Search

While converging research suggests that increased white matter hyperintensity load is associated with poorer cognition, and the presence of hypertension is associated with increased white matter hyperintensity load, the relationship among hypertension, cognition and white matter hyperintensities is not well understood. We sought to determine the effect of white matter hyperintensity burden on the relationship between hypertension and cognition in individuals with post-stroke aphasia, with the hypothesis that white matter hyperintensity load moderates the relationship between history of hypertension and cognitive function. Health history, Fazekas scores for white matter hyperintensities and Wechsler Adult Intelligence Scale Matrix Reasoning subtest scores for 79 people with aphasia collected as part of the Predicting Outcomes of Language Rehabilitation study at the Center for the Study of Aphasia Recovery at the University of South Carolina and the Medical University of South Carolina were analysed retrospectively. We found that participants with a history of hypertension had increased deep white matter hyperintensity severity ( P < 0.001), but not periventricular white matter hyperintensity severity ( P = 0.116). Moderation analysis revealed that deep white matter hyperintensity load moderates the relationship between high blood pressure and Wechsler Adult Intelligence Scale scores when controlling for age, education, aphasia severity and lesion volume. The interaction is significant, showing that a history of high blood pressure and severe deep white matter hyperintensities together are associated with poorer Matrix Reasoning scores. The overall model explains 41.85% of the overall variation in Matrix Reasoning score in this group of participants. These findings underscore the importance of considering cardiovascular risk factors in aphasia treatment, specifically hypertension and its relationship to brain health in post-stroke cognitive function., Competing Interests: The authors report no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

In post-stroke aphasia, language improvements following speech therapy are variable and can only be partially explained by the lesion. Brain tissue integrity beyond the lesion (brain health) may influence language recovery and can be impacted by cardiovascular risk factors, notably diabetes. We examined the impact of diabetes on structural network integrity and language recovery. Seventy-eight participants with chronic post-stroke aphasia underwent six weeks of semantic and phonological language therapy. To quantify structural network integrity, we evaluated the ratio of long-to-short-range white matter fibers within each participant's whole brain connectome, as long-range fibers are more susceptible to vascular injury and have been linked to high level cognitive processing. We found that diabetes moderated the relationship between structural network integrity and naming improvement at 1 month post treatment. For participants without diabetes (n = 59), there was a positive relationship between structural network integrity and naming improvement (t = 2.19, p = 0.032). Among individuals with diabetes (n = 19), there were fewer treatment gains and virtually no association between structural network integrity and naming improvement. Our results indicate that structural network integrity is associated with treatment gains in aphasia for those without diabetes. These results highlight the importance of post-stroke structural white matter architectural integrity in aphasia recovery., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: There is growing interest in the measurement of growth differentiation factor 15 (GDF-15) in a range of disorders associated with cachexia. We undertook studies to determine whether a common histidine (H) to aspartate (D) variant at position 202 in the pro-peptide (position 6 in the mature peptide) interfered with its detection by 3 of the most commonly used immunoassays., Methods: Three synthetic GDF-15-forms (HH homo-, HD hetero-, and DD-homodimers) were measured after serial dilution using Roche Elecsys®, R&D QuantikineTM ELISA, and MSD R&D DuoSet® immunoassays. GDF-15 concentrations were measured by the Roche and the MSD R&D immunoassays in 173 genotyped participants (61 HH homozygotes, 59 HD heterozygotes, and 53 DD homozygotes). For the comparative statistical analyses of the GDF-15 concentrations, we used non-parametric tests, in particular Bland-Altman difference (bias) plots and Passing-Bablok regression. The bioactivity of the 2 different homodimers was compared in a cell-based assay in HEK293S-SRF-RET/GFRAL cells., Results: The Roche assay detected H- and D-containing peptides similarly but the R&D reagents (Quantikine and DuoSet) consistently underreported GDF-15 concentrations in the presence of the D variant. DD dimers had recoveries of approximately 45% while HD dimers recoveries were 62% to 78%. In human serum samples, the GDF-15 concentrations reported by the R&D assay were a median of 4% lower for HH, a median of 36% lower for HD, and a median of 61% lower for DD compared to the Roche assay. The bioactivities of the HH and DD peptides were indistinguishable., Conclusions: The D variant of GDF-15 substantially affects its measurement by a commonly used immunoassay, a finding that has clear implications for its interpretation in research and clinical settings., Competing Interests: Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: M. Ratcliff is an AstraZeneca employee; M. Hyvönen is a CSO (0.1 FTE time commitment, paid as salaried employee) and founder of Qkine. Qkine produces and sells GDF-15 protein; A. Backmark is an Astrazeneca employee; R. Roth is employed by AstraZeneca; L. Jermutus is an AstraZeneca employee. Consultant or Advisory Role: M. Hyvönen, consultancy contract through Cambridge University Technical Services of Ansh Labs LLC, which develops GDF-15 ELISA kits; M. Blueher received consulting fees from Amgen (personal honoraria), AstraZeneca (personal honoraria), Bayer (personal honoraria), Boehringer-Ingelheim (personal honoraria), Daiichi-Sankyo (personal honoraria), Lilly (personal honoraria), Novo Nordisk (personal honoraria), Novartis (personal honoraria), Pfizer (personal honoraria), and Sanofi (personal honoraria), and participated on a Data Safety Monitoring Board or Advisory Board with Boehringer-Ingelheim and Eli Lilly; N. Sattar received consulting fees from Afimmune, Amgen, AstraZeneca, Boehringer Ingelheim, Eli Lilly, Hanmi Pharmaceuticals, Merck Sharp & Dohme, Novartis, Novo Nordisk, Pfizer, and Sanofi. Stock Ownership: M. Ratcliff received stock/stock options paid to himself; A. Backmark is a shareholder in AstraZeneca; R. Roth has stock/stock options in AstraZeneca; L. Jermutus has stock/stock options in AstraZeneca; M. Hyvönen, major shareholder for Qkine Ltd. Honoraria: P. Welsh has honoraria from Novo Nordisk; M. Blueher, Amgen, AstraZeneca, Bayer, Boehringer-Ingelheim, Daiichi-Sankyo, Lilly, Novo Nordisk, Novartis, Pfizer, and Sanofi. Research Funding: Y. Karusheva received funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) (project number 447713146); M. Ratcliff received a BBSRC iCase PhD studentship with AstraZeneca. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through CRC 1052, project number 209933838, subproject B1 to M. Blueher and by Deutsches Zentrum für Diabetesforschung (DZD, Grant: 82DZD00601) to M. Blueher and E. Guiu-Jurado. E. Guiu-Jurado, Deutsches Zentrum für Diabetesforschung e.V. (DZD) Postdoctoral fellowship, Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through CRC 1052, project number 209933838, Helmholtz Institute for Metabolic, Obesity and Vascular Research (HI-MAG) of the Helmholtz Zentrum München at the University of Leipzig and University Hospital Leipzig; N. Sattar received grants contracts from AstraZeneca, Boehringer Ingelheim, Novartis, and Roche Diagnostics and funding support from the British Heart Foundation Research Excellence Award (RE/18/6/34217); P. Welsh has grants/contracts with Roche Diagnostics, AstraZeneca, Boehringer Ingelheim, and Novartis. The research was funded by the MRC grants MC.UU.00014.1 and MC.UU.00014.5 and supported by the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014*). Expert Testimony: None declared. Patents: None declared. Other Remuneration: M. Hyvönen, royalties from Qkine Ltd for technologies for production of TGF-β growth factors, including GDF-15., (© American Association for Clinical Chemistry 2022.)

Aims: Non-compaction cardiomyopathy is a devastating genetic disease caused by insufficient consolidation of ventricular wall muscle that can result in inadequate cardiac performance. Despite being the third most common cardiomyopathy, the mechanisms underlying the disease, including the cell types involved, are poorly understood. We have previously shown that endothelial cell-specific deletion of the chromatin remodeller gene Ino80 results in defective coronary vessel development that leads to ventricular non-compaction in embryonic mouse hearts. We aimed to identify candidate angiocrines expressed by endocardial and endothelial cells (ECs) in wildtype and LVNC conditions in Tie2Cre;Ino80fl/fltransgenic embryonic mouse hearts, and test the effect of these candidates on cardiomyocyte proliferation and maturation., Methods and Results: We used single-cell RNA-sequencing to characterize endothelial and endocardial defects in Ino80-deficient hearts. We observed a pathological endocardial cell population in the non-compacted hearts and identified multiple dysregulated angiocrine factors that dramatically affected cardiomyocyte behaviour. We identified Col15a1 as a coronary vessel-secreted angiocrine factor, downregulated by Ino80-deficiency, that functioned to promote cardiomyocyte proliferation. Furthermore, mutant endocardial and endothelial cells up-regulated expression of secreted factors, such as Tgfbi, Igfbp3, Isg15, and Adm, which decreased cardiomyocyte proliferation and increased maturation., Conclusions: These findings support a model where coronary endothelial cells normally promote myocardial compaction through secreted factors, but that endocardial and endothelial cells can secrete factors that contribute to non-compaction under pathological conditions., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Background: Eosinophilic esophagitis is a chronic inflammatory gastrointestinal disease with a high prevalence in younger, atopic males. In our clinical practice, we observed a striking preponderance of patients having a high educational background. The purposes of this study were first to assess the level of education of eosinophilic esophagitis patients and second to compare the findings to patients with inflammatory bowel disease, another chronic immune-mediated condition of the gastrointestinal tract, and with the Swiss general population., Methods: Using a questionnaire, we assessed the educational level of adult patients who have attended Swiss Eosinophilic Esophagitis Clinics in the past. In addition, the educational level of the parents was assessed as well. We calculated the proportions of patients and parents who have obtained a higher educational level. Data from the Swiss Inflammatory Bowel Disease Cohort Study and from the Swiss general population served as confirmation and as comparison, respectively., Results: A total of 277 successfully contacted patients (response rate 69.1%; mean age 51.1 years, 73% male) participated. A significantly higher proportion of surveyed eosinophilic esophagitis patients had a high International Standard Classification of Education level (66.8%, P < 0.001) compared with inflammatory bowel disease patients (n = 2534; 34.2%, P < 0.001) and to the Swiss general population (n = 6,066,907; 30.5% P < 0.001)., Conclusion: Our analysis confirms the clinical observation that eosinophilic esophagitis patients have a significantly higher educational level compared with the general population and to patients with other chronic inflammatory diseases of the gastrointestinal tract. As a limitation, this impressive finding remains on a purely descriptive level., (© The Author(s) 2021. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glaucophyta are members of the Archaeplastida, the founding group of photosynthetic eukaryotes that also includes red algae (Rhodophyta), green algae, and plants (Viridiplantae). Here we present a high-quality assembly, built using long-read sequences, of the ca. 100 Mb nuclear genome of the model glaucophyte Cyanophora paradoxa. We also conducted a quick-freeze deep-etch electron microscopy (QFDEEM) analysis of C. paradoxa cells to investigate glaucophyte morphology in comparison to other organisms. Using the genome data, we generated a resolved 115-taxon eukaryotic tree of life that includes a well-supported, monophyletic Archaeplastida. Analysis of muroplast peptidoglycan (PG) ultrastructure using QFDEEM shows that PG is most dense at the cleavage-furrow. Analysis of the chlamydial contribution to glaucophytes and other Archaeplastida shows that these foreign sequences likely played a key role in anaerobic glycolysis in primordial algae to alleviate ATP starvation under night-time hypoxia. The robust genome assembly of C. paradoxa significantly advances knowledge about this model species and provides a reference for exploring the panoply of traits associated with the anciently diverged glaucophyte lineage., (© The Author(s) 2019. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Inflammation is an important biological process involved in many target organ toxicities. However, there has been little consensus on how to represent inflammatory processes using the adverse outcome pathway (AOP) framework. In particular, there were concerns that inflammation was not being represented in a way that it would be recognized as a highly connected, central node within the global AOP network. The consideration of salient features common to the inflammatory process across tissues was used as a basis to propose 3 hub key events (KEs) for use in AOP network development. Each event, "tissue resident cell activation", "increased pro-inflammatory mediators", and "leukocyte recruitment/activation," is viewed as a hallmark of inflammation, independent of tissue, and can be independently measured. Using these proposed hub KEs, it was possible to link together a series of AOPs that previously had no shared KEs. Significant challenges remain with regard to accurate prediction of inflammation-related toxicological outcomes even if a broader and more connected network of inflammation-centered AOPs is developed. Nonetheless, the current proposal addresses one of the major hurdles associated with representation of inflammation in AOPs and may aid fit-for-purpose evaluations of other AOPs operating in a network context.

Objectives: Small sample size limits the number of immunostains that may be attempted in colorectal carcinoma (CRC) biopsy specimens. We investigated the utility of dual stain with special AT-rich sequence binding protein 2 (SATB2) or caudal-type homeobox 2 (CDX2) and cytokeratin 20 (CK20) or villin in identifying CRC., Methods: Tissue microarrays with 222 CRCs and 375 other carcinomas were built. Dual stain was performed pairing nuclear stains CDX2 or SATB2 with CK20 or villin., Results: All four single stains showed excellent sensitivity (93%-99%) but variable specificity (56%-88%) for CRC. All four dual stains also showed excellent sensitivity (90%-96%) while much improved specificity (88%-98%) compared with single stains. SATB2 dual stain (with CK20 or villin) showed a higher specificity than CDX2 dual stain (with CK20 or villin) with a comparable sensitivity., Conclusions: SATB2 dual stain shows the greatest potential clinical utility in identifying CRC and is superior to CDX2 dual stain. More important, SATB2 dual stain could be helpful for specimens with limited tissues or those having a nonclassic staining pattern.

The ecological prominence of diatoms in the ocean environment largely results from their superior competitive ability for dissolved nitrate (NO 3 - ). To investigate the cellular and genetic basis of diatom NO 3 - assimilation, we generated a knockout in the nitrate reductase gene ( NR -KO) of the model pennate diatom Phaeodactylum tricornutum In NR -KO cells, N-assimilation was abolished although NO 3 - transport remained intact. Unassimilated NO 3 - accumulated in NR -KO cells, resulting in swelling and associated changes in biochemical composition and physiology. Elevated expression of genes encoding putative vacuolar NO 3 - chloride channel transporters plus electron micrographs indicating enlarged vacuoles suggested vacuolar storage of NO 3 - Triacylglycerol concentrations in the NR -KO cells increased immediately following the addition of NO 3 - , and these increases coincided with elevated gene expression of key triacylglycerol biosynthesis components. Simultaneously, induction of transcripts encoding proteins involved in thylakoid membrane lipid recycling suggested more abrupt repartitioning of carbon resources in NR -KO cells compared with the wild type. Conversely, ribosomal structure and photosystem genes were immediately deactivated in NR -KO cells following NO 3 - addition, followed within hours by deactivation of genes encoding enzymes for chlorophyll biosynthesis and carbon fixation and metabolism. N-assimilation pathway genes respond uniquely, apparently induced simultaneously by both NO 3 - replete and deplete conditions., (© 2017 American Society of Plant Biologists. All rights reserved.)

Objectives: Determination of the primary site of origin for mucinous neoplasms identified in the peritoneal and/or pelvic cavities may be challenging, with major differential diagnoses including appendiceal mucinous neoplasm (AMN) and ovarian mucinous neoplasm (OMN). Special AT-rich sequence binding protein 2 (SATB2) has been shown to be highly selectively expressed in the lower gastrointestinal tract, including the appendix., Methods: We investigated the utility of a dual stain (DS) with SATB2 or caudal type homeobox 2 (CDX2) and cytokeratin 20 (CK20) or villin in distinguishing AMNs from OMNs. Tissue microarrays with 40 AMNs and 18 OMNs were stained with SATB2 or CDX2 paired with either CK20 or villin., Results: SATB2 single stain showed a good sensitivity of 83% and the highest specificity of 78% for AMNs over OMNs among all four stains. DS with SATB2 and villin showed an identical sensitivity of 78% but specificity increased to 94%, while DS with SATB2 and CK20 showed a sensitivity of 80% and a specificity of 100%. In contrast, DS with CDX2 and CK20/villin showed slightly higher sensitivity but much lower specificity., Conclusions: DS with SATB2/CK20 shows the greatest potential clinical utility in distinguishing AMNs from OMNs and is superior to DS with CDX2/CK20. Importantly, DS could be helpful for specimens with limited tissues., (© American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Aims: To investigate the production of soluble cross-reacting material 197 (CRM 197 ) in Escherichia coli, a safe and effective T-cell-dependent protein carrier for polysaccharides used in the manufacture and application of multivalent conjugate vaccines., Methods and Results: The use of co-expression of a sulphydryl oxidase (SOX) and protein disulphide isomerase for the production of soluble CRM 197 in E. coli is described. CRM 197 contains two disulphide bonds, which are normally unable to form in the reducing environment of the E. coli cytoplasm. It was found that co-expression yielded soluble CRM 197 , at a production rate ~10% of the production of insoluble CRM 197 , in equivalent small-scale cultures. Structural analysis of the purified CRM 197 compared to CRM 197 commercially produced in cultures of recombinant Pseudomonas fluorescens indicated that the E. coli soluble protein compares favourably on all structural levels., Conclusions: SOX and protein disulphide isomerase are enzymes involved in the formation of intra-protein disulphide bonds, and can influence the tertiary structure of the protein being produced, resulting in increased solubility due to the correct folding of the protein. Their use enabled the production of soluble untagged CRM 197 in E. coli, which was previously unachievable., Significance and Impact of the Study: Previous literature reports have shown that CRM 197 can be expressed in E. coli, though only in an insoluble form, or in soluble form as a fusion protein. It is currently commercially produced in cultures of recombinant P. fluorescens. The use of a widely used, well-characterized expression host such as E. coli, rather than P. fluorescens broadens the applicability of the production technology, and the production system described here is worthy of further investigation for scaled up manufacture of CRM 197 ., (© 2017 The Society for Applied Microbiology.)

Objective: To identify predictors of later study withdrawal among participants active in The Environmental Determinants of Diabetes in the Young (TEDDY) for 1 year. , Methods: Multiple logistic regression was used to discriminate 3,042 children active in TEDDY for the first 3 years from 432 children who withdrew in Years 2 or 3. Predictor variables were tested in blocks-demographic, maternal lifestyle behaviors, stress and child illness, maternal reactions to child's increased diabetes risk, in-study behaviors-and a final best model developed. , Results: Few demographic factors predicted study withdrawal. Maternal lifestyle behaviors, accuracy of the mother's risk perception, and in-study behaviors were more important. Frequent child illnesses were associated with greater study retention. , Conclusions: Demographic measures are insufficient predictors of later study withdrawal among those active in a study for at least 1 year; behavioral/psychological factors offer improved prediction and guidance for the development of retention strategies., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Pediatric Psychology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Sexually transmitted infectious (STI) colitis often raises concern for inflammatory bowel disease (IBD). In this study, we compare histologic features of IBD with STI colitis caused by syphilis and lymphogranuloma venereum., Methods: The STI colitis group included 10 unique colorectal biopsy specimens in patients with clinically confirmed syphilis and/or lymphogranuloma venereum. The STI biopsy specimens were compared with patients matched for age, sex, and site with Crohn disease (n = 10) or ulcerative colitis (n = 10). All IBD controls had an established history of IBD (up to 276 months of follow-up, mean follow-up = 102 months)., Results: Discriminating features (P < .05) of STI colitis included its exclusive identification in human immunodeficiency virus-positive men who have sex with men, anal pain, and anal discharge. STI colitis contained the triad of (1) minimal active chronic crypt centric damage, (2) a lack of mucosal eosinophilia, and (3) submucosal plasma cells, endothelial swelling, and perivascular plasma cells. Nondiscriminating features (P > .05) included rectal bleeding, endoscopic appearance, skip lesions, ulcerations, aphthoid lesions, granulomata, foreign body giant cells, neural hyperplasia, fibrosis, and lymphoid aggregates., Conclusions: While STI colitis shares many overlapping features with IBD, histologic and clinical discriminating features may be helpful when confronted with that differential diagnosis., (Copyright© by the American Society for Clinical Pathology.)

Background: The prognosis of Mycobacterium abscessus infections is poor due to the lack of effective drug treatment. The objective of this study was to set up an animal model suitable to test antibiotic activity against M. abscessus., Methods: The following mouse strains were evaluated: Swiss, BALB/c, C57BL/6, nude, beige, A/J, and GKO. Antibiotic activity was tested for clarithromycin, amikacin, cefoxitin, tigecycline, and bedaquiline (TMC207). Finally, we evaluated the 3-drug combination clarithromycin, cefoxitin, and amikacin., Results: Nude and GKO mice fulfilled criteria for the model but only nude mice offered sufficient availability for large therapeutic experiments. Among the 3 drugs usually combined for treatment of M. abscessus infection, cefoxitin was the most active because it improved survival and reduced bacillary loads in spleen whereas clarithromycin and amikacin prevented death but had little impact on bacillary loads. The triple-drug combination was not more active than cefoxitin alone. Tigecycline displayed bactericidal activity whereas bedaquiline was almost inactive., Conclusions: Nude mice are an adequate model for in vivo chemotherapy studies. Among tested drugs, cefoxitin and tigecycline showed promising in vivo activity against M. abscessus. The best drug combination remains to be determined.

Objectives: Besifloxacin is a new fluoroquinolone in development for ocular use. We investigated its mode of action and resistance in two major ocular pathogens, Streptococcus pneumoniae and Staphylococcus aureus, and in the reference species Escherichia coli., Methods: Primary and secondary targets of besifloxacin were evaluated by: (i) mutant selection experiments; (ii) MIC testing of defined topoisomerase mutants; and (iii) inhibition and cleavable complex assays with purified S. pneumoniae and E. coli DNA gyrase and topoisomerase IV enzymes., Results: Enzyme assays showed similar besifloxacin activity against S. pneumoniae gyrase and topoisomerase IV, with IC(50) and CC(25) of 2.5 and 1 microM, respectively. In contrast to ciprofloxacin and moxifloxacin, besifloxacin was equally potent against both S. pneumoniae and E. coli gyrases. DNA gyrase was the primary target in all three species, with substitutions observed at positions 81, 83 and 87 in GyrA and 426 and 466 in GyrB (E. coli numbering). Topoisomerase IV was the secondary target. Notably, resistant mutants were not recovered at 4-fold besifloxacin MICs for S. aureus and S. pneumoniae, and S. aureus topoisomerase mutants were only obtained after serial passage in liquid medium. Besifloxacin MICs were similarly affected by parC or gyrA mutations in S. aureus and S. pneumoniae and remained below 1 mg/L in gyrA-parC double mutants., Conclusions: Although mutant selection experiments indicated that gyrase is a primary target, further biochemical and genetic studies showed that besifloxacin has potent, relatively balanced activity against both essential DNA gyrase and topoisomerase IV targets in S. aureus and S. pneumoniae.

The eukaryotic RecA homologs Rad51 and Dmc1 are essential for strand exchange between homologous chromosomes during meiosis. All members of the RecA family of recombinases polymerize on DNA to form helical nucleoprotein filaments, which is the active form of the protein. Here we compare the filament structures of the Rad51 and Dmc1 proteins from both human and budding yeast. Previous studies of Dmc1 filaments suggested that they might be structurally distinct from filaments of other members of the RecA family, including Rad51. The data presented here indicate that Rad51 and Dmc1 filaments are essentially identical with respect to several structural parameters, including persistence length, helical pitch, filament diameter, DNA base pairs per helical turn and helical handedness. These data, together with previous studies demonstrating similar in vitro recombinase activity for Dmc1 and Rad51, support the view that differences in the meiotic function of Rad51 and Dmc1 are more likely to result from the influence of distinct sets of accessory proteins than from intrinsic differences in filament structure.

The tomato resistance genes Cf-4 and Cf-9 confer specific, hypersensitive response-associated recognition of Cladosporium carrying the avirulence genes Avr4 and Avr9, respectively. Cf-4 and Cf-9 encode type I transmembrane proteins with extracellular leucine-rich repeats (LRRs). Compared with Cf-9, Cf-4 lacks two LRRs and differs in 78 amino acid residues. To investigate the relevance of these differences for specificity, we exchanged domains between Cf-4 and Cf-9, and mutant constructs were tested for mediating the hypersensitive response by transient coexpression with either Avr4 or Avr9. We show that the number of LRRs is essential for both Cf-4 and Cf-9 function. In addition, Cf-9 specificity resides entirely in the LRR domain and appears to be distributed over several distant LRRs. In contrast, Cf-4 specificity determinants reside in the N-terminal LRR-flanking domain and three amino acid residues in LRRs 13, 14, and 16. These residues are present at putative solvent-exposed positions, and all are required for full Cf-4 function. Finally, we show that Cf-9 carrying the specificity determinants of Cf-4 has recognitional specificity for AVR4. The data indicate that diversifying selection of solvent-exposed residues has been a more important factor in the generation of Cf-4 specificity than has sequence exchange between Cf-4 progenitor genes. The fact that most variant residues in Cf-4 are not essential for Cf-4 specificity indicates that the diverse decoration of R proteins is not fully adapted to confer recognition of a certain avirulence determinant but likely provides a basis for a versatile, adaptive recognition system.

Within the maize leaf primordium, coordinated cell division and differentiation patterns result in the development of two morphologically and biochemically distinct photosynthetic cell types, the bundle sheath and the mesophyll. The bundle sheath defective2-mutable1 (bsd2-m1) mutation specifically disrupts C4 differentiation in bundle sheath cells in that the levels of bundle sheath cell-specific photosynthetic enzymes are reduced and the bundle sheath chloroplast structure is aberrant. In contrast, mesophyll cell-specific enzymes accumulate to normal levels, and the mesophyll cell chloroplast structure is not perturbed. Throughout mutant leaf development, the large and small subunits of ribulose bisphosphate carboxylase are absent; however, both rbcL and RbcS transcripts accumulate. Moreover, chloroplast-encoded rbcL transcripts accumulate ectopically in mesophyll cells. Although the bundle sheath cell chloroplast structure deteriorates rapidly when plants are exposed to light, this deterioration is most likely a secondary effect resulting from cell-specific photooxidative damage. Therefore, we propose that the Bsd2 gene plays a direct role in the post-transcriptional control of rbcL transcript accumulation and/or translation, both in bundle sheath and mesophyll cells, and an indirect role in the maintenance of bundle sheath cell chloroplast structure.

A system is described for isolating temperature-sensitive mutants of Saccharomyces cerevisiae with defects in early meiotic events. We used an otherwise haploid strain disomic (n+1) for chromosome III, and heteroallelic at the leucine-2 locus. Meiotic development was initiated by exposure of the strain to acetate sporulation medium, and monitored by the appearance of leucine-independent intragenic recombinants. Mutant isolation was based on the recovery of thermally induced defects in recombination. The temperature-sensitive characteristic was included to allow eventual characterizations of the temporal period during meiosis when each gene performs its essential function. Following mutagenesis with either ethyl methane sulfonate or nitrosoguanidine individual clones were tested at 34 degrees and 24 degrees for acetate-induced recombination. Starting with 2700 clones, derived from cells that survived mutagenic treatment, we isolated 48 strains with thermally induced lesions in recombination. In the majority of mutants premeiotic replication occurred normally, or nearly normally, at the restrictive temperature, indicating that the meiotic cycle was initiated and that there was a defect in an event required for intragenic recombination. We also detected mutants where the thermally induced lesion in recombination resulted from temperature-sensitive premeiotic DNA synthesis.