Reversible cryogenic blockade of neural function in the brain of unrestrained animals.

Reversible cryogenic blockade of neural systems in the brain may be accomplished by local cooling. A small cryoprobe is described which may be implanted in the brain of an unrestrained, behaving animal. Cooling may be restricted to the tip of the cryoprobe and the temperature of the tip and the shaft may be regulated and monitored independently by miniature thermocouples and appropriate control and indicator devices. Electrophysiological results are presented which show that the cryogenic blocking effect may be localized to within a few millimeters of the tip of the cryoprobe and that the size of the region blocked is proportional to the tip temperature. The system described has been shown to be effective in both electrophysiological and behavioral studies.

Pyroxferroite: Stability and X-ray Crystallography of Synthetic Ca0.15Fe0.85SiO3 Pyroxenoid.

Synthetic Ca(0.15)Fe(0.85)SiO(3) pyroxenoid has the same (pyroxmangite) structure and very nearly the same composition as pyroxferroite, a new mineral found in Apollo 11 lunar samples. The synthetic material is not stable below pressures of approximately 10 kilobars. It appears likely that the lunar pyroxferroite has persisted in a metastable state for some billions of years.

Brain wave components of the contingent negative variation in humans.

In a contingent negative variation paradigm with two stimuli paired at an interstimulus interval of 4 seconds, two distinct waveforms having functional and topographic differences are observed. An early wave is maximal over the frontal cortex and is elicited by the warning stimulus. A later wave, maximal over the motor cortex, precedes the imperative stimulus and is identified with preparation for motor response.

Response plasticity of lateral geniculate neurons during and after pairing of auditory and visual stimuli.

Neurons of the lateral geniculate nucleus, responsive only to visual stimulation, show response plasticity during and after pairing of auditory and visual stimuli. Modal response histograms reveal a gradual decrement in the number of spikes at interstimulus pairing intervals of 0 and 100 milliseconds but not 500 milliseconds. This plasticity effect, limited to tonic units, may persist for 2 to 3 minutes after termination of click-flash pairing.

Synthesis of the contingent negative variation brain potential from noncontingent stimulus and motor elements.

Slow shifts in brain potential (commonly called the contingent negative variation), obtained during a warned reaction-time task with a foreperiod of 1 second, were compared with waveforms synthesized by the addition of separately obtained potentials associated with individual (nonpaired) sensory stimuli and self-initiated motor movements. The synthesized waveforms match closely the actual contingent negative variation, suggesting that it is constituted largely of separate, noncontingent elements related to sensory and motor processes.

Leftward ribosome frameshifting at a hungry codon.

Previous experiments have shown that limitation for certain aminoacyl-tRNA species results in phenotypic suppression of a subset of frameshift mutant alleles, including members in both the (+) and (-) incorrect reading frames. Here, we demonstrate that such phenotypic suppression can occur through a ribosome reading frame shift at a hungry AAG codon calling for lysyl-tRNA in short supply. Direct amino acid sequence analysis of the product and DNA sequence manipulation of the gene demonstrate that the ribosome frameshift occurs through a movement of one base to the left, so as to decode the triplet overlapping the hungry codon from the left or 5' side, followed by continued normal translation in the new, shifted reading frame.

On the mechanism of leftward frameshifting at several hungry codons.

We have used lacZ reporter genes to assess leftward ribosome frameshifting on sequences containing the quadruplet U UUC followed by several different triplets coding for lysine, isoleucine, or leucine. Limitation for lysine-tRNA provokes leftward frameshifting when the slippery quadruplet is followed by either lysine codon aag or aaa, but not when followed by an isoleucine or leucine codon. Limitation for isoleucine provokes frameshifting when the quadruplet is followed by either isoleucine codon aua or auc, but not when it is followed by a lysine codon. We conclude that the quadruplet promotes shifting when the ribosome is stalled at any "hungry" codon immediately after it. Changing the quadruplet to U AGC, at which peptidyl-tRNA cognate to the AGC triplet will be mismatched at all three anticodon positions if it slips left, abolishes frameshifting when the ribosome is stalled at the next position. We conclude that the U UUC quadruplet promotes frameshifting by virtue of its ability to pair with a left-slipped peptidyl-tRNA. The frameshift promoted by isoleucine-tRNA limitation of the U UUC aua sequence was analyzed by amino acid sequencing of the protein product. It occurs through reading of the Cau histidine codon overlapping the hungry codon from the left. This result rules out a "simultaneous slippage" type of mechanism. It strongly suggests instead that starvation-promoted frameshifting occurs primarily by slippage of peptidyl-tRNA just upstream of the stall site, followed by decoding of the triplet overlapping the stall site from the left or 5' side. A secondary finding is that the last base of the "hungry" codon has a moderate effect on its shiftiness, aag being shiftier than aaa, and aua being shiftier than auc.

On the mechanism of ribosomal frameshifting at hungry codons.

In a few, rather rare cases, frameshift mutant alleles are phenotypically suppressed during limitation for particular aminoacyl-tRNA species. The simplest interpretation is compensatory ribosome frameshifting at a "hungry" codon in the vicinity of the suppressed frameshift mutation. We have now tested this interpretation directly by obtaining amino acid sequence data on such a phenotypically suppressed protein. We used a plasmid-borne lacZ gene, engineered to be in the (+) reading frame. Its background leakiness is increased by two orders of magnitude during lysyl-tRNA limitation. The enzyme made under this condition has the amino acid sequence expected from the DNA sequence up to the first lysine codon, then shifts in the (-) direction to recreate the correct lacZ reading frame. The lysine is replaced by serine, presumably due to cognate reading of an overlapping AGC codon displaced by one base to the 3' side of the AAG codon. When the 3' overlapping codon is AGA or AGG, there is no ribosome frameshifting; when it is AGU (read by the same serine tRNA) there is frameshifting, although less efficiently than in the case of AGC. The mechanism of cognate overlapping reading contradicts more elaborate models that two of the authors have suggested previously. However, the possibility remains that there is more than one mechanism of ribosome frameshifting at hungry codons.

On the role of the P-site in leftward ribosome frameshifting at a hungry codon.

Previous work characterized ribosomal frameshifting within the sequence C UUC AAG provoked by lysyl-tRNA limitation. The ribosome frameshift is one base to the left of the AAG lysine codon, as shown by dotted overlining above. We now show that the frequency of this leftward ribosome frameshift is strongly influenced by the identity of the bases two, three and four positions to the left of the actual frameshift site. The nature of these influences coincides exactly with the possibilities of base-pairing between the sequence and the anticodon of the P-site peptidyl-tRNA when shifted one base to the left just upstream of the frameshift site. We conclude that a peptidyl shift in the P-site is intimately involved in leftward frameshifting in the adjacent A site when it codes for an aminoacyl-tRNA in short supply.

Enhanced ribosome frameshifting in stationary phase cells.

We have examined the effect of growth phase in Escherichia coli on the translation of a plasmid-borne lacZ gene in which active enzyme synthesis requires a leftward frameshift. During the log phase of growth, the differential rate of enzyme synthesis is very low. It increases by about two orders of magnitude during the small amount of protein synthesis which occurs at the end of log phase and the early part of stationary phase. The increase is sufficient to increase the enzyme's specific activity in crude extracts to 30 times more than it would be if the log-phase differential rate continued unchanged. No such large increase is observed with a zero-frame lacZ+ control gene on the same plasmid under the control of the same promoter; a significant but much smaller increase is observed with a zero-frame control containing an in-frame terminator triplet in the region of the required frameshift. Protein sequence analysis of the enzyme made from the frameshift reporter in stationary cells shows that the increased enzyme synthesis is due to frameshifting, and not due to termination and reinitiation. The frameshift occurs at or right after the sequence U UUC AAG, an intrinsically shifty site.

On the role of the starved codon and the takeoff site in ribosome bypassing in Escherichia coli.

Translating ribosomes can skip over stretches of messenger RNA and resume protein chain elongation after a "bypassed" region. We have previously shown that limitation for isoleucyl-tRNA can initiate a ribosome bypass when an AUA codon is in the ribosomal A-site. We have now generalized this effect to other "hungry" codons calling for four different limiting aminoacyl-tRNA species, suggesting that a pause at any A-site will have this effect. We have assessed bypassing in a large family of reporters with nearly every different triplet in the "takeoff site", i.e. the P-site on the 5' side of the hungry codon, and an identical "landing site" codon 16 nucleotides downstream. The different takeoff sites vary over a factor of 50 in bypassing proficiency. At least part of this variation appears to reflect stability of the codon Colon, two colons anticodon interaction at the takeoff site, as indicated by the following: (a) the bypassing proficiency of different tRNAs shows a rough correlation with the frequency of A Colon, two colons U as opposed to G Colon, two colons C pairs in the codon Colon, two colons anticodon association; (b) specific tRNAs bypass more frequently from codons ending in U than from their synonym ending in C; (c) an arginine tRNA with Inosine in the wobble position which reads CGU, CGC, and CGA bypasses much more frequently from the last codon than the first two synonyms.

Inseparability of X-Heterochromatic Functions Responsible for X:Y Pairing, Meiotic Drive, and Male Fertility in Drosophila melanogaster.

Deficiencies encompassing part or all of the X heterochromatin of Drosophila melanogaster have been linked to three abnormalities in male meiosis and spermatogenesis: X-Y nondisjunction, skewed sperm recovery ratios favoring sperm with reduced chromatin content, and sterility in males carrying either a Y-autosome translocation or mal( +)Y. In this study, 18 X heterochromatic deficiencies of varying sizes were tested in XY males for their spermatogenic phenotypes. All 18 proved to be either mutant for all three phenotypes or wild type for all three. Although variable among mutant deficiencies, expression levels of all three phenotypes were strongly correlated. Deficiencies that cause high levels of nondisjunction also cause severe recovery ratio distortion and are completely sterile in conjunction with mal(+) Y. Low nondisjunction deficiencies cause comparable mild effects for the other phenotypes. The same deficiencies were also tested in males carrying a large heterochromatic free X duplication Dp(1; f)3. For all deficiencies which induce nondisjunction in XY males, the Y and free duplication pair regularly and the X fails to pair in XYDp males. Drive levels are constant across deficiencies in these males. Thus elimination of variability in the pairing phenotype also eliminates variability in sperm recovery ratios.

Male-sterilizing interactions between duplications and deficiencies for proximal X-chromosome material in Drosophila melanogaster.

The genetic limits of sixty-four deficiencies in the vicinity of the euchromatic-heterochromatic junction of the X chromosome were mapped with respect to a number of proximal recessive lethal mutations. They were also tested for male fertility in combination with three Y chromosomes carrying different amounts of proximal X-chromosome-derived material (BSYy+, y+Ymal126 and y+Ymal+). All deficiencies that did not include the locus of bb and a few that did were male-fertile in all male-viable Df(1)/Dp(1;Y) combinations. Nineteen bb deficiencies fell into six different classes by virtue of their male-fertility phenotypes when combined with the duplicated Y chromosomes. The six categories of deficiencies are consistent with a formalism that invokes three factors or regions at the base of the X, one distal and two proximal to bb, which bind a substance critical for precocious inactivation of the X chromosome in the primary spermatocyte. Free duplications carrying these regions or factors compete for the substance in such a way that, in the presence of such duplications, proximally deficient X chromosomes are unable to command sufficient substance for proper control of X-chromosome gene activity preparatory to spermatogenesis. We conclude that there is no single factor at the base of the X that is required for the fertility of males whose genotype is otherwise normal.

Cytogenetic analysis of a segment of the Y chromosome of Drosophila melanogaster.

Males carrying a large deficiency in the long arm of the Y chromosome known to delete the fertility gene kl-2 are sterile and exhibit a complex phenotype: (1) First metaphase chromosomes are irregular in outline and appear sticky; (2) spermatids contain micronuclei; (3) the nebenkerns of the spermatids are nonuniform in size; (4) a high molecular weight protein ordinarily present in sperm is absent; and (5) crystals appear in the nucleus and cytoplasm of spermatocytes and spermatids. In such males that carry Ste+ on their X chromosome the crystals appear long and needle shaped; in Ste males the needles are much shorter and assemble into star-shaped aggregates. The large deficiency may be subdivided into two shorter component deficiencies. The more distal is male sterile and lacks the high molecular weight polypeptide; the more proximal is responsible for the remainder of the phenotype. Ste males carrying the more proximal component deficiency are sterile, but Ste+ males are fertile. Genetic studies of chromosome segregation in such males reveal that (1) both the sex chromosomes and the large autosomes undergo nondisjunction, (2) the fourth chromosomes disjoin regularly, (3) sex chromosome nondisjunction is more frequent in cells in which the second or third chromosomes nondisjoin than in cells in which autosomal disjunction is regular, (4) in doubly exceptional cells, the sex chromosomes tend to segregate to the opposite pole from the autosomes and (5) there is meiotic drive; i.e., reciprocal meiotic products are not recovered with equal frequencies, complements with fewer chromosomes being recovered more frequently than those with more chromosomes. The proximal component deficiency can itself be further subdivided into two smaller component deficiencies, both of which have nearly normal spermatogenic phenotypes as observed in the light microscope. Meiosis in Ste+ males carrying either of these small Y deficiencies is normal; Ste males, however, exhibit low levels of sex chromosome nondisjunction with either deficient Y. The meiotic phenotype is apparently sensitive to the amount of Y chromosome missing and to the Ste constitution of the X chromosome.

Segmental aneuploidy and the genetic gross structure of the Drosophila genome.

By combining elements of two Y-autosome translocations with displaced autosomal breakpoints, it is possible to produce zygotes heterozygous for a deficiency for the region between the breakpoints, and also, as a complementary product, zygotes carrying a duplication for precisely the same region. A set of Y-autosome translocations with appropriately positioned breakpoints, therefore, can in principle be used to generate a non-overlapping set of deficiencies and duplications for the entire autosomal complement.-Using this method, we have succeeded in examining segmental aneuploids for 85% of chromosomes 2 and 3 in order to assess the effects of aneuploidy and to determine the number and location of dosage-sensitive loci in the Drosophila genome (Figure 5). Combining our data with previously reported results on the synthesis of Drosophila aneuploids (see Lindsley and Grell 1968), the following generalities emerge.-1. The X chromosome contains no triplo-lethal loci, few or no haplo-lethal loci, at least seven Minute loci, one hyperploid-sensitive locus, and one locus that is both triplo-abnormal and haplo-abnormal. 2. Chromosome 2 contains no triplo-lethal loci, few or no haplo-lethal loci, at least 17 Minute loci, and at least four other haplo-abnormal loci. 3. Chromosome 3 contains one triplo-lethal locus that is also haplo-lethal, few or no other haplo-lethal loci, at least 16 Minute loci, and at least six other haplo-abnormal loci. 4. Chromosome 4 contains no triplo-lethal loci, no haplo-lethal loci, one Minute locus, and no other haplo-abnormal loci.-Thus, the Drosophila genome contains 57 loci, aneuploidy for which leads to a recognizable effect on the organism: one of these is triplo-lethal and haplo-lethal, one is triplo-abnormal and haplo-abnormal, one is hyperploid-sensitive, ten are haplo-abnormal, 41 are Minutes, and three are either haplo-lethals or Minutes. Because of the paucity of aneuploid-lethal loci, it may be concluded that the deleterious effects of aneuploidy are mostly the consequence of the additive effects of genes that are slightly sensitive to abnormal dosage. Moreover, except for the single triplo-lethal locus, the effects of hyperploidy are much less pronounced than those of the corresponding hypoploidy.

Effects of intraoperative plasmapheresis on blood loss in cardiac surgery.

Intraoperative platelet-rich plasmapheresis allows autotransfusion of fresh, undamaged platelets and clotting factors at the completion of the operation. To evaluate this technology, we randomly assigned 100 consecutive patients who were to undergo an elective coronary bypass procedure and had normal clotting studies into the experimental (plasmapheresis) or the control group. Characteristics of both groups were similar, including average age (61.4 years versus 61.3 years [experimental versus control group]), sex (78% male versus 74% male), preoperative weight (80.9 kg versus 80.2 kg), preoperative red cell mass (1,989 mL versus 1,890 mL), perfusion time (102 minutes versus 106 minutes), and coagulation studies. Both internal mammary arteries were used in 68% of the patients. All patients had preoperative and postoperative blood volume determinations and complete clotting studies. Sixty-two variables related to bleeding were analyzed. Strict indications for transfusion were a hemoglobin level less than 7 g/100 mL in patients younger than 70 years and a hemoglobin level less than 8 g/100 mL in patients older than 70 years. The group receiving intraoperative plasmapheresis had a significant reduction in operative red cell mass loss (1,050 +/- 43 mL versus 1,226 +/- 61 mL; p = 0.021), a reduction in the average homologous transfusion (0.67 +/- 0.15 unit versus 1.8 +/- 0.25 units; p = 0.0002), and an increase in the percentage of patients not requiring blood transfusions (66% versus 32%; p = 0.001). This technique is useful in reducing postoperative blood loss and homologous transfusions.

Influences of hypothalamic stimulation upon septal and hippocampal electrical activity in the cat.

Spontaneous patterns of hippocampal EEG and septal cell activity were studied in immobilized cats, and the influences of high frequency stimulation of medial hypothalamus (MH) and lateral hypothalamus (LH) were determined. Septal cells were divided into 3 classes on the basis of their discharge patterns: (1), rhythmic bursting (2), non-rhythmic bursting and (3), non-bursting, and the relationship of these discharge patterns to hippocampal theta rhythm was analyzed. Rhythmic bursting cells displayed close frequency and phase relations to hippocampal theta rhythm and were located chiefly in the diagonal band of Broca. Cells of the other two categories were found both within and outside of the diagonal band region.

A prospective study of aspirin's effect on red blood cell loss in cardiac surgery.

The effect of aspirin on red blood cell (RBC) loss and blood transfusions was evaluated prospectively in 100 consecutive patients, with normal bleeding times, undergoing elective coronary artery bypass (CABG) surgery. Patients taking 85-325 mgm of aspirin daily up to or within 48 hours of surgery (the "aspirin" group) were compared to patients not taking aspirin or those who had discontinued aspirin at least 4 days before surgery (the "no-aspirin" group). RBC loss was determined by measuring preoperative and postoperative RBC volume using RISA and 51Cr techniques. There were no significant differences, respectively, between the aspirin and no-aspirin groups for: RBC loss (1158 +/- 67 ml vs 1129 +/- 47 ml, p = 0.737), chest tube drainage (925 +/- 31 ml vs 844 +/- 70 ml, p = 0.553), and gm% discharge Hemoglobin (Hgb) (9.94 +/- 0.32 vs 9.49 +/- 1.4, p = 0.0148). Strict criteria for blood transfusions were employed: (1) intraoperative hematocrit of less than 21%, (2) postoperative Hgb of less than 7 gm% for patients less than 70 years old and (3) postoperative Hgb of less than 8 gm% for patients greater than 70 years old. There were no significant differences, respectively, between the aspirin and no-aspirin groups for units of blood transfused (1.32 +/- vs 1.21 +/- 0.20, p = 0.843) and patients not receiving transfusions during the entire hospitalization (44% vs 50%). Patients taking 85-325 mgm of aspirin with a normal bleeding time undergoing elective CABG did not have increased RBC loss or increased transfusion requirements. These results indicate it is not necessary to delay elective CABG surgery for the purpose of discontinuing aspirin.