Use of a modified rotor and enlarged separation chamber for isolation of human granulocytes by counterflow centrifugation-elutriation

Human granulocytes were isolated from leukapheresis blood by counterflow centrifugation-elutriation using the Beckman J21B centrifuge and JE6 rotor. Attempts to increase the absolute number of human granulocytes recovered by a single- or double-chamber procedure revealed the physical limitations of the chamber capacity in granulocyte isolation. To improve the absolute number of granulocytes from leukapheresis blood, an enlarged separation chamber and accompanying rotor were fabricated. The enlarged chamger has three times the capacity of the standard 4.5-ml Beckman chamber. Aside from increased yield of granulocytes, the 13.2-ml chamber affords a 25% saving in isolation time compared to the Beckman chamber for isolation of a comparable number of cells. In vitro analysis of the isolated granulocytes for viability, latex bead ingestion, and chemotactic response indicate no apparent loss of granulocyte function as a result of the isolation procedure.     

Effect of storage conditions on function of granulocytes

The effect of collection technique, anticoagulant, pH, glucose, and temperature on in vitro granulocyte function were studied after 24 hr of storage in the liquid state. Collection by CL did not adversely affect granulocyte function, however, cells collected by FL had accelerated loss of bactericidal activity and chemotactic response. Citrate anticoagulants provided better maintenance of bacteridical activity, NBT reduction, and chemotactic response than heparin, EDTA, and ion-exchange anticoagulants. Chemiluminescence was well maintained when the initial pH of the preservative solution (CPD plasma) was between 6.5 and 8.0 but maintenance of chemotaxis required pH of 7.0–7.5. Glucose concentrations of 80–1000 mg/dl provided adequate maintenance of chemiluminescence and chemotaxis. Bacterial killing was well maintained by storage at either 1–6 or 20–24 °C. Storage at 1–6 °C caused decreased chemotaxis, decreased ability of granulocytes to adhere and spread on a foreign surface, and a decreased intravascular recovery and shortened half-life after transfusion. Although short-term liquid storage may be practical, at present, granulocytes should be transfused as soon as possible after collection.     

Visualization of protein-SDS complexes in polyacrylamide gels by chilling

A method is described which gives direct visualization of protein bands in sodium dodecyl sulfate electrophoresis gels. The procedure consists of chilling the gels to 0–4°C and observing the white opaque bands which correspond to the stained protein bands. Ovalbumin can be detected by this method at a minimum concentration of 0.2 μg per mm² of gel surface area.     

The combined determination of indolyl-3-acetic and abscissic acid in plant materials

A method for isoelectric focusing of antibodies in agarose gels with ampholytes synthesized in the laboratory from pentaethylenehexamine is presented. The ampholytes are easy to prepare, give results comparable to those with commercial ampholytes, and are much less expensive. Substituting agrarose bonded to plastic film for the polyacrylamide gels on glass plates commonly used offers many advantages and enhances the usefulness of isoelectric focusing as a tool for studying antibody molecules.     

Reduction by atropine of phencyclidine hypertension and apneusis

The aim of this study was to assess the possible role of a central cholinergic component phencyclidine (PCP)-induced hypertension. Sprague-Dawley rats, lightly anesthetized with urethane, exhibited a dose related pressor response following 0.1–1.0 mg/kg PCP i.v. After i.v. atropine pre-treatment, the PCP dose-response curve was shifted to the right, and the magnitude of the pressor responses was reduced by about 50%. In addition, atropine reduced the incidence of apneusis, but had no effect on the bradycardia that accompanied the pressor responses. Methylatropine (i.v.) did not reduce the PCP pressor responses, nor did it prevent the apneusis induced by PCP. These results suggest that in addition to its direct pressor effects the activation of central cholinergic systems contribute significantly to the cardiovascular and respiratory toxicity induced by PCP.     

Stereoselective preparation of deuterated reduced nicotinamide adenine nucleotides and substrates by enzymatic synthesis.

A-Side (4-R)-(4-²H)-reduced nicotinamide adenine dinucleotide (NADD) was prepared by a stepwise oxidation of ethanol-d₆ to acetate in the presence of NAD, alcohol dehydrogenase, and aldehyde dehydrogenase. The B-side (4-S) isomer of NADD was prepared using the glucose dehydrogenase activity of glucose-6-phosphate dehydrogenase to oxidize to oxidize glucose-1-d in 40% dimethyl aulfoxide. Subsequent purifieation of the reduced nucleotides was achieved using a column of strongly basic polystyrene macroporous resin (AG MP-1) eluted with 0.2 m LiCl, pH 10, and applying the pooled NADD peak to a polyacrylamide gel (Bio-Gel P-2) column. The final $\text{A}{}_{260}\text{A}{}_{340}$ ratio obtained for these preparations was below 2.3. Preparation of the deuterated reduced nucleotides in this manner allows production of specifieally deuterated substrates by coupled enzymatic synthesis. L-Malate-2-d was prepared by coupled synthesis of A-side NADD to the reduction of oxaloacetate by the A-side enzyme malate dehydrogenase.     

Radiometric assay for minute quantities of l-fucose

A radiometric assay has been developed for l-fucose which is capable of measuring quantities of this deoxysugar as low as 25 pmol. The assay couples l-fucose dehydrogenase to l-glutamate dehydrogenase and l-glutamate decarboxylase to yield radioactive CO₂ which is collected and counted as a measure of l-fucose content. The assay eliminates high backgrounds observed with fluorescence assays.     

Block by phencyclidine of acetylcholine and barium induced adrenal catecholamine secretion

Activation of the sympathetic system by phencyclidine (PCP) should result in catecholamine release from the adrenals. However, adrenalectomy does not reduce PCP-induced hypertension. In an attempt to rectify this inconsistency, the direct effects of PCP on the bovine adrenal medulla were examined. At (3×10^?6M), PCP reduced the acetylcholine-(ACh)-induced catecholamine release by 50%. Surprisingly, barium-induced secretion of catecholamines was also reduced by PCP. ACh-induced catecholamine release was not altered by 10^?3M 4-aminopyridine (4 AP), the potassium channel blocker. Thus, calcium antagonist actions of PCP and consequent block of catecholamine secretion from adrenal medulla may explain the lack of effect of adrenalectomy on PCP-induced hypertension. Possible contributions of calcium and/or potassium channel blockade to other manifestations of PCP overdosage are discussed.     

Identification and mass analysis of human fibrinogen molecules and their domains by scanning transmission electron microscopy

The domainal substructure and molecular conformation of human fibrinogen have been investigated by evaluating scanning transmission electron microscopic images of freeze-dried or negatively contrasted native fibrinogen (fractions I-4 and I-9), glutaraldehyde-treated fibrinogen, or plasmic core fragments D₁ and E₂. Although some unstained freeze-dried native or glutaraldehyde-treated fibrinogen molecules were relatively compact and even occasionally spheroidal, typical images were elongated symmetrical tridomainal structures 460 Å ± 20 Å in length; frequently they were bent into a variety of elongated though non-linear arrangements. Their identification as monomolecular forms of fibrinogen by scanning transmission electron microscopic mass measurements resolved uncertainties relating to the identity of such objects as single molecules. The central domains of fraction I-4 molecules had a greater mass than those of fraction I-9 (1.01 × 10⁵M_rversus 7.5 × 10 M_r, respectively). This difference accounted for the observed mass difference between fraction I-4 and fraction I-9 molecules (i.e. 3.27 × 10⁵M_rversus 2.97 × 10⁵M_r, respectively) and suggested that the COOH-terminal region of the Aα chain (major portions of which are always absent from fraction I-9 molecules) is situated within the mass integration radius for the central domain. When the COOH-terminal region of the Aα chain was present it appeared in negative stain as a thread-like structure originating between the middle and outer domains and extending toward the central domain, sometimes appearing to wind around the long axis.The outer domains of negatively stained molecules resembled negatively stained images of fragment D₁ and could frequently be resolved into at least two discrete subdomains, forming an oblong structure usually canted at an angle of ～120 ° to 150 ° relative to the long axis. Our findings are consistent with prevailing tridomainal structural models of fibrinogen and suggest that these molecules are flexible and may exist in unfolded configurations, or as relatively compact, partially or completely folded forms.     

Sodium periodate stimulation of human lymphocytes : A comparison between normal and chronic lymphocytic leukemia lymphocytes

Lymphocytes from healthy donors and from patients with chronic lymphocytic leukemia (CLL) were stimulated to divide with sodium periodate. The time of maximal response of normal lymphocytes to sodium periodate (NaIO₄) was earlier than that observed to phytohemagglutinin (PHA), but the magnitude was lower. In comparison, CLL lymphocytes responded to NaIO₄ more extensively and earlier than to PHA.     

In vitro and in vivo evaluation of cis-methyl-phencyclidine (cis-MPCP) as a potential antagonist of phencyclidine (PCP)

In four preparations/tests (isolated nerve, venticular strip, rotarod, and mouse acute lethality), cis-N-phenyl-4-methylcyclohexyl piperidine (cis-MPCP) was consistently less active than PCP and trans-MPCP. As expected, cis-MPCP, at 10^?4M, which did not depress the action potential evoked on frog sciatic nerves, reduced by half both the nerve block and prolongation of relative refractory period caused by PCP. However, cis-MPCP at 10^?6M, which by itself had little effect, failed to reduce the positive inotropic effect of PCP on the field-stimulated rat ventricular strip. Cis-MPCP also failed to decrease the ataxic effect of 6 mg/kg PCP (ED80) in the mouse rotarod test. Finally, at a dose that was neither ataxic nor lethal to mice (20 mg/kg), cis-MPCP failed to reduce the 24-hour LD₅₀ of PCP. These data suggest that the actions of PCP are mediated through a multiple receptor system.     

Preparative isoelectric focusing with Pevikon as supporting medium

A preparative isoelectric focusing method is described in detail that uses Pevikon instead of Sephadex as a supporting medium. Separation is demonstrated in a human serum protein preparation that contained α₁-antitrypsin, transferrin and α₂-macroglobulin. Pevikon has some advantages over Sephadex in this type of isoelectric focusing.     

Characterization of a macrophage chemotactic lymphokine produced by purified protein derivative stimulation in vitro and in vivo

Using an immunoadsorbent column conjugated with anti-macrophage chemotactic factor-c (anti-MCF-c), MCF-c which has been separated and highly purified from a delayed-type hypersensitivity reaction (DHR) site, shares common antigenicity with the major macrophage chemotactic lymphokine released from purified protein derivative (PPD)-stimulated lymphocytes and also macrophage chemotactic lymphokine from phytohemagglutinin (PHA)-stimulated lymphocytes. Using a combination of the immunoadsorbent column and Sephadexgel chromatography these two lymphokines are purified to homogeneity from PPD- or PHA-stimulated guinea pig lymphocyte culture supernatants. These observations, taken in conjunction with the similarity in biological activities, physicochemical properties, and antigenicities, suggest that these two mediators are very similar, or possibly identical. MCF-c with chemotactic activity for macrophages seemed to exist as complexes with serum protein at the skin site of PPD-induced DHR in guinea pigs. The active substance, separated from the complexes under acid conditions, is indistinguishable from the major macrophage chemotactic lymphokine released by PPD stimulation with respect to antigenicity, heat stability, and non-diffusibility. They both have a molecular weight of about 12, 500. The chemotactic lymphokine formed comparable complexes with serum protein under neutral conditions; however, this complex dissociated in acid without loss of activity.     

DNA-binding domains of human fibronectin

Here we report the use of the technique of transferring proteins from polyacrylamide gels to nitrocellulose sheets to identify the DNA-binding domains of human plasma fibronectin. After separation of fibronectin and its tryptic digest on a sodium dodecylsulfate-polyacrylamide gel, the polypeptides were transferred to nitrocellulose sheets and the DNA-binding polypeptides were identified by incubation of the nitrocellul$\text{o}$ se sheets with ³²P-labeled human DNA followed by autoradiography.The following results were obtained: a) only two fibronectin trypsin resistant peptides (50 and 60 kd) showed DNA-binding capacity; b) in competition experiments a 300 fold excess of E. coli DNA did not inhibit the human DNA-human fibronectin binding, indicating the specificity of the interaction.     

Qualitative patterns of protein synthesis in the preimplantation mouse embryo: I. Normal pregnancy

Qualitative patterns of protein synthesis in preimplantation mouse embryos were examined by SDS-polyacrylamide-gel electrophoresis followed by autoradiography. The results demonstrate that the qualitative pattern of protein synthesis in newly fertilized eggs (day 1) is very similar to the protein pattern obtained from ovulated, unfertilized eggs. By late day 1 or early day 2, most of these “maternal” proteins are no longer being synthesized by the embryo, and many new autoradiographic bands are apparent. The most intriguing aspect of this study is the observation that all major changes in the qualitative pattern of protein synthesis take place between fertilization and the four- to eight-cell stage (day 3). From early day 3 onward, the qualitative pattern of protein synthesis remains essentially unchanged.Many of the major autoradiographic bands observed in mouse embryos from the four- to eight-cell stage and onward are also observed in protein patterns obtained from blastocyst-stage rabbit embryos. The changing patterns of protein synthesis revealed in this study occur before any gross differentiation of the embryos is evident (delineation of the inner cell mass and trophoblast) and before a marked increase in the relative rate of incorporation of l-[³⁵S]methionine takes place. However, the qualitative changes in the pattern of protein synthesis do coincide with a period of extensive fine structural differentiation.     

Immunochemical distinctions among histones and their variants in a solid-phase radioimmunoassay

A solid-phase radioimmunoassay allows detection of small structural differences in histones. In this assay, histones are absorbed to plastic tubes; the coated tubes bind antibody from the IgG fraction of antihistone rabbit antiserum, and the bound rabbit IgG is detected with ¹²⁵I-labeled purified goat anti-rabbit IgG. H2a-anti-H2a and H2b-anti-H2b systems showed no cross-reactivity with each other. H2a variants (H2a.1 and H2a.2) showed some cross-reaction but were distinguished reciprocally by corresponding antisera even though they differ in only two amino acids. This quantitative distinction was detected with a wide range of IgG concentrations, whereas only low concentrations revealed the differences in complement fixation assays. Histones in solution competed with the tube-absorbed histone for binding of the IgG.     

The effects of the acute administration of buprenorphine hydrochloride on the release of anterior pituitary hormones in the rat: evidence for the involvement of multiple opiate receptors

Multiple opiate receptor agonists and antagonists have been found to produce different patterns of anterior pituitary hormone release. The present studies examined the pattern of anterior pituitary hormone release produced by buprenorphine. The effects of the kappa agonist ethylketocyclazocine on thyroid stimulating hormone release were also examined. Following buprenorphine, serum levels of corticosterone and luteinizing hormone were not changed while growth hormone release was stimulated in a dose-dependent manner. Prolactin release was stimulated after the lowest dose of buprenorphine while the highest dose induced a fall in serum prolactin. Similar biphasic effects on thyroid stimulating hormone were seen after either buprenorphine or ethylketocyclazocine. The results provide support for the role of multiple opiate receptors in opiate-induced changes in anterior pituitary hormone release.