Lactobacillus plantarum Lp62 exerts probiotic effects against Gardnerella vaginalis ATCC 49154 in bacterial vaginosis.

The severe side-effects elicited by conventional antibiotic therapy and the recurrence of Bacterial vaginosis-associated bacteria and bacterial resistance have led to the development of novel alternative therapies, among which genital probiotics are widely used. In this study, we aimed to evaluate the antimicrobial activities of Lactobacillus plantarum Lp62 and its supernatant against Gardnerella vaginalis, using both in vitro and in vivo approaches. In vitro assays were used to evaluate the viability of the strain and the antimicrobial activities of the supernatant in different pH ranges. An in vivo assay was performed on female BALB/c mice, wherein the animals were divided into eight groups: four control groups and four treated groups (for curative and preventive therapies). After infecting and treating the mice, the animals were killed to quantify the bacterial load using qPCR, evaluate leucocyte cellular response, determine vaginal cytokine levels and perform cytokine tissue gene expression. Our analyses revealed significant activity of the strain and its supernatant against G. vaginalis. Preliminary in vitro tests showed that the strain grew with equal efficiency in different pH ranges. Meanwhile, the presence of halo and inhibition of pathogen growth established the significant activity of the supernatant against G. vaginalis. We observed that both micro-organisms are resident bacteria of mouse microbiota and that the lactobacilli population growth was affected by G. vaginalis and vice versa. We also observed that the treated groups, with their low bacterial load, absence of leucocyte recruitment, reduced cytokine levels in the vaginal lavage and normalized cytokine gene expression, successfully controlled the infection.

Effects of preheating and precooling on the hardness and shrinkage of a composite resin cured with QTH and LED.

The aim of this study was to evaluate in vitro the hardness and shrinkage of a pre-cooled or preheated hybrid composite resin cured by a quartz-tungsten-halogen light (QTH) and light-emitting diode (LED) curing units. The temperature on the tip of the devices was also investigated. Specimens of Charisma resin composite were produced with a metal mold kept under 37°C. The syringes were submitted to 4°C, 23°C, and 60°C (n=20) before light-curing, which was carried out with the Optilux 501 VCL and Elipar FreeLight 2 units for 20 seconds. The specimens were kept under 37°C in a high humidity condition and darkness for 48 hours. The Knoop hardness test was carried out with a 50 gram-force (gf) load for 10 seconds, and the measurement of the shrinkage gap was carried out using an optical microscope. The data were subjected to analysis of variance and the Games-Howell test (α=0.05). The mean hardness of the groups were similar, irrespective of the temperatures (p>0.05). For 4°C and 60°C, the top surface light-cured by LED presented significantly reduced shrinkage when compared with the bottom and to both surfaces cured by QTH (p<0.05). It was concluded that the hardness was not affected by pre-cooling or preheating. However, polymerization shrinkage was slightly affected by different pre-polymerization temperatures. The QTH-curing generated greater shrinkage than LED-curing only when the composite was preheated. Different temperatures did not affect the composite hardness and shrinkage when cured by a LED curing unit.

Surface AFM microscopy of unworn and worn samples of silicone hydrogel contact lenses.

PURPOSE: To evaluate the qualitative and quantitative topographic changes in the surface of worn contact lenses (CLs) of different materials using atomic force microscopy (AFM). METHODS: The topography of five different CL materials was evaluated with AFM over a surface of 25 microm(2) according to previously published experimental setup. Average roughness (R(a)) and root mean square (Rms) values were obtained for unworn and worn samples. RESULTS: The R(a) value increased for balafilcon A (11.62-13.68 nm for unworn and worn samples, respectively), lotrafilcon A (3.67-15.01 nm for unworn and worn samples, respectively), lotrafilcon B (4.08-8.42 nm for unworn and worn samples, respectively), galyfilcon A (2.81-14.6 nm for unworn and worn samples, respectively), and comfilcon A (2.87-4.63 nm for unworn and worn samples, respectively). Differences were statistically significant for all lenses except Rms and R(a) for comfilcon A, and R(a) parameter for balafilcon A (p > 0.05). The least relative increase was observed for some balafilcon A samples and for some of these samples the roughness decreased after the lenses had been worn. CONCLUSION: The changes in surface roughness between unworn and worn lenses are different for different silicone-hydrogel materials. Overall all CLs increased the degree of surface roughness after being worn, even for very short periods of time. However, for samples of balafilcon A, roughness increases at a lower extent or even can decrease as compared to unworn samples of the same material due to filling of the macropores.

Dynamic in vitro dehydration patterns of unworn and worn silicone hydrogel contact lenses.

PURPOSE: To evaluate the effect of wear on dynamic in vitro dehydration of silicone hydrogel (Si-Hi) contact lens (CL) using a previously described gravimetric procedure. METHODS: Five different silicone hydrogel (Si-Hi) contact lenses (CL) were evaluated after being worn by patients under daily wear conditions for 15 days to 1 month. Lenses were conditioned and disinfected with a multipurpose solution after each day of wear. Lenses were left to dehydrate in an analytical balance under controlled conditions of temperature and relative humidity and the results compared with data from new lens samples of the same power and material. Several quantitative parameters were obtained and compared between worn and unworn samples. RESULTS: The quantitative parameters derived from the dehydration curves showed statistically significant differences between worn and unworn lenses regarding the initial dehydration rate and ability to maintain their original hydration. Worn lenses showed shorter phase I duration (decreased by 30 to 60% compared to unworn samples), a significantly faster initial dehydration rate (increased by about 1%/minute), and lower water retention index (decreased by 10 to 20%) as derived from the initial cumulative dehydration. All the differences were statistically significant for all lenses (p < 0.05). CONCLUSION: The initial dehydration rates were significantly increased in all CL after wear. This could be of clinical interest because it represents the average initial dehydration rates during the first moments when the lens is left to dehydrate after a blink. This study suggests that even after the lenses had been equilibrated in saline solution for several days, the materials lose their ability to retain water.

Technical note: a comparison of central and peripheral intraocular pressure using rebound tonometry.

PURPOSE: To compare central and peripheral intraocular pressure (IOP) readings obtained with rebound tonometry. METHODS: Intraocular pressure was measured on the right eye of 153 patients (65 males, 88 females), aged from 21 to 85 years (mean +/- S.D., 55.5 +/- 15.2 years) with the ICare rebound tonometer at centre, and 2 mm from the limbus (in the nasal and temporal regions along the 0-180 degrees corneal meridian). RESULTS: Intraocular pressure values obtained with the ICare were 14.9 +/- 2.8; 14.1 +/- 2.5 and 14.5 +/- 2.7 mmHg at centre, nasal and temporal corneal locations, respectively. On average, nasal and temporal IOP readings were 0.75 and 0.37 mmHg lower than the central reading (p < 0.05 and p > 0.05, respectively). A highly significant correlation was found between central and peripheral measurements in nasal (r(2) = 0.905; p < 0.001) and temporal (r(2) = 0.879; p < 0.001) regions along the horizontal meridian. Almost 80% of patients presented nasal IOP values within +/-1 mmHg of the central value. CONCLUSIONS: Intraocular pressure values measured with the ICare rebound tonometer on the nasal corneal region is slightly lower on average and highly correlated with IOP values recorded at corneal centre. Both nasal and temporal readings are in good agreement with central IOP, and could be used to obtain a reliable estimate of rebound IOP in corneas where central readings cannot be taken.

Refractive, biometric and topographic changes among Portuguese university science students: a 3-year longitudinal study.

PURPOSE: The aim of this study was to investigate the changes in refractive, biometric and topographic ocular parameters among university students in Portugal during a 3-year period. METHODS: A 3-year longitudinal study comprised 118 Portuguese university students from the School of Science (34 males and 84 females; mean age 20.6 +/- 2.3 years). Ocular refraction, corneal curvature (CR) and eccentricity, and A-scan biometry were conducted under cycloplegia. The sphero-cylindrical refractive results were converted into vector representations (M, J(0) and J(45)) for statistical analysis. Myopia was defined as M < or = -0.50 D, emmetropia as M > -0.50 D and < +0.50 D and hyperopia as M > or = +0.50 D. RESULTS: At the beginning of the study sphero-cylindrical refraction (M) ranged from -6.75 to +3.00 D, with a mean value of 0.23 +/- 1.46 D [mean +/- standard deviation (S.D.)]. Eighty-three students presented astigmatism with a mean value (+/-S.D.) of -0.52 +/- 0.41 D, and a maximum of -2.25 D. After 3 years the mean refractive change for the M component was -0.29 +/- 0.38 D (p < 0.001) and non-significant changes of 0.02 +/- 0.16 D (p = 0.281) for the J(0) component and 0.01 +/- 0.09 D (p = 0.784) for the J(45) component. Prevalence of myopia increased by 5.1%, while the prevalence of hyperopia decreased by 9.4%. Myopia progression > or =0.5 D was observed in 22% of the population. Axial length, vitreous chamber depth and lens thickness increased significantly while anterior chamber depth and central CR did not change significantly. CONCLUSIONS: This study shows a change in refraction towards myopia accompanied by a vitreous chamber elongation in a Portuguese population comprising science students during the first three years of their university course. Younger students were more likely to show clinically significant myopia progression.

Technical note: Accuracy and repeatability of a new portable ultrasound pachymeter.

PURPOSE: To assess the accuracy and repeatability of central corneal thickness (CCT) measurements taken with a new portable ultrasound (US) pachymeter. METHODS: Central thickness measurements were taken with a portable pachymeter (SP-100 Handy; Tomey, Nagoya, Japan) and a conventional US pachymeter (Nidek UP-1000; Nidek Technologies, Gamagori, Japan) from 57 right corneas of 57 young adults (19 males, 38 females) aged 18-44 years (mean +/- S.D., 22.95 +/- 3.92). Three repeated measures were obtained and then compared to obtain the repeatability of each instrument and the agreement between the pachymeters. The three readings taken with the portable pachymeter were compared against each other in order to evaluate intra-session repeatability and bias of each individual measurement with respect to the mean of three. RESULTS: Mean values of CCT were 537 +/- 35 microm for conventional and 534 +/- 35 microm for the new portable pachymeter. A high agreement was found between the two instruments (mean difference = 2.58 microm; 95% CI 1.41-3.75 microm) with only two eyes presenting differences larger than +/-8.6 microm which represents 95% CI in the Bland-Altman plots which represents 1.6% of the mean CCT. The first reading taken showed the highest agreement with the mean value for the portable pachymeter. CONCLUSIONS: The instrument tested in this study is able to take reliable measurements of corneal thickness even if a single reading is considered. Intra-session repeatability was very high, as was also the agreement between the average of three readings taken with the two US pachymeters.

Age differences in central and peripheral intraocular pressure using a rebound tonometer.

AIM: To evaluate the influence of age on the measurements and relationships among central and peripheral intraocular pressure (IOP) readings taken with a rebound tonometer. METHODS: The IOPs were measured using the ICare rebound tonometer on the right eyes of 217 patients (88 men and 129 women) aged 18-85 years (mean 45.9 (SD 19.8) years), at the centre and at 2 mm from the nasal and temporal limbus along the horizontal meridian. Three age groups were established: young (< or =30 years old; n = 75), middle aged (31-60 years old; n = 77) and old patients (>60 years old; n = 65). RESULTS: A high correlation was found between the central and peripheral IOP readings, with the central readings being higher than the peripheral ones. Higher IOP values for the central location were found in the younger patients. Older patients had significantly lower temporal IOP readings than those for the remaining two groups (p<0.001), whereas no significant differences were found among groups when IOP was measured at the central and nasal locations. A significant decrease was observed in the nasal and temporal IOP readings as the age increased (p = 0.011 and 0.006, respectively). CONCLUSION: Older patients had lower IOP values than the middle-aged and younger patients in the temporal peripheral location. A negative correlation was found between age and IOP by rebound tonometry in the corneal periphery but not in its centre.

Non-contact tonometry synchronized with cardiac rhythm and its relationship with blood pressure.

PURPOSE: The main objectives of this study were to determine the differences between non-synchronized intraocular pressure (IOP_N) and intraocular pressure readings synchronized with cardiac pulse and try to determine if these parameters are related to blood pressure values. METHODS: One hundred and sixty-five right eyes from 165 volunteers (107 females, 58 males) aged from 19 to 73 years (mean +/- S.D., 29.93 +/- 11.17) were examined with the Nidek NT-4000, a new non-contact tonometer that allows the measurement of IOP synchronized with the cardiac rhythm. IOP measurements in the four different modes of synchronization were taken in a randomized order. Three measures of each parameter were taken and then averaged. The blood pressure was determined three times with a portable manometer and mean values of systolic and diastolic pressure and the pulse rate were computed. Mean arterial pressure (MAP) was determined as being 1/3 of systolic plus 2/3 of diastolic blood pressure. RESULTS: The mean +/- S.D. values for the standard intraocular pressure (IOP_N: 14.76 +/- 2.86), intraocular pressure in the systolic instant or peak (IOP_P: 14.99 +/- 2.85), intraocular pressure in the middle instant between heartbeats or middle (IOP_M: 14.68 +/- 2.76), and intraocular pressure in the diastolic instant or bottom (IOP_B: 13.86 +/- 2.61) were obtained. The IOP_P was higher than the remaining values. A significant difference in mean IOP existed between IOP_B and the remaining modes of measuring (p < 0.05). Differences were statistically significant for all pair comparisons involving IOP_B. Arterial blood pressure values were systolic 125.5 +/- 14.22, diastolic 77.7 +/- 8.38 and MAP 93.64 +/- 9.44 mmHg. The pulse rate was 77.3 +/- 12.6 beats per minute. Except for the MAP (p = 0.025) there was no significant correlation between different IOP values and systolic or diastolic blood pressure, or pulse rate. CONCLUSIONS: NT-4000 is able to differentiate IOP values when synchronized with the cardiac rhythm and those differences are expected to be within a range of +/-2.5 to +/- 3.0 mmHg. IOP_B seems to be the parameter whose value differs from the non-synchronized and the remaining synchronized parameters in a significant way. Other than a weak association with MAP, no significant correlation between IOP and BP was found. The measurements of IOP readings for the three modes are consistent with timings during the cardiac cycle and IOP pulse cycle.

The influence of cycloplegia in objective refraction.

The purpose of this study was to compare refractions measured with an autorefractor and by retinoscopy with and without cycloplegia. The objective refractions were performed in 199 right eyes from 199 healthy young adults with a mean age of 21.6 +/- 2.66 years. The measurements were performed first without cycloplegia and repeated 30 min later with cycloplegia. Data were analysed using Fourier decomposition of the power profile. More negative values of component M and J(0) were given by non-cycloplegic autorefraction compared with cycloplegic autorefraction (p < 0.0001). However more positive values for the J(45) vector were given by non-cycloplegic autorefraction, although this difference was not statistically significant (p = 0.233). By retinoscopy, more negative values of component M were obtained with non-cycloplegic retinoscopy (p < 0.0001); for the cylindrical vectors J(0) and J(45) the retinoscopy without cycloplegia yields more negative values (p = 0.234; p = 0.112, respectively). Accepting that differences between cycloplegic and non-cycloplegic retinoscopy are only due to the accommodative response, the present results confirm that when performed by an experienced clinician, retinoscopy is a more reliable method to obtain the objective starting point for refraction under non-cycloplegic conditions.

A comparison of the ARK-700A autokeratometer and Medmont E300 corneal topographer when measuring peripheral corneal curvature.

PURPOSE: The purpose of this study was to evaluate if corneal topogometry measurements as taken with the ARK-700A autokeratometer are comparable with those measured by the Medmont E300 videokeratoscope at corresponding locations. METHODS: Central and peripheral radius and eccentricity were measured in 122 right eyes of young normal subjects using autokeratometry and videokeratoscopy, obtained in a random order. RESULTS: Curvature measurements obtained with the ARK700A and Medmont E300 correlated well for the central cornea. Larger differences were observed between peripheral autokeratometry readings and the empirically determined corresponding locations with the videokeratoscope. Correlations between the instruments are inconsistent, resulting in unacceptable confidence intervals. Corneal eccentricity was significantly different between the instruments for the vertical (t = 2.4; p = 0.018) and for the horizontal meridians. In the first case, the difference between the averaged values was not clinically significant, but in the horizontal meridian the AK significantly overestimated eccentricity values (t = -11.5; p < 0.001) with differences which were clinically significant. CONCLUSIONS: Central corneal curvature data obtained by ARK700A and Medmont E300 can be interchanged but the same is not true of peripheral determinations of corneal shape. ARK700A probably measures peripheral corneal shape within an elliptical region between 5 and 7 mm in diameter, with the major axis in the vertical meridian.

Clinical performance of non-contact tonometry by Reichert AT550 in glaucomatous patients.

Measuring intraocular pressure (IOP) by non-contact tonometry (NCT) has been demonstrated to be a valid and reliable technique to be used in primary eye care; it is easier to use, it does not transmit infectious diseases, and it is not necessary to use anaesthetic or staining eye drops. Recently, a new NCT device has showed an excellent level of agreement with Goldmann tonometry, but there are no records of its performance in glaucomatous eyes. To rectify this, IOP was measured in twenty-two patients (44 eyes) receiving medical treatment to control elevated IOP, with AT550 and Goldmann tonometry. Mean values of IOP were 18.98 +/- 2.77 and 19.08 +/- 3.02 mmHg using Goldmann and AT550, respectively. Plots of differences against means displayed good agreement (mean difference +/- limits of agreement, -0.09 +/- 3.30); this value was not significantly different from zero (t-test for dependent samples, p = 0.709). In conclusion, IOP values as measured with the AT550 NCT are clinically comparable with those obtained with Goldmann tonometry in glaucomatous patients. This validates this NCT not only for screening of IOP but to follow-up glaucomatous patients with a rapid, non-invasive method.

Clinical performance of the Reichert AT550: a new non-contact tonometer.

The aim of the present study was to assess the level of accuracy for measurements of intra-ocular pressure (IOP) obtained with a new non-contact tonometer (NCT) the Reichert AT550. Measurements were compared against those obtained with the Reichert Xpert Plus, Goldmann applanation tonometer and Perkins tonometer. Thirty-five university students were assessed with the four tonometers in a randomised order, with non-contact tonometry performed first. Each of the four measurement devices had its own trained clinical observer. Plots of differences of IOP as a function of the mean for each pair of instruments were obtained. No statistically significant differences were found when comparing the AT550 NCT with contact applanation tonometry (AT) (p> 0.05), displaying the closest level of agreement (as represented by the lowest mean difference and the narrowest confidence interval) with the Goldmann tonometer (limits of agreement, 0.12+/-2.17). In conclusion, readings of IOP with the AT550 NCT are clinically comparable with those obtained with Goldmann tonometry in a population with IOP within the normal range.

Measurement of corneal thickness by videopachymetry: preliminary results.

Corneal thickness measurements find applications in areas such as diagnosis and management of corneal disorders and corneal surgery. We present a technique for the measurement of corneal thickness using a CCD camera mounted on a slit-lamp biomicroscope and common image processing software. Thickness measurements were performed on three RGP contact lenses of known thickness with an average error of 5 microm, SD of 8 microm. Measurements of a living cornea yielded a thickness of 505 microm and a SD of 8 microm for 10 consecutive measurements; this is an acceptable value.

Prevalence of anti-Leishmania donovani antibody among Brazilian blood donors and multiply transfused hemodialysis patients.

The prevalence of anti-Leishmania donovani antibodies was investigated in 1,500 Brazilian blood donors and multiply transfused hemodialysis patients. Sera were tested using the fucose-mannose ligand (FML) ELISA, which was shown to have 100% sensitivity and 96% specificity for kala-azar. Among 1,194 volunteer blood donors, seroreactivity was 9%, increasing to 25% in a periurban kala-azar focus. However, higher positivity (37%) was found in multiply transfused hemodialysis patients from Natal, where kala-azar is constantly present in low numbers (endemic), with sporadic outbreaks in localized regions (endemic and epidemic). Risk factors included blood transfusion, which was significantly associated with the presence of anti-Leishmania antibodies (chi2 = 8.567, P < 0.005), but did not include potential exposure to sandfly bites (chi2 = 0.033, P > 0.1). The prevalence significantly decreased to 7% in hemodialysis patients from Rio de Janeiro, where kala-azar is only occasionally seen, and was 0% in patients undergoing continuous ambulatorial peritoneal dialysis. The prospective analysis of 27 FML-seroreactive donors from Natal revealed amastigotes of Leishmania in the bone marrow of one subject while four had clinical complaints, including splenomegaly and hepatosplenomegaly. Our results point to the need for control of blood transfusion as a possible route for transmission of kala-azar in endemic areas.

Gravidade da doença renal cística adquirida determina melhora da anemia da insuficiência renal crônica / Severety of acquired cystic kidney disease determines improvimet in chronic kidney failure anemia

Objetivo. Identificar correlaçao entre duraçao do tratamento dialítico, gravidade da doença renal cística adquirida (DRCA) medida pelo tamanho dos cistos e seu efeito sobre a correçao espontânea da anemia. Material e Métodos. Foram selecionados dez pacientes, seis do sexo masculino e quatro do feminino, com mais de cinco anos em tratamento dialítico. Nenhum paciente selecionado tinha doença renal policística bilateral como doença primária. A avaliaçao renal foi feita com ulra-sonografia. DRCA foi caracterizada pela presença de quatro ou mais cistos em cada rim. Os maiores cistos foram medidos para efeito de correlaçao. O diagnóstico da anemia foi estabelecido pelo valor do hematócrito e da hemoglobina séricos. Foram analisados, também, uréia, creatinina, albumina, ferro, capacidade total de combinaçao do ferro e o percentual de saturaçao da transferrina séricos. Resultados. Os pacientes estavam adequadamente dialisados (uréia e creatinina séricas = 98,7 + 35mg/dL e 9,7 + 2,7 mg/dL, respectivamente), com bom estado nutricional (albumina sérica = 4,5 + 0,5g/dL), e tinham razoável reserva de ferro (ferro sérico = 80 + 34mg/dL). A prevalência de DRCA foi de 80 por cento. Nao detectamos nenhum sinal ultra-sonográfico de malignidade nesses cistos. Houve correlaçao significante entre tempo de diálise e hematócrito (R = 0,70: p<0,05). O tamanho dos cistos teve correlaçao direta e significante com os valores do hematócrito (R = 0,74; p < 0,05). Conclusoes. Os resultados mostram que a melhora espontânea da anemia observada nos pacientes em diálise crônica se correlaciona de forma significante com a gravidade da doença renal cística adquirida. Isso sugere uma atividade funcional dos cistos renais na produçao de eritropoetina.

[Severity of acquired cystic kidney disease determines improvement in chronic kidney failure anemia]. / Gravidade da doença renal cística adquirida determina melhora da anemia da insuficiência renal crônica.

OBJECTIVE: To identify a correlation between dialysis treatment duration and severity of acquired cystic disease of the end-stage kidney measured by cyst sizes; and assess its effect on spontaneous improvement of anemia. MATERIALS AND METHODS: Ten patients, 6 males and 4 females were selected, who have been on hemodialytic treatment for more than 5 years. There was no patient selected with autosomal dominant polycystic kidney disease. The renal evaluation has been made through ultrasonographic studies. Acquired cystic disease of the end-stage kidney was characterized by finding four or more cysts in both kidneys. The largest cysts were measured for correlation effect. The diagnosis of anemia was established by hematocrit and hemoglobin serum values. It has also been analyzed serum urea, creatinine, albumin, iron, total iron-binding capacity and the per cent saturation of serum transferrin were tested. RESULTS: The patients were properly dialysed (serum urea and creatinine = 98.7 +/- 35 mg/dL e 9.7 +/- 2.7 mg/dL, respectively). There were in good nutritional shape (serum albumin = 4.5 +/- 0.5 g/dL) and had normal serum iron level (serum iron = 80 +/- 34 mg/dL). The prevalence of acquired renal cystic disease was 80%. No finding of malignancy has been detected in these cysts. There was a significant correlation between time on dialysis treatment and hematocrit values (R = 0.70; p < 0.05). Cyst sizes had a direct and significant correlation with hematocrit levels (R = 0.74; p < 0.05). CONCLUSION: These results show that spontaneous improvement on anemia seen in patients on chronic dialysis has a significant correlation with the severity of acquired cystic disease of the end-stage kidney. Our data suggest a functional role of acquired kidney cysts on endogenous erythropoietin production.

Targeting of chimeric G alpha i proteins to specific membrane domains.

Heterotrimeric guanine nucleotide-regulatory (G) proteins are associated with a variety of intracellular membranes and specific plasma membrane domains. In polarized epithelial LLC-PK1 cells we have shown previously that endogenous G alpha i-2 is localized on the basolateral plasma membrane, whereas G alpha i-3 is localized on Golgi membranes. The targeting of these highly homologous G alpha i proteins to distinct membrane domains was studied by the transfection and expression of chimeric G alpha i proteins in LLC-PK1 cells. Chimeric cDNAs were constructed from the cDNAs for G alpha i-3 and G alpha i-2 and introduced into a pMXX eukaryotic expression vector containing a mouse metallothionein-I promoter. Stably transfected cell lines were produced that expressed either G alpha i-2/3 or G alpha i-3/2 chimeric proteins. Chimeric and endogenous G alpha i proteins were detected in cells using specific carboxy-terminal peptide antibodies. Immunofluorescence staining was used to localize endogenous and chimeric G alpha i proteins in LLC-PK1 cells. The staining of chimeric proteins was detected as an increased intensity of staining on membranes containing endogenous G alpha i proteins. Using confocal microscopy and image analysis we localized G alpha i-2 to a specific sub-domain of the lateral membrane of polarized cells, the chimeric G alpha i-3/2 protein was then shown to colocalize with endogenous G alpha i-2 in the same lateral plasma membrane domain. The chimeric G alpha i-2/3 protein colocalized with endogenous G alpha i-3 on Golgi membranes in LLC-PK1 cells. These results show that chimeric G alpha i proteins were targeted to the same membrane domains as endogenous G alpha i proteins and the specificity of their membrane targeting was conferred by the carboxy-terminal end of the proteins. These data provide the first evidence for specific targeting information contained in the carboxy termini of G alpha i proteins, which appears to be independent of amino-terminal membrane attachment sites in these proteins.

Binding of the cytosolic p200 protein to Golgi membranes is regulated by heterotrimeric G proteins.

The formation of vesicles for protein trafficking requires the dynamic binding of cytosolic coat proteins onto Golgi membranes and this binding is regulated by a variety of GTPases, including heterotrimeric G proteins. We have previously shown the presence of the pertussis toxin-sensitive G alpha i-3 protein on Golgi membranes and demonstrated a functional role for G alpha i-3 in the trafficking of secretory proteins through the Golgi complex. We have also described a brefeldin A-sensitive phosphoprotein, p200, which is found in the cytoplasm and on Golgi membranes. The present study investigates the role of heterotrimeric G proteins in the regulation of p200 binding to Golgi membranes. An in vitro binding assay was used to measure the binding of cytosolic p200 to LLC-PK1 cell microsomal membranes and to purified rat liver Golgi membranes in the presence of specific activators of G proteins. The binding of p200 to Golgi membranes was compared to that of the coatomer protein beta-COP, for which G protein-dependent membrane binding has previously been established. Membrane binding of both p200 and beta-COP was induced maximally by activation of all G proteins in the presence of GTP gamma S. More selective activation of the heterotrimeric G proteins, with AlFn or mastoparan, also induced membrane binding of p200 and beta-COP. Pertussis toxin pretreatment of Golgi membranes, to selectively inactivate G alpha i-3, reduced the AlFn and mastoparan-induced binding of p200 to Golgi membranes, whereas no significant effect of pertussis toxin on beta-COP binding was found in this assay. The effect of pertussis toxin thus implicates G alpha i-3, as one component of a regulatory pathway, in the binding of cytosolic p200 to Golgi membranes. The effects of AlFn and pertussis toxin on p200 membrane binding were also shown in intact cells by immunofluorescence staining. AlFn treatment of cells induced translocation of p200 from the cytoplasm onto the Golgi complex, resulting in a conformational change in some Golgi membranes. The translocation of p200 was blocked by pretreatment of intact NRK cells with pertussis toxin. The data presented here support the conclusion that the binding of the p200 protein to Golgi membranes involves regulation by the pertussis toxin-sensitive heterotrimeric G proteins, specifically the G alpha i-3 protein.

Entry of cholera toxin into polarized human intestinal epithelial cells. Identification of an early brefeldin A sensitive event required for A1-peptide generation.

The effect of brefeldin-A (BFA), a reversible inhibitor of vesicular transport, on cholera toxin (CT)-induced Cl- secretion (Isc) was examined in the polarized human intestinal cell line, T84. Pretreatment of T84 monolayers with 5 microM BFA reversibly inhibited Isc in response to apical or basolateral addition of 120 nM CT (2.4 +/- 0.5 vs. 68 +/- 3 microA/cm2, n = 5). In contrast, BFA did not inhibit Isc responses to the cAMP agonist VIP (63 +/- 7 microA/cm2). BFA had no effect on cell surface binding and endocytosis of a functional fluorescent CT analog or on the dose dependency of CT induced 32P-NAD ribosylation of Gs alpha in vitro. In contrast, BFA completely inhibited (> 95%) the ability of T84 cells to reduce CT to the enzymatically active A1-peptide. BFA had to be added within the first 10 min of CT exposure to inhibit CT-elicited Isc. The early BFA-sensitive step occurred before a temperature-sensitive step essential for apical CT action. These studies show that sequential steps are required for a biological response to apical CT: (a) binding to cell surfaces and rapid endocytosis; (b) early, BFA-sensitive vesicular transport essential for reduction of the A1-peptide; and (c) subsequent temperature-sensitive translocation of a signal (the A1-peptide or possibly ADP-ribose-Gs alpha) to the basolateral domain.