Transplacental IgG Subclass Concentrations in Pregnancies at Risk of Haemolytic Disease of the Newborn

The relationship of haemolytic disease of the newborn (HDN) to the transplacental passage of the four IgG subclasses was assessed at varous gestational ages by comparing the maternal and fetal IgG subclass concentrations in 34 pregnancies at risk of HDN with those in 30 pregnancies not at risk. Higher maternal and fetal IgG1 levels were attained in pregnancies at risk of HDN than in pregnancies not at risk. In contrast, a slight decrease in maternal IgG2 and IgG4 levels occurred in pregnancies at risk of HDN, as compared with a slight rise in maternal IgG2 and IgG4 levels in pregnancies not at risk of HDN. Changes in fetal IgG2 and 4 concentrations in either type of pregnancy were very similar, showing only slight increases between the 19th and 34th week of gestation. A slight decrease in maternal IgG3 occurred in both types of pregnancy. In contrast, higher and fairly steady levels of fetal IgG3 were observed in fetuses not at risk of HDN throughout gestation, when compared with those in 'at risk' pregnancies. However, the statistical reliability of these results is not clear since only small numbers of samples were tested and because wide variations in IgG concentrations were observed. The IgG subclass concentrations in 50 paired maternal and cord blood samples were also measured and revealed that IgG1 levels were substantially higher in cord rather than maternal blood; cord and maternal IgG2, 3 and 4 levels, on the other hand, were fairly similar.     

Acid-base changes in milk and blood of rats in acidosis and alkalosis.

Lactating white rats (Rattus norvegicus) were subjected to metabolic and respiratory acidosis and metabolic alkalosis. Before and during the various treatments, the acid-base status of heart blood and milk was determined. Acute metabolic acidosis lowered the pH of plasma and milk; Pco(2) and bicarbonate concentrations in plasma were lowered, and in milk Pco(2) was raised and the bicarbonate concentration remained unchanged. Respiratory acidosis and acetazolamide caused a drop in blood pH and in blood and milk bicarbonate concentrations; milk pH remained unchanged, but Pco(2) was raised in both plasma and milk. Acute metabolic alkalosis raised the blood pH and milk Pco(2); plasma Pco(2) and bicarbonate concentrations in blood and milk remained unchanged. The data show that greater changes occur in acid-base parameters of blood than milk when animals are exposed to acidifying and alkalinizing stimuli.     

The effects of adenosine on transepithelial resistance and sodium uptake in the inner medullary collecting duct

It has been previously demonstrated that adenosine induces natriuresis when administered directly into the renal circulation of the rat. It was postulated that the mechanism was inhibition of tubule Na⁺ reabsorption. In the current study, the hypothesis was tested that adenosine inhibits ion reabsorption across the inner medullary collecting duct (IMCD), a tubule segment which is rich in adenosine receptors. IMCD epithelium from rat kidney was grown in primary culture as a confluent monolayer on Costar filters, allowing selective access to the basolateral and apical surfaces of the cells. Transepithelial resistance was taken as a measure of epithelial permeability and ion conductance. Na⁺ uptake was studied using ²²Na⁺ and used to determine the permeability of the epithelial monolayer specifically to Na⁺. Exposure of the basolateral aspect of the monolayer to adenosine (10⁻⁸–10⁻⁷ M) increased transepithelial resistance in a dose- and time-dependent manner; in parallel, adenosine (10⁻⁷–10⁻⁶ M) reduced apical Na⁺ uptake from 20±5 to 10±2 nmol/cm². 1,3-Dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX, 5×10⁻⁹ M), an adenosine antagonist with selectivity for the A₁ receptor, inhibited the rise in transepithelial resistance and the decrease in Na⁺ uptake following the addition of adenosine. The effects of adenosine on transepithelial resistance were reproduced with the A₁ receptor selective adenosine analogue N ⁶-cyclohexyladenosine (CHA, 10⁻⁸ –10⁻⁷ M) but not with the A₂ selective analogues, 5′-N-ethylcarboxamidoadenosine (NECA) or CGS 21680. CHA (10⁻⁷ M) inhibited apical Na⁺ uptake by 50%, an effect abolished by PACPX. The effects of adenosine on transepithelial resistance and Na⁺ uptake were inhibited, but only in part, by amiloride. These data suggest that adenosine inhibits ion movement, specifically apical Na⁺ uptake, across the IMCD epithelium and that this effect is mediated by A₁ receptors from the basolateral aspect of the cell. The results are consistent with the hypothesis that adenosine inhibits Na⁺ reabsorption across the IMCD.     

Ras gene product expression in blood and marrow smears of patients with acute leukemia: importance of fixation

Activation of ras protooncogenes by any of several possible mutations in codons 12, 13 or 61 has been demonstrated in a variety of human malignancies, including acute non-lymphoblastic leukemia (ANLL). In situ staining for the ras gene product, p21, has been demonstrated in carcinomas of several sites. High levels of p21 expression have been associated with histologic anaplasia in prostate cancer and regional lymph node metastasis in breast cancer. We examined 16 marrow aspirates and blood smears from patients with acute leukemia, predominantly ANLL, and eight controls. Marrow aspirates or blood were smeared on glass slides and fixed immediately in 10% buffered formalin. p21 was examined with avidin-biotin linked immunoperoxidase visualization. Particular attention must be paid to antibody selection and fixation protocol to demonstrate p21, owing to its rapid degradation ex vivo. Three of 16 patients exhibited occasional high p21 expression primarily in leukemic blasts, but in no case were more than 10% of blast cells positive. Normal reticuloendothelial and myeloid cells occasionally exhibited mild to moderately heavy staining, but megakaryocytes, erythroid precursors, lymphocytes and plasma cells were consistently negative. Most patients, 5 normal volunteers and 3 patients with non-malignant disease, exhibited no reactivity, or only a faint blush. These data suggest that while point mutation and concomitant activation of c-N-ras occurs regularly in ANLL, high levels of ras p21 expression are rarely found with this technique.     

A benign congenital myopathy in an inbred Samaritan family.

We describe a novel form of myopathy in a mother and her two daughters from an inbred Samaritan family. The patients displayed severe neonatal hypotonia, lethargy and dysmorphic features. Motor milestones were delayed; however, the hypotonia and muscle weakness gradually improved during the first 2 years of life and independent walking was achieved by 18 months. The mother at the age of 23 years shows myopathic facies and minimal proximal weakness. Her intelligence is normal. Her muscle biopsy revealed central nuclei and disruption of the intermyofibrillary network with moth eaten and spiral fibers. Mutations in SMN, MTM1 and the myotonic dystrophy genes were excluded. We suggest this is a new benign form of congenital myopathy. Inheritance is probably autosomal recessive.     

Stability and specificity of heterodimer formation for the coiled‐coil neck regions of the motor proteins Kif3A and Kif3B: the role of unstructured oppositely charged regions

Abstract: We investigated the folding, stability, and specificity of dimerization of the neck regions of the kinesin‐like proteins Kif3A (residues 356–416) and Kif3B (residues 351–411). We showed that the complementary charged regions found in the hinge regions (which directly follow the neck regions) of these proteins do not adopt any secondary structure in solution. We then explored the ability of the complementary charged regions to specify heterodimer formation for the neck region coiled‐coils found in Kif3A and Kif3B. Redox experiments demonstrated that oppositely charged regions specified the formation of a heterodimeric coiled‐coil. Denaturation studies with urea demonstrated that the negatively charged region of Kif3A dramatically destabilized its neck coiled‐coil (urea_1/2 value of 3.9 m compared with 6.7 m for the coiled‐coil alone). By comparison, the placement of a positively charged region C‐terminal to the neck coiled‐coil of Kif3B had little effect on stability (urea_1/2 value of 8.2 m compared with 8.8 m for the coiled‐coil alone). The pairing of complementary charged regions leads to specific heterodimer formation where the stability of the heterodimeric neck coiled‐coil with charged regions had similar stability (urea_1/2 value of 7.8 m ) to the most stable homodimer (Kif3B) with charged regions (urea_1/2 value of 8.0 m ) and dramatically more stable than the Kif3A homodimer with charged regions (urea_1/2, value of 3.9 m ). The heterodimeric coiled‐coil with charged extensions has essentially the same stability as the heterodimeric coiled‐coil on its own (urea_1/2 values of 7.8 and 8.1 m , respectively) suggesting that specificity of heterodimerization is driven by non‐specific attraction of the oppositely unstructured charged regions without affecting stability of the heterodimeric coiled‐coil.     

High performance liquid chromatographic analysis of insulin degradation products from a cultured kidney cell line

The kidney is a major site for insulin removal and degradation, but the subcellular processes and enzymes involved have not been established. We have examined this process by analyzing insulin degradation products by HPLC. Monoiodoinsulin specifically labeled on either the A14 or B26 tyrosine residue was incubated with a cultured kidney epithelial cell line, and both intracellular and extracellular products were examined on HPLC. The products were then compared with products of known structure generated by hepatocytes and the enzyme insulin protease. Intracellular and extracellular products were different, suggesting two different degradative pathways, as previously shown in liver. The extracellular degradation products eluted from HPLC both before and after sulfitolysis similarly with hepatocyte products and products generated by insulin protease. The intracellular products also eluted identically with hepatocyte products. Based on comparisons with identified products, the kidney cell generates two fragments from the A chain of intact insulin, one with a cleavage at A13-A14 and the other at A14-A15. The B chain of intact insulin is cleaved in a number of different sites, resulting in peptides that elute identically with B chain peptides cleaved at B9-B10, B13-B14, B16-B17, B24-B25, and B25-B26. These similarities with hepatocytes and insulin protease suggest that liver and kidney have similar mechanisms for insulin degradation and that insulin protease or a very similar enzyme is involved in both tissues.