Forensic microbiology and bite marks: a systematic review.

The forensic role of microbiology in bite mark analysis as evidence in a court of law has not yet been explored, as the analysis of bite marks is mostly morphology-based. The aim of this systematic review is to investigate if the analysis of the oral microbiota may be helpful as a complementary forensic tool. Articles were searched on the PubMed database, using predefined data fields and keywords. The final selection included a total of 6 papers (out of 42). Our results indicated that the Streptococcus genus is a key player in the analysis of bite mark microbiology from a forensic perspective and its genomic analysis may facilitate the association of a bite mark to the perpetrator. However, much more research is still needed before this forensic strategy can be applied in real scenarios. There is a need to optimize and standardize the methods of microbiome analysis and to determine several factors that may influence the results, such as the frequency of bacterial genotypes in the human population and the temporal stability of the oral microbiome on human skin.

Characterization of lip prints in a Portuguese twins' population.

Lip print patterns are referred to as unique to each individual, but controversy exists surrounding twins. In this study, the lip prints of 19 pairs of monozygotic and 47 pairs of dizygotic twins were studied. The left lower lip was photographed and the furrows were classified using Renaud's classification. Results showed the same lip pattern was found only in one monozygotic pair (5.3%) and in 4 dizygotic pairs (8.5%), and no significant statistical differences were found between groups (p > 0.05). In monozygotic twins only type C furrows presence displayed statistical significant differences (p=0.034). As for dizygotic twins, there were statistical significant differences in the frequency of type A (p=0.005) and type G furrows (p=0.018). As for the most common types, both groups displayed a higher prevalence of vertical furrows (type B: 97.4% and 96.8%, type A: 86.8% and 87.2%, in monozygotic and dizygotic, respectively). The least frequent furrow type was type I and type E in monozygotic (2.6% and 5.3%, respectively) and types E, F and I, in dizygotic (6.4%, 7.4%. and 7.4%, respectively). Our results seem to point out that lip print patterns should be useful carefully in twins' identification.

Post-mortem microbiology in sudden death: sampling protocols proposed in different clinical settings.

BACKGROUND: Autopsies, including minimally invasive autopsies, are a powerful tool for determination of the cause of death. When a patient dies from an infection, microbiology is crucial to identify the causative organism. Post-mortem microbiology (PMM) aims to detect unexpected infections causing sudden deaths; confirm clinically suspected but unproven infection; evaluate the efficacy of antimicrobial therapy; identify emergent pathogens; and recognize medical errors. Additionally, the analysis of the thanatomicrobiome may help to estimate the post-mortem interval. AIMS: The aim was to provide advice in the collection of PMM samples and to propose sampling guidelines for microbiologists advising autopsy pathologists facing different sudden death scenarios. SOURCES: A multidisciplinary team with experts in various fields of microbiology and autopsies on behalf of the ESGFOR (ESCMID - European Society of Clinical Microbiology and Infectious Diseases - study group of forensic and post-mortem microbiology and in collaboration with the European Society of Pathology) developed this narrative review based on a literature search using MedLine and Scopus electronic databases supplemented with their own expertise. CONTENT: These guidelines address measures to prevent sample contamination in autopsy microbiology; general PMM sampling technique; protocols for PMM sampling in different scenarios and using minimally invasive autopsy; and potential use of the evolving post-mortem microbiome to estimate the post-mortem interval. IMPLICATIONS: Adequate sampling is paramount to identify the causative organism. Meaningful interpretation of PMM results requires careful evaluation in the context of clinical history, macroscopic and histological findings. Networking and closer collaboration among microbiologists and autopsy pathologists is vital to maximize the yield of PMM.

Oral yeast colonization throughout pregnancy.

BACKGROUND: Recent studies suggest that placenta may harbour a unique microbiome that may have origin in maternal oral microbiome. Although the major physiological and hormonal adjustments observed in pregnant women lead to biochemical and microbiological modifications of the oral environment, very few studies evaluated the changes suffered by the oral microbiota throughout pregnancy. So, the aim of our study was to evaluate oral yeast colonization throughout pregnancy and to compare it with non-pregnant women. MATERIAL AND METHODS: The oral yeast colonization was assessed in saliva of 30 pregnant and non-pregnant women longitudinally over a 6-months period. Demographic information was collected, a non-invasive intra-oral examination was performed and saliva flow and pH were determined. RESULTS: Pregnant and non-pregnant groups were similar regarding age and level of education. Saliva flow rate did not differ, but saliva pH was lower in pregnant than in non-pregnant women. Oral yeast prevalence was higher in pregnant than in non-pregnant women, either in the first or in the third trimester, but did not attain statistical significance. In individuals colonized with yeast, the total yeast quantification (Log10CFU/mL) increase from the 1st to the 3rd trimester in pregnant women, but not in non-pregnant women. CONCLUSIONS: Pregnancy may favour oral yeast growth that may be associated with an acidic oral environment.

The Oral Microbiome in Health and Its Implication in Oral and Systemic Diseases.

The oral microbiome can alter the balance between health and disease, locally and systemically. Within the oral cavity, bacteria, archaea, fungi, protozoa, and viruses may all be found, each having a particular role, but strongly interacting with each other and with the host, in sickness or in health. A description on how colonization occurs and how the oral microbiome dynamically evolves throughout the host's life is given. In this chapter the authors also address oral and nonoral conditions in which oral microorganisms may play a role in the etiology and progression, presenting the up-to-date knowledge on oral dysbiosis as well as the known underlying pathophysiologic mechanisms involving oral microorganisms in each condition. In oral pathology, oral microorganisms are associated with several diseases, namely dental caries, periodontal diseases, endodontic infections, and also oral cancer. In systemic diseases, nonoral infections, adverse pregnancy outcomes, cardiovascular diseases, and diabetes are among the most prevalent pathologies linked with oral cavity microorganisms. The knowledge on how colonization occurs, how oral microbiome coevolves with the host, and how oral microorganisms interact with each other may be a key factor to understand diseases etiology and progression.

The Role of the Gut Microbiome on Chronic Kidney Disease.

Chronic kidney disease (CKD) is estimated to affect nearly 500 million people worldwide and cardiovascular (CV) disease is a major cause of death in this population. However, therapeutic interventions targeting traditional CV risks are not effective at lowering the incidence of CV events or at delaying the progression of the disease in CKD patients. In recent years, disturbances of normal gut microbiome were recognized in the pathogenesis of diverse chronic diseases. Gut dysbiosis is being unraveled in CKD and pointed as a nontraditional risk factor for CV risk and CKD progression. The most often reported changes in gut microbiome in CKD are related to the lower levels of Bifidobacteriaceae and Lactobacillaceae and to higher levels of Enterobacteriaceae. Although metagenomics brought us an amplified vision on the microbial world that inhabits the human host, it still lacks the sensitivity to characterize the microbiome up to species level, not revealing alterations that occur within specific genus. Here, we review the current state-of-the-art concerning gut dysbiosis in CKD and its role in pathophysiological mechanisms in CKD, particularly in relation with CV risk. Also, the strategies towards prevention and treatment of gut dysbiosis in CKD progression will be discussed.

Unpredictable susceptibility of emerging clinical moulds to tri-azoles: review of the literature and upcoming challenges for mould identification.

Tri-azoles represent the front-line drugs for the treatment of mould diseases; nevertheless, some emerging moulds, such as Fusarium spp., Scedosporium spp., Mucorales and others, may be less susceptible or resistant to these antifungals. A review of the literature was conducted on the susceptibility of rare moulds to the tri-azoles itraconazole, posaconazole and voriconazole. Particular attention was paid to isolates identified by molecular analyses. The range of susceptibility values described for the three tri-azoles was frequently large (from 0.06 to >16), and a high variability was found within each species; isolates were rarely reported as entirely susceptible to all tri-azoles. In addition, the susceptibility of 76 emerging moulds from our collection (including Hypocreales, Dothideomycetes, Scedosporium spp., Mucorales and rare Aspergillus spp.) to itraconazole and voriconazole was determined by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 and European Committee for Antimicrobial Susceptibility Testing (EUCAST) methods. Susceptibility discrepancies (of two dilutions) were found comparing CLSI and EUCAST for Dothideomycetes; the values for the remaining moulds were similar. More practical, faster and inexpensive susceptibility tools are welcome for testing emerging moulds, as these tests still represent a critical tool to support clinicians on the selection of proper antifungal treatment. The susceptibility of emerging moulds to tri-azoles cannot be predicted exclusively following mould identification, as the isolates' susceptibilities showed highly variable values. Some emerging moulds still remain very difficult to identity, even following standard molecular analyses which result in complex fungal collections. This fact limits the definition of epidemiological cut-offs and clinical breakpoints that are still imperative for emerging moulds.

Wiki as a tool for microbiology teaching, learning and assessment.

INTRODUCTION: Evidence suggests that cooperative learning and peer-assessment fosters students' ability to work with others and may lead to better cognitive outcomes and higher achievement. This work aimed to assess the use of an online collaborative tool for the teaching/learning and assessment of Microbiology. MATERIALS AND METHODS: A total of 144 students were grouped and assigned to create wiki entries as well as to peer review wikis created by colleagues (peer-assessment process) using the Wiki module from Moodle Virtual Learning Environment (MVLE). MVLE actions log was used for tracking students' activity. RESULTS: The number of student's actions within wiki did not present a strong correlation with wiki scores, so it should not be used as a heavy evaluation parameter. The amount of work developed between members of the same group differed significantly, suggesting that final scores should be attributed individually. When peer-assessment process was implemented, the number of editing actions increased, suggesting that the peer-assessment strategy encourages the development of a better work. The vast majority of students execute the work in the last 10% of the period assigned for task development, which can be counter-productive for a truly collaborative work. CONCLUSIONS: Wiki revealed to be a useful tool for Microbiology teaching/learning and assessment, promoting collaborative work, promoting virtual mobility and facilitating the real-time monitoring of the students' work. This pedagogical project promoted also the involvement of students in their assessment process, encouraging their critical sense and quest for Excellency.

Does the chemistry in the saliva of Down syndrome children explain their low caries prevalence?

AIM: The present study focuses on the relationship between dental caries and saliva components such as phosphate, calcium, potassium, chloride as well as α-amylase in children with Down syndrome. MATERIALS AND METHODS: Forty-five Caucasian sibling pairs, with the mean age of 13±4 years compose the final sample. Stimulated whole saliva was collected from DS children and their siblings and an automatic analyser quantified the biochemical parameters. RESULTS: Down syndrome children presented lower caries rates. The salivary concentration of calcium, phosphate, potassium and chloride did not differ between DS and sibling children. In respect to α-amylases, the absolute salivary concentration as well as salivary secretion rate was similar between DS and sibling controls. CONCLUSION: In conclusion, no correlation between dental caries and salivary ionic composition as well as α-amylase secretion rate was found in DS children.

Periodontal inflammation in renal transplant recipients receiving everolimus or tacrolimus - preliminary results.

OBJECTIVE: To compare oral health status between renal transplant recipients (RTRs) receiving tacrolimus (Tac) or everolimus (ERL) as immunosuppressive therapy. DESIGN: This study is a cross-sectional study. METHODS: Thirty-six RTRs receiving Tac and 22 RTRs receiving ERL were included in the study. Age, gender, time since transplant and pharmacological data were recorded for both groups. Oral health status was assessed through the evaluation of teeth, periodontal parameters as well as saliva flow rate and pH. RESULTS: RTRs receiving ERL were older than those receiving Tac. No differences were found between groups concerning oral hygiene habits, oral symptoms, smoking habits, unstimulated and stimulated saliva flow rate and pH, clinical attachment level or the number of decayed, missing and filled teeth. However, RTRs receiving ERL presented lower visible plaque index and lower values for bleeding on probing when compared to RTRs receiving Tac. In addition, RTRs receiving ERL presented a gingival index varying from normal to moderate inflammation whereas RTRs receiving Tac presented a gingival index varying from mild to severe inflammation. CONCLUSIONS: RTRs receiving ERL have lower periodontal inflammation when compared to RTRs receiving Tac.

Renal dopaminergic system activity in rat remnant kidney up to twenty-six weeks after surgery.

AIMS: In 3/4 nephrectomized (3/4nx) rats the renal dopaminergic system was suggested to be involved in the adaptive increase of sodium excretion two weeks after renal mass ablation. The aim of the present study was to evaluate the renal adaptations in sodium handling and renal dopaminergic system activity in 3/4nx rats up to twenty-six weeks after surgery. MAIN METHODS: The rats were placed in metabolic cages for the collection of 24 h urine for evaluation of sodium, dopamine, dopamine precursor and metabolites. Blood pressure, aromatic L-amino acid decarboxylase (AADC) activity in proximal tubules and the effect of dopamine D(1) receptor selective antagonist (Sch-23390) on natriuresis was evaluated. KEY FINDINGS: A time-dependent increase in both systolic and diastolic blood pressure was observed in 3/4nx rats, and this was accompanied by a decrease in urinary levels of dopamine and in renal AADC activity at twenty-six weeks after renal mass ablation. In contrast to what has been found two weeks after renal mass ablation, the natriuretic response to volume expansion was progressively reduced in 3/4nx rats at ten and twenty-six weeks after surgery and this was accompanied by insensitivity of natriuresis to Sch-23390. SIGNIFICANCE: In conclusion the renal dopaminergic system activity is compromised in 3/4nx rats in a time-dependent manner after renal mass ablation. It is suggested that this may contribute to compromise sodium excretion and increase blood pressure, in chronic renal insufficiency.

Cardiac remodeling and dysfunction in nephrotic syndrome.

There is an increased incidence of heart disease in patients with chronic nephrotic syndrome (NS), which may be attributable to the malnutrition and activated inflammatory state accompanying the sustained proteinuria. In this study, we evaluated renal function, cardiac morphometry, contractile function, and myocardial gene expression in the established puromycin aminonucleoside nephrosis rat model of NS. Two weeks after aminonucleoside injection, there was massive proteinuria, decreased creatinine clearance, and a negative sodium balance. Skeletal and cardiac muscle atrophy was present and was accompanied by impaired left ventricular (LV) hemodynamic function along with decreased contractile properties of isolated LV muscle strips. The expression of selected cytokines and proteins involved in calcium handling in myocardial tissue was evaluated by real time polymerase chain reaction. This revealed that the expression of interleukin-1beta, tumor necrosis factor-alpha, and phospholamban were elevated, whereas that of cardiac sarco(endo)plasmic reticulum calcium pump protein was decreased. We suggest that protein wasting and systemic inflammatory activation during NS contribute to cardiac remodeling and dysfunction.

Renal dopaminergic system activity in uninephrectomized rats up to 26 weeks after surgery.

BACKGROUND: Dopamine of renal origin exerts natriuretic and diuretic effects by activating D1-like receptors located at various regions in the nephron. Two weeks after uninephrectomy the renal dopaminergic system was suggested to be involved in the adaptative increase of sodium excretion. AIM: The aim of the present study was to evaluate the renal adaptations in sodium handling and renal dopaminergic system activity in uninephrectomized (Unx) rats up to 26 weeks after the surgery. RESULTS: A time-dependent increase in both systolic and diastolic blood pressure was observed in Unx rats up to 26 weeks after uninephrectomy. This was accompanied by a compensatory increase in aromatic L-amino acid decarboxylase at 2 weeks but not 10 and 26 weeks after uninephrectomy. In contrast to what has been found 2 weeks after uninephrectomy, at 10 and 26 weeks after surgery the natriuretic response to volume expansion was reduced in Unx rats and this was accompanied by insensitivity of natriuresis to dopamine D1 receptor selective antagonist (Sch23390). CONCLUSION: A time-dependent decrease in dopamine sensitive natriuresis is observed in Unx rats throughout the 26 weeks after uninephectomy. It is suggested that this may contribute to compromise sodium excretion and increase blood pressure.

Morphologic, molecular and hemodynamic cardiac alterations in a model of nephropathy induced by puromycin aminonucleoside.

INTRODUCTION: Proteinuria and decreased glomerular filtration rate are assuming increased importance in defining cardiovascular risk in chronic renal insufficiency. The aim of this work was to study morphologic, molecular and hemodynamic cardiac alterations in an animal model of proteinuria and renal insufficiency induced by puromycin aminonucleoside (PAN). METHODS: Normotensive rats (n = 14) were injected with PAN (150 mg/kg, i.p.) or with vehicle. Blood pressure was measured daily and the animals were placed in metabolic cages for evaluation of urinary excretion of sodium, protein and creatinine. Fourteen days after PAN administration left ventricular hemodynamics were evaluated through a pressure tip micromanometer and heart morphology was examined. Transmural samples of left ventricle were then taken for mRNA quantification of SERCA2a, phospholamban (PLB), insulin-like growth factor 1 (IGF-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: The animals treated with PAN presented a decrease in creatinine clearance (14th day: 2.24 +/- 0.32 vs. 4.51 +/- 1.08 ml/min) and an increase in proteinuria (14th day: 51.0 +/- 9.0 vs. 3.8 +/- 0.7 mg/mg creatinine), without changes in systolic (14th day: 151 +/- 7 vs. 141 +/- 6 mmHg) or diastolic blood pressure (14th day: 85 +/- 7 vs. 86 +/- 3 mmHg), These alterations were accompanied by cardiac atrophy with decreased left ventricular contractility. A reduction in the SERCA2a/PLB mRNA ratio was observed without significant alteration in the expression of IGF-1 in the left ventricle. CONCLUSIONS: PAN-induced nephropathy is accompanied by cardiac atrophy, left ventricular dysfunction and alterations in the expression of genes involved in myocardial calcium kinetics. These findings were not accompanied by increases in blood pressure and may contribute to our understanding of the increased cardiovascular risk in chronic renal insufficiency.

Blunted renal dopaminergic system activity in HgCl2-induced membranous nephropathy.

The present study evaluated the possible role of the renal dopaminergic system in the sodium retention of HgCl2-induced nephrotic syndrome. The time courses of urinary excretion of sodium, protein, dopamine and the precursor l-3,4-dihydroxyphenylalanine (L-Dopa) were evaluated in HgCl2-treated and control rats up to day 21. The renal aromatic l-amino acid decarboxylase (AADC) activity, the enzyme responsible for the synthesis of renal dopamine, was evaluated during negligible proteinuria accompanied with enhanced sodium retention (day 7), increased proteinuria accompanied with greatest sodium retention (day 14) as well as during increased proteinuria accompanied with negative sodium balance (day 21). Also, the influence of volume expansion (VE, 5% bw) and the effects of the D1-like agonist fenoldopam (10 microg kg bw(-1) min(-1)) on natriuresis and on proximal tubular Na+,K+-ATPase activity were examined on day 14. The daily urinary dopamine output and urinary dopamine/L-Dopa ratios were reduced in HgCl2-treated rats from day 2 and beyond. This was accompanied by a marked decrease in renal AADC throughout the study. During VE, the fenoldopam-induced inhibition of proximal tubular Na+,K+-ATPase activity was similar between HgCl2-treated and control rats. However, the urinary sodium excretion during fenoldopam infusion was markedly increased by 60% to 120% in control rats but was not altered in HgCl2-treated rats. It is concluded that HgCl2 nephrosis is associated with a blunted renal dopaminergic system activity. However, the lack of renal dopamine in HgCl2 nephrosis does not appear to be related with the overall renal sodium retention in a state of proteinuria.

Jejunal dopamine and Na,K+-ATPase activity in nephrotic syndrome.

BACKGROUND: The occurrence of complementary functions in sodium transport between the intestine and the kidney was suggested to occur when the renal function is immature or compromised and jejunal dopamine has been implicated in this renal-intestinal cross-talk. The jejunal sodium transport was not previously evaluated in the nephrotic syndrome. METHODS: We examined the jejunal Na(+),K(+)-ATPase activity and the role of dopamine in puromycin aminonucleoside (PAN) and HgCl(2)-induced nephrotic syndrome rat models. RESULTS: In both nephrotic syndrome rat models, the jejunal Na(+),K(+)-ATPase activity was reduced during greatest sodium retention and ascites accumulation (PAN nephrosis, day 7; HgCl(2) nephrosis, day 14), whereas during enhanced sodium excretion and ascites mobilization the jejunal Na(+),K(+)-ATPase activity was increased in HgCl(2) nephrosis (day 21) and was similar to controls in PAN nephrosis (day 14). In both PAN- and HgCl(2)-induced nephrosis, the jejunal aromatic L-amino acid decarboxylase (AADC) activity, the enzyme responsible for the synthesis of jejunal dopamine, did not differ from controls. In addition, the jejunal Na(+),K(+)-ATPase activity was not sensitive to inhibition by dopamine (1 microM) in both experimental groups throughout the study. CONCLUSIONS: In the nephrotic syndrome the jejunal Na(+),K(+)-ATPase activity may respond in a compensatory way to changes in extracellular volume, through dopamine-independent mechanisms.

Renal dopaminergic system activity in the rat remnant kidney.

BACKGROUND: Renal dopamine exerts natriuretic and diuretic effects by activating D1-like receptors. Uninephrectomy results in increased renal dopaminergic activity and dopamine-sensitive enhanced natriuresis. METHODS: The present study evaluated renal adaptations in sodium handling and the role of dopamine in rats submitted to (3/4) nephrectomy: right nephrectomy and excision of both poles of the left kidney ((3/4)nx rats). RESULTS: Two weeks after surgery the absolute urinary levels of dopamine were markedly reduced in (3/4)nx rats whereas the urinary dopamine excretion per % of residual nephrons was significantly increased in the remnant kidney of (3/4)nx rats. The V(max) values for renal aromatic L-amino acid decarboxylase, the enzyme responsible for the synthesis of renal dopamine, were decreased in (3/4)nx rats. Renal catechol-O-methyltransferase activity, the enzyme responsible for the methylation of dopamine, was increased in (3/4)nx rats whereas the renal activities of monoamine oxidases A and B did not differ between (3/4)nx and Sham animals. Volume expansion (5% body weight) resulted in similar natriuretic responses in (3/4)nx and Sham rats. During D1 antagonist administration (Sch-23390, 30 microg x h(-1) x kg(-1)) the natriuretic response to volume expansion was reduced in (3/4)nx rats more pronouncedly than in Sham animals. CONCLUSION: The decrease in absolute renal dopamine output in (3/4)nx rats is related with reduced renal synthesis and enhanced O-methylation of the amine. However, this is accompanied in (3/4)nx rats by increased renal dopamine excretion per residual nephrons and dopamine-sensitive enhanced natriuresis.

Differences in the renal dopaminergic system activity between Wistar rats from two suppliers.

AIM: Dopamine of renal origin reduces tubular sodium reabsorption and controls blood pressure. The present study evaluated renal dopaminergic activity and its response to uninephrectomy in Wistar Han rats from two suppliers, Harlan (W-H) and Charles River (W-CR). RESULTS: After uninephrectomy, the fractional excretion of sodium (FENa+) increased in both W-CR and W-H rats (W-CR: from 0.17 +/- 0.01 to 0.27 +/- 0.02%; W-H: from 0.39 +/- 0.04 to 0.54 +/- 0.04%, P < 0.05); however, in W-CR rats the FENa+ was lower than in W-H rats in both Sham and uninephrectomized (Unx) animals (P < 0.05). Systolic blood pressure in Unx W-CR rats was higher than in Unx W-H animals (131 +/- 3 vs. 122 +/- 2 mmHg, P < 0.05). Uninephrectomy was accompanied in W-H rats by increases in urinary levels (nmol g kidney(-1) day(-1)) of dopamine (10.3 +/- 0.5 vs. 8.3 +/- 0.7, P < 0.05) and 3,4-dihydroxyphenylacetic acid (DOPAC) (30.5 +/- 3.7 vs. 21.3 +/- 1.4, P < 0.05) and increases (P < 0.05) in maximal velocity values (Vmax in nmol mg prot(-1) 15 min(-1), 325 +/- 12 vs. 265 +/- 3) for renal aromatic L-amino acid decarboxylase (AADC), the enzyme responsible for the synthesis of renal dopamine. By contrast, in W-CR rats uninephrectomy did not change either the urinary levels of dopamine (7.1 +/- 0.5 vs. 7.6 +/- 0.7) and DOPAC (25.0 +/- 1.9 vs. 24.8 +/- 4.1) or AADC activity (Vmax 199 +/- 3 vs. 193 +/- 9). The Vmax values for renal AADC in W-CR rats were lower than those found in corresponding W-H animals. CONCLUSION: Wistar rats from different suppliers represent an important source of variability in the renal dopaminergic system activity. This may contribute to differences in sodium balance and blood pressure control in response to uninephrectomy.

Regulation of apical transporter of L-DOPA in human intestinal Caco-2 cells.

The present study examined the nature of the apical inward L-3,4-dihydroxyphenylalanine (L-DOPA) transporter in human intestinal epithelial Caco-2 cells, and whether protein kinases modulate the activity of this transporter. The apical inward transfer of L-DOPA was promoted through an energy-dependent and sodium-insensitive transporter (Km=33 microM; Vmax=2932 pmol/mg protein/6 min). This transporter was insensitive to N-(methylamino)-isobutyric acid, but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH; IC50=83 microM). The organic cation inhibitor decynium 24 failed to affect the accumulation of L-DOPA, whereas the organic anion inhibitor 4,4'-diisothiocynatostilbene-2,2'-disulphonic acid (DIDS) competitively inhibited L-DOPA uptake (IC50=83 microM). However, the apical-to-basal and basal-to-apical transepithelial transport and the cell accumulation of [3H]-PAH was close to that of [14C]-sorbitol and insensitive to DIDS (300 microM). Modulators of protein kinase A (PKA) [cyclic adenosine monophosphate (cAMP), forskolin, H-89 and cholera toxin], protein kinase G (PKG) [cyclic guanosine monophosphate (GMP), zaprinast, LY 83583 and sodium nitroprusside] and protein kinase C (PKC) (phorbol 12,13-dibutirate and chelerythrine) failed to affect the accumulation of L-DOPA. The Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA uptake (IC50s of 53 and 252 microM, respectively), but the rise of intracellular Ca2+ by A23187 (1 microM) and thapsigargin (1 microM) played no role on L-DOPA uptake. It is concluded that Caco-2 cells take up L-DOPA over the apical cell border through the sodium-independent and pH-sensitive L-type amino acid transporter.