Foetal development of the human gluteus maximus muscle with special reference to its fascial insertion.

The human gluteus maximus muscle (GMX) is characterised by its insertion to the iliotibial tract (a lateral thick fascia of the thigh beneath the fascia lata), which plays a critical role in lateral stabilisation of the hip joint during walking. In contrast, in non-human primates, the GMX and biceps femoris muscle provide a flexor complex. According to our observations of 15 human embryos and 11 foetuses at 7-10 weeks of gestation (21-55 mm), the GMX anlage was divided into 1) a superior part that developed earlier and 2) a small inferior part that developed later. The latter was adjacent to, or even continuous with, the biceps femoris. At 8 weeks, both parts inserted into the femur, possibly the future gluteal tuberosity. However, depending on traction by the developing inferior part as well as pressure from the developing major trochanter of the femur, most of the original femoral insertion of the GMX appeared to be detached from the femur. Therefore, at 9-10 weeks, the GMX had a digastric muscle-like appearance with an intermediate band connecting the major superior part to the small inferior mass. This band, most likely corresponding to the initial iliotibial tract, extended laterally and distally far from the muscle fibres. The fascia lata was still thin and the tensor fasciae latae seemed to develop much later. It seems likely that the evolutionary transition from quadripedality to bipedality and a permanently upright posture would require the development of a new GMX complex with the iliotibial tract that differs from that in non-human primates. (Folia Morphol 2018; 77, 1: 144-150).

Influence of glistenings on the optical quality of acrylic foldable intraocular lens.

AIMS: To assess the influence of glistenings on the optical quality of acrylic foldable intraocular lens. METHODS: Several degrees of glistenings in the optic were experimentally created by immersing the lens in water at 37 degrees C for 48 hours and then at 25 degrees C for 24 hours. Optical bench tests were carried out in water including measurements of spectral transmittance with the spectrophotometer, intensity of forward scattering using the integrating sphere photometer, modulation transfer function, and resolving power at various contrasts with and without the veiling glare light source. RESULTS: Glistenings of 1+ to 4+ degrees were created, among which the 4+ glistenings seemed to be extremely intense and thought to be beyond the range of clinical settings. Clinically feasible level of glistenings, up to 3+, did not adversely influence spectral transmittance, scattering, modulation transfer function, and resolving power at various contrasts. The 4+ glistenings caused mild to moderate deteriorations of the optical quality of the lens. CONCLUSION: The optical quality of the acrylic foldable intraocular lens is not significantly affected by the level of glistenings usually seen in the clinical setting.

Opposing regulation of B cell receptor-induced activation of mitogen-activated protein kinases by CD45.

In this study, we examined the contribution made by CD45 to B cell antigen receptor (BCR)-induced activation of mitogen-activated protein kinase (MAPK) family members. We found that CD45 negatively regulated BCR-induced c-Jun NH(2)-terminal kinase (JNK) and p38 activation in immature WEHI-231 cells, whereas in mature BAL-17 cells, CD45 positively regulated JNK and p38 activation and negatively regulated extracellular signal-regulated kinase activity. Furthermore, cooperative action of JNK and p38 dictated BCR-induced inhibition of growth. Thus, CD45 appears to differentially regulate BCR-induced activation of MAPK members, and can exert opposing effects on JNK and p38 in different cellular milieu, controlling the B cell fate.

CD45 is required for CD40-induced inhibition of DNA synthesis and regulation of c-Jun NH2-terminal kinase and p38 in BAL-17 B cells.

Stimulation of B cell antigen receptor (BCR) may induce proliferation, differentiation, or apoptosis, depending upon the maturational stage of the cell and the presence or absence of signals transmitted via coreceptors. One such signal is delivered via CD40; for instance, ligation of CD40 rescues B cells from BCR-induced apoptosis. Here we show that, in contrast to WEHI-231 cells, CD40 ligation did not reverse BCR-induced growth inhibition in the BAL-17 mature B cell line and CD40 ligation itself inhibited proliferation. This inhibitory signaling was not observed in CD45-deficient cells. Further analyses demonstrate that transfection of dominant-negative form of SEK1 or treatment with SB203580 strongly reduced CD40-induced inhibition of BAL-17 proliferation, suggesting a requirement for c-Jun NH2-terminal kinase and p38 in CD40-induced inhibition of proliferation. Interestingly, CD40-initiated activation of c-Jun NH2-terminal kinase and p38 was enhanced and sustained in CD45-deficient cells, and these phenotypes were reversed by transfecting CD45 gene. However, CD40-mediated induction of cell surface molecules was not affected in CD45-deficient cells. Taken collectively, these results suggest that CD45 exerts a decisive effect on selective sets of CD40-mediated signaling pathways, dictating B cell fate.

Src homology region 2 (SH2) domain-containing phosphatase-1 dephosphorylates B cell linker protein/SH2 domain leukocyte protein of 65 kDa and selectively regulates c-Jun NH2-terminal kinase activation in B cells.

Src homology region 2 (SH2) domain-containing phosphatase-1 (SHP-1) is a cytosolic protein tyrosine phosphatase containing two SH2 domains in its NH2 terminus. That immunological abnormalities of the motheaten and viable motheaten mice are caused by mutations in the gene encoding SHP-1 indicates that SHP-1 plays important roles in lymphocyte differentiation, proliferation, and activation. To elucidate molecular mechanisms by which SHP-1 regulates BCR-mediated signal transduction, we determined SHP-1 substrates in B cells using the substrate-trapping approach. When the phosphatase activity-deficient form of SHP-1, in which the catalytic center cysteine (C453) was replaced with serine (SHP-1-C/S), was introduced in WEHI-231 cells, tyrosine phosphorylation of a protein of about 70 kDa was strongly enhanced. Immunoprecipitation and Western blot analyses revealed that this protein is the B cell linker protein (BLNK), also named SH2 domain leukocyte protein of 65 kDa, and that upon tyrosine phosphorylation BLNK binds to SHP-1-C/S in vitro. In vitro kinase assays demonstrated that hyperphosphorylation of BLNK in SHP-1-C/S-expressing cells was not due to enhanced activity of Lyn or Syk. Furthermore, BCR-induced activation of c-Jun NH2-terminal kinase was shown to be significantly enhanced in SHP-1-C/S transfectants. Taken collectively, our results suggest that BLNK is a physiological substrate of SHP-1 in B cells and that SHP-1 selectively regulates c-Jun NH2-terminal kinase activation.

CD45 negatively regulates lyn activity by dephosphorylating both positive and negative regulatory tyrosine residues in immature B cells.

Using CD45-deficient clones from the immature B cell line, WEHI-231, we previously demonstrated that CD45 selectively dephosphorylates the Src-family protein tyrosine kinase Lyn and inhibits its kinase activity. To further define the mechanisms of CD45 action on Lyn, we metabolically labeled Lyn from CD45-positive and -negative WEHI-231 cells and analyzed cyanogen bromide fragments by SDS-PAGE analysis. Phosphoamino acid analysis confirmed that Lyn is tyrosine phosphorylated with little serine or threonine phosphorylation. In CD45-negative cells, two bands at 8.2 and 4.1 kDa were phosphorylated in the absence of B cell Ag receptor (BCR) ligation. The 8.2-kDa band corresponded to a fragment containing the positive regulatory site (Tyr397), as assessed by its size and its phosphorylation in an in vitro kinase assay. The 4.1-kDa band was phosphorylated by COOH-terminal Src kinase, suggesting that it contains the COOH-terminal negative regulatory site (Tyr508). CD45 was also shown to dephosphorylate autophosphorylated Lyn in vitro. Thus, CD45 dephosphorylates not only the negative but also the positive regulatory tyrosine residues of Lyn. Furthermore, coimmunoprecipitations using anti-Igalpha Ab demonstrated that Lyn associated with the resting BCR was constitutively phosphorylated and activated in CD45-negative cells. In the parental cells, both regulatory sites were phosphorylated on BCR ligation. Taken collectively, these results suggest that CD45 keeps both BCR-associated and total cytoplasmic pools of Lyn in an inactive state, and a mechanism by which Lyn is activated by relative reduction of CD45 effect may be operative on BCR ligation.

Requirement of PEST domain tyrosine phosphatase PEP in B cell antigen receptor-induced growth arrest and apoptosis.

Signaling events leading to B cell growth or apoptosis are beginning to be unravelled, but detailed information is still lacking. To identify signaling molecules involved in B cell antigen receptor (BCR)-initiated pathways, we used the immature B cell line, WEHI-231, to investigate protein tyrosine phosphatases (PTP) whose expression was modulated by BCR ligation. Among the PTP cloned by reverse transcription-PCR, mRNA expression of the proline-, glutamic acid-, serine- and threonine-rich (PEST) domain phosphatase (PEP) was selectively elevated 3.1-fold within 3 h after anti-IgM antibody stimulation. In contrast, expression of another PEST domain phosphatase, PTP-PEST, was unaffected. Western blot analysis revealed that 71% of PEP was located in the cytosolic fraction, while 29% was in the membrane fraction. To examine the direct contribution made by PEP to BCR-initiated signal transduction, we transfected an antisense PEP cDNA into WEHI-231 cells. Two stable clones were established in which PEP expression was reduced by 34% and 47%, respectively. Strikingly, BCR-mediated inhibition of DNA synthesis was significantly rescued in the clones, and G1 phase cell cycle arrest and apoptosis were almost completely ablated. Considered collectively, these results indicate that PEP is a positive, crucial regulator in determining B cell fate triggered by BCR engagement.

A novel 66-kilodalton protein complexes with Rrn6, Rrn7, and TATA-binding protein to promote polymerase I transcription initiation in Saccharomyces cerevisiae.

We report the cloning of RRN11, a gene coding for a 66-kDa protein essential for transcription initiation by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. Rrn11 specifically complexes with two previously identified transcription factors, Rrn6 and Rrn7 (D. A. Keys, J. S. Steffan, J. A. Dodd, R. T. Yamamoto, Y. Nogi, and M. Nomura, Genes Dev. 8:2349-2362, 1994). The Rrn11-Rrn6-Rrn7 complex also binds the TATA-binding protein and is required for transcription by the core domain of the Pol I promoter. Therefore, we have designated the Rrn11-Rrn6-Rrn7-TATA-binding protein complex the yeast Pol I core factor. A two-hybrid assay was used to demonstrate involvement of short leucine heptad repeats on both Rrn11 and Rrn6 in the in vivo association of these two proteins. This assay also verified the previously described strong association between Rrn6 and Rrn7, independent of the Rrn6 leucine repeat.

On-off switching of enzymatic reaction by recombinant calmodulin on a solid-phase matrix.

A fusion protein consisting of human calmodulin (CaM) and glutathione S-transferase (GST) was produced by gene fusion. The fusion protein was overexpressed in Escherichia coli as a soluble form and purified with one-step affinity chromatography using glutathione-Sepharose. The protein had the modulating activity of CaM and the binding capability to glutathione of GST. Phosphodiesterase, which is a CaM dependent enzyme, was activated by the fusion protein, with the Ca2+ level equal to the level equivalent to a native CaM. Furthermore, CaM could be immobilized on a solid-phase matrix through the use of GST moiety while its modulating activity was retained. Phosphodiesterase activity was switched on and off by the immobilized CaM with or without Ca2+, and repeated use of CaM was demonstrated.

Two different cellular redox systems regulate the DNA-binding activity of the p50 subunit of NF-kappa B in vitro.

The NF-kappa B/Rel/Dorsal (NRD) transcription factor family binds target DNA sequences through their conserved N-terminal basic region that contains a single cysteine residue flanked by basic residues. This cysteine residue plays a critical role in the regulation of the DNA-binding activity of NRD members, since chemical modifications of this residue modulate the DNA-binding activity of NRD members. Here we show that cellular factors regulate the DNA-binding activity of NRD members in vitro by reduction-oxidation (redox) mechanisms. Two cellular redox systems, thioredoxin/thioredoxin reductase and apurinic/apyrimidinic endonuclease (also called Redox factor-1), independently, as well as, synergistically stimulate the DNA-binding activity of bacterially synthesized (recombinant) p50, one of the subunits of NF-kappa B that is a major NRD factor inducible in various types of cells. Since the mutation of the conserved residue (Cys61) in the N-terminal basic region of p50 impairs the stimulation of p50 DNA-binding activity by these redox factors, the regulation of p50 DNA-binding activity by these redox factors is mediated through this cysteine residue. It is, therefore, possible that these two cellular redox systems could play independent, as well as synergistic roles in the regulation of NF-kappa B functions in vivo through the redox control of their DNA-binding activity.

A lymphoid cell-specific nuclear factor containing c-Rel-like proteins preferentially interacts with interleukin-6 kappa B-related motifs whose activities are repressed in lymphoid cells.

The proto-oncoprotein c-Rel is a member of the nuclear factor kappa B transcription factor family, which includes the p50 and p65 subunits of nuclear factor kappa B. We show here that c-Rel binds to kappa B sites as homodimers as well as heterodimers with p50. These homodimers and heterodimers show distinct DNA-binding specificities and affinities for various kappa B motifs. In particular, the c-Rel homodimer has a high affinity for interleukin-6 (IL-6) and beta interferon kappa B sites. In spite of its association with p50 in vitro, however, we found a lymphoid cell-specific nuclear factor in vivo that contains c-Rel but not p50 epitopes; this factor, termed IL-6 kappa B binding factor II, appears to contain the c-Rel homodimer and preferentially recognizes several IL-6 kappa B-related kappa B motifs. Although it has been previously shown that the IL-6 kappa B motif functions as a potent IL-1/tumor necrosis factor-responsive element in nonlymphoid cells, its activity was found to be repressed in lymphoid cells such as a Jurkat T-cell line. We also present evidence that IL-6 kappa B binding factor II functions as a repressor specific for IL-6 kappa B-related kappa B motifs in lymphoid cells.

Involvement of a NF-kappa B-like transcription factor in the activation of the interleukin-6 gene by inflammatory lymphokines.

Interleukin-6 (IL-6) is one of the major mediators of inflammation, and its expression is inducible by the other inflammatory lymphokines, interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). We demonstrate that a common IL-6 promoter element, termed inflammatory lymphokine-responsive element (ILRE), is important for induction of IL-6 gene expression by IL-1 and TNF-alpha despite possible differences in the mechanisms of action of these lymphokines. Remarkably, the ILRE sequence, located between -73 to -63 relative to the mRNA cap site, is highly homologous to NF-kappa B transcription factor-binding motifs and binds an IL-1-TNF-alpha-inducible nuclear factor; the sequence specificities, binding characteristics, and subcellular localizations of this factor are indistinguishable from those of NF-kappa B. In addition, mutations of the ILRE sequence which impair the binding of this nuclear factor abolished the induction of IL-6 gene expression by IL-1 and TNF-alpha in vivo. These results indicate that a nuclear factor indistinguishable from NF-kappa B is involved in the transcriptional activation of the IL-6 gene by IL-1 and TNF-alpha.

Characterization of three monoclonal antibodies against C3 with selective specificities.

We raised 15 mouse monoclonal antibodies against human C3 from two separate fusions. Mouse plasmacytoma cells were fused with spleen cells from mice immunized either with a mixture of C3, C3b and C3c, or with a mixture of C3dg and C3d. Three of the 15 monoclonals were characterized in detail. N-7A reacted with native C3 as well as C3b and C3c. Two other monoclonals, C-5G and G-3E, recognized neoantigenic determinants on C3c and C3dg, respectively. In ELISA, C-5G reacted with C3b and C3c but not with native C3 nor with C3dg; and on the other hand G-3E reacted only with C3dg. The selective specificities of these monoclonals were further confirmed in a binding assay to C3 fragments formed on cellular intermediates. C-5G bound exclusively to EC3b, and G-3E bound to EiC3b, EC3dg and EC3d. N-7A bound only very poorly to EC3b. C-5G inhibited both the haemolytic activity of C5 convertase and also the CR1-mediated rosette formation of B-enriched peripheral mononuclear cells with EC3b. G-3E inhibited the CR2-mediated EC3dg-rosette formation of Raji cells. These monoclonals, with selective specificities, can distinguish the state of activation and degradation of the C3 molecule.

Functional and antigenic properties of complement receptor type 2, CR2.

This is the first report demonstrating that C3d receptor (CR2) has functional activity in regulating complement cascade. Purified CR2 was examined for its cofactor activity in factor I-mediated cleavage of membrane-bound iC3b. CR2 plus C3b inactivator (I) released C3c from EA 125I-iC3b, and the release was inhibited when CR2 was preincubated with OKB7 monoclonal anti-CR2. Furthermore, immunoelectroblotting analysis showed crossreactivity of CR2 with 57H anti-CR1. These results indicate that CR2 has functional and antigenic similarity to CR1, thus providing a supporting evidence for placement of CR2 as a member of the recently defined gene family of C3- and C4-regulatory proteins composed of CR1, C4-binding protein, and factor H.

A solid-phase anti-C3 assay for detection of immune complexes in six distinguished forms.

A solid-phase anti-C3 assay detecting immune complexes associated with C3 fragments is described. Two monoclonal antibodies against C3 fragments are used; one recognizes an epitope expressed on C3b, C3c and C3i, the other recognizes an epitope expressed on C3dg and iC3b. Combining these monoclonal anti-C3s with antibodies against class-specific immunoglobulins in enzyme-linked immunosorbent assay (ELISA), immune complexes (ICs) are detected in six distinguished forms; C3b-IgG-IC, iC3b/C3dg-IgG-IC, C3b-IgA-IC, iC3b/C3dg-IgA-IC, C3b-IgM-IC and iC3b/C3dg-IgM-IC. About three-fourths of the plasma samples from patients with active SLE or IgA-nephritis were found positive in one or more types of ICs. IgA-type ICs were found very frequently in patients with autoimmune or renal diseases.