Isolation of a Toxoplasma gondii cyclin by yeast two-hybrid interactive screen

GAL4-based yeast two-hybrid cDNA libraries from Toxoplasma gondii RH strain were constructed and screened for interactors of a putative T. gondii cdc2-related kinase, TgCRK2. A screen of 3.2 million transformants yielded a single yeast clone that harbored a protein fusion capable of specifically interacting with TgCRK2. Sequencing revealed the cDNA insert (TgCYC1) had homology to the cyclin class of proteins. The TgCYC1 cDNA fragment was used to probe a conventional T. gondii cDNA library and a 2.65 kb cDNA coding for a predicted protein of 582 amino acids was obtained. Based on comparison with a 5'-RACE product from tachyzoite mRNA, the 2.65 kb cDNA for TgCYC1 appeared to be complete. TgCYC1 had the highest similarity to Plasmodium falciparum CYC1 and displayed sequence characteristics that place it in the cyclin H class of eukaryotic cyclins. In synchronous tachyzoite populations the level of TgCYC1 mRNA was unchanged indicating it is not cell cycle regulated at the mRNA level. TgCYC1 rescues the G(1)/S cyclin cell cycle defect in S. cerevisiae strain DL1 demonstrating that this apicomplexan cyclin can function in an established heterologous model system.     

Pseudorandom testing technique for the characterization of local hemodynamic control

Summary In order to investigate the low frequency properties of renal and femoral hemodynamic variables, pseudorandom testing techniques were used. The arterial flow of each bed, in separate experiments, was modulated by a low amplitude signal based on a pseudorandom binary sequence (PRBS) generated by digital computer.The cross-correlation functions between input flow and arterial pressure, venous pressure, and venous flow exhibit damped oscillations in all cases. These responses are parameterized in terms of a damping ratio () and an undamped natural frequency _n for a second order model. The parameters of the model are dependent upon the state of the bed as defined by mean arterial and venous pressures, mean flow through the bed, resistance, and oxygen consumption.The results of this study offer further insight into the dynamic low frequency autoregulation phenomenon for the renal and femoral beds of the dog.Supported by NIH Grant HE 11747.     

Two dynamically distinct inhibitory networks in layer 4 of the neocortex

Normal operations of the neocortex depend critically on several types of inhibitory interneurons, but the specific function of each type is unknown. One possibility is that interneurons are differentially engaged by patterns of activity that vary in frequency and timing. To explore this, we studied the strength and short-term dynamics of chemical synapses interconnecting local excitatory neurons (regular-spiking, or RS, cells) with two types of inhibitory interneurons: fast-spiking (FS) cells, and low-threshold spiking (LTS) cells of layer 4 in the rat barrel cortex. We also tested two other pathways onto the interneurons: thalamocortical connections and recurrent collaterals from corticothalamic projection neurons of layer 6. The excitatory and inhibitory synapses interconnecting RS cells and FS cells were highly reliable in response to single stimuli and displayed strong short-term depression. In contrast, excitatory and inhibitory synapses interconnecting the RS and LTS cells were less reliable when initially activated. Excitatory synapses from RS cells onto LTS cells showed dramatic short-term facilitation, whereas inhibitory synapses made by LTS cells onto RS cells facilitated modestly or slightly depressed. Thalamocortical inputs strongly excited both RS and FS cells but rarely and only weakly contacted LTS cells. Both types of interneurons were strongly excited by facilitating synapses from axon collaterals of corticothalamic neurons. We conclude that there are two parallel but dynamically distinct systems of synaptic inhibition in layer 4 of neocortex, each defined by its intrinsic spiking properties, the short-term plasticity of its chemical synapses, and (as shown previously) an exclusive set of electrical synapses. Because of their unique dynamic properties, each inhibitory network will be recruited by different temporal patterns of cortical activity.     

f1 Coat protein synthesis and altered phospholipid metabolism in f1 infected Escherichia coli

The phospholipid composition of cytoplasmic membranes prepared from bacteria grown in the presence of [³²P]phosphate and infected with f1 wild type and f1 amber mutant bacteriophages was determined. Ninety minutes after infection with f1 amber mutants in genes 1, 3, 4, 5, 6, and 7 the percentage of cardiolipin was increased from the level in uninfected bacteria of 5% to about 20–35%, and the percentage of phosphatidylethanolamine was decreased from 70% to about 50–60%. The phospholipid composition of cytoplasmic membranes from bacteria infected with a phage containing an amber mutation in the coat cistron (gene 8) did not differ from that of uninfected bacteria. Although late in infection there were no detectable alterations in phospholipid metabolism in wild type infected bacteria, transient alterations in phospholipid metabolism occurred in these bacteria 10 to 20 min after infection. During this time period, the f1 coat protein was found to be rapidly synthesized but was not being packaged into mature phage and released from bacteria. Both the long-term alterations of phospholipid metabolism found in the amber mutant infected bacteria and the transient alterations found in wild-type infected bacteria resulted from an increase in the rate of synthesis of phosphatidylglycerol and cardiolipin and a decrease in the rate of synthesis of phosphatidylethanolamine. These results are discussed in terms of the relationship between the accumulation of f1 coat protein in infected bacteria and the observed alterations in phospholipid metabolism.     

Supported planar bilayers in studies on immune cell adhesion and communication

Supported planar bilayers have been used extensively in immunology to study molecular interactions at interfaces as a model for cell-cell interaction. Examples include Fc receptor-mediated adhesion and signaling and formation of the immunological synapse between T cells and antigen-presenting cells. The advantage of the supported planar bilayer system is control of the bilayer composition and the optical advantages of imaging the cell-bilayer or bilayer-bilayer interface by various types of trans-, epi- and total internal reflection illumination. Supported planar bilayers are simple to form by liposome fusion and recent advances in micro- and nanotechnology greatly extend the power of supported bilayers to address key questions in immunology and cell biology.     

A completed screen for mutations of the rhodopsin gene in a panel of patients with autosomal dominant retinitis pigmentosa.

Recently it has been demonstrated that some families with autosomal dominant retinitis pigmentosa (adRP) have mutations in the rhodopsin gene while others do not. Previously we have identified six such mutations in seven adRP families in this laboratory, one of which was previously described in US patients. We now present a completed screen of the rhodopsin gene in a panel of 39 adRP families, by a rapid screening technique which will be of use for routine diagnosis. Nine different mutations were ultimately found, in a total of twelve of the 39 families. These include the six previously identified mutations, in codons 68-71, 190, 211, 255, 296 and 347, two new ones in codons 53 and 106, and another mutation first identified in a single US patient, in codon 58. Thus approximately 30% of adRP families have 'Rhodopsin RP' while the remainder probably have a defect elsewhere in the genome. Of those families in which rhodopsin mutations have been found, four have been classified D type, three as sectorial RP and the remainder are of uncertain classification. All families excluded from chromosome 3q by linkage have been classified R type. These data suggest a correlation between clinical sub-classification and the underlying rhodopsin/non-rhodopsin heterogeneity.     

Severe classical congenital muscular dystrophy and merosin expression

Vajsar J, Chitayat D, Becker LE, Ho M, Ben-Zeev B, Jay V. Severe classical congenital muscular dystrophy and merosin expression. Clin Genet 1998: 54: 193–198. 0 Munksgaard, 1998
It has been suggested that patients with autosomal recessive merosin deficient congenital muscular dystrophy (CMD), as opposed to the merosin positive cases form a homogeneous subgroup of a clinically more severe form of CMD. We examined merosin expression in muscle biopsies from five children with the severe classical form of CMD. Merosin deficiency was found only in 1 patient, a 6–year-old female, with abnormal brain myelination. However, her initial biopsy did not reveal the classical picture of dystrophy. The four merosin positive cases exhibited severe muscle weakness but their brain imagings were normal. There were no familial cases, except for the mother of 1 patient who had a milder form of the disease, suggesting an autosomal dominant mode of inheritance.
In contrast to previous reports, the merosin deficient CMD cases were rare in our group. Furthermore, merosin positive cases were also associated with severe phenotype suggesting that a severe phenotype is not exclusive to merosin deficient cases. Finally, the absence of merosin in a neonate with hypotonia and weakness can be helpful in making a definitive diagnosis of CMD, even though the dystrophic process may not be evident yet and histology may be non-specific.     

CD28-dependent killing by human YT cells requires phosphatidylinositol 3-kinase activation

CD28/B7 interactions have been demonstrated to provide a co-stimulatory signal for the generation of CD8⁺ cytotoxic T lymphocytes in the absence of CD4⁺ T helper cells. The CD28 signals required for induction of cytotoxicity have yet to be described. To investigate further the biochemical signaling pathways associated with CD28-dependent cytotoxicity, we have studied the human thymic leukemia cell line, YT. YT cells kill B7⁺ targets in a non-major histocompatibility complex (MHC)-restricted, CD28-dependent manner. CD28 ligation on the surface of YT cells caused a rapid increase in the tyrosine phosphorylation of four major cellular substrates with masses estimated to be 110, 95, 85, and 44 kDa. The 110 and 85 kDa substrates were identified as the catalytic and regulatory subunits, respectively, of phosphatidylinositol 3-kinase (PI3-K). Engagement of CD28 caused the rapid receptor association and activation of PI3-K but did not activate phospholipase C_γ. CD28-induced tyrosine phosphorylation and PI3-K activation was independent of p56^lck protein tyrosine kinase (PTK) activity (previously reported to be associated with CD28) and was insensitive to inhibition by the PTK inhibitor herbimycin A. Two structurally and mechanistically dissimilar inhibitors of PI3-K, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) also failed to block CD28-dependent tyrosine phosphorylation events or the association of PI3-K with the CD28 receptor. However, both drugs inhibited CD28-dependent cytotoxicity and CD28 receptor associated PI3-K activity with IC₅₀ values similar to the reported IC₅₀ values for PI3-K inhibition. Although herbimycin A did not significantly block the observed CD28-dependent tyrosine phosphorylation or PI3-K activation, herbimycin did block CD28-dependent cytotoxicity in a dose-dependent manner. These data support a role for PI3-K activation in the CD28-dependent initiation of cytotoxic effector function and suggest that a herbimycin sensitive step(s) is either CD28-independent, resides within a PI3-K-independent CD28 signaling pathway, or is downstream of CD28-dependent PI3-K activation.     

Recruitment and baseline epidemiologic profile of participants in the first phase 3 HIV vaccine efficacy trial

OBJECTIVE: To describe recruitment and baseline epidemiologic characteristics of volunteers in the first phase 3 placebo-controlled trial of a recombinant gp120 HIV vaccine (AIDSVAX B/B). METHODS: Volunteers were gay/bisexual men or women at risk for sexually transmitted HIV infection. Recruitment strategies, demographics, and risk factors were assessed. HIV status was determined by standard HIV-1 antibody assays. Seronegative/viremic HIV infection at enrollment was determined using the HIV-1 nucleic acid test. RESULTS: From June 1998 through October 1999, 5417 of 7185 volunteers screened were enrolled at 61 sites in the United States, Canada, and The Netherlands. Successful recruitment methods included distribution of study information at gay venues, advertising and media coverage, and referrals from volunteers. Most volunteers were altruistically motivated, men (98%), young (median, 36 years), white (83%), well educated (61% college education or more), and at high risk for HIV during the 6 months before enrollment. At baseline, 14 were HIV infected (12 were seronegative but viremic; 2 were seropositive and viremic). CONCLUSION: Men and women at high risk for sexually transmitted HIV infection were successfully recruited for the first phase 3 HIV vaccine efficacy trial. Knowledge of recruitment and baseline epidemiologic characteristics of participants in this trial will provide valuable guidance for designing and conducting future trials.