Diurnal variation and age-related changes of bone turnover markers in female Göttingen minipigs.

We investigated diurnal variation and age-related changes in bone turnover markers in female Gottingen minipigs. Ten females, 6-9 months of age, were used for confirmation of diurnal variation. Blood was collected at 3 h intervals for 24 h, and bone-specific alkaline phosphatase and intact osteocalcin (OC) levels were determined by enzyme immunoassay and radioimmunoassay, respectively. Urine was collected at 3 h intervals for 24 h using a tray attached to the bottom of the cage. The levels of N-terminal telopeptide of type I collagen (NTX) were determined by enzyme immunoassay. Pyridinoline and deoxypyridinoline were measured by high performance liquid chromatography. OC and NTX exhibited diurnal variation (Kruskal-Wallis test, P < 0.05), with the highest and lowest levels at 18:00 h (76.7 +/- 26.2 ng/ml) and 06:00 h (44.3 +/- 10.3 ng/ml), and at 03:00-05:59 h (550.4 +/- 82.4 nmol/micromol Cr) and 12:00-14:59 h (297.8 +/- 152.5 nmol/micromol Cr), respectively. In the study of age-related changes, blood and urine samples from 66 females (age range, 3-76 months) were examined to determine the bone turnover markers. All markers showed high correlations with age (0.569 < R(2) < 0.818). High levels of bone turnover markers were observed in young animals, decreasing with age (Kruskal-Wallis test, P < 0.01). The diurnal variation and age-related changes revealed in the present study will be useful in studies of bone diseases using female Gottingen minipigs.

Overexpression of lipoprotein lipase improves insulin resistance induced by a high-fat diet in transgenic rabbits.

AIMS/HYPOTHESIS: Dysfunctions of lipoprotein lipase (LPL) have been found to be associated with dyslipidaemias, atherosclerosis, obesity and insulin resistance. There are two conflicting hypotheses regarding the roles of LPL in glucose metabolism and insulin resistance. Whether systemically increased LPL activity would be beneficial or detrimental to insulin sensitivity is yet to be resolved. To address this issue, we studied transgenic rabbits overexpressing human LPL transgene. METHODS: LPL transgenic and control rabbits were fed a 10% high-fat diet (HFD) for 16 weeks. To evaluate glucose metabolism, we compared plasma levels of glucose and insulin in transgenic rabbits with control rabbits and performed an intravenous glucose tolerance test. In addition, we measured adipose tissue accumulation in HFD-fed rabbits. RESULTS: Increased LPL activity in transgenic rabbits resulted in a significant reduction of plasma triglycerides and non-esterified fatty acids, but not in basal levels of glucose and insulin. HFD feeding induced an elevation of plasma glucose levels accompanied by hyperinsulinaemia in control rabbits, but was significantly inhibited in transgenic rabbits. The intravenous glucose tolerance test showed that transgenic rabbits had faster glucose clearance associated with lower levels of insulin secretion than control rabbits. In addition, there was a significant reduction of body adipose tissue in transgenic rabbits compared with in control rabbits fed an HFD. Scanning electron microscopic examination revealed that adipocytes in transgenic rabbits were predominately small cells. CONCLUSIONS/INTERPRETATION: Our results showed that systemically increased LPL activity improves insulin resistance and reduces adipose accumulation in transgenic rabbits, indicating that systemic elevation of LPL may have potential benefits for the treatment of insulin resistance and obesity.

Distribution and enzyme histochemical characterisation of mast cells in cats.

Mast cells from 15 different cat organs were examined in terms of distribution and protease activity. The number of mast cells in each site was found to vary when visualised by metachromatic staining using Alcian Blue. Enzyme histochemical analysis revealed the existence of two subtypes of mast cells. These were categorised based on protease content, i.e. whether the mast cells contained chymase or tryptase. Tryptase-positive mast cells were clearly identifiable in every organ examined, whereas chymase-containing mast cells were predominantly observed in the ear (skin), tongue, spleen, and submucosa of the stomach and rectum. The chymase-reactive cells were not detected in the heart, or in the muscularis or serosa of the duodenum, jejunum, ileum or rectum. In addition, we suggest the existence of another subtype of mast cell containing both chymase and tryptase and localised within the ear (skin), tongue, spleen and submucosa of the rectum.

Location of haemagglutinin in bacterial cells of Fusobacterium necrophorum subsp. necrophorum.

The location of haemagglutinin (HA) of Fusobacterium necrophorum subsp. necrophorum VPI 2891 strain was investigated by immunofluorescence, confocal laser scan microscopy and immunoelectron microscopy. The immunofluorescence study demonstrated the fluorescence specific for the HA on the bacterial cells and confocal laser scan microscopy indicated similar fluorescence around the cross section of the bacterial cell. The immunoelectron microscopic study also revealed that the protein A-gold conjugates were located around the bacterial surfaces. These findings suggest that HA is one of the components of the cell surfaces of F. necrophorum subsp, necrophorum.

No effect of voluntary exercise on ovarian follicle in rats.

The present study was to clarify the relationship between voluntary exercise and follicular growth or ovulation. Rats kept for 4 weeks in a rotating drum with free access to the wheel, food and water ran 2-12 km per day. The number of ova shed after superovulation treatments and the number of non-atretic follicles were not influenced by voluntary exercise. These experiments demonstrate that spontaneous voluntary exercise does not affect either the number of ova shed or the number of non-atretic follicles in superovulating rats or control rats.

Establishment and characterization of a new canine B-cell leukemia cell line.

A new cell line derived from a spontaneous canine leukemia was established and designated GL-1. The cells have been cultured in a floating fashion and passaged for over two years. They were round with rich cytoplasm containing many rough endoplasmic reticula and mitochondria. Peroxidase staining was negative. The nuclei of many cells were round, but segmented nuclei were seen frequently. The doubling time of the cells was 27.3 hr and they had 78 chromosomes. Surface marker analysis using monoclonal antibodies (MABs) and flowcytometry revealed that GL-1 possessed CD45 and surface IgG. However, the cells did not react with MABs detecting T-cell markers. These results indicate that GL-1 has a lymphocytic lineage and is derived from a B-cell leukemia.

Differential freezing tolerance of rat pancreatic islets depending on their size variation.

We have investigated the freezing tolerance of rat pancreatic islets. Freshly isolated rat pancreatic islets were divided into three groups based on their longest diameter (small; 100 - 200 microns, medium; 201 - 300 microns, large; > 300 microns). They were then cryopreserved at a slow cooling rate (-0.3 degrees C/min) in the presence of dimethyl sulfoxide (Me2SO) or ethylene glycol (EG). After storage at -196 degrees C for 1 - 4 weeks, they were thawed and their ability to secrete insulin in response to fluctuations in glucose concentration was examined during three consecutive static incubations in vitro (1st; 2.8 mM, 2nd; 16.7 mM, 3rd; 2.8 mM). Morphological examination of the beta-granule population was determined by image analysis, and correlation with islets size was analyzed. The amount of insulin released from large-sized islets was significantly suppressed in EG (p < 0.05) and Me2SO (p < 0.01) groups compared to unfrozen islets. However, the mean volume of the large-sized islets isolated from one rat accounted for 43.0% of the total volume. On the other hand, the amount of insulin released from small- and medium-sized islets did not differ from those of unfrozen islets, and their mean volumes were 13.2 and 43.8% respectively. The percentage of cells with beta-granules was significantly correlated with size in both EG (r = -0.52) and Me2SO (r = -0.35) groups, but no significant correlation was observed in the unfrozen islets groups. These findings suggest that large-sized islets are more susceptible to freezing injury than small- or medium-sized islets. Moreover, the volume distribution of isolated islets indicated that it may be important to retain the ability of insulin secretion from the large-sized islets.

Flow cytometric analysis and immunohistochemistry of delayed-type hypersensitivity responses in mice immunized with rat hepatocytes.

Delayed-type hypersensitivity (DTH) responses to rat hepatocytes (HCs) in mice were investigated by flow cytometric analysis and immunohistochemistry with monoclonal antibodies directed against murine class II, CD4, and CD8 antigens. Mice were immunized subcutaneously (s.c.) with 10(6) rat HCs (referred to as s.c.-immunized mice), and control mice were injected s.c. with sterile Hanks' solution (non-immunized mice). Four days later, 10(5) rat HCs were injected into the footpad of s.c.-immunized mice and non-immunized mice. The DTH response in s.c.-immunized mice significantly increased after challenge when compared to that in non-immunized mice. The numbers of class II+, CD4+, and CD8+ cells in the footpad, and CD4+ and CD8+ cells in the inguinal lymph node of s.c.-immunized mice significantly increased during the DTH response. An increase in the number of CD4+ cells in the footpad of s.c.-immunized mice after challenge was more significant than that of non-immunized mice. The number of CD4+ cells increased more markedly in the footpad of s.c.-immunized mice as compared to that of CD8+ cells. Furthermore, immunohistochemical studies of the footpad of s.c.-immunized mice showed more severe infiltration of CD4+ cells rather than CD8+ cells at the injection site of rat HCs. These results suggest that the DTH response in the footpad of mice immunized with rat HCs is associated with severe infiltration of CD4+ cells.

Implication of activation of intraperitoneal macrophages with bio-degradable microspheres.

To estimate the possibility that biological degradable starch microspheres (DMS) activate abdominal or intraperitoneal macrophages (IMP), two sizes of DMS (Spherex, Pharmacia, Sweden) were injected into the peritoneum of the ICR mice of 4 to 8 weeks of age. Three days after the injection, peritoneal fluid was collected and incubated for one hour at 37 degrees C under 5% CO2. The cells which adhered to the petri dish were IMP, to which DMS was added for 18 hrs. The cultured IMP were observed by scanning electron microscope (SEM) and the ratio of the active type to the total number of IMP was counted as an index of the effect of DMS to IMP. The activation effect of DMS on the incubated IMP was significant in the group which was cultured with 2 microns DMS after the 45 microns DMS injection. That indicated the possible DMS function as a potential IMP activating factor (MAF).

Quantitative and morphological studies on the influence of zinc deficiency on the liver of pregnant rats.

The effect of zinc deficiency on trace metals in the liver, spleen, kidney, pancreas and duodenum was investigated in the control and zinc-deficient rats at 17 days and 20 days of pregnancy. Zinc-deficient rats fell into limosis after 5 days of pregnancy. The contents of zinc, iron, copper and manganese in the maternal tissues were measured by colorimetry with 5NPPF. The morphological changes of the liver were observed by light and electron microscopy. The contents of zinc in the pancreas and duodenum were less in the zinc-deficient group than in the control at both 17 days and 20 days of pregnancy. The contents of the copper and manganese in the liver, kidney, pancreas, duodenum and spleen, however, were not significantly different from the control. The contents of iron in the liver, spleen and kidney in the deficient group increased greatly at 17 days and 20 days of pregnancy compared with the control group. The combination rate of transferrin with iron also increased significantly in the deficient group. Staining with Berlin blue or Turnbull's blue showed intense reaction to iron around the interlobular connective tissue of the liver in the zinc deficient group at 20 days of pregnancy. Ultrastructurally, the liver of the zinc-deficient rats showed the decrease of glycogen granules and increase of lipid-like granules and lysosomes with various sizes and electron densities. These findings suggest that zinc deficiency causes the increase of iron contents in the various organs during pregnancy, and that there is an intimate interrelationship between zinc and iron in the metabolism of iron during pregnancy.

Parasitological observations of chronic diarrhoea in suspected AIDS adult patients in Kinshasa (Zaire).

The parasitological profile of chronic diarrhoea in 46 Zairian adults suspected of Aids demonstrated that the frequency of protozoa was five times higher than that of helminths; 86% of the protozoa were sporozoa: Isospora belli was the most frequent (19%), followed by Cryptosporidium isolated for the first time in Zaire (8%) and Blastocystis hominis (2%). 37 of the 46 patients were immunodeficient.

PIP: Chronic diarrhea occurs in close to 90% of acquired immunodeficiency syndrome (AIDS) cases Zaire, but there has been no systematic analysis of the parasite species. In the present study, 46 adults with a history of episodic diarrhea (mean duration, 18 months) and clinical symptoms suggestive of AIDS underwent fecal diagnosis. In the 19 stool samples that were positive for parasites, protozoa were 5 times more common than helminths. 13 (86%) of the 15 protozoa-positive samples contained sporozoa. Of these, Isospora belli was the most common (19%), followed by Cryptosporidium (8%). Laboratory analysis and clinical symptoms revealed that 37 of these 46 chronic diarrhea patients were, in fact, immunosuppressed. Of the 9 immunocompetent patients, 5 had parasite- free stools. Significant was the finding that all 4 patients with cryptosporidiosis and 8 of the 9 patients with isosporosis were immunodeficient. Overall, these findings suggest that the parasitological profile of chronic diarrhea differs between immunosuppressed and immunocompetent African patients.