Saccharomyces cerevisiae can respond in a complex way to the presence of different energy sources. While yeast can metabolize many different sugars, glucose provides the highest energy yield. To achieve this energy efficiency, yeast cells rely on specialized enzymes and metabolic states that mediate glucose metabolism by sensing and responding to this sugar. The presence of glucose triggers a rapid and dramatic change in the expression of about a quarter of S. cerevisiae's 5,500 genes.An organism's survival depends on developing effective strategies for identifying and adapting to available sources of food. Even organisms as small as the budding yeast Many of the cellular participants in the glucose “sense-and-response” pathway have been identified, but their exact relationships remain unknown. The gene expression response to glucose has been previously characterized through the use of a cDNA microarray, which allows simultaneous assessment of the transcriptional state of every gene. In research reported in this issue, James Broach and his colleagues at Princeton University have attempted to connect the individual components in this complex pathway by performing microarray analysis on a series of mutants (yeast strains with defects in specific proteins). They link certain portions of the response to known proteins and begin to understand how the pieces of this pathway fit together.The researchers initially focused on two proteins, called Ras2 and Gpa2. These proteins have been previously implicated in the transcriptional response to glucose and are members of a well-established family of signaling proteins. To investigate the roles of these proteins in the response, the researchers used mutants of Ras2 and Gpa2 that could be activated on demand and then performed microarray analysis to see which genes responded to activation of these proteins. They found that even in the absence of glucose, activation of either protein induced a transcriptional profile almost identical to the profile generated in yeast exposed to glucose. This shows that the complex and dramatic transcriptional response to glucose can be recapitulated by the activation of a single protein.The Ras2/Gpa2 pathway, however, is not the whole story. The group then went on to show that not all glucose-responsive genes are regulated in the same manner. They found that another pathway, independent of Ras2 and Gpa2, is able to elicit a portion of the transcriptional response to glucose. The partial redundancy of these pathways is a curious phenomenon and bears further investigation.The authors have achieved an initial step in mapping the topology of the intricate signaling pathway (or pathways) involved in the response to glucose. Furthermore, the approach—that of using microarray analysis of mutants in a pathway to deduce the mechanisms of regulation—will be useful in efforts to map other complex responses in yeast as well as in higher organisms.


Dr. Gerberding outlines critical steps for arresting the HIV/AIDS epidemic


Enterococcus faecalis and Enterococcus faecium and is associated with a high mortality rate. Enterococcus casseliflavus has been implicated in a wide variety of infections in humans, but never in meningitis.Enterococcal meningitis is an uncommon disease usually caused by E. casseliflavus. The patient was successfully treated with meropenem and ampicillin-sulbactam.A 77-year-old Italian female presented for evaluation of fever, stupor, diarrhea and vomiting of 3 days duration. There was no history of head injury nor of previous surgical procedures. She had been suffering from rheumatoid arthritis for 30 years, for which she was being treated with steroids and methotrexate. On admission, she was febrile, alert but not oriented to time and place. Her neck was stiff, and she had a positive Kernig's sign. The patient's cerebrospinal fluid was opalescent with a glucose concentration of 14 mg/dl, a protein level of 472 mg/dl, and a white cell count of 200/μL with 95% polymorphonuclear leukocytes and 5% lymphocytes. Gram staining of CSF revealed no organisms, culture yielded E. casseliflavus can be inserted among the etiologic agents of meningitis. Awareness of infection of central nervous system with Enterococcus species that possess an intrinsic vancomycin resistance should be increased. Enterococcal meningitis is an uncommon disease accounting for only 0.3% to 4% of cases of bacterial meningitis which is nevertheless associated with a high mortality rate. It has been described most frequently in patients with neurosurgical conditions , although it can also occur as a "spontaneous" infection complicating remote enterococcal infections such as endocarditis or pyelonephritis .Enterococcus faecalis and Enterococcus faecium are the two species most frequently isolated during the course of meningitis (76%–90% and 9–22% respectively). Enterococcus casseliflavus, first considered as a subspecies of E. faecium, is a motile enterococcus that produces a yellow pigment in agar and often has a VanC phenotype determining an intrinsic low level resistance to vancomycin. It has been implicated in a wide variety of infections in humans, especially immunocompromised hosts, but to the best of our knowledge it has never been associated to meningitis [ningitis .E. casseliflavus that was believed to originate from the gut in an old patient with bowel erosions.We describe here a case of enterococcal meningitis caused by A 77-year-old Italian female presented for evaluation of fever, stupor, diarrhea and vomiting of 3 days duration. She had no urinary symptoms. There was no history of head injury nor of previous surgical procedures. She had been suffering from rheumatoid arthritis for 30 years, for which she was being treated with steroids and methotrexate; other medical problems were insulin-dependent diabetes and moderate renal failure.On admission, she was febrile , alert but not oriented to time and place. Her neck was stiff, and she had a positive Kernig's sign. A CT brain scan showed an increase in subarachnoid space and in the volume of the ventricular system.Laboratory examinations revealed a white blood cell count of 15,100/μL with 70% neutrophils and 23% lymphocytes. Results of urinalysis were unremarkable. Cultures of blood and urine were drawn and subsequently resulted negative.The patient's cerebrospinal fluid (CSF) was opalescent with a glucose concentration of 14 mg/dl, a protein level of 472 mg/dl, and a white cell count of 200/μL with 95% polymorphonuclear leukocytes and 5% lymphocytes. Gram staining of CSF revealed no organisms. Pending the culture results the patient was empirically treated with intravenous meropenem, cotrimoxazole, acyclovir and dexamethasone.E. casseliflavus that was identified using the Vitek-2 system (bioMérieux-Vitek) on the basis of 6.5% NaCl tolerance, bile-esculin hydrolysis, and growth rate at 45°C, arginine hydrolysis, methyl-a-D-glucopyranoside testing, and acid production from ribose, motility testing, and yellow pigmentation testing.Culture of CSF yielded The isolate was sensitive to penicillin, ampicillin, ampicillin-sulbactam, imipenem, teicoplanin, tetracyclines and linezolid; it exhibited intermediate sensitivity to vancomycin , trimethoprim-sulfamethoxazole , levofloxacin , norfloxacin , ciprofloxacin  and quinupristin-dalfopristin ; it was resistant to clindamycin  and showed high resistance to gentamicin, streptomycin and kanamycin .The patient became afebrile 48 hours after the beginning of antibiotic therapy with rapid improvement of her mental status and disappearance of meningeal signs (within 36 hours).Once the organism was identified (4 days later), trimethoprim-sulfamethoxazole and acyclovir were discontinued and ampicillin-sulbactam (3 g every 6 hours) was added. After 2 weeks of antibiotic therapy the patient was discharged in good health with sterilization of the CSF culture. An echocardiogram revealed no vegetations whereas a colonoscopy examination showed two ulcerative lesions associated with two polyps, oedema and multiple punctuate erosions.Enterococcus identified as E. gallinarum have been previously documented; they occurred in patients with ventriculoperitoneal shunts for hydrocephalus [E. casseliflavus and E. gallinarum are responsible for 1–2% of all enterococcal infections and are characterized by the fact that they possess intrinsic low-level vancomycin resistance [E. gallinarum, and the VanC-2/3 ligase is specific for E. casseliflavus [Enterococcal infections of the central nervous system are quite rare and according to a MEDLINE search of the English literature only three cases of CNS infection by a motile cephalus ,7. E. casistance . The Vanliflavus . Organisliflavus ,9,10.E. casseliflavus it is important to remember that most strains are susceptible to penicillin and ampicillin. Combination therapy of ampicillin with an aminoglycoside such as gentamicin or streptomycin is considered the standard therapy of enterococcal meningitis due to ampicillin-susceptible strains [E. casseliflavus are beta-lactamase negative. There was not therefore a clear indication for the use of combination therapy with meropenem and a beta-lactamase inhibitor in the case reported.Despite the intrinsic low-level vancomycin resistance exhibited by  strains . MeropenE. casseliflavus.Our patient had gastrointestinal signs and symptoms, and colonoscopy revealed multiple erosions, which probably were the portal of entry of In fact, enterococcal meningitis may appear as a complication of diverse gastrointestinal diseases such as enterocolitis, peritonitis, abdominal surgery, or bowel carcinoma. A case of bowel erosions as the portal of entry of enterococcal meningitis has been previously reported .E. gallinarum and E. casseliflavus colonize the gastrointestinal tracts of both hospitalized individuals and nonhospitalized healthy ones [E. gallinarum and E. casseliflavus in healthy subjects who were administered oral vancomycin [E. gallinarum and E. casseliflavus/flavescens are part of the normal stool flora of the general population; this has perhaps impacted the ability of researchers to detect specific risk factors [Several studies have demonstrated that thy ones ,12,13. Tncomycin . Our pat factors .Although our patient had significant underlying conditions this case of meningitis was mild and had most of the typical features of "spontaneous" enterococcal meningitis .E. gallinarum and E. casseliflavus has been associated with high mortality, but it is difficult to attribute their mortality directly to infection or to the underlying conditions of the patient.The clinical significance of the enterococci that are intrinsically resistant to vancomycin has not been fully established yet. Infection with E. casseliflavus meningitis and it is the fourth documented so far with a motile Enterococcus species. Awareness of infection of central nervous system with Enterococcus species that possess an intrinsic vancomycin resistance should be increased.This case is the first report of The author(s) declare that they have no competing interests.IC and RDL carried out the clinical study of the patient and conceived of the study. GS and GBC carried out the microbiologic studies. AT and AC carried out the clinical study of the patient, conceived of the study and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The evidence base of clinical interventions in paediatric hospitals of developing countries has not been formally assessed. We performed this study to determine the proportion of evidence-based therapeutic interventions in a paediatric referral hospital of a developing countryThe medical records of 167 patients admitted in one-month period were revised. Primary diagnosis and primary therapeutic interventions were determined for each patient. A systematic search was performed to assess the level of evidence for each intervention. Therapeutic interventions were classified using the Ellis score and the Oxford Centre for Evidence Based Medicine Levels of EvidenceAny dehydration due to diarrhoea (59 cases) and pneumonia (42 cases) were the most frequent diagnoses. Based on Ellis score, level I evidence supported the primary therapeutic intervention in 21%, level II in 73% and level III in 6% cases. Using the Oxford classification 16%, 8%, 1% and 75% therapeutic interventions corresponded to grades A, B, C, and D recommendations, respectively. Overall, according to Ellis score, 94% interventions were evidence based. However, out of the total, 75% interventions were based on expert opinion or basic sciences. Most children with mild to moderate dehydration (52 cases) were inappropriately treated with slow intravenous fluids, and most children with non-complicated community acquired pneumonia (42 cases) received intravenous antibioticsMost interventions were inappropriate, despite the availability of effective therapy for several of them. Diarrhoeal dehydration and community acquired pneumonia were the most common diagnoses and were inappropriately managed. Existing effective interventions for dehydration and pneumonia need to be put into practice at referral hospitals of developing countries. For the remaining problems, there is the need to conduct appropriate clinical studies. Caution must be taken when assigning the level of evidence supporting therapeutic interventions, as commonly used classifications may be misleading Previous studies have shown that medical interventions based on scientific evidence range from 10 to 80% . These sIn paediatric practice, the proportion of evidence based interventions reported ranges from 75 to 91% . These sThe assessment of quality of care at referral level for severely ill children is an important component of the efforts for reducing the child mortality rate in poor countries, for reducing the burden on health systems and for investing money in favour of high priority health interventions ,10. ThusReferral care provided to children hospitalized in the paediatric department of the Instituto Especializado de Salud del Niño (IESN) was assessed for evidence base. IESN is a national paediatric hospital with more than 500 inpatient beds, serving mostly patients from deprived socioeconomic areas of Lima and inner cities of the country.The clinical records of 195 children aged 1 month through 16 years old and hospitalized during January 2003 were initially revised.One of the investigators (NYC) assessed the clinical records of children and assigned to each one a primary diagnosis and one or more primary therapeutic interventions, on the basis of the main clinical features and/or definitive diagnostic laboratory aids. Patients in whom a primary diagnosis was not possible to determine or those without a clear diagnosis were excluded.The primary intervention was defined as the treatment or other manoeuvre that represented the most important attempt to cure, alleviate, or care for the patient in respect of his or her primary diagnosis .To determine the level of evidence for each primary intervention, Cochrane reviews were searched. If there was not such a systematic review, a search through PubMed (MEDLINE) was performed by one of the investigators (NYC). All Cochrane reviews were searched through their own search tools. For PubMed, the period of search was 1966 through 2002. The key words used included those related to the primary diagnosis . Limits: "Title", "All child: 0–18 years". Publication type was sequentially searched for "Practice Guideline", "Meta-Analysis", "Randomized Controlled Trial" and "Review". Articles in English or Spanish were included. The National Guideline Clearinghouse was additionally visited for additional references. Published recommendations for judging the quality of guidelines were used for deciding the selection of the guidelines . In addiThe level of evidence assigned to interventions was based on Ellis score  and the Oxford Centre for Evidence Based Medicine Levels of Evidence  ,12. ElliAs a next step to our study, we planned the dissemination of the results among the hospital policy makers and the suggestion of corrective courses of action for those interventions needing improvement.Results of the search strategies for PubMed are included as an appendix See . A guideOverall, one hundred and ninety five clinical records were revised. Twenty eight clinical records were excluded because of undefined primary diagnosis Table . One hunOut of 167 primary interventions, 21% were supported by level I evidence, 73% were level II, and 6% were ranked as level III, according to Ellis classification .Most assessed interventions were considered as level II Table . DiarrhoOvertly unsubstantiated therapy according to Ellis classification is shown in Table Comparison of grade of recommendation of the prescribed intervention with the appropriate one is shown in Table In this study 94% of therapeutic interventions were evidence-based by Ellis score. It may seem encouraging that more than 90% of therapeutic decisions in a referral paediatric hospital of a developing country are evidence based. However, the level II of evidence from Ellis includes interventions based in cohort studies, case-control studies, case series, expert's opinion, and even those based in basic sciences. We attempted therefore to classify the primary interventions according to more specific criteria. Using the Oxford classification, 75% of therapeutic interventions were based in expert opinions or in basic sciences (Grade of Recommendation D).Some limitations of our study include the possible author' bias when assigning the primary diagnosis and primary intervention. The assignment of diagnosis by the clinician may have been influenced by both the choice of treatment and the available evidence. Only one of us classified the primary intervention. In addition, we evaluated only a primary intervention for a single primary diagnosis. Actually, many patients had more than one diagnosis and obviously more than one therapeutic intervention.We used as evidence-base for rating the interventions assessed in our study, guidelines and evidence-based resources published in the developed world. This raises the issue of whether they are fully applicable to our setting. The most prevalent conditions found were diarrhoeal dehydration and community acquired pneumonia. For both of them we used British produced guidelines ,21 becauRegarding community acquired pneumonia, the British guidelines recommend antibiotic treatment for all children with pneumonia, due to the difficulties in identifying the aetiology, and they also specify criteria for hospitalization . These rWe acknowledge that the evidence derived from studies performed in developed countries should be translated with caution to developing settings. However, when the native research is scarce or of low quality, we think that the transfer of knowledge from the developed countries is an acceptable approach, as far as the particular characteristics of patients in developing countries are considered on an individual basis.Dehydration due to diarrhoea and pneumonia were the most frequent diagnoses. Oral rehydration for diarrhoeal dehydration and antibiotics for pneumonia are considered as interventions with sufficient evidence for implementing them widely ,10. In oIn addition to their enormous potential for saving lives, outpatient antibiotic therapy for pneumonia and outpatient oral rehydration can drastically reduce the rate of hospitalizations, the hospital stay, the hospital mortality rate, and the costs incurred. At our hospital, the mean stay time for hospitalized children is 4.7 days, and the mean crude mortality rate is 3.6%. We estimated the cost of managing hospitalized children with pneumonia and diarrhoeal dehydration as US$ 10.6/day and US$ 8.6/day, respectively. These costs are referred only to hospital bed and laboratory tests. A substantial amount of money could be saved treating these conditions on an outpatient basis.We planned the dissemination of our results among the hospital policy makers. The tools that will be suggested for improving the standards include the development and systematic application of locally produced guidelines and/or the adaptation of published guidelines. A useful alternative that has been experienced for several years at our inpatient ward unit is to make available personal computers connected to Internet for attending physicians, residents and interns, and to encourage the use of online evidence-based resources. This last alternative may work better, particularly where there are motivated physicians who are able to lead the efforts for improving the health care standards. However, the ultimate decision to systematically introduce and monitor the suggested changes will rest on hospital managers. Such changes should also depend on taking into account the role of several other determinants of the clinical decision making by individual practitioners, such as continuous training, motivation, time, availability of drugs, equipments and supplies, supervision, and long-term health system strengthening strategies.Caution must be taken when assigning the level of evidence that supports therapeutic interventions, as commonly used classifications may be misleading. Existing effective interventions for dehydration and pneumonia are not being implemented in developing countries, even at referral level, and thus there is the need to change the current medical interventional behaviours. For the remaining diagnoses, the majority of assessed interventions were based on weak or non-existent evidence, highlighting the need to conduct appropriate clinical studies.The author(s) declare that they have no competing interests.NYC and LH conceived and designed the study. All authors analyzed and interpreted the data and contributed to earlier drafts of the manuscript.The pre-publication history for this paper can be accessed here:"Appendix: Literature search results" details of the Medline (PubMed) search strategy and of the results are provided in this additional file. The selected articles are in black, bold characters. In addition, the search results from the National Guideline Clearinghouse website are provided for painful crisis in sickle cell anaemia.Click here for file


The amino acid composition of a low molecular weight chromium binding peptide (LMWCr), isolated from bovine liver, is reportedly E:G:C:D::4:2:2:2, though its sequence has not been discovered. There is some controversy surrounding the exact biochemical forms and the action of Cr(III) in biological systems; the topic has been the subject of many experimental reports and continues to be investigated. Clarification of Cr-protein interactions will further understanding Cr(III) biochemistry and provide a basis for novel therapies based on metallocomplexes or small molecules.Oryza sativa). A broader search for pentameric sequences (and assuming a disulfide dimer) corresponding to the stoichiometric ratio E:D:G:C::2:1:1:1, within the set of human proteins and the set of proteins in, or related to, the insulin signaling pathway, yields a match at an acidic region in the α-subunit of the insulin receptor . A synthetic peptide derived from this sequence binds chromium(III) and forms a metal-peptide complex that has properties matching those reported for isolated LMWCr and Cr(III)-containing peptide fractions.A genomic search of the non-redundant database for all possible decapeptides of the reported composition yields three exact matches, EDGEECDCGE, DGEECDCGEE and CEGGCEEDDE. The first two sequences are found in ADAM 19  proteins in man and mouse; the last is found in a protein kinase in rice (The search for an acidic decameric sequence indicates that LMWCr may not be a contiguous sequence. The identification of a distinct pentameric sequence in a significant insulin-signaling pathway protein suggests a possible identity for the LMWCr peptide. This identification clarifies directions for further investigation of LMWCr peptide fractions, chromium bio-coordination chemistry and a possible role in the insulin signaling pathway. Implications for models of chromium action in the insulin-signaling pathway are discussed. EECGD-, residues 175–184) and conforms to the predicted stoichiometry. Further, visible absorption data and electron paramagnetic measurements of the synthesized disulfide linked dimeric Cr-peptide complex are identical to that of the biologically isolated fractions.Type II diabetes continues to grow as a worldwide epidemic; it is expected that this disease will surpass 300 million cases by 2025 -5. Undernr) protein database for their occurrence. Based on a simple model where all amino acids are equally possible and about 1.5 million sequences of average length 200 present in the nr database, the E-value for an independent exact match is about 0.55 .Recent reports ,21,24, unr database corresponding to 3 unique permutations: EDGEECDCGE, DGEECDCGEE and CEGGCEEDDE. One match, CEGGCEEDDE, was found only in one sequence annotated as a putative protein kinase in rice (Oryza sativa). The peptide sequences, EDGEECDCGE and DGEECDCGEE, both matched the same region of the disintegrin domain of the human and mouse versions of ADAM 19  [bos taurus. This acid-rich sequence region (Table We found perfect matches in the D: 8728) . Since tnr database is much higher, ~104. Restricting this search to the human proteome gave rise to 439 (expected: ~102) matches, with no reason to prefer one over the other.Noting that the reported stoichiometry consists of even numbers of amino acids, we further considered the possibility that LMWCr might be a disulfide-linked dimer, with each monomeric unit having the composition, E:D:G:C::2:1:1:1. Such a hypothesis is consistent with the limited resolution of the experimental data used to derive the stoichiometry  and yielConsidering that LMWCr might be a subsequence of a protein related to "insulin" or known to be involved in the insulin signalling pathway, we compiled a set of 96 such sequences. This set comprised two components: 1) proteins playing a role in the insulin signaling pathway (23 protein sequences were selected) derived from pathway charts, and 2) the set of all protein sequences derived from SwissProt for the search, "insulin + human" (78 entries). The two components were compared for redundancy and the duplicates removed (5 instances).-1). Comparison of the pentapeptide set to a more detailed insulin signaling pathway construct [A cross comparison of the resulting 439 entries using the BLAST query method versus the set of 96 pathway and insulin-related proteins results in a unique match for one of the 60 pentapeptides, EECGD, within the insulin receptor (INSR), residues 180–184  complexes, i.e. proteolysis may lead to a group of similar peptides that differ by one or more amino acid residues on either side of the Cr binding site. This interpretation differs significantly from reports on the isolation of LMWCr, which attempt to avoid a proteolytic product.This sub-sequence lies in the extracellular α-subunit of INSR in an acid-rich region (-NKDDNEECGD-) towards the end of the L1 domain, and at the start of the "cysteine-rich region". A BLAST query  for all KECGDLCPGTL-. The cysteine in -KECGD- forms a disulfide bond with another cysteine residue 28 positions away in the crystal structure. However, we do not know if this is due to the crystallization conditions or representative of the natural form. Further, the insulin receptor possesses 3 additional cysteine residues compared to the insulin-like growth factor receptor, thus the pattern of pairing of cysteine residues to form disulfide bonds may be different in the two receptor types. The observed difference may be great enough to preclude Cr(III) acting on the insulin-like growth factor receptor.A crystal or solution structure of this region of the insulin receptor has not been determined. However, the crystal structure of a homologous molecule, the insulin-like growth factor receptor  has been published ,31. Sequ2) and generated the disulfide-linked dimer via air oxidation. This peptide was subjected to conditions for the reconstitution of apo-LMWCr to generate a holo-peptide complex. Incubation of the peptide with fresh solutions of CrCl3 results in a clear gray-green solutions of a Cr-peptide complex with visible electronic spectra 2 and (AcNEECGD-CONH2)2 indicate that Cr exists in trinuclear arrays in these complexes. A preliminary spectrum is shown in Figure The identified sequence in hand, we synthesized the pentapeptide -peptide complexes must be fully addressed, along with thermodynamic and kinetic aspects of Cr(III) binding and transfer.in vitro studies suggest that Cr(III) replacement in the physiological iron transport and storage apparatus may lead to some small beneficial outcome for certain diseases [in vitro experiments (including our own) may be finally ascertained only after the fact.Models of non-toxicological action of Cr(III) in biological systems may broadly fall into three categories: structural, redox, and iron homeostasis. The earliest models , and thodiseases . The bioextracellular model for Cr(III) biochemistry with respect to insulin signaling may be plausible (see Supporting Information). Such a structural model would include the known aspects of INSR cycling and insulin degradation [intracellular mechanisms for LMWCr action [Although unexpected, the results in this report and a critical review of other literature ,11-18, sradation , and incradation  of a terr action ,18, redor action , and ther action . In addir action  may satiThis model is a parsimonious alternative to current proposals of Cr action in the insulin signaling pathway. However, this model points directly back to significant kinetic and a thermodynamic questions about Cr(III) in biological systems. For instance, what is the physical form of Cr in the bloodstream? How is Cr(III) transported and exchanged between ligands in the serum? Is transport specific? What structure/activity relationship exists in Cr(III) complexes to allow their transport across biological membranes? Thermodynamically, a hydrolyzed, multinuclear Cr cluster should predominate at neutral pH, but transport by transferrin would presumably be in the mononuclear Fe binding sites. Alternatively, Cr(III) clusters may be transported non-specifically in serum by proteins, possibly including transferrin and serum albumin. At this point, there exist significant gaps in understanding the possible biochemistry of Cr(III) and what molecular processes it may affect.The proposed extracellular model of Cr(III) action in this report is upstream of IRS1, a therapeutic target of White and others ,8, and mA bioinformatic search for an acidic decameric sequence matching reported stoichiometries of LMWCr amino acid composition indicates that the peptide may not be a contiguous sequence. An expanded search localized a pentameric sequence in the insulin receptor and suggests a possible identity for the Cr(III)-containing peptide fractions derived from liver. Disulfide linked penta- and hexameric peptides based on the identified sequence bind Cr(III) in a similar fashion to LMWCr fractions reported in the literature.nr database was downloaded and Perl scripts used to search for exact matches corresponding to all unique permutations of EEEEGGCCDD. In a separate search, a set of 78 unique protein sequences from Swiss-Prot were obtained as a result of using the query "insulin human." This set was searched for exact matches corresponding to all unique permutations of EEGCD. In addition, the human proteome was also downloaded and searched for matches to the same set of pentameric peptides.The 2, was synthesized by continuous-flow automated solid-phase synthesis on a Perseptive Biosystems Pioneer Peptide Synthesis System. Peptide synthesis was performed by using standard Fmoc-protection strategies with TBTU/DIEA activation strategy. Typically, a 0.5 mmol scale, using Rink amide resin (loading ~0.65 mmol/g) and four times excess of the other reagents  were used. After the solid-phase synthesis was complete, acetylation at the amino terminus was carried out for 2 hours (50:50:1:: acetic anhydride:dimethylformamide:pyridine), and the resulting peptide cleaved from the resin. The peptide was cleaved from the solid phase resin by adding a slurry of 94% trifluoroacetic acid, 2.5% of water, 2.5% of ethanedithiol, and 1% of triisopropylsilane to the reaction vessel and shaking it for 5 h. The trifluoroacetic acid, containing the peptide, was filtered off by vacuum, and the resin was washed 2–3 times with trifluoroacetic acid. The filtrate was evaporated under nitrogen gas until the volume was reduced to 15 mL. 30 mL of ice-cold diethyl ether was added to the filtrate, causing the peptide to precipitate, and the mixture centrifuged to form a pellet of the peptide. The diethyl ether was decanted and the peptide pellet was washed with diethyl ether three times. Finally, the peptide was dissolved in 20 mL water containing 0.1% trifluoroacetic acid and extracted with diethyl ether three times. The aqueous portion was collected and freeze-dried to give the synthetic peptide. AcEECGD-CONH2, has a retention time of 5.3 minutes on a 250 mm × 4.5 mm C-18 reversed phase HPLC column using a gradient of 5 to 20% acetonitrile in 0.1% trifluoroacetic acid/water mobile phase running at 1 mL per minute and detected at a wavelength of 220 nm.The peptide, AcEECGD-CONH2, was dissolved to 10 mg/mL in a 0.1 M solution of ammonium carbonate at pH 7 and allowed to oxidize under ambient air for 48 hours. The product was isolated by lyophilization and analyzed by HPLC. The disulfide peptide dimer, (AcEECGD-CONH2)2, has a retention time of 6.0 minutes using the above conditions. The product, (AcEECGD-CONH2)2, has a calculated mass of 1183.35141 and exhibits a mass of 1183.3477 Daltons when analyzed by electrospray mass spectrometry.The synthetic peptide, AcEECGD-CONH2)2 was weighed and dissolved in 30 mL of water. A portion of this solution (5 mL) was taken up in a 25 mL tube, and 4 equivalents of chromium(III) chloride were added as a solid or in an aqueous solution. The reaction of the two components took several minutes and was monitored by ultraviolet-visible spectrophotometry. The chromium peptide complex, Cr3O(AcEECGD-CONH2)2, exhibits characteristic spectral absorbance features at 432 nm and 615 nm. The features are consistent with chromium bound to oxygen atom donors and similar to the reported spectrum of LMWCr [Fifteen milligrams of  complexes and drafted the manuscript, tables and graphics. All authors read and approved the final manuscript.2 and proposed Cr(III) model at INSR. Supporting tables (2) include FASTA sequences for insulin signaling map (20 proteins) and pentameric peptides found in genomic search (439 entries)Supporting figures (3) include HPLC and MS data for AcEECGD-CONHClick here for file


DJ-1 were recently described in two families with autosomal recessive Parkinsonism, one of which is a large deletion that is likely to lead to loss of function. Here we show that embryonic stem cells deficient in DJ-1 display increased sensitivity to oxidative stress and proteasomal inhibition. The accumulation of reactive oxygen species in toxin-treated DJ-1-deficient cells initially appears normal, but these cells are unable to cope with the consequent damage that ultimately leads to apoptotic death. Furthermore, we find that dopamine neurons derived from in vitro–differentiated DJ-1-deficient embryonic stem cells display decreased survival and increased sensitivity to oxidative stress. These data are consistent with a protective role for DJ-1, and demonstrate the utility of genetically modified embryonic stem cell–derived neurons as cellular models of neuronal disorders.The hallmark of Parkinson's disease (PD) is the selective loss of dopamine neurons in the ventral midbrain. Although the cause of neurodegeneration in PD is unknown, a Mendelian inheritance pattern is observed in rare cases, indicating a genetic factor. Furthermore, pathological analyses of PD substantia nigra have correlated cellular oxidative stress and altered proteasomal function with PD. Homozygous mutations in  Dopaminergic neurons, derived from DJ-1-deficient embryonic stem cells, display decreased survival and increased sensitivity to oxidative stress Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by rigidity, slowed movement, gait difficulty, and tremor at rest . The patα-synuclein lead to a rare familial form of PD  . Of note, a mutation that encodes a similarly truncated protein (at the human DJ-1 exon 7 splice acceptor) has been described in a patient with early-onset PD  cell clone, F063A04, that harbors a retroviral integration at the .gsf.de) . This inonset PD .DJ-1 allele, clone F063A04 was exposed to a high dose of the antibiotic G418, which selects cells that are homozygous for the neomycin resistance gene insertion (DJ-1 locus) were identified by Southern blotting -2,5-diphenyltetrazolium bromide [MTT] by metabolic enzymes) in triplicate polymerase-1 (PARP) in a pattern indicative of an apoptotic death program  and FACS analysis. Initial ROS accumulation (at 15 min after stimulation) appeared unaltered in the DJ-1-deficient cells in comparison to control heterozygous cells , a DN-specific toxin that enters DNs through the dopamine transporter and leads to oxidative stress and apoptotic death . DJ-1-deDJ-1. DJ1-shRNA virus-infected cells displayed efficient silencing of DJ-1 gene expression to 10%–20% of control vector-infected cultures .To confirm the role of DJ-1 in primary midbrain DNs, DJ-1 expression was inhibited by RNA interference (RNAi) in embryonic day 13.5 (E13.5) murine primary midbrain cultures by lentiviral transduction of short hairpin RNAs (shRNAs)  Rubinso. E13.5 mblotting Q). TransDJ-1 shRNA virus and with control vector displayed similar numbers of TH-positive neurons in the absence of exposure to H2O2   . Total esurvival T. These 2O2, but they are unable to cope with the consequent toxicity, culminating in apoptosis. Additionally, we find that DJ-1 deficiency sensitizes cells to the proteasomal inhibitor lactacystin but not other toxic stimuli such as tunicamycin. Proteasomal inhibition induces the accumulation of short-lived and misfolded cytoplasmic proteins, leading to oxidative stress and apoptosis  for 18–36 h according to the manufacturer's instructions . The antiserum was affinity-purified on a peptide-coupled Sulfolink column  according to the manufacturer's instructions. Antibody was used at a dilution of 1:200 for immunohistochemistry and Western blotting as described . Immunoh4 cells/well) were treated for 15 h with H2O2 in ES cell medium deficient in β-mercaptoethanol . Dopamine uptake assays were performed as described  treatment, the drug was diluted in the differentiation medium  and mediDJ-1 shRNA vector was generated by insertion of annealed oligonucleotides 5′-TGTCACTGTTGCAGGCTTGGTTCAAGAGACCAAGCCTGCAACAGTGACTTTTTTC-3′ and 5′-ACAGTGACAACGTCCGAACCAAGTTCTCTGGTTCGGACGTTGTCACTGAAAAAAGAGCT-3′ into the LentiLox vector . For expression of DJ-1 in ES cell rescue experiments, DJ-1 was cloned into the expression vector pcDNA3.1  containing a Flag peptide sequence at the N-terminus using standard cloning techniques. Flag-L166P DJ-1 (pcDNA3) was generated by PCR-mediated mutagenesis.The cDNA for human 5 cells per well), grown for 24 h, and then treated with 10 μM H2O2 as indicated. Cells were lysed in 200 μl lysis buffer and cleared lysate was prepared as described above. An aliquot of 40 μl from each time point was added to 2 M HCl (120 μl) with or without 10 μM 2,4-dinitrophenyl-hydrazine and incubated for 1 h at 24 °C with shaking. Proteins were then TCA-precipitated and resuspended in 200 μl of 6 M guanidinium chloride. Absorbance was measured at 360 nm, and DNP-conjugated samples were normalized for protein concentration with the underivitized control samples.For protein carbonyl quantitation , cells wFigure S1DJ-1 cDNA in WT (+/+), heterozygous (+/–), and knockout (–/–) cultures. Each expression value was normalized to that of β-actin and expressed relative to the respective value of the WT (+/+) control. These gene expression patterns were replicated in at least three independent PCR experiments. Total RNA from ES cells differentiated with the SDIA method for 18 days was isolated using the Absolutely RNA Miniprep kit . Synthesis of cDNA was performed using the SuperScript first strand synthesis system for RT-PCR (Invitrogen). Real-time PCR reactions were optimized to determine the linear amplification range. Quantitative real-time RT-PCRs were performed (Stratagene MX3000P) using the QuantiTect SYBR Green PCR Master Mix  according to the manufacturer's instructions. DJ-1 primer sequences were 5′-CGAAGAAATTCGATGGCTTCCAAAAGAGCTCTGGT-3′ and 5′-CAGACTCGAGCTGCTTCACATACTACTGCTGAGGT-3′; primers used for β-actin were 5′-TTTTGGATGCAAGGTCACAA-3′ and 5′-CTCCACAATGGCTAGTGCAA-3′. For quantitative analyses, PCR product levels were measured in real time during the annealing step, and values were normalized to those of β-actin.(A) Real-time PCR analyses of DJ-1 (625 bp) and β-actin (350 bp).(B) Ethidium bromide staining of the PCR products obtained after 29 cycles for (704 KB EPS).Click here for additional data file.Figure S2DJ-1 heterozygous cells (solid bar) and DJ-1-deficient knockout clone 32 (open bar) after exposure to CuCl2 or staurosporine at the doses indicated.(A and B) Cell viability of (C) MTT values of untreated DJ-1-deficient ES cell clones and the control heterozygous cells. Assays were performed exactly as in (D) MTT values of untreated DJ-1-deficient ES cells transfected with vector alone or various DJ-1-encoding plasmids. Transfection and expression of WT DJ-1 or mutant forms of DJ-1 does not alter the basal metabolic activity or viability of the cells.DJ-1 or the L166P mutant.(E) Western blotting of extracts from ES cells transfected with vectors harboring WT human (545 KB EPS).Click here for additional data file.Figure S3Both cell cultures were differentiated by SDIA for 18 DIV. Cells were fixed with 4% paraformaldehyde and stained with mouse monoclonal antibodies against HB9  and rabbit polyclonal antibodies against GABA  as in (5.5 MB TIF).Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession numbers of the genes discussed in this paper are α-synuclein (NM_000345), Parkin (AB009973), and DJ-1 (AB073864).The GenBank (


Plasma levels of tumor necrosis factor (TNF)-α and of C-reactive protein (CRP) are elevated in smokers. Previous studies failed to show an association between the G-308A polymorphism in the promoter region of the TNF-α gene and coronary artery disease (CAD). We investigated whether smoking would interact with the TNF-α G-308A polymorphism in determining plasma levels of TNF-α and CRP.Study participants with a complete data set in terms of smoking and the TNF-α G-308A polymorphism were 300 middle-aged male and female industrial employees. After excluding 24 irregular smokers, analyses were performed on 198 "non-smokers" (life-long non-smokers or subjects who quit smoking >6 months ago) as compared to 78 "regular smokers" (subjects currently smoking >3 cigarettes/day). All subjects had a fasting morning blood draw to measure plasma levels of TNF-α and CRP by high-sensitive enzyme-linked immunosorbent assays.1,250 = 5.67, p = .018) but not for genotype . The interaction-term between genotype and smoking status was not significant . The fully adjusted model with plasma TNF-α failed to show significant main effects for smoking and genotype, as well as for the smoking-status-genotype interaction.The cardiovascular risk factor adjusted analysis regressing log-transformed CRP levels against smoking status, genotype, and smoking-status-genotype interaction revealed a significant main effect for smoking status (FThe findings suggest that the TNF-α G-308A polymorphism does not mediate the effect of smoking on plasma CRP levels. It remains to be seen whether other genetic polymorphisms along the inflammatory pathway may modulate vascular risk in smokers. Coronary artery disease (CAD) is associated with chronic inflammatory processes in which blood-derived macrophages play a key role . Tumor nSmoking is one of the strongest cardiovascular risk factors for atherosclerotic diseases . Severalin vivo.Another candidate polymorphism that might mediate the cardiovascular risk with smoking, and that is at the initiation of the inflammatory cascade, is the G-308A polymorphism of the TNF-α gene promoter site. The TNF-α G-308A polymorphism is a single base pair polymorphism located at position 308 in the TNF-α gene that maps to human chromosome 6 (p21.1-p21.3) resulting in substitution of the nucleotide adenine (A) for guanine (G) . In the in vitro [While the TNF-α G-308A polymorphism has been associated with increased production of TNF-α in vitro ,18, previn vitro -25. Howein vitro -28.If alleles are randomly distributed between smokers and non-smokers, case-control studies not explicitly investigating the possible environmental-genetic interaction might fail to unravel the role of genetic polymorphisms in CAD. Therefore, we speculated that smoking would affect plasma TNF-α and CRP levels depending on the TNF-α genotype. If so, the genotype might affect the rate of disease progression rather than the existence of atherosclerotic lesions.The study was conducted as part of a larger project in an airplane manufacturing plant in southern Germany. From a total of 1,760 employees, participation was offered to a representative sample of 647 men and women. Of those, 537  volunteered to participate. The Institutional Review Board approved the study protocol. All 537 subjects completed questionnaires on medical and psychosocial health status. A subsample of 332 subjects agreed to have a variety of biological measures assessed. Three hundred of these individuals had complete data on smoking status, smoking history and the TNF-α G-308A gene polymorphism.Based on self-reported smoking history, we categorized life-long non-smokers or those, who had quit smoking for at least 6 months as „non-smokers" (n = 198), smokers who were currently smoking >3 cigarettes per day as "regular smokers" (n = 78), and smokers reporting to consume up to 3 cigarettes/day or who had stopped smoking for less than 6 months as „irregular smokers" (n = 24). Because of the resulting small numbers of irregular smokers, the latter 24 individuals were excluded from further analyses. Of the remaining 276 individuals, we excluded those with a history suggestive of symptomatic atherosclerotic disease, individuals reporting intake of drugs or conditions that might affect CRP levels (including chronic inflammatory diseases such as active rheumatoid arthritis), and subjects for whom CRP data were missing because of occasional assay problems. Subjects reporting a positive history of elevated blood glucose were not excluded. This selection procedure resulted in a final dataset of 261 subjects.Data were collected on two occasions: First, subjects completed a medical questionnaire and examination. The medical assessment consisted of a 96-item questionnaire assessing the medical history and smoking behavior. The questionnaire was based on the Nurses Health Study  with queAfter completion of questionnaires, subjects had a 15-min rest period while sitting. Thereafter, systolic and diastolic blood pressure (BP) were measured twice within 5 min by sphygmomanometry, and the average of the two readings was computed. The waist-to-hip ratio was calculated based on waist circumference (as measured at its narrowest point between the ribs and iliac crest) and hip circumference . Blood samples after overnight fasting were collected within one day to two weeks after having obtained medical data. Blood sampling was scheduled two hours after awakening to minimize circadian variation in variables of interest.Venous blood was obtained using cooled (4°C) citrate tubes for the TNF-α assay. Plasma was snap-frozen after centrifugation until further processing. We chose high-sensitive enzyme-linked immunosorbent assays (ELISA) to measure plasma concentrations of TNF-α  and of CRP . In contrast to standard CRP assays, the high-sensitivity assay for CRP allows stratification of subjects with CRP levels below the range used for infectious disease workup . MoreoveTo determine the TNF-α -308 G/A gene polymorphism, we extracted genomic DNA from the leukocyte-containing pellets remaining after centrifugation of coagulated blood using the QIAmp DNA Blood Mini Kit . The TNF-α G-308A polymorphism was assessed by fluorescent real-time polymerase chain reaction with melting curve analysis on a LightCycler  using the TNF-α G-308A ToolSet for LightCycler  containing specific primers and fluorescent mutation detection oligonucleotide probes, in conjunction with the Roche Light Cycler Hyb Probe Master Mix  according to the manufacturer's protocols. For statistical analyses, we used the following groups: a) the GG variant, and b) the rarer AA and GA genotypes combined.Descriptive data are presented as means ± SD or as median and interquartile range for severely skewed data. To approximate a normal distribution, we log transformed TNF-α and CRP values. General linear models were employed to elucidate the proportion of variance explained of log-transformed plasma TNF-α and CRP values (dependent variables). Independent variables were smoking status (regular smokers vs. non-smokers), gene variant, and an interaction term between smoking status and gene variant. Following this crude analysis, we entered possible covariates  into the equation. Results were considered statistically significant at the p ≤ .05 level; all tests were 2-tailed. To minimize possible type II errors when assessing interaction terms, we considered interaction terms when the F-statistic on the interaction term had a p-value < 0.2 . All regTable After excluding irregular smokers, we found the GG wild type polymorphism in 203 subjects (74%); GA heterozygote were 70 participants (25%) and AA homozygote were 3 participants (1%). Calculated allele frequencies amounted to 0.86 for the G allele and to 0.14 for the A allele. Regular smokers were slightly though not significantly more frequent amongst subjects with the GG genotype than amongst individuals carrying the GA or AA genotype . Table 1,250 = 5.67, p = .018), but not for genotype . The interaction-term between genotype and smoking status failed to gain significance , indicating that the effect of smoking on plasma levels of CRP is not affected by the TNF-α -308G/A polymorphism . Of the considered covariates, gender , age , and HDL-C  were associated with CRP levels, but not BP or LDL-C. Men, older individuals, and those with lower HDL-C had higher plasma levels of CRP.The adjusted analysis regressing log-transformed CRP levels against smoking status, genotype, and smoking-status-genotype interaction revealed a main effect for smoking status  and for smoking , as well as for the smoking-status-genotype interaction. However, the adjusted model revealed a significant main effect for HDL-C , suggesting that individuals with higher plasma levels of TNF-α had lower HDL-C. Post-hoc analyses revealed no interaction between smoking status and HDL-C or genotype and HDL-C.The fully adjusted model with plasma levels of TNF-α also failed to show significant main effects for genotype (FGenes, health-behavior and the psychosocial environment interact to determine whether or not, and, if at all, how rapidly silent atherosclerosis will progress to the clinical manifestation of CAD ,35. The While plasma levels of the proinflammatory cytokine TNF-α are regulated by several polymorphisms of the TNF-α gene , it is tvitro upon stimulation with lipopolysaccharide than cells from individuals being homozygous for the G allele [Interestingly, blood cells from individuals who carry the A allele of the TNF-α G-308A gene polymorphism express more TNF-α in G allele . DespiteG allele -25. TherIn spite of two recent studies, which found an interaction between smoking and polymorphisms of molecules participating in the inflammatory response ,15, the It must be noted that our findings are preliminary, and, they do not allow us to reject the overall hypothesis of a smoking-gene interaction modifying inflammatory processes contributing to atherosclerosis initiation and progression ,15. For in vitro studies [The lack of a difference in plasma TNF-α levels between subjects with the A allele as compared to those homozygous for the G allele stands in contrast to previous  studies ,18. Howe studies  may playOur study suggests that both plasma TNF-α and CRP levels are not regulated by an interaction between smoking and the G-308A polymorphisms of the TNF-α gene promoter site. We thus remain far from adopting a clinical practice that would counsel smokers to quit smoking based on a particular gene polymorphism. Nonetheless, our results do not refute the overall hypothesis that genetic polymorphisms along the inflammatory pathway may account for the differential effect of tobacco consumption on the cardiovascular risk in individuals who smoke.The authors declare that they have no competing interests.MG participated in the design of the study and drafted the first version of the manuscript.JF participated in the design of the study, in data acquiring, performed the statistical analyses, and critically revised the manuscript.FM carried out and supervised the molecular genetic studies.RvK participated in the design of the study and wrote the final version of the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Biologists have long known that the African great apes  are our closest relatives, evolutionarily speaking. The recent release of the chimp draft genome sequence confirms this relationship at the nucleotide level, showing that human and chimp DNA is roughly 99% identical. Given the genetic similarity between human and nonhuman primates, the next big challenge is to identify those changes in the human genotype (the genetic complement of an organism) that generated the complex phenotype  that distinguishes humans from the great apes. For example, modern humans have larger brains and a larger cerebral cortex than both nonhuman primates and their forebears, the early hominids. Elucidating the molecular mechanisms that account for this expansion will provide insight into brain evolution.ASPM gene cause microencephaly, a rare incurable disorder characterized by an abnormally small cerebral cortex. Since the microencephalic brain is about the same size as the early hominid brain, researchers hypothesized that ASPM—whose normal function is unclear—may have been a target of natural selection in the expansion of the primate cerebral cortex. Last year, researchers showed that selective pressure on the ASPM gene correlated with increased human brain size over the past few million years, when humans and chimps diverged from their common ancestor. Now, Vladimir Larionov and colleagues report that the selective pressure began even earlier—as far back as 7–8 million years ago, when gorillas, chimps, and humans shared a common ancestor.One way to figure out which genes are involved in a physiological process is to analyze mutations in the genotype that generate an abnormal phenotype. Such efforts are easier in the relatively rare instance that one gene affects a single trait. Mutations in the ASPM gene, including promoter and intronic (noncoding) sequences, from chimpanzees, gorillas, orangutans, and rhesus macaques. They sequenced these YACs to determine the complete genomic sequence of the ASPM gene from each species. Next, they characterized sequence changes among these species, based on whether the resulting substitutions in amino acids produced changes in the ASPM protein, to determine how fast the protein was evolving. Larionov and colleagues found that different parts of the protein evolved at different rates, with the rapidly evolving sequences under positive selection  and the slowly evolving sequences under “purifying” selection (significant disruptions were jettisoned). Positive selection on genes is one important way to drive evolutionary change.The researchers used a newly developed technology  to extract specialized cloning agents in yeast  containing the entire ASPM gene, Larionov and colleagues show that the increase in human brain size—which began some 2–2.5 million years ago—happened millions of years after the gene underwent accelerated selective pressure. The ASPM gene, they conclude, likely plays a significant role in brain evolution. The next big challenge will be identifying the forces that preferentially acted on the human genotype to kick-start the process of brain expansion, forces that promise to shed light on what makes us human. New genomic technologies like TAR-cloning will likely accelerate this process.By reconstructing the evolutionary history of the


Vascular smooth muscle cells (VSMC) lining the vessel wall respond to growth factors and other stimuli released by injured cells. However, the extracellular matrix (ECM) may differentially modulate VSMC responses to these growth factors, such as proliferation, migration and adhesion. Our previous reports of low-level expression of one ECM molecule, laminin-5, in normal and injured vessels suggest that laminin-5, in addition to growth factors, may mediate VSMC response following vascular injury. To elucidate VSMC response on laminin-5 we investigated-the role of platelet-derived growth factor (PDGF-BB) in activating the mitogen-activated protein kinase (MAPK) signaling cascade as a possible link between growth-factor initiated phenotypic changes in vitro assays we assessed rat vascular smooth muscle cell (rVSMC) responses plated on laminin-5 to the addition of exogenous, soluble PDGF-BB. Our results indicate that although laminin-5 induces haptotactic migration of rVSMC, the addition of PDGF-BB significantly increases rVSMC migration on laminin-5, which is inhibited in a dose-dependent manner by the MAPK inhibitor, PD98059, and transforming growth factor (TGF-β1). In addition, PDGF-BB greatly reduces rVSMC adhesion to laminin-5, an effect that is reversible by MAPK inhibition or the addition of TGF-β1. In addition, this reduction in adhesion is less significant on another ECM substrate, fibronectin and is reversible using TGF-β1 but not MAPK inhibition. PDGF-BB also strongly increased rVSMC proliferation on laminin-5, but had no effect on rVSMC plated on fibronectin. Finally, plating rVSMC on laminin-5 did not induce an increase in MAPK activation, while plating on fibronectin or the addition of soluble PDGF-BB did.Using a system of These results suggest that rVSMC binding to laminin-5 activates integrin-dependent intracellular signaling cascades that are different from those of fibronectin or PDGF-BB, causing rVSMC to respond more acutely to the inhibition of MAPK. In contrast, our results suggest that fibronectin and PDGF-BB may activate parallel, reinforcing intracellular signaling cascades that converge in the activation of MAPK and are therefore less sensitive to MAPK inhibition. These results suggest a partial mechanism to explain the regulation of rVSMC behaviors, including migration, adhesion, and proliferation that may be responsible for the progression of restenosis. Angioplasty is a procedure designed to treat vascular stenosis, blockage(s), or atherosclerotic lesions, but it may also, simultaneously, cause damage to the integrity of the blood vessel wall. Restenosis is the subsequent narrowing and occlusion of the blood vessel in response to the injury or damage sustained during angioplastic procedures such as balloon dilation . During The characteristic response of VSMC, endothelial cells, platelets, and macrophages at the site of injury is the release of specific soluble growth factors which include platelet-derived growth factor (PDGF), transforming growth factor (TGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) -4. VSMC To date, the precise molecular mechanisms that link growth factor-initiated intracellular signaling to ECM-mediated adhesion, migration, and proliferation of VSMC are still unknown. Our previous reports of low-level laminin-5 expression in the intima of normal vasculature and an increased expression of laminin-5 in the neointima of injured vessels suggest that laminin-5, in addition to PDGF and TGF, may mediate VSMC responsiveness following vascular injury -13.in vitro assays to study the role of laminin-5 in modulating these behaviors in rat vascular smooth muscle cells (rVSMC). We report here that PDGF induces differential responses in rVSMC behaviors on laminin-5, but not on fibronectin. In addition, we find that the PDGF-induced responses on laminin-5 are inhibited in a dose-dependent manner by an inhibitor of the mitogen-activated protein kinase (MAPK) pathway, PD98059, but not on fibronectin.To further elucidate VSMC response to growth factors and the intracellular signaling cascades that may be linked to ECM-mediated adhesion, we used in vitro may be the result of different signaling pathways that are initiated by integrin-mediated adhesion to laminin-5. We suggest that rVSMC binding to laminin-5 may initially activate a MAPK-independent signaling cascade that may make these cells more responsive to MAPK inhibition. In contrast, rVSMC binding to fibronectin may activate a signaling pathway shared by PDGF that ultimately converges in MAPK activation. These results suggest that a complex interaction of ECM and growth factors may closely regulate VSMC behavior following vascular injury and these studies may directly identify define molecular targets that may reduce the incidence of restenosis following angioplasty.These differences in MAPK-sensitive rVSMC responses We have previously reported that laminin-5 expression by rat vascular smooth muscle cells (rVSMC) is upregulated by platelet-derived growth factor (PDGF-BB) and that laminin-5 specifically enhances PDGF-BB-stimulated rVSMC migration . In addiThe level of rVSMC migration was observed over 18 hours in transwell migration filters in the presence or absence of laminin-5, with and without PDGF-BB or serum. Maximal cell migration (chemotaxis) was obtained using cell migration media containing ten (10) percent fetal calf serum (FCS). Similar to our previous reports, laminin-5-coated wells induced a greater than four-fold increase in rVSMC migration over that measured in naked plastic controls in three independent experiments as shown in Figure The mitogen-activated protein kinase (MAPK) pathway is known to be activated by PDGF-BB-stimulation in VSMC . To explTo determine if this modulation is restricted to MEK/MAPK-inhibition, we tested the effects of adding exogenous transforming growth factor TGF-β1) for its ability to modulate the PDGF-stimulated increase in rVSMC migration on laminin-5. Our results indicated that the addition of TGF-β1 was able to reduce laminin-5-stimulated rVSMC migration to levels approximating the levels observed in naked plastic controls over all tested ranges (5 – 50 ng/mL), although the most statistically distinct reduction was found over the range of 5 – 25 ng/mL, as shown in Figure  for its in vitro adhesion assays were performed. In the absence of exogenous growth factor stimulation, laminin-5- and fibronectin-coated wells (20 μg/mL) sustained an approximate two and a half-fold increase in rVSMC adhesion compared with negative controls, as shown in Figure To determine whether or not the PDGF-BB-stimulated increase in rVSMC migration correlates with a reduction in rVSMC adhesion to laminin-5, thirty-minute ed wells 0 μg/mL sTo investigate if the relationship between the PDGF-BB-stimulated increase in migration and corresponding decrease in adhesion of rVSMC on laminin-5 may be related to MAPK activation, cells were pre-treated with PD98059 (40 ng/mL) for twenty (20) minutes prior to assay and the adhesion media was supplemented with PD98059 (40 ng/mL). The addition of exogenous PD98059 (40 ng/mL) restored the PDGF-BB-stimulated reduction (25 ng/mL PDGF-BB) in rVSMC adhesion to laminin-5, to approximately eighty-five (85) percent of laminin-5 controls . The addition of PD98059, however, did not restore the thirty (30) percent PDGF-BB-stimulated reduction in rVSMC adhesion on fibronectin.To determine if this modulation is restricted to MEK/MAPK-inhibition, we tested the effects of adding exogenous transforming growth factor (TGF-β1). Our results indicated that the addition of TGF-β1 (25 ng/mL) was able to restore the PDGF-BB-stimulated reduction in rVSMC adhesion on laminin-5 by roughly the same level as PD98059, eighty two (82) percent versus eighty five (85) percent, respectively. In contrast to PD98059, the addition of TGF-β1 (25 ng/mL) was sufficient to restore the PDGF-BB-stimulated reduction in rVSMC adhesion on fibronectin.in vitro proliferation assays to determine the relative effects of the extracellular matrix (ECM) and exogenous growth factors described above. First, to test the effects of the ECM substrate on rVSMC proliferation in the absence of exogenous growth factors, quiescent cells were plated in cell-culture plates either coated with or without laminin-5 or fibronectin, in serum-free Dulbecco's Modified Eagle's Medium (DMEM) for one (1) to six (6) days.Based upon our observations of rVSMC migration and adhesion, we performed 0(G1) between forty eight (48) and seventy two (72) hours as determined by flow cytometry and determination of [3H] thymidine-labeled nuclei [To induce quiescence, rVSMC were incubated for forty eight (48) hours without mitogen  at 37°C and quiescence was verified by proliferation controls, cultured for forty eight (48) hours at 37°C with mitogen stimulus as shown in Figure d nuclei .Our results suggest that culture of rVSMC plated on exogenous laminin-5 (coated at a concentration of 20 μg/mL) did not significantly increase cellular proliferation compared with naked plastic controls over a period of four (4) days, as shown in Figure Next, to test the modulating effects of exogenous growth factors on rVSMC proliferation when cultured on these ECM substrates, quiescent cells were plated in cell-culture plates either naked or coated with laminin-5 or fibronectin, in the presence of TGF-β1 or PDGF-BB, both at a concentration of 25 ng/mL, from one (1) to six (6) days. Our results indicated that the addition of exogenous TGF-β1 (25 ng/mL) to the cell culture medium was sufficient to induce a suppressing effect on rVSMC proliferation on naked plastic, as well as laminin-5 and fibronectin, to levels approximating the quiescent growth controls.The addition of PDGF-BB was sufficient to induce an increase in rVSMC proliferation on laminin-5 of nearly one-half (46%) over laminin-5 DMEM  controls . However, our results indicate that the addition of exogenous PDGF-BB (25 ng/mL) did not have a statistically significant effect on cells plated on fibronectin.To examine MAPK activation, rVSMC were plated onto laminin-5- or fibronectin-coated plates, then lysed after thirty minutes and prepared for immunoblotting. Our results indicated that laminin-5 did not induce a detectable increase in MAPK activation over a thirty (30) minute time interval, as shown in Figure The addition of growth factors, such as PDGF-BB and TGF-β1, have been associated with increases in p44/p42 (ERK1/2) or MAPK activation -18. To din vitro VSMC mitogen and may be responsible for initiating the phenotypic changes in VSMC migration and proliferation during restenosis in vivo [PDGF-BB is an  in vivo ,20. Our  in vivo -13.in vitro [The current study augments the body of evidence that suggests VSMC growth is influenced by an ECM-VSMC interaction  and thatin vitro ,8,10. Moin vitro, driven by PDGF-BB responsiveness, can be blocked by MAPK inhibition (via MEK1 inhibitor: PD98059) on laminin-5, but not on fibronectin.Because PDGF-BB stimulation increases the overall levels of intracellular MAPK, as well as MAPK phosphorylation, we sought to explore this signaling cascade to determine its role in modulating these rVSMC behaviors on laminin-5 . SpecifiAn additional signaling regulator, TGF-β1, has been implicated in the negative regulation and decreased rate of proliferation of VSMC stimulated with serum or PDGF -28. Our 1 phase of mitosis.Several lines of evidence now suggest that the anti-mitogenic effects of TGF-β1 may be dissociated from inhibition of ERK1/2 signaling pathways ,18. ThesAlthough our previous reports linked ERK1/2 to rVSMC adhesion and migration, these studies did not examine the possibility for differential signaling initiated by rVSMC binding to laminin-5 or fibronectin ,13. Expain vitro. Based upon these findings, we postulate that PDGF-BB and laminin-5 binding may initially activate different intracellular signaling cascades, causing rVSMC to be more responsive to the inhibition of MEK1 and MAPK on laminin-5 than those activated on fibronectin, as outlined in Figure Our results indicate that laminin-5 activates different intracellular signaling pathways from those of fibronectin and PDGF-BB in rVSMC and that binding to laminin-5 may modulate rVSMC behaviors that are distinctive from those modulated by fibronectin. Although binding of rVSMC to laminin-5 may not cause an initial increase in MAPK activation levels or proliferation, laminin-5 can augment PDGF-BB-stimulated proliferation and migration of rVSMC In contrast to laminin-5, fibonectin and PDGF-BB may have parallel, reinforcing roles in MAPK activation. Our analysis of the effects of TGF-β1 demonstrated that TGF-β1 does not strongly activate MAPK in rVSMC, but rather strongly inhibits the effects of fibronectin and PDGF-BB-stimulation on laminin-5. Our results support the previous findings that TGF-β1 may inhibit mitogenesis and other VSMC behaviors via mechanisms independent of MAPK activation.These results suggest that a clear understanding of the roles and contributions of each ECM may provide new insights into the mechanisms of regulating rVSMC cell behaviors, including migration, adhesion, and proliferation. Further analysis of the events that trigger and sustain the underlying cellular mechanisms of rVSMC behaviors may help aid in the design of more effective therapies for the treatment of restenosis.2 in humidified chambers. Cells were maintained in DMEM High Glucose, supplemented with 10% fetal bovine serum and 1% L-glutamine (29.2 mg/mL), penicillin G , and streptomycin sulfate  (GPS) from Irvine Scientific . Rat aortic smooth muscle cell explants were a gift from RC Smith and were isolated and passaged as previously described [0(G1) arrest can routinely be induced by incubation of cells for 72 h. in low-mitogen (0.5% FBS) medium [Cells were maintained in 100 mm × 20 mm Corning tissue-culture dishes  at 37°C and 5% COlutamine .2 mg/mL,) medium ,30, thes5 in each of 96-transwell chamber filters (100 μL of 1.2 × 106 cells/mL solution) with and without ECM in the presence or absence of PDGF-BB at the indicated concentrations (5–25 ng/mL) and allowed to migrate for 18 hours at 37°C. Where applicable, the medium was supplemented with PD98059 (MEK1-inhibitor) at the indicated concentration. Cells were counted at the end of an 18-hour interval as indicated, quantified with the following modification. 30 minutes prior to measuring migration, 5 μM calcein AM from Molecular Probes  was added to the migration wells at 37°C. To quantitate migration, cells were removed from the top of the filter with cotton-tipped applicators and fluorescence of the incorporated calcein was measured from the bottom of the filter with a fluorescence plate reader. Relative fluorescence values for each experimental condition are expressed relative to controls and untreated samples.Cell migration assays were performed in Costar transwell filter plates either coated with purified matrix (laminin-5 or fibronectin) at a protein concentration of 20 μg/mL for one hour (60 min.) at room temperature, 25°C, and washed twice with phosphate-buffered saline 0.2% Tween-20 and 5% skim milk (PBST) prior to assay as previously described ,32. Cell5 in each of 96-transwell chamber filters (100 μL of 1.2 × 106cells/mL solution) with and without ECM-coating (described above) in the presence or absence of PDGF-BB (25 ng/mL), TGF-β1 (25 ng/mL), or both, and allowed to attach for 30 minutes at 37°C. Where applicable, cells were first incubated for 20 minutes with PD98059 (40 ng/mL), a MEK1 inhibitor from New England Biolabs  at 37°C and the adhesion assay culture medium was supplemented with PD98059 at 40 ng/mL. Following adhesion, non-adherent cells were removed by suspending plates upside down in a rotating tank of PBS for 10 minutes at room temperature, 25°C. Adherent cells were then fixed and stained and the relative absorbance was measured using a TECAN-SPECTRAFluor spectrophotometer  at 595 nm.Cell adhesion assays were performed as previously described ,32 using5 in 100 mm2 cell culture plates with and without fibronectin or laminin-5 and allowed to attach overnight (12 h.) at 37°C. Cells were then starved in serum-free DMEM for 48 hours to induce quiescence at 37°C, as outlined in Cell Culture Methods below. The medium was then replaced with fresh medium containing 25 ng/mL of TGF-β1 or PDGF-BB obtained from Calbiochem  and incubated at 37°C. Cells were removed from culture wells with trypsin/EDTA and counted using trypan blue stain from Gibco Life Technologies  and a VWR Scientific Counting Chamber  at 24 hour intervals, from 1 – 6 days.Tissue culture plates were coated with purified fibronectin from Calbiochem  or laminin-5 from Demos  at a 20 μg/mL protein concentration for 1 hour (60 min.) at room temperature, 25°C as previously described . Cells wQuiescent VSMC were pre-treated with culture medium containing 10% FCS, PDGF-BB (25 ng/mL), TGF-β1 (25 ng/mL), or plated on laminin-5- or fibronectin-coated tissue culture plates with DMEM for 30 minutes at 37°C. Subconfluent cell cultures were then lysed using ice-cold, 1X lysis buffer  and boiled in SDS Boiling Buffer prior to analysis as previously described . Proteint distribution. All samples were measured using two-tailed t tests as departure from normality can make more of a difference in a one-tailed than in a two-tailed t test. So long as the sample size is even moderate (>20) for each group, quite severe departures from normality make little practical difference in the conclusions reached from these analyses [The differences between untreated and treated cell populations were measured using a analyses .The author(s) declare that they have no competing interests.KK carried out the migration, adhesion, and proliferation assays, the Western Blot analysis and assisted with experimental design. GEP conceived, monitored, and coordinated the experimental design. Both KK and GEP contributed equally to the writing of this manuscript.


Genomic sequence variation is the hallmark of life and is key to understanding diversity and adaptation among the numerous microorganisms on earth. Analysis of the sequenced microbial genomes suggests that genes are evolving at many different rates. We have attempted to derive a new classification of genes into three broad categories: lineage-specific genes that evolve rapidly and appear unique to individual species or strains; highly conserved genes that frequently perform housekeeping functions; and partially variable genes that contain highly variable regions, at least 70 amino acids long, interspersed among well-conserved regions. The latter we term segmentally variable genes (SVGs), and we suggest that they are especially interesting targets for biochemical studies. Among these genes are ones necessary to deal with the environment, including genes involved in host–pathogen interactions, defense mechanisms, and intracellular responses to internal and environmental changes. For the most part, the detailed function of these variable regions remains unknown. We propose that they are likely to perform important binding functions responsible for protein–protein, protein–nucleic acid, or protein–small molecule interactions. Discerning their function and identifying their binding partners may offer biologists new insights into the basic mechanisms of adaptation, context-dependent evolution, and the interaction between microbes and their environment. Segmentally variable genes show a mosaic pattern of one or more rapidly evolving, variable regions. Discerning their function may provide new insights into the forces that shape genome diversity and adaptation Microbes occupy almost every habitable niche in the biosphere, highlighting their enormous capability for adaptation and survival. This adaptive ability has been refined during millennia of evolution and has resulted in genes that evolve at very different rates. Some, such as housekeeping genes that code for the essential biochemical functions of the organism, are now evolving rather slowly. Others that have to defend against potentially lethal attack by viruses or toxins and adapt to varying environmental conditions, often evolve more rapidly . PathogeAn example of an SVG family is provided by the cytosine-5 DNA methyltransferases . These eTo the best of our knowledge, there has been no systematic attempt to identify, catalog, and classify similar SVGs in the sequenced microbial genomes. Nor has any attempt been made to find potentially common functions among genes displaying this property. Since it is known that many genes involved in defense mechanisms, such as the DNA methyltransferases and the antigens exposed on the surface of bacteria, show such variability , it is thttp://geneva.bu.edu) is also provided with results for several selected genomes, including Escherichia coli, Helicobacter pylori, Neisseria meningitidis, and several others. Each gene is accompanied with schematic diagrams from Pfam . SVGs are defined as genes containing at least one highly variable region, containing more than 70 amino acids, interspersed among well-conserved regions. In any single SVG family, the length of the variable region can differ only within a certain range  space, where the x-axis shows the length of the variable region and the y-axis shows the number of possible homologs of each gene. Lineage-specific genes , which in this case recognizes the DNA sequence CCTC  and C-terminus (approximately 100 amino acids). VacB has been shown to encode a 3′–5′ exoribonuclease and is necessary for expression of virulence  to 8.6 MB(Streptomyces coelicolor)  and a low percentage of lineage-specific genes (0.2%). However, when a closely related species, M. pneumoniae, is excluded from the database, its proportion of lineage-specific genes rises to 14%, while the proportion of SVGs remains unchanged. In general, the genomic proportion of SVGs is less affected by the database content.The number of SVGs increases as genome sizes vary, from 0.5 MBlicolor) . For mosIn the following sections, we have selected several SVG families to demonstrate the functional implication of segmental variability.Ralstonia solanacearum [Rs] and Ralstonia metallidurans [Rm] in , e.g., genes encoding methyl-accepting chemotactic protein (MCP)  than in other genomes (see the gene list of V. cholerae at http://geneva.bu.edu), which must correspond to its expanded ability to detect different chemical signals and find favorable environments. Although a few conserved motifs have been detected in the sensing region  and a response regulator. Most HKs are membrane-bound, homodimeric proteins with an N-terminal periplasmic sensing domain and a C-terminal cytoplasmic kinase domain. HKs usually possess a highly variable sensing domain , while the cytoplasmic kinase domain is quite conserved. By diversifying the sensing domain, microorganisms can develop different two-component modules to respond to different signals and interact with small molecules from the exterior. g region , the exaThe biggest family of SVGs is cell envelope-related, including the ATP-binding cassette transporters (ABC transporters), outer-membrane proteins, and virulence-related gene products. For membrane proteins, since part of their sequences are exposed to the outside of the cell and interact directly with the environment, one might hypothesize that the variable portions have evolved rapidly to deal with the changing environmental conditions.hetA and spaB shown in E. coli has been solved (E. coli BtuB overlaps with the 22-strand β-barrel (position 150–360), while the N-terminal hatch domain (position 6–132) and the extreme C-terminal TonB-box domain (position 550–614) are conserved among many homologs  and a large (over 300 amino acids) variable integral membrane domain. Two examples from this group are the multidrug-resistance genes n solved . The varhomologs . The exthomologs  and possgyrB) and DNA topoisomerase A (topA), whose competing actions control the degree of DNA supercoiling  is variable both in sequence content and in length A. The corculosis A. Interelymerase  and in Dlymerase . Since Rlymerase . Deletioresponse . All togVariable regions are sometimes found in DNA processing enzymes with essential and conserved functions. One example is DNA polymerase I, which has a variable region between the conserved C-terminal 5′–3′ polymerase domain and the N-terminal 5′–3′ exonuclease domain. In some polymerases, this region encodes a 3′–5′ exonuclease activity for proofreading replication errors, and conserved motifs can be observed . HoweverE. coli ProRS are known to be involved in the posttransfer editing for mischarged Ala-tRNAPro  . This fa-tRNAPro . ProRS i-tRNAPro . Partialcylation , suggestbilities . As a reN-acetyl muramyl pentapeptide; SpsA: unknown). Owing to the lack of homology in the acceptor binding domains, the substrate specificities encoded by these regions for most GTs are still unknown.Variable regions exist in carbohydrate metabolizing enzymes, such as glycosyltransferases (GTs) and glycoside hydrolases (GHs), which respectively catalyze the biosynthesis of diverse glycoconjugates and their selective cleavage . Many paProkaryotic transcriptional regulators form another large group of SVGs. Transcription regulators are usually two-domain proteins with one binding to DNA and one binding to ligand. The DNA-binding domains, which usually interact with DNA via helix–turn–helix, zinc-finger, or other modes, are more conserved than ligand-binding domains. Based on the characteristic conserved DNA-binding domains, transcriptional regulators can be classified into many different families . Even wiH. pylori: 17 genes; N. meningitidis: 32 genes; V. cholerae: 69 genes, etc.; see http://geneva.bu.edu for the full list). In contrast to lineage-specific hypothetical genes, these hypothetical genes contain conserved domains, which suggest their functional importance. Although most of the conserved domains in these hypothetical genes have currently unknown function, there are a few exceptions. Among them are the prokaryotic mechanosensitive channel proteins, which respond to external osmotic pressure . The central regions (approximately 150 amino acids) of these genes are conserved while both the N-terminal region (approximately 100 amino acids) and the C-terminal region (approximately 100 amino acids) are variable . H. pylori is one of the few microbes that can colonize the highly acidic gastric environment , which appear to be segmentally variable. The unique pattern of cell surface glycosylation in S. pneumoniae has been under extensive investigation and plays an important role in pathogenesis . In 2 test. For all genomes examined, there is a strong association between gene duplication and SVGs, suggesting an SVG is more likely to have originated from a duplicated gene.In H. pylori strain 26695, gene products of HP1299 (253 amino acids) and HP1037 (357 amino acids) both have a conserved domain  that is characteristic of the methionyl aminopeptidase (map) family   that is variable among its similar genes  binding  . HP1299 map gene , while tar genes . Additiofunction . A functfunction . In mostA major fraction of bioinformatics research on sequence analysis has focused on the conserved regions in proteins, trying to hypothesize the role of the protein by identifying sequence motifs that have been shown experimentally to correlate with a specific function. Some work has gone into cataloging the groups of lineage-specific proteins that show no similarity to other proteins in GenBank , but theIn this paper we have initiated an effort to identify SVGs, which contain both well-conserved regions and highly variable regions. By looking carefully at a few specific examples where functional information is available from experimental data, we find that the variable region often seems to play a key role in mediating interactions with other molecules, both large and small. Sometimes the variable portions are involved in biological processes with a component of interaction between the cell and agents from the external environment. For instance, the DNA methyltransferases are part of a defense system that recognizes and clears invading foreign DNA; membrane-bound sensory HKs and mechanosensitive ion channels, etc., monitor changes of living conditions. Sometimes the variable portions are involved in intracellular processes that appear to have lineage-specific features. Thus, the variable regions inside DNA GyrB and several types of AARSs probably determine the specificity of substrate recognition. The detailed factors that introduce the molecular variability may go well beyond our explanations here and likely vary from case to case. Some variable regions may have diverged a long time ago and are now kept constant, while others may keep changing. In all of these cases, SVGs are exceptionally worthy targets of further experimental investigation, and such investigations can be greatly aided by the presence of the conserved regions that may suggest a preliminary function to be tested.H. pylori, none of them had hits with a disrupting stop codon in the variable region; 20 of them have hits with genes showing insertions/deletions that cause frame shifts in the variable region. However, the real number is likely to be much fewer, since, based on previous work, many of them may be the results of sequencing errors . We have also examined the amino acid composition, codon usage, and GC content in the variable regions and the conserved regions of the same SVG. While there is no significant deviation of amino acid composition and GC content between the two regions in general, codon usage appears to be biased in the variable regions (data not shown).SVGs usually account for 10%–20% of the total genes in a microbial genome. Currently, we think of the class of lineage-specific genes as being the key factor that distinguishes one strain or species from another. The class of SVGs that we have defined in this paper must now be added to this collection of lineage-specific genes by virtue of the unique segments that constitute their variable regions. They also appear to provide functional elements that help to differentiate among strains and species. This point is well illustrated by considering the restriction-modification systems. Here, the DNA methyltransferases, which have a variable region responsible for DNA recognition, are members of the SVG class. With the help of their companion restriction endonucleases, which typically appear as lineage-specific genes, they serve to keep foreign, unmodified DNA sequences from entering the genome. In this case, the synergy of function provided by members of the two classes highlights the key role that both sets of genes must play in defining the individuality of a strain or species.Our analysis to date is limited to prokaryotes and archaea where SVGs are transcribed and translated as contiguous genomic segments. In eukaryotes, alternative RNA splicing introduces substantial additional complexity into the interpretation of gene structure and protein product, thereby rendering impossible the simple analysis we have applied here. It is tempting to consider alternative splicing as a highly evolved control mechanism to introduce the variability we find in the SVGs and thereby achieve the functional diversity necessary for cell survival under different conditions. In eukaryotes, alternatively spliced exons can be introduced in response to the functional demands of different cell types by merely juggling protein coding regions in the genome, thereby creating an SVG structure. If this view is correct, then it reinforces and highlights the importance of these SVGs to the workings of the cell.In this paper we have provided an initial glimpse of SVGs, which appear to provide an important genetic layer in the adaptation of cells to novel environments and hazardous pathogens. We have focused attention on the biological significance of these genes, especially those that have highly diverged segments. We are currently trying to develop a more refined classification of these genes so as to explore the functional significance of the variability. We would like to know whether extreme variability is required for diverse function or whether more modest variation is sufficient. Such questions require that we can first distinguish positive selection acting on these variable regions from neutral evolution leading to gene decay and eventual loss. Since the variable regions we report are often not amenable to current tools available for alignment, we are exploring new methods that will help us to assess whether positive selection is driving the evolution of these genes.In summary, we have identified an extremely useful way of classifying genes that leads to the identification of those with a high priority for both experimental and computational research.Our method for detecting SVGs includes several steps: (1) identification of similar genes followed by query-anchored multiple alignment using nongapped BLAST ; (2) taxE-value is less than 1E-10. Here we use a strict E-value threshold to avoid possible functional divergence among the homologs. Accordingly, we use the BLOSUM80 scoring matrix in the BLASTP search, although the result does not change dramatically if BLOSUM62 is used. Second, the overall length of the hit sequence does not differ significantly from the query sequence. We define the gap content (GapC) between two sequences:Given a gene, we start by searching for all its similar genes in the nonredundant database  using nongapped BLAST . We use L,l are the lengths of the protein sequences of two genes. It is a measure of the smallest percentage of gaps needed to be introduced into the pairwise alignment. Sequences with a high GapC value indicate significantly different domain structures, possibly owing to domain insertions or losses, and thus are excluded from the set of similar genes. In our current implementation, we require that GapC must be less than 0.2.where Similar genes reported by BLASTP are not evenly distributed among different species. In many cases, highly similar genes from different strains of the same species or highly similar paralogous genes from a particular strain tend to introduce bias into the dataset. We adopted a simple taxonomy clustering by using the NCBI Taxonomy Database  to reducWe collapse all the similar genes from the same species into a single group. Then we choose the gene with the best similarity score to the query sequence as the representative of that species for later calculations. The definition of species follows the hierarchical taxonomy used in the NCBI Taxonomy database (superkingdom → phylum → class → subclass → order → family → genus → species → no rank [strain]). By doing taxonomy clustering, we are able to collect a less biased sample of similar genes from different species.http://geneva.bu.edu). Two unaligned regions in two sequences are considered as the variable regions if they are bounded by similar HSPs at both ends . To avoid the possibility of a large segment containing insertions or deletions, we again require that GapC be less than 0.2 between these two unaligned regions.Query-anchored multiple alignment after taxonomy clustering is performed by aligning the HSPs reported by nongapped BLAST (see (m) it is inside an HSP region and the number of times (n) it is inside a variable region. A high ratio of n over m + n suggests that this position is inside the variable region most of the time. We estimate the statistical significance  of the variability for each position by a binomial distribution:For each amino acid position in the query gene, we can count the number of times q is the probability of an amino acid position being inside a HSP region. We estimate q by averaging the proportion of HSP in each hit sequence among all hits. If the p-value calculated using the above formula is less than the significance level, which we set at 0.05, we then consider this position as a variable position; otherwise, it is a conserved position. A consecutive run of variable positions forms a variable region. The next question is how long the variable region should be to be considered meaningful, as opposed to functionally unimportant regions such as linker regions, which are usually short. From our experience, there is no clear decision boundary between the length of the region and its functional importance. Any choice of cutoffs would have to balance between false positives and false negatives. However, previous studies on the length distribution of protein domains has shown that the most likely length of a protein domain is around 70 amino acids, and regions shorter than this are less likely to form a functional domain (H. pylori.where l domain . Based on over (m + n) for each position and plotting it. We call such plots variability profiles. Sample variability profiles are shown in A direct way of visualizing the variability of a protein sequence is by calculating the ratio of http://geneva.bu.edu. SVGs for several representative genomes are listed there. For SVG lists in other genomes, please request more information from Y. Zheng at E-mail: zhengyu@bu.edu. All the case examples mentioned throughout the paper and Supporting Information have been compiled into one Web page, http://geneva.bu.edu/paper03.html, with hyperlinks. Readers can follow each hyperlink to access additional information from Pfam, BLOCKS, PRINTS, COG, and nongapped BLAST for each gene.We provide a static collection of segmentally variable genes at our Web site, Figure S1Conservation score is plotted under the alignment . The conserved portions are as follows: N-terminal domain, extreme C-terminal domain, and a segment between N-terminal and C-terminal domain. The variable domain  overlaps with the transmembrane 22-strand β-barrel regions.(2.69 MB EPS).Click here for additional data file.Figure S2H. pylori ProRS. The blue segments are HSPs reported by nongapped BLAST. The yellow segments are the variable region. The gray region is the gap-rich region . See http://geneva.bu.edu/paper03.html for a high-resolution Web figure.The query protein is (4.71 MB EPS).Click here for additional data file.Figure S3(3.12 MB EPS).Click here for additional data file.Figure S4YhdY is currently annotated as a hypothetical protein and contains a conserved domain for mechanosensitive proteins  and two variable domains (N- and C-termini).(2.86 MB EPS).Click here for additional data file.Figure S5map). This is an example of a fully conserved gene.It is the methionine aminopeptidase (type Ia (1.87 MB EPS).Click here for additional data file.Figure S6It is currently annotated as “conserved hypothetical protein.” The N-terminal region is variable. The conserved C-terminal domain is characteristic of methionine aminopeptidase.(2.22 MB EPS).Click here for additional data file.Figure S7Shown as a histogram. Only variable regions inside fully conserved genes and SVGs are included. Pink line shows the domain size distribution in 3D-structure database (data from (643 KB EPS).Click here for additional data file.Table S1(62 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/GenBank/) accession numbers for the genes discussed in atpA (2314285), atpD (2314283), atpG (2314284), dnaX (2313841), flgK (2314271), ftsK (2314237), gyrB (2313611), hetA (2314367), HP1450 (2314626), infB (2314195), M.hpyAVIB , mutS (2313742), NQO3 (2314431), NQO8 (2314432), polA (2314647), rps4 (2314460), spaB (2313717), spoT (2313901), tlpA (2313179), and tlpC (2313162).The GenBank (Agrobacterium tumefaciens (15890351), B. subtilis (16079962), Enterococcus faecalis (8100675), E. coli K12 (16128553), L. innocua (16801788), Mycobacterium leprae (15826988), M. tuberculosis CDC1551 (15840173), Nitrosomonas europaea (22955201), Nostoc sp. PCC 7120 (17228666), P. syringae pv. syringae B728a (23470301), Ralstonia metallidurans (22980570), R. solanacearum (17548875), Synechococcus sp. PCC 7942 (21954778), and Thermotoga maritime (15644402); in case studies, B. subtilis yhdY (2633299), E. coli b1330 (1787591), H. pylori cytosine-specific DNA methyltransferase (2313124), H. pylori HP1299 (2314463), H. pylori HP1037 (2314181), H. pylori prolyl-tRNA synthetase (2313329), and H. pylori VacB (2314413).The GenBank accession numbers for the genes discussed in