Estudio de la frecuencia de parpadeo y evaluación de la película lagrimal en superficie ocular de cobayos / Eye blinking frequency studies and tear film evaluation in guinea pig's ocular surface

OBJETVOS: La Queratopatía Climática Esferoidea (QCE) es una enfermedad degenerativa de la córnea humana caracterizada por la agregación de proteínas bajo el epitelio y una progresiva opacidad corneal, causada por condiciones ambientales desfavorables. Si bien esta patología fue descripta por primera vez hace más de cien años, en la actualidad no existe un modelo experimental de QCE que permita avanzar en el conocimiento de la etiopatogenia de la misma para plantear posibles tratamientos. En el intento por desarrollar este modelo en cobayos y debido a la escasa bibliografía disponible que describa la anatomía, fisiología y superficie ocular de estos animales, realizamos diferentes tests fisiológicos de superficie ocular en cobayos y lo compararemos con el humano. MÉTODOS: Para estos estudios se utilizaron 15 cobayos. Se realizaron videograbaciones de los mismos en un ambiente de tranquilidad y bajo ciertos estímulos para estudiar la dinámica de parpadeo y sueño. Con el fin de analizar la producción y la estabilidad del film lagrimal, se realizaron test de Schirmer, medición de la altura del menisco lagrimal inferior por OCT visante y tiempo de ruptura precorneal (TRP) respectivamente. RESULTADOS: El cobayo parpadea de forma completa y parcial, siendo más frecuente ésta última modalidad. La frecuencia y tipo de parpadeos/ minuto fue: Total= 2.35± 0.87; completos= 0.68±0.43; parciales=1.49±0.75. Su frecuencia de parpadeo es 5 veces menor que en humanos. La respuesta palpebral frente a diferentes estímulos externos fue escasa. Los cobayos no poseen hábitos nocturnos como muchos roedores y duermen por cortos períodos. A menudo no cierran completamente los ojos. Test de Schirmer cobayos: 8.14±1.86 mm/5 min (Humanos: 11.9 ± 6.79 mm/5 min). Altura menisco inferior cobayos: 0,206 ± 0,059 mm (Humanos: 0,277 ± 0,033 mm). TRP cobayos: 2-3 min. (Humanos: 10-15 seg). CONCLUSIONES: Los resultados obtenidos muestran que el cobayo, a diferencia de humanos, tiene una frecuencia de parpadeo menor. A su vez, esto se puede correlacionar con una producción de lágrima disminuida (objetivada con Test de Schirmer) y una mayor estabilidad de la película lagrimal, como lo evidencia el prolongado TRP. (AU)

PURPOSE: Climatic spheroidal keratophaty (CSK), also known as climatic droplet keratopathy (CDK), is a degenerative human corneal disease characterized by protein aggregation under the corneal epithelium that leads to a progressive corneal opacity. Of unknown etiology, it is related to harsh environmental conditions. Although CSK was described many years ago and no experimental model of the disease is still available, we perform different physiological test on the guinea pig´s ocular surface and compare the results with human parameters. METHODS: 15 guinea pigs were used for this study. Video recordings of their behavior in a quiet environment and under certain stimulus were performed to study the blinking and sleeping dynamic. Test de Schirmer, Inferior tear meniscus height measurement, Break up time (BUT) test were performed, with the aim of analyze the tear film production and stability. RESULTS: The guinea pig blinks in both a complete and a partial way, being more frequent the last one. The blink type and frequency were: Total = 2.35± 0.87; complete= 0.68±0.43; partial=1.49±0.75. The blink frequency is 5 times smaller than in humans. The eyelid response against different external stimuli was poor. The night habits of this animals were different compared with other rodents, they sleep for a short periods of time. They often do not close their eyes completely. Schirmer test in guinea pigs was 8.14±1.86 mm/5 min (humans: 11.9 ± 6.79 mm/5 min). Inferior tear meniscus height measurement in guinea pigs was 0,206 ± 0,059 mm (humans: 0,277 ± 0,033 mm). BUT in Guinea Pig: 2-3 min. (Humans: 10-15 seg). CONCLUSIONS: Guinea Pigs have a short blink frequency unlike humans, a decreased tear production, and an increased tear film stability. (AU)

Effects produced by different types of laser in cornea of Guinea pigs: Identification of a laser capable of producing superficial lesions without leaving scars.

PURPOSE: Climatic droplets keratopathy (CDK) is closely associated with superficial corneal erosions and lack of protective mechanisms against the harmful effects of ultraviolet radiation (UVR) during a prolonged period of time. One of the difficulties in studying the pathogenic mechanisms involved in this human disease is the lack of an experimental animal model. In this paper, a study is conducted on the effects of 4 types of lasers at various powers and time conditions on the normal guinea pig corneas in order to select only one laser condition that reversibly injures the epithelium and superficial stroma, without leaving scarring. METHODS: Damage was induced in the cornea of Guinea pigs using different powers and exposure times of 4 types of laser: argon, CO2, diode and Nd-Yag, and any injuries were evaluated by biomicroscopy (BM) and optical microscopy. Corneas from other normal animals were exposed to argon laser (350 mW, 0.3s, 50 µm of diameter), and the induced alterations were studied at different times using BM, optical coherence tomography (OCT) and transmission electron microscopy (TEM). RESULTS: Only argon laser at 350 mW, 0.3s, 50 µm of diameter produced epithelium and superficial stroma lesions. Some leukomas were observed by BM, and they disappeared by day 15. Corneal thickness measured by OCT decreased in the eyes treated with argon laser during the first week. Using TEM, different ultra structural alterations in corneal epithelium and stroma were observed during the early days, which disappeared by day 15. CONCLUSIONS: It was possible to develop reproducible corneal epithelium and anterior stroma injuries using Argon laser at 350 mW, 0.3s, 50 µm of diameter. In vivo and in vitro studies showed that injured corneas with these laser conditions did not leave irreversible microscopic or ultra structural alterations. This protocol of corneal erosion combined with exposure to UVR and partial deficiency of ascorbate in the diets of the animals for an extended period of time has been used in order to try to develop an experimental model of CDK.

On the Cornea of Healthy Merino Sheep: A Detailed Ex Vivo Confocal, Histological and Ultrastructural Study.

Our study performed qualitative and quantitative studies on the corneal ultrastructure of healthy female Merino sheep of ages 4 months and 6 years old from the Argentinean Pampa. The corneas were evaluated using ex vivo laser-scanning confocal microscopy, light microscopy and transmission electron microscopy. Those studies allowed us to obtain detailed images of the corneal layers as well as quantitative data of the cellular and sub-basal nerve densities in the cornea from sheep of different ages. The density of the corneal cells was significantly different in the anterior versus the posterior epithelium and stroma. Moreover, the density of the epithelial, stromal cells and endothelial cells, as well as the sub-basal nerve density were significantly lower in adult than in young animals. Our work provided a wide-ranging description of the corneal ultrastructure of healthy female Merino sheep, which adds to the current knowledge about the ophthalmological aspects of this species and undoubtedly benefits veterinarians.

Estudio del polimorfismo del gen de TNF-α, de dicha citocina, y de MCP-1 en pacientes con queratopatía climática esferoidea / Studies of TNF-α gene polymorphism, TNF-α and MCP-1 cytokines in patients with spheroidal climatic keratopathy

Objetivo. Investigar polimorfismo de nucleótidos únicos (SNP) en la posición -308 (G/A) del gen TNF-α y la participación de las citocinas TNF-α y MCP-1 en pacientes con queratopatía climática esferoidea (QCE) y en controles sanos. Materiales y métodos. Participaron 15 pacientes con QCE y 15 individuos sanos del departamento El Cuy, Provincia de Río Negro. Todos ellos, luego de firmar el consentimiento informado, recibieron un examen oftalmológico completo y se recolectaron muestras de sangre y lágrima para realizar diferentes estudios. EL ADN genómico fue obtenido de sangre de todos los individuos mediante el método de salting out y posteriormente amplificado y estudiado mediante reacción en cadena de la polimerasa (PCR) con el sistema de amplificación refractaria a la mutación (ARMS). También se investigaron concentraciones de algunas citocinas proinflamatorias en lágrimas y en sobrenadante de cultivo de células epiteliales corneales humanas (CECH) tratadas o no con radiación ultravioleta B (RUV-B). Resultados. Los resultados de SNP en la posición -308 (G/A) del gen TNF-α (frecuencia alélica y genotípica) indicaron ausencia de diferencias significativas entre pacientes y controles sanos. Fenotípicamente ambos grupos de individuos serían bajos o intermedios productores in vitro de la citocina TNF-α. Sin embargo en las lágrimas de pacientes con QCE se detectaron concentraciones significativamente superiores de TNF-α, IL-1ß y MCP-1 (citocinas proinflamatorias) que en lágrimas de individuos controles sanos (p<0,0001) En la periferia y limbo de la córnea las células dendríticas (CD) incrementaron significativamente con el progreso de la enfermedad (p<0,05). La contribución del epitelio corneal en el proceso inflamatorio fue investigada utilizando CECH expuestas o no a 10 mJ/cm2 de RUV-B. A pesar de la presencia de gelatinasas, IL-6 e IL-8 en sobrenadantes de cultivos obtenidos a las 48 horas (datos no mostrados) no observamos niveles detectables de TNF-α, IL-1ß ni MCP-1. Conclusión. Este trabajo aporta nuevos datos para aumentar los conocimientos sobre los mecanismos inmunológicos involucrados en la etiopatogenia y progresión de la QCE. Demostramos que las citocinas proinflamatorias MCP-1 y TNF-α están significativamente elevadas en lágrimas de individuos con QCE, como se observó previamente con IL-1ß. MCP-1 sería la responsable del aumento de CD en córnea periférica y limbo de estos pacientes a medida de que la enfermedad avanza. El hallazgo de que estas citocinas no pudieron ser detectadas en cultivos de CECH estresadas con RUV-B implica que otras células son las responsables de su producción o que además de RUV-B otros factores son necesarios para iniciar esta cascada de eventos que se observan en esta hipersensibilidad corneal humana(AU)

Purpose. To investigate Single Nucleotide Polymorphism (SNP) at -308 position (G/A) of TNF-α gen and involving of TNF-α and MCP-1 cytokines in Climatic Droplet Keratopathy (CDK) patients and healthy controls. Materials and methods. Fifteen patients with CDK and fifteen healthy controls from departamento El Cuy, province of Rio Negro were involved in this study. After informed consent was obtained from all participants, they had a complete eye examination and then tear and blood samples were collected to perform different assays. DNA was obtained from blood of all individuals using the method of "salting out" and then amplified and studied performing the polymerase chain reaction (PCR) with Amplification-refractory Mutation System (ARMS). Furthermore, some cytokines concentrations were measured in tears and supernatants from human corneal epithelial cells (HCEs) exposed or not to UVR-B radiation. Results. Analysis from SNP at position -308 (G/A) of TNF-α gen (allelic and genotypic frequency) showed no significant differences between patients and healthy controls. Phenotypically both groups of individuals would be low or intermediate in vitro producers of TNF-α cytokine. However, in tears from CDK's patients we detected significantly higher concentrations of TNF-α, IL-1ß and MCP-1 (pro-inflammatory cytokines) than in healthy control subjects tears (p<0.0001). At the corneal peripheral / limbus area, dendritic cells (DCs) increased significantly with the progression of the disease (p<0.05). The corneal epithelium contribution to the inflammatory process was investigated using HCEs exposed or not to 10 mJ/cm2 of UV radiation­B (UVR-B). Despite the presence of gelatinases, IL-6 and IL-8 in culture supernatants obtained after 48 hours (data not shown), detectable levels of TNF-α, IL-1ß and MCP-1 were not detected. Conclusion. This study provides new insights to increase our knowledge about the immunological mechanisms involved in the etiopathogenesis and progression of CDK. We showed that pro-inflammatory cytokines MCP-1 y TNF-α were significantly increased in tears from CDK's patients, as previously described with IL-1ß. MCP-1 would be responsible for the increasing of DCs on the corneal peripheral / limbus area of these subjects as the disease progresses. The fact that these cytokines could not be detected in cultures of HCEs stressed with UVR-B implies that other cells are responsible for their production or, in addition to UVR-B, other factors are necessary to initiate the cascade of events observed in this human corneal hypersensitivity. (AU)

Participación de componentes inmunológicos en la etiopatogenia de la queratopatía climática esferoidea / Involvement of immune components in the etiopathogenia of climatic droplet keratopathy

Objetivo. Investigar si componentes de la inmunidad innata están involucrados en la iniciación/perpetuación de las anormalidades estructurales observadas en la capa de Bowman y el estroma superficial de la córnea de pacientes con queratopatía climática esferoidea (QCE). Materiales y métodos. En el estudio participaron 8 pacientes con QCE y 12 individuos sanos del Departamento El Cuy, Provincia de Río Negro, y 10 individuos sanos de la ciudad de Córdoba. Todos ellos, luego de firmar el consentimiento informado, recibieron un examen oftalmológico completo y se recolectaron muestras de lágrima para estudiar las concentraciones de diferentes citocinas, niveles y formas de metaloproteinasas de matriz (MMPs), y el inhibidor natural de MMPs (TIMP-1). Se realizó microscopía confocal in vivo (MCF) en algunos pacientes y controles. Biopsias de córneas provenientes de pacientes que fueron tratados con queratoplastia penetrante también fueron estudiadas mediante inmunohistoquímica (IHQ). Resultados. Los resultados de MCF indicaron claramente una progresión en la cantidad de depósitos a nivel subepitelial, a medida que la enfermedad avanza. El daño progresivo de las fibras nerviosas sub basales y estromales en los estadios 2 y 3 se correlaciona con pérdida de la sensibilidad corneal. Además de estas alteraciones, observamos que el número de células dendríticas (CD) en el limbo corneal aumentó significativamente a medida que la QCE progresa. En lágrimas de pacientes con QCE se detectaron concentraciones significativamente superiores de citocinas proinflamatorias (IL1ß e IL-8) que en individuos controles (p<0,005). No se halló IL-2, IL-17, IL-4, IL-13 ni IL-10 en pacientes y ni controles. Las actividades de gelatinasas (MMP-9 y -2) fueron significativamente mayores en QCE que en los controles (p<0,001), mientras que los niveles de TIMP-1 fueron significativamente menores en los pacientes (p<0,05). La concentración de MMP-8 fue mayor en controles pero los niveles de esta colagenasa-2 fueron 30 veces superiores, tanto en QCE como controles, con respecto a los valores de los individuos de un centro urbano. Mediante IHC observamos reactividad para MMP-9 en la mayoría de las células epiteliales, solamente en córneas con QCE. Conclusión. Demostramos un rol protagónico del eje citocinas proinflamatorias - gela-tinasas en el desarrollo de la QCE. Los altos niveles de IL-1ß e IL-8 en lágrimas de pacientes facilitan la producción de MMP-8 y gelatinasas, y los efectos de las mismas se exacerban, ya que los pacientes tienen bajos niveles de sus inhibidores naturales (TIMP-1). La MMP-9, además de degradar componentes de la matriz extracelular, cataliza la activación postranscripcional de IL-1ß, potenciando el proceso inflamatorio. Estos resultados son los primeros en explicar mecanismos inmunológicos involucrados en la etiopatogénesis de la QCE y aportan nuevas alternativas para el desarrollo de terapias preventivas utilizando inhibidores de IL-1ß y/o gelatinasas(AU)

Objective. To investigate whether components of innate immunity are involved in the initiation / perpetuation of the structural abnormalities observed in Bowman's layer and superficial stroma of the córnea of patients with Climatic droplet keratopathy (CDK). Materials and Methods. The study included 8 CDK patients and 12 healthy individuals from Department El Cuy, Province of Río Negro, and 10 healthy subjects from the city of Córdoba. All of them, after signing informed consent, received a thorough eye exam and tear samples were collected to study the concentrations of different cytokines, and levels and forms of matrix metalloproteinases (MMPs) and their natural inhibitor (TIMP-1). In vivo confocal microscopy (CFM) was performed in some patients and controls. Corneal biopsies from CDK patients treated with penetrating keratoplasty were also studied by immunohistochemistry (IHC). Results. CFM results clearly indicated a progression in the amount of deposits at corneal sub epithelial level as the disease progresses. The progressive damage in the nerve plexus in stages 2 and 3 correlated with a loss of corneal sensitivity. In addition to these alterations, we observed that the number of dendritic cells (DC) in the limbus increased significantly as the disease progresses.In tears of patients with CDK we detected significantly higher concentrations of pro-inflammatory cytokines (IL-1ß and IL-8) than in control subjects (p < 0.005). We found no IL-2, IL-17, IL-4, IL-13 and IL-10 in patients and controls. The activities of gelatinases (MMP-9 and -2) were significantly higher in CDK than in controls (p < 0.001), while TIMP-1 levels were significantly lower in patients (p < 0.05). The concentration of MMP-8 was higher in controls, but levels of this collagenase-2 were 30 times higher, both in CDK and controls, with respect to MMP-8 values of individuals inhabiting an urban area. By IHC we observed reactivity for MMP-9 in most epithelial cells only in CDK corneas. Conclussion. We demonstrated a key role of the axis pro-inflammatory cytokines ­ gelatinases in the development of CDK. High levels of IL-1ß and IL-8 in tears of patients facilitate the production of MMP-8 and gelatinases, and the effects of these molecules are exacerbated because patients have low levels of their natural inhibitors (TIMP-1). Since MMP-9 besides degrading extracellular matrix components, catalyzes the post translational activation of IL-1ß, the inflammatory process is fuelled. These results are the first to explain immunological mechanisms involved in the pathogenesis of the QCE and provide new alternatives for the development of preventive therapies using inhibitors of IL-1ß and / or gelatinases.(AU)

Cavidad oral: importante sitio de vigilancia inmunológica / Oral cavity: important site for immune surveillance

La cavidad oral es el principio del tracto digestivo y uno de los sitios del cuerpo más expuestos al ingreso de todo tipo de patógenos, tanto del aire como de los alimentos. Aunque varios excelentes artículos han examinado distintos aspectos de tejidos linfoides asociados a mucosas (MALT), no hay suficiente información acerca de la respuesta inmune en la cavidad oral. En esta revisión destacamos algunos aspectos sobre la anatomía / histología de la cavidad oral, estructuras asociadas y células o moléculas con crucial función inmunológica contra antígenos que ingresan en la boca. Los estudios sobre la mucosa oral han adquirido mucha notoriedad últimamente debido a que ofrece una excelente accesibilidad y evita la degradación de las proteínas y péptidos. En la cavidad bucal se puede generar una respuesta inmune apropiada contra microorganismos, en donde además de la IgA salival muchas otras moléculas son liberadas y cumplen un rol protagónico. A pesar de todos estos factores de defensa, existen momentos en donde el individuo se encuentra más expuesto, dependiendo de la edad, factores hormonales, genéticos, hábitos de fumar y la actividad física, ya que todo esto modifica la tasa de flujo salival, tasa de secreción y concentración de IgA salival y demás proteínas. (AU)

The oral cavity is the beginning of the digestive tract and one of the most exposed body sites to the entry of all types of pathogens in the air as food. Although several excellent articles have examined various aspects of mucosa-associated lymphoid tissue (MALT), there is insufficient information about the oral cavity immune response. In this review we highlight some aspects of the anatomy / histology of the oral cavity, associated structures and cells or molecules with crucial immunological function against antigens that enter the mouth. Studies on the oral mucosa have gained much notoriety lately because it offers excellent accessibility and prevents the degradation of proteins and peptides. In the oral cavity can generate an appropriate immune response against microorganisms, where salivary IgA in addition to many other molecules are released and play a role. Despite these defense factors, there are moments where the individual is more exposed, depending on age, hormonal factors, genetic, smoking habits and physical activity, and that this changes the rate of salivary flow rate secretion and concentration of salivary IgA and other proteins.(AU)

VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation.

Plant embryogenesis is intimately associated with programmed cell death. The mechanisms of initiation and control of programmed cell death during plant embryo development are not known. Proteolytic activity associated with caspase-like proteins is paramount for control of programmed cell death in animals and yeasts. Caspase family of proteases has unique strong preference for cleavage of the target proteins next to asparagine residue. In this work, we have used synthetic peptide substrates containing caspase recognition sites and corresponding specific inhibitors to analyse the role of caspase-like activity in the regulation of programmed cell death during plant embryogenesis. We demonstrate that VEIDase is a principal caspase-like activity implicated in plant embryogenesis. This activity increases at the early stages of embryo development that coincide with massive cell death during shape remodeling. The VEIDase activity exhibits high sensitivity to pH, ionic strength and Zn(2+) concentration. Altogether, biochemical assays show that VEIDase plant caspase-like activity resembles that of both mammalian caspase-6 and yeast metacaspase, YCA1. In vivo, VEIDase activity is localised specifically in the embryonic cells during both the commitment and in the beginning of the execution phase of programmed cell death. Inhibition of VEIDase prevents normal embryo development via blocking the embryo-suspensor differentiation. Our data indicate that the VEIDase activity is an integral part in the control of plant developmental cell death programme, and that this activity is essential for the embryo pattern formation.

Spatial and temporal expression of two cytosolic glutamine synthetase genes in Scots pine: functional implications on nitrogen metabolism during early stages of conifer development.

Ammonium assimilation during the initial stages of Scots pine growth involves two cytosolic glutamine synthetase (GS, EC: 6.3.1.2) isoenzymes encoded by separate genes, GS1a and GS1b. GS1a was most exclusively expressed in photosynthetic tissues of the seedling whereas GS1b was expressed ubiquitously showing higher levels in non-photosynthetic tissues such as root and hypocotyl. Temporal expression analysis has shown that when germination starts GS1b is the predominant form in the embryo, however, its relative abundance in the tissue decreased in the postgerminative stages when green cotyledons are developed. In contrast GS1a was present at a low level in the embryo but its abundance increased markedly during germination and seedling growth. These data suggest that GS1a and GS1b genes display different and non-redundant roles in the nitrogen metabolism of conifers. The precise localization of individual transcripts by in situ hybridization strongly supports this possibility. GS1 gene products are mainly expressed in different cellular types: GS1a in chlorophylic parenchyma and GS1b in the vascular bundles of all tissues examined in the seedling. Our data support that glutamine biosynthesis in pine seedlings follows a different pattern related to angiosperms involving two cytosolic GS proteins: one of them a typical cytosolic GS which may be involved in the generation of glutamine for N transport and a second cytosolic GS generating amino donors for the biosynthesis of major N compounds in photosynthetic tissues, a closer role to angiosperm chloroplastic GS. The results are discussed with regard to recent studies on N mobilization and metabolism during the initial stages of conifer development.

Characterization of rpoB gene mutations in rifampicin resistant Mycobacterium tuberculosis strains isolated from pulmonary tuberculosis patients at 5 Mexican public hospitals.

OBJECTIVE: To characterize the rpoB gene mutations of the rifampicin-resistant M. tuberculosis strains isolated in pulmonary tuberculosis patients from Mexico. MATERIAL AND METHODS: Thirty-seven clinical M. tuberculosis isolates cultured on Löwenstein-Jensen media and obtained from consecutive tuberculosis patients in 5 public hospitals were analyzed by PCR and the INNO-LiPA Rif TB for amplification and detection of mutations associated with rifampicin resistance, respectively. RESULTS: Twenty-three out of 37 isolates (62.2%) were found to be wild type (rifampicin susceptible), while 14 isolates (37.8%) contained mutations associated with rifampicin resistance. Seven out of the 37 isolates (18.9%) had a delta S1 mutation, in the nucleotide position number 511; one (2.7%) had a R4b mutation, in nucleotide H526D; five (13.5%) contained a R5 mutation, in nucleotide S531L; and one (2.7%) showed a double mutation delta S1/R4b. CONCLUSION: According to the marker used (rifampicin resistance), at least five different strains of M. tuberculosis circulate among pulmonary tuberculosis patients in Mexico. rpoB gene mutations associated with rifampicin resistance are common in Mexico. A single mutation in nucleotide 511 was the most frequently observed, followed by single mutations in nucleotides S531L and H526D.

Expression analysis of a cytosolic glutamine synthetase gene in cotyledons of Scots pine seedlings: developmental, light regulation and spatial distribution of specific transcripts.

The expression of a cytosolic glutamine synthetase (GS1; EC 6.3.1.2) gene was examined in cotyledons of Scots pine seedlings. Light strongly stimulated GS1 mRNA accumulation during development. Similarly, steady-state levels of GS1 transcripts increased in dark-grown seedlings transferred to light and decreased in dark-adapted seedlings. Light/dark adaptation affected rbcS and lhcb2 mRNA levels and chlorophyll contents in the same manner. Light-grown seedlings in the presence of the herbicide norflurazon showed a drastic decrease in mRNA for GS and photosynthetic proteins, whereas the effect of the herbicide on mitochondrial beta-ATP synthase mRNA was limited. These results indicate that factors associated with developing chloroplasts could be required for maximal GS1 gene expression during seedling development. The level of GS polypeptide, determined by immunoblot, was up-regulated during seedling development in the light or dark. However, the levels of the polypeptide detected were unaltered by the light/dark adaptation treatments. The analysis of GS1 mRNA association with polysomes indicated that the discrepancies between GS protein and mRNA levels are not a result of a differential translational rate of the transcript in darkness relative to light. Two GS isoproteins with different isoelectric point were resolved by two-dimensional PAGE in light- and dark-germinated plants. The relative abundance of one of them was markedly affected by light and correlated with the observed changes in GS mRNA, suggesting that the other form is not a product derived from the detected transcript. In situ hybridization of cotyledon sections showed the presence of GS1 mRNAs in mesophyll and phloem cells confirming gene expression in photosynthetic tissues. High levels of transcript were detected also in meristematic cells of apical primordia. These data suggest a dual role for the GS1 gene associated with chloroplast development/activity and glutamine biosynthesis for nitrogen mobilization during early growth of Scots pine.

Population structure and genetic divergence in Anopheles nuneztovari (Diptera: Culicidae) from Brazil and Colombia.

Anopheles nuneztovari is considered an important vector of human malaria in several localities in Venezuela and Colombia. Its status as a vector of human malaria is still unresolved in areas of the Brazilian Amazon, in spite of have been found infected with Plasmodium sp.. For a better understanding of the genetic differentiation of populations of A. nuneztovari, electrophoretic analysis using 11 enzymes was performed on four populations from Brazil and two from Colombia. The results showed a strong differentiation for two loci: alpha-glycerophosphate dehydrogenase (alpha-Gpd) and malate dehydrogenase (Mdh) from 16 loci analyzed. Diagnostic loci were not detected. The populations of A. nuneztovari from the Brazilian Amazon showed little genetic structure and low geographic differentiation, based on the F(IS) (0.029), F(ST) (0.070), and genetic distance (0.001-0.032) values. The results of the isozyme analysis do not coincide with the indication of two lineages in the Amazon Basin by analysis of mitochondrial DNA, suggesting that this evolutionary event is recent. The mean F(ST) value (0.324) suggests that there is considerable genetic divergence among populations from the Brazilian Amazon and Colombia. The genetic distance among populations from the Brazilian Amazon and Colombia is ranges from 0.047 to 0.148, with the highest values between the Brazilian Amazon and Sitronela (SIT) (0.125-0.148). These results are consistent with those observed among members of anopheline species complexes. It is suggested that geographic isolation has reduced the gene flow, resulting in the genetic divergence of the SIT population. Dendrogram analysis showed three large groups: one Amazonian and two Colombia, indicating some genetic structuring. The present study is important because it attempted to clarify the taxonomic status of A. nuneztovari and provide a better understanding of the role of this mosquito in transmission of human malaria in northern South America.

A simple method for cultivating freshwater copepods used in biological control of Aedes aegypti.

A simple method for indoor and outdoor cultivation of Mesocyclops aspericornis, Macrocyclops albidus and Mesocyclops n. sp. copepods is presented. This method utilizes Chilomonas sp., Paramecium caudatum and fresh lettuce as food sources for copepod cultures. Steps for initiating and maintaining copepod cultures are provided.

Mosquito vector control and biology in Latin America--a second symposium.

The second Spanish language symposium presented by the American Mosquito Control Association (AMCA) was held as part of the 58th Annual Meeting in Corpus Christi, TX in March 1992. The principal objective, as it was for the 1991 symposium, was to increase and stimulate greater participation in the AMCA by vector control specialists and public health workers from Latin America. This publication includes summaries of 25 individual presentations that were given in Spanish. The symposium included the following topics: biology and chemical control of Aedes aegypti and anopheline vectors of malaria; field and laboratory studies of biological control agents for Aedes aegypti; community participation in the prevention of dengue; and other various aspects of the biology of other medically important arthropods (e.g., Simulium ochraceum, Lutzomyia and Culicoides).

Genetic structure of natural populations of Anopheles albimanus in Colombia.

Electrophoretic and cytogenetic studies were undertaken on the population structure of Anopheles albimanus from 11 localities in Colombia, 3 from northern (Atlantic coast) and 8 from southern (Pacific coast) regions. Of the 25 allozyme loci examined, significant allele frequency differences were observed at 4 loci: hydroxy acid dehydrogenase (Had-1) and 3 esterases (Est-2, Est-4 and Est-6). The northern populations had higher variability, with 55% polymorphic loci, a mean heterozygosity of 20.4% and a mean of 3.0 alleles per locus. These values for southern populations were 24%, 9.1% and 1.5%, respectively. There were neither diagnostic loci nor clinal effect on frequencies of allozymes. Except for a small inversion on the X chromosome in low frequency in certain populations, all populations were homosequential in chromosomal banding patterns. Hybrids from matings between natural populations and the Gainesville laboratory strain were fully fertile. Estimates of genetic similarities (0.95-0.97 among southern and 0.99-1.00 among northern populations) suggest a lack of significant genetic differentiation among distant populations in this species. Based on the chromosomal, hybridization and electrophoretic data, we concluded that mosquitoes from the 11 collections were conspecific populations of An. albimanus.

Indoor resting heights of some anophelines in Colombia.

The nocturnal, indoor resting behavior of female anophelines in Colombia was studied. Anopheles darlingi and An. marajoara had a tendency to rest close to the ground, but An. oswaldoi and An. rangeli rested higher up. This behavior was independent of bloodfed status (except for An. oswaldoi) and whether the surface had been sprayed with DDT. With this information it should be possible to modify insecticide applications to coincide with the resting preferences of these species.

First record of DDT resistance in Anopheles darlingi.

DDT susceptibility of Anopheles darlingi was compared from the southeastern and northwestern regions of Colombia. Mosquitoes from southeastern Colombia (Puerto Lleras) were fully susceptible to DDT but in the northwest (Quibdo) LT50s were 14.3 times higher. This resistance appeared to be restricted to the Quibdo area. This is the first time that the DDT resistance has been detected in this important malaria vector.

Natural control of larval Anopheles albimanus (Diptera: Culicidae) by the predator Mesocyclops (Copepoda: Cyclopoida).

Forty-two ground water sites on the Pacific and Atlantic coasts of Colombia were sampled quantitatively with a plankton net to assess the abundance of mosquito larvae and associated fauna. Many of the sites contained substantial numbers of Anopheles albimanus Wiedemann larvae, but the larvae were virtually absent wherever there were large populations of the copepods Mesocyclops venezolanus, M. longisetus, or M. aspericornis. Mesocyclops were observed to prey readily upon first-instar An. albimanus larvae in the laboratory, but smaller genera of cyclopoid copepods did not kill An. albimanus larvae. We conclude that Mesocyclops may prove useful for biological control of Anopheles.

Irritability to DDT of natural populations of the primary malaria vectors in Colombia.

Natural populations of Anopheles albimanus, An. darlingi and An. nuneztovari were tested for their irritability to DDT in different regions of Colombia. Individual anophelines were exposed to filter papers impregnated with 2% DDT and irritability was determined based on the number of flights a mosquito made in 15 min. All three species showed variability in their irritability to DDT. Anopheles albimanus showed irritability only in Bolivar, but not in three other regions. Anopheles darlingi showed no irritability in neither the Atlantico nor Llanos regions, while An. nuneztovari showed irritability in the Oriente but not in Uraba. The irritability to DDT shown in some populations of An. albimanus and An. nuneztovari may reduce the effectiveness of residual applications of this insecticide by causing the mosquitoes to seek untreated surfaces and/or leave the house to rest outdoors and thus avoid a lethal dose.

Genetic variability and mapping of peptidase-4 in Anopheles albimanus.

Field-collected and laboratory populations of Anopheles albimanus were analyzed for the presence of variability for the enzyme, peptidase. Four zones of peptidase electromorphs were observed. The Peptidase-4 (Pep-4) locus was analyzed genetically and assigned to a location adjacent to the centromere on the right arm of chromosome 2 by employing crosses involving morphological mutants, allozyme markers, a holandric translocation, and pericentric inversions. The gene sequence (and map distances) on chromosome 2 beginning from the left arm is: brown larva--40--ebony (eb)--centromere--pep-4--?--bent(be)--?--Glutamate oxaloacetate transaminase (Got)--11--Glucose oxidase-2(Go-2)--17--green larva (gl)--9--amber (am)--2--propoxur resistance (prr)--2--red eye (re)--21--6-Phosphogluconate dehydrogenase (6-Pgd)--1--yellow. The crossover frequencies between eb and Pep-4 and Pep-4 and Got were 34 and 16, respectively.

Trypanosoma magdalenae sp. n (Protozoa: Kinetoplastida) from a freshwater teleost, Petenia kraussii, in Colombia.

A new, monomorphic trypanosome, Trypanosoma magdalenae sp. n. was found in five of 38 fish, Petenia kraussii, from the Río Magdalena in Colombia, South America. It is the first trypanosome species designated from freshwater teleosts in Colombia. The trypomastigotes measured in 42.4 micron +/- 2.05 SD (range, 39.8 - 45.6) by 2.1 micron +/- 0.20 (2.0 - 2.5). Their nuclear index was 1.6 micron +/- 0.22 (1.25 - 1.84) and their kinetoplastic index equaled 1.1 micron +/- 0.02 (1.08 - 1.15). Infection intensity was usually limited to two or three flagellates per 40 microliter of packed blood cells.