Human lactoferrin triggers a mitochondrial- and caspase-dependent regulated cell death in Saccharomyces cerevisiae.

We have previously shown that the antifungal activity of human lactoferrin (hLf) against Candida albicans relies on its ability to induce cell death associated with apoptotic markers. To gain a deeper understanding of the mechanisms underlying hLf-induced apoptosis, we characterized this cell death process in the well-established Saccharomyces cerevisiae model. Our results indicate that hLf induces cell death in S. cerevisiae in a manner that requires energy and de novo protein synthesis. Cell death is associated with nuclear chromatin condensation, preservation of plasma membrane integrity, and is Yca1p metacaspase-dependent. Lactoferrin also caused mitochondrial dysfunction associated with ROS accumulation and release of cytochrome c. Pre-incubation with oligomycin, an oxidative phosphorylation inhibitor, increased resistance to hLf and, accordingly, mutants deficient in the F1F0-ATP synthase complex were more resistant to death induced by hLf. This indicates that mitochondrial energetic metabolism plays a key role in the killing effect of hLf, though a direct role of F1F0-ATP synthase cannot be precluded. Overexpression of the anti-apoptotic protein Bcl-xL or pre-incubation with N-acetyl cysteine reduced the intracellular level of ROS and increased resistance to hLf, confirming a ROS-mediated mitochondrial cell death process. Mitochondrial involvement was further reinforced by the higher resistance of cells lacking mitochondrial DNA, or other known yeast mitochondrial apoptosis regulators, such as, Aif1p, Cyc3p and Aac1/2/3p. This study provides new insights into a detailed understanding at the molecular level of hLf-induced apoptosis, which may allow the design of new strategies to overcome the emergence of resistance of clinically relevant fungi to conventional antifungals.

Genetic diversity of wild grapevine populations in Spain and their genetic relationships with cultivated grapevines.

The wild grapevine, Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi, considered as the ancestor of the cultivated grapevine, is native from Eurasia. In Spain, natural populations of V. vinifera ssp. sylvestris can still be found along river banks. In this work, we have performed a wide search of wild grapevine populations in Spain and characterized the amount and distribution of their genetic diversity using 25 nuclear SSR loci. We have also analysed the possible coexistence in the natural habitat of wild grapevines with naturalized grapevine cultivars and rootstocks. In this way, phenotypic and genetic analyses identified 19% of the collected samples as derived from cultivated genotypes, being either naturalized cultivars or hybrid genotypes derived from spontaneous crosses between wild and cultivated grapevines. The genetic diversity of wild grapevine populations was similar than that observed in the cultivated group. The molecular analysis showed that cultivated germplasm and wild germplasm are genetically divergent with low level of introgression. Using a model-based approach implemented in the software structure, we identified four genetic groups, with two of them fundamentally represented among cultivated genotypes and two among wild accessions. The analyses of genetic relationships between wild and cultivated grapevines could suggest a genetic contribution of wild accessions from Spain to current Western cultivars.

Potassium efflux induced by a new lactoferrin-derived peptide mimicking the effect of native human lactoferrin on the bacterial cytoplasmic membrane.

A 31-amino acid synthetic peptide (NH(2)-FFSASCVPGADKGQFPNLCRLCAGTGENKCA-COOH) was chemically synthesized based on the amino acid sequence of a region of human lactoferrin homologous to other sequences present in the N- and C-lobes of all members of the transferrin family proteins. The peptide, termed kaliocin-1, and lactoferrin showed a bactericidal effect in assays performed in low-ionic-strength conditions. This is the first time that it is shown that the antimicrobial effect of lactoferrin depends on the extracellular cation concentration. The antimicrobial effect of kaliocin-1 was lower than that of human lactoferrin, but their activities were inhibited by Na(+) or K(+) in a cation concentration-dependent manner. In addition, the peptide was able to mimic native lactoferrin, inducing K(+)-efflux and a selective dissipation of the transmembrane electrical potential of Escherichia coli cells without causing extensive damage to the outer and inner bacterial membranes. In contrast, the peptide, but not lactoferrin, was able to permeabilize different ions through liposomal membranes. The hypothetical interaction of kaliocin-1 with a bacterial membrane compound is discussed based in the different ion flux induced on cellular and artificial membranes as well as data from circular dichroism assays. Kaliocin-1 was not cytotoxic and could be a suitable model for the design of analogs able to mimic the antibacterial effect of human lactoferrin.

[Right sensory-motor syndrome as the presentation of a spontaneous cervico-thoracic epidural hematoma]. / Síndrome sensitivomotor derecho como presentación de un hematoma epidural cervicotorácico espontáneo.

INTRODUCTION: A spontaneous cervical epidural hematoma is an infrequent cause of cord compression. The commonest clinical presentations are with paraparesia and tetraparesia. Transient hemiparesia is very rare and a sensory-motor syndrome is exceptional. CLINICAL CASE: A 38 year old man had sudden onset of spontaneous interscapular vertebral pain with bilateral root radiation. A few minutes later he started to have weakness and sensory loss in his right limbs. On clinical examination there was pain on pressure over the spinal apophyses of the T4 and T5 vertebrae, hemiparesia and hemi-hypoalgesia of the right limbs. After ten hours this had all returned to normal. MRI of the cord showed an epidural hematoma extending from C6 to T2. Coagulation studies and spinal arteriography were normal. CONCLUSIONS: The sensory motor syndrome is an unusual form of presentation of a spontaneous cervico-thoracic epidural hematoma. Root pain is a symptom of great value for orientation of a syndrome which would otherwise seem to be of central origin. The explanation for this clinical findings may be compression of both lateral cord pathways due to their particular blood distribution. Regarding therapeutic approach, this should be conservative, with close observation to see whether it will resolve spontaneously in a short period of time.

Antimicrobial susceptibilities of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens spp. isolated in Spain.

The susceptibilities of 143 Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens isolates to 18 antimicrobial agents were tested. All P. gingivalis isolates were susceptible. In contrast, some Prevotella spp. (17%) were resistant to beta-lactams, erythromycin, clindamycin, or tetracycline and carried resistance genes, ermF or tetQ, or beta-lactamases.

Evaluation of the antimicrobial effect of lactoferrin on Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens.

The antimicrobial effect of lactoferrin (apoLf) on the oral, black-pigmented anaerobes Porphyromonas gingivalis, Prevotella intermedia and P. nitrescens has been studied. ApoLf did not kill any of these species but it did inhibit the growth of P. gingivalis, while iron-saturated Lf (FeLf) had no effect. The other two species were unaffected by apoLf. This growth inhibitory effect of apoLf could not be explained on the basis of chelation of inorganic iron, since growth of P. gingivalis occurred in the presence of ethylenediamine di-o-hydroxyphenylacetic acid provided haemin was added. Both apoLf and FeLf reduced haemin uptake by all three species and caused the release of cell-bound haemin in a dose-dependent manner. In addition, haemin reduced the binding of both apoLf and FeLf to P. intermedia and P. nigrescens but stimulated the binding of Lf by P. gingivalis. These data suggest that Lf forms complexes with haemin in solution and competes for the binding of haemin to certain cell receptors, possibly lipopolysaccharides, but this is not sufficient to inhibit growth of the bacteria. P. gingivalis appears to bind Lf-haemin complexes, as well as haemin alone, which may facilitate access of the Lf to the outer and cytoplasmic membranes of P. gingivalis, so disrupting function.

[The one and a half vertical syndrome as the only evidence of bilateral thalamo-subthalamic infarction]. / Síndrome del uno y medio vertical como única manifestación de un infarto talamosubtalámico bilateral.

INTRODUCTION: The one and a half vertical syndrome consists of paralysis of upward vertical conjugated gaze and monocular paralysis of downward gaze or vice-versa. It occurs as a consequence of a mesencephalodiencephalic lesion, either unilaterally or bilaterally, due to effects on structures such as the interstitial nucleus of Cajal, posterior commissure and rostral interstitial nucleus of the medial longitudinal bundle. The arterial supply of the structures involved in the supranuclear control of vertical gaze depends on the paramedian thalamic arteries (or posterior thalamo-subthalamic arteries) and the paramedian peduncular arteries. CLINICAL CASE: We present the case of a 58 year old woman with non-insulin dependent diabetes mellitus who presented with sudden onset of diplopia of vertical gaze. On examination there was paralysis of superior vertical conjugated gaze and monocular paralysis of ocular infraversion (one and a half syndrome) with no other pathological findings. Cerebral angioresonance and Doppler studies of the territory supplied by the posterior cerebral artery were normal. CONCLUSIONS: Both unilateral and bilateral mesencephalodiencephalic infarcts usually present clinically as disorders of consciousness and of conduct (the almost universal form of presentation), involvement of the pyramidal tract, the third cranial nerves and a supranuclear disorder of vertical gaze. The unusual feature of the case we present is a bilateral thalamo-subthalamic infarct which presented solely as a one and a half vertical syndrome with no clinically apparent effect on anatomically adjacent structures. We have not found any similar cases in the literature.

Endarteritis and mycotic aortic aneurysm caused by an oral strain of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans was isolated from blood cultures of a 33-year-old febrile patient with a previously undiagnosed coarctation of the aorta. Subgingival samples from diseased periodontal pockets revealed the presence of A. actinomycetemcomitans. An infected (mycotic) aortic aneurysm and endarteritis were diagnosed and surgically treated. The identity of blood and oral clinical isolates of A. actinomycetemcomitans was supported by genetic analysis, including fingerprinting by restriction fragment length polymorphism, ribotyping, and random amplified polymorphic DNA; biotyping; and antibiogram typing. These data strongly suggest that the periodontal pockets were the primary source of A. actinomycetemcomitans endarteritis in this case.

[Neuroimaging of hypoglycemia]. / Neuroimagen en la hipoglucemia.

INTRODUCTION: Sustained hypoglycemia causes serious cerebral damage. The cortex, hippocampus and basal ganglia are particularly vulnerable to this. Exceptionally, there have been communications regarding neuroimaging visualization of cerebral lesions attributable to hypoglycemia only. We present the case of a woman who suffered hypoglycemic coma with permanent neurological damage. Lesions were seen on magnetic resonance (MR) and cranial computerized tomography (CT). CLINICAL CASE: A 22 year old woman with no vascular risk factors was admitted to hospital in hypoglycemic coma, after attempting suicide with oral antidiabetic drugs. The duration of the coma was unknown. On admission the glycaemia was 28 mg/dl. Cranial CT scan was normal during the first 24 hours. Cerebral MR scan one week late showed hyperintense lesions in T2, basal ganglia and left hippocampus. The cranial CT scan one year later showed diffuse atrophy, with bilateral lesions of low attenuation in the basal ganglia and dilatation of the ventricular system. CONCLUSIONS: It is unusual to see lesions secondary to hypoglycemia on neuroimaging investigations. The etiopathogenic mechanism is still unknown and it has generally been described in diabetic patients. In our case these lesions can only be attributed to hypoglycemia. MR is more sensitive than cranial CT scan for the detection of these lesions during the acute phase.

A beta-lactamase belonging to group 2e from oral clinical isolates of Prevotella intermedia.

A beta-lactamase in oral clinical isolates of Prevotella intermedia that hydrolyzed cefuroxime and cephalothin with rates of 600 and 53.3 respectively, relative to that for cephaloridine (100), was characterized as a 2e-cephalosporinase. Inhibition was observed by clavulanic acid (IC50 0.72 microM), tazobactam (IC50 0.21 microM) and sulbactam (IC50 0.07 microM) and was not inhibited by cloxacillin, EDTA, NaCl or p-CMB. The pI and pH optima were 4.7 and 5.4, respectively.

A new clinical tool for gait evaluation in Parkinson's disease.

This study was devised to check the feasibility and validity of a rating scale specifically designed to evaluate gait impairment in Parkinson's disease (RSGE). Demographic data, a brief questionnaire on general aspects influencing gait and mobility, a battery of scales (Barthel Index; Hoehn and Yahr staging; and Northwestern University Disability, Schwab and England, and Unified Parkinson's Disease Rating Scale [UPDRS]), and timed tests ("Up and Go" and "Steps x Seconds" tests) were recorded under protocol, as was the RSGE-Version 1.0 (23 items in four subscales). Fifty patients enrolled at two centers were included. Twenty-five (50%) were simultaneously (though independently) evaluated by three examiners, in order to determine the interrater reliability. The mean age of the patients was 67.6 +/- 11.16 years, with a mean 8.18 +/- 5.58 years of disease duration. Motor fluctuations were present in 48% of patients. The RSGE Cronbach's alpha was 0.94. Only the item "Dyskinesias" was not correlated with the RSGE total sum. The item "Axial rigidity" showed a fair interrater reliability (kappa = 0.30). However, most of the RSGE items (16/23, 70%) had kappa > or = 0.65. The convergent validity with the applied scales was very high (Spearman r = 0.74-0.90, p < 0.001). The highest correlation (0.90) was obtained with the UPDRS. Also, the RSGE correlation with timed tests was very satisfactory ("Up and Go" = 0.81; "Steps x Seconds" = 0.70; both, p < 0.001). Factor analysis of the RSGE disclosed four dimensions explaining 68% of the variance. The RSGE-Version 1.0 proved to be a valid instrument. The reliability of some items has to be improved, however.

[Isolation of Haemophilus influenzae and Haemophilus parainfluenzae in genitourinary infections: a 4-year review]. / Aislamiento de Haemophilus influenzae y Haemophilus parainfluenzae en infecciones genitourinarias: una revisión de 4 años.

BACKGROUND: Haemophilus spp. had been previously suggested as a potential pathogen in genitourinary infections that could be sexually transmitted. In order to check that suggestions we have determined the incidence, pathogenic role, possible sexual transmission and susceptibility to antibiotics in isolates of Haemophilus parainfluenzae and Haemophilus influenzae from genital tract infections. The microbiological samples were taken during a period of four years from patients attended in a Service of Sexual Transmission Diseases and the data were further reviewed. METHODS: The study included 5,572 genital specimens from 2,182 women prostitutes with different genitourinary infections and from 825 men with urethritis. Microbiological samples were cultured in a non-specific media for genital pathogens and species of Haemophilus spp. and clinical circumstances of isolation were evaluated. Susceptibility tests were performed by using a standard microdilution test. RESULTS: Haemophilus spp. was isolated in 155 samples (2.8%) using a non-selective culture method. H. parainfluenzae was isolated in 100 cases (64.5%), Haemophilus influenzae in 45 cases (29%) and Haemophilus spp. in 10 strains (6.4%). Haemophilus spp. was isolated as a sole pathogen in men with urethritis (8 cases), epididymo-orchitis (2 cases), cervicitis and/or vaginitis (9 cases) and Bartholin's Abscess (2 cases). The most frequent biotypes were H. parainfluenzae biotype II (43%) and III (19%), and H. influenzae biotype IV (35.5%). Beta lactamase activity and ampicillin resistance were present in 29% of the H. parainfluenzae strains and in the 26.7% of clinical isolates of H. influenzae. CONCLUSIONS: 1) Haemophilus spp. was isolated from genitourinary infections at a low frequency in the studied group. 2) The pathogenic role of Haemophilus spp. was suggested when was isolated as a sole pathogen present from some infections of the genitourinary tract such as urethritis in men and Bartholin's abscess in women. 3) The susceptibility to antibiotics in the clinical isolates of Haemophilus spp. from genitourinary infections was similar previously reported studies performed in Spain.

Reliability of biologic indicators in a mail-return sterilization-monitoring service: a review of 3 years.

Most mail-return sterilization-monitoring services use spore strips to test sterilizers in dental clinics, but factors such as delay caused by mailing to the laboratory could cause false negatives. The aims of this study were to determine the influence of poststerilization time and temperature on the biologic indicator recovery system and to evaluate sterilization failure and its possible causes in dental clinics subscribing to a mail-return sterilization-monitoring service. Spore strips used in independent tests revealed the poststerilization time and temperature after a 7-day delay to have no significant influence. Sixty-six dental clinics that received quarterly biologic indicators to evaluate the effectiveness of their sterilizers had sterilization failure rates of 28.7% in 1992, 18.1% in 1993, and 9.1% in 1994, a statistically significant decrease in sterilization failure during the 3-year period. The usual causes of failure were operator error in wrapping of instruments, loading, operating temperature, or exposure time.