Impaired induction of the apoptosis-protective protein Bcl-xL in activated PBMC from asymptomatic HIV-infected individuals

Progression to AIDS in asymptomatic HIV-infected individuals is characterized by a gradual but progressive loss of CD4+ T cells. While the mechanisms underlying this decline are currently unknown, recent evidence suggests that these cells are abnormally sensitive to apoptosis in response to activation signals. Recent work has implicated downregulation of Bcl-2 with the increased spontaneous apoptosis in lymphocytes from HIV-infected patients. We have evaluated the roles of the apoptosis-protective proteins Bcl-2 and Bcl-x in stimulated PBMC from asymptomatic HIV-infected and HIV-uninfected individuals. We found that Bcl-2 was constitutively expressed in PBMC from both HIV-infected and uninfected samples. However, Bcl-x induction was delayed and responses were decreased in stimulated HIV-infected samples. Additionally, single-cell intracellular staining of Bcl-x revealed a significant inverse correlation between PWM-induced Bcl-x expression and apoptosis (r = -0.695, P = 0.05). This was confirmed at the single-cell level in direct experiments when stimulated cells were sorted based on Bcl-x induction and then measured for apoptosis. Furthermore, low Bcl-x expression was not due to reduced lymphocyte activation following PWM stimulation. Our data indicate that the induction of Bcl-x is markedly impaired in asymptomatic HIV-infected patients and that stimuli which induce inadequate expression of Bcl-x are associated with increased levels of apoptosis in these cells.     

Vitamin and mineral transfer during fetal development and the early postnatal period in pigs

There are periods during pregnancy when sows may have a temporally high requirement for certain vitamins and minerals. Proteins transferring retinol and Fe to the developing pig fetus have been discovered, whereas transport mechanisms for other vitamins and minerals are probably present but have not yet been identified. Sow body tissues can serve as a reservoir for many micronutrients, but it is not known whether these reserves can supply an adequate quantity during critical fetal developmental periods. There is a low placental transfer of vitamin E to the fetus even if the dietary concentration fed to a gestating animal is high, but colostrum and milk concentrations can be increased when the nutrient is fed to sows. If the dam's diet contains inadequate Ca or P, the concentration of these elements in the developing fetus and milk will not be affected. Consequently, sow bone demineralization will occur under conditions of dietary inadequacy of Ca and P. Other nutrients can be depleted from sow tissue reservoirs over several parities (e.g., Se), resulting in low quantities being provided in the milk for nursing pigs. Scientific information involving adequate vitamin and mineral nutrition for female pigs to improve conception rate and embryonal survival that will result in optimum fetal and postnatal pig development can be considered to be in its infancy.     

Determination of aldehydes and other lipid peroxidation products in biological samples by gas chromatography-mass spectrometry

The extremely broad spectrum of the biological effects of aldehydic lipid peroxidation products has necessitated the development of a technique that can quantitate all of the aldehydes formed in biological materials. The proposed method is based on the use of O-(2, 3, 4, 5, 6-pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA.HCl) to form the O-pentafluorobenzyl-oxime (PFB-oxime) derivatives of 22 saturated and unsaturated aldehydes (C2-C12) including hexanal, 4-hydroxy-non-2-enal (HNE), and malondialdehyde (MDA), followed by trimethylsilylation of the hydroxyl group to trimethylsilyl (TMS) ethers. The PFB-oxime-TMS derivatives were analyzed by capillary column gas chromatography-negative-ion chemical ionization mass spectrometry (GC-NICIMS) with ammonia as reagent gas. Quantitation was achieved using benzaldehyde-ring-D5 as an internal standard in selected ion recording (SIR) mode. Standard curves were linear (r > 0.99) for all individual aldehydes. The detection limit was between 50 and 100 fmol per 1 microliter injected aldehyde. Recovery of all aldehydes from urine, plasma, and tissue homogenate was over 85%, except HNE, trans-2-octenal and trans-2,-cis-6-nonadienal from plasma and tissue sample, which were between 60 and 80%, suggesting these aldehydes may bind to protein and lipid components, especially to SH groups of proteins. The high sensitivity of this method allows the measurement of physiological aldehyde levels in biological samples. The products of aldehyde metabolism can also be measured by this assay.     

Gene mapping from a bovine 1;29 DNA library prepared with chromosome microdissection

The relative concentrations of IgM and IgG antibodies (Ab) to Schistosoma mansoni soluble egg antigen (SEA) were evaluated in paired samples of venous blood sera and buffer-eluates of capillary blood drops dried on filter papers. The samples were obtained from school children at early and chronic stages of schistosomiasis diagnosed on the basis of history, clinical symptomatology and parasitological criteria. Enzyme-linked immunosorbent assay (ELISA), simultaneously performed, revealed paired samples to display comparable Ab levels in all cases. Samples from children with early schistosomiasis had specific IgM:IgG ratios greater than 1 [optical densities (O.D.) in sera and blood eluates of 0.77 +/- 0.32 and 0.68 +/- 0.30, respectively for IgM and 0.52 +/- 0.25 and 0.50 +/- 0.25 for IgG]. This ratio, however, was less than 1 in samples from chronically infected children (O.D. of 0.20 +/- 0.11 and 0.20 +/- 0.11 for IgM and 0.69 +/- 0.33 and 0.73 +/- 0.32 for IgG). The specific advantages of this simplified technique are the use of anti-SEA Abs in fingerstick blood eluates, rather than sera of venous blood to serologically diagnose schistosomiasis and to differentiate early from chronic infections particularly when used for mass screening, such as epidemiologic surveys.     

Inhibition of restriction endonuclease activity by DNA binding fluorochromes

Activity of type II restriction endonuclease is affected by many common factors including buffer composition and sequences flanking the recognition site (Brabec et al., Eur.J. Biochem. 216, 183, 1993). The successful development of Optical Mapping (Schwartz et al., Science, 262, 110, 1993; Meng et al., Nature Genet. 9, 432, 1995; Wang and Schwartz, PNAS, 1995 Cai et al., PNAS, 92, 5164, 1995) relied on optimization of light microscope-based imaging of fluorescently labeled DNA molecules during restriction endonuclease digestion. Little was known about the effects of commonly used DNA-fluorochromes on restriction endonuclease activity. Thus, we developed an enzyme activity assay using lambda bacteriophage DNA or adenovirus-2 DNA to evaluate the effects of five DNA binding fluorochromes (4'-6-daimidine-2-phenylindole (DAPI), ethidium bromide (EtdBr), ethidium bromide homodimer (EthD-1), bis-benzimide (H33258) and benzothiazolium-4-quinolinium dimer (TOTO-1)) on the enzymatic activities of eleven type II restriction endonucleases (Asc I, Csp I, Dra I, EcoR I, Hha I, Hind III, Not I, Rsr II, Sfi I, SgrA I and Sma I). We found that the minor groove binding fluorochrome, DAPI, did not measurably inhibit activity of this group, with the exception of Dra I. Similarly, another minor groove binding fluorochrome H33258 inhibited Dra I and Not I (slightly). The three intercalating fluorochromes EtdBr, EthD-1 and TOTO-1, however, variably inhibited the other enzymes. Since Beta-mercaptoethanol (Beta-ME) is used to discourage photodamage of stained DNA molecules, we also assessed its effect on restriction endonuclease activity. Interestingly, Dra I, Hind III, Sfi I and Sma I retained full activities at high concentration of Beta-ME (5%), but Asc I, Csp I, Not I, Rsr II and SgrA I showed varying sensitivities to the Beta-ME. Isoschizomers Csp I and Rsr II behaved differently to both fluorochromes and Beta-ME. The results presented here should provide a basis for further development of new Optical Mapping-based techniques requiring fluorescence labeling of other actively imaged enzymatic reactions.     

General purpose inference engine for canonical graph models

The design and implementation of a General Purpose Inference Engine for canonical graph models that is both flexible and efficient is addressed. Conventional inference techniques (e.g. forward chaining, backward chaining and mixed strategies) are described, and new modes of flexibility through the provision of inexact matching between data and assertions/rules are explained. In GPIE, scanning/searching of the rules in the rule base is restricted to a minimum during execution, but at the expense of compilation of the rule set prior to execution. The generality of the rule set is transparent to the inference engine, thereby permitting reasoning at various levels. This research demonstrates that a graph-based inference engine offering flexible control structures and inxact matching can complement intermediate notations, such as conceptual graphs, offering the expressive power of a rich knowledge representation formalism. The availability of an extendible graph processor for building appropriate canonical graph models presents the exciting prospect of a general purpose reasoning engine.     

Critical issues regarding AIDS among injecting drug users

The paper identifies and reviews some critical issues in the field of human immunodeficiency virus (HIV) transmission among intravenous drug users. First, it discusses political denial and compartmentalization of the problem, giving an example from the United States that illustrates the lack of a coherent national strategy. It then reviews the role that stereotypes play in policy-making and points out that behaviour change can be considerable, giving details of successful safer injection programmes. The conditions that foster injection as a mode of ingesting drugs are reviewed, as in the role of drug trans-shipment patterns, particularly as a possible conduit of HIV. Finally, the role of prisons as places for the spread of HIV, and therefore for its prevention, is discussed.