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Objectives: 1. To quantify the percentage of prescriptions of low therapeutic value, and the associated pharmaceutical expenditure, which the specialist incurs for the general practitioner. 2. To determine which are the therapeutic groups of low therapeutic value most often delegated in this way, and so determine the profile of the specialists who recommend most medication with insufficient benefits., Design: A descriptive, crossover, prospective study based on all the prescriptions of the month of March (21 days) to 4156 patients from the town of Es Castell (5720 inhabitants), Menorca., Material and Methods: The variables to be evaluated were defined and classified in scales. They included prescription, packages, working age/pensioner, origin (General Practitioner, Specialist, Private, Emergency), cost of the prescription, specialties broken down into anatomical-therapeutic groups, whether it was chronic or acute medication, and whether it was of high or low therapeutic value., Results: Of the 3599 packages prescribed, 1993 were generated from outside the PC practice (55.3%). Of the 6069411 pesetas of expenditure in the period studied, 38.5% was due to general practitioners, 59.1% specialists, and 2.3% emergency services. 12.7% (456) of the packages prescribed were of low therapeutic value, of which 52.6% corresponded to the general practitioners' prescriptions, 45.6% to the specialists' and 4.5% to private practice. However, of the 523224 pesetas of expenditure, 62% was for specialists' prescriptions and only 36% for the general practitioners'. On comparing the profiles of prescriptions of low therapeutic value coming from specialists and direct from Primary Care, differences were found. The "cardiovascular" group accounted for 40.9% of specialist prescriptions and only 11.3% of G.P. ones; "central nervous system" (psychiatry, neurology) accounted for 24.5% and 12.1%, respectively. "Respiratory", however, was the opposite: 3.8% specialist and 25% G.P. Similarly the "others" category and 18.8% for specialists against 37.1% for G.P.s., Conclusions: It can be inferred that there is shared responsibility in the public health system between specialists and G.P.s for prescribing products of low therapeutic value. However, since the medication that specialists prescribe is dearer, they cause more expenditure than the packages of low therapeutic value, which they delegate to G.P.s.

We have previously shown that the maize (Zea mays L.) storage prolamine gamma-zein, accumulates in endoplasmic reticulum-derived protein bodies in transgenic plants of Arabidopsis thaliana (L.) ecotype R + P. The retention of gamma-zein in the endoplasmic reticulum was found to be mediated by structural features contained in the polypeptide, an N-terminal proline-rich and a C-terminal cysteine-rich domain which were necessary for the correct retention and assembly of gamma-zein within protein bodies (M.I. Geli et al., 1994, Plant Cell 6: 1911-1922). In the present work we incorporated in the gamma-zein gene lysine-rich coding sequences which were positioned after the N-terminal proline-rich domain and at five amino-acid residues from the C-terminus. The targeting of lysine-rich gamma-zeins was analyzed by expression of chimeric genes regulated by the cauliflower mosaic virus (CaMV) 35S promoter in transgenic Arabidopsis plants. The lysine-rich gamma-zeins were detected by immunoblotting and we found that these proteins were modified posttranslationally to reach their mature form. Subcellular fractionation and immunocytochemical studies demonstrated that glycosylated lysine-rich gamma-zeins were secreted to the cell wall of transgenic Arabidopsis leaf cells.

Density functional theory (DFT) calculations have been carried out on the [2+2] and [2+3] addition of ethylene to Cr=O bonds in CrO(2)Cl(2). The [2+3] addition was found to be more favorable with an electronic reaction enthalpy of -9.6 kcal/mol and an activation barrier of 15.8 kcal/mol. The corresponding values for the [2+2] addition were 15.4 and 27.9 kcal/mol, respectively. It was concluded that epoxides formed in the reaction between CrO(2)Cl(2) and olefins are unlikely to originate from a [2+2] addition path to a Cr=O bond as previously suggested. The two addition reactions were analyzed by the intrinsic reaction coordinate method, and comparisons were made to a previous study of [2+2] and [2+3] addition by ethylene to OsO(4). Qualitative considerations were used to rationalize why the addition of ethylene to metal-oxygen bonds in tetrahedral d(0) oxo complexes seems more prone to proceed by a [2+3] mechanism.

Background: The high prevalence of betathalassemia minor and the presence of several cases of thalassemia major in Menorca (Spain) led us, ten years ago, to set up a population based prevention campaign, following the basic schedule of those already taking place in other areas, specially in Italy., Patients and Methods: The target population was the children in the last year of mandatory school, when they are 13 to 14 years old. The campaign started in the school year 1986-1987 and in the 1995-1996 course we reached the tenth yearly campaign. The campaign was structured in three stages: the first one included all information and educational aspects; the second step corresponded to blood sampling and detection of carriers; finally, results were reported to the individuals and genetic counseling was given. For evaluation purposes, we have monitored participation rates, the proportion of detected carriers which present to haematology visit and the trend in the proportion of carriers which previously knew their status., Results: A total of 8,591 children were screened, accounting for a 83.8% participation rate. The observed prevalence was 26.7/1000 (95% CI, 23.5-29.9/1000). We also detected 9 cases of alfathalassemia and 4 cases of deltabetathalassemia., Conclusions: Our results show that participation rates may be reached through school screening campaigns for thalassemia. After ten years of campaign, the number of not previously known cases has become small, indicating that the campaign screening to relatives of detected carriers and a higher consciousness level in the general population have given the expected effect. In the last 14 years, not a single homozigote case has been born, although we cannot conclude that this has been only due to the campaign.

Background: The G6PD deficiency is a red cell enzymopathy very frequent in certain Mediterranean countries. In Menorca (Balearic Islands), a relatively high incidence of favism carried us to study the prevalence of this alteration, taking advantage of the "Campaign for detection of heterozygous beta-thalassaemia to prevent the homozygous beta-thalassaemia" that we make annually., Methods: We studied a total of 1139 school boys between 13-14 years old for three consecutive school years. We used the methylene blue as screening test and the deficiency of G6PD was confirmed with enzymatic quantification in the haemolysate. We also analysed the clinical manifestations and studied the relatives., Results: We have confirmed 11 cases of G6PD deficiency (prevalence of 9.7/1000), all of them native of the island. The clinical manifestations were: in 6 cases (54.5%) no clinical manifestations were found, 5 cases (45.4%) had presented neonatal jaundice and 2 cases (18.2%) had suffered a favism crisis. The study of relatives permitted us to analyse 26 additional samples (17 women and 9 men), detecting in 8 of them (4 women and 4 men) the enzymopathy ., Conclusions: The prevalence of G6PD deficiency in Menorca is one of the highest in Spain. Most of the carriers are asymptomatic, the most important clinical manifestations being the neonatal jaundice and favism. The screening test used is efficient for unmistakable hemizygotes detection.

During maize seed development, endosperm cells synthesize large amounts of storage proteins, alpha-, beta-, and gamma-zeins, which accumulate within endoplasmic reticulum (ER)-derived protein bodies. The absence of lysine in all zein polypeptides results in an imbalance in the amino acid composition of maize seeds. We modified the maize gamma-zein gene through the introduction of lysine-rich (Pro-Lys)n coding sequences at different sites of the gamma-zein coding sequence. Maize endosperms were transiently transformed by biolistic bombardment with Lys-rich gamma-zein constructs under the control of the 1.7 kb gamma-zein seed-specific promoter and the cauliflower mosaic virus (CaMV) 35S promoter. When (Pro-Lys)n sequences were inserted contiguous to or in substitution of the Pro-Xaa region of the gamma-zein, high levels of protein were observed. In contrast, when (Pro-Lys)n sequences were inserted five residues from the C-terminal, the transcript was present but modified protein was not detected. These results suggest that only an appropriate positioning of Lys-rich inserts leads to the modified molecule displaying correct folding and stability. Subcellular localization analyses and immunoelectron microscopy studies on isolated protein bodies demonstrated that modified gamma-zeins accumulate within these organelles and co-localized with endogenous alpha- and gamma-zeins. The studies reported here show the feasibility of manipulating the gamma-zein gene in order to obtain stable and correctly targeted Lys-rich zeins in maize seeds.

When we stimulated 3T6 fibroblasts, we observed a mobilization of arachidonic acid (AA) from phospholipids. The magnitude of this response decreased as the cells became confluent and the change coincided with a decrease in the percentage of cells in growth phases (G2 + M); this was not a consequence of the time in culture or a factor in the growth medium. Preconfluent fibroblasts incubated with the calcium ionophore A23187 (1 microM), 4beta-phorbol-12-myristate acetate (PMA, 10 microM), bradykinin (10 microM) or fetal calf serum (20%) released 38.8%, 62.5%, 11.2% and 45.6% [3H]AA, respectively. Confluent cells stimulated under the same conditions released only 16.9%, 1.5%, 0.5% and 18.5% [3H]AA, respectively. This decreasing mobilization of AA was demonstrated using metabolic labelling and measurement of prostaglandin PGE2. The decrease was not due to a changing pool of AA. [3H]AA release from each phospholipid decreased with confluence. Conversion of confluent cells to the proliferative phenotype by mechanical wounding of the monolayer increased the release of [3H]AA. This effect is consistent with regulated, growth-dependent changes in the activity of phospholipase A2, a process regulated by changes in phosphorylation of the enzyme. The increased release of [3H]AA from preconfluent, actively dividing cells may have important physiological implications and may help elucidate mechanisms regulating release of AA.

Objective: To assess the incidence, mortality, and survival of lung cancer in Menorca (Balearic Islands, Spain)., Methods: All cases detected between 1990 and 1993, were collected retrospectively, using the cancer registry methodology., Results: The incidence rate observed was 47.7 cases per 100,000 inhabitants per year, while the mortality rate was 44.2. Mean age of patient was 67.5 years 90% of whom were male. The most frequently observed histological type was the epidermoid carcinoma (43% of cases), followed by the small-cell carcinoma (20%) and the adenocarcinoma (19%). Mean survival, over a total of 141 cases studied, was about 9 months (281 days)., Conclusion: Incidence rates adjusted by world population show that Menorca lung cancer incidence rates are in the upper range among Spanish regions.

[gamma]-Zein is a maize storage protein synthesized by endosperm cells and stored together with [alpha]- and [beta]-zeins in specialized organelles called protein bodies. Previous studies have shown that in maize there is only one type of protein body and it is derived directly from the endoplasmic reticulum (ER). In this article, we describe the domains of [gamma]-zein involved in ER retention and the domains involved in protein body formation. To identify the signal responsible for [gamma]-zein retention in ER-derived protein bodies, DNAs encoding various deletion mutants of [gamma]-zein were constructed and introduced into Arabidopsis as a heterologous system. By using pulse-chase experiments and immunoelectron microscopy, we demonstrated that the deletion of a proline-rich domain at the N terminus of [gamma]-zein puts an end to its retention in the ER; this resulted in the secretion of the mutated protein. The amino acid sequence of [gamma]-zein necessary for ER retention is the repeat domain composed of eight units of the hexapeptide PPPVHL. In addition, we observed that only those [gamma]-zein mutants that contained both the proline-rich repeat domain and the C-terminal cysteine-rich domain were able to form ER-derived protein bodies. We suggest that the retention of [gamma]-zein in the ER could be a result of a protein-protein association or a transient interaction of the repeat domain with ER membranes.

A case-control study on diet and gastric cancer, carried out in selected areas of four regions of Spain (Aragon, Castile, Catalonia, and Galicia) in 1988 and 1989, included 354 cases of histologically confirmed gastric adenocarcinoma and 354 controls matched by age, sex, and area of residence. Cases and controls were selected from 15 hospitals, representing most of the hospital facilities in the study areas. Usual diet was estimated by means of a dietary history questionnaire administered by interview. An increased risk of gastric cancer was observed for high consumption of exogenous nitrosamines (odds ratio = 2.1 for the highest quartile of consumption versus the lowest; p for linear trend = 0.007), nitrites, fat, and cholesterol. However, in a multivariate regression model, the effect of fat and cholesterol disappeared. An inverse association with the risk for gastric cancer was seen for high intake of fiber, vitamin C, folate, carotene, and nitrates. High consumption of vitamin C seemed to neutralize the increased risk related to simultaneous consumption of nitrosamines. For histologic type, the authors found no meaningful differences in the effect of most of the nutrients between intestinal and diffuse cancers. Their findings are consistent with previously reported results about the protective effect of fruit and vegetables and the increased risk associated with foods that are important sources of nitrites and preformed nitrosamines.

The maize abscisic acid (ABA)-responsive rab17 mRNA and Rab17 protein distribution in maize embryo tissues was investigated by in situ hybridization and immunocytochemistry. rab17 mRNA and Rab17 protein were found in all cells of embryo tissues. Synthesis of rab17 mRNA occurred initially in the embryo axis. As maturation progressed, rab17 mRNA was detectable in the scutellum and accumulated in axis cells and provascular tissues. However, the response to exogenous ABA differed in various embryo cell types. The Rab17 protein was located in the nucleus and in the cytoplasm, and qualitative differences in the phosphorylation states of the protein were found between the two subcellular compartments. Based on the similar domain arrangements of Rab17 and a nuclear localization signal (NLS) binding phosphoprotein, Nopp140, interaction of Rab17 with NLS peptides was studied. We found specific binding of Rab17 to the wild-type NLS of the SV40 T antigen but not to an import incompetent mutant peptide. Moreover, binding of the NLS peptide to Rab17 was found to be dependent upon phosphorylation. These results suggest that Rab17 may play a role in nuclear protein transport.

In order to examine the role of cysteine (Cys)-rich domains in the accumulation of maize (Zea mays L.) gamma-zein within the endoplasmic-reticulum-derived protein bodies, we studied the localization of gamma-zein and of two truncated forms of gamma-zein in Xenopus laevis oocytes. The two derivatives were constructed from a DNA encoding the gamma-zein: one by deletion of the Pro-X linker region (21 amino acids) and the other by deletion of the Cys-rich domain (94 amino acids). In-vitro-synthesized transcripts were injected into oocytes and the distribution of the translation products was then analyzed. The entire gamma-zein and both truncated forms of the gamma-zein had accumulated efficiently in microsomes and no traces of secretion were observed. We suggest that neither C-terminal Cys-rich nor Pro-X domains are essential for gamma-zein retention in oocyte vesicles. Therefore, structural features derived from disulphide bonds are not necessary for gamma-zein targeting on the endoplasmic reticulum.

The dietary pattern of a population sample of males selected as a control group for a case-control study on bladder cancer carried out in four different regions of Spain is presented. Out of 807 population controls initially selected, 530 were interviewed, 465 males and 65 females. The method of selection of the study subjects and the diet assessment method are described. Our results confirm the Mediterranean pattern of the Spanish diet, with an important consumption of fresh fish, fruits and vegetables, and the use of vegetable oils, specially olive oil, for cooking and seasoning, accompanied with a high polyunsaturated/saturated ratio (0.7). The consumption of butter, cheese and other dairy products, on the other hand, is very low. Estimated total caloric intake is relatively low if compared with international figures, although questionnaire base assessment may seriously underestimate caloric intake, as it is the absolute amount of intake of lipids and carbohydrates. In relative terms, however, lipids provide the highest percentage of calories.

The selection of the best control group is a major concern in the design of any case-control study, because of its enormous implications on both interval validity and cost. We compare the dietary habits of hospital and neighbourhood control groups which had both been used in a case-control study on bladder cancer. The analysis is based on 485 matched sets. No differences were observed between the two groups in terms of nutrient consumption nor in consumption of specific foods or food groups. Alcohol consumption represents an exception. This may be attributed to the use of incorrect exclusion criteria given that alcohol consumption was not one of the major determinants of the original study. We conclude that, provided correct exclusion criteria are used, hospital controls may be a suitable as neighbourhood controls in the assessment of dietary risk factors in case-control studies in Spain.

Hydroxyproline-rich glycoproteins (HRGPs) fromZea mays have been immunolocalized in the cell wall of root tip cells using ultrathin sections and antibodies ellicited against the purified protein. The accumulation of mRNA corresponding to this protein was studied using the cDNA probe. Maximum accumulation of the mRNA was found in tissues with a high proportion of dividing cells such as those in the root tip of young maize seedlings and a close relationship with cellular division was also observed in in-vitro cultures. However, the level of the mRNA in elongating tissues was minimal, as shown by studies carried out on the elongation zones of root tips and coleoptiles. The mRNA was induced by stress conditions, particularly by wounding young leaves and coleoptiles. It is concluded that in maize this group of proline-rich cell-wall proteins accumulates during cell division and not during cell elongation or differentiation, and participates in the stress-response mechanisms of the plant.

The ABA-induced MA12 cDNA from maize, which encodes a set of highly phosphorylated embryo proteins, was used to isolate the corresponding genomic clone. This gene, called RAB-17 (responsive to ABA), encodes a basic, glycine-rich protein (mol. wt. 17,164) containing a cluster of 8 serine residues, seven of them contiguous. It is a homologue of the rice RAB-21 gene (Mundy J, Chua NH, EMBO J 7; 2279-2286, 1988). Phosphoamino acid analysis of the isolated protein indicates that only the serine residues are phosphorylated and a putative casein-type kinase phosphorylatable sequence was identified in the protein. The pattern of expression and in vivo phosphorylation of the RAB-17 protein was studied during maize embryo germination and in calli of both meristematic or embryonic origin. ABA treatment induced the synthesis of RAB-17 mRNA and protein in calli, however, the RAB-17 proteins were found to be highly phosphorylated only in embryos.

Experiments to establish the site of biosynthesis and the possible translocation into microsomes of glutelins-2 (28 kD G2) and low molecular weight zeins (10, 14, 15 kD Z2) have been carried out. Free and membrane-bound polyribosomes as well as microsomal membranes were isolated from immature endosperms of W64A Zea mays L. In vitro translation studies were carried out in the presence and in the absence of membranes using [(35)S]-methionine or [(35)S]-cysteine as precursors. Cell-free translation products were characterized by electrophoretic mobility, solubility and antigenic properties. The results obtained indicate that 28 kD G2 and low molecular weight zeins are primarily synthesized on membrane-bound polysomes. From experiments using proteinase K as a probe, we also conclude that these proteins are translocated into microsomes where they accumulate. The translocated and pre-28 kD G2 proteins do not present changes in the apparent molecular weight. However we show that there are differences in their isoelectric points, a fact that indicates the existence of 28 kD G2 processing.

Accumulation of the 28 KD protein of the glutelin-(G2) fraction was followed in developing maize endosperm, using sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and peak integration of scanned gels. 28 KD glutelin-2 could already be observed from 15 days after pollination and its accumulates reached a plateau during the second half of the development period. The process of biosynthesis of 28 KD glutelin-2 and zeins occurs in a parallel way. Subcellular fractions obtained from linear sucrose gradient centrifugation of developing maize endosperms were analyzed by SDS-PAGE and immunoblotting using a serum reacting against glutelin-2 and 14 KD Z2. Glutelin-2 was found to be present in the protein bodies when subcellular fractionation was carried out without dithiothreitol (DTT). The presence of a reducing agent causes the elution of glutelin-2 from protein bodies. Immunocytochemical labelling using the protein A-colloidal gold technique in protein bodies incubated with anti-G2 IgG revealed that G2 is located mainly in the periphery of protein bodies. These results are interpreted as indicating a structural role for glutelins in protein bodies.

We show that maize storage protein translocation across microsomal membranes is mediated by signal recognition particles (SRPs) similar to those described in animal systems (Dobberstein, B. (1978) Hoppe-Seyler's Z. Physiol. Chem. 252, 955-962; Walter, P., and Blobel, G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7112-7116). We have prepared a high salt extract from endosperm cell homogenates, from which a ribosome-free fraction was obtained. This fraction is enriched in an SRP-like factor which apparently corresponds to a ribonucleoprotein particle that sediments at about 12 S. The RNA moiety of this 12 S particle is complex, showing a three-band electrophoretic pattern and sedimenting at about 8 S. The fraction restores translocation competence of salt-washed maize microsomes as tested by using a pre-zein message. In contrast to canine SRPs, the maize SRP-like component does not cause a translation arrest of maize storage proteins (zein) in a wheat germ cell-free system.

The oil-stop procedure applied in transport studies on whole cells was adapted for isolated plasma membrane vesicles. The advantages of the proposed technique with respect to the commonly used filtration method are the reduction of the period of the termination step, the increase in the allowed membrane content of the samples and the absence of the leakage of substrates from the intravesicular space.

Two-dimensional gel electrophoresis with non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide gels in the second dimension has been used for the analysis of organ-specific proteins in maize. The method has been used to study the whole protein pattern of developing organs and adult leaves as well as protein patterns of in vitro translation. Examples of two-dimensional immunoblotting and in vitro translation of endosperm-specific proteins are also shown. Two-dimensional gel electrophoresis appears as an essential analytical step in the identification of organ-specific proteins and for the detection of the protein products related to organ-specific genes identified by other means.

Storage proteins of maize (Zea mays L.) were studied in germinated seeds, as were the proteins of protein bodies isolated from endosperms at different germination times. Major endosperm storage proteins were degraded in a sequential way, glutelin 2 being hydrolysed faster than zein 1. Immunocytochemical labelling of the different protein bodies using the antisera anti-glutelin 2 and anti-zein 1 indicates that the protein bodies were degraded by progressive hydrolysis from their surface. The digestion of glutelin 2 correlated with the disappearance of the protein-body membranes.