Recessive dystrophic epidermolysis bullosa associated with mesangioproliferative glomerulonephritis and multifocal necrotizing leucoencephalopathy of the pons.

We report a woman with recessive dystrophic epidermolysis bullosa (RDEB) in whom there was prolonged sepsis and death at age 22 years. Autopsy revealed multiple epidermolytic skin lesions with chronic ulceration, mesangioproliferative glomerulonephritis and multifocal necrotizing leucoencephalopathy (MNL) of the pons. The latter two conditions may have been mediated by sepsis-associated cytokines. Although mesangioproliferative glomerulonephritis has previously been described in association with RDEB, to our knowledge this is the first report of MNL in a patient with RDEB.

Five cases of calciphylaxis and a review of the literature.

Calciphylaxis is a rare phenomenon of cutaneous necrosis that typically occurs in association with renal failure and has a poor prognosis. We report 5 new cases of calciphylaxis that illustrate the important clinical and histopathologic features of the disease. All patients had end-stage renal failure at the time that purpuric plaques and nodules were noted; these subsequently progressed to necrotic ulcers with eschars. All skin biopsy specimens showed varying degrees of calcification of the medial layer of blood vessel walls in the dermis and subcutaneous fat. Neither the product of serum calcium and phosphorus concentrations nor parathyroid hormone levels correlated temporally with the clinical observations in every case, emphasizing the importance of clinical-histopathologic correlation. Although certain features of calciphylaxis in humans resemble the animal model originally proposed, there are also some crucial differences. We review the pathogenesis, epidemiology, clinical and histopathologic features, and treatment of this disease.

LAD-1 is absent in a subset of junctional epidermolysis bullosa patients.

The anchoring filament protein LAD-1 has been recently identified as the target of autoantibodies in the acquired blistering disorder linear IgA bullous dermatosis. Because this protein appears to be involved in the process of dermal-epidermal cohesion, this study sought to determine the involvement of LAD-1 in the pathology of junctional epidermolysis bullosa (JEB). To this end, 44 patients with a variety of subtypes of JEB were analyzed by indirect immunofluorescence microscopy with antibodies to LAD-1, BP180, and laminin-5. We found that only patients with generalized atrophic benign epidermolysis bullosa (GABEB) contained LAD-1 defects. Of the 16 GABEB patients studied, 13 showed absent or greatly reduced expression of LAD-1 (including 2 patients with a peculiar interrupted staining pattern) and 3 patients showed defects of laminin-5 expression with normal LAD-1 expression. Patients who showed LAD-1 defects also showed abnormal expression of BP180. Keratinocytes were cultured from the skin of two GABEB patients and analyzed by indirect immunofluorescent microscopy. One culture demonstrated defects of BP180 and LAD-1 expression (which was also verified by radioimmunoprecipitation assay), and one culture showed decreased laminin-5 expression but normal BP180 and LAD-1 expression. Thus, these studies demonstrate that: (i) LAD-1 and BP180 are normally expressed in all subtypes of JEB except GABEB, (ii) the majority of GABEB patients show absent or near absent expression of both LAD-1 and BP180 but normal expression of laminin-5, and (iii) a smaller subset of GABEB patients show normal LAD-1 and BP180 expression but express persistent but reduced levels of laminin-5.

Beyond survival: children's growth for national development.

PIP: The international community, in collaboration with national governments, is making strides towards saving children's lives. These critically important survival interventions rescue children; they constitute essential but insufficient action. The major challenge of the future is to move beyond survival. Poor children saved from the clutches of death face undernutrition, high morbidity and deprived environments--forces that rob the children of their growth potential. They become physically and mentally less productive and are more likely to perpetuate the poor growth syndrome in the next generation. Beyond survival lies the pursuit of growth. The task is to unlock children's potential. 40% of the world's children under 5 years of age, 141 million, are chronically undernourished. The concept that growth-retarded children are stunted but healthy and therefore need no attention is false; for these children and their countries, small is unhealthy. Growth promotion strategies can lead to stronger, smarter and healthier children and adults. A country's human capital is enhanced. Investing in children's growth accelerates national development. The primary purpose of the paper is to set forth the economic rationale that will motivate governments to undertake growth promotion strategies. The main humanitarian and political arguments are also indicated.^ieng

Human keratinocytes contain mRNA indistinguishable from monocyte interleukin 1 alpha and beta mRNA. Keratinocyte epidermal cell-derived thymocyte-activating factor is identical to interleukin 1.

Keratinocytes produce an IL-1 like factor termed epidermal cell-derived thymocyte-activating factor (ETAF). In this study, we show that ETAF and IL-1 are identical by the following criteria: Both normal and malignant human keratinocytes contain mRNAs identical to monocytic IL-1 alpha and IL-1 beta mRNA, as determined by an S1 nuclease protection assay; and IL-1 activity in medium conditioned by these cells can be neutralized by antibodies specific for human IL-1. The IL-1 alpha and IL-1 beta mRNAs can be identified in cultured human keratinocytes in the absence of identifiable stimulation; this basal level of mRNA can be further induced to accumulate with certain defined stimuli. Cultured normal human keratinocytes (HFKs) contain 2-4 times more IL-1 alpha than IL-1 beta mRNA; in contrast, human peripheral blood monocytes contain 10-20 times more IL-1 beta than IL-1 alpha mRNA. The IL-1 activity released by these HFK can be neutralized by an antibody that neutralizes both alpha and beta IL-1, but not by an antibody that neutralizes only IL-1 beta. While human monocytes produce a large excess of IL-1 beta after appropriate stimulation, these data suggest that IL-1 alpha is a major (and may be the predominant) form of IL-1 produced by human keratinocytes.

Cyclic AMP-dependent protein kinase in calf-snout epidermis.

The biochemical characteristics of cyclic AMP-dependent protein kinase in calf-snout epidermis were investigated. The activity of cyclic AMP-dependent protein kinase was higher in the lower layer than the upper layer of epidermis. The supernatant of homogenates of the lower layer of calf-snout epidermis was fractionated by DEAE-cellulose chromatography and contained two major peaks of protein kinase activity stimulated by cyclic AMP. This chromatographic pattern is similar to that referred to as Type I and Type II of cyclic AMP-dependent protein kinase in bovine muscle. Both peaks of cyclic AMP-dependent protein kinase in calf-snout epidermis could phosphorylate keratin polypeptides in vitro. The phosphorylation reaction was activated by cyclic AMP and inhibited by a heat-stable inhibitor of cyclic AMP-dependent protein kinase. When Type II enzyme of cyclic AMP-dependent protein kinase was incubated with [gamma-32P]ATP in the absence of substrates, such as histone or keratin polypeptides, the 54,000 dalton protein was phosphorylated and this autophosphorylation was inhibited by the addition of 10 microM cyclic AMP. These results suggest that cyclic AMP-dependent protein kinase in calf-snout epidermis has properties similar to those in bovine muscle and plays an important role in the phosphorylation of keratin polypeptides in calf-snout epidermis.

Identification of the major postsynaptic density protein as homologous with the major calmodulin-binding subunit of a calmodulin-dependent protein kinase.

The major postsynaptic density protein (mPSDp), comprising greater than 50% of postsynaptic density (PSD) protein, is an endogenous substrate for calmodulin-dependent phosphorylation as well as a calmodulin-binding protein in PSD preparations. The results in this investigation indicate that mPSDp is highly homologous with the major calmodulin-binding subunit (p) of tubulin-associated calmodulin-dependent kinase (TACK), and that PSD fractions also contain a protein homologous with the sigma-subunit of TACK. Homologies between mPSDp and a 63,000 dalton PSD protein and the rho- and sigma-subunits of TACK were established by the following criteria: (1) identical apparent molecular weights; (2) identical calmodulin-binding properties; (3) manifestation of Ca2+-calmodulin-stimulated autophosphorylation; (4) identical isoelectric points; (5) identical calmodulin binding and autophosphorylation patterns on two-dimensional gels; (6) homologous two-dimensional tryptic peptide maps; and (7) similar phosphoamino acid-specific phosphorylation of tubulin. The results suggest that mPSDp is a calmodulin-binding protein involved in modulating protein kinase activity in the postsynaptic density and that a tubulin kinase system homologous with TACK exists in a membrane-bound form in the PSD.

Phosphorylation of keratin polypeptides.

The phosphorylation of keratin polypeptides was examined in calf snout epidermis. When slices of epidermis were incubated in the medium containing 32Pi, the radioactivity was incorporated into several proteins. The predominant phosphorylated proteins migrated in SDS-polyacrylamide gels with apparent molecular weights between 49000 and 69000 and coincided with keratin polypeptides. The extent of keratin phosphorylation was not altered in the presence of dibutyryl cyclic AMP or reagents which elevate intracellular cyclic AMP. When homogenates of epidermis were incubated with [gamma-32P]ATP, keratin polypeptides were the predominant species phosphorylated as was also observed in epidermal slices. The presence of cyclic AMP or heat-stable inhibitor of cyclic AMP-dependent protein kinase in the reaction mixture did not affect the phosphorylation of keratin polypeptides, although the phosphorylation of exogenously-added histone was stimulated and inhibited, respectively, by these additions. Keratin polypeptides extracted from calf snout epidermis by 8 M urea were phosphorylated by incubation with [gamma-32P]ATP and cyclic AMP-dependent protein kinase from calf snout epidermis or bovine heart. No proteins were phosphorylated without the addition of the enzymes. The presence of cyclic AMP in the reaction mixture stimulated the keratin phosphorylation, and further addition of heat-stable protein kinase inhibitor reduced this stimulation.

Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins.

Tubulin is a major substrate for endogenous Ca2+-calmodulin-dependent phosphorylation in synaptic cytoplasm. The present study details the purification to apparent homogeneity and characterization of a brain cytosolic Ca2+-calmodulin-dependent kinase which phosphorylates tubulin and microtubule-associated proteins as major substrates. The cytosolic kinase system, purified by sequential chromatography on phosphocellulose resin, calmodulin-affinity resin, and Fractogel TSK HW-55, chromatographs as a homogeneous complex of approximately 600,000 Da on Sephacryl S-300. This calmodulin-dependent kinase possesses a group of properties which specifically characterize this enzyme system: 1) the enzyme contains two calmodulin-binding doublets, rho and sigma, of approximately 52,000 and 63,000 Da, respectively; 2) both the rho and the sigma subunits demonstrate isoelectric points between 6.7 and 7.2; 3) both the rho and sigma subunits demonstrate autophosphorylation; 4) both the rho and sigma subunits show significant homologies as assessed by tryptic peptide fingerprints; 5) in the absence of substrate, both the rho and sigma subunits manifest lower mobility autophosphorylated species; 6) the kinase phosphorylates beta-tubulin equally on threonine and serine residues. Substrate specificity, kinetic parameters, calmodulin-binding properties, subunit composition, and subunit isoelectric points clearly differentiate this enzyme from other previously reported calmodulin-dependent kinases.

Congenital cutis laxa associated with exocrine pancreatic insufficiency.

We report a patient who has congenital cutis laxa and exocrine pancreatic insufficiency. Collagen analysis has provided evidence for defective elastin cross linking, possibly related to a mild copper deficiency. Connective tissue anomalies, in the presence of poor growth and/or symptoms of malabsorption should alert the physician to investigate pancreatic function. Adequate replacement of pancreatic enzymes will result in normal growth and development.

Psoralen-DNA cross-linking photoadducts in dyskeratosis congenita: delay in excision and promotion of sister chromatid exchange.

Dyskeratosis congenita is a rare X-linked recessive disease, characterized by mucosal leukokeratosis, nail dystrophy, telangiectasia, reticulated hyperpigmentation, pancytopenia, and a heightened susceptibility to infection and malignancy. We exposed cultured fibroblasts and peripheral leukocytes from normal persons and from 2 unrelated young adult men with dyskeratosis congenita to 4,5',8-trimethylpsoralen and ultraviolet light. We than compared certain of their responses. Labeled DNA from fibroblasts exposed to 4,5',8-trimethylpsoralen and ultraviolet light showed fast-sedimenting DNA, a pattern we interpreted as evidence that cross-linking, psoralen-DNA photoadducts had been formed by the treatment. Fast-sedimenting DNA persisted for 24 hr in dyskeratosis congenita cells but disappeared from normal cells during a 24-hr repair period. Cultured peripheral blood leukocytes from persons with this syndrome similarly exposed to 4,5',8-trimethylpsoralen and ultraviolet light developed more sister chromatid exchanges than did cells from normal persons. These data suggest that a heightened susceptibility in DNA cross-links may be of fundamental importance in the etiology of dyskeratosis congenita.

Nuclear nonhistone proteins in murine melanoma cells. I. Quantitative isolation and fractionation.

Nuclear nonhistone proteins (NHP's) have been implicated as regulatory agents involved in controlling genetic expression. Utilizing murine melanoma cells, we describe a method for isolating and fractionating NHP's which greatly increases the yield of these proteins as well as the level of resolution required for detecting small differences in particular NHP's. Mouse melanoma cells were grown in medium labeled with [(3)H]leucine. Following 48 hr of incubation, the cells were harvested and nuclei isolated. The NHP's were extracted from the nuclei in a series of steps which yielded four major fractions: NHP(1), NHP(2), NHP(3), NHP(4). This method solubilized 80-90% of the protein from the nuclear homogenate. The NHP fractions were then separated on DEAE-cellulose columns in a series of salt steps increasing in concentration from 0.05 to 0.50 M NaCl, followed by steps of 2 M NaCl and 4 and 7 M guanidine-hydrochloride. The 40 NHP fractions eluted from these columns were further separated on polyacrylamide-SDS gels and ranged in molecular weight from 9000 to 110,000 daltons. Differences were observed in the electrophoretic pattern of each of these 40 fractions. The high resolution of these fractionation procedures greatly enhances the possibility of observing small changes in proteins which may play a role in gene regulation.

Regulation of melanocyte stimulating hormone action at the receptor level: discontinuous binding of hormone to synchronized mouse melanoma cells during the cell cycle.

Melanocyte stimulating hormone coupled to Sepharose effects an increase in tryosinase (EC 1.14.18.1; monophenol monoxygenase) activity of cultivated mouse melanoma cells. Synchronized cells are found to respond to melanocyte stimulating hormone only in the G2 phase of the cell cycle, although their response to cyclic AMP is independent of position in the cell cycle. The binding of (125)I-labeled melanocyte stimulating hormone occurs predominantly in G2. These observations are satisfied by a model in which the hormone can activate adenylate cyclase (EC 4.6.1.1) by binding to a melanocyte stimulating hormone receptor only in G2; the events distal to cyclic AMP production can occur throughout the cell cycle.