Biomedical subjects
Publications and source records attributed to I Diaz.
Subzonal insemination was the first micromanipulation technique used in the treatment of male factor sterility. Analysis of the sperm pathology prior to patient inclusion in the program allowed us to study gamete interaction in the human and the early embryogenesis. A series of 523 cycles and 3,027 micro-injected oocytes is reported.
The barley genes HvLtp4.2 and HvLtp4.3 both encode the lipid transfer protein LTP4 and are less than 1 kb apart in tail-to-tail orientation. They differ in their non-coding regions from each other and from the gene corresponding to a previously reported Ltp4 cDNA (now Ltp4.1). Southern blot analysis indicated the existence of three or more Ltp4 genes per haploid genome and showed considerable polymorphism among barley cultivars. We have investigated the transient expression of genes HvLtp4.2 and HvLtp4.3 following transformation by particle bombardment, using promoter fusions to the beta-glucuronidase reporter sequence. In leaves, activities of the two promoters were of the same order as those of the sucrose synthase (Ss1) and cauliflower mosaic virus 35S promoters used as controls. Their expression patterns were similar, except that Ltp4.2 was more active than Ltp4.3 in endosperm, and Ltp4.3 was active in roots, while Ltp4.2 was not. The promoters of both genes were induced by low temperature, both in winter and spring barley cultivars. Northern blot analysis, using the Ltp4-specific probe, indicated that Xanthomonas campestris pv. translucens induced an increase over basal levels of Ltp4 mRNA, while Pseudomonas syringae pv. japonica caused a decrease. The Ltp4.3-Gus promoter fusion also responded in opposite ways to these two compatible bacterial pathogens, whereas the Ltp4.2-Gus construction did not respond to infection.
The gene Itr1, encoding trypsin inhibitor BTI-CMe, has been obtained from a genomic library of Hordeum vulgare L. The gene has no introns and presents in its 5'-upstream region 605 bp that are homologous to the long terminal repeats (LTR) of the 'copia-like' retro-transposon Bare-1. Functional analysis of the Itr1 promoter by transient expression in protoplasts derived from different barley tissues, has shown that in this system the Itr1 promoter retains its endosperm specifity and the trans-regulation mediated by the Lys3a gene. The proximal promoter extending 343 bp upstream of the translation initiation ATG codon is sufficient to confer full GUS expression and for endosperm specifity. In protoplasts derived from the lys3a mutant, Risø 1508, GUS activity was less than 5% of that obtained with the same constructs in the protoplasts of wild-type Bomi from which it derives. Gel retardation experiments, after incubation with proteins obtained from both types of endosperm nuclei, also show differential patterns. Possible reasons for these differences are discussed.
BACKGROUND/AIMS: Results of several studies on DNA ploidy as a prognostic indicator in hepatocellular carcinoma are contradictory. The present study analysed the correlations between DNA ploidy of resected hepatocellular carcinoma and tumour characteristics, tumour recurrence, risk factors and survival. METHODS: Tumoural DNA ploidy of hepatocellular carcinomas from 37 patients with cirrhosis who underwent curative tumour resection was studied by flow cytometry. RESULTS: A diploid pattern was found in 23 hepatocellular carcinomas (62.2%) and an aneuploid pattern in 14 (37.8%). The tumour recurrence rate did not differ statistically between diploid (69.6%) and aneuploid (50%) hepatocellular carcinomas. The only prognostic variable with significant difference in DNA pattern was the histologic tumour type; the majority of non-trabecular tumours were aneuploid while most trabecular hepatocellular carcinomas had a diploid DNA pattern. Actuarial survival at 1, 2, 3 and 4 years of patients with diploid and aneuploid tumours was 69.6%, 40.6%, 16.2% and 0%, and 69.3%, 59.4%, 49.5% and 32.9%, respectively (log rank p = 0.1927). CONCLUSION: These results indicate that DNA ploidy has no prognostic value in hepatocellular carcinoma.
Tissue-specific expression of the gene coding for trypsin inhibitor BTI-CMe in barley (Itr1) occurs during the first half of endosperm development. In transgenic tobacco, the Itr1 promoter drives expression of the beta-glucuronidase reporter gene not only in developing endosperm but also in embryo, cotyledons and the meristematic intercotyledonary zone of germinating seedlings. A promoter fragment extending 343 bp upstream of the translation initiation ATG codon was sufficient for full transgene expression, whereas, the proximal 83 bp segment of the promoter was inactive. Possible reasons for the differences in expression patterns are discussed.
Thionins are shown to form disulphide linkages with other proteins. The reaction with bacterial enzymes beta-glucuronidase and neomycin phosphotransferase II could be prevented and reversed with dithiothreitol and blocked with N-ethylmaleimide. Other cysteine-rich low-molecular-weight toxic peptides from plants (LTP-3 from barley and P19 from potato) did not react as the thionins. Certain cysteine-containing proteins, such bovine serum albumin, ovalbumin and cytochrome c, reacted with thionins, while others, including carbonic anhydrase, soybean trypsin inhibitor, bovine-lung trypsin inhibitor and phosphorylase B did not. Selectivity of the reaction with a periplasmic component of the phytopathogenic bacterium Pseudomonas solanacearum was also shown.
The MYB.Ph3 protein recognized two DNA sequences that resemble the two known types of MYB DNA binding site: consensus I (MBSI), aaaAaaC(G/C)-GTTA, and consensus II (MBSII), aaaAGTTAGTTA. Optimal MBSI was recognized by animal c-MYB and not by Am305 from Antirrhinum, whereas MBSII showed the reverse behaviour. Different constraints on MYB.Ph3 binding to the two classes of sequences were demonstrated. DNA binding studies with mutated MBSI and MBSII and hydroxyl radical footprinting analysis, pointed to the N-terminal MYB repeat (R2) as the most involved in determining the dual DNA binding specificity of MYB.Ph3 and supported the idea that binding to MBSI and MBSII does not involve alternative orientations of the two repeats of MYB.Ph3. Minimal promoters containing either MBSI and MBSII were activated to the same extent by MYB.Ph3 in yeast, indicating that both types of binding site can be functionally equivalent. MYB.Ph3 binding sites are present in the promoter of flavonoid biosynthetic genes, such as the Petunia chsJ gene, which was transcriptionally activated by MYB.Ph3 in tobacco protoplasts. MYB.Ph3 was immunolocalized in the epidermal cell layer of petals, where flavonoid biosynthetic genes are actively expressed. This strongly suggests a role for MYB.Ph3 in the regulation of flavonoid biosynthesis.
Acute sinusitis is one of the most commonly observed entities in clinical practice. Despite the frequency of the disease, diagnosis and therapy often remain empiric. Most cases are secondary to sinus ostia obstruction associated with the common cold or allergies. Maxillary sinusitis is most common. Because of the proximity of vital anatomic structures and venous drainage systems, serious complications frequently arise from sphenoid, frontal, and ethmoid sinusitis. Clinical signs and symptoms most helpful in the diagnosis of maxillary sinusitis are the presence of a maxillary toothache, lack of improvement with decongestants, a purulent nasal discharge, cough, purulent secretions observed on nasal examination, abnormal transillumination, and sinus tenderness. Plain film radiographs are helpful, but do not adequately visualize the anterior ethmoid sinuses. Computed tomography provides superior visualization, but cost remains prohibitive for routine cases. Most maxillary sinusitis in adults is secondary to Streptococcus pneumoniae or Hemophilus influenzae. Moroxella catarrhalis is common in children. Staphylococcus aureus is observed more frequently in frontal or sphenoid disease. Most patients with acute sinusitis are treated without microbiological diagnosis and respond well to commonly used oral antimicrobials with activity against the usual pathogens. Complications of sinusitis include meningitis, periorbital infections, subdural empyema, epidural abscess, brains abscess, cavernous sinus thrombosis, and osteomyelitis.
Activity of neomycin phosphotransferase II (NPTII; gene, neo; five cysteines) in tobacco protoplasts transfected with fusions of the octopine TR2' or cauliflower mosaic virus 35S promoter and the neo gene, with or without a signal peptide, increased up to 8-fold in response to externally added dithiothreitol at concentrations that did not affect protoplast viability (up to 2.5 mM). Activity of phosphinothricin acetyltransferase (PAT; gene, bar; one cysteine) expressed under control of the TR1' or 35S promoter was not similarly affected, thus excluding a redox modulation of transcription as the mechanism of NPTII activation by dithiothreitol. Western-blot analyses showed an increase in the amount of protein in response to dithiothreitol, whereas neither the steady-state level of NPTII mRNA nor the specific activity of the purified enzyme was affected. The same type of modulation was observed for transiently expressed beta-glucuronidase (nine cysteines) produced from a fusion with the 35S promoter, with or without a signal peptide. Limitation of cotranslational and/or early posttranslational steps by excessively oxidizing sulfhydryl/disulfide redox potentials is postulated to explain the low net accumulation of cysteine-rich proteins of bacterial origin (i.e., NPTII and beta-glucuronidase) when expressed in plant protoplasts, and the marked increase in such proteins in response to externally added dithiothreitol.
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Glomus tumors occurring in the external genitalia of two women aged 53 and 46 years, respectively, are reported. One of the tumors was clitoral and the other arose in the periurethral area of the vulva. The diagnosis in one case was supported by immunocytochemical analysis. Electron microscopy was performed on both tumors. Only two glomus tumors involving the female external genitalia have been reported in the literature. The tumors we describe appeared to be morphologically similar to the glomus tumors that typically arise in the distal extremities.
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Thionins cause the irreversible inactivation of beta-glucuronidase (GUS) in vitro in a dose- and time-dependent manner. The enzyme is also sensitive to externally added thionins when expressed in the cytoplasmic compartment of tobacco protoplasts transformed with the Gus gene under the 35S promoter of the cauliflower mosaic virus. In protoplasts transformed with the Gus gene fused to a signal peptide, where GUS is translocated into the lumen of the endoplasmic reticulum, the activity is significantly increased both by externally-added and by transiently-expressed thionin, suggesting that it interferes with GUS secretion.
Tomato and tobacco plants were inoculated with either of two strains of tomato aspermy virus, 1-TAV or V-TAV, and each of six isolates of cucumber mosaic virus satellite RNA (CMV-satRNA), B1, B2, B3, Ix, or WL2. Ribonuclease protection assays, used to detect total satRNA and encapsidated satRNA, revealed that G-satRNA generated new satellite RNA not of the inoculated sequence. The other CMV-satRNAs were compared for their ability (1) to replicate, (2) to modulate symptoms, (3) to reduce TAV accumulation, and (4) to alter the extent of encapsidation of TAV genomic RNAs. The fraction of B2- and B3-satRNAs encapsidated was greater for 1-TAV than for V-TAV, although spread and accumulation of the satRNA were similar for both helper viruses. These results suggest that CMV-satRNA may spread in a nonencapsidated form. Accumulation of CMV-satRNA in systemically infected leaves was detected for all inoculum combinations except V-TAV and Ix-satRNA, for which the satellite RNA increased only in protoplasts and inoculated leaves of tobacco or tomato. In such inoculated leaves, Ix-satRNA was not detected in capsids. Thus the effectiveness of the TAV helpers of CMV-satRNAs may be controlled in at least some instances by the extent of satRNA spread or encapsidation rather than by the efficiency of satRNA replication. In contrast to infections initiated by inoculation of CMV and CMV-satRNA, inoculation of 1-TAV or V-TAV and CMV-satRNA did not alter the relative amounts of viral genomic RNAs encapsidated or result in accumulation of large amounts of double-stranded satRNA.
Between 1938 and 1985, 73 patients with ureteral tumors underwent surgery at Puigvert Foundation (1 case with bilateral synchronous tumors). Sixty-three patients were males and 10 females. Ages ranged between 49 and 78 (mean of 62) years. Tumoral stages were: pTa 5 cases; pT1 56 cases; pT2 10 cases, and pT3 2 cases. Radical treatment was performed in 38 cases and conservative treatment in 35 cases. Follow-up ranged between 2 and 20 (mean of 7) years. The overall survival rate at 5 years was 100, 82 and 50% for stages pTa, pT1 and pT2-3, respectively (p less than 0.05). Analyzed together, the actuarial survival at 5 years was 90.3% with conservative surgery and 67.8% with radical surgery (p not significant). The survival by stages for the patients treated with radical surgery was: 100% in pTa; 69.6% in pT1, and 57.1% in pT2-3. For those treated conservatively, survival was 100% in pTa, 95.8% in pT1 and 33.3% in pT2. Thus patients presenting with infiltrating tumors should not be treated conservatively, while patients with superficial lesions had a similar evolution whether treated by conservative or radical surgery.
We have measured the growth rates and elongation rates for different proteins in wild-type, miaA, rpsL, and miaA, rpsL double mutants of Escherichia coli in the presence as well as the absence of streptomycin. The data show that while miaA and rpsL mutants inhibit elongation rates to equivalent levels, miaA inhibits the growth rate twice as effectively as does rpsL. The double mutant is more effectively inhibited than either single mutant and Sm repairs in part the growth rate as well as protein elongation rates. The data suggest that the conditional streptomycin-dependent phenotype of the double mutant cannot be due simply to the depressed polypeptide elongation rates of the double mutant.
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Petrullo et al. (1983) have studied the consequences of combining a mutation (rpsL-) that normally generates streptomycin resistant (Smr) ribosomes with a mutation (miaA-) that leads to loss of a tRNA hypermodification. They found surprisingly that such doubly mutant bacteria become streptomycin dependent (Smd). Here, we show in vitro that ribosomes purified from an Smr mutant behave very like Smd ribosomes when they are combined with tRNA from an miaA- mutant. Our analysis suggests that proofreading becomes excessively intense when the mutant components are combined, and that this reduces the efficiency of translation to the very low levels characteristic of Smd ribosomes. We show that Sm increases the efficiency of translation in vitro by suppressing the proofreading flows. We suggest that this will explain the growth stimulatory effect of Sm on the rpsL-, miaA- double mutants.