMR techniques. The protein is found to contain two antiparallel beta-sheets each of four beta-strands with the beta-sheets being connected by a single disulfide linkage. No evidence for any regular helical structure is found. Amide proton-solvent-exchange rate constants and 3JHN alpha coupling constants are evaluated.

Renal function, protein excretion, and pathology of Balkan endemic nephropathy. II. Protein excretion.

The urinary proteins of 40 patients with Balkan endemic nephropathy from the Tuzla region were examined using ultrathin-layer SDS pore-gradient polyacrylamide gel electrophoresis in combination with silver staining. The typical urinary protein spectrum contained immunoglobulin G, Tamm-Horsfall protein, transferrin, albumin, beta 2-microglobulin (beta 2m), immunoglobulin light chains, retinol-binding protein, and alpha 1-microglobulin (alpha 1m). Densitometric measurements were used to derive glomerular tubular protein ratios (GTPR) and to characterize protein excretion patterns in the 28 patients who excreted more than 150 mg/liter of protein. Results showed that proteinuria of Balkan nephropathy is predominantly tubular, consisting of low-molecular-weight species. The most commonly identified proteins were alpha 1m, light chains, retinol binding protein, and beta 2m. The pattern of proteinuria based on GTPR did not correlate with the underlying histology or the degree of renal failure. These findings, using the ultrathin-layer SDS pore-gradient method of protein separation, more accurately demonstrates the low-molecular-weight proteinuria characteristic for the early stages of BEN.

Survey of small molecule and ion binding to beta 2-microglobulin--possible relation to BEN.

Using a new method which permits rapid detection of ligand binding to cross linked protein films and crystals, we evaluated a spectrum of ligand binding to cross linked amorphous films of beta 2-microglobulin purified from urine of Balkan endemic nephropathy patients. Among more than 50 substances studied, including amino acids, fatty acids, sugars, nucleotides, nucleosides, and different inorganic ions, only polyvalent cations, such as Cu2+, Ca2+, Zn2+, Be2+ and La3+, were found to strongly bind to human beta 2-microglobulin with dissociation constants in the range 10(-6) to 10(-4) M. These cations can release beta 2-microglobulin from HLA complexes leading to an increased beta 2-microglobulin level in serum. These observations suggest a possible mechanism for the release of beta 2-microglobulin to make it available to exert toxic effects on the kidney as a potential pathogenesis of Balkan endemic nephropathy.

D2O-induced cell excitation.

The effects of deuterium oxide (D2O) on giant internodal cells of the fresh water alga Chara gymnophylla, were investigated. D2O causes membrane excitation followed by potassium leakage. The primary effect consists of an almost instantaneous membrane depolarization resembling an action potential with incomplete repolarization. A hypothesis was proposed which deals with an "osmotic stress" effect of D2O on membrane ion channels followed by the suppression of the electrogenic pump activity. The initial changes (potential spike and rapid K+ efflux) may represent the previously undetected link between the D2O-induced temporary arrest of protoplasmic streaming and the early events triggered at the plasma membrane level as the primary site of D2O action.

Adsorption of human beta 2-microglobulin at a water/mercury interface.

Adsorption of human beta 2-microglobulin from a neutral solution of 0.15 M NaCl on a mercury surface was studied at 25 degrees C by measurement of the differential capacity of the electrical double layer. From the diffusion-controlled adsorption kinetics, the surface concentration and hence the area occupied by the adsorbed beta 2-microglobulin molecule were determined at various potentials of the mercury surface. The results indicate unfolding or flattening of beta 2-microglobulin molecules adsorbed in particular on the electrically uncharged surface. The extent of this interfacial conformational rearrangement was reduced with growing positive or negative surface charge density.

Interaction of fatty acid sodium salts with sodium deoxycholate.

Interaction of homologous fatty acids (C3-C18) with sodium deoxycholate was investigated. From NMR and ultrasonic results it was found that short chain homologues (up to C9) do not participate in the formation of mixed micelles with sodium deoxycholate. Fatty acid homologues with longer chains (starting with C9) form mixed micelles by "burying" hydrophobic chains in hydrophobic environment of a sodium deoxycholate micelle.