Selective Brain Cooling Reduces Water Turnover in Dehydrated Sheep

In artiodactyls, arterial blood destined for the brain can be cooled through counter-current heat exchange within the cavernous sinus via a process called selective brain cooling. We test the hypothesis that selective brain cooling, which results in lowered hypothalamic temperature, contributes to water conservation in sheep. Nine Dorper sheep, instrumented to provide measurements of carotid blood and brain temperature, were dosed with deuterium oxide (D₂O), exposed to heat for 8 days (40◦C for 6-h per day) and deprived of water for the last five days (days 3 to 8). Plasma osmolality increased and the body water fraction decreased over the five days of water deprivation, with the sheep losing 16.7% of their body mass. Following water deprivation, both the mean 24h carotid blood temperature and the mean 24h brain temperature increased, but carotid blood temperature increased more than did brain temperature resulting in increased selective brain cooling. There was considerable inter-individual variation in the degree to which individual sheep used selective brain cooling. In general, sheep spent more time using selective brain cooling, and it was of greater magnitude, when dehydrated compared to when they were euhydrated. We found a significant positive correlation between selective brain cooling magnitude and osmolality (an index of hydration state). Both the magnitude of selective brain cooling and the proportion of time that sheep spent selective brain cooling were negatively correlated with water turnover. Sheep that used selective brain cooling more frequently, and with greater magnitude, lost less water than did conspecifics using selective brain cooling less efficiently. Our results show that a 50kg sheep can save 2.6L of water per day (~60% of daily water intake) when it employs selective brain cooling for 50% of the day during heat exposure. We conclude that selective brain cooling has a water conservation function in artiodactyls.     

Helical motion analysis of the knee--I. Methodology for studying kinematics during locomotion

A technique for investigating the three-dimensional kinematics of knee motion during dynamic functional tasks has been developed. It involves the combined usage of a six degree of freedom goniometer and helical motion analysis. A detailed procedure for coordinate system alignment and calibration must be followed. Once established this entire procedure is routinely implementable. Ensemble averages from multiple walking strides reveal that this technique is sensitive enough to differentiate between the kinematics of an uninjured and injured knee.     

Influence of template strandedness on in vitro replication of mutagen-damaged DNA

We analyzed the ability of DNA polymerases to bypass damage on single- and double-stranded templates. In vitro DNA synthesis was studied on UV-irradiated and polyaromatic hydrocarbon reacted (benzo[a]pyrenediol epoxide and oxiranylpyrene) double-stranded templates by a protocol involving initiation on a uniquely nicked circular double-stranded template. The template was prepared by treating single-stranded (+)M13mp2 circular strands with mutagen and then hybridizing with restricted M13 RFmp2, followed by isolation of the nicked RFII forms. The protocol permits either (+), (-), or both strands to carry lesions. We found that the rules for termination and bypass of lesions previously observed with single-stranded DNA templates also hold for double-stranded templates. Termination of synthesis occurs primarily one nucleotide 3' to the lesion in the template strand. Bypass of UV-induced lesions can be followed in a series of three partial reactions in the presence of Mn2+ and dGMP, which relax the specificity of nucleotide insertion and 3'----5' exonuclease activity, respectively. There is no evidence for greater permissivity of bypass in double-as opposed to single-stranded templates. As with single-stranded templates, purines and preferentially deoxyadenosine (dA) are inserted opposite lesions. Lesions in the nontemplate strand elicit neither termination nor pausing. The addition of Rec A protein resulted in a measurable increase of bypass in this system.     

Comparison of the precursor and mature forms of rat heart mitochondrial malate dehydrogenase

The complete amino acid sequence of mitochondrial malate dehydrogenase from rat heart has been determined by chemical methods. Peptides used in this study were purified after digestions with cyanogen bromide, trypsin, endoproteinase Lys C, and staphylococcal protease V-8. The amino acid sequence of this mature enzyme is compared with that of the precursor form, which includes the primary structure of the transit peptide. The transit peptide is required for incorporation into mitochondria and appears to be homologous to the NH2-terminal arm of a related cytoplasmic enzyme, pig heart lactate dehydrogenase. The amino acid differences between the rat heart and pig heart mitochondrial malate dehydrogenases are analyzed in terms of the three-dimensional structure of the latter. Only 12/314 differences are found; most are conservative changes, and all are on or near the surface of the enzyme. We propose that the transit peptide is located on the surface of the mitochondrial malate dehydrogenase precursor.     

Mutation induced in vitro on a C-8 guanine aminofluorene containing template by a modified T7 DNA polymerase

We reacted uracil-containing M13mp2 DNA with N-hydroxy-2-aminofluorene to produce a template with N-(deoxyguanosin-8-yl)-2-aminofluorene adducts. This template was hybridized to a non-uracil-containing linear fragment from which the lac z complementing insert had been removed to produce a gapped substrate. DNA synthesis using this substrate with the modified T7 DNA polymerase Sequenase led to an increase in the number and frequency of lac- mutations observed. Escherichia coli DNA polymerase I (Kf) did not yield a comparable increase in mutation frequency or number even though both Sequenase and the E. coli polymerase had similar, low, 3'----5' exonuclease activities as compared to T4 DNA polymerase. We did not observe an increase in mutations when synthesis was attempted on a template reacted with N-acetoxy-2-(acetylamino)fluorene to give N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene adducts. Both E. coli and T7 enzymes terminate synthesis before all (acetylamino)fluorene lesions. Only some of the putative aminofluorene adducts produced strong termination bands, and there was a difference in the pattern generated by Sequenase and E. coli pol I (Kf) using the same substrate. Analysis of the mutations obtained from Sequenase synthesis on the aminofluorene-containing templates indicated a preponderance of -1 deletions at G's and of G----T transversions.     

Complete congruence between morphological and rbcL-based molecular phylogenies in birches and related species (Betulaceae).

Estimations of phylogenies from morphological and molecular data often show contrasting results. We compared morphological and molecular phylogenies in an ancient family of woody dicots, the Betulaceae (birch family). The phylogeny of the family was estimated from parsimony analysis of morphological characters in the genera Alnus, Betula, Carpinus, Corylus, Ostrya, and Ostryopsis and from parsimony and distance-matrix analyses of DNA sequences of the chloroplast gene encoding the large subunit of ribulose-1,5-biphosphate carboxylase (rbcL) in the genera Alnus, Betula, Carpinus, Corylus, and Ostrya and in two outgroups, Quercus and Liquidambar. The topologies obtained by the different methods were completely congruent, and bootstrapping strongly supported the division of the family Betulaceae into two major clades, Betuleae (Alnus and Betula) and Coryleae (other members). Only slightly more homoplasy was present in the rbcL sequence data set than in the morphological set. Relative-rate tests indicated that the Coryleae clade had a faster rate of rbcL evolution than did the Betuleae clade. Heterogeneity of rates of morphological evolution also paralleled those for rbcL.     

Molecular basis of organ-specific selection of viral variants during chronic infection.

Viral variants of different phenotypes are present in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the Armstrong strain of lymphocytic choriomeningitis virus. The CNS isolates are similar to the parental virus and cause acute infections in adult mice, whereas the lymphoid isolates cause chronic infections associated with suppressed T-cell responses. In this study, we provide a molecular basis for this organ-specific selection and identify a single amino acid change in the viral glycoprotein that correlates with the tissue specific selection and the persistent and immunosuppressive phenotype of the variants. This phenylalanine (F)-to-leucine (L) change at position 260 of the viral glycoprotein was seen in the vast majority (43 of 47) of the lymphoid isolates, and variants with L at this residue were selected in spleens of persistently infected mice. In striking contrast, isolates with the parental sequence (F at residue 260) predominated (48 of 59 isolates) in the CNS of the same carrier mice. Complete nucleotide sequence analysis of the major structural genes of several independently derived (from different mice) spleen isolates showed that these variants were greater than 99.8% identical to the parental virus. In fact, the only common change among these spleen isolates was the F----L mutation at residue 260 of the glycoprotein. These results show that an RNA virus can exhibit minimal genetic drift during chronic infection in its natural host, and yet a single or few mutations can result in the organ-specific selection of variants that are markedly different from the parental virus.     

Flavivirus enzyme-substrate interactions studied with chimeric proteinases: identification of an intragenic locus important for substrate recognition.

The proteins of flaviviruses are translated as a single long polyprotein which is co- and posttranslationally processed by both cellular and viral proteinases. We have studied the processing of flavivirus polyproteins in vitro by a viral proteinase located within protein NS3 that cleaves at least three sites within the nonstructural region of the polyprotein, acting primarily autocatalytically. Recombinant polyproteins in which part of the polyprotein is derived from yellow fever virus and part from dengue virus were used. We found that polyproteins containing the yellow fever virus cleavage sites were processed efficiently by the yellow fever virus enzyme, by the dengue virus enzyme, and by various chimeric enzymes. In contrast, dengue virus cleavage sites were cleaved inefficiently by the dengue virus enzyme and not at all by the yellow fever virus enzyme. Studies with chimeric proteinases and with site-directed mutants provided evidence for a direct interaction between the cleavage sites and the proposed substrate-binding pocket of the enzyme. We also found that the efficiency and order of processing could be altered by site-directed mutagenesis of the proposed substrate-binding pocket.