The mitogenic principle of Escherichia coli lipoprotein: B-lymphocyte mitogenicity of N-palmitoyl-cysteine and N-palmitoyl-glutamic acid α-methyl esters

N-Palmitoyl-cysteine methyl ester and N-palmitoyl-glutamic acid α-methyl ester, which are analogues to the lipophilic N-terminal part of the lipoprotein from the outer membrane of $\text{Escherichia coli}$, were synthesized and tested for biological activity in an $\text{in}\text{vitro}$ lymphocyte culture system: in spleen cells from several inbred mouse strains the fatty acid derivatives exhibited mitogenic activity towards B-lymphocytes comparable to the effect of lipoprotein, as measured by ³H-thymidine incorporation and by hemolytic plaque assays. These results confirm former investigations, which have shown that the mitogenic principle of the lipoprotein molecule resides in its N-terminal fatty acid-containing part. The proper dispersion of the water-insoluble substances was critical for their mitogenic activity. Optimal mitogenicity was obtained by sonicating the substances at concentrations of 0.1 mg/ml in culture medium.     

Transfer of cultured bovine embryos

A total of 126 bovine embryos were surgically collected from 16 superovulated donor heifers 5 days after estrus and randomly selected for either immediate transfer to synchronized recipients or $\text{in}$$\text{vitro}$ culture at 37°C for 24 hours and subsequent transfer. Twenty-four of 56 (42.8%) embryos maintained for 24 hours in Ham's F10 medium supplemented with 10% heat treated fetal calf serum (HTFCS) and transferred to 32 recipients produced live calves. Survival of 70 noncultured embryos transferred to 35 recipients was 55.7% (39 calves). The percentages of recipients that were diagnosed pregnant at 42 days with cultured and control embryos were 59.4% ($\text{19}\text{32}$) and 74.3% ($\text{26}\text{35}$), respectively. No statistical difference was observed between the $\text{in}$$\text{vitro}$ cultured and control embryos for viability following transfer to recipient females.In a second study, Day 7 embryos maintained in Ham's F10 medium supplemented with 10% HTFC serum for various culture periods were tested for viability following nonsurgical transfer to recipient females. A total of $\text{1}\text{5}$, $\text{1}\text{3}$ and $\text{0}\text{4}$ embryos cultured for 24, 48 and 72 hours, respectively, resulted in pregnant recipients following transfer.     

Commercial splitting of bovine embryos

Independent studies were undertaken at Alberta Livestock Transplants (ALT) and at Select Embryos, Inc. (SEI) to develop procedures for splitting bovine embryos. At both locations embryos were recovered seven days after the onset of estrus and superovulation. Initially, survival after splitting was evaluated by culture $\text{in}$$\text{vitro}$ for 18 to 24 hours. Culturing half or demi-embryos without a zona pellucida at ALT resulted in 15% survival compared to 35% survival when both halves were in separate zonae. Culturing demi-embryos on a monolayer of luteal cells at SEI did not improve survival $\text{in}$$\text{vitro}$. In fertility trials, best results were obtained at ALT when both demi-embryos within separate zonae were nonsurgically transferred into separate uterine horns of the same recipient (55% pregnancy rate) and at SEI when one demi-embryo was surgically transferred into the uterine horn ipsilateral to the corpus luteum (65% pregnancy rate). Culturing demi-embryos more than 4 hours reduced fertility at both locations. Splitting embryos was a worthwhile addition to the commercial ET programs and further trials are in progress to improve survival $\text{in}$$\text{vitro}$ and pregnancy rates.     

A new method for thawing frozen ram semen

The purpose of this work was to facilitate the on-farm use of frozen semen by initially thawing the straws in laboratory treated sperm (TS) rather than on-farm control sperm (CS), as is usually done. After thawing, TS was diluted, centrifuged, and extended in skim milk for storage at +15° C until utilized 3 to 6 hours later. $\text{In}$$\text{vitro}$: immediately after preparation and addition of skim milk for TS and thawing for CS, the percentage of stained cells and abnormal cells was higher (P < 0.01) in TS than in CS. In contrast, following a 3 hour incubation, TS and CS had the same proportion of motile cells. $\text{In}$$\text{vivo}$: fertility and prolificacy of FGA + PMSG-treated ewes were slightly higher following AI (1 AI/female) with TS than with CS: 52.4% $\text{vs}$ 44.2% and 155.0% $\text{vs}$ 148.0%, respectively. Fertility was also higher (P < 0.01) with fresh semen than with TS, but the difference was only 9.2 points (70.3% $\text{vs}$ 61.1% for the respective 798 and 242 ewes inseminated once). Prolificacy rates were similar (164.3% $\text{vs}$ 167.6%).     

Intraperitoneal injection of zymosan in mice induces pain,inflammation and the synthesis of peptidoleukotrienes and prostaglandin E2

Intraperitoneal injection of zymosan in mice induced rapid extravasation and accumulation of plasma proteins in the peritoneal cavity. Neutrophils began to appear in the peritoneal cavity after a lag period of approximately 3 hours. The injected mice exhibited a pain response (writhing) during the first 30 minutes after injection, but writhing ceased before protein or cell accumulation had reached maximum levels. The injection of zymosan induced synthesis of PGE₂ (measured by RIA) which reached maximum levels of 30 minutes, then declined slowly. Peptido-leukotriene levels (detected by bioassay, RIA and HPLC) increased rapidly after injection, reached a peak within an hour of injection and declined to undetectable levels within 4 hours. The early peptido-LT was predominantly LTC₄, while later, LTE₄ was the major component. LTD₄ levels remained low throughout and no LTB₄ was detected at any time. Indomethacin treatment elevated levels of peptido-LTs, recued PGE₂ levels and inhibited writhing. Phenidone reduced peptido-LT levels. $\text{In}$$\text{vitro}$ studies demonstrated that zymosan stimulates LTC4 synthesis by peritoneal cells whereas LTE₄, LTD₄, LTB₄ or monoHETES were not detectable (using HPLC methods). The source of enzymes responsible for the $\text{in}$$\text{vivo}$ metabolism of LTC₄ to LTD₄ and LTE₄ could not be identified.     

Perturbation of the phagocytic response in P388D1 cultured macrophages by agents altering cell calcium

Phagocytosis in adherent P388D₁ (D₁) cells was monitored utilizing formalin treated $\text{Listeria}$$\text{monocytogenes}$ ($\text{Lm}$) previously labeled with ¹²⁵iododeoxyuridine. The dependence of this phagocytic process on calcium was studied by using several agents which alter calcium metabolism. The calcium antagonist ruthenium red (RR) produced a dose and time dependent stimulation (60–70%) of $\text{Lm}$ phagocytosis by D₁ cells. Utilizing another calcium antagonist, D-600, a prolonged inhibition (4 hours) of phagocytosis (40%) was observed. The addition of the cation ionophore A23187 produced a transient stimulatory increase (38% at 2 hours) in the phagocytic response. The concomitant addition of RR and D-600 did not alter the phagocytosis of $\text{Lm}$ by D₁ cells as compared to control cells. However, this complete drug/drug antagonism was not seen with the combinations of A23187 and D-600 or RR and A23187. The addition of A23187 and D-600 resulted in a time dependent inhibition of phagocytosis which did not become maximal until 3 to 4 hours. A23187 and RR produced a time independent stimulation of phagocytosis which was significantly less than that which was observed for RR alone, but was of longer duration than the response produced by A23187 alone. The use of these calcium probes in the P388D₁ macrophage model suggests a role for calcium in the phagocytic process.     

Simian virus 40 rapidly lowers cAMP levels in mouse cells

The addition of SV40 to contact inhibited $\text{Balb}\text{3T3}$ cells causes a 2-fold decrease in intracellular cAMP levels. The levels reach a minimum 3 hours after virus addition, and after a few hours begin to rise toward normal. No significant changes in cAMP levels are observed after cells are exposed to UV-inactivated virus or are mock-infected. This is the earliest known effect of SV40 infection. We propose that SV40 induces host DNA synthesis by lowering cAMP levels.     

The measurement of 18-hydroxy-11-deoxycorticosterone in human plasma by radioimmunoassay

A radioimmunoassay method for the measurement of plasma levels of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) has been developed. The antiserum against 18-OH-DOC was produced in rabbits immunized against 18-OH-DOC-3-oxime-bovine serum albumin. Plasma (1–2 ml) was extracted with dichloromethane and chromatographed on paper. The purified extracts were incubated with antiserum at a $\text{1}\text{22,000}$ dilution for $\text{1}\text{2}$ hour at 37°C and for 2 hours at 4°C. Saturated ammonium sulfate was used to separate free from bound 18-OH-DOC. 1, 2-³H-18-OH-DOC was added to all samples to correct for losses and to determine the percent free. Pyridine (0.1%) was added to solvents to maintain the stability of 18-OH-DOC. Recovery after extraction was 58 ± 8 (S.D.)%. The accuracy and precision of the method were acceptable, and a sensitivity of 2 pg per sample enabled the measurement of very low levels of 18-OH-DOC. High specificity was demonstrated by a low blank value (0 ± 0.2 pg) and by demonstrating that alternative paper chromatography separation systems gave results not differing significantly from those obtained by the present method. The mean 8AM plasma 18-OH-DOC level was 8.5 ± 1.2 ng per 100 ml in 18 normotensive control subjects. There was a marked response of plasma 18-OH-DOC to ACTH stimulation and dexamethasone suppression and a significant increase after 3 hours upright posture.     

Comparison of invivo and invitro uterine contractility in the ewe

Uterine contractions were observed $\text{in}$$\text{vivo}$ by laparotomy and exposure of the uterus. Ten hours after the beginning of estrus, an average of 39 contractions per 10 min originated in the posterior ends of the uterine horns and moved toward the oviducts, while an average of, 13 contractions originated in the anterior ends of the horns and moved toward the cervix. Two days later (58 hr after the beginning of estrus), the contractions had changed in origin and direction; only 6 contractions originated in the posterior ends of the horns and moved anteriorly, while 39 originated in the anterior ends and moved posteriorly.Experiments were done to determine whether the change in origin of contractions $\text{in}$$\text{vivo}$ was reflected in the contractility of strips of myometrium in a tissue bath. The number and amplitude of contractions were recorded from strips of circular and of longitudinal myometrium taken from the posterior and anterior ends of the uterine horns at 10 and 58 hr after the beginning of estrus. The myometrial strips contracted approximately 3 to 4 times per minute regardless of the time after the beginning of estrus, the end of the uterine horn from which the tissue was taken, or whether the contracting muscle was circular or longitudinal. Thus, the physiological mechanisms that controlled the number and origin of uterine contractions $\text{in}$$\text{vivo}$ did not maintain that control over myometrial tissue $\text{in}$$\text{vitro}$.     

Induction of ornithine decarboxylase in macrophages by bacterial lipopolysaccharides (LPS) and mycobacterial cell wall material

Lipopolysaccharide from $\text{E. Coli}$ (LPS) and BCG cell walls (BCGcw) are recognized immunoadjuvants that directly stimulate some macrophage functions. The macrophage cell line J774.1 and peritoneal exudate cells (PEC) from mice can be stimulated by LPS or other adjuvants $\text{in vitro}$ to synthesize and release protein factor(s) that activate thymus-derived lymphocytes. We have utilized J774.1 cells and PEC to demonstrate that an increase in ornithine decarboxylase (ODC) activity is a marker of early biochemical changes in adjuvant-stimulated macrophages. BCGcw and LPS increased ODC within 2 hours in J774.1 cells as well as murine peritoneal exudate macrophages. Maximal increases in ODC were detected 4 hours after the addition of adjuvants to J774.1 cells. The marked increases (12–23 fold) in ODC observed with BCGcw (20 μg/ml) did not appear to involve an effect on cell proliferation which was suppressed by this adjuvant. Cycloheximide inhibited the induction of ODC by LPS and BCGcw in the macrophage cell line. Evidence that the induction of ODC may be promoted by an increase in cyclic AMP was provided by experiments demonstrating that prostaglandin E₁ (PGE₁) and 8-bromo-adenosine-3′:5′-monophosphate (8Br-cyclic AMP) can mimic the effects of LPS and BCGcw in J774.1 cells. These observations indicate that one of the early biochemical changes in macrophages promoted by adjuvants is an induction of ODC.     

Amino acid substitution of mating factor of Saccharomyces cerevisiae structure-activity relationship.

Analogs of the mating factor of $\text{Saccharomyces cerevisiae}$, Trp¹- Trp³-Leu-Gln-Leu⁶-Lys⁷-Pro⁸-Gly-Gln-Pro¹¹-Met¹²-Tyr¹³, from which amino acids were eliminated or substituted for other amino acid, were synthesized. These analogs showed lower biological activity than the natural mating factor if assayed after 6 hours incubation with $\text{a}$-mating type cells of $\text{S. cerevisiae}$. However, if assayed after 24 or 48 hours incubation, the situation changed, i.e. the analogs in which Leu⁶ or Lys⁷ were replaced by the corresponding D-isomer, showed higher mating factor activity than the unsubstituted mating factor. The same result was obtained with the analogs in which Met was replaced by norleucine.     

Metabolism of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by cell cultures

The metabolism of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine (platelet-activating factor) was studied using various cultured cell lines. All incubations were done in the presence of bovine serum albumin and serum-free media, since albumin eliminates the adsorption of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine to cultureware and serum enzymes interfere. Human leukemia (HL-60) cells, MDCK canine kidney cells, and transformed and nontransformed clones of mouse C3H1OT1/2 cells display varying rates of uptake, degradation, and capacities for reacylation of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine. HL-60 cells displayed the highest uptake rate (24.6 pmol/mg cell protein/15 min). Whereas C3H10T1/2 cells in culture showed uptake rates comparable to other cells tested, they displayed a relative metabolic inertness towards 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine.     

Synchronization of estrus in cyclic beef heifers with the prostaglandin analog alfaprostol

Seventy-eight Hereford-Angus crossbred heifers were injected intramuscularly twice with 6 mg of alfaprostol^b in 6 ml of propylene glycol. On each representative day of a 20-day estrous cycle (estrus = Day 0), either three or four heifers received their first injection. The second injection was given 12 days after the first, regardless of the response to the first injection. Thirty-nine heifers were not treated. The first alfaprostol injection reduced serum progesterone to less than 1 ng/ml in all heifers injected after Day 4. A total of 79.5% ($\text{62}\text{78}$) of the heifers exhibited estrus by five days after the first injection. Average interval from injection to estrus was 63 hours. The second injection occurred on Days 6 through 16 for all but one heifer, with 75.6% ($\text{59}\text{78}$) falling on Days 8 through 11 of the estrous cycle. Estrus was detected in 93.6% ($\text{73}\text{78}$) of the heifers within five days after the second injection, with an average interval to estrus of 66 hours.Day of cycle at second injection did not affect the interval to estrus. Conception occurred in 79.4% ($\text{58}\text{73}$) of the heifers inseminated in the five days after the second injection. Occurrence of estrus and conception was no different in treated heifers after five days of the insemination period than in nontreated heifers after 21 days of the insemination period, where 94.9% ($\text{37}\text{39}$) were observed in estrus and 83.8% ($\text{31}\text{37}$) conceived. Overall percent conception for a 55-day insemination period was 89.7 ($\text{70}\text{78}$) for treated and 87.2 ($\text{34}\text{39}$) for nontreated heifers. Day of cycle at first or second injection did not affect conception after the second injection. Some signs of estrus were observed in 11 of the 16 heifers injected before Day 5.A second trial to determine if alfaprostol induced luteolysis early in the cycle was conducted. Twenty purebred Angus, Hereford, or Simmental heifers received either one or two injections of alfaprostol on either Day 1, 2, 3, or 4. Only five heifers showed any signs of estrus, and the three that were inseminated did not conceive. Subsequent cycle length indicated that luteolysis occurred in only one heifer.Data suggest that alfaprostol is an effective luteolytic agent in cyclic beef heifers after Day 4 and that two injections 12 days apart will effectively synchronize estrus in heifers when distributed throughout the cycle at the first injection without affecting conception rate.     

Photoperiodism in the common coturnix (Coturnix c. japonica) male

Testicular growth of young and mature coturnix ($\text{Coturnix c.}$$\text{japonica}$) on intermittent light was observed. Five experiments were designed to determine if intermittent symmetrical photoperiods were more effective when they occurred during the photosensitive phase. A brief review of night interruption and resonance experiments with male birds is also given.All birds received a total of 12 hours of light per day. The number of light cycles per 24 hours and hours of light and dark per cycle were as follows: 1(LD12:12), 2(LD6:6), 3(LD4:4) and 4(LD3:3). Testicular growth differed among groups depending on the number of photoperiods received. The most effective light treatment was the 4(LD3:3) and least effective was 1(LD12:12). These results are interpreted on the basis of a circadian rhythm in photosensitivity.     

Concanavalin A-mediated agglutinability of Balbc3T3 cells grown in media supplemented with different phosphatidylcholines

$\text{Balb}\text{c3T3}$ cells were grown near confluency in media supplemented with 10% fetal calf serum and than exposed for 24 hours to media containing different phosphatidylcholines bound to delipidated fetal calf serum. Compared to cells grown in regular media, 3T3 cells exposed to media containing dioleoyl-phosphatidylcholine dramatically increased their agglutinability by Concanavalin A. Exposure to several other phosphatidylcholines had no effect.     

Changes in the lectin binding capacities of hepatoma cells after treatment with chondroitinase.

Binding capacities of cells of ascites hepatoma, AH 130 FN, towards lectins were examined before and after treatment with chondroitinase AC. Chondroitin sulfate A was removed from the cells by the enzyme treatment, and the binding capacity of the cells towards $\text{Ricinus communis}$ agglutinin (RCA) increased remarkably while the binding constant was unchanged, whereas that towards Concanavalin A (ConA) remained practically unchanged.     

Proteins with haemagglutinin activity in larvae of the Colorado beetle, Leptinotarsa decemlineata

The haemagglutinating activity of larval haemolymph of Leptinotarsa decemlineata against red blood cells of various origins has been examined. This activity appeared to be unspecific, since all the different types of erythrocytes were agglutinated by a haemolymph dilution of $\text{1}\text{128}$ to $\text{1}\text{512}$. Only horse erythrocytes were agglutinated to a greater degree ($\text{1}\text{3200}$. Red blood cells became much more sensitive after treatment with trypsin, while formol fixation also resulted in a better agglutinability. Sulphated polysacchrides (heparin, mucin, dextran sulphate) were good inhibitors of the haemagglutination reaction. A weaker inhibition was obtained with hexosamines. As demonstrated by immunoelectrophoresis, two haemagglutinins occur in larval haemolymph. One is specific for larvae and pupae, and is therefore called the larval-pupal haemagglutinin. It is absent in adults. The second haemagglutinin is the well-known chromoprotein 2, which is present in all developmental stages, including the egg, where it constitutes an important element of yolk proteins. The affinity of chromoprotein 2 toward dextran sulphate was confirmed by precipitation tests in agarose.     

Metabolism of 25-hydroxy-vitamin D3 by primary cultures of chick kidney cells

The primary culture of kidney cells from vitamin D deficient chicks is described. After four days in culture the cells reach confluency and retain their ability to metabolize 25-hydroxyvitamin D₃ to 1,25-dihydroxyvitamin D₃. Addition of one unit of bovine parathyroid hormone to the culture medium for 48 hours prior to assay had no effect on the cells' ability to produce 1,25-dihydroxy vitamin D₃, whereas after 24 hours in the presence of 5×10^?8$\text{M}$ 1,25-dihydroxyvitamin D₃ the cells produced not this metabolite, but 24,25-dihydroxyvitamin D₃. This cell culture system will allow the investigation of the regulation of renal 25-hydroxyvitamin D₃ metabolism under controlled $\text{in vitro}$ conditions.     

Effect of ammonia and glutamine on macromolecule synthesis and breakdown during sporulation of Saccharomyces cerevisiae.

The effect of two known inhibitors of sporulation in yeast, ammonia and glutamine, on certain biochemical events during sporogenesis have been studied using sporulating $\text{a}\text{α}$ and non sporulating $\text{α}\text{α}$ cells. Both strains gave similar results on the increase in dry cell weight, protein and RNA breakdown and the suppression of the intensive RNA and protein syntheses occurring after 4 hours. The inhibitory effect of ammonia and glutamine on RNA and protein syntheses is reversible under the same conditions which do so for sporulation.     

Studies on the modification of the cellular response to injury. IV. Protective effect of extracellular acidosis against anoxia,thermal, and p-chloromercuribenzene sulfonic acid treatment of isolated rat liver cells

Isolated rat liver cells were exposed $\text{in}$$\text{vitro}$ to ageing in an aerobic condition and to the effects of anoxia, thermal and $\text{p}$-chloromercuribenzene sulfonic (PCMBS) acid treatment, The survival of the cells was studied at normal and acidic pH using vital dye staining, intracellular concentration of potassium and ATP as indicators of viability. In aerobic conditions at pH 7.4, control cells lost their viability within approximately 6 hours. Extracellular acidosis not only prolonged the life span of isolated control hepatocytes against mechanical and other possible stress factors associated with the incubation and the absence of substrates in the medium, but also protected the cells significantly against various other superimposed injurious effects. These observations augment our previous observations on Ehrlich ascites tumore cells and lend further support to the hypothesis that extracellular acidosis, a common phenomenon associated with cell injury, is not harmful to cell survival $\text{in}$$\text{vitro}$ evidently prolongs it. The mechanism(s) behind this effect is not fully understood but it is suggested that extracellular acidosis stabilizes, and thereby protects cellular membrane systems making them more resistant to various deteriorating effects.