Monte Carlo simulations and experimental results on neutron production in the uranium spallation target QUINTA irradiated with 660 MeV protons.

The activation experiment was performed using the accelerated beam of Phasotron accelerator at the Joint Institute for Nuclear Research (JINR). The natural uranium spallation target QUINTA was irradiated with protons with energy 660 MeV. Monte Carlo simulations of neutron production were performed using the Geant4 code. The number of leakage neutrons from the sections of the uranium target surrounded by the lead shielding and the number of leakage neutrons from lead were determined. The total number of fissions in the setup QUINTA was determined. Experimental values of reaction rates for the produced nuclei in the 127I sample were obtained and several values of reaction rates were compared with the results of simulations. Experimentally determined fluence of neutrons in energy interval 10-175 MeV using the (n,xn) reactions in the 127I(NaI) sample was compared with the results of simulations. Possibility of transmutation of the long-lived radionuclide 129I in the QUINTA setup was estimated.

Molecular characterization of the Thermomonospora curvata aglA gene encoding a thermotolerant alpha-1,4-glucosidase.

The cloning, sequencing and structural characterization of a gene encoding a thermostable alpha-1,4-glucosidase from Thermomonospora curvata is described. DNA sequence analysis revealed four open reading frames designated aglA, aglR, aglE and aglF. The aglA gene encodes a thermostable alpha-1,4-glucosidase from T. curvata and is situated between two genes, aglR and aglE. Genes aglA, aglE and aglF are transcribed in the same direction, while aglR is transcribed in the opposite direction. By comparing the amino acid sequence of the alpha-1,4-glucosidase from T. curvata with other alpha-glucanases, it appears that the enzyme is a member of the alpha-amylase family. The proteins of this family have an (alpha/beta)8 barrel super secondary structure. The topology of the alpha-1,4-glucosidase was predicted by computer-assisted analysis. The topology of the secondary structures of the alpha-1,4-glucosidase resembles the structure of barley alpha-amylase, but the primary structure resembles most closely the oligo-1,6-glucosidase from Bacillus cereus. Putative catalytic residues (D221, E281 and D343) and calcium binding residues (N116, E179, D191, H224 or G225) are proposed.

Cloning and characterization of the pknA gene from Streptomyces coelicolor A3(2), coding for the Mn2+ dependent protein Ser/Thr kinase.

A gene pknA, coding for an eukaryotic-type protein Ser/Thr kinase, was cloned from the Streptomyces coelicolor A3(2) chromosome. The PknA protein kinase, containing the C-terminal eukaryotic-type kinase domain with an N-terminal extension, was expressed in Escherichia coli and Streptomyces lividans. The affinity purified MBP-PknA fusion protein was assayed for kinase activity that showed its ability to autophosphorylate in vitro in the presence of [gamma-32P]ATP. The activity was Mn2+ dependent. The preautophosphorylated kinase phosphorylated at least two proteins (sizes 30 and 32 kDa) in the S. coelicolor J1501 cell-free extracts of all developmental stages. The larger of them was also phosphorylated in vitro by an endogenous protein kinase in late stages extracts, but not earlier. Although Mn2+ dependent protein phosphorylation has previously been described in Streptomyces, this is the first report of a gene encoding such an enzyme in this genus.

WD-repeat protein encoding genes among prokaryotes of the Streptomyces genus.

Southern hybridization with probes designed for detection of WD-repeats coding sequences gave positive results in 21 streptomycete strains indicating that WD-repeats encoding genes are massively spread among streptomycetes. One of them, the wdlA gene of Streptomyces lincolnensis, codes for a 971 amino acid protein with seven WD-repeats in its C-terminus, two transmembrane domains and an ATP/GTP binding site upstream of the WD-repeat region.

The genes lmbB1 and lmbB2 of Streptomyces lincolnensis encode enzymes involved in the conversion of L-tyrosine to propylproline during the biosynthesis of the antibiotic lincomycin A.

The genes lmbA,B1,B2 in the lincomycin A production gene cluster of Streptomyces lincolnensis were shown to form a common transcription unit with the promoter located directly upstream of lmbA. The proteins LmbB1 (mol. mass, 18 kDa) and LmbB2 (mol. mass 34 kDa), when over-produced together in Escherichia coli, brought about enzyme activities for the specific conversion of both L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) to a yellow-colored product. The LmbB1 protein alone catalyzed the conversion of L-DOPA, but not of L-tyrosine. The purified LmbB1 protein showed a Km for L-DOPA of 258.3 microM. The L-tyrosine converting activity could not been demonstrated in vitro. The preliminary interpretation of these data suggests that the protein LmbB1 is an L-DOPA extradiol-cleaving 2,3-dioxygenase and that the protein LmbB2, either alone or in accord with LmbB1, represents an L-tyrosine 3-hydroxylase. This sequence of putative oxidation reactions on L-tyrosine seems to represent a new pathway different from the ones catalyzed by mammalian L-tyrosine hydroxylases or the wide-spread tyrosinases. The protein LmbA seemed not to be involved in this process. The labile, yellow-colored product from L-DOPA could not be converted to a picolinic acid derivative [3-(2-carboxy-5-pyridyl)alanine] in the presence of ammonia. Therefore, it probably is not a derivative of a cis, cis-3-hydroxymuconic acid semialdehyde; instead, its speculative structure represents a heterocyclic precursor of the propylhygric acid moiety of lincomycin A.

Effects of temperature and novobiocin on the expression of calf prochymosin gene and on plasmid copy number in recombinant Escherichia coli.

Escherichia coli strain HB101 harboring an expression plasmid bearing calf prochymosin gene under the control of the tac promoter was grown in the presence of IPTG with or without novobiocin at 28 and 40 degrees C, respectively. The differential rates of synthesis of prochymosin inclusions, and, for comparison, of beta-lactamase and beta-galactosidase, as well as plasmid copy number, were determined during the first hours of steady state growth. At 28 degrees C the induced expression of prochymosin gene was almost blocked. Addition of novobiocin did not alleviate this effect. In fact, it strengthened it, and we conclude that both these additive inhibitory effects are a consequence of the decrease in negative superhelical tension of plasmid DNA to an insufficient level. At 40 degrees C the differential rate of prochymosin synthesis was markedly enhanced. Since the copy number of the expression plasmid increased approximately to the same extent, we conclude that an increase in gene dose is the cause. The stimulation of cloned heterologous gene expression at 40 degrees C and inhibition at 28 degrees C may be conveniently used in biotechnological-scale cultivations of some recombinant bacteria.

Constitution of the metabolic type of streptomycetes during the first hours of cultivation.

Using the examples of biosynthesis of streptomycin, bialaphos, actinorhodin, oligoketides and autoregulators during the first hours of streptomycete cultivation, it is stressed that the external environment in cooperation with the internal metabolic abilities of the cell determines the metabolic type that would develop during the life cycle of the producing streptomycetes. If we accept that a certain metabolic type (from the point of view of the production of secondary metabolites) was determined already during the first hours of cultivation of the microorganisms, we must also admit that the availability of primary metabolites in the so-called production phase of growth (stationary phase, idiophase, etc.) is to a certain extent determined by the very early stages of strain development.

[Phalloplethysmography findings in pedophilia and exhibitionism offenders]. / Falopletysmografické nálezy u pachatelu pedofilne exhibicionistických deliktu.

BACKGROUND: Exhibitionist behaviour focused on children (usually girls) is one of the most frequent forms of sexual child abuse. Exposure in the presence of children signalizes possible pedophil inclinations of the offender. The present paper presents the results of PPG examinations of these offenders with special attention to assessment how often these men have an objective phallographic pedophil orientation. The results of PPG examinations in this group of sexual delinquents are also compared with the findings in a control group of men who report a heterosexual orientation and preference of objects of suitable age. METHODS AND RESULTS: Using an electrocapacitance phalloplythysmographic apparatus the authors examined 50 men (mean age 36.3 years, range 17-67 years) who committed exhibitionist activities in the presence of girls under 12 years. The PPG examination was made under standard conditions; all examined subjects were shown sets of coloured slides with adult and child objects of hetero and homosexual character. In the same way a control group was examined formed by 50 male volunteers of similar age and social background. The men in the control group responded more frequently and more merkedly by positive vasomotor reactions to adult heterosexual objects than subjects who committed pedophil exhibitionist offences. The latter reacted significantly more often to child objects of both sexes. In more than half the delinquent group (in 26 men) there was an undefined differentiation of age as regards objects of female sex and 6 (12%) men of this group had preference for heterosexual child objects, i.e. an obvious pedophil orientation. In men of the control group only in one case there was an indication of an ill defined differentiation of age, in all the others an unequivocal or clearly indicated preference of heterosexual objects was found. CONCLUSIONS: PPG examinations confirmed that only a minor part of subjects committing pedophil exhibitionist offences have a deviant orientation. It was revealed that the most frequent phallographic finding in this group of sexual delinquents is an ill defined differentiation of age in objects of female sex. The investigation confirmed the importance of phalloplethysmography in elucidating sexually motivated structures of offenders of pedophil sexual activities.

A deduced Thermomonospora curvata protein containing serine/threonine protein kinase and WD-repeat domains.

The gene pkwA coding for a typical WD-repeat protein was found in the chromosome of the bacterium Thermomonospora curvata CCM 3352. Until now WD-repeat proteins were through to be confined to eukaryotes.

[Occurrence of deviant tendencies in phalloplethysmographic and vulvoplethysmographic examinations in sexually dysfunctional patients (case reports)]. / Výskyt deviantních tendencí pri PPG a VPG vysetrení sexuálne dysfunkcních pacientu (kazuistické príklady).

The author demonstrates on four case-histories the presence of sexual deviant trends as the cause of sexual dysfunctions in conventional heterosexual relations in men and women and the irreplaceable part played by vulvo- or phalloplethysmography used for their detection. In a man treated for some time on account of impaired erectile potency and in a woman who visited the surgery with her husband and presented complaints diagnosed as secondary frigidity and anorgasmy, PPG or VPG examination revealed a homosexual orientation. Both these patients knew of their homosexual orientation but did not tell anybody. The mentioned male patient intentionally dissimulated homosexuality, the woman attempted in vain to achieve heterosexual adaptation. In two young men with primary disorders of erectile potency PPG examination revealed sadomasochistic tendencies. In the first one the impaired erectility created the impression of "debutant's failure" when starting a partnership, in the second one it seemed to be due to adverse psychosocial factors. During an interview following the examination both described spontaneously and in detail erotic dreams and masturbation fantasies with sadomasochistic contents. They were not aware of the association of their specific sexual motivation structure. It was typical for all four patients that they never disclosed previously their deviant sexual tendencies and only the specific situation during the PPG or VPG examination, which they had voluntarily after previous instruction, they were motivated to confess their problems.

[Vulvar plethysmographic findings in transsexual women]. / Vulvopletysmografické nálezy u transsexuálních zen.

BACKGROUND: Transsexualism is a complicated diagnostic category. From the differential diagnostic aspect it is necessary, among others, to rule out homosexuality. The objective of the present work was to express objectively vulvo-plethysmographic reactions in transsexual women and to assess whether (or how) these reactions differ from findings in women with a homosexual orientation and to evaluate subsequently possibilities of vulvo-plethysmography in the differential diagnosis of homosexuality and transsexualism. METHODS AND RESULTS: Using an electro-capacitance vulvo-plethysmographic apparatus (designed by Figar et al.), 25 women (mean age 27.6 years, range 18-39 years) with an inverse sexual orientation--transsexuals--were examined. The results were compared with a group of 50 women, matched for age, who reported a homosexual orientation. On evaluation of the total number of recorded positive reactions to heterosexual and homosexual categories of exposed erotic stimuli the difference between the two groups on projection of 3 from 4 categories of these stimuli was highly significant (p < 0.001, chi 2 test). The differences were revealed in particular when evaluating preferences of pictures of sexual activities of partners. While in homosexually oriented women, as expected, preference of homosexual activities was recorded (74%) or a not clear-cut differentiation of these stimuli (26%), in 88% of transsexual women there was a more or less markedly expressed preference of heterosexual activities. CONCLUSIONS: Significant differences were found in results of vulvo-plethysmographic examinations of transsexual and homosexual women. This confirmed the expedience of using electro-capacitance vulvo-plethysmography for the differential diagnosis of homosexual and transsexual women.

Improved procedure for a high-yield recovery of enzymatically active recombinant calf chymosin from Escherichia coli inclusion bodies.

The high-yield recovery of enzymatically active recombinant calf chymosin from Escherichia coli inclusion bodies was achieved by optimization of solubilization and renaturation conditions. The solubilization was carried out in 8 M urea at various pHs, at various temperatures, and for various periods of time. The following values were found optimal: 1 h at 31 degrees C, pH 10.4. For successful correct refolding of solubilized prochymosin molecules it was found to be necessary to dilute the solution into an alkaline buffer (pH 10.7) in such a way that the final concentration of urea did not exceed 0.32 M and that of protein 0.275 mg/ml. Our optimized procedure gives about eight times higher yields of enzymatically active chymosin than the current published methods.

Characterization of the alpha-amylase-encoding gene from Thermomonospora curvata.

The nucleotide sequence of a 3007-bp DNA fragment from Thermomonospora curvata CCM3352 containing the coding and regulatory region of the alpha-amylase-encoding gene (tam) was determined. Primer extension mapping was used to determine the 5' end of the transcript, and it was demonstrated that the gene is transcribed from a unique promoter which is also functional in Streptomyces lividans TK24. Transcription of tam in T. curvata was induced by maltose, even in the presence of glucose. In S. lividans, tam was expressed constitutively. The deduced amino acid sequence reveals a considerable similarity with alpha-amylases from streptomycetes.

Large-scale isolation and partial characterization of plasmid DNA from B. larvae.

Large-scale preparation of plasmid DNA from two Bacillus larvae strains 423 and 728, honey-bee pathogens, is described. The isolated plasmid DNAs were analyzed by restriction enzyme mapping. No difference in the resulting maps was found for six restriction enzymes. The plasmid DNAs were also compared by Southern blot hybridization and by electron microscopy. The results confirmed the identity of these plasmid DNAs. All these data suggest that B. larvae strains harbor the same plasmid.

Transformation of Streptomyces lincolnensis protoplasts with plasmid vectors.

A method for the preparation and regeneration of protoplasts of Streptomyces lincolnensis is described. Mycelium in the early exponential phase appeared to be most suitable for this purpose and yielded up to 25% regenerated intact cells. Transformation of S. lincolnensis protoplasts was achieved using broad-host-range streptomycete plasmid vectors pIJ622, pMP66, pRS410 and pIJ943 constructed from replicons pIJ101, pSLG33 and SCP2. The efficiency of transformation was 3.10(3) transformants per micrograms plasmid DNA when (2-5).10(7) recipient protoplasts were used. Interspecific transformations showed that there is no efficient restriction system in S. lincolnensis that would limit the transfer of genetic information from S. lividans or E. coli.

[Phalloplethysmographic findings in homosexual pedophile offenders]. / Falopletysmografické nálezy u pachatelu homosexuálne pedofilních deliktu.

Using phalloplethysmographic tests comprising 20 coloured slides of four categories the authors examined 50 homosexual pedophil delinquents and a matched group of heterosexual men. The findings of the two compared groups differed significantly on exposure to three categories of erotic stimuli--adult heterosexual and homosexual objects and child homosexual objects. Only on projection of child heterosexual objects there were no statistically significant differences in the number and magnitude of positive vasomotor reactions recorded in the two groups. Men in the control group responded more frequently and more positively to adult heterosexual objects than homosexual pedophil delinquents. The latter responded more frequently positively to adult and child homosexual objects. In more than one quarter of the delinquents (13 men) a preference of child objects was found, i.e. a deviant pedophil orientation. In seven men the preference of child objects was without sexual differentiation, six men preferred homosexual child objects.

[Vulvoplethysmographic reaction in homosexually-oriented women]. / Vulvopletysmografické reakce u homosexuálne orientovaných zen.

The author examined, using an electrocapacitance VPG apparatus, 50 women reporting homosexual orientation. The results were compared with a control group of 50 women volunteers who reported a defined heterosexual orientation and had no marked complaints and problems in their sexual life. The general vasomotor reactivity in response to erotic stimuli did not differ in the two groups of women (the number of recorded vasomotor reactions differed only insignificantly). During evaluation of the total number of positive reactions to heterosexual and homosexual categories of stimuli the difference between the two groups was highly significant (similarly as during evaluation of the number of positive reactions which were described as major reactions). Women of the homosexually oriented group responded as expected, significantly more frequently and more markedly to homosexual stimuli. As compared with the control group of heterosexually oriented women, they had a generally poorer capacity to differentiate between different categories of erotic stimuli and a lower reactivity during projection of slides of homosexual partnership activities than during projection of slides of homosexual objects (nudes). In all examined women of the control group an unequivocal heterosexual orientation was recorded, in the group of homosexual women a more or less defined homosexual orientation was confirmed. In both groups was a high correlation between the subjectively reported sexual orientation and the results of the VPG examination. The authors confirmed that it is possible to use electrocapacitance vulvoplethysmography as evidence of the homosexual orientation in women and that this psychophysiological examination method is relatively reliable in the diagnosis of female homosexuality.