Chromosomal insertions localized around oriC affect the cell cycle in Escherichia coli.

The present work reports the effects of localized insertions around the origin of Escherichia coli chromosome, oriC, on cell cycle parameters. These insertions cause an increase of the C period with an inverse correlation to the distance from oriC. In addition, Omega insertion near oriC causes an increase in the number of replication forks per chromosome, n, and Tn10 insertion causes a decrease in growth rate. We found that the same insertion positioned in another region of the chromosome, outside of oriC, has a negligible effect on the C period. Marker frequency analysis suggests a slower replication velocity along the whole chromosome. We propose that the insertions positioned at less than 2 kbp from oriC could create a structural alteration in the origin of replication that would result in a longer C period. Flow cytometry reveals that asynchrony is not associated with these alterations.

A calcium-binding protein that may be required for the initiation of chromosome replication in Escherichia coli.

Starvation for isoleucine but not for other amino acids in an ilv- strain or the addition of valine in an ilv+ strain inhibits initiation of chromosome and minichromosome replication in stringent (Rel+) Escherichia coli, but it does not inhibit replication in relaxed (relA) mutants (Guzman et al, 1988). From these results, we concluded that, (1) oriC initiation of replication is inhibited by ppGpp, and (2) isoleucine is not needed for the protein synthesis required at initiation. These results led us to find an isoleucine-free protein whose de novo synthesis is the sole protein synthesis requirement for oriC initiation. We also present evidence that this protein may be a calcium-binding protein located at 73 min in the genetic map.

Thiourea derivatives and their nickel(II) and platinum(II) complexes: antifungal activity.

We have synthesized two thiourea derivatives of methyl anthranilate (1, 2) and their complexes with nickel (3) and platinum(II) (4). We have also prepared the complexes of nickel(II) with two benzoylthiourea derivatives (5, 6). The obtained compounds were characterized by elemental analysis, spectroscopic methods (FT-IR, UV-vis, NMR), mass spectrometry and thermal analysis. Compound 1, C(20)H(23)N(3)O(2)S, crystallizes in monoclinic space group P21/n, with Z=4, and unit cell parameters, a=8.8042(4) A, b=7.6608(3) A, c=28.834(2) A, alpha=gamma=90 degrees, beta=90.94(1) degrees. Compound 2, C(20)H(21)N(3)O(3)S, crystallizes in monoclinic space group P21/c, with Z=4, and unit cell parameters, a=7.7345(4) A, b=8.6715(4) A, c=29.113(2) A, alpha=gamma=90 degrees, beta=90.67(1) degrees. Compound 5, C(24)H(30)N(4)NiO(2)S(2), crystallizes in monoclinic space group P21/n, with Z=4, and unit cell parameters, a=10.4317(8) A, b=18.517(2) A, c=13.299(1) A, alpha=gamma=90 degrees, beta=104.53(1) degrees. Compound 6, C(25)H(28)Cl(2)N(4)NiO(4)S(2), crystallizes with a molecule of CH(2)Cl(2) in triclinic space group P-1, with Z=2, and unit cell parameters, a=10.362(1) A, b=11.849(2) A, c=12.536(2) A, alpha=90.04(2) degrees, beta=84.73(1) degrees, gamma=113.43(2) degrees. Compounds 1 and 2 show antifungal activity against the major pathogens responsible for important plant diseases (Botrytis cinerea, Colletotrichum fragariae, Fusarium oxysporum and Phoma betae). The antifungal activity is practically the same for morpholine and ethyl derivatives.

Chloride and ethyl ester morpholine thiourea derivatives and their Ni(II) complexes. Crystal and molecular structures of the thiourea derivative L-leucine methyl ester and its complexes with Cu(II) and Pt(II). Growth of the pathogenic fungus Botrytis cinerea.

We have synthesized a series of ligands (1, 3, 4, 6 and 7) and some of their complexes with Ni(II), Cu(II) and Pt(II) (2, 5, 8 and 9). These compounds were studied and characterized by elemental analysis, IR and UV-Vis spectra, conductivity measurements in solution, FAB+/MS, 1H and 13C NMR, ESR, etc. Compound 7 crystallized in the orthorhombic space group P2(1)2(1)2(1), with Z = 4. Unit cell parameters were as follows: a = 21.307(2) A, alpha = 90 degrees, b = 12.498(1) A, beta = 90 degrees, c = 7.7232(4) A, gamma = 90 degrees. For seven of these compounds, the antifungal activity of a major pathogen responsible for important crop damage was studied. In general, inhibition by the ligands was higher than that of the complexes. When the thiourea was linked to some diethyl groups, the compounds showed higher antifungal activity than the morpholine groups. Compound 3 achieved total inhibition (100%).

Mutation and DNA replication in Escherichia coli treated with low concentrations of N-methyl-N'-nitro-N-nitrosoguanidine.

N-Methyl-N'-nitro-N-nitrosoguanidine (nitrosoguanidine) causes an unexpectedly high frequency of closely linked double mutants because of its specificity for chromosome regions in replication. Low nitrosoguanidine concentrations (I mug/ml) in liquid cultures allow replications at the normal rate and are mutagenic. It was expected that mutations would be spread over the chromosome as it replicated, but a high frequency of closely linked double mutants was found. If a thymine auxotroph is grown in the presence of 5-bromodeoxyuridine (BUdR) and nitrosoguanidine, then exposed to 313-nm radiation (which destroys BUdR-substituted DNA), the mutation frequency is much higher among survivors than among non-irradiated cells. It is concluded that nitrosoguanidine inhibits DNA replication in a small fraction of the population and that mutations are induced in that same fraction. Nitrosoguanidine treatment leads to a high frequency of closely linked double mutants under all known conditions.

Preparation and characterization of tetrachlorocobaltates(II) of alpha,omega-alkylenediammonium. Magnetic and thermal properties. Crystal structure of

Tetrachlorocobaltates(II) of diprotonated alpha,omega-diaminoalkanes with the formula [NH(3)(CH(2))(n)NH(3)]CoCl(4), where n = 5 (cadaverine; 1,5-pentanediammonium tetrachlorocobaltate), 8 (1,8-octanediammonium tetrachlorocobaltate) and 10 (1,10-decanediammonium tetrachlorocobaltate), were prepared. The compounds were studied by mass spectrometry, FT-IR and visible spectroscopy, magnetic susceptibility techniques and thermal analysis. The compounds contain the tetrahedral tetrachlorocobaltate(II) ion and the corresponding diprotonated diamine (cadaverine, 1,8-octamethylenediamine and 1,10-decamethylenediamine). The compound corresponding to cadaverine crystallizes in the monoclinic space group P2(1)/c, with lattice parameters a = 7.1633 (7), b = 15.940 (3), c = 11.137 (2) Å, beta = 98.44 (1) degrees and Z = 4. Its crystal structure contains slightly distorted tetrahedral CoCl(4)(2-) ions: the largest difference in Co-Cl bond lengths is 0.029 Å and the largest difference in Cl-Co-Cl angles is 7.76 degrees. The compound also contains diprotonated cadaverine ions. An extensive hydrogen-bonding network connects these ions. The slightly positive deviations of the magnetic susceptibility from the Curie-Weiss law are in agreement with the (4)A(2) ground state for the tetrachlorocobaltate anion. The compounds with eight and ten C atoms show phase transitions in the solid state and a greater complexity is observed in their differential scanning calorimetry curves. Unfortunately, no suitable single crystals of these could be obtained.

[Autoimmune hepatitis and anti-Ro antibodies. Is there a link?]. / Hepatitis autoinmune y anticuerpos anti-Ro positivos. ¿Alguna relación?

Antinuclear antibodies (ANA) are considered to be markers of autoimmune disease. Several specific reactivities of ANA are known, among them the Ro (SS-a) complex. Although the presence of anti-Ro antibodies is widely known in systemic connective tissue diseases such as lupus erythematous or Sjögren's syndrome, few studies have attempted to link this finding with autoimmune hepatitis (AIH). We present a case of acute AIH in a 55-year-old woman who tested positive for anti-Ro 60 kD antibodies. The Ro (SS-A) antigen is a complex ribonucleoprotein whose structure is still the subject of debate. Two fractions, of 50 and 60 kD, have been identified. A review of the literature revealed frequencies of 21-34% for anti-Ro 52 kD and of 9-13-6% for anti-Ro 60 kD in AIH. Although no relationship has been demonstrated between this complex and the etiopathogenic mechanisms or clinical patterns of AIH, we propose that multicenter investigations with large series should be performed before the possible involvement of anti-Ro antibodies in these diseases is definitively ruled out.

[Prognostic value of DNA ploidy and nuclear morphometry in metastatic prostate cancer]. / Valor pronóstico de la ploidía del ADN y la morfometría nuclear en el cáncer de próstata metastásico.

PURPOSE: To assess the prognostic value of DNA ploidy and nuclear morphometry in metastatic prostate cancer after androgenic deprivation treatment. METHODS: Fifty four patients with prostate cancer and bone metastases who had undergone androgenic suppression treatment were retrospectively studied. The deoxyribonucleic acid (DNA) content was analysed by flow cytometry. Nuclear morphometry characterized 14 nuclear descriptors. The study also included age, Gleason score, T classification, haematocrite, serum albumin, serum alkaline phosphatase, serum prostatic acid phosphatase and the amount of metastatic foci detected during radioisotope bone scan. Univariate survival analyses were performed and Cox's proportional hazards model was used to identify significant prognostic factors. To assess how the experimental factors improve the capacity of the classical factors for predicting the patients who reach median survival, logistic regression multivariate analysis was performed for the classical prognostic factors only and after added experimental variables (DNA content and Nuclear Area). RESULTS: The univariate survival analyses assigned a prognostic value to T category, level of albumin, alkaline phosphatase, Gleason score, bone scan, DNA ploidy and mean nuclear area. In the case of the Cox regression model only Gleason score, bone scan, mean nuclear area and DNA ploidy provided independent prognostic information. In logistic regression for classic prognostic factors only Gleason score is significant (sensibility 89.3%, specificity 64%). However, when the experimental factors are added, in addition to Gleason score, radioisotope bone scan and DNA ploidy are of prognostic value (sensibility 90% and specificity 72%). CONCLUSIONS: The study of DNA content and nuclear morphometry in the primitive tumor provides independent prognostic information in survival analysis for patients with metastatic prostate cancer. However, there is limited improvement with respect to the classical factors in predicting survival. This questions its utility in the daily clinical usage.

[Sarcomatoid renal carcinoma]. / Carcinoma renal sarcomatoide.

Study of 250 pieces from nephrectomies performed in our service due to renal carcinoma, 10 (4%) of which where of the sarcomatoid histological variant. In 2 of the 10 cases, tumour was confined within the renal capsule at the time of surgery. These were the only cases where no disease progression was seen after treatment. A survival study using the Kaplan-Meter method was conducted. Median survival of patients with sarcomatoid pattern was 6 months, showing a significant difference with that obtained in other histological patterns. DNA content of tumoral cells was assessed by flow cytometry, which displayed a diploid pattern in 7 patients and aneuploid in 3. The 2 cases with no disease progression were diploid. We conducted nuclear cytomorphometry studies which showed significant differences in the mean nuclear perimeter between progressive and non-progressive tumours.

[Polycythemia in newborn infants. II. Umbilical cord and capillary hematocrits]. / Policitemia en el recién nacido. II. Hematócritos del cordón y capilares.

Actual values for capillary hematocrits at 24 and 48 hours of life, through a hospital sample, representative of our population, were obtained; results were 58 +/- 7% (X +/- 1 DS) for both. These results were correlated to the initial hematocrit (50 +/- 5%), with had been reported in the first part of the series. The relationship between capillary hematocrits and the initial one, was very good, and between the capillary ones, was oven better. This relationship persisted after distributing the sample in four groups: Apgar, crying (if it presented before or after clamping) and the features of amniotic fluid. This demonstrates that capillary hematocrits, are as reliable as venous, central and peripheral hematocrit (in this case, from umbilical chord blood), contrary to what has been previously reported in literature. The presence of chord loop or circular, was analyzed also, when it was possible.

[Spasticity and progressive ataxia due to vanishing white matter]. / Espasticidad y ataxia progresiva por sustancia blanca evanescente.

TITLE: Espasticidad y ataxia progresiva por sustancia blanca evanescente.

On the origin and evolution of the genetic code.

Two ideas have essentially been used to explain the origin of the genetic code: Crick's frozen accident and Woese's amino acid-codon specific chemical interaction. Whatever the origin and codon-amino acid correlation, it is difficult to imagine the sudden appearance of the genetic code in its present form of 64 codons coding for 20 amino acids without appealing to some evolutionary process. On the contrary, it is more reasonable to assume that it evolved from a much simpler initial state in which a few triplets were coding for each of a small number of amino acids. Analysis of genetic code through information theory and the metabolism of pyrimidine biosynthesis provide evidence that suggests that the genetic code could have begun in an RNA world with the two letters A and U grouped in eight triplets coding for seven amino acids and one stop signal. This code could have progressively evolved by making gradual use of letters G and C to end with 64 triplets coding for 20 amino acids and three stop signals. According to proposed evidence, DNA could have appeared after the four-letter structure was already achieved. In the newborn DNA world, T substituted U to get higher physicochemical and genetic stability.

The effect of nitrosoguanidine upon DNA synthesis in vitro.

Both the polymerase and the exonuclease activities of DNA polymerase III are inactivated by treatment with nitrosoguanidine. The treatment of the DNA template with the mutagen does not affect the template in supporting DNA synthesis. No effect of nitrosoguanidine upon fidelity of replication in vitro was detected.

Direct procedure for the determination of the number of replication forks and the reinitiation fraction in bacteria.

A direct method for calculating the average number of replication forks per chromosome in an exponentially growing bacterial culture and the fraction of reinitiation after an inductive treatment of the initiation step is presented. This method has allowed the development of REPLICON, a computer program designed for the resolution of the algorithm and simulation of the bacterial chromosome replication. Using REPLICON the following parameters can be obtained: average number of replication forks per chromosome, time required for the complete replication cycle, average amount of DNA per nucleotide, gene frequency of any chromosomal locus and reinitiation fraction. The use of this analysis also permits the determination of the uni- or bidirectionality of replication.

A temperature upshift induces initiation of replication at oriC on the Escherichia coli chromosome.

A temperature upshift of 10 or more degrees in the growth temperature of a bacterial culture causes induction of extra rounds of chromosome replication. This heat-induced replication (HIR) initiates at oriC, is transitory, requires RNase H1 and RecA proteins and requires neither RNA polymerase activity nor de novo protein synthesis. The number of origins activated by heat is growth rate and temperature differential dependent. An origin activation higher than 20% increases the DNA:mass ratio around twofold, and this value is kept constant for the subsequent generations of growth at 41 C. We have also shown that HIR is neither related to SDR nor induced by the heat shock response. We suggest that a thermodynamic alteration of oriC structure or of membrane fluidity could explain the observed HIR.

Determining the optimal thymidine concentration for growing Thy- Escherichia coli strains.

Changes of thymidine concentration in the growth medium affect the chromosome replication time of Thy- strains without at the same time causing a detectable difference in the growth rate (R. H. Pritchard and A. Zaritsky, Nature 226:126-131, 1970). Consequently, the optimal thymidine concentration cannot be determined by ascertaining which concentration produces the highest growth rate. Here we present a method for determining the optimal thymidine concentration of any Thy- Escherichia coli strain. Using this method, we found that the E. coli "wild-type" strain MG1655 has a partial Thy- phenotype.