Comparative migration of B- and T-Lymphocytes in the rat spleen and lymph nodes.

B-lymphocytes were obtained either by thoracic duct cannulation of thymectomized, irradiated rats or by isolation of complement-receptor-bearing lymphocytes from normal rats. They were labeled in vitro with [³H]-leucine and injected iv into syngeneic recipients from which samples of spleen and lymph node were taken at intervals from 15 min to 48 hr after injection. The sites of initial localisation of B- and T-lymphocytes were identical suggesting that the cells migrated into both organs by a common entrance. The two cell types remained closely associated for several hours in the paracortex of lymph nodes and at the periphery of the periarteriolar lymphoid sheath of the spleen. After 1–6 hr, B-cells segregated from T-cells by moving on into the adjacent part of the lymphocyte corona in the follicular area. By 24 hr, B-cells were evenly distributed throughout the corona. A definite minority of B-cells but no T-cells were seen within the germinal centres. In the spleen, T-cells moved into the central area of the periarteriolar sheath before returning to the blood. The immunological significance of the routes of B- and T-cell migration is discussed.     

Lymphokines in sensitized rats. III. Influence of prednisolone treatment of lymph node and spleen architecture in sensitized rats and upon MIF release in vitro.

The influence of prednisolone (corticosteroid, C.S.) treatment upon established cell-mediated immunity (CMI) induced by complete Freund's adjuvant (CFA) has been studied in rats by using in vitro migration inhibitory factor (MIF) assays, type IV skin reactions, and regional lymph node and spleen histology. Additionally, changes in the mononuclear-polymorphonuclear ratio of peripheral blood and T-cell accumulation in bone marrow in response to C.S. treatment have been determined. These results have been evaluated by comparison with equivalent experiments upon animals treated with anti-rat lymphocyte serum (ALS), oxisuran, or 2-[(methylsulfinyl)-acetyl]pyridine, which selectively suppresses CMI. The results suggest the existence of a population of “educated” T-cells in the thymic cortex of sensitized rats, and they suggest that prolonged C.S. administration does not suppress T-effector cells involved in established CMI but, rather, affects lymphocyte and monocyte migration patterns, including the migration of educated T-cells from the thymic cortex into other tissue compartments.     

In vitro induction of IgM synthesis and secretion in rabbit spleen cells by soluble Concanavalin A

Rabbit spleen cells are not activated by Concanavalin A (Con A) conjugated to Sepharose 4B but are stimulated by soluble Con A which induces DNA and protein synthesis. At optimal concentration (5 μg/ml) one notes an increased intracellular protein and IgM synthesis and then secretion. This increase in protein synthesis is seen at all phases of the culture. At the intracellular level, IgM is found in the form of 7S molecules and a significant proportion of polymers with a centrifugation constant smaller than 19S. Fully assembled 19S polymers are found in the fluid phase. These results are compatible with a model of cellular cooperation, the basis of which is not the presentation of the inducer (mitogen or antigen) by a cell type to another, but rather the secretion of mediators by one of the cell populations making other cells responsive to the inducer.     

Norepinephrine-like effects of neuropeptide Y on LH release in the rat

Neuropeptide Y, a recently isolated neuropeptide exhibited norepinephrine-like effects on LH release after intracerebroventricular administration at doses from 0.5 to 10 micrograms. While it promptly suppressed LH release in ovariectomized rats, there was a dose-related stimulation of LH secretion in ovarian steroid primed-ovariectomized rats. In view of the evidence that neuropeptide Y may coexist with adrenergic neurotransmitters, these findings suggest that it may play a role in regulation of LH release in the rat, either independently or in concert with catecholamines.     

Effects of maternal diabetes on the development of carbohydrate metabolizing enzymes in fetal rat liver

Effects of streptozotocin-induced maternal diabetes on fetal hepatic carbohydrate-metabolizing enzyme development and hormonal status has been explored in the rat. Hepatic glycogen synthase a activity of the normal fetus rose to a maximum at 20 days of gestation, then fell prior to parturition. In fetuses of diabetic mothers, this prepartum decline was curtailed, resulting in enhanced synthase a activity and increased glycogen content in fetal livers at term. Elevation in hepatic synthase a in fetuses of diabetic mothers was due, not to altered interconversion between existing synthase a and b, but to equivalent increases in both forms of the enzyme. Both hepatic and free plasma corticosterone levels were elevated in fetuses of diabetic mothers and may be responsible for the enhanced development of total glycogen synthase observed in these fetuses. In normal fetuses hepatic phosphofructokinase and pyruvate kinase activities also rose to maxima at 20 days, then declined prior to term. In fetuses of diabetic mothers pyruvate kinase activity attained higher than normal maximal levels and phosphofructokinase activity fell more gradually, thus resulting in elevations in both enzyme activities at term. Augmentations in these glycolytic enzymes are compatible with hyperinsulinemia observed in fetuses of diabetic mothers. The following conclusions may be drawn from these findings. During late fetal life developmental patterns of rate-limiting hepatic glycogen-synthesizing and glycolytic enzymes are adapted to glucose utilization. In the normal fetus these patterns reverse at term, thereby promoting glucose mobilization, which prepares the fetus for abrupt deprivation of maternal glucose at birth. Maternal diabetes results in retardation of these reversal processes, presumably due to elevations in fetal glucocorticoid and insulin levels. Glycogenolytic and glucogenic capacities are thereby impaired in these fetuses.     

Automated fluorometric amino acid analysis: the determination of proline and hydroxyproline.

A fluorometric method for the automated determination of the imino acids proline and hydroxyproline has been developed. The assay is based on the postcolumn reaction of the imino acids with alkaline sodium hypochlorite, which yields oxidation products amenable to detection with fluorogenic amine reagents. The method is simple and can be adapted readily to high-sensitivity amino acid analyzers which use o-phthalaldehyde for detection. As little as 10 pmol proline and 20 pmol hydroxyproline can be determined accurately. Thus the full array of natural imino and amino acids can now be determined on a high-sensitivity amino acid analyzer using o-phthalaldehyde.     

Corticosterone decreases the efficacy of adrenaline to affect passive avoidance retention of adrenalectomized rats

Short-term (48h) adrenalectomy (ADX) resulted in a deficit in the retention of a passive avoidance response. An inverted U-shaped dose-response relationship was found following immediate post-learning administration of adrenaline (A). A in a dose range of 0.005 - 5 micrograms/kg s.c. facilitated later retention. While corticosterone (CS) replacement alone had no effect, pretreatment with CS (300 micrograms/kg) was followed by a shift in the dose-response curve of A in ADX rats. Ten thousand times higher doses of A were required to improve retention behavior. Administration of the potent synthetic glucocorticoid dexamethasone failed to affect the responsiveness to A. It is concluded that corticosterone decreases the efficacy by which adrenaline affects later retention behavior of ADX rats. The specificity of corticosterone in this interaction suggests the involvement of the corticosterone receptor system which has its predominant localization in hippocampal neurons.     

A new fluorometric method for the determination of pyridoxal 5′-phosphate

A new fluorometric method using semicarbazide for the determination of pyridoxal and pyridoxal 5′-phosphate (PLP) in whole blood, red cells and plasma has been developed. Semicarbazide breaks the Schiff base of PLP and proteins by “trans-Schiffization” reaction and forms semicarbazone of PLP. The semicarbazone of PLP emits strongly at 460 nm when excited at 380 nm. Several metabolic intermediates were tested for the possible interference. Only pyridoxal was found to interfere. The interference can be corrected since pyridoxal emits at 380 nm when excited at 320 nm. Using this method we found that rabbit red cells in vivo are freely permeable to PLP.     

RNA sequence determination in the nanogram range by a combination of in vitro labeling procedures.

Recently developed methods which allow one to read RNA sequences directly from polyacrylamide gels do not always provide unequivocal results. A combination of primary and secondary in vitro 5′-labeling, as presented here, is methodically and in its results equivalent to fingerprinting and sequencing techniques developed for in vivo labeled RNA. 5 S RNA was used to demonstrate the applicability and reliability of this combination of postlabeling procedures: 5 μg RNA was partially digested, and the resulting overlapping fragments were 5′-³²P-labeled with T4 phage-induced polynucleotide kinase in vitro. After two-dimensional polyacrylamide gel electrophoresis and carrier-free electrophoretic elution, the labeled long fragments, obtained in the 10-ng range, were completely degraded with RNase T₁ and RNase A, respectively. These digests were again ³²P-phosphorylated with T4 kinase and lead to fingerprints which allowed the deduction of the nucleotide sequences of the corresponding long fragments.     

U-60, 257 inhibits O3-induced bronchial hyperreactivity in the guinea pig

We studied the effects on ozone-induced airway hyperreactivity of U-60, 257, a pyrroloprostacyclin shown to inhibit leukotriene C/D biosynthesis in vitro. A group of 5 guinea pigs were pretreated with U-60, 257 (5 mg/kg IV), and studied before and 30 min after a 15 min exposure to 3.0 ppm ozone. These animals were compared to a similarly exposed group that was untreated (n=10). Reactivity was determined by measuring specific airway resistance (SRaw) upon intravenous acetylcholine infusion in unanesthetized, spontaneously breathing animals. Prior to ozone exposure, we found that U-60, 257 treatment did not affect either SRaw or muscarinic reactivity. After exposure to 3.0 ppm, all untreated guinea pigs showed substantial muscarinic hyperreactivity. In contrast, no significant change in SRaw or muscarinic reactivity occurred after ozone in any animal pretreated with U-60, 257. We conclude that ozone-induced bronchial hyperreactivity in the guinea pig rapidly develops after a brief, high level exposure. This effect may be mediated, in part, by leukotrienes generated upon ozone exposure.     

A non-peptide morphine-like compound from brain

A non-peptide morphine-like compound was extracted from calf brain and partially purified by immunoabsorption to antimorphine antibodies. The material was able to depress electrically stimulated contractions of the myenteric plexus-longitudinal muscle of the isolated guinea pig ileum. This effect could be prevented and reversed by naloxone and by antimorphine antibodies. Biological activity and morphine-like immunoreactivity were not affected by treatment with proteolytic enzymes. These results represent the first independent confirmation of the non-peptide morphine-like compound reported by Gintzler $\text{et al}$.     

A high-sampling-rate automated continuous-flow fluorometric technique for the analysis of nanogram levels of histamine in biological samples

An automated continuous-flow technique of the modified fluorometric method of Shore was devised to obtain a high sampling rate (60/h) and a sensitive measurement of the histamine content of biological samples. The volumes of samples range from 50 to 500 microliter. A linear relationship is obtained from 0 to 5 micrograms/ml (histamine base) with a good specificity. The limit of detection is 25 pg (actual amount). The coefficient of variation is less than or equal to +/- 5% for concentrations of less than 2 ng/ml and from +/- 0.2 to +/- 2% for higher concentrations. With this technique more sensitive, more specific, and twice as fast as similar ones, histamine content in 350-400 unknowns can be measured routinely in a working day. It has been used for more than 4 years and has proven to be a reliable and useful tool for the numerous research studies in which histamine is involved: immunology, allergy, pharmacology, dermatology, cancer, nutrition.     

Changes in the morphology of spermatozoa during their passage through the genital tract in dairy bulls with normal and impaired spermat ogenesis

An investigation was conducted on changes in sperm morphology along the excurrent duct of bulls. The study comprised 20 bulls with proven fertility and normal semen picture, and 10 bulls with pathologic semen. The morphology of spermatozoa was evaluated on ejaculates and on postslaughter collected contents from the excurrent duct. The incidence of pathologic sperm heads decreased along the duct in both groups of bulls. The main decrease generally occurred in caput epididymidis. In bulls with pathologic semen, the decrease continued in lower regions of epididymis and was deferens. The rate and pattern of sperm removal seem to primarily depend on the quality of spermatozoa entering the excurrent duct. The removal was clearly selective and is assumed to be associated with phagocytosis of spermatozoa mainly in the efferent ductules and proximal part of caput epididymis. Proximal cytoplasmic droplets were present on almost all sperm in the efferent ductules. The incidence decreased during passage along the genital tract. Migration of cytoplasmic droplets from a proximal to a distal position took place between regions C and D of the caput epididymidis. The incidence of middle-piece abnormalities decreased during passage along the genital tract, while the incidence of sperm tail abnormalities increased in the corpus and cauda epididymidis in all bulls.     

B cells with or without C3 receptor: Murine B-cell subsets highly and lowly responsive to anti-Ig stimulation

Bone marrow-derived lymphocytes (B cells) with or without receptors for a third component of complement (CR) were studied in their responsiveness to the F(ab′)₂ fragment of antiimmunoglobulins (anti-Ig). Spleen cells from C57BL/6J mice were fractionated by the centrifugation over Ficoll-Hypaque density gradient after they were rosetted with erythrocyte-antibody complement complexes. The cells in the interface fraction responded poorly to anti-Ig, while the cells in the pellet fraction responded well. The low responsiveness of CR(?) B cells was confirmed by assaying the responsiveness of cells passed through a Sephadex G-10-complement column. Reduced response of CR(?) B cells could not be explained by the depletion of helper or accessory cells. The relationship between CR, B-cell differentiation and proliferative capacity of B cells is discussed.     

Reduced ability of naloxone to stimulate LH and testosterone release in aging male rats; possible relation to increase in hypothalamic met5-enkephalin

Blood luteinizing hormone (LH) and testosterone levels are lower in old than in young male rats. The specific opiate antagonist, naloxone, previously shown to increase serum LH in mature male rats, exhibited relatively little ability to raise serum LH and testosterone levels in old (18–20 mo) as compared to young (4–5 mo) male rats. The brain opiate, met⁵-enkephalin, which depresses LH, was found to be significantly higher in the hypothalamus of old than of young male rats. These observations suggest that hypothalamic opiates may be partially responsible for the lower serum LH and testosterone levels in old male rats, and for reduced release of these hormones in response to naloxone administration.     

Proton relaxation rate and fluorometric studies of manganese and rare earth binding to concanavalin A

The binding of Mn²⁺, Ca²⁺, and rare earth ions to apoconcanavalin A has been studied by water proton relaxation enhancement, electron paramagnetic resonance spectroscopy, and fluorescence spectroscopy. An electron paramagnetic resonance and water proton relaxation rate study of the titration of apoconcanavalin A with Mn²⁺ gives evidence of two equivalent binding sites per monomer with K_D = 50 μm ± 4 μm. When a similar Mn²⁺ titration of apoconcanavalin A is performed in the presence of Ca²⁺ ion, very little free Mn²⁺ is detected by electron paramagnetic resonance until the two Mn²⁺ binding sites per monomer are filled. The substitution of a rare earth ion for Ca²⁺ ion in the above experiment often resulted in a slight displacement of Mn²⁺ from the transition metal site as detected by electron paramagnetic resonance. A water proton relaxation rate study of the titration of apoconcanavalin A with Gd³⁺ reflects two binding sites with a K_D = 40 μm ± 4 μm and two with a K_D = 200 μm ± 50 μm. The fluorescence emission spectrum of concanavalin A (λ_em = 340 nm) is slightly quenched by the addition of Tb³⁺ while Tb³⁺ fluorescence is greatly enhanced. A fluorometric titration of apoconcanavalin A with Tb³⁺ also reflects two sites with a K_D = 40 μm ± 15 μm and two with a K_D = 270 μm ± 50 μm.     

A random walk model of ovum transport

This paper discusses the random nature of ovum transport, and presents a Brownian Motion model of ovum transport in the ampulla and isthmus. A new explanation of the delay in transport at the ampullary-isthmic junction, based on widely differing diffusion coefficients for the ampulla and isthmus, is proposed.     

Release of cholecystokinin-like immunoreactivity from slices of dorsal and ventral striatum of rat brain

Dorsal and ventral striatum may differ in their neuronal organisation and function. In a comparative in vitro study, we investigated the release of cholecystokinin-like immunoreactivity from slices of dorsal and ventral striatum, respectively. Release of immunoreactivity was induced by veratridine. The dopamine D₂-receptor agonist RU 24926 enhanced, while substance P reduced the release from slices of dorsal striatum. The two agents had no effect on the release of immunoreactivity from slices of ventral striatum. It is concluded that the discrepancy in the modulation of the release of cholecystokinin-like immunoreactivity reflects differences in neuronal organisation in both functionally important areas.     

The effect of local application of homocysteine on neuronal activity in the central nervous system of the rat

Homocysteine, a monocarboxylic, sulfur-containing amino acid, produces convulsions in rats and mice when administered systemically. Convulsions and high serum concentrations of homocysteine are among the symptoms that characterize patients with homocystinuria, a hereditary disorder of amino acid metabolism. In order to evaluate the effects of homocysteine on the central nervous system directly, extracellular recordings were made from neurons in rat cerebral cortex, cerebellum and midbrain during local application of homocysteine by pressure ejection or iontophoresis. Both methods of drug delivery produced dose-dependent increases in the activity of neurons in every area tested. Activity was increased by D, L-homocysteine and L-glutamate in 67 percent of cells tested with both drugs. The doses required to produce equivalent excitations in this group of cells were similar, suggesting that homocysteine is at least as potent as glutamate. The excitatory effects of both homocysteine and glutamate were antagonized by local application of betaine, a biological methyl donor which blocks convulsions produced by systematic administration of pentylenetetrazol and electroshock as well as homocysteine. The effects of local application of homocysteine were also blocked by local application of the glutamate antagonist glutamate diethylester (GDEE). In 6 of 7 cells tested, GDEE appeared to preferentially affect homocysteine-induced excitations. These data indicate that homocysteine has an excitatory action on neurons, a finding which may account for some of the symptoms associated with certain disorders of amino acid metabolism.