Roles of specific amino acids in the N terminus of Pseudomonas aeruginosa flagellin and of flagellin glycosylation in the innate immune response

The Toll-like receptor 5 (TLR5) binding site has been predicted to be in the N terminus of the flagellin molecule. In order to better define the interaction between the N-terminal amino acids of Pseudomonas aeruginosa flagellin and TLR5, site-specific mutations were generated between residues 88 and 97 of P. aeruginosa PAK flagellin as well as outside of this region. The mutant flagellins were expressed in Escherichia coli BL21(plysS), purified by affinity chromatography, and passed through a polymyxin B column to remove contaminating lipopolysaccharide (LPS). Their ability to stimulate interleukin-8 (IL-8) release from A549 cells was examined. The cloned mutated genes were used to complement a PAK fliC mutant in order to test for effects on motility and on IL-8 release by purified flagellar preparations. All the mutations, single or double, in the predicted TLR5 binding region reduced IL-8 signaling to less than 95% of the wild-type flagellin levels, but the single mutation outside the binding region had no effect. Changes made at two amino acid sites resulted in loss/reduction of motility; however, changes made at single sites, i.e., Q83A, L88A, R90A, M91A, L94A, and Q97A, had no effect on motility. The mutated genes encoding two of the motile but poorly signaling flagellins had no compensatory mutations to allow motility. Thus, while it is speculated that pathogen-associated molecular patterns (PAMPs) have evolved in locations that are essential to maintain function, it appears that there is tolerance for at least single amino acid changes in the PAMP of P. aeruginosa flagellin. The purpose of flagellin glycosylation in P. aeruginosa is unknown. In order to examine its role, if any, in signaling an inflammatory response, we used whole flagella from the motile chromosomal mutant strains PAKrfbC and PAO1rfbC, which are defective in flagellin glycosylation. IL-8 release from A549 cells stimulated with nonglycosylated flagellar preparations (having less then 1 picogram of LPS/mug) was significantly reduced compared to their respective wild-type flagellar preparations, indicating a role of flagellar glycosylation in the proinflammatory action of Pseudomonas flagellin. The basis of the latter activity is unknown, since the glycosylation sites are found in the D3 domain of flagellins and the TLR5 binding site is located in the D1 domain. Thus, P. aeruginosa flagellin has evolved additional flagellar signaling mechanisms over that described for Salmonella flagellin.     

Length heteromorphisms of fluorescent (f) and non-fluorescent (nf) segments of human Y chromosome: classification, frequencies, and incidence in normal Caucasians.

Sixty normal male Caucasians were selected to study the length of the Y chromosome. QFQ banding was performed. Chromosomes 19 and 20 (F) and Y were measured directly from the film. Y/F, f/F, and nf/F indices (f = fluorescent; nf = non-fluorescent segment) were determined. The length of the Y chromosome was classified into 5 groups; very small, small, average, large, and very large with Y/F indices of less than 0.8, 0.81--0.94, 0.95--1.09, 1.1--1.23, and greater than 1.23, respectively. The frequencies of Y/F indices for these groups were 0 (0%), 9 (15.0%), 40 (66.7%), 8 (13.3%), and 3 (5.0%), respectively. The most frequent class was 0.95--1.09 and was defined as the 'average' Y/F index for the human Y chromosome. The variation in the total length of the Y chromosome was accounted for by variations in the length of the non-fluorescent as well as the fluorescent segments. No relation between f and nf segments was observed. The mean Y/F, f/F, and nf/F indices were 1.022, 0.441, and 0.574, respectively.     

A human recessive neurosensory nonsyndromic hearing impairment locus is a potential homologue of the murine deafness (dn) locus

A locus for recessive neurosensory nonsyndromic hearing impairmentmaps to chromosome 9q13–q21 in two regionally separateconsanguineous families from India. Each family demonstratesa LOD score greater than 4.5 to this region. D9S15, tightlylinked to the Friedreich's ataxia locus, a region that has beendefined with over 1 Mb of YAC contig information and severalexpressed sequences, is one of the flanking markers. In mice,the deafness (dn) locus maps to mouse chromosome 19 and flankingloci are syntenic to human chromosome 9q11–q21. The dnmouse is a potential model for the hearing impairment foundin both these families.     

Waves earlier than P3 are more informative in putative subcortical Dementias: A study with mapping and neuropsychological techniques

Summary Thirty subjects (normal controls, patients with putative subcortical dementia and non-demented patient controls) were studied using advanced neurophysiological (16 scalp-electrode positions, computer-assisted brain electrical activity mapping, auditory oddball paradigm) and neuropsychological techniques. Our study suggests that waves earlier than P₃ (N₁, P₂ and N₂) are all correlated with global measures of cognitive functions. They are, however, differentially correlated with specific measures of cognitive functions, N₁ and P₂ with mental speed and N₂ with short-term memory. The abnormalities of these waves (earlier than P₃) may be an electrophysiologic marker of dementia in patients with putative subcortical states.     

Early epidermal destruction with subsequent epidermal hyperplasia is a unique feature of the papilloma-independent squamous cell carcinoma phenotype in PKCepsilon overexpressing transgenic mice

Protein kinase C epsilon (PKCepsilon) overexpressing transgenic (PKCepsilon Tg) mice develop papilloma-independent squamous cell carcinomas (SCC) elicited by 7,12-dimethylbenz[a]anthracene (DMBA) tumor initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) tumor promotion. We examined whether epidermal cell turnover kinetics was altered during the development of SCC in PKCepsilon Tg mice. Dorsal skin samples were fixed for histological examination. A single application of TPA resulted in extensive infiltration of polymorphonuclear neutrophils (PMNs) into the epidermis at 24 h after TPA treatment in PKCepsilon Tg mice while wild-type (WT) mouse skin showed focal infiltration by PMNs. Complete epidermal necrosis was observed at 48 h in PKCepsilon Tg mice only; at 72 h, epidermal cell regeneration beginning from hair follicles was observed in PKCepsilon Tg mice. Since the first TPA treatment to DMBA-initiated PKCepsilon Tg mouse skin led to epidermal destruction analogous to skin abrasion, we propose the papilloma-independent phenotype may be explained by death of initiated interfollicular cells originally destined to become papillomas. Epidermal destruction did not occur after multiple doses of TPA, presumably reflecting adaptation of epidermis to chronic TPA treatment. Prolonged hyperplasia in the hair follicle may result in the early neoplastic lesions originally described by Jansen et al. (2001) by expanding initiated cells in the hair follicles resulting in the subsequent development of SCC.     

Non-fermenters in human infections

One thousand and one hundred thirty non-fermenting gram negative bacteria were isolated from various samples. Of these, Pseudomonas aeruginosa was the commonest isolate (72.83%) followed by Acinetobacter anitratus (8.4%), Alcaligenes faecalis (7.6%), Acinetobacter lwoffi (4.4%), Pseudomonas flourescens (2.4%), Schwanella putrefaciens (1.6%), Stenotrophomonas maltophilia (1.6%), Pseudomonas putida (0.4%), Bravundimonas vesicularis (0.4%) and Flavobacterium meningosepticum (0.4%). Antibiotic sensitivity pattern showed multiple drug resistance pattern with majority of the isolates being resistance to two or more drugs.     

Primary adenocarcinoma (intestinal type) of nasal septum--a case report

Primary nasal septal adenocarcinoma is a rare lesion. A sporadic case of septal adenocarcinoma in a 22 years old male is being described along with review of literature.     

Carcinoid tumors of the lung: a diagnostic challenge in bronchial washings

Bronchial washings are used routinely in the diagnosis of lung tumors. However, unlike other tumors, the diagnosis of bronchial carcinoids on bronchial washings is difficult. We reviewed 17 cases of histologically proven bronchial carcinoids from the files of the cytology laboratory over a period of 15 yr (1986–2001). The bronchial washings and histology sections of all the cases were reviewed separately by two independent observers and the results tabulated. Two cases had inadequate bronchial washings for evaluation and were excluded from the study. A growth was identified on bronchoscopy in 13 of 15 cases. Initial cytologic diagnoses were ?adenocarcinoma/?carcinoid and suspicious of carcinoid in one case each. However, on review, tumor was identified in 10 of 13 cases initially considered to be negative. The possible reasons for a false‐negative report on initial cytology include the paucity of tumor cell fragments in the bronchial washings (5 of 12 cases showing only one to two tumor fragments) and their bland appearance, often being mistaken for benign columnar cells. This study highlights the potential pitfalls in the diagnosis of bronchial carcinoids on bronchial washings and underlines the importance of a diligent search in cases with high clinical suspicion and positive bronchoscopic findings. Diagn. Cytopathol. 2004;30:62–66. © 2004 Wiley‐Liss, Inc.     

Alterations in hepatic kinase activity following whole body gamma-irradiation of mice

The chronological activation of the signaling molecules following whole body gamma-irradiation was investigated in mouse liver. The activity of two kinases, tyrosine kinase and protein kinase C (PKC), was found to respond differently to gamma-irradiation. Tyrosine kinase was found to respond to much lower doses of irradiation (10 cGy), whereas PKC was found to be activated at comparatively higher doses (3 Gy). Tyrosine kinase showed a sharp activation at 30 min and then a decline to normal values at 1 h. Activation of PKC was apparent at as early as 15 min of irradiation and showed a maximal increase at 30 min. This was followed by a decline to normal values at 1 h. The response of the whole organ was found to be different from that of reported effects on a single cell. These results suggest that the data obtained from the single-cell studies would have limited application in the experiments involving the whole animal. Interruption of these signals at various steps is currently being used to manipulate tumor response to radiotherapy. In such cases, the difference in response of a single cell and a whole animal must be considered.     

Clinical study of tetanus neonatorum

Seventy six cases of neonatal tetanus were studied. There was preponderance of males. Short incubation period, short period of onset, low birth weight, presence of fever and tachycardia were associated with a poor prognosis. The cases were divided into 2 groups of 46 and 30. First group was given TIG while second group was given ATS. All other therapies were kept identical. Mortality rate in two groups was 97.5% and 96.5%. Corrected mortality due to tetanus per se was 22.5% and 28.5% (p-0.5). Fifteen cases (32.7%) and 12 cases (40%) in group A & B respectively were born to mothers who had already received one to three doses of tetanus toxoid. The potency of the vaccine should be ensured before it is administered to mothers, through proper maintenance of cold chain. It is concluded that TIG is not superior to ATS in managing moderate and severe grade cases.