We tested the hypothesis that dopamine-dependent motor learning mechanism underlies the long-duration response to levodopa in Parkinson disease (PD) based on our studies in a mouse model. By data-mining the motor task performance in dominant and nondominant hands of the subjects in a double-blind randomized trial of levodopa therapy, the effects of activity and dopamine therapy were examined.
We data-mined the Earlier versus Later Levodopa Therapy in Parkinson's Disease (ELLDOPA) study published in 2005 and performed statistical analysis comparing the effects of levodopa and dominance of handedness over 42 weeks.
The mean change in finger-tapping counts from baseline before the initiation of therapy to predose at 9 weeks and 40 weeks increased more in the dominant compared to nondominant hand in levodopa-treated subjects in a dose-dependent fashion. There was no significant difference in dominant vs nondominant hands in the placebo group. The short-duration response assessed by the difference of postdose performance compared to predose performance at the same visit did not show any significant difference between dominant vs nondominant hands.
Active use of the dominant hand and dopamine replacement therapy produces synergistic effect on long-lasting motor task performance during “off” medication state. Such effect was confined to dopamine-responsive symptoms and not seen in dopamine-resistant symptoms such as gait and balance. We propose that long-lasting motor learning facilitated by activity and dopamine is a form of disease modification that is often seen in trials of medications that have symptomatic effects.
The purpose of this study was to test the reliability and validity of a brief language usage scale as a measure of acculturation in 197 Central American immigrant women. This study presents an analysis of cross-sectional survey data collected during face-to-face interviews conducted in Spanish as part of the program evaluation of the Infant Feeding for Hispanic Supplemental Nutrition Program for Women, Infants, and Children (WIC) Populations a Peer Education Model. The Short Acculturation Scale, a four-item language usage scale exploring the participants’ language preferences, was used as a measure of acculturation. The participant’s age, length of time in the United States, and perceived social support for breastfeeding were used as validation measures. Results demonstrated good internal reliability for the acculturation summary scale. Consistent with previous studies, significant correlations (p < 0.01) were found between acculturation and mother’s age, perceived social support for breastfeeding, and mother’s length of time in the United States. The reliability and validity data from this group of Central American immigrants support the continued use of this brief measure of acculturation in diverse Latino subpopulations when multidimensional measures are neither practical nor feasible.
Hispanic; Latino; acculturation
Purpose. To introduce a new anterior segment optical coherence tomography parameter, trabecular-iris circumference volume (TICV), which measures the integrated volume of the peripheral angle, and establish a reference range in normal, open angle eyes. Methods. One eye of each participant with open angles and a normal anterior segment was imaged using 3D mode by the CASIA SS-1000 (Tomey, Nagoya, Japan). Trabecular-iris space area (TISA) and TICV at 500 and 750 µm were calculated. Analysis of covariance was performed to examine the effect of age and its interaction with spherical equivalent. Results. The study included 100 participants with a mean age of 50 (±15) years (range 20–79). TICV showed a normal distribution with a mean (±SD) value of 4.75 µL (±2.30) for TICV500 and a mean (±SD) value of 8.90 µL (±3.88) for TICV750. Overall, TICV showed an age-related reduction (P = 0.035). In addition, angle volume increased with increased myopia for all age groups, except for those older than 65 years. Conclusions. This study introduces a new parameter to measure peripheral angle volume, TICV, with age-adjusted normal ranges for open angle eyes. Further investigation is warranted to determine the clinical utility of this new parameter.
Purpose. To evaluate the normal aging effects on trabecular meshwork (TM) parameters using Fourier domain anterior segment optical coherence tomography (ASOCT) images. Patients and Methods. One eye from 45 participants with open angles was imaged. Two independent readers measured TM area, TM length, and area and length of the TM interface shadow from 3 age groups (18–40, 41–60, and 61–80). Measurements were compared using stepwise regression analysis. Results. The average TM parameters were 0.0487 (±0.0092) mm2 for TM area, 0.5502 (±0.1033) mm for TM length, 0.1623 (±0.341) mm2 for TM interface shadow area, and 0.7755 (±0.1574) mm for TM interface shadow length. Interobserver reproducibility coefficients ranged from 0.45 (TM length) to 0.82 (TM area). TM area and length were not correlated with age. While the TM interface shadow length did not correlate with age, the TM interface shadow area increased with age. Race, sex, intraocular pressure, and gonioscopy score were not correlated with any TM parameters. Conclusion. Although the TM measurements were not correlated with age, the TM interface shadow area increased with age. Further study is required to determine whether there is any relationship between the age-related ASOCT findings of the TM interface shadow area and physiologic function.
Purpose. To evaluate potential risk factors for developing tube shunt exposure in glaucoma patients. Patients and Methods. Forty-one cases from 41 patients that had tube shunt exposure from 1996 to 2005 were identified from the Robert Cizik Eye Clinic and Bascom Palmer Eye Institute. Each case was matched with 2 controls of the same gender and with tube shunts implanted within 6 months of the index case. Conditional logistic regression was used to determine risk factors. Results. The study cohort includes a total of 121 eyes from 121 patients. The mean age was 63.6 ± 19.7 years, ranging from 1 to 96 years. The average time to exposure was 19.29 ± 23.75 months (range 0.36–85.74 months). Risk factors associated with tube exposure were Hispanic ethnicity (P = 0.0115; OR = 3.6; 95% CI, 1.3–9.7), neovascular glaucoma (P = 0.0064; OR = 28.5; 95% CI, 2.6–316.9), previous trabeculectomy (P = 0.0070; OR = 5.3; 95% CI, 1.6–17.7), and combined surgery (P = 0.0381; OR = 3.7; 95% CI, 1.1–12.7). Conclusions. Hispanic ethnicity, neovascular glaucoma, previous trabeculectomy, and combined surgery were identified as potential risk factors for tube shunt exposure. These potential risk factors should be considered when determining the indication for performing tube shunt implantation and the frequency of long-term followup.
The Internet is bringing fundamental changes to medical practice through improved access to health information and participation in decision making. However, patient preferences for participation in health care vary greatly. Promoting patient-centered health care requires an understanding of the relationship between Internet use and a broader range of preferences for participation than previously measured.
To explore (1) whether there is a significant relationship between Internet use frequency and patients’ overall preferences for obtaining health information and decision-making autonomy, and (2) whether the relationships between Internet use frequency and information and decision-making preferences differ with respect to different aspects of health conditions.
The Health Information Wants Questionnaire (HIWQ) was administered to gather data about patients’ preferences for the (1) amount of information desired about different aspects of a health condition, and (2) level of decision-making autonomy desired across those same aspects.
The study sample included 438 individuals: 226 undergraduates (mean age 20; SD 2.15) and 212 community-dwelling older adults (mean age 72; SD 9.00). A significant difference was found between the younger and older age groups’ Internet use frequencies, with the younger age group having significantly more frequent Internet use than the older age group (younger age group mean 5.98, SD 0.33; older age group mean 3.50, SD 2.00; t
436=17.42, P<.01). Internet use frequency was positively related to the overall preference rating (γ=.15, P<.05), suggesting that frequent Internet users preferred significantly more information and decision making than infrequent Internet users. The relationships between Internet use frequency and different types of preferences varied: compared with infrequent Internet users, frequent Internet users preferred more information but less decision making for diagnosis (γ=.57, P<.01); more information and more decision-making autonomy for laboratory test (γ=.15, P<.05), complementary and alternative medicine (γ=.32, P<.01), and self-care (γ=.15, P<.05); and less information but more decision-making autonomy for the psychosocial (γ=-.51, P<.01) and health care provider (γ=-.27, P<.05) aspects. No significant difference was found between frequent and infrequent Internet users in their preferences for treatment information and decision making.
Internet use frequency has a positive relationship with the overall preferences for obtaining health information and decision-making autonomy, but its relationship with different types of preferences varies. These findings have important implications for medical practice.
patient-centered care; patient preference; shared decision-making; patient participation; Internet
Purpose. To evaluate outcomes of peripheral iridotomy (PI) for initial management of primary angle closure suspects (PACS), chronic primary angle closure (CPAC), and chronic primary angle closure glaucoma (CPACG). Patients and Methods. Seventy-nine eyes with PACS, CPAC, or CPACG and better than 20/50 visual acuity that underwent PI as initial management were included. Eyes with previous acute angle closure attacks, laser trabeculoplasties, surgeries, or intraocular injections were excluded. Additional treatments, glaucomatous progression, intraocular pressure, visual acuity, and the number of medications were evaluated. Results. The mean followup was 57.1 ± 29.0 months (range 13.8–150.6 months). Sixty-eight eyes (86.1%) underwent additional medical, laser, or surgical treatment. Forty eyes (50.6%) underwent lens extraction due to reduced visual acuity. The mean 10× logMAR visual acuity score for all patients significantly declined from 0.94 ± 1.12 at baseline to 1.83 ± 3.49 (N = 79, P = 0.0261) at the last followup. Conclusions. Most patients who undergo PI for CPAC spectrum will require additional intervention for either IOP lowering or improvement of visual acuity. This suggests that a procedure that not only deepens the angle but also lowers IOP and improves visual acuity would be desirable as further intervention could be avoided. Evaluation of techniques that achieve all 3 goals is warranted.
Purpose. To investigate age- and position-related changes of anterior chamber angle anatomy in normal, healthy eyes. Patients and Methods. Thirty subjects were separated into a younger and older cohort. The superior and inferior anterior chamber angles of the eyes were measured in supine and sitting positions by ultrasound biomicroscopy (UBM) with bag/balloon technology. Statistical analysis was used to evaluate positional and age-related changes in angle morphology. Results. In the younger cohort, no location or positional differences in angle anatomy were observed. In the older cohort, the inferior quadrant was significantly narrower than the superior quadrant (P = 0.0186) in the supine position. This cohort also demonstrated an interaction effect between position and location. In the older cohort, the angle was deeper inferiorly while the subject was sitting but was deeper superiorly while the subject was supine.
Conclusion. Comparison of positional variations in anterior chamber angle anatomy as measured by UBM has recently become possible. This study found that age-related positional changes in the anterior chamber angle anatomy exist in normal healthy eyes.
Purpose. Tube-related exposure is a known complication of glaucoma drainage device (GDD) surgery. Our objective is to report the early (approximately 1 year) tube exposure rate of implants covered with a keraSys (IOP Inc., Costa Mesa, CA, USA) tissue reinforcement graft. Patients and Methods. A retrospective, noncomparative, consecutive case series of 42 eyes with GDD implantation with keraSys patch grafts was performed. Main outcome measurements included patch-related complications: patch exposure, tube exposure, wound dehiscence, and patch migration. Results. Forty-two eyes were followed for an average of 15.24 ± 10.44 months (range 1.0–32.3 months). Four (10%) eyes experienced patch-related complications: two with exposure 8 months postoperatively, one with exposure 13 months postoperatively, and one with exposure 4 weeks postoperatively. Conclusion. The effectiveness of the keraSys patch graft is limited by the higher than expected early exposure rate found in this case series. These results should be confirmed in other studies.
To compare the results of Glaucoma Progression Analysis (GPA, Carl Zeiss Meditec, Dublin, CA) to subjective expert consensus in the detection of glaucomatous visual field progression.
Retrospective, observational case series.
100 eyes of 83 glaucoma patients.
Five serial Humphrey visual fields from 100 eyes of 83 glaucoma patients were evaluated by five masked glaucoma subspecialists for determination of progression. Four months later, with a randomly reordered patient sequence, the same visual field series were re-evaluated by the same graders, at which time they had access to the Glaucoma Progression Analysis (GPA) printout.
Main Outcome Measures
The level of agreement between majority expert consensus and GPA, both before and after access to GPA data, was assessed using kappa statistics.
On initial review and on re-evaluation with access to the GPA printout, the level of agreement between majority expert consensus and GPA was fair (kappa = 0.52, 95% confidence interval [CI] = 0.35 – 0.69 and 0.62, 95% CI = 0.46 – 0.78, respectively). Expert consensus was more likely to classify a series of fields as showing progression than was GPA (p ≤ 0.002). There was good agreement between expert consensus on initial review and reevaluation 4 months later (kappa = 0.77, 95% CI = 0.65 – 0.90).
The level of agreement between majority expert consensus of subjective determination of visual field progression and GPA is fair. In cases of disagreement with GPA, the expert consensus classification was usually progression. Access to the results of GPA did not significantly change the level of agreement between expert consensus and the GPA result; however, expert consensus did change in 11 of 100 cases.
To determine the efficacy and safety of diode transscleral cyclophotocoagulation (TSCPC) after tube shunt failure.
Patients and Methods
The patient population consisted of 32 eyes of 31 patients with uncontrolled glaucoma. Each eye had a previously implanted aqueous tube shunt and was currently on maximally tolerated medication. Each eye also underwent TSCPC treatment using the Iridex (Mountain View, CA) diode laser with a maximum of 360 degrees of treatment. All 31 charts were reviewed for data pertaining to demographics, treatment, ocular history, and follow-up clinical examinations. Safety was evaluated by complication data. Efficacy was evaluated in terms of TSCPC treatment parameters (number of laser applications, laser power, application duration, and degrees of ciliary body treated), intraocular pressure (IOP), number of hypotensive medications, and any further treatment required.
With a mean (SD) follow-up of 17.1 (16.3) (median = 11.7) months from the last treatment, the mean IOP decreased from 28.6 (10.2) mmHg to 16.8 (7.5) mmHg (35% reduction) at 3 months (n = 30, p < 0.0001) and to 14.7 (7.9) mmHg (43% reduction) at 1 year (n = 13, p < 0.0001). Complications included hypotony (n = 4), hyphema (n = 2), failed corneal transplant (n = 1), and loss of light perception (n = 5).
TSCPC has a significant ocular hypotensive effect on glaucoma refractory to both tube shunt and medical therapy. The safety of this intervention remains unclear in this high risk patient population and warrants further study.
transscleral; cyclophotocoagulation; tube shunt; diode
Purpose. To evaluate intraobserver and interobserver agreement in locating the scleral spur landmark (SSL) and anterior chamber angle measurements obtained using Fourier Domain Anterior Segment Optical Coherence Tomography (ASOCT) images. Methods. Two independent, masked observers (SR and AZC) identified SSLs on ASOCT images from 31 eyes with open and nonopen angles. A third independent reader, NPB, adjudicated SSL placement if identifications differed by more than 80 μm. Nine months later, SR reidentified SSLs. Intraobserver and interobserver agreement in SSL placement, trabecular-iris space area (TISA750), and angle opening distance (AOD750) were calculated. Results. In 84% of quadrants, SR's SSL placements during 2 sessions were within 80 μm in both the X- and Y-axes, and in 77% of quadrants, SR and AZC were within 80 μm in both axes. In adjudicated images, 90% of all quadrants were within 80 μm, 88% in nonopen-angle eyes, and 92% in open-angle eyes. The intraobserver and interobserver correlation coefficients (with and without adjudication) were above 0.9 for TISA750 and AOD750 for all quadrants. Conclusions. Reproducible identification of the SSL from images obtained with FD-ASOCT is possible. The ability to identify the SSL allows reproducible measurement of the anterior chamber angle using TISA750 and AOD750.
Androgen-deprivation therapy is well-established for treating prostate cancer but is associated with bone loss and an increased risk of fracture. We investigated the effects of denosumab, a fully human monoclonal antibody against receptor activator of nuclear factor-κB ligand, on bone mineral density and fractures in men receiving androgen-deprivation therapy for nonmetastatic prostate cancer.
In this double-blind, multicenter study, we randomly assigned patients to receive denosumab at a dose of 60 mg subcutaneously every 6 months or placebo (734 patients in each group). The primary end point was percent change in bone mineral density at the lumbar spine at 24 months. Key secondary end points included percent change in bone mineral densities at the femoral neck and total hip at 24 months and at all three sites at 36 months, as well as incidence of new vertebral fractures.
At 24 months, bone mineral density of the lumbar spine had increased by 5.6% in the denosumab group as compared with a loss of 1.0% in the placebo group (P<0.001); significant differences between the two groups were seen at as early as 1 month and sustained through 36 months. Denosumab therapy was also associated with significant increases in bone mineral density at the total hip, femoral neck, and distal third of the radius at all time points. Patients who received denosumab had a decreased incidence of new vertebral fractures at 36 months (1.5%, vs. 3.9% with placebo) (relative risk, 0.38; 95% confidence interval, 0.19 to 0.78; P = 0.006). Rates of adverse events were similar between the two groups.
Denosumab was associated with increased bone mineral density at all sites and a reduction in the incidence of new vertebral fractures among men receiving androgen-deprivation therapy for nonmetastatic prostate cancer. (ClinicalTrials.gov number, NCT00089674.)
Glaucoma is a collection of diseases characterized by multifactorial progressive changes leading to visual field loss and optic neuropathy most frequently due to elevated intraocular pressure (IOP). The goal of treatment is the lowering of the IOP to prevent additional optic nerve damage. Treatment usually begins with topical pharmacological agents as monotherapy, progresses to combination therapy with agents from up to 4 different classes of IOP-lowering medications, and then proceeds to laser or incisional surgical modalities for refractory cases. The fixed combination therapy with the carbonic anhydrase inhibitor dorzolamide hydrochloride 2% and the beta blocker timolol maleate 0.5% is now available in a generic formulation for the treatment of patients who have not responded sufficiently to monotherapy with beta adrenergic blockers. In pre- and postmarketing clinical studies, the fixed combination dorzolamide–timolol has been shown to be safe and efficacious, and well tolerated by patients. The fixed combination dorzolamide–timolol is convenient for patients, reduces their dosing regimen with the goal of increasing their compliance, reduces the effects of “washout” when instilling multiple drops, and reduces the preservative burden by reducing the number of drops administered per day.
dorzolamide; timolol; glaucoma; ocular hypertension; elevated IOP; fixed combination therapy
ocular blood flow' glaucoma
With the quantity of genomic data increasing at an exponential rate, it is imperative that these data be captured electronically, in a standard format. Standardization activities must proceed within the auspices of open-access and international working bodies. To tackle the issues surrounding the development of better descriptions of genomic investigations, we have formed the Genomic Standards Consortium (GSC). Here, we introduce the minimum information about a genome sequence (MIGS) specification with the intent of promoting participation in its development and discussing the resources that will be required to develop improved mechanisms of metadata capture and exchange. As part of its wider goals, the GSC also supports improving the ‘transparency’ of the information contained in existing genomic databases.
Recent research has established correlations between stress, anxiety, insomnia and excess body weight and these correlations have significant implications for health. This study measured the effects of a proprietary blend of extracts of Magnolia officinalis and Phellodendron amurense (Relora®) on anxiety, stress and sleep in healthy premenopausal women.
This randomized, parallel, placebo controlled clinical study was conducted with healthy, overweight (BMI 25 to 34.9), premenopausal female adults, between the ages of 20 and 50 years, who typically eat more in response to stressful situations and scores above the national mean for women on self-reporting anxiety. The intervention was Relora (250 mg capsules) or identical placebo 3 times daily for 6 weeks. Anxiety as measured by the Spielberger STATE-TRAIT questionnaires, salivary amylase and cortisol levels, Likert Scales/Visual Analog Scores for sleep quality and latency, appetite, and clinical markers of safety. The study was conducted by Miami Research Associates, a clinical research organization in Miami, FL.
The intent-to-treat population consisted of 40 subjects with 26 participants completing the study. There were no significant adverse events. Relora was effective, in comparison to placebo, in reducing temporary, transitory anxiety as measured by the Spielberger STATE anxiety questionnaire. It was not effective in reducing long-standing feelings of anxiety or depression as measured using the Spielberger TRAIT questionnaire. Other assessments conducted in this study including salivary cortisol and amylase levels, appetite, body morphology and sleep quality/latency were not significantly changed by Relora in comparison to placebo.
This pilot study indicates that Relora may offer some relief for premenopausal women experiencing mild transitory anxiety. There were no safety concerns or significant adverse events observed in this study.
Because sildenafil citrate is a treatment, not a cure, for erectile dysfunction (ED), many men may choose to use it for an extended period. Men with ED who had previously completed 1 of 4 double-blind trials with short-term open-label extension (combined duration, 0.9–1.2 years) were eligible for this 4-year, open-label, extension study, which assessed the safety and effectiveness of flexible doses (25, 50, and 100 mg sildenafil) used as needed. Adverse events that were serious or led to dosing changes or discontinuation (temporary or permanent) were recorded. Many of the 979 participants (mean age, 58 [range, 27–82] years; mean ED duration, 4.5 years) had concomitant hypertension (28%), diabetes (22%), or hyperlipidemia (14%). Overall, 37 (3.8%) had treatment-related adverse events (none serious) requiring dosage change or discontinuation and 62 (6.3%) discontinued because of insufficient response. At each yearly assessment, more than 94% of participants responded affirmatively to the questions: “Are you satisfied with the effect of treatment on your erections?” and “If yes, has treatment improved your ability to engage in sexual activity?” These results argue against the loss of tolerability or the development of tachyphylaxis over a prolonged period of as needed, flexible-dose sildenafil treatment of men with ED.
sildenafil citrate; erectile dysfunction; tolerability; effectiveness
Six environmental fosmid clones from Antarctic coastal water bacterioplankton were completely sequenced. The genome fragments harbored small-subunit rRNA genes that were between 85 and 91% similar to those of their nearest cultivated relatives. The six fragments span four phyla, including the Gemmatimonadetes, Proteobacteria (α and γ), Bacteroidetes, and high-G+C gram-positive bacteria. Gene-finding and annotation analyses identified 244 total open reading frames. Amino acid comparisons of 123 and 113 Antarctic bacterial amino acid sequences to mesophilic homologs from G+C-specific and SwissProt/UniProt databases, respectively, revealed widespread adaptation to the cold. The most significant changes in these Antarctic bacterial protein sequences included a reduction in salt-bridge-forming residues such as arginine, glutamic acid, and aspartic acid, reduced proline contents, and a reduction in stabilizing hydrophobic clusters. Stretches of disordered amino acids were significantly longer in the Antarctic sequences than in the mesophilic sequences. These characteristics were not specific to any one phylum, COG role category, or G+C content and imply that underlying genotypic and biochemical adaptations to the cold are inherent to life in the permanently subzero Antarctic waters.
Serial analysis of gene expression (SAGE) is a powerful approach for the identification of differentially expressed genes, providing comprehensive and quantitative gene expression profiles in the form of short tag sequences. Each tag represents a unique transcript, and the relative frequencies of tags in the SAGE library are equal to the relative proportions of the transcripts they represent. One of the major obstacles in the preparation of SAGE libraries from microorganisms is the requirement for large amounts of starting material (i.e., mRNA). Here, we present a novel approach for the construction of SAGE libraries from small quantities of total RNA by using Y linkers to selectively amplify 3′ cDNA fragments. To validate this method, we constructed comprehensive gene expression profiles of the toxic dinoflagellate Pfiesteria shumwayae. SAGE libraries were constructed from an actively toxic fish-fed culture of P. shumwayae and from a recently toxic alga-fed culture. P. shumwayae-specific gene transcripts were identified by comparison of tag sequences in the two libraries. Representative tags with frequencies ranging from 0.026 to 3.3% of the total number of tags in the libraries were chosen for further analysis. Expression of each transcript was confirmed in separate control cultures of toxic P. shumwayae. The modified SAGE method described here produces gene expression profiles that appear to be both comprehensive and quantitative, and it is directly applicable to the study of gene expression in other environmentally relevant microbial species.
The attenuation and immunogenicity of two novel Salmonella vaccine strains, Salmonella enterica serovar Typhi (Ty2 ΔaroC ΔssaV, designated ZH9) and S. enterica serovar Typhimurium (TML ΔaroC ΔssaV, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 107, 108, or 109 CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 108 and two of three receiving 109 CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 108 and 109 CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 109 CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi.
To identify the major outer surface proteins of Streptococcus agalactiae (group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as S. agalactiae specific, 27 have been identified. Six of these proteins, previously unidentified in S. agalactiae, were sequenced and cloned. These were ornithine carbamoyltransferase, phosphoglycerate kinase, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, purine nucleoside phosphorylase, enolase, and glucose-6-phosphate isomerase. Using a gram-positive expression system, we have overexpressed two of these proteins in an in vitro system. These recombinant, purified proteins were used to raise antisera. The identification of these proteins as residing on the outer surface was confirmed by the ability of the antisera to react against whole, live bacteria. Further, in a neonatal-animal model system, we demonstrate that some of these sera are protective against lethal doses of bacteria. These studies demonstrate the successful application of proteomics as a technique for identifying vaccine candidates.
Planktonic crenarchaeotes are present in high abundance in Antarctic winter surface waters, and they also make up a large proportion of total cell numbers throughout deep ocean waters. To better characterize these uncultivated marine crenarchaeotes, we analyzed large genome fragments from individuals recovered from a single Antarctic picoplankton population and compared them to those from a representative obtained from deeper waters of the temperate North Pacific. Sequencing and analysis of the entire DNA insert from one Antarctic marine archaeon (fosmid 74A4) revealed differences in genome structure and content between Antarctic surface water and temperate deepwater archaea. Analysis of the predicted gene products encoded by the 74A4 sequence and those derived from a temperate, deepwater planktonic crenarchaeote (fosmid 4B7) revealed many typical archaeal proteins but also several proteins that so far have not been detected in archaea. The unique fraction of marine archaeal genes included, among others, those for a predicted RNA-binding protein of the bacterial cold shock family and a eukaryote-type Zn finger protein. Comparison of closely related archaea originating from a single population revealed significant genomic divergence that was not evident from 16S rRNA sequence variation. The data suggest that considerable functional diversity may exist within single populations of coexisting microbial strains, even those with identical 16S rRNA sequences. Our results also demonstrate that genomic approaches can provide high-resolution information relevant to microbial population genetics, ecology, and evolution, even for microbes that have not yet been cultivated.
Vestimentiferan tubeworms thriving in sulfidic deep-sea hydrothermal vents and cold seeps are constrained by their nutritional reliance on chemoautotrophic endosymbionts. In a recent phylogenetic study using 16S ribosomal DNA, we found that endosymbionts from vent and seep habitats form two distinct clades with little variation within each clade. In the present study, we used two different approaches to assess the genetic variation among biogeographically distinct vestimentiferan symbionts. DNA sequences were obtained for the noncoding, internal transcribed spacer (ITS) regions of the rRNA operons of symbionts associated with six different genera of vestimentiferan tubeworms. ITS sequences from endosymbionts of host genera collected from different habitats and widely distributed vent sites were surprisingly conserved. Because the ITS region was not sufficient for distinguishing endosymbionts from different habitats or locations, we used a DNA fingerprinting technique, repetitive-extragenic-palindrome PCR (REP-PCR), to reveal differences in the distribution of repetitive sequences in the genomes of the bacterial endosymbionts. Most of the endosymbionts displayed unique REP-PCR patterns. A cladogram generated from these fingerprints reflected relationships that may be influenced by a variety of factors, including host genera, geographic location, and bottom type.