Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

Evidence for additional mediator in prostaglandin-induced choleresis

PGA1 and PGF2alpha were administered to isolated perfused porcine and canine livers to determine whether these hormones could induce hepatic choleresis. PGA1 (25 microgram/kg/10 min) decreased portal venous resistance, but had no effect on bile flow, oxygen, pyruvate, or lactate consumption in canine livers. PGF2alpha increased portal venous resistance and weight gain while decreasing bile flow and oxygen consumption in canine livers. At high doses (50 microgram/kg/10 min) these effects resulted in irreversible outflow block. At low doses (5 microgram/kg/10 min) these trends were reversible. Porcine livers did not exhibit the outflow block syndrome after PGF2alpha administration (100 microgram/kg/10 min); however, choleresis was not observed. Thus, the in-vivo choleretic effects of prostaglandins previously reported are probably mediated partially or wholly by extrahepatic release of other hormones, neurological stimulation or alterations in mesenteric blood flow.     

Effects of growth hormone-releasing factor and somatostatin on growth hormone secretion in hypophysial stalk-transected beef calves

The effects of growth hormone-releasing factor (GHRF) on growth hormone (GH) secretion were studied in beef calves after hypophysial stalk transection (HST). Peripheral GH concentration during surgery was elevated for 60 min after the initiation of anesthesia to 15 ng/ml, which was greater than plasma levels after HST and during the recovery period (0-30 hr mean, 3 ng/ml; P less than 0.05). Episodic GH secretion normally seen in sham-operated controls (SOC) was abolished after HST. Before HST, calves responded to 80% of the GHRF challenges, whereas after HST calves responded to every challenge of GHRF with an increase in plasma GH. A dose of 0.067 microgram human pancreatic (hp) hpGHRF(1-40)OH/kg body wt 3 days after HST increased plasma GH to 55 ng/ml from a control period mean of 5 ng/ml (P less than 0.04). On Day 8, HST calves received two injections of 0.067 microgram hpGHRF/kg body wt at 3-hr intervals, with feeding 70 min after the first injection. During two preinjection control periods, basal GH averaged less than 4 ng/ml and increased to 17 (P less than 0.02) and 9 (P less than 0.04) ng/ml immediately after the first and second injection of hpGHRF, but the response declined over the 8-day period after surgery. On Days 19 and 20, the HST calves were infused iv with 0.033 and 0.067 microgram somatostatin(SS)-14 (SRIH)/kg body wt, during which a pulse injection of 0.067 microgram hpGHRF/kg body wt was administered. GH increased to 9 and 5 ng/ml during the 0.033- and 0.067-microgram SRIH infusions after GHRF; no somatotropic rebound was observed after the SRIH was discontinued as was seen in the animals while the hypothalamic-hypophysial connections were intact. Five and six months after HST the responses to two analogs of rat hypothalamic GHRF were similar to those in SOC calves. These results indicate that HST calves responded to exogenous GHRF with an abrupt increase in plasma GH, but GH response to GHRF during SRIH infusion was greatly inhibited.     

In vitro development of sheep preantral follicles

Preantral ovarian follicles isolated from prepubertal sheep ovaries were individually cultured for 6 days in the presence of increasing doses of FSH (ranging from 0.01 to 1 microg/ml) and under two different oxygen concentrations, 20% and 5% O2. Follicle development was evaluated on the basis of antral cavity formation as well as the presence of healthy cumulus oocyte complexes. Follicle growth was enhanced by FSH addition to culture medium, while the use of a low oxygen concentration slightly stimulated this process. However, when follicles were cultured in the presence of high doses of FSH (1 microgram/ml) and under low oxygen concentration, a high proportion of them showed the presence of an antral cavity and of a healthy cumulus-oocyte complex. In addition, under this specific culture condition sheep preantral follicles released higher levels of estradiol as compared to those secreted at lower FSH concentrations or under 20% O2. When the meiotic competence of oocytes derived from follicles cultured at 1 microgram/ml FSH was assessed, no significant difference was recorded between the two oxygen groups. These results show that the culture conditions here identified are beneficial to in vitro growth and differentiation of sheep preantral follicles.     

Increased production of aflatoxins by Aspergillus parasiticus Speare in the presence of rubratoxin B.

The influence of rubratoxin B, a metabolite of Penicillium rubrum Stoll, on the growth and aflatoxin production of a strain of Aspergillus parasiticus Speare grown in the chemically defined medium of Reddy et al. (Appl. Microbiol. 22:393-396, 1971) was studied. After 4 days of incubation on a rotary shaker at 25 degrees C, the presence of 10 microgram/ml caused 45 to 50% reduction in dry weight production, although at the same concentration of rubratoxin B, the reduction of growth after 10 days was only 15%. In the presence of 50 microgram/ml there was a reduction in dry weight production of 94% after 4 days of incubation, and it was still 86% after 8 days. Rubratoxin B concentrations of 50 microgram/ml and higher usually caused a reduction in aflatoxin production in the medium comparable with the reduction in biomass, but at concentrations as low as 10 microgram/ml, there was a pronounced increase in the production of aflatoxins, especially of G1, despite the reduction in biomass. The ecological significance of these observations is discussed.     

Insulin-like growth factor 1 can decrease degradation and promote synthesis of proteoglycan in cartilage exposed to cytokines.

A model system of explanted cartilage has been used in vitro to determine whether insulin-like growth factor 1 (IGF 1), which promotes matrix formation is effective in the presence of cytokines such as interleukin 1 (IL1) and tumour necrosis factor (TNF), which induce net matrix depletion. IGF 1 induced a dose-dependent 2.5-fold stimulation of proteoglycan synthesis, with a half-maximal dose of 25 ng/ml. A similar relative increase occurred in response to IGF 1 (10-100 ng/ml) in cartilage cultured also with IL1 or TNF (5-500 pM). There was no detectable qualitative change in the average molecular size or charge of the aggregating proteoglycan synthesized by explants exposed to IGF 1 alone or with IL1 or TNF. The increased production of prostaglandin E2, which is initiated when IL1 or TNF bind to the chondrocytes, was the same in the presence or absence of IGF 1. The time taken for 50% of pre-labelled proteoglycan to be released from the explants (t1/2) increased in the presence of IGF 1 (100 ng/ml) from 21 to 32 days in control cultures and from 8 to 26 days in cartilage cultured with IL1 (50 pM). It is concluded that IGF 1 enhances the synthesis of aggregating proteoglycan in cartilage exposed to cytokines and can directly decrease both the basal and the cytokine-stimulated degradation of proteoglycan in cartilage.     

Pentagastrin as a stimulator of immunogenesis

It has been shown that subcutaneous injection of pentagastrin to mice in doses 0.01-5 micrograms per animal during 10 days resulted in a considerable stimulation of the immune response to sheep red blood cells (SRBC). Pentagastrin in doses of 5 and 1 micrograms per animal was demonstrated to have the highest immunostimulating effect. These doses increased the production of IGM-antibody-forming cells 2.2-2.7-fold and produced a twofold elevation of the antibody titer. Pentagastrin did not influence the immune response to thymus independent Vi-antigen. The in vitro treatment of mouse bone marrow cells with pentagastrin (0.1 or 0.01 microgram/ml) increased the number of Thy-1 positive cells from 0 to 16-17%. Pentagastrin at a dose of 0.001 microgram/ml was not effective.     

The effects of prostaglandin A1 and prostaglandin B1 on the differentiation of cartilage in the chick embryo

Summary In organ cultures of chick embryonic limb rudiments the mean length of explants treated with 25g/ml prostaglandin B₁ (PGB₁) was significantly smaller than that of paired controls (P<0.001) after 4, 6 and 8 days in vitro. The deceleration of linear growth was constant during 8 days in vitro. Growth inhibition was confirmed by a statistically significant decrease in explant dry weight after 8 days of culture. However, PGB₁ caused no observable alteration in the histological structure of the explants. The possible role of PGB₁ in the physiological control of cartilage growth is postulated. Explants similarly treated with prostaglandin A₁ (PGA₁) at concentrations of 15 g/ml for 8 days or 20g/ml for 4 and 8 days exhibited comma and inverted commas phenomena, caused by the intermingling of chondroblasts from the epiphyseal and flattened-cell zones, which thus ceased to be distinct entities. Adenylate cyclase in the plasma membrane may be involved in this disturbance of cartilage differentiation.     

Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos.

The objective of this study was to examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization. Oocytes were aspirated from 2-5 mm follicles of ovaries collected at a local abattoir. The oocyte-cumulus complexes (OCCs) were cultured for 23-25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 micrograms/ml luteinizing hormone (LH), 0.5 microgram/ml follicle-stimulating hormone (FSH), and 1 microgram/ml estradiol. After maturation for 23-25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum-free embryo culture medium (HECM-1/BSA) for 8 days, after which embryo development was assessed. Five media (SFRE, MEM alpha, TCM199, MEM alpha/+, RPMI:MEM alpha) better supported normal oocyte maturation as determined by embryo development to the two-cell (76-82%), morula/blastocyst (25-32%), and blastocyst (12-19%) stages. Oocytes that were matured in Waymouth's medium MB 752/l or Ham's F-12 had a significantly reduced incidence of cleavage to the two-cell stage (52% and 37%, respectively), which was not attributed to failure of fertilization. Of the eggs that did cleave to the two-cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of the hypothalamic-pituitary-axis in the ovine fetus: ontogeny of action of ovine corticotropin-releasing factor

In samples from twenty chronically cannulated ovine fetuses the plasma immunoreactive adrenocorticotrophin (ACTH) concentrations were 12.5 +/- 3.2(8), 15.2 +/- 4.1(9) and 21.2 +/- 5.6(8) pg/ml at periods, prior to parturition, of -30 to -35, -25 to -29 and -20 to -24 days respectively. Values are mean +/- SEM (number of samples). These values were not significantly different from each other but were significantly lower (P less than 0.02) than values in the next two age groups -36.0 +/- 4.9(7) pg/ml at -19 to -15 days, and 39.6 +/- 6.6(11) pg/ml at -14 to -9 days. A further significant increase (P less than 0.05) occurred in the -8 to -3 day period, ACTH being 53.9 +/- 5.4(12) pg/ml. On day of delivery two samples had values of 325 and 360 pg/ml. A single injection, intravenously of 1.0 microgram ovine corticotrophin-releasing factor (O-CRF), caused a significant increase in fetal plasma ACTH concentrations in fetuses of -6 to -23 days prior to delivery but not in fetuses -24 to -35 days prior to parturition. The maximum values of ACTH after O-CRF were significantly greater in fetuses -2 to 0 days prior to parturition than in younger fetuses (P less than 0.01). In 6 experiments in 4 fetuses (parturition -1 to -13 days) the effect of 1.0 microgram O-CRF persisted for at least 2.5 h. The results support the hypothesis that the pituitary release of ACTH changes sensitivity to hypothalamic O-CRF at least twice during the last fifth of gestation; an increasing sensitivity is seen as term approaches.     

Bombesin inhibits insulin release from isolated pancreatic islets of rats in vitro.

The effect of bombesin on insulin release from isolated pancreatic islets of rats was examined in vitro. Bombesin, at the doses ranging from 10 ng/ml to 1 microgram/ml, significantly inhibited 16.7 mM glucose-induced insulin release, while bombesin had no inhibitory effect on insulin release at 8.3 mM and 3.3 mM glucose. Moreover, bombesin also suppressed insulin release elicited by 10 mM arginine at the doses of 100 ng/ml and 1 microgram/ml. These results indicate that bombesin has a direct inhibitory action on insulin release.     

Teratogenic effects of chlorambucil on in vivo and in vitro organogenesis in mice.

Pregnant ICR/DUB mice were each given a single oral injection of chlorambucil (14.2 or 20 mg/kg) on the 10th, 11th, 12th, or 13th day of gestation (plug day = 1st day). Fetuses examined on the 18th day were decreased in weight and had tail, cranial, and limb defects. They type and frequency of malformations differed according to the dosage and day of treatment. Limb defects resulted from treatment on the 11th or 12th days of gestation and tail defects from treatment on all days. Control limb buds from 12th day embryos cultured for 6 days in serum-supplemented BGJ medium containing 0.5-2 mug/ml chlorambucil were retarded in development and had cartilage abnormalities. The extent of the deformities was dose related. Limb buds were also taken from embryos 24 h after in vivo exposure to teratogenic doses of chlorambucil and cultured in control medium. After 6 days in culture these limbs also had growth impairment and cartilage abnormalities. The defects in limbs exposed in vitro were similar to those in limbs exposed in vivo.     

Modulation of human peripheral blood neutrophil apoptosis by ultraviolet C-range radiation and by gamma-irradiation

It was found that the irradiation with in vitro UVC (254 nm) in the dose range of 6-600 J/m2 accelerates the apoptosis of human peripheral blood neutrophils in a dose-dependent manner, with saturation occurring at UVC doses of 250-300 J/m2. gamma-Irradiation with a dose of 2 Gy accelerates the apoptosis of neutrophils, whereas the irradiation with doses of 10 and 20 Gy suppresses it (by 9 h of cultivation). Lipopolysaccharide (1 microgram/ml) suppresses the UVC-induced apoptosis of neutrophils.     

Inhibition of luteinizing hormone secretion during quiescent interval of testicular androgen production in immature mice

We reported [1] that the proliferation of seminal vesicle cells in mice takes place largely in the neonatal (days 0-15) and pubertal (days 25-35) periods and that between neonatal and pubertal proliferations, a quiescent interval of cell proliferation due to markedly diminished secretion of androgens occurs. The present study was carried out to investigate the mechanism for this quiescent interval of Leydig cell activity. Serum LH concentrations were moderate (0.29 ng NIH-LH-S1/ml) at 8 days of age, low (0.13 ng/ml) at 18 days, and high (0.78-0.60 ng/ml) at 30, 40 and 60 days. The LH level on day 18 was almost the same as that found in hypophysectomized adult mice (0.12 ng/ml). These changes with age in serum LH concentrations paralleled those for serum total androgen (testosterone plus 5 alpha-androgens) concentrations. The injection of HCG (1 IU/day) or LH releasing hormone (0.1 or 0.4 microgram/6h) for 1 or 2 days resulted in significant and marked increases on day 18 in testicular and serum androgen levels and/or the proliferation of seminal vesicle cells measured with 5-[125I]iodo-2'-deoxyuridine uptake by the whole seminal vesicles. These findings lead to the hypothesis that the quiescent interval of testicular androgen production due to inhibition of pituitary LH secretion occurs around day 20 in mice.     

Isolation, purification, and antibiotic activity of o-methoxycinnamaldehyde from cinnamon.

o-Methoxycinnamaldehyde has been isolated and purified from powdered cinnamon. The compound inhibits the growth and toxin production of mycotoxin-producing fungi. The substance completely inhibited the growth of Aspergillus parasiticus and A. flavus at 100 microgram/ml and A. ochraceus and A. versicolor at 200 microgram/ml. It inhibited the production of aflatoxin B1 by over 90% at 6.25 microgram/ml, ochratoxin A at 25 microgram/ml, and sterigmatocystin at 50 microgram/ml. The substance also displayed a strong inhibitory effect on the growth of five dermatophytoses species, e.g., Microsporum canis (minimum inhibitory concentration, 3.12 to 6.25 microgram/ml). However, no antibacterial effect was observed at concentrations as high as 50 microgram/ml.     

Isolation of the homogeneous constituents from non-diffusible sugar-peptide (NSP) fraction originating from bovine plasma--II. The influence on cell growth of BHK-21, BHK/RSV and PR-RSH cells in vitro

1. The sugar-peptide aggregate isolated from the NSP fraction of bovine blood, added in concentration 20 microgram/ml, decrease of [3H]thymidine incorporation and cell proliferation of BHK-21 and BHK/RSV cells in vitro to approx. 25% of untreated control. 2. Two of four peptide constituents of the aggregate show distinct and extremely different effects on cell growth in vitro. The effects of peptide D mol. wt 5000-6000 is characteristic for peptide growth factors, while the peptide G mol. wt 1200-1800 decreases rapidly the growth of all investigated kinds of cells to 8-16% relative to untreated control. 3. The NSP fraction contains also two non-peptide constituents, which inhibit the growth of Rous sarcoma virus transformed cells to a statistically significant degree and have no effect on the growth of BHK-21 cells.     

Effect of treatment with submaximal and excessive doses of caerulein on pancreatic growth in newborn rats

Different groups of CFY female newborn rats were treated with saline, or 1 microgram/kg or 100 micrograms/kg doses of caerulein given s. c. 3 x/day. Application of 100 micrograms/kg dose of caerulein for 3 days stimulated pancreatic growth inducing pancreatic hyperplasia; both (1 and 100 micrograms/kg) doses evoked increase in trypsin/DNA ratio inducing pancreatic hypertrophy in 4-days-old rats. Using the indices as before application of 1 microgram/kg caerulein for 10 days stimulated pancreatic growth and both (1 and 100 micrograms/kg) doses elicited glandular hypertrophy in 11-days-old rats. In 24-old-rats the 1 microgram/kg doses of caerulein given for 3 days stimulated pancreatic growth and induced pancreatic hypertrophy, the 100 micrograms/kg doses of the peptide given for 3 days, however, evoked pancreatic aplasia and atrophy.     

The effects on the male genital organs and dogs of a new antifertility compound KABI 1774

The effects of KABI 1774, a new antifertility compound, on the genital organs of rabbits and dogs were studied by means of electron microscopy. KABI 1774 (2,6-cis-diphenylhexamethylcyclo-tetrasiloxane) was administered daily in a dose of 2 mg/kg body weight to rabbits for 2, 5, 10, 15, 20, 25, and 30 days, and in doses of 10 mg/kg and 250 mg/kg body weights to dogs for 40 days. In rabbits, cell demise was observed in the epididymes, especially in the middle caput and corpus after 2, 5, and 10 days, and was sometimes seen in the accessory gland. After 15 days, spermatozoa in the corpus did not demonstrate normal maturation changes, and after 20 days, spermatozoa in the cauda epididymis were dead. Between 15 and 25 days, the seminiferous tubules showed arrested spermatogenesis, followed by degeneration of all spermatids and many spermatocytes. The epithelium of the pelvic urethra was infiltrated with granulocytes. In dogs, the effects on the seminife rous epithelium ranged from arrested spermatogenesis to complete degeneration of most germ cells. Atrophy of the interstitial cells, shrinkage of the nucleus, and considerable loss of the specialized cytoplasmic ultrastructure were observed. There was some cell death and highly marked atrophy of the epithelium. Generally, no spermatozoa were observed. Marked epithelial atrophy was present in the prostate gland, and a few areas also showed epithelial metaplasia. The findings are con sistent with the concept that the cyclotetrasiloxane compound mimics estrogens in its antigonadotropic effect, though a direct antiandrogenic effect on the epididymes may also be involved.     

Experimental studies on the pathogenesis of ochronotic arthropathy. The effects of homogentisic acid on adult and fetal articular chondrocyte morphology, proliferative capacity and synthesis of proteoglycans in vitro

Lapine adult and fetal articular chondrocytes in monolayer culture were employed as an experimental model for studying the effects of homogentisic acid on chondrocyte morphology, proliferation and synthesis of proteoglycans. Concentrations of homogentisic acid in the range from 0.1 to 100 micrograms/ml were investigated and a cytotoxic effect was detected with concentrations of 5 micrograms/ml and above. Thus, with adult articular chondrocytes this effect was seen between 36 and 48 h of subculture with 5 micrograms/ml or more of homogentisic acid present from the beginning of subculture. In fetal articular chondrocyte cultures a concentration of 1 microgram/ml elicited a statistically significant reduction (13%) in cell proliferation without altering sulphated macromolecular synthesis. Experiments with 3H-thymidine autoradiography to measure the pulse labeling index (LI) in fetal chondrocytes in vitro showed that the 5 micrograms/ml concentration of homogentisic acid present for 21 h from the beginning of subculture gave a LI of 2%, compared with 25% for controls. Thus, homogentisic acid can reduce chondrocyte proliferative capacity before morphological alterations can be detected by light microscopy. No significant alterations in the synthesis of proteoglycans were detected at concentrations below the cytotoxic level. The homogentisic acid-induced cytotoxicity and reduction of proliferation in chondrocytes represents a possible pathway by which cartilage is damaged in ochronotic arthropathy.     

Effects of phenytoin on acetylcholinesterase activity and cell protein in cultured chick embryonic skeletal muscle

Cultured pectoral muscle from 11-day-old chick embryos was treated for 48 h with phenytoin (diphenylhydantoin, DPH) in concentrations ranging from 15 to 270 microgram/ml on days 7-9 in vitro. Acetylcholinesterase (AChE, EC 3.1.1.7), creatine phosphokinase (CPK, EC 2.7.3.2), and lactic dehydrogenase (LDH, EC 1.1.1.27) activities, [3H]leucine incorporation into protein, and total protein of the cultures decreased in a dose-related manner with DPH concentrations of 30 microgram/ml and greater. Total AChE activity and AChE activity released into the medium were specifically decreased with 15 microgram DPH per millilitre. In cultures treated chronically with 15 microgram DPH per millilitre on days 5-13 in vitro, total AChE activity and AChE activity released into the medium were 66.0 +/- 13.2 and 64.7 +/- 11.8% of untreated controls, respectively, but cellular AChE activity, cell protein, and [3H]leucine incorporation into protein were unaffected. The results indicate that DPH specifically decreases the total net synthesis of AChE activity by a direct action on cultured chick embryo muscle.