Deoligomerization of human tumor necrosis factor alpha (TNF), spiked with 125I-labeled form, was studied quantitatively using size-exclusion chromatography and off-line monitoring with a gamma-counter. A detailed investigation of the oligomeric state of TNF was carried out as a function of its own concentration (0.3-7500 nM referred to the subunit, M(r) 17,000) in the absence or in the presence of various amounts (10, 100, 1000 microM) of suramin, an inhibitor of TNF biological activity in vitro, which promotes TNF deoligomerization. The dependence of trimeric form content on total TNF concentration was modeled with a sequential dissociation process (trimer-->dimer-->monomer) assuming an identical dissociation constant for each step, Kd1 = 0.2 nM. This model was used as the simplest for data fitting although, generally, no chromatographic resolution of dimeric species could be obtained. Best fitting of all data could be achieved with a model including a conformational change of TNF trimer into a state more prone to deoligomerization (Kd2 = 400 nM), which was favored by suramin binding. A kinetic study of TNF dissociation by the same method produced values for the deoligomerization rate of trimer: on the average, koff approximately 4 x 10(-5) S-1 (t1/2 approximately 5 h) between 4 and 20 degrees C with little dependence on suramin concentration; at 37 degrees C, a sizable increase is observed in the presence of 1 mM suramin (koff = 2.3 x 10(-4) S-1, t1/2 = 0.8 h). Data of suramin inhibition on TNF receptor binding, as obtained after incubation times much shorter than the above half-life of trimer, indicate that suramin binding to TNF trimer is the early mechanism of receptor binding inhibition.

The significance of the erythrocyte abnormalities described in rats and humans with spontaneous hypertension is far from clear. This study, in two highly inbred strains of rats, was designed to evaluate whether these abnormalities are primary and thus genetically related to hyper-tension. The Milan hypertensive strain (MHS) and its normotensive control strain (MNS) were used to carry out two types of experiments. In two groups of lethally irradiated (MHS x MNS) F, hybrids, bone marrow from MHS or MNS was transplanted. The differences in red cell function between the recipients of bone marrow from MHS and recipients of bone marrow from MNS were similar to those existing between the parental donor MHS and MNS: Na +-K + cotransport was increased (p < 0.02) and intracellular Na + content (p < 0.05) and cell volume (p < 0.02) were decreased in MHS. The same pattern was observed when this experiment was repeated in different groups of F, hybrids. In individuals of the segregating F 2 population, obtained by crossing the (MHS x MNS) F, hybrids, there was a positive correlation (p < 0.001) between the red blood cell Na +-K + cotransport and the mean blood pressure. These results indicate that the erythrocyte abnormalities may well be genetically associated with the primary cause of spontaneous hypertension in rats. Because of the many similarities demonstrated when young prehypertensive MHS or humans prone to develop hypertension are compared with their respective controls, it is possible that the findings described here in rats are relevant to human essential hypertension. (Hypertension 7: 319-325, 1985) KEY WORDS • red blood cell • genetic hypertension • cell volume • ion transport T HE cause of essential hypertension is not known. It is generally accepted that hyperten-sion develops when there is increased genetic sensitivity to certain environmental stimuli 1 and that red blood cell (RBC) membrane functions are abnormal in humans with essential hypertension 2 " 6 and rats with spontaneous hypertension. 7 '" It is not clear whether these alterations in red cell cation transport are the result of chance genetic selection unrelated to the cause of hypertension, are secondary to the hyperten-sive disease, or are the direct phenotypic expression of the same genetic abnormalities responsible for the increased susceptibility to hypertension. We must determine which of these possibilities is responsible if we wish to use the RBC transport systems as probes to study the genetic mechanisms of arterial hypertension. We have approached these questions by using two highly inbred strains of rats, the Milan hypertensive strain (MHS, inbreeding coefficient 0.988) and its nor-motensive control (MNS, inbreeding coefficient 0.989). Previous studies 12 " 14 with MHS and MNS showed many pathophysiological similarities between their hypertension and essential hypertension in humans. Kidney cross-transplantation and renal function studies suggested that the hypertension of MHS might originate from a primary increase in the transport of Na...

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Over-expression of the HER-2 oncogene correlates with poor prognosis in breast and ovarian carcinomas. Using a sensitive immunohistochemical assay, we have detected low levels of gp185HER-2 in intradermal nevi (78%) and in primary (75%) and metastatic melanomas (58%). The HER-2 gene product expressed by cultured melanoma cells had the expected molecular weight, but no levels of tyrosine phosphorylation could be detected. Consistently, we were unable to inhibit in vitro growth of melanoma cells with an anti-gp 185HER-2 MAb, in conditions in which the growth of SKBr-3 breast-carcinoma cells was severely impaired. However, immunotoxins to gp 185HER-2 were able to kill gp185HER-2-positive melanoma cells. These data indicate that low levels of gp185HER-2 are expressed by the melanocyte lineage, with no correlation with transformation or tumor progression. Nevertheless, gp185HER-2 appears a suitable target for immunotherapy of cutaneous melanoma.

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