Amplified fragment length polymorphism analysis of the VNTR locus D1S80 in central Spain

Summary The polymorphism of the D1S80 locus has been analyzed in a population sample of 203 unrelated individuals living in Madrid (central Spain) by PCR and subsequent semi-dry discontinuous polyacrylamide gel electrophoresis (Tris-chloride/Tris-glycine buffer system) followed by silver staining, The electrophoretic system described in this study offers high resolution in the separation of the different D1S80 alleles allowing the detection of microvariability around the allele T22 in the spanish population. Twenty different alleles containing 17–40 repeats of the basic 16bp unit were distinguished. The alleles T18 and T24 were found to be relatively common in Spain, as in other populations:, with frequencies of 0.224 and 0.372, respectively. No evidence of significant deviations from Hardy-Weinberg equilibrium was found in these preliminary population data.     

Enzymatic synthesis of penicillins

Different penicillins (phenylacetyl, 2-hydroxyphenylacetyl, 4-hydroxyphenylacetyl, phenoxyacetyl and 2-thiopheneacetylpenicillin) have been synthesized "in vitro" by direct N-acylation of 6-aminopenicillanic acid (6-APA) with the acyl group of several acyl-CoA derivatives. The enzyme that catalyzes these reactions, acyl-CoA: 6-APA acyltransferase of Penicillium chrysogenum, was purified to homogeneity (374-fold) and its amino acid composition is given. This protein accepts as substrates several aliphatic acids and different aromatic acids with the only requirement that an acetyl-CoA moiety must be present in the substrate molecule. Shortening or lengthening of the acyl moiety prevents the 6-APA-N-acylation reaction. The presence of an amino group in the alpha-position of the acetyl group does not allow this molecule to be used as substrate. However, different substitutions in the phenyl group (hydroxylation of the carbons 2 and 4) or its replacement by another aromatic ring (thiophene) were accepted with varying reactions rates in the acylation reaction when a 176-fold purified acyltransferase was employed. The homogeneity pure enzyme accepts as substrate thiophene acetyl-CoA but it did not 2-hydroxyphenyl and 4-hydroxyphenylacetyl-CoA. The presence of an oxygen atom between the aromatic and the acetyl moieties did not affect the catalysis.     

Spike patterning by Ca2+-dependent regulation of a muscarinic cation current in entorhinal cortex layer II neurons

In entorhinal cortex layer II neurons, muscarinic receptor activation promotes depolarization via activation of a nonspecific cation current (I(NCM)). Under muscarinic influence, these neurons also develop changes in excitability that result in activity-dependent induction of delayed firing and bursting activity. To identify the membrane processes underlying these phenomena, we examined whether I(NCM) may undergo activity-dependent regulation. Our voltage-clamp experiments revealed that appropriate depolarizing protocols increased the basal level of inward current activated during muscarinic stimulation and suggested that this effect was due to I(NCM) upregulation. In the presence of low buffering for intracellular Ca(2+), this upregulation was transient, and its decay could be followed by a phase of I(NCM) downregulation. Both up- and downregulation were elicited by depolarizing stimuli able to activate voltage-gated Ca(2+) channels (VGCC); both were sensitive to increasing concentrations of intracellular Ca(2+)-chelating agents with downregulation being abolished at lower Ca(2+)-buffering capacities; both were reduced or suppressed by VGCC block or in the absence of extracellular Ca(2+). These data indicate that relatively small increases in [Ca(2+)](i) driven by firing activity can induce upregulation of a basal muscarinic depolarizing-current level, whereas more pronounced [Ca(2+)](i) elevations can result in I(NCM) downregulation. We propose that the interaction of activity-dependent positive and negative feedback mechanisms on I(NCM) allows entorhinal cortex layer II neurons to exhibit emergent properties, such as delayed firing and enhanced or suppressed responses to repeated stimuli, that may be of importance in the memory functions of the temporal lobe and in the pathophysiology of epilepsy.     

Wilson disease: novel mutations in the ATP7B gene and clinical correlation in Brazilian patients

Wilson disease (WD) is a rare inherited autosomal recessive disorder caused by a defect in a metal transporting P-type ATPase, resulting in copper overload in various tissues and cells. The aim was to assess both the phenotype in Brazilian WD patients and the corresponding ATP7B genotype. Sixty subjects belonging to 46 pedigrees diagnosed as WD were included in this study. Direct sequencing of all 21 exons within ATP7B and their flanking introns was performed. Demographic, clinical, laboratory and histopathological data at the time of diagnosis were obtained. We identified twenty-five mutations, twelve of them reported for the first time. The c.3402delC mutation had the highest allelic frequency (30.8%), followed by the c.2123T>C (p.L708P) (16.7%). Exons 8 and 15 were the site of 62.5% of the mutations. The common European mutation c.3207C>A (p.H1069Q) was not present at all. Phenotype varied greatly among individuals with the same ATP7B genotype. Our data confirm the heterogeneity of ATP7B genotype in Brazilian WD patients. The mutational spectrum is compatible with the Brazilian history of Mediterranean immigration; however, new mutations, and different frequencies and phenotype associated with the previously known mutations characterize this population. Exons 8 and 15 should be preferentially screened in WD cases from Brazil. Phenotype variation among subjects with the same ATP7B genotype suggests that modifying factors play an additional role in the pathogenesis of WD.     

Phage types and genotypes of shiga toxin-producing Escherichia coli O157:H7 isolates from humans and animals in spain: identification and characterization of two predominating phage types (PT2 and PT8)

Phage typing and DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) were used for the epidemiological subtyping of a collection of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999. Phage typing distinguished a total of 18 phage types among 171 strains isolated from different sources (67 humans, 82 bovines, 12 ovines, and 10 beef products). However, five phage types, phage type 2 (PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11 strains), and PT54 (16 strains), accounted for 68% of the study isolates. PT2 and PT8 were the most frequently found among strains from both humans (51%) and bovines (46%). Interestingly, we detected a significant association between PT2 and PT14 and the presence of acute pathologies. A group of 108 of the 171 strains were analyzed by PFGE, and 53 distinct XbaI macrorestriction patterns were identified, with 38 strains exhibiting unique PFGE patterns. In contrast, phage typing identified 15 different phage types. A total of 66 phage type-PFGE subtype combinations were identified among the 108 strains. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type. The most common phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and 11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef product strains). Nine (29%) of 31 human strains showed phage type-PFGE pattern combinations that were detected among the bovine strains included in this study, and 26 (38%) of 68 bovine strains produced phage type-PFGE pattern combinations observed among human strains included in this study, confirming that cattle are a major reservoir of strains pathogenic for humans. PT2 and PT8 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused.     

Subcutaneous cryptococcosis due to Cryptococcus diffluens in a patient with sporotrichoid lesions case report, features of the case isolate and in vitro antifungal susceptibilities.

Environmental fungi, in particular primary pathogens and Cryptococcus spp. can be responsible for skin lesions mimicking sporotrichosis. In this paper, we report a case of subcutaneous cryptococcosis in an apparently healthy, young male patient due to a non-C. neoformans Cryptococcus species, C. diffluens. The isolate showed in vitro phenotypic switching that may affect virulence and host inflammatory and immune responses, and in vitro resistance to amphotericin B and 5-flucytosin. This species shares several phenotypic traits with C. neoformans, and, therefore, decisive diagnosis should be based on biopsy and culturing results followed by molecular identification.     

Regulation of tumor necrosis factor (TNF)-alpha synthesis and TNF receptors expression in T lymphocytes through the CD2 activation pathway.

The involvement of the CD2 (T11) molecule, an alternative activation pathway for T lymphocytes, in the regulation of tumor necrosis factor (TNF)-alpha/TNF receptor system in human T lymphocytes has been investigated. It has been found that both TNF-alpha synthesis and secretion were induced after incubation of purified T lymphocytes with the appropriate mitogenic combination of antibodies specific for two different epitopes on the CD2 molecule. Moreover, TNF-alpha secretion was also observed by activation of T lymphocytes either through CD3 or CD69 molecular pathways, or with other stimulating agents such as Ca2+ ionophore in combination with phorbol esters. The expression of TNF receptors has been studied in both nonactivated and CD2-activated T lymphocytes. Unstimulated T cells weakly expressed a functional 75-kDa receptor form, whereas they lacked detectable levels of the 55-kDa receptor form. Triggering of T cell activation through the CD2 molecule also markedly increased the expression of the p75-kDa TNF receptor form, but did not exert any inductive effect on the expression of the p55-kDa TNF receptor. In addition, we have found that TNF-alpha enhanced the proliferative response triggered by the mixture of anti-CD2 monoclonal antibodies. Taken together, these results support a role for the CD2 activation pathway in the functional regulation of TNF-alpha/TNF receptor system in T lymphocytes, and reinforce the view of CD2 as an alternative pathway for regulation of the cytokine network that modulates the function of T lymphocytes.     

Cell type determines the relative proportions of (-) and (+) strand RNA during poliovirus replication.

To examine the influence of the host cell type on poliovirus RNA synthesis we compared the levels of (-) and (+) strand poliovirus RNA during infection of epithelial (HeLa and HEp-2), leukocytic (U-937, HL-60 and K-562) and nerve (IMR-32) cells. The levels of (-) strand RNA were higher in IMR-32, U-937, K-562 or HL-60 cells than those in HeLa or HEp-2 cells. By contrast, (+) strand RNA content was greater in HeLa or HEp-2 cells. Although significant levels of (+) strand RNA were detected in U-937, K-562 and HL-60 cells, no viral protein synthesis was detected by polyacrylamide gel electrophoretic analysis of metabolically labelled proteins. The molar ratio of poliovirus (-) and (+) RNAs was 2-3 fold higher in IMR-32, U-937 and K-562 cells than in HeLa or HL-60 cells and 5-6 fold higher than in HEp-2 cells. Differentiation of HL-60 cells with a variety of inducers produced differential effects on poliovirus (-) and (+) RNA content and modified the molar ratio of (-)/(+) strand RNAs. These findings indicate that host cell components play a critical role in the regulation of the amount of poliovirus (-) and (+) strand RNAs synthesized during infection.     

Identification of a 7S globulin as a novel coconut allergen.

BACKGROUND: Coconut (Cocos nucifera) is a monocotyledonous plant of the Arecaceae family. Allergy to coconut is infrequent, with only 5 cases reported so far in the medical literature. OBJECTIVE: To identify coconut allergens in 2 patients allergic to this food. METHODS: We describe 2 patients allergic to coconut: an adult pollen-allergic patient monosensitized to coconut who presented with severe oropharyngeal symptoms and a child with a previous allergy to walnut, not allergic to pollen, who developed anaphylaxis on coconut ingestion. Both patients had positive skin prick test results and serum specific IgE (CAP) to coconut. IgE sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting was performed to identify the allergens involved, and a strong IgE binding band detected in both patients was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS). Stability to pepsin digestion of the coconut extract and its cross-reactivity with tree nuts were studied. RESULTS: An immunoblot showed an almost identical profile of IgE binding proteins in the coconut extract in both patients who reacted strongly to a band of approximately 29 kDa. The peptide analysis by MALDI-TOF MS of this band obtained the sequence GHGKREDPEKR. The protein with the highest correlation with this peptide was found to be a 7S globulin from Elaeis guineensis, another oil palm species also belonging to the Arecaceae family. The 29-kDa band was digested by pepsin in less than 1 minute. Cross-reactivity among coconut, walnut, and hazelnut was demonstrated by CAP inhibition in patient 2. CONCLUSION: We have identified a 7S storage protein as a novel coconut allergen.