Release of lutropin (LH) and follitropin (FSH) in cattle after administration of a new gonadoliberin (GnRH) analogue in comparison with the gonadoliberin decapeptide.

A gonadoliberin (GnRH) analogue nonapeptide (Hoe 766) was administered intramuscularly in concentrations between 2.5 and 50 μg to m?ture cows in order to study the response of lutropin (LH) and follitropin (FSH). Results were compared with those from experiments of the GnRH decapeptide (Hoe 471). Plasma LH and FSH were radio-immunologically determined. Increasing doses of GnRH analogue up to 15–20 μg caused an approximately linear increase in total plasma LH and FSH until the response reached a plateau. With these amounts peak values were about 60 fold higher for LH and 3.5 fold higher for FSH than basal levels about 135 minutes after injection. Higher values lasted for more than 6 h for LH and about 5 h for FSH. The LH response was much greater and more prolonged than for FSH.Doses of the nonapeptide analogue 50 to 70 times lower than the GnRH decapeptide provoked about the same height and duration of LH and FSH response.     

Colony formation by subpopulations of human T lymphocytes. III. Antigenic phenotype and function of colony-forming cells, colony cells, and their expanded progeny

The antigenic phenotype of individual PHA-induced T lymphocyte colonies was studied with a direct immunofluorescence technique using fluorescein-labeled anti-Leu-2a and anti-Leu-3a antibodies. Of the colonies grown from mononuclear peripheral blood cells 85% were Leu-3a+ (inducer/helper phenotype), 12% were Leu-2a+ (suppressor/cytotoxic phenotype), and 3% contained equal numbers of Leu-2a+ and Leu-3a+ cells. Fluorescence-activated cell sorter (FACS) separated T-cell subsets showed that Leu-2a+ cells and Leu-3a+ cells form exclusively Leu-2a+ and Leu-3a+ colonies, respectively. Leu-3a+ cells formed colonies in both the absence and presence of conditioned medium (PHA-CM), whereas colony formation by Leu-2a+ cells was absolutely dependent on PHA-CM. Mixing experiments with FACS-separated T-cell subsets showed that Leu-2a+ cells inhibit colony formation by Leu-3a+ cells in a cell dose-dependent manner both in the presence and absence of PHA-CM. Phenotype analysis of individual colonies from mixing experiments strongly suggested monoclonal proliferation in the present colony assay system. The majority of expanded T-cell colonies showed helper activity in a reverse hemolytic plaque-forming B-cell assay, although to a lesser degree as compared to that of freshly isolated T lymphocytes.     

Determination of cobalamins in biological material. II. The cobalamins in human plasma and erythrocytes after desalting on nonpolar adsorbent material, and separation by one-dimensional thin-layer chromatography

An improved method for determination of cobalamins in biological materials is described. The cobalamins are extracted with ethanol after preincubation with cadmium acetate, which inhibits nonspecific adsorption of hydroxocobalamin to proteins. The extracts are desalted on Amberlite XAD-2 columns, which at least doubles the capacity of the analysis, as compared to the previous phenol procedure. The cobalamins are separated by one-dimensional thin-layer chromatography. Bioautography with Escherichia coli strain 113-3 is performed after the light-sensitive cobalamins and hydroxocobalamin have been converted to sulfitocobalamin. This is done in order to ensure comparable growth responses to equal amounts of the cobalamins. Reference intervals of plasma and erythrocytes of healthy blood donors for methyl-, 5'-deoxyadenosyl-, cyano-, hydroxo/aquo-, and sulfitocobalamin are presented. The differences between the present and previous results are discussed.     

Ascorbic acid modulation of splenic cell cyclic GMP metabolism.

Chronic ascorbate deprivation of guinea pigs decreased splenic cell cyclic GMP levels (80%); ascorbate (1 mM) addition to these cells $\text{in vitro}$ restored the cellular concentration to control levels. Splenic cells from non-scorbutic animals also exhibited increases in cyclic GMP levels in response to exogenous ascorbate whereas thiol reducing agents diminished cellular cyclic GMP concentration. Agents that inhibit the propagation of free radicals prevented this cellular effect of ascorbate while agents known to interfere with or promote H₂O₂ production had no effect. Guanylate cyclase activity in cell lysates increased after treatment of intact cells with ascorbate; dithiothreitol reversed this effect. Ascorbate also enhanced guanylate cyclase activity in cell lysates. The results suggest that oxidizing equivalents in the form of the monoanionic free radical of ascorbate alter cyclic GMP metabolism in these cells by activating guanylate cyclase via a mechanism involving oxidation of a cyclase-related component.     

Use of a serum gradient for separation of polykaryocytes from single cells after fusion with Sendai virus

After cell fusion, polykaryocytes and heterokaryocytes were separated from single cells by a calf serum gradient. Fractions enriched with polykaryocytes were thus obtained. Heterokaryocytes with a prevalence of nuclei from either parental cell were distributed in different fractions of the gradient. The efficiency of the separation procedure is evaluated and its possible application to problems in cell hybridization techniques is discussed.     

B cell and T cell lymphopenia in systemic lupus erythematosus

The absolute number and proportion of B and T lymphocytes in the peripheral blood was determined in 16 active and 11 inactive patients with systemic lupus erythematosus. The total number of B and T lymphocytes was significantly reduced when compared to normals. The population of thymus-derived cells seems to be the one predominately affected. These abnormalities improve, but are not completely reversed, when patients are in remission.     

Nucleosome structure in Aspergillus nidulans

The structure of chromatin from Aspergillus nidulans was studied using micrococcal nuclease and DNAase I. Limited digestion with micrococcal nuclease revealed a nucleosomal repeat of 154 base pairs for Aspergillus and 198 base pairs for rat liver. With more extensive digestion, both types of chromatin gave a similar quasi-limit product with a prominent fragment at 140 base pairs. The similarity of the two limit digests suggests that the structure of the 140 base pair nucleosome core is conserved. This implies that the difference in nucleosome repeat lengths between Aspergillus and rat liver is caused by a difference in the length of the DNA between two nucleosome cores. Digestion of Aspergillus chromatin with DNAase I produced a pattern of single-stranded fragments at intervals of 10 bases which was similar to that produced from rat liver chromatin.     

Mitochondrial regulation in sea urchins. I. Mitochondrial ultrastructure transformations and changes in the ADP:ATP ratio at fertilization.

Mitochondrial ultrastructural transformations have been examined in intact eggs and embryos from three sea urchins, Arbacia punctulata, Strongylocentrotus purpuratus, and Lytechinus pictus. Following fertilization, naturally occurring ultrastructural transformations (designated as condensed to orthodox) were observed to occur in mitochondria of all three families. The ratios of the soluble ADP and ATP pools were examined in eggs of S. purpuratus before and after fertilization in order to test whether the ultrastructural transformations reflect a decrease in relative size of the ADP pool following fertilization. Our data indicate that there is a decrease in the ADP:ATP ratio at fertilization. These findings and their implications are discussed with respect to presently accepted theories on mitochondrial regulation.     

Airway constrictor effects of leukotriene D4 in dogs with hyperreactive airways

Leukotriene D₄ (5 μg/ml) aerosol constricts airways of dogs with nonspecific airway hyperreactivity but not of mongrel dogs which lack nonspecific airway hyperreactivity. R_L increased 200 + 25% and C_dyn decreased to 77 ± 5% of the pre-challenge value. LTD₄ (10 μg/ml) produced no further increase. Atropine (0.2 mg/kg) prevented the increase in R_L and decrease in C_dyn, suggesting that part of the effect of LTD₄ on airways is neurally mediated.     

Lymphocyte function in experimental African trypanosomiasis. VIII. Loss of suppressor T cell function in lymph nodes

The immunosuppression that occurs in mice experimentally infected with African trypanosomiasis has been examined further. In the present study we have examined lymph node cells from Trypanosoma rhodesiense-infected C57Bl/6J mice for the ability to produce mitogen induced antigen-nonspecific suppressor T cells (Ts). Inguinal, mesenteric, and brachial lymph node cells were harvested from uninfected control mice and from mice at different periods of infection. These cells were cultured with or without concanavalin A (Con A) for 48 hr to induce Ts activity. After stimulation, the control and infected lymph node cells were passed over Sephadex G-10 columns to remove suppressor macrophages that arise during the infection from Con A-induced Ts. The column passed cells were then added to normal mouse responder spleen cells in a primary in vitro antibody response culture system with sheep erythrocytes (SRBC) as antigen. The resultant plaque-forming cell responses to SRBC indicated that Ts function was not induced in infected lymph node cell populations. However, early in the infection, a stimulatory signal was provided by both the untreated and Con A-treated infected lymph node cells, which was lost in the terminal stage. Determinations of T cell subpopulations revealed that the infected Lyt 2.2-bearing subpopulation was not significantly altered from normal controls. We conclude that T. rhodesense infected mice fail to mount normal lymph node cell antigen nonspecific Ts responses and that this loss of activity may be due to an intrinsic dysfunction in the suppressor T cell population.     

Nature of T-cells resident in spleens of thymectomized,lethally irradiated,bone marrow-reconstituted mice

Adult thymectomized, lethally irradiated, bone marrow-reconstituted (ATxXB) mice that had been weakly primed with SRBC or HRBC between thymectomy and irradiation were shown to retain antigen-specific immunological memories for at least 1–5 months after bone marrow reconstitution. This could be shown by anamnestic antibody response in vivo as well as by proliferative response of the spleen cells to the test antigens in vitro. Spleen cells taken from ATxXB mice showed a reduced but significant proliferative response to nonspecific T-cell mitogens, in particular to Con A, in vitro. Treatment of the donor bone marrow cells used for reconstitution of ATxXB mice with anti-Thy 1.2 sera + C′ did not affect the generation of immunological memories nor the magnitude of the proliferative response of spleen cells to nonspecific T-cell mitogens in vitro, indicating that the cells responsible for such functions were host derived. Finally, the antibody-forming capacity of spleen cells derived from SRBC-primed ATxXB mice to the test antigen in vitro was completely abrogated by exposure to 450 R, whereas the helper function of the same cell suspension remained unaffected even after exposure to 1000 R. Implication of these findings on the nature of T cells resident in spleens of ATxXB mice was discussed.     

An analysis of sandbathing and grooming in the kangaroo rat (Dipodomys merriami)

The sandbathing and grooming behaviour of ten kangaroo rats (Dipodomys merriami) were recorded on sand and woodchip substrates after periods of 0,1,5 and 10 days without sand. Sandbathing is restricted to the sandy substrate. Grooming occurs on both, but with a higher frequency on sand. Increases in both grooming and sandbathing occur with increasing sand deprivation, but the temporal patterning does not change. D. merriami tends to alternate sandbathing components in contrast to other Dipodomys species. Lipid on the pelage increases noticeably with sand deprivation and decreases during a sandbathing bout; sand appears to be removed from the pelage by shaking and grooming. These findings suggest a three-process system for care of the body surface.     

Ovarian hormones and the duration of sexual receptivity in the female golden hamster

The induction of sexual receptivity and its maintenance after copulation in ovariectomized female golden hamsters (Mesocricetus auratus) was found to be a function of the levels of ovarian hormones administered. Various combinations of estradiol benzoate (between 0.6 and 666 μg) and progesterone (between 0.05 and 5.0 mg) were administered in two experiments. Although some animals responded at 0.6 μg, higher levels of estradiol benzoate (1–6 μg or more) were more effective in inducing levels of lordosis equivalent to those seen in intact females in natural estrus. After mating, a depression in lordosis was observed in both ovariectomized and intact females. However, in ovariectomized females (excluding animals that did not respond initially) the duration of postcopulatory receptivity was a function of the level of progesterone administered. High levels of progesterone tended to prolong slightly the duration of postcopulatory receptivity.     

Isolation and properties of four alpha-amylase isozymes from human submandibular saliva

Four isoamylases have been isolated from human submandibular secretions by gel filtration and isoelectric focusing. The isozymes (1A, 1B, 2A, 2B) were each purified about 8-fold and each yielded one major band on disc gel electrophoresis. In all cases the major protein band contained more than 95% of the protein and amylase activity recovered. The isoenzymes, in order of their relative positions on the polyacrylamide gels (from the anodal end), their isoelectric points, and percentage distribution in the submandibular secretion are as follows: isozyme 2A, pH 5.9, 9%; isozyme 1A, pH 5.9, 18%; isozyme 2B, pH 6.4, 63%; isozyme 1B, pH 6.4, 10%. Amino acid analyses showed that the protein compositions of the four isoamylases were essentially the same. Possible differences were noted in aspartic acid, serine, glutamic acid, and proline contents. Molecular weights, determined by SDS disc gel electrophoresis, were 57,000 for 1A and 1B, and 54,000 for 2A and 2B. This molecular weight difference is attributed mainly to the presence of bound carbohydrate on isozymes 1A and 1B. Gas Chromatographic analysis was used for determining the carbohydrate compositions. Molar ratios of sugars were similar for both glycoprotein amylases (moles sugar/mole enzyme): glucosamine, 3; mannose, 3; galactose, 2; fucose, 3. Isoamylase 1A, which had more carbohydrate than 1B, also contained about 2 moles of N-acetylneuraminic acid. Sialic acid was not detected in isozyme 1B.     

Mass production of animal cells in nude mice with retention of cell specific markers.

Biochemical analysis of macromolecular constituents of somatic cells in culture frequently requires the production of large quantities of cells from small initial populations, often a laborious and expensive procedure. Here we show that nude mice, because of their genetic immune deficiency, provide a relatively simple and reliable means of growing large quantities of cells irrespective of the species or tissue of origin. Most established animal cell lines are capable of growing as large tumors in these athymic mice. The passage of mammalian cells in nude mice does not cause either the loss or modification of specific biochemical, chromosomal, antigenic or other cellular markers, nor the induction of malignant transformation of the host cells. Tumors formed by the injected cells grow as localized, encapsulated masses, and host cell admixture due to elements of the vascular system in the tumor is variable but not extensive. Pure cultures of the original cell type can easily be recovered from the tumors if they are derived from previously established cell lines. The use of nude mice for large scale growth of animal cells is particularly advantageous for cell lines which are not adapted to mass suspension culture for highly differentiated functional tumor cells which cannot be grown in vitro.     

Cytolytic antibodies to murine lymphomas elicited by immunization with allogenic and syngenic tumors

C57BL/6 mice immunized with allogenic L#2 tumor cells or syngenic EL-4 tumor cells produced lytic antibodies directed against cell-surface antigens present on EL-4 tumor cells but absent from normal C57BL/6 tissues. Such immunized C57BL mice also exhibited prolonged survival after challenge with syngenic EL-4 tumor cells.     

Countercurrent distribution of lymphocytes from human peripheral blood in an aqueous two-phase system. I. Separation into subsets of lymphocytes bearing distinctive markers

Lymphocytes from human peripheral blood have been separated by countercurrent distribution in a charged aqueous two-phase system composed of Dextran T 500 and polyethylene glycol 6000 with a cell yield of 59–88% and viability above 90%. A highly reproducible partition pattern was seen with four distinct peaks. Lymphocytes with surface membrane immunoglobulin (SmIg) were located in the first part of the distribution corresponding mainly to peak I. T lymphocytes as detected by E rosetting and α-naphthyl acetate esterase (ANAE) staining showed a broad distribution with a maximum in peaks II and III. ANAE-negative lymphocytes were seen in both extremes of the distribution, corresponding to B cells in the first part and to a population of E^? and SmIg^? lymphocytes in the last part. Monocytes were present in all fractions with some enrichment in peaks II–IV. Lymphocytes with low-affinity Fc receptors were found in B-cell-containing fractions in the first part of the distribution, but also in the last part. Lymphocytes with high-affinity Fc receptors were detected mainly in peak IV. It is thus demonstrated that peripheral blood lymphocytes can be fractionated into subpopulations enriched in cells with characteristic markers.     

Testicular testosterone concentration and in vitro response to HCG in normal and in testosterone immunized rabbits.

The testosterone concentration, the in vitro response to HCG and the percentage Leydig cells in testes of normal and of testosterone-3-BSA immunized rabbits were determined. Following immunization all three parameters increased in the same order of magnitude (1.8-2.6fold). The results indicate that active immunization with testosterone has no deleterious effects on the endocrine capacity of the Leydig cells. The observed functional and morpholigical alterations of the testes are due solely to increased trophic hormone secretion from the pituitary caused by antibody binding of circulating androgens. The basic testosterone concentration in the testes of the control rabbits were in the range of values reported for other species.     

Reversal of social deficits produced by isolation rearing in monkeys

A major body of research has clearly demonstrated that rearing monkeys for at least the first 6 months of life in total social isolation from conspecifics produces profound psychopathology, including both massive social deficits and ideosyncratic self-directed behaviors rarely seen in normal monkeys. Previously, isolation-induced psychopathology was thought to be permanent, and various theoretical explanations, e.g. critical periods and “emergence trauma” were posited to account for such phenomena. Recent research at Wisconsin has indicated that the isolation syndrome in monkeys is, in fact, reversible, rendering the above theoretical positions inadequate. In this paper, the procedures for successful rehabilitation of isolate-reared monkeys are described and an alternative theoretical explanation presented. Implications of the theory and data are discussed.     

Use of preformed cell aggregates and layers to measure tissue-specific differences in intercellular adhesion

The binding of aggregates formed from various 7-day chick embryo tissues to cultured cell layers was analyzed 24 hr following trypsin dissociation of the tissues. The proprotion of aggregates binding is independent of the number of aggregates added, and changes with time over 60 min in a manner consistent with a first-order process. The adhesive parameter measured, the percentage of aggregates binding to cell layers per unit time, varies slightly with aggregate size but is not dependent upon the probability of collision of the aggregate with the layer. The rate of binding and the effect of modifiers of binding (temperature, inhibitors of oxidative metabolism and glutaraldehyde) are substantially different for neural retina interactions than for liver or heart interactions, suggesting that retina cells may form intercellular bonds via a mechanism distinct from that of liver or heart cells. The rate of binding between like tissue types is, with one exception, greater than between unlike types. Glutaraldehyde treatment of only one of the reactants abolishes this adhesive specificity. Aggregate binding provides a means of quantitatively assessing intercellular adhesion which has the advantage of reducing the effects of trypsinization on measurements of adhesion, and therefore lends itself to the investigation of cellular consequences of adhesion.