Thymus-independent T-cell differentiation in vitro

The generation of large quantities of novel human T-cell clones ex vivo would make a wide range of gene- and immuno-therapies for tumours and viral infections possible. Several techniques have been described to generate, in vitro and in vivo (using xenogenic hosts), mature T cells from fetal-neonatal and adult human CD34+ cells. All these techniques are cumbersome and cannot be easily translated into clinical protocols because they involve co-cultivation of CD34+ cells with thymic fragments from either human or murine fetuses. We report that the mononuclear cells of human cord blood contain a cell population that supports the differentiation of CD34+ cells into CD4+ or CD8+ naive T cells in serum-deprived cultures stimulated with stem cell factor and interleukin 7. CD4+ or CD8+ CD45RA+ TCRalphabeta+ T cells were continuously produced in vitro over a period of 20 d under these conditions. The generation of T cells in these cultures was a dynamic process and clones of T cells expressing new T-cell receptor beta-chain rearrangements were generated over time. These results pave the way for the development of very simple culture conditions for ex-vivo production of naive helper or cytotoxic T cells which could be very useful for gene- and immuno-therapy of human diseases.     

The CRY1 blue light photoreceptor of Arabidopsis interacts with phytochrome A in vitro

Plants have at least two major photosensory receptors: phytochrome (absorbing primarily red/far-red light) and cryptochrome (absorbing blue/UV-A light); considerable physiological and genetic evidence suggests some form of communication or functional dependence between the receptors. Here, we demonstrate in vitro, using purified recombinant photoreceptors, that Arabidopsis CRY1 and CRY2 (cryptochrome) are substrates for phosphorylation by a phytochrome A-associated kinase activity. Several mutations within the CRY1 C terminus lead to reduced phosphorylation by phytochrome preparations in vitro. Yeast two-hybrid interaction studies using expressed C-terminal fragments of CRY1 and phytochrome A from Arabidopsis confirm a direct physical interaction between both photoreceptors. In vivo labeling studies and specific mutant alleles of CRY1, which interfere with the function of phytochrome, suggest the possible relevance of these findings in vivo.     

Induction of ermAMR from a clinical strain of Enterococcus faecalis by 16-membered-ring macrolide antibiotics

We cloned the MLSB resistance determinant by PCR from a clinical isolate of Enterococcus faecalis 373, which is induced more strongly by a 16-membered-ring macrolide, tylosin, than by erythromycin. To elucidate the molecular basis of resistance of E. faecalis 373, we analyzed the cloned gene, designated ermAMR, by site-directed mutagenesis and reporter gene assay. Our results showed that an arginine-to-cysteine change in the seventh codon of the putative leader peptide endowed tylosin with resistance inducibility and that TAAA duplication enabled the control region to express the downstream methylase gene at a drastically increased level.     

Clonidine reduces dopamine and increases GABA in the nucleus accumbens: an in vivo microdialysis study

The spectrum of infectious disease (ID) emergencies in hospitalized patients was assessed in a prospective study of 3,626 inpatient ID consultations in a 1,350-bed teaching hospital. ID emergencies, defined by a need or anticipated need for advanced life support or by irreversible organ damage leading to permanent functional loss, were encountered in 175 patients. Infections of the central nervous system (26.3%), cardiovascular system (14.9%), alimentary system (13.1%), and lower respiratory tract (7.4%) and adverse reactions to antimicrobial agents (7.4%) were most common. In 18.9% of the cases, the referring clinicians were unaware of the emergency at the time of referral. Drug reactions (46.1%), severe alimentary and peritoneal infections (32.0%), upper respiratory tract infections (28.6%), and skin and soft-tissue infections (27.3%) were most frequently missed. The emergency ID conditions were not recognized because they had an atypical presentation (51.5%), were not commonly seen in the referring specialty (24.2%), were due to rare organisms (15.2%), or had unusual anatomical sites of involvement (9.1%). A close liaison between clinicians and the ID team is crucial for recognition of ID emergencies at their early stages so that appropriate investigations and management can be instituted expediently, before the occurrence of irreversible damage.     

Strategies to manipulate reliability: impact on statistical associations

OBJECTIVE: To examine the effects of improving measurement reliability on associations between risk factors and childhood psychiatric disorder. METHOD: Data were from a general population sample of parents (N = 211) with children aged 6 to 16 years. Reliability of measurement was improved in three ways: by increasing the number of items in a scale (internal-consistency reliability), by averaging assessments of the same variables collected on two different occasions, and by constructing latent variable measures. To assess the effects of improving reliability, selected risk factors were regressed on parental assessments of childhood oppositional defiant disorder (ODD) and overanxious disorder (OAD). RESULTS: Improving reliability led to systematic increases in the magnitude of standardized regression coefficients between family dysfunction and ODD (beta = .30-.51) and between family dysfunction and OAD (beta = .24-.48). In multiple regression, improving reliability served to strengthen the specificity of associations between ODD, OAD, and family dysfunction and maternal depressed mood. Although latent variable methods produced the largest associations, the standard errors of these estimates were also larger, resulting in wider confidence intervals and slightly larger significance values. CONCLUSIONS: Improving reliability of measurement results in larger associations between risk factors and childhood disorder and may increase the opportunity of revealing differential associations between variables.     

A novel technique for isolation of pure sperm heads from disintegrated mammalian spermatozoa

The phenomenon of differential charge distribution on sperm surface membrane has been utilised here in a low e.m.f. (electro motive force) capillary electrophoresis system to effect separation of sperm heads from disintegrated mixed spermatozoal subfractions. Washed caudal sperm of goat (Black Bengal variety) and ejaculated washed human sperm were fractionated by sonication into head, mid-piece and tail portions. Routine techniques of density gradient centrifugation on Percoll and/or sucrose were performed with sonicated spermatozoa for separation into their respective subfractions. The products obtained were not free of contamination in either case. Mixed sperm fractions when subjected to the afore mentioned modified capillary electrophoresis technique only the head pieces exhibited high affinity for migration towards the cathode terminal. With this method around 50% of the total sperm heads were separated and collected in absolutely pure form at the cathode side within 2 hrs. at 150 volts (V) and 1.5 milliampere (mA) current at 37.5 degrees C. A 4 cm. long capillary tube with a bore size 1.2 mm. was used for this purpose.