1,25-dihydroxyvitamin D3 production and vitamin D3 receptor expression are developmentally regulated during differentiation of human monocytes into macrophages

It has been well established that human mononuclear phagocytes have the capacity to produce 1,25-dihydroxy-vitamin D3 [1,25(OH)3D3] and express the vitamin D receptor (VDR). However, 1 alpha-hydroxylase activity and VDR receptor expression during differentiation of monocytes (MO) into mature macrophages (MAC) have not been previously examined. The in vitro maturation of blood MO can serve as a model for the in vivo transformation of immature blood MO into MAC. Here, when cultured in the presence of serum, MO undergo characteristic changes in morphology, antigenic phenotype, and functional activity consistent with their differentiation into MAC. We serially measured 1,25(OH)2D3 and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] synthesis, specific [3H]-1,25(OH)2D3 binding, and VDR mRNA levels during in vitro maturation of MO into MAC and correlated these functions with maturation-associated changes in the phenotype (MAX.1 and CD71) and secretory repertoire (interleukin-1 beta [IL-1 beta], neopterin) of the cells. MO showed only little conversion of 25-(OH)D3 into 1,25(OH)2D3 (1.4 +/- 0.4 pmol/10(6) cells/6 h, n = 5) that increased gradually during maturation into MAC at day 8 of culture (5.3 +/- 4.3 pmol/10(6) cells/6 h, n = 5). Interferon-gamma (IFN-gamma) increased baseline 1,25(OH)2D3-synthesis approximately twofold during all phases of differentiation. The time course of increased 1,25(OH)2D3-synthesis correlated with enhanced secretion of neopterin and expression of MAX.1 and CD71. The addition of exogenous 1,25(OH)2D3 did not influence constitutive 1,25(OH)2D3 synthesis, but IFN-gamma-stimulated production was suppressed to baseline levels. Exogenous 1,25(OH)2D3 also stimulated 24,25(OH)2D3 synthesis in freshly isolated MO (from 1.0 +/- 0.8 pmol/6 h to 5.6 +/- 0.9 pmol), whereas matured MAC showed no 24,25(OH)2D3 synthesis. Furthermore, we examined the expression of the VDR during the differentiation process. VDR mRNA and protein were constitutively expressed in MO, whereas VDR was downregulated in mature MAC on both the mRNA and protein levels. Homologous upregulation of VDR protein by 1,25(OH)2D3 occurred in MO and, to a lesser degree, in MAC. In contrast, VDR mRNA concentrations were not influenced by 1,25(OH)2D3. Taken together, our results show that MO into MAC differentiation in vitro is associated with (1) an enhanced capacity to synthesize 1,25(OH)2D3, (2) a loss of 24,25(OH)2D3-synthesizing activity, and (3) a decrease in the expression of VDR mRNA and protein. Because 1,25(OH)2D3 was shown to induce differentiation of MO into MAC, our data sugest an autoregulatory mechanism of MO/MAC generation by 1,25(OH)2D3.     

Spinal co-administration of peptide substance P antagonist increases antinociceptive effect of the opioid peptide biphalin

Intrathecal injection of 0.25 micrograms of undecapeptide substance P antagonist (SPA) produced transient antinociception with a peak effect at 5 min. Increasing the SPA dose resulted in neurotoxicity. Intrathecal injection of the opioid peptide biphalin (BIP) produced antinociception for over 3 hrs without neurotoxicity. Co-administration of SPA (at subtoxic doses) increased BIP's antinociceptive effect. Naltrexone reversed analgesia due to BIP alone as well as after BIP+SPA.     

Buspirone''s efficacy in organic-induced aggression

The wild-type tumor suppressor protein p53 is a short-lived protein that plays important roles in regulation of cell cycle, differentiation, and survival. Mutations that inactivate or alter the tumor suppressor activity of the protein seem to be the most common genetic change in human cancer and are frequently associated with changes in its stability. The ubiquitin system has been implicated in the degradation of p53 both in vivo and in vitro. A mutant cell line that harbors a thermolabile ubiquitin-activating enzyme, E1, fails to degrade p53 at the nonpermissive temperature. Studies in cell-free extracts have shown that covalent attachment of ubiquitin to the protein requires the three conjugating enzymes: E1, a novel species of ubiquitin-carrier protein (ubiquitin-conjugating enzyme; UBC),E2-F1, and an ubiquitin-protein ligase, E3. Recognition of p53 by the ligase is facilitated by formation of a complex between the protein and the human papillomavirus (HPV) oncoprotein E6. Therefore, the ligase has been designated E6-associated protein (E6-AP). However, these in vitro studies have not demonstrated that the conjugates serve as essential intermediates in the proteolytic process. In fact, in many cases, conjugation of ubiquitin to the target protein does not signal its degradation. Thus, it is essential to demonstrate that p53-ubiquitin adducts serve as essential proteolytic intermediates and are recognized and degraded by the 26S protease complex, the proteolytic arm of the ubiquitin pathway. In this study, we demonstrate that conjugates of p53 generated in the presence of purified, E1, E2, E6-AP, E6, ubiquitin and ATP, are specifically recognized by the 26S protease complex and degraded. In contrast, unconjugated p53 remains stable. The ability to reconstitute the system from purified components will enable detailed analysis of the recognition process and the structural motifs involved in targeting the protein for degradation.     

Membrane electrical properties associated with insulin receptor downregulation in human erythrocytes

We reviewed 29 consecutive patients after cemented femoral revision of cemented hip arthroplasties for osteolysis. After an average follow-up of 8.5 years, osteolysis had recurred in only two cases (6.9%) and 25 femoral components (86%) remained well fixed.     

Conservative initial treatment for liver abscesses in children

A total of 124 children aged less than 14 years with a liver abscess were seen in a 16-year period (1974-1990) and treated by non-operative initial management. Of the abscesses 98 occurred in the right liver and 26 in the left. The abscesses were solitary in 93 patients. Overall, 77 of the solitary and 21 of the multiple abscesses were confined to the right liver. In 78 of the right-sided and 20 of the left-sided abscesses the infection was primarily pyogenic in nature with Staphylococcus aureus being the usual organism cultured. The remainder were of amoebic origin. Clinical features were similar in patients with amoebic and pyogenic abscesses. Clinical and ultrasonographic follow-up demonstrated successful non-operative management and healing in 37 per cent of all patients submitted to an initial protocol of medical supportive care and antibiotic therapy. Of the multiple abscesses 60 per cent responded to non-operative management. Fourteen of the 16 solitary left-sided liver abscesses required drainage and three left-sided abscesses ruptured before drainage. Patients with a solitary left-sided abscess warrant early operative intervention.     

Measurement of creatine kinase Z in human sera using a DEAE-cellulose mini-column method

A DEAE-cellulose mini-column method has been developed which allows for the quantitation in human serum of creatine kinase Z, a sub-band of creatine kinase first described by Lim ((1975) Clin. Chem. 21, 975, Abstract 181) and Sax et al. (Sax, S.M., Moore, J.J., Giegel, J.L. and Welsh, M. (1976) Clin. Chem. 22, 87). We have shown that creatine kinase Z is rather unstable in nature, and converts to a form which electrophoreses with creatine kinase MM on agarose gel electrophoresis. CK-Z is not present in normal human serum. CK-Z is present in human heart extracts, in patients with myocardial infarcts and in patients with skeletal muscle trauma. In infarct patients CK-Z levels paralleled changes in the CK-MB levels. CK-Z ranged in activity from 8.8-67.2 I.U. whereas CK-MB ranged from 29.6-121.6 I.U. in infarct patients. CK-Z and CK-MB activity were measured at or close to the peak rise in total CK activity.     

Immunoperoxidase localisation of human placental lactogen: a marker for the placental origin of the giant cells in ''syncytial endometritis'' of pregnancy

Various common C4 gene products were isolated from serum by immunoprecipitation. After reduction the C4 alpha-, beta-, and gamma-polypeptide chains were studied by two-dimensional electrophoresis. Isoelectrofocusing was performed in the first dimension and sodium dodecyl sulphate polyacrylamide gradient gel electrophoresis in the second. The charge differences behind the electrophoretic C4 polymorphism were shown to reside in the 95,000-u (atomic mass units) alpha-chain. Charge variation closely mirroring the alpha-chain differences were also found in a 49,000-u fragment of the alpha-chain, most probably C4d. The basic beta-chain could not be studied in detail, but no differences were observed with regard to molecular weight or charge of the gamma-chains of the different C4 gene products.     

Periosteal migration in the growing mandible: an animal model

Migration of mandibular periosteum and attached musculature was tracked along the inferior border of the ramus in growing and nongrowing guinea pigs (Cavia porcellus) over a 6-week period. Particulate metallic growth-tracing implants were placed through the bony mandible and adjacent musculature at two anteroposterior locations and two bony reference markers were placed anteriorly. Quantification from weekly radiographs of growing animals showed marked posterior migration of the periosteum, whereas in nongrowing animals there was negligible periosteum movement. Significantly greater migration occurred in posterior (6.37 +/- 0.76 mm) implants relative to the anterior implants (3.45 +/- 0.86 mm, p < 0.001). The neutral zone, where little periosteal migration occurs, was calculated to be approximately at the anteroposterior center of the molar tooth row. Analysis of the orientation of the medial pterygoid muscle relative to the mandible showed that muscle fibers on average become more horizontal. Thus, the study found differential anteroposterior migration of the mandibular periosteum in growing animals and correlative changes in orientation of the medial pterygoid muscle.