Class I and class II restriction pattern polymorphisms associated with independently derived RT1 haplotypes in inbred rats

This communication reports the DNA level identification of class I and class II sequences associated with 20 RT1 haplotypes which have been assigned previously to eight RT1 groups. Sixteen to 22 bands in genomic blots hybridized with the mouse pH-2III class I cDNA probe. Only the three RT1 ^khaplotypes associated with identical class I restriction fragment patterns. Differences in restriction bands between putatively identical RT1 haplotypes were either less than or equal to 6%, or greater than 50%, suggesting a relatively high level of recombination between serologically identified RT1.A genes and the majority of class I sequences. Restriction fragment patterns associated with three RT1 ^uhaplotypes differed by less than 6%. However, intra-RT1 ^a,intra-RT1 ^b,and intra-RT1 ^lrestriction fragment differences were between 50 and 64%. In specific cases, different RT1 haplotypes associated with identical class I restriction patterns, e.g., RT1 ^m(MNR) and RT1 ^d(MR); higher resolution confirmed the difference (two bands) between RT1 ^mand RT1 ^d.Results of hybridization with the human DC₁ probe confirmed that the AVN RT1 ^aand NSD RT1 ^bhaplotypes were generated by recombinations within the vicinity of the RT1.B : RT1.D regions. These results demonstrate that a previous classification of RT1 haplotypes was incomplete and did not include the majority of class I and class II sequences which distinguish RT1 haplotypes.     

Fatty-acid desaturation and microsomal lipid fatty-acid composition in experimental hyperthyroidism.

We have studied the influence of experimental hyperthyroidism in the rat on the synthesis of unsaturated fatty acids and on liver microsomal lipid fatty-acid composition. Tri-iodothyronine treatment (25 micrograms/100 g body weight) daily for 3 weeks caused no significant changes in delta 9 (stearate) desaturation but a 24% decrease in delta 6 (linoleate) desaturation. Much larger doses of tri-iodothyronine increased delta 9 desaturation. Liver microsomal fatty-acid composition in hyperthyroidism is altered with significantly increased proportions of stearate and arachidonate and decreased proportions of palmitate, palmitoleate, linoleate (C18:2) and eicosa-8,11,14-trienoate (C20:3). These changes, other than the decreases proportion of C20:3 fatty acid, which may be due to the diminished delta 6 desaturase activity, cannot be attributed to changes in fatty-acid desaturation. Most of these changes were also found to be due not simply to the decreased weight gain or the increased food intake of the hyperthyroid animals. Only the decreased C18:2 fatty-acid proportions could be mimicked by restricting food intake of control animals and none of the changes were prevented by restricting food intake of hyperthyroid animals. Thus most of the changes in microsomal lipid fatty-acid composition are likely to be due to a thyroid hormone effect on peripheral lipid mobilization or lipid degradation.     

Aberrant Pregnancy Adaptations in the Peripheral Immune Response in Type 1 Diabetes: A Rat Model

Introduction

Despite tight glycemic control, pregnancy complication rate in type 1 diabetes patients is higher than in normal pregnancy. Other etiological factors may be responsible for the development of adverse pregnancy outcome. Acceptance of the semi-allogeneic fetus is accompanied by adaptations in the maternal immune-response. Maladaptations of the immune-response has been shown to contribute to pregnancy complications. We hypothesized that type 1 diabetes, as an autoimmune disease, may be associated with maladaptations of the immune-response to pregnancy, possibly resulting in pregnancy complications.

Methods

We studied pregnancy outcome and pregnancy-induced immunological adaptations in a normoglycemic rat-model of type 1 diabetes, i.e. biobreeding diabetes-prone rats (BBDP; 5 non-pregnant rats, 7 pregnant day 10 rats and 6 pregnant day 18 rats) , versus non-diabetic control rats (i.e. congenic non-diabetic biobreeding diabetes-resistant (BBDR; 6 non-pregnant rats, 6 pregnant day 10 rats and 6 pregnant day 18 rats) and Wistar-rats (6 non-pregnant, 6 pregnant day 10 rats and 5 pregnant day 18 rats)).

Results

We observed reduced litter size, lower fetal weight of viable fetuses and increased numbers of resorptions versus control rats. These complications are accompanied by various differences in the immune-response between BBDP and control rats in both pregnant and non-pregnant animals. The immune-response in non-pregnant BBDP-rats was characterized by decreased percentages of lymphocytes, increased percentages of effector T-cells, regulatory T-cells and natural killer cells, an increased Th1/Th2-ratio and activated monocytes versus Wistar and BBDR-rats. Furthermore, pregnancy-induced adaptations in BBDP-rats coincided with an increased Th1/Th2-ratio, a decreased mean fluorescence intensity CD161a/NKR-P1b ratio and no further activation of monocytes versus non-diabetic control rats.

Conclusion

This study suggests that even in the face of strict normoglycemia, pregnancy complications still occur in type 1 diabetic pregnancies. This adverse pregnancy outcome may be related to the aberrant immunological adaptations to pregnancy in diabetic rats.     

Enzymes for Pancreatic Islet Isolation Impact Chemokine-Production and Polarization of Insulin-Producing β-Cells with Reduced Functional Survival of Immunoisolated Rat Islet-Allografts as a Consequence

The primary aim of this study was to determine whether normal variations in enzyme-activities of collagenases applied for rat-islet isolation impact longevity of encapsulated islet grafts. Also we studied the functional and immunological properties of rat islets isolated with different enzyme preparations to determine whether this impacts these parameters. Rat-islets were isolated from the pancreas with two different collagenases with commonly accepted collagenase, neutral protease, and clostripain activities. Islets had a similar and acceptable glucose-induced insulin-release profile but a profound statistical significant difference in production of the chemokines IP-10 and Gro-α. The islets were studied with nanotomy which is an EM-based technology for unbiased study of ultrastructural features of islets such as cell-cell contacts, endocrine-cell condition, ER stress, mitochondrial conditions, and cell polarization. The islet-batch with higher chemokine-production had a lower amount of polarized insulin-producing β-cells. All islets had more intercellular spaces and less interconnected areas with tight cell-cell junctions when compared to islets in the pancreas. Islet-graft function was studied by implanting encapsulated and free islet grafts in rat recipients. Alginate-based encapsulated grafts isolated with the enzyme-lot inducing higher chemokine production and lower polarization survived for a two-fold shorter period of time. The lower survival-time of the encapsulated grafts was correlated with a higher influx of inflammatory cells at 7 days after implantation. Islets from the same two batches transplanted as free unencapsulated-graft, did not show any difference in survival or function in vivo. Lack of insight in factors contributing to the current lab-to-lab variation in longevity of encapsulated islet-grafts is considered to be a threat for clinical application. Our data suggest that seemingly minor variations in activity of enzymes applied for islet-isolation might contribute to longevity-variations of immunoisolated islet-grafts.     

Kinetic properties of DM-nitrophen binding to calcium and magnesium

Caged-Ca(2+) compounds such as nitrophenyl-EGTA (NP-EGTA) and DM-nitrophen (DMn) are extremely useful in biological research, but their use in live cells is hampered by cytoplasmic [Mg(2+)]. We determined the properties of Ca(2+) release from NP-EGTA and DMn by using Oregon green BAPTA-5N to measure changes in [Ca(2+)] after ultraviolet flash photolysis in vitro, with or without Mg(2+) present. A large fraction (65%) of NP-EGTA, which has a negligible Mg(2+) affinity, uncages with a time constant of 10.3 ms, resulting in relatively slow increases in [Ca(2+)]. Uncaging of DMn is considerably faster, but DMn has a significant affinity for Mg(2+) to complicate the uncaging process. With experimentally determined values for the Ca(2+) and Mg(2+) binding/unbinding rates of DMn and NP-EGTA, we built a mathematical model to assess the utility of NP-EGTA and DMn in rapid Ca(2+)-uncaging experiments in the presence of Mg(2+). We discuss the advantages and disadvantages of using each compound under different conditions. To determine the kinetics of Ca(2+) binding to biologically relevant Ca(2+) buffers, such as Ca(2+)-binding proteins, the use of DMn is preferable even in the presence of Mg(2+).     

Early stimulation of rat liver microsomal protein synthesis after tri-iodothyronine injection in vivo.

In an effort to determine the physiological significance of previous studies showing stimulation of microsomal protein synthesis by thyroxine added in vitro, an early effect of tri-iodothyronine injected in vivo was sought. Tri-iodothyronine (25 micrograms/100 g) administered to euthyroid rats stimulated microsomal protein synthesis in vitro within 3--6 h. This effect occurred much earlier than the 26 h lag previously reported after tri-iodothyronine administration to hypothyroid rats. This early effect of tri-iodothyronine on protein synthesis is prevented by alpha-amanitin, suggesting that it is dependent on RNA synthesis. The failure to find a direct effect in vivo of tri-iodothyronine on translation casts doubt on the physiological significance of previous studies that have shown a direct stimulation of translation by thyroxine added in vitro.     

Binding of antigen in the absence of histocompatibility proteins by arsonate-reactive T-cell clones

Inducer T-cell clones reactive to the p-azobenzenearsonate (arsonate) hapten possess binding sites for radioactive arsanylated proteins, which are not present on clones with other antigen specificities. Binding occurred in the absence of histocompatibility proteins. Binding was specific for the p-azobenzenearsonate hapten, since unconjugated proteins and proteins conjugated to the nonactivating o-azobenzenearsonate hapten neither bound to the clones nor competed binding of radioactive antigen. One of the clones was studied in more detail, using a panel of structural analogs of arsonate conjugated to the carrier protein ovalbumin. All conjugates that activated the clone in the presence of antigen-presenting cells also competed binding of radioactive antigen in the absence of antigen-presenting cells. Nonactivating conjugates did not compete binding. Based on evidence in this and the succeeding paper (Rao et al., accompanying paper), we suggest that these arsonate-binding sites may include the physiological antigen receptors of arsonate-reactive T-cell clones.     

Effects of piperine analogues on stimulation of melanocyte proliferation and melanocyte differentiation

A wide range of piperine analogues has been synthesised in order to undertake a structure-activity study of their ability to stimulate melanocyte proliferation. Results demonstrate that an aromatic ring containing at least one ether function and a carbonyl group containing side chain is essential for this activity. A number of highly active piperine analogues have been identified, for instance 1-(3,4-methylenedioxyphenyl)-penta-2E,4E-dienoic acid methyl ester (5a), 1-E,E-piperinoyl-isobutylamine (4f) and 1-(3,4-methylenedioxyphenyl)-pentanoic acid cyclohexyl amide (20). A selection of analogues has also been evaluated for their effect on melanocyte morphology and melanogenesis. The piperine analogues altered cell morphology by increasing dendrite formation leading to bi-, tri- and quadripolar cells. These same analogues were found to increase total melanin in cell cultures, although melanin content per cell was not significantly altered from control in the presence of these compounds.