Recombination between HLA-A and C and between HLA-B and complement locus C4 in the same individual

In a French family with 2 parents and 5 children a crossing over was found in the HLA region on both of the parental haplotypes of one of the children. The following markers were studied: HLA-A, B, C,DR, DQ(MB), DP(SB), complement allotypes C4 and Bf and glyoxalase I polymorphism. In the third child, the paternal haplotype had a recombination between HLA-A and HLA-C and the maternal haplotype a recombination between HLA-B and complement locus C4. Mixed lymphocyte cultures confirmed the serological findings and non-HLA markers (blood groups and immunoglobulin allotypes) showed no evidence of extrapaternity. The family also demonstrates a probable duplication of the C4B1 gene in one of the paternal haplotypes.     

Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana

All of the protein products of Turnip crinkle virus (TCV; Tombusviridae, Carmovirus) were tested for their ability to suppress RNA silencing of a reporter gene after transient expression in Agrobacterium-infiltrated Nicotiana benthamiana leaves. Only the capsid protein, P38, showed suppression activity, although this was not obvious when P38 was expressed as part of a TCV infection of the same tissues. When P38 was expressed from a PVX vector, symptoms with enhanced severity that correlated with increased PVX RNA accumulation were observed. This contradiction between ectopic expression of P38 and TCV infection could be accounted for if the active determinant of suppressor activity within P38 was sequestered within the capsid protein structure. The N-terminal 25 amino acids were shown to be important for this activity. This region forms part of the unexposed R-domain that interacts with the RNA within the virus particle. This observation throws light on some of the complex biology exhibited by TCV.     

Principal neuron spiking: neither necessary nor sufficient for cerebral blood flow in rat cerebellum

Neuronal activity, cerebral blood flow, and metabolic responses are all strongly coupled, although the mechanisms behind the coupling remain unclear. One of the key questions is whether or not increases in spiking activity in the stimulated neurons are sufficient to drive the activity-dependent rises in cerebral blood flow (CBF) that form the basis of the signals used in functional neuroimaging such as the blood oxygen level-dependent (BOLD) signal. To this end the present study examined the effect of enhanced spike activity per se on CBF in rat cerebellar cortex under conditions of disinhibition, achieved by blocking GABA_A receptors using either bicuculline or picrotoxin. Purkinje cell spiking activity and local field potentials were recorded by glass microelectrodes, and laser Doppler flowmetry was used to monitor CBF. Disinhibition increased Purkinje cell spiking rate to 200–300% of control without incurring any increase in basal CBF. This demonstrates that increased spike activity per se is not sufficient to affect basal CBF. The neurovascular coupling between excitatory synaptic activity and CBF responses evoked by inferior olive (climbing fibre) stimulation was preserved during disinhibition. Thus, the unchanged basal CBF in the presence of the dramatic rise in Purkinje cell spiking rate was not explained by impaired synaptic activity–CBF coupling. On the basis of our previous and the present studies, we conclude that increased spiking activity of principal neurons is neither sufficient nor necessary to elicit CBF responses and in turn BOLD signals, and that activation-dependent vascular signals reflect excitatory synaptic activity.     

Clone-based systematic haplotyping (CSH): a procedure for physical haplotyping of whole genomes

We present a novel methodology to determine the phase of single-nucleotide polymorphisms (SNPs) on a chromosome, which we term clone-based systematic haplotyping (CSH). The CSH procedure is based on separating the allelic chromosomes of a diploid genome by fosmid/cosmid cloning, and subsequent SNP typing of 96 clone pools, each representing approximately 10% of the genome. The pools are screened by PCR for the sequence of interest, followed by SNP typing on the PCR products using the GOOD assay. We demonstrate that by CSH, the haplotype of SNPs separated by more than 50 kilobases can definitely be assigned. We propose this method as being suitable for constructing maps of ancestral haplotypes, analysis of complex diseases, and for diagnosis of rare defects in which the molecular haplotype is crucial. In addition, by amplifying the initial DNA by many orders of magnitude, the original DNA resource is effectively immortalized, enabling the haplotyping of hundreds of thousands of SNPs per individual.     

Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody.Using this technique we have been able to identify the precipitate arc in crossed immunoelectrophoresis of major histocompatibility complex (MHC) class I molecules in a mixture of all detergent solubilized cell membrane molecules by means of a monoclonal antibody, the specificity of which was known independently to be against MHC class I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains.     

Relative contributions of innate and acquired host responses to bacterial control and arthritis development in Lyme disease

TLR2(-/-)/scid double-mutant mice were infected with B. burgdorferi to assess the relative importance of acquired and innate host defenses. Although spirochete levels at 4 weeks were lower in TLR2(-/-) mice than in TLR2(-/-)/scid mice, the increased arthritis severity of TLR2 (Toll-like receptor 2)-deficient mice was reduced by the presence of the scid mutation.     

Potassium and sodium shifts during in vitro isometric muscle contraction,and the time course of the ion-gradient recovery

Intracellular potassium ([K⁺]_i), interstitial potassium ([K⁺]_inter), intracellular sodium ([Na⁺]_i), and resting membrane potential (RMP) were measured before and after repetitive stimulation of mouse soleus and EDL (extensor digitorum longus) muscles. At rest, RMP was –69.8 mV for soleus and –74.9 mV for EDL (37°C). [K⁺]_i was 168 mM and 182 mM, respectively. In soleus, free [Na⁺]_i was 12.7 mM. After repetitive stimulation (960 stimuli) RMP had decreased by 11.9 mV for soleus and by 18.2 mV for EDL. [K⁺]_i was reduced by 32 mM and 48 mM, respectively, whereas [K⁺]_inter was doubled. In soleus [Na⁺]_i had increased by 10.6 mM, demonstrating that the [K⁺]_i-decrease is three times higher than the [Na⁺]_i-increase. It is concluded that this difference reflects different activity induced movements of Na and K, and that the difference is not due to the Na/K pumping ratio. The possible involvement of the potassium loss in muscle fatigue is discussed. After stimulation RMP recovered with a time constant of 0.9 min for soleus and 1.5 min for EDL. Within the first minutes after stimulation the intracellular potassium concentration increased by 20.4 mM/min for soleus and 21.7 mM/min for EDL. Free [Na⁺]_i decreased with less than 10 mM/min. The mechanisms underlying the different rate of changes are discussed.Parts of this work have been published in preliminary form (Juel and Sjøgaard 1984)     

A comparison of micropuncture and lithium clearance methods in the assessment of renal tubular function in rats with diabetes insipidus

The lithium clearance technique has been proposed as a non-invasive method whereby fluid delivery from the pars recta and pars convoluta of proximal tubules can be measured as C_Li and C_IN [0.78 C_Li/C_IN+0.22], respectively [12], C_Li being the clearance of lithium and C_IN that of inulin. In the present study, fluid delivery from proximal tubules was estimated simultaneously by micropuncture and lithium clearance techniques in anaesthetized Brattleboro rats with diabetes insipidus, under control conditions and following chronic treatment with hydrochlorothiazide. Absolute deliveries from the proximal convoluted tubules as determined by the micropuncture and lithium clearance methods were 437 and 427 μl/min, respectively, in untreated animals and 348 and 355 μl/min, respectively, in thiazide-treated animals. The individual results obtained by the two methods showed a high degree of correlation (r=0.85,P<0.001). In untreated Brattleboro rats, proximal fluid delivery as estimated by both the micropuncture and lithium clearance techniques showed significant (P<0.001) correlations with urine flow rate. These results provide further evidence for the acceptance of lithium clearance as a valid estimate of proximal tubular fluid delivery.