A Quick-Freeze Method for Making Smear Slides Permanent

Many types of smear slides can be made permanent rapidly and effectively by substituting for the usual dehydration series a single-step process of freezing the slide on a block of dry ice, placing it immediately in 95% or absolute alcohol, and then mounting it. Advantages of the technic are its speed, the ease of separation of cover slip from slide with a minimum loss of cells, and the superiority of the resulting permanent slides.     

Computer models of a new deoxy-sickle cell hemoglobin fiber based on x-ray diffraction data.

A new x-ray fiber diffraction pattern from deoxygenated sickle cell erythrocytes has been observed. It displays 14 layer lines with a 109 A periodicity compared with the 64 A periodicity of the "classic" sickle cell hemoglobin (HbS) fiber. These data and association energy calculations serve as a basis for computer model building. Systematic searches over four-dimensional parameter space yielded twelve protofilament models that satisfy the following constraints: (a) two HbS molecules be related by twofold screw symmetry with a translational repeat of 109 A; (b) at least one of the substituted residues in HbS, val beta 6, should participate in intermolecular contacts; and (c) the energy of intermolecular interaction be less than -24 kcal/mol. Each of the protofilament models is a zigzag mono-strand that stands in contrast to the double-stranded protofilament of the "classic" fiber. Fiber models were constructed with each of the 12 protofilament models, pseudo-hexagonally packed. Searches of variable packing parameters showed four fiber models with minimal protofilament association energies and minimal differences between calculated transforms and observed data. The R-factor was less than 0.24 for each of these four models. In three of the fiber models the protofilament association energy is between -(93 and 130) kcal, and in a fourth, the energy is -64 kcal. One protofilament model constituted three distinct fiber models of the lower energy class, and a second protofilament model packed with a higher association energy into a fourth fiber model. The selection of a unique fiber model from among these four cannot be made because of the limited available data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analytical techniques for boron and boron 10 analysis in a solid experimental tumor EO. 771

Summary If a tumor can be preferentially loaded with a suitable boron-10 compound and irradiated with thermal neutrons, malignant cells can be selectively destroyed via the-particle + Li 7-nucleus from the reaction¹⁰B(n,)⁷Li.Neutron capture therapy with two boron-10 amino acid analogs of low toxicity has been tested in recent years: (a) trimethylamine-carboxyborane, (A3) and (b) amine-carboxyborane, (A7). Now the boron-10 glycineamide analog (A8), amineboryl-carboxamide has been synthesized; it contains 13.81% boron (90% Boron 10+10% Boron 11) and shows a very low toxicity in mice. The effects of this compound were tested on the syngeneic solid adenocarcinoma EO 771 on the right hind leg of male C57 BL/6J mice under standard conditions, by measuring tumor volume growth delay and cell cycle changes using flow cytometry. Boron distribution between tumor and muscle was analyzed by emission spectroscopy with inductively coupled plasma (ICP) following injection of a suspension of peanut oil emulsion. In addition, boron-10 concentration in the tumor were analyzed with prompt-activation analysis and neutron capture radiography (Kodak-Pathé LR 115) at the MRR reactor in Brookhaven after i.p. injection of 0.4 mg/g A8.Application of A8 alone (0.4 mg/g i.p.) or thermal neutron irradiation of the tumor EO. 771 produced a tumor growth delay of 1–2 days for tumor volume doubling. Application of the boron 10 glycine-amide analog A8 i.p. plus 510¹² n/cm² resulted in a growth delay of 3–6 days.In contrastintratumoral application of A8 plus 410¹² n/cm² neutrons gave a growth delay of 7–14 days; the fraction of (G2 + M) cells rose from 35% (neutrons alone) to 52%, as evaluated from flow cytometry.Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthday     

Paired sequence difference in ribosomal RNAs: evolutionary and phylogenetic implications

Ribosomal RNAs have secondary structures that are maintained by internal Watson-Crick pairing. Through analysis of chordate, arthropod, and plant 5S ribosomal RNA sequences, we show that Darwinian selection operates on these nucleotide sequences to maintain functionally important secondary structure. Insect phylogenies based on nucleotide positions involved in pairing and the production of secondary structure are incongruent with those constructed on the basis of positions that are not. Furthermore, phylogeny reconstruction using these nonpairing bases is concordant with other, morphological data.      

The genetic basis of negative selection of Tcrb-Vll+ T cells

Non-H-2 genes responsible for negative selection of Tcrb-V 11⁺ T cells were examined using backcross mice of various strains with C58, which does not delete Tcrb-V 11⁺ T cells. Two independently segregating genes were found: one leading to partial deletion was closely linked toLy-2/Ly-3 on chromosome 6, and the second giving virtually complete deletion has not yet been mapped. The A strain had only the former, whereas BALB/c, BALBK, B10.BR, CBA-T6, C3H/He, and DBA/2 expressed both of these genes. Although a gene(s) of the NIH strain led only to partial deletion, the chromosomal localization of the gene(s) has not yet been determined: no informative polymorphic molecules are expressed from genes on chromosome 6 of this strain.     

Modulation of bovine luteal cell synthetic capacity by interferon-gamma

Previous work from our laboratory has demonstrated that major histocompatibility complex (MHC) antigens are expressed on cultured bovine luteal cells following exposure to the T lymphocyte-derived cytokine, interferon-gamma (IFN-gamma). In light of these actions of IFN-gamma, it was of interest to investigate the effects of this cytokine on other aspects of luteal function. Therefore, bovine luteal cells were cultured for 7 days in the presence or absence of IFN-gamma, and luteal progesterone (P4), prostaglandin F2 alpha (PGF2 alpha), and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) production were evaluated. After a 24-h exposure to IFN-gamma (100 U), both PGF2 alpha and 6-keto-PGF1 alpha production were decreased approximately 50% (p less than 0.05). However, as time in culture progressed, IFN-gamma markedly increased the synthesis of both prostaglandins approximately 400% above controls (p less than 0.05). Stimulation of prostaglandin production by IFN-gamma was abrogated by the addition of exogenous P4. During the period of IFN-gamma-stimulated prostaglandin synthesis, LH-stimulated P4 production was inhibited by IFN-gamma treatment. However, the suppression of P4 production by IFN-gamma was not mediated by the increase in prostaglandins since concomitant treatment with indomethacin did not reverse the inhibition of steroidogenesis. These results suggest that IFN-gamma, in addition to an indirect role in promoting immune response mechanisms, may also directly affect luteal function by enhancing luteal prostaglandin synthesis and by inhibiting luteal steroidogenesis.     

Measurement of Respiratory Volume for Virus Retention Studies in Mice

A pressure plethysmograph for measuring respiratory volume in mice during exposure to virus aerosols is described. The respiratory frequency and tidal volume were measured, and from these data the minute ventilation was calculated. The mean respiratory frequency of adult, male mice was 255 per min; the mean tidal volume of 0.18 ml was inversely related to respiratory frequency. The standardized mean minute ventilation rate was 1.46 ml per g of body weight. The respiratory frequency and tidal volume of CD-1 and HA/ICR strains of mice of the same age were similar. The respiratory retention rate for a 2.7-mum aerosol of vesicular stomatitis virus was 41%, and 58% of the virus retained was found in the trachea and lung.     

Developing the ethics of implementation research in health

Implementation research (IR) is growing in recognition as an important generator of practical knowledge that can be translated into health policy. With its aim to answer questions about how to improve access to interventions that have been shown to work but have not reached many of the people who could benefit from them, IR involves a range of particular ethical considerations that have not yet been comprehensively covered in international guidelines on health research ethics. The fundamental ethical principles governing clinical research apply equally in IR, but the application of these principles may differ depending on the IR question, context, and the nature of the proposed intervention. IR questions cover a broad range of topics that focus on improving health system functioning and improving equitable and just access to effective health care interventions. As such, IR designs are flexible and often innovative, and ethical principles cannot simply be extrapolated from their applications in clinical research. Meaningful engagement with all stakeholders including communities and research participants is a fundamental ethical requirement that cuts across all study phases of IR and links most ethical concerns. Careful modification of the informed consent process may be required in IR to permit study of a needed intervention. The risks associated with IR may be difficult to anticipate and may be very context-specific. The benefits of IR may not accrue to the same groups who participate in the research, therefore justifying the risks versus benefits of IR may be ethically challenging. The expectation that knowledge generated through IR should be rapidly translated into health policy and practice necessitates up-front commitments from decision-makers to sustainability and scalability of effective interventions. Greater awareness of the particular ethical implications of the features of IR is urgently needed to facilitate optimal ethical conduct of IR and uniform ethical review.     

Differential effect of imipenem treatment on wild-type and NK cell-deficient CD8 knockout mice during acute intra-abdominal injury

CD8 knockout mice depleted of natural killer (NK) cells by treatment with anti-asialoGM1 (CD8KO/alphaAsGM1 mice) are resistant to injury caused by cecal ligation and puncture (CLP). However, CLP-induced injury is complex. Potential sources of injury include bacterial dissemination, cecal ischemia, and translocation of bacterial toxins. We treated wild-type and CD8KO/alphaAsGM1 mice with imipenem after CLP to decrease bacterial dissemination. Additional mice were subjected to cecal ligation without puncture of the cecal wall or cecal ligation and removal of cecal contents. Imipenem treatment decreased bacterial counts by at least two orders of magnitude. However, all wild-type mice, whether treated with saline or imipenem, died by 42 h after CLP and exhibited significant hypothermia, metabolic acidosis, and high plasma cytokine concentrations. Wild-type mice subjected to cecal ligation without puncture also died, despite very low bacterial counts in blood, but wild-type mice subjected to cecal ligation and washout of cecal contents survived. In CD8KO/alphaAsGM1 mice subjected to CLP, imipenem treatment increased survival from 50% to 100%. After cecal ligation without puncture, long-term survival was 80-90% in CD8KO/alphaAsGM1 mice. Hypothermia, metabolic acidosis, and cytokine production were attenuated in CD8KO/alphaAsGM1 mice compared with wild-type controls. These results indicate that bacterial dissemination is not a major source of injury in wild-type mice after CLP, but the presence of gut flora in the cecal lumen is required for induction of systemic inflammation after cecal injury. CD8KO/alphaAsGM1 mice are resistant to the systemic manifestations of cecal injury.