Localization of HArep-containing genes on the chromosome of Porphyromonas gingivalis W83

We have mapped a group of virulence genes of Porphyromonas gingivalis to a single large fragment of the genome. These genes (rgpA, kgp, and hagA) all contain a consensus repeat sequence (HArep). rgpA and kgp encode cysteine proteases with Arg-X and Lys-X specificity, respectively, and hagA encodes a hemagglutinin. Genomic DNA fragments separated by pulse-field gel electrophoresis were blotted and probed in order to localize the genes to a 0.25-Mb NheI fragment of the P. gingivalis W83 genome. Further hybridization analyses with single- and double-restriction digestion allowed us to generate a physical map of the fragment and determine the precise locations of the protease and hemagglutinin genes. In addition, we found an insertion-like sequence, IS195, near the ends of the 0. 25-Mb NheI fragment. A similarly sized fragment carrying HArep sequences was also demonstrated in the P. gingivalis W12 and W50 genomes.     

Cloning and characterization of a new protease gene (prtH) from Porphyromonas gingivalis.

Porphyromonas gingivalis has been implicated as a contributing etiological agent of adult periodontitis and generalized forms of early-onset periodontitis. Proteases of P. gingivalis may contribute to its pathogenicity by destroying connective tissue as well as inactivating key plasma proteins that might mediate protective host functions. In order to explore this problem, antiserum raised against membrane vesicles of P. gingivalis W83 was used to screen a genomic library of strain W83 constructed by using the lambda DASH vector system. A recombinant phage (lambda 34) expressing a P. gingivalis protease from the library was identified and characterized. Casein substrate zymography of lambda 34 lysates revealed a protease with an apparent molecular mass of 97 kDa. The gene encoding this protease was designated prtH. It was localized to a 3.7-kb HindIII-BamHI fragment and specified an enzyme which hydrolyzed the human C3 complement protein under defined conditions. The nucleotide sequence of this 3.7-kb fragment was determined, and one 2.9-kb open reading frame (992 amino acids) corresponding to a 110-kDa protein was detected, suggesting it might be a precursor of the 97-kDa active protease. prtH is not similar to any previously cloned protease gene from P. gingivalis.     

Genetic Transformation of Streptococcus sanguis (Challis) with Cryptic Plasmids from Streptococcus ferus

By using the basic methodology initially published by Kretschmer et al. (J. Bacteriol. 124:225-231, 1975), we have been able to introduce phenotypically cryptic plasmids from Streptococcus ferus (formerly Streptococcus mutans subsp. ferus) into Streptococcus sanguis by genetic transformation. In this system, the entry of the cryptic plasmids is selected indirectly. This is effected with transforming deoxyribonucleic acid mixtures in which the cryptic plasmid deoxyribonucleic acid is present in an approximate 10-fold molar excess with respect to a plasmid (pVA1) known to confer erythromycin resistance. Under such conditions, 5 to 10% of the pVA1-containing erythromycin-resistant transformants were cotransformed with cryptic plasmid deoxyribonucleic acid. pVA1 may be selectively eliminated by growth of its S. sanguis host strain at 42°C, enabling the construction of isogenic strains with and without S. ferus cryptic plasmids. Comparative physiological studies of such strains have failed to reveal any plasmid-conferred phenotypes in S. sanguis. With this procedure, we have been able to physically separate two small cryptic plasmids (2.4 × 10⁶ and 2.8 × 10⁶ daltons) of S. ferus. Although these plasmids were found naturally to exist in a single S. ferus host, they were able to replicate independently of one another in S. sanguis. Restriction enzyme fingerprinting indicated that these plasmids did not share a common ancestry.     

Genetic analysis of fructan-hyperproducing strains of Streptococcus mutans.

Fructan polymer, synthesized from sucrose by the extracellular fructosyltransferase of Streptococcus mutans, is thought to contribute to the progression of dental caries. It may serve as an extracellular storage polysaccharide facilitating survival and acid production. It may also have a role in adherence or accumulation of bacterial cells on the tooth surface. A number of clinical isolates of S. mutans which produce large, mucoid colonies on sucrose-containing agar as a result of increased production of fructan have been discovered. By using eight independent isolates, we sought to determine if such fructan-hyperproducing strains represented a genetically homogeneous group of organisms. Restriction fragment patterns of total cellular DNA were examined by using pulsed-field and conventional gel electrophoresis. Four genetic types which appeared to correlate with the serotype of the organism and the geographic site of isolation were evident. Southern blot analysis of several genetic loci for extracellular enzymes revealed some minor differences between the strains, but the basic genomic organizations of these loci were similar. To evaluate whether the excess fructan produced by these strains enhanced the virulence of these organisms in the oral cavity, it was of interest to create mutants deficient in fructosidase (FruA), the extracellular enzyme which degrades this polymer. The fruA gene was inactivated by allelic exchange in two fructan-hyperproducing strains as well as in S. mutans GS5, a strain which does not hyperproduce fructan. All of the fruA mutant strains were devoid of fructan hydrolase activity when levan was used as a substrate. However, the fructan-hyperproducing strains retained the ability to hydrolyze inulin, suggesting the presence of a second fructosidase with specificity for inulin in these strains.     

The Caregiver Quality of Life Cystic Fibrosis (CQOLCF) Scale: Modification and validation of an instrument to measure quality of life in cystic fibrosis family caregivers

Objective: Modify the Caregiver Quality of Life Index – Cancer (CQOLC) scale (Weitzner, Jacobsen et al. Qual Life Res 2000; 8: 55–63) as the Caregiver Quality of Life Cystic Fibrosis (CQOLCF) scale, validate it with cystic fibrosis (CF) family caregivers, and assess caregiver quality of life QOL with patient disease severity. Methods: Following modifications, 100 family caregivers were administered the CQOLCF. Construct validity was assessed by the Medical Outcomes Study Short Form (SF-36) and the Beck Depression Inventory (BDI). Pulmonary function scores and hospitalizations assessed relationship between caregiver QOL and patient disease severity. Results: Split-half reliability was 0.862 and internal consistency (Cronbach's ) was 0.909. As expected, there were relatively high correlations with the mental health (0.634) and emotional distress (–0.687); and low correlations with physical health (0.049). ANCOVA examined differences in the CQOLCF totals based on disease severity, controlling for age. Significant mean differences existed (days hospitalized F = 3.010, sig. = 0.022). Discussion: The CQOLCF appears to be a valid, reliable, and internally consistent disease-specific scale with CF family caregivers. Future research recommendations include administering the CQOLCF to an increased study sample to explore item factor analysis.     

Kikuchi-Fujimoto disease (histiocytic necrotizing lymphadenopathy) clinicopathologic and immunohistochemical study of 14 cases and its differential diagnosis with other reactive and neoplastic necrotizing lymphadenopathies

Kikuchi-Fujimoto disease also known as histiocytic necrotizing lymphadenopaty (HNL) is a rare entity, originally described in Japanese population, although currently it has been described all over the world. It is more frequent in young women and it is usually located in cervical lymph nodes. We report 14 cases of HNL in Mexican population, their clinicopathological and immunohistochemical study as well as a comparative study with other necrotizing lymphadenopaties due to B or T-cell lymphomas, tuberculosis, Epstein Barr virus infection, and non-specific necrosis. In our study we found that there was more expression of the immunomarkers CD68, MPO, CD123 and antikerat in OSCAR in the cases of HNL in contrast with the lesser or even null expression of the same markers in the necrotized lymph nodes of the comparative study group.     

Training NIH K Award Recipients: The Role of the Mentor

Mentors play important roles in training new investigators. This study was designed to determine characteristics of NIH mentored K award recipients and their mentors, their interpersonal interactions, and the factors, which influence satisfaction within this relationship. Method: A survey of 3027 NIH mentored K recipients and 1384 mentors was conducted in 2009. Nine hundred twenty‐nine (30.7%) of the K recipients and 448 (32.4%) mentors completed the survey. Results: The gender of K respondents was evenly divided while the mentors were 72.1% male. The overall rating of their mentors was positive. Ideally, both thought the mentor should be important in research training; however, in actual practice, both rated the importance as lower. A total of 88.2% of recipients were satisfied with their relationship. Although the number of black K recipients was low, this group was more likely to be dissatisfied with the mentor relationship (6/29 or 20.7%) than their white counterparts. The frequency of meeting or communicating was correlated with K recipient satisfaction. Conclusions: Overall K recipients are satisfied with their mentor relationships. Although the number of black K recipient respondents was small, the higher level of mentor dissatisfaction should be further evaluated. Qualities of mentors, including the frequency of interactions and accessibility, can influence satisfaction. Clin Trans Sci 2012; Volume 5: 386–393     

Extracardiac left to right shunt in a patient with biventricular postinfarction rupture and pseudoaneurysm

A 68 year old man had a diaphragmatic myocardial infarction and 9 months later was admitted with severe congestive heart failure (functional class IV). Cardiac catheterization demonstrated a postinfarction pseudoaneurysm. Because of a massive left to right shunt (pulmonary to systemic flow ratio = 2.7), concomitant rupture of the ventricular septum was suspected. At surgery the pseudoaneurysm communicated with the right ventricle through two different orifices and with the left ventricle through another ostium. The ventricular septum was intact. Therefore, the shunt was extracardiac through the pseudoaneurysm (left ventricle----pseudoaneurysm----right ventricle). The unique combination of lesions allowed the patient to survive. The false aneurysm was excised and primary repair was performed in the orifices of the right and left ventricular walls. The postoperative course was uneventful and 10 months later the patient was in functional class I.     

A Three-year Study of Obesity and its Relationship to High Blood Pressure in Adolescents

A sample of 356 matched cases were tracked for three years during high school. Measures taken included self-reported behavior and clinical measures of height, weight, skinfold, blood pressure, and body mass index. Data were analyzed by ethnic group, age, and sex groups. Three year (1981-1982 to 1984-1985) trends for students who were overfat, overweight, and obese revealed: a relatively greater proportion of female to male students were overfat as seniors; overweight trends for each of the four groups (ethnic group and gender) were stable over the study period; a sharp increase of obesity trends among black females was observed; and significant positive relationships existed between Percent Ideal Body Weight, skinfold thickness, Body Mass Index, and blood pressure among females of both ethnic groups. The hypothesis that the early onset of obesity is an indicator of obesity in older adolescents was supported. Students classified at risk as freshmen are more likely to remain at risk as seniors.