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Proteins and peptides in mammals are based exclusively on L-amino acids. Recent investigations show that D-amino acids exhibit physiological effects in vivo, despite of their very small quantities. We have investigated the hepatoprotective effects of the Land D-enantiomers of alpha-melanocortin peptide (alpha-MSH). The results showed that peptide-enantiomerism is related to the protective effects of melanocortin peptides in vivo. L-alpha-MSH exhibited potent hepatoprotective effect in the experimental model of acetaminophen induced hepatotoxicity in male CBA mice, while its D-mirror image was inefficient. Furthermore, the antibody to the L-peptide did not recognize the D-structure. The results indicate that the opposite peptide configuration may be used to modulate its function and metabolism in vivo and in vitro.

Biđin Z., I. Lojkic, M. Mikec, B. Pokric: Naturally Occurring Egg Drop Syndrome Infection in Turkeys. Acta Vet. Brno 2007, 76: 415-421. A decrease in the egg quality, production, fertility and hatchability without serious clinical signs of illness was recorded in turkey fl ocks in Croatia at the beginning of 2002. It was assumed that the egg drop syndrome virus might be one of the etiological agents responsible for the abnormalities in the egg production. The systematic serological monitoring, using a haemagglutination inhibition test, showed that the antibodies to the egg drop syndrome virus existed in 94.4 and 55.1% of the sera analysed in 2002 and 2003, respectively. The haemagglutination inhibition titres ranged from 16 to 128. The sera samples were randomly collected from 11 - to 46-week-old hens from the affected fl ocks. The serological evidence of the egg drop syndrome virus infection was confi rmed by detection of the presence of the virus genome in the turkey sera by the polymerase chain reaction. Vaccination of the 18- and 25-week-old turkey hens against the egg drop syndrome virus started in March 2003. After this period, the presence of antibodies to the egg drop syndrome virus (the haemagglutination inhibition titres between 16 and 256) was found in 96.7% of the analysed sera, while the egg production reached normal or higher values for the Nicholas hybrid line of turkeys. Croatia, egg drop syndrome virus, haemagglutination inhibition, Meleagris gallopavo

A theoretical approach for the determination of the equilibrium constant, Ka, of the reaction between a multideterminant antigen (Ag) and specific polyclonal antibodies (Ab) forming the insoluble Ab/Ag immune complex, is derived. The constant can be expressed as a function of the two accessible experimental parameters, the precipitating concentration of the antigen and the Ab/Ag molar ratio. For this purpose Ab/Ag immune complex must be prepared at equivalence, and equilibrium between precipitated and soluble species must be reached. The proposed method is experimentally tested on the system human serum albumin (HSA) and polyclonal rabbit antibodies. The Ab/Ag precipitates are prepared by the direct mixing of biological fluids in which immunoreacting components naturally occur. Previous separation, purification, or labeling of immunoreacting components are not required. The conditions for the precipitation of Ab/Ag complexes at equivalence, the stoichiometric composition or the average number of Ab molecules bound to one Ag molecule, and the solubility of the immunoprecipitating components are determined by a rectangular two-dimensional double immunodiffusion. Since the solubility determined under the conditions of a double immunodiffusion is a result of the interaction of the global diffusion of the precipitating components and particle growth kinetics, it mostly refers to the dynamic conditions. To find the solubility under equilibrium conditions, it is sufficient to determine the minimal factor by which the solutions of both immunoprecipitating components should be diluted so that no precipitate is formed upon their mixing at equivalence. The dilution factor is determined by a measurement of the laser light scattering of the immunoprecipitating systems prepared with serially diluted Ag and Ab solutions.

Summary: The lack of control of tumour behaviour is manifested in different ways, depending primarily on the type of tumour. This results in numerous problems of tumour diagnosis and therapy. In the case of "benign" tumours, like pituitary adenomas, in vitro studies are often used for evaluation of the tumour. The use of tissue explant cultures of human pituitary adenomas and the comparison of the feature of cultured tumours with their behaviour in vivo showed that corticotropin is released not only from the tumours associated with Gushing^ disease, but also from clinically non-functioning tumours. Hence, it was supposed that the release of corticotropin in vivo from non-secreting tumours is probably under the influence of certain neuroendocrine and/or systemic humoral factors. To test this possibility, samples of 22 tumours were cultured in plain culture medium or in the presence of the "human plasma ultrafiltrate bioactive fraction" (tentatively termed as TBP) prepared by anion-exchang e chromatography. In the presence of TBP the release of corticotropin was strongly inhibited in adenomas showing relatively high spontaneous secreting activity in vitro (> 200 ng/1 in 24 hours), while immunohistochemistry of these tumours indicated accumulation of corticotropin inside the cells. In contrast, TBP stimulated corticotropin release from tumours that showed relatively low basic corticotropin release (< 200 ng/1 in 24 hours), with no obvious change in cellular corticotropin immunoreactivity. Such a dual activity of TBP was not observed for 8 samples of adenomas cultured in the presence of surrounding pituitary tissue, probably because TBP did not affect corticotropin secretion by the normal pituitary cells (as indicated by immunohistochemistry). From these results, it appears that TBP could be one of the humoral factors involved in the regulation of corticotropin release from pituitary adenoma tissue. Its possible involvement in the regulation of corticotropin release from normal pituitary tissue, however, is uncertain.

A method for simultaneous qualitative and quantitative characterization of virus envelope proteins and virus-neutralizing antibodies is described. The quantitative estimate is based solely on the precipitation of the antibody/antigen (Ab/Ag) complexes at equivalence under equilibrium conditions. The qualitative analysis is performed by determining the diffusion coefficients, i.e., molecular masses of antigen and antibodies. Isolation, purification, labelling of antigen and antibodies and use of any standards are not required. The analyses are carried out directly in crude biological fluids in which antigen and antibodies naturally occur. The results obtained are independent on the avidity of the Ab/Ag system to be analysed. The method was tested by using antigenic subunits of Newcastle disease virus (NDV) and four chicken anti-NDV immune sera of different avidities.

The two-dimensional double immunodiffusion, the so-called 'two-cross' method, is introduced for determination of the size of antigenic subunits released after solubilization of enveloped viruses with non-ionic detergents. The method enables the determination of diffusion coefficients and calculation of relative molecular masses of immunoreacting components without using any standard and without prior isolation, purification or labelling of material to be analysed. The molecular masses of surface antigenic fragments of the Newcastle disease virus (NDV) disrupted by non-ionic detergent Triton X-100 were determined. Two antigenic fragments, the larger having a molecular mass between 350 and 187 kDa and the smaller from 140 to 86 kDa, were released by the action of 0.1-1.0% detergent at 20 degrees C. One fragment, whose molecular mass varied from 210 to 187 kDa, was obtained at 37 degrees C after treatment of NDV with 0.2-1.0% detergent, respectively The detergent disruption of purified NDV is studied for comparison. No difference was found whether purified or NDV in allantoic fluid was subjected to the same detergent extraction.

The insoluble immune complexes (ICs) were prepared under the conditions of double immunodiffusion in gel, using the suspension of the ultrasound treated PK 15 cell line infected with porcine parvovirus (PPV) containing both viral particles and viral proteins, as well as pig or rabbit anti PPV polyclonal immune sera. The immunodiffusion performed in an agarose gel allows only viral subunits with a molecular mass equal to or less than 1000 KKD, rather than the viral particles, to diffuse through the gel and reach the point where the immunoprecipitate is to be formed. The immunoprecipitation under the conditions of the diffusion ensures the optimal, i.e., equimolar ratio of both immunoprecipitating components, antibody/antigen in the IC. The sodium dodecyl sulfate polyacrylamide gel electrophoresis and the Western blot analyses showed the ICs were composed of two proteins, a protein which molecular mass corresponded to the VP2 of the PPV and a protein with a molecular mass of the IgG. This suggests that the ICs are mainly composed of the VP2 antigen and IgG class antibodies. The potency of the IC vaccines prepared in the form of a water in oil in water emulsion (WOWE) was compared with that of a commercially available, inactivated oil vaccine. The vaccination of gilts, 6 weeks before mating, with the IC containing allogeneic pig antibodies, resulted in the development of high and long lasting anti PPV antibody titers, similar to those generated by the licensed vaccine (probability > 0.01). The content of the virus material administered by the IC was more than twice lower than that in the licensed vaccine. Neither systemic nor local reactions were observed in the gilts during the period of the trial with the IC vaccine. The number of viable piglets per litter varied between 9 and 12 and no signs of the PPV infection were detected. Rabbits were used as one of the alternative laboratory animal models accepted for the testing of the vaccine against the PPV. The rabbit humoral immune response generated by the IC containing the allogeneic antibodies was higher than that generated by the ICs containing the xenogeneic pig antibodies. It was similar to that generated by two times higher content of the virus material administered by a commercially available vaccine. The IC based vaccines belong to non replicating, subunit vaccines which are both ecologically convenient and the safest vaccines of all.

The hydropathic profile, secondary structure, epitope and binding site of the a-melanotropin molecule were investigated. It was shown that the standard algorithm according to Kyte and Doolittle may be combined with complex methods of the secondary structure prediction to extract the information relevant for the epitope location and modeling. The binding ligand-receptor motifs of the hormone were investigated by means of the SSpro8 method. The Molecular Recognition Theory combined with an NCBInr protein database search was applied to find the possible paratope (receptor) structures for the predicted a-melanotropin epitope (ligand). The described concept constitutes a useful and simple set of procedures for deriving new biologically active peptides and antibodies and also for performing modulation of peptide-receptor interaction.

Molecular Recognition Theory is based on the finding of Blalock et al. (Biochem. Biophys. Res. Commun. 121 (1984) 203-207; Nature Med. 1 (1995) 876-878; Biochem. J. 234 (1986) 679-683) that peptides specified by the complementary RNAs bind to each other with higher specificity and efficacy. This theory is investigated considering the interaction of the sense peptides coded by means of messenger RNA (read in 5'--3' direction) and antisense peptides coded in 3'--5' direction. We analysed the hydropathy of the complementary amino acid pairs and their frequencies in 10 peptide-receptor systems with verified ligand-receptor interaction. An optimization procedure aimed to reduce the number of possible antisense peptides derived from the sense peptide has been proposed. Molecular Recognition Theory was also validated by an "in vivo" experiment. It was shown that 3'--5', peptide antisense of alpha-MSH abolished its cytoprotective effects on the gastric mucosa in rats. Molecular Recognition Theory could be useful method to simplify experimental procedures, reduce the costs of the peptide synthesis, and improve peptide structure modelling.

The activities of two enzymes from phase I of biotransformation, aniline hydroxylase and ethylmorphine N-demethylase were investigated in turkey embryos and poults. Measurements were performed five times prior to hatching, at the 18th, 22nd, 24th, 26th and 27th day of embryos’ life and five times after hatching, at the 1st, 5th, 9th, 16th and 26th day of turkeys’ life. Ten days prior to hatching the enzyme activities in embryo livers amounted to 116.8 ± 1.6 nmol p-aminophenol/g liver/30 min, and 222.8 ± 25.0 nmol formaldehyde/g liver/30 min for aniline hydroxylase and ethylmorphine Ndemethylase, respectively. A three- and two-fold increase in aniline hydroxylase and ethylmorphine N-demethylase activities was observed immediately after hatching (1st day) in relation to 27-day-old embryos, respectively. Thereafter activities continued to rise and in 26-day-old poults they amounted to 672.9 ± 10.3 nmol p-aminophenol/g liver/30 min, and 2672.6 ± 27.9 nmol formaldehyde/g liver/30 min. Body masses, as well as absolute and relative liver masses were measured after hatching. The ratio between liver/body mass showed that relative liver mass increased from the 1st to the 9th day of life, followed by a constant slight decrease until the 26th day of life. These results generated in physiological conditions indicate that biotransformation phase I reactions are higher at the end of first month of turkey life than those in turkey embryos and in newly-hatched turkey. Thereby, liver capacityfor hydroxylation and N-demethylation of xenobiotics and endogenous compounds gradually increase in young turkey., U puranskih zametaka i purića istražene su aktivnosti dva enzima iz I. faze biotransformacije: anilin hidroksilaze i etilmorfin N demetilaze. Mjerenja su provedena pet puta prije leženja, i to 18., 22., 24., 26. i 27. dana razvitka zametka, te pet puta poslije leženja, 1., 5., 9., 16. i 26. dana života purića. Aktivnost jetrene anilin hidroksilaze u osamnaestodnevnih zametaka bila je 116.8 ± 1.6 nmol p-aminofenola/g jetre/30 min, a aktivnost etilmorfin N-demetilaze 222.8 ± 25.0 nmol formaldehida/g jetre/30 min. Dvostruko odnosno trostruko povećanje aktivnosti tih enzima izmjereno je neposredno nakon leženja u odnosu na 27 dana stare zametke. Nakon toga, aktivnosti enzima uglavnom su i dalje rasle, a najviše su bile zadnjeg dana mjerenja, tj. 26. dana života: 672.9 ± 10.3 nmol p-aminofenola/ g jetre/30 min i 2672.6 ± 27.9 nmol formaldehida/g jetre/30 min. Tjelesne mase purića te apsolutne i relativne mase jetre mjerene su nakon leženja, neposredno prije uzorkovanja jetre za enzimske analize. Vrijednosti relativne mase jetre u purića uvećavale su se od 1. do 9. dana života, nakon čega su se smanjivale do kraja promatranog razdoblja. Rezultati istraživanja aktivnosti navedenih enzima, iz I. faze biološkog prijetvora u fiziološkim uvjetima pokazuju da su njihove vrijednosti više u purića u dobi od mjesec dana nego u netom izleženih purića i puranskih zametaka, što je znak da se postupno uvećava kapacitet jetre za hidroksilaciju i N-demetilaciju endogenih i organizmu stranih tvari.

Infectious bursal disease viruses (IBDVs) in 26 IBDV-positive bursa samples collected in Croatia during the period 1996-2000 and in two commercially available vaccines were differentiated by the presence or absence of the CfoI, SacI, SspI, StuI, and TaqI restriction sites in the 422-bp fragment of segment A of the VP2 gene (nt 732-1153). The fragments from 14 (54%) field isolates were TaqI+ StuI+ SspI+ and SacI- CfoI-, indicating their very virulent (vv) character. The presence of CfoI restriction site in 10 (38%) field isolates is uncommon for vvIBDV strains. It was detected in only the 88180 vvIBDV strain. Nevertheless, these isolates can be classified as vv strains according to TaqI+ StuI+ SspI+ SacI- restrictions. Two SacI+ StuI+ CfoI+ TaqI- SspI- field isolates (8%) could be classified as non-vvIBDVs. The StuI+ restriction is common to vvIBDV strains. However, the StuI recognition sequence is present in the F52/70 classic European and 002-73 attenuated strains as well. The SacI+ CfoI+ StuI- SspI- restrictions and the lack of the TaqI restriction at nt position 832 show that the IBDV in GUMBOKAL IM-SPF vaccine corresponds to the attenuated and/or vaccine strains. The TaqI restriction at nt position 875 suggests that the IBDV in GUMBOKAL SPF vaccine could belong to the mild strains.

MATH/CHEM/COMP 2001 Dubrovnik International Course & Conference (MCC 2001)

We used the rat model of ethanol induced gastric lesions to measure cytoprotective effects of neuropeptides met-enkephalin and alpha-melanocyte stimulating hormone (alpha-MSH). The lesions were induced with i.g. application of 1 ml 96% ethanol. The peptides were given i.p. 1 h before the ethanol. Sacrifice was made 1 h after ethanol application and hemorrhagic gastric area was assessed in mm(2). alpha-MSH and met-enkephalin exhibited significant and additive cytoprotective effects. The protective effects of alpha-MSH were significantly stronger than of met-enkephalin. Almost total absence of lesions was obtained with met-enkephalin and alpha-MSH mixture 10:1 (10 mg/kg met-enkephalin and 1 mg/kg alpha-MSH). The addition of indomethacin (5 mg/kg s.c.) almost completely abolished the effect of met-enkaphalin, while alpha-MSH mediated cytoprotection was weakened but still present. Interestingly, indomethacin also blocked almost completely the cytoprotective effects of met-enkephalin and alpha-MSH mixture. The latter result may have a practical consequence for the clinical trials in which met-enkephalin and alpha-MSH could be used in combination with non-steroidal anti-inflammatory drugs.

Reverse transcription-polymerase chain reaction (RT-PCR) followed by restriction fragment-length polymorphism (RFLP) analysis was tested in order to determine its accuracy and sensitivity for differentiation of infectious bursal disease virus (IBDV) strains. Two commercial vaccine strains and two Croatian field isolates were analysed. RT-PCR assay was used for amplification of a variable part of VP2 gene (422 bp) encoding for major neutralizing epitopes. Amplified VP2 fragments were further characterized by RFLP analysis using the SacI and TaqI restriction endonucleases. Both field isolates showed RFLP pattern similar to highly virulent European and Asian strains. Both vaccine strains had RFLP profiles different from that of the field isolates. Vaccinal strain 2512 had RFLP pattern similar to mild vaccinal strain pbg-98, and strain VMG-91 was similar to other classical vaccinal strains of intermediate pathogenicity., Procijenjena je točnost i osjetljivost metode temeljene na razlici dužina restrikcijskih odsječaka DNA (RFLP, restriction fragment lenght polymorphism) za razlikovanje sojeva virusa zarazne bolesti burze (VZBB). Lančanom reakcijom polimerazom (PCR, polymerase chain reaction) uz prethodnu reverznu transkripciju (RT, reverse transcription) umnožen je dio promjenljivog dijela VP2 gena koji kodira glavne virusne neutralizacijske epitope. Nastali produkti veličine 422 parova baza (bp, base pair) pocijepani su restrikcijskim endonukleazama SacI i TaqI. Restrikcijski profili nastali enzimskim cijepanjem uspoređeni su s podatcima navedenim u banci gena. Metoda je testirana na dva komercijalna cjepiva proizvedena u Hrvatskoj (Gumbokal® SPF, soj 2512 i Gumbokal® IM SPF, soj VMG 91) te dva hrvatska terenska izolata VZBB (IBDV/96/01 i IBDV/ 97/02). Oba su terenska izolata pokazala RFLP profil jednak vrlo virulentnim europskim i azijskim sojevima VZBB. Oba cijepna soja imala su različit RFLP profil od terenskih izolata. Cijepni soj 2512 ima RFLP profil jednak blagim cijepnim sojem pbg-98, a VMG 91 je sličan poznatim intermedijarnim sojevima VZBB.

We applied the machine learning procedure to the analysis of serum and aqueous humor albumin, IgG and interferon-γ patterns. The data were analyzed with respect to the cataract, type of diabetes, retinopathy and blood-aqueous humor barrier damage. Intraocular production of IgG was detected in diabetic patients, since the IgG index values were increased in some diabetic groups (especially those with type I diabetes and retinopathy). Comparison of numerical methods and clonal IgG detection suggested that intraocular IgG patterns in diabetes are predominately polyclonal. Interferon-γ (IFN-γ) was pathological in serum and aqueous humor of patients with type I diabetes and to a lesser extent in the type II diabetes group. Machine learning system based on C5.0 classifier extracted 98.5% accurate rules for discrimination of the senile cataract group, diabetes group and diabetic retinopathy group. Our results confirm that the combination of immunochemical and numerical methods with a proper statistical analysis may contribute to the diagnosis of the pathologic ocular immune response and the related retinal or vascular disorders in diabetes. Significant savings in laboratory material and efforts may be obtained by means of the machine learning based optimization of the tests.

Immunogenicity of an experimental vaccine against Newcastle disease (ND) was tested on chickens and turkeys under laboratory and field condition, respectively.Vaccine, prepared in form of a water-in-oil-in-water (WOW), contained detergent solubilized ND virus. 21-day-old chickens, hatched from SPF eggs, were intramuscularlly (i.m.) vaccinated with 0.2 ml vaccine containing 1, 1/25, 1/50 and 1/100 viral doses. Also, 21-day-old light laying chickens received i.m. 0.2 ml vaccine containing 1, 1/5 1/25 and 1/50 viral doses. Control groups were not vaccinated. Three weeks postvaccination, chickens were i.m. challenge infected with 106 ELD50 Herts33 strain of NDV. The vaccinal dose recommended by Eu.Ph for safetly chicken protection was detrmined according to the Spaerman-Kärber methode and it amounted to 0.5 ml vaccine with 50 PD50/dose. Turkey breeders, vaccinated with 1.0 ml vaccine at the age of 16 weeks developed a high level of NDV antibodies persisting till the end of experiment, i.e. turkey age of 50 weeks. This suggest that no additional vaccination is requred during the whole laying period.

The Newcastle disease virus (NDV) was solubilized with 10% w/v Tween 80 and inactivated with 0.05% v/v formalin. The average molecular mass of the released antigenic subunits was 307 kD. The tested vaccine was prepared in the form of a water-in-oil-in-water emulsion (WOWE) vaccine. The oil-to-aqueous ratio was 1:2. The solubilized NDV was administered alone or built in a tetravalent WOWE vaccine. A dose of the monovalent vaccine containing an equivalent of 44.7 microliters of detergent-treated NDV-allantoic fluid (NDV-AF) was sufficient for the complete protection of the commercially available chickens vaccinated at the age of 5 wk and challenged 7 wk later. The anti-NDV-free chickens, vaccinated at 4 wk of age and challenged 2 wk postvaccination, were 100% and 73% protected by a vaccinal dose containing 178.6 and 89.3 microliters of detergent-treated NDV-AF, respectively. Commercially available light pullets, primary vaccinated with live lentogenic NDV vaccine, generated a protective level of NDV antibodies after revaccination with WOWE vaccine containing 89.3 microliters of detergent treated NDV-AF. Laying hens were revaccinated under field conditions at the beginning of the laying cycle by the tetravalent vaccine. A vaccinal dose/bird containing 11.2 microliters of detergent-treated NDV-AF elicited a long-lasting high level of NDV neutralizing antibodies.

A method for the exact and simultaneous measurements of immunoprecipitable antigens and antibodies in native biological fluids is described. It is based solely on the microgravimetric analysis of at least two immunoprecipitates of different stoichiometric compositions. The volume ratios of antigen and antibody solutions for the preparation of the immunoprecipitates at equivalence are determined by the two-cross immunodiffusion technique. The proposed procedure has advantages over other methods for quantitative determination of proteins, since the separation, purification and labelling of immune components, as well as the use of standards, are not required. The method was tested by using human serum albumin (HSA) and three rabbit antiHSA immune sera of various avidities. Immunoprecipitates of different stoichiometric compositions were prepared at pH 5.0, 5.5, 7.0 and 8.6. The minimal precision of the method was±0.2 μ g of protein. The coefficients of variation were 0.14 to 0.38% for HSA and 0.03 to 0.08% for antiHSA antibody.

In order to determine the mutations responsible for virulence, three Croatian field infectious bursal disease viruses (IBDV), designated Cro-Ig/02, Cro-Po/00, and Cro-Pa/98 were characterized. Coding regions of both genomic segments were sequenced, and the nucleotide and deduced amino acid sequences were compared with previously reported full-length sequenced IBDV strains. Phylogenetic analysis, based on the nucleotide and deduced amino acid sequences of polyprotein and VP1, was performed. Eight characteristic amino acid residues, that were common to very virulent (vv) IBDV, were detected on polyprotein: 222A, 256I, 294I, 451L, 685N, 715S, 751D, and 1005A. All eight were found in Cro-Ig/02 and Cro-Po/00. C-Pa/98 had all the characteristics of an attenuated strain, except for glutamine on residue 253, which is common for vv, classical virulent, and variant strains. Between less virulent and vvIBDV, three substitutions were found on VP5: 49 GRR, 79 IRF, and 137 RRW. In VP1, there were nine characteristic amino acid residues common to vvIBDV: 146D, 147N, 242E, 390M, 393D, 511S, 562P, 687P, and 695R. All nine residues were found in Cro-Ig/02, and eight were found in Cro-Po/00, which had isoleucine on residue 390. Based on our analyses, isolates Cro-Ig/02 and Cro-Po/00 were classified with vv IBDV strains. Cro-Pa/98 shared all characteristic amino acid residues with attenuated and classical virulence strains, so it was classified with those.

Precipitating titers and immunochemical titers obtained in a wide range of antigen-to-antibody concentration ratios by the two-cross immunodiffusion technique are compared with the corresponding laser light scatter precipitin curves. The two-cross immunodiffusion technique has also been applied to investigate whether polyethylene glycol of molecular mass 6000 and dextrans of molecular masses from 10,000 to 2,000,000 enhance the immunoprecipitation processes of the system human serum IgG-rabbit immune serum at pH 5.5 and 8.1 at 20 degrees C. It was found that the significant increase of precipitating titers of both precipitating components in the presence of polyethylene glycol is a consequence of a strong decrease of solubility of the primary antigen-antibody complex. The decrease of solubility does not affect the immunochemical titer of the immune serum, indicating stoichiometrical invariance of the precipitate at the equivalence. The apparent strong decrease of diffusion coefficients of both antigen and antibody in 20- and 40-g/liter polyethylene glycol solution is attributed to increase of viscosity of the solutions and to a partial self-association of protein molecules due to steric exclusion. In 40-g/liter polyethylene glycol solutions at pH 5.5 every fourth molecular entity of antigen and every third molecular entity of antibody are present in the form of a two-molecular self-associate, whereas in 20-g/liter polyethylene glycol solutions only 1% of antigen molecules and 8% of antibody molecules are associated. With the increase of pH to 8.1 the self-association of protein molecules is strongly further enhanced. Dextrans in 20-g/liter solutions, without regard to their relative molecular masses, do not influence precipitating titers and solubility of the antigen-antibody system at equivalence and do not enhance self-association of protein molecules. The strong decrease of diffusion coefficients of immunoglobulin G antigen and antibodies in dextran solutions is solely attributed to the increase of viscosity of the dextran solutions; hence there was no evidence of interaction of dextrans with serum IgG proteins.

The two-cross technique, a new two-dimensional double-diffusion technique in gelplates, has been applied for simultaneous determination of precipitating titers and diffusion coeffients of antigen and antibody in body fluids. The advantage of this technique is that it works without using any standard solution and ensures conditions of “time-invariant sink”. The theory of the technique has been verified by experimental results on the precipitating system human serum-rabbit anti-human IgG in phosphate-buffered saline solution at pH 7.4. The results obtained using several modes of calculations from experimental parameters have been compared and found satisfactory. The accuracy and reproducibility of the results have been confirmed. It has been found that at 20°C the diffusion coefficient of human IgG in 10-times-diluted serum is (4.4 ± 0.2) × 10 −7 cm 2 s −1 , while the diffusion coefficient of rabbit anti-human IgG in a purified preparation is (2.9 ± 0.2) × 10 −7 cm 2 s −1 . The critical precipitating concentration of human IgG against rabbit anti-human IgG is invariable to concentration and amounts to 0.174 ± 0.03 mg/100 ml at pH 7.4.

To obtain electrophoretic mobilities referring to free solution, relative or absolute correction factors should be used to correct apparent mobility data obtained in a porous supporting medium. The absolute correction factor gives a direct measure of the texture or porosity of the support. The significance and determination of the correction factors are explained in detail, theoretically and experimentally. Another important characteristic of the porous supporting medium is the acid—base dissociation at its interface, resulting in electroosmotic flow dependent on pH. Filter paper cellulose is dissociated, through at least three dissociation constants, over the entire pH range except at the isoelectric point, pH 1.10.

The “two-cross” technique, a new two-dimensional immunodiffusion technique, is applied to the study of immunosystems human serum transferrin: a preparation of rabbit antibodies, human serum immunoglobulin A-rabbit antiserum, and human chorionic somatomammotropin-rabbit antiserum. By using this technique it is possible to determine simultaneously the diffusion coefficients and the precipitating titers of the precipitating components and the immunochemical titers of antibody or antisera. The investigations were performed in 0.15 mol dm −3 phosphate-buffered saline solutions in the pH range 5.0–8.6 in 1% (w/v) agarose gel at 20.0 ± 0.1°C. The results obtained for the diffusion coefficients and for the precipitating titers indicated that the reversible self-association of antigens and antibodies with the increasing pH of the medium is accompanied by the decreasing solubility of the precipitating components in the immunoprecipitating system. The changes in immunochemical titers with increasing pH were used to explain the changes in the antigen-to-antibody ratios at equivalency.

Publisher Summary This chapter discusses the two-cross immunodiffusion technique for determining the diffusion coefficients and precipitating titers of antigen and antibody. The two-cross immunodiffusion technique is essentially a two-dimensional double immunodiffusion procedure. The diffusion proceeds simultaneously from two crosses, each consisting of four troughs cut at a right angle in a gel plate. The troughs of each cross are filled in alternate order with antigen solution and antibody solution. The concentrations of the diffusing components are adjusted to obtain a narrow and sharp precipitation band in the gel plate. The two-cross immunodiffusion technique is restricted to the antigen and antibody concentrations that give visible precipitin bands. The consistency of the results over time obtained for precipitating titers, critical precipitating concentrations, and diffusion coefficients for the immunoprecipitating system human IgG in serum-rabbit antibody preparation is discussed in the chapter. It is found that the antigen-to-antibody ratio in the precipitate at the equivalency for the system IgG-anti-IgG is constant from pH 5 up to pH 7, and then increases slightly.

The mechanism of natural precipitation processes has been studied using as a model system the one-dimensional double diffusion of the precipitating components in a gel. It was found, theoretically and experimentally, that precipitation under conditions of double diffusion occurs when the equivalent concentrations of both precipitating components are equal to a “critical” concentration. Under the experimental conditions used here the conventional solubility law does not apply. A general relation is deduced which enables the estimation of the relative error in the determination of the critical concentration for precipitation. Theoretical treatments of other authors are discussed.

DNA microarray analysis is a laboratory technique that permits simultaneous measurement of messenger RNA expression for larger number of genes. The original motivation behind development of this technology was automated discovery of gene networks. However, statistical basis of DNA microarray data analysis is still controversial. The most important statistical problems are: normalization methods, a huge number of genes (variables) versus a small number of observations (the dimensionality of the data), multiple comparison and low power. In our paper we explain how statistical limitations prevented DNA microarray to fulfil its original role.

Auxins are the first identified plant hormones that control and regulate plant growth and development. It is known that Auxin Binding Protein1 (ABP1) binds auxins with high affinity and it is a strong candidate for one of the auxin receptors. In order to understand molecular mechanism of auxin binding to ABP1 series of molecular dynamics (MD) simulations of ABP1 and its complexes with auxin-related compounds have been made. Beside the standard MD simulations at room temperature, simulations in which temperature was shortly increased on 500 K have also been made (altogether 70 ns of MD simulations was accomplished). Entrance and exit of auxin-related compounds from auxin binding pocket of ABP1 have been investigated using random expulsion molecular dynamics (REMD) simulations. Results of MD and REMD simulations enabled hypothesis about ABP1 molecular mechanism. Two different conformations of ABP1 are identified, one of which is stabilized by auxin presence. In all simulations the same network of water molecules leading from the bulky water to the ABP1 active site was formed, which pointes to the possible role that water molecules have in the ABP1 mechanism of working. Simulations of auxin exit from ABP1 binding site revealed two main routes by which auxin molecules enter and leave the ABP1 binding site. Considering assumed orientation of ABP1 to the membrane, one of them leads into membrane and the other to the lumen of ER or citosol, depending on the ABP1 location

Crown and thiacrown ethers are an important and widely used class of organic molecules particularly for chemical separations because of their selective binding of metal ions. Here we investigate the complexation of Cu, Cd and Hg cations with following structurally related thiacrown ether macrocycles in the liquid phase and gas phase mass spectrometry. Special attention was paid to selectivity of formation and stability of the monomeric products, as well as to the addition of second crown ether molecule.

We present new method that enables simple prediction of alpha- and beta-protein folding types from the nucleotide and amino acid sequences. SMO machine learning algotithm for the support vector machines was used for the classification. Relative frequency patterns of particular codons proved to be sufficient for the accurate SMO based discrimination of alpha- and beta- protein folding types. The method is not dependent on the physicochemical parameter analysis of the nucleotide or amino acid sequences. Overall prediction results and procedure cross-validations indicated that this procedure enables simple, quick and accurate prediction of the protein folds on a PC. The mehod may be used for detecting the type of the protein folding from the newly sequenced exon regions.