Targeted delivery of anti-cancer growth inhibitory peptides derived from human alpha-fetoprotein: review of an International Multi-Center Collaborative Study.

The alpha-fetoprotein derived growth inhibitory peptide (GIP) is a 34-amino acid peptide composed of three biologically active subfragments. GIP-34 and its three constituent segments have been synthesized, purified, and studied for biological activity. The GIP-34 and GIP-8 have been characterized as anticancer therapeutic peptides. An multicenter study was initiated to elucidate the means by which these peptide drugs could be targeted to tumor cells. The study first established which cancer types were specifically targeted by the GIP peptides in both in vitro and in vivo investigations. It was next demonstrated that radiolabeled peptide ((125)I GIP-34) is specifically localized to rodent breast tumors at 24 h post-injection. The radionuclide studies also provided evidence for a proposed cell surface receptor; this was confirmed in a further study using fluorescent-labeled GIP-nanobeads which localized at the plasma membrane of MCF-7 breast cancer cells. Finally, it was readily demonstrated that GIP conjugated to either fluorescein or doxorubicin (DOX) underwent tumor cell uptake; subsequently, DOX-GIP conjugates induced cytotoxic cell destruction indicating the utility of GIP segments as cancer therapeutic agents. Following a discussion of the preceding results, a candidate cell surface receptor family was proposed which correlated with previous published reports for a putative AFP/GIP receptor.

Alpha- and beta-forms of gutta-percha in products for root canal filling.

AIM: This study was undertaken to evaluate which materials were in the alpha-form of gutta-percha as claimed, and which were in the more conventional beta-form, and to explore the effect of heating on the materials. METHODOLOGY: Samples of gutta-percha without chemical additives, and dental gutta-percha formulations which included (i) two products previously studied; (ii) 12 newer products; and (iii) one newer product that had been stored at high temperature, were analysed by simultaneous differential thermal analysis and thermogravimetry. RESULTS: It was found that only four of the newer materials contained the alpha-form; all the rest comprised beta-gutta-percha. No weight loss was found for any material under the conditions of the present experiments. A typical heating cycle up to 130 degrees C caused changes in material behaviour - that is, on reheating fewer endothermic peaks were present. Storage of gutta-percha samples for 10 years under ambient temperature and storage in a heater at 80 degrees C appeared to have no effect on properties. CONCLUSIONS: It was concluded that heating dental gutta-percha to 130 degrees C causes physical changes; this was not seen with chemically pure gutta-percha. The presence of additives in the dental samples altered material behaviour.

Regulatory mechanisms controlling expression of the DAN/TIR mannoprotein genes during anaerobic remodeling of the cell wall in Saccharomyces cerevisiae.

The DAN/TIR genes of Saccharomyces cerevisiae encode homologous mannoproteins, some of which are essential for anaerobic growth. Expression of these genes is induced during anaerobiosis and in some cases during cold shock. We show that several heme-responsive mechanisms combine to regulate DAN/TIR gene expression. The first mechanism employs two repression factors, Mox1 and Mox2, and an activation factor, Mox4 (for mannoprotein regulation by oxygen). The genes encoding these proteins were identified by selecting for recessive mutants with altered regulation of a dan1::ura3 fusion. MOX4 is identical to UPC2, encoding a binucleate zinc cluster protein controlling expression of an anaerobic sterol transport system. Mox4/Upc2 is required for expression of all the DAN/TIR genes. It appears to act through a consensus sequence termed the AR1 site, as does Mox2. The noninducible mox4Delta allele was epistatic to the constitutive mox1 and mox2 mutations, suggesting that Mox1 and Mox2 modulate activation by Mox4 in a heme-dependent fashion. Mutations in a putative repression domain in Mox4 caused constitutive expression of the DAN/TIR genes, indicating a role for this domain in heme repression. MOX4 expression is induced both in anaerobic and cold-shocked cells, so heme may also regulate DAN/TIR expression through inhibition of expression of MOX4. Indeed, ectopic expression of MOX4 in aerobic cells resulted in partially constitutive expression of DAN1. Heme also regulates expression of some of the DAN/TIR genes through the Rox7 repressor, which also controls expression of the hypoxic gene ANB1. In addition Rox1, another heme-responsive repressor, and the global repressors Tup1 and Ssn6 are also required for full aerobic repression of these genes.

Induction and repression of DAN1 and the family of anaerobic mannoprotein genes in Saccharomyces cerevisiae occurs through a complex array of regulatory sites.

The DAN/TIR mannoprotein genes of Saccharomyces cerevisiae (DAN1, DAN2, DAN3, DAN4, TIR1, TIR2, TIR3 and TIR4) are expressed in anaerobic cells while the predominant cell wall proteins Cwp1 and Cwp2 are down-regulated. Elements involved in activation and repression of the DAN/TIR genes were defined in this study, using the DAN1 promoter as a model. Nested deletions in a DAN1/lacZ reporter pinpointed regions carrying activation and repression elements. Inspection revealed two consensus sequences subsequently shown to be independent anaerobic response elements (AR1, consensus TCGTTYAG; AR2, consensus AAAAATTGTTGA). AR1 is found in all of the DAN/TIR promoters; AR2 is found in DAN1, DAN2 and DAN3. A 120 bp segment carrying two copies of AR1 preferentially activated transcription of lacZ under anaerobic conditions. A fusion of three synthetic copies of AR1 to MEL1 was also expressed anaerobically. Mutations in either AR1 site within the 120 bp segment caused a drastic loss of expression, indicating that both are necessary for activation and implying cooperativity between adjacent transcriptional activation complexes. A single AR2 site carried on a 46 bp fragment from the DAN1 promoter activated lacZ transcription under anaerobic conditions, as did a 26 bp synthetic AR2 fragment fused to MEL1. Nucleotide substitutions within the AR2 sequence eliminated the activity of the 46 bp segment. Ablation of the AR2 sequences in the full promoter caused a partial reduction of expression. The presence of the ATTGTT core (recognized by HMG proteins) in the AR2 sequence suggests that an HMG protein may activate through AR2. One region was implicated in aerobic repression of DAN1. It contains sites for the heme-induced Mot3 and Rox1 repressors.

Role of nitric oxide in the synthesis of guanidinosuccinic acid, an activator of the N-methyl-D-aspartate receptor.

BACKGROUND: We propose that reactive oxygen and argininosuccinic acid (ASA) form guanidinosuccinic acid (GSA). An alternative to this hypothesis is the so-called guanidine cycle, which consists of a series of hydroxyurea derivatives that serve as intermediates in a pathway leading from urea to GSA. We compare the role of the guanidine cycle to that of nitric oxide (NO) in the synthesis of GSA. METHODS: The members of the guanidine cycle (hydroxyurea, hydroxylamine plus homoserine, L-canaline, and L-canavanine) were incubated with isolated rat hepatocytes. The known NO donors, NOR-2, NOC-7, and SIN-1, were incubated with ASA in vitro. Ornithine, arginine, or citrulline, which increase arginine, a precursor of NO, were incubated with isolated rat hepatocytes. GSA was determined by high-performance liquid chromatography. RESULTS: None of guanidine cycle members except for urea formed GSA. SIN-1, which generates superoxide and NO formed GSA, but other simple NO donors, did not. Both carboxy-PTIO, a scavenger of NO, and dimethyl sulfoxide, a hydroxyl radical scavenger, completely inhibited GSA synthesis by SIN-1. GSA formation by SIN-1 reached a maximum at 0.5 mmol/L and decreased at higher concentrations. GSA synthesis, stimulated by urea in isolated hepatocytes, was inhibited by ornithine, arginine, or citrulline with ammonia, but not by ornithine without ammonia, where arginine production is limited. CONCLUSION: GSA is formed from ASA and the hydroxyl radical. When arginine increased in hepatocytes, GSA synthesis decreased. These data suggest that increased NO, which results from high concentrations of arginine, or SIN-1 scavenges the hydroxyl radical. This may explain the decreased GSA synthesis in inborn errors of the urea cycle where ASA is decreased, and also the diminished GSA excretion in arginemia.

Intersubjectivity and narcissism in group psychotherapy: how feedback works.

In group psychotherapy, inter-subjectivity is complicated by the number and quality of therapist-member, member-member, and member-group relations. Inter-member feedback structures the relational process. However, fears of narcissistic injury engender resistance to this form of engagement. The nature of the narcissistic belief system that motivates such resistance is discussed. Then, two models of feedback are presented: the cybernetic and the inter-subjective. In the cybernetic model, feedback is intended to inform recipients about themselves and to change their behavior accordingly. As such, this model is consistent with narcissistic beliefs in the power of others' perceptions to control one's being, identity, or value. The inter-subjective model focuses, instead, on what feedback tells recipients about their donors' worlds. This model and some of its parameters are exemplified clinically.

Self-structure and self-transformation in group psychotherapy.

This article first outlines a theory of self-structure as a hierarchically organized multiplicity of versions of self. It then describes self-transformation as a two-part process: (Part 1) the articulation and strengthening of individual self-boundaries, and (Part 2) the reclaiming of split-off, denied, or projected aspects of self. Clinically, both parts are products of the communicative interaction among members, the therapist, and the group as a whole. A parallel conception of group development posits that the group, as an object and as a social system, also needs to: (a) articulate and strengthen its boundaries so that it may (b) contain the sustained interdependent, sometimes conflictual, interactivity among members that is essential to the self-reclaiming process.

Betacellulin-Pseudomonas toxin fusion proteins bind but are not cytotoxic to cells expressing HER4; correlation of EGFR for cytotoxic activity.

Betacellulin (BTC) is a member of the EGF ligand family that directly binds to both EGFR and HER4 and induces the growth of certain epithelial cell types. Fusion proteins composed of the terminal 48 or 50 amino acids of mature betacellulin and a binding defective form of Pseudomonas exotoxin (BTC-TX48 and BTC-TX50, respectively), have been produced. BTC-TX50 induced tyrosine phosphorylation of both EGFR and HER4, whereas BTC-TX48 induced phosphorylation of HER4 but to a much lesser extent EGFR, indicating that the presence of two additional amino acid residues, Arg62 and Lys63, contribute to full kinase activity. BTC-TX50 was up to 300-fold more active at inhibiting protein synthesis than BTC-TX48 on cell lines expressing EGFR, most likely due to the >tenfold higher affinity of BTC-TX50. MDA-MB-453 breast carcinoma cells which express HER4 but not EGFR, were not sensitive to either BTC-TX form. These data indicate that despite the ability of BTC-TX to bind and phosphorylate HER4, it was only cytotoxic to cells expressing EGFR. The inability of BTC-TX to kill cells was likely due to its failure to internalize through HER4.

Role of epidermal growth factor receptor family members in growth and differentiation of breast carcinoma.

Members of the epidermal growth factor (EGF) family of tyrosine kinase receptors are involved in the regulation of cell growth and differentiation, and are found to be expressed in many types of cancers. Activation of these receptors can be elicited by multiple ligands, resulting in the formation of a spectrum of heterodimer complexes and a number of biological outcomes. A clear demonstration of biological activation by a single complex has been difficult to address because of the endogenous expression of HERs (human EGF-like receptors) in many cell lines. We have generated a collection of cell lines expressing all HERs alone or in all pairwise combinations in a clone of NIH 3T3 cells (3T3-7d) devoid of detectable EGF receptor family members. Transformation, as measured by growth in soft agar, only occurred in cells expressing two different HER family members. Transformation with activated Neu and the rate of in vivo tumour formation were also correlated with the expression of multiple HERs in the same cell. To further our understanding of the role of heterodimer signalling, we demonstrated that, within a breast carcinoma cell line, activation of HER-3 results in cellular differentiation, prolonged activation of extracellular-signal-related kinase 1 (ERK1) activity and an increase in p21CIP1/WAF1 nuclear staining. In contrast, activation of HER-4 is mitogenic, induces transient activation of ERK1 activity and decreases the nuclear staining of p21CIP1/WAF1. These differences in biochemical and biological responses are correlated with the contrasting abilities of HER-3 and HER-4 to be down-regulated from the cell surface. The cell-surface localization of HER-3 does not change in response to ligand, whereas activation of HER-4 results in a loss of cell-surface staining followed by accumulation into a perinuclear compartment.

The DAN1 gene of S. cerevisiae is regulated in parallel with the hypoxic genes, but by a different mechanism.

The DAN1 gene is expressed under anaerobic conditions in yeast and completely repressed during aerobic growth. The function of the gene is unknown, and genetic disruption had no effect on fitness which could be detected, even upon prolonged anaerobic growth. Expression of DAN1 was constitutive in a heme-deficient strain, indicating that heme participates in repression. Expression was blocked by heme in anaerobic medium, suggesting that heme acts as a negative co-effector rather than through its metabolic functions, i.e., in the production of a co-effector. Expression of DAN1 was regulated in parallel with the hypoxic gene ANB1, showing identical kinetics of induction and dose response to heme. However, unlike ANB1, DAN1 is not regulated by the repressor of the hypoxic regulon, ROX1, as shown by observation of normal aerobic repression of DAN1 in a strain carrying a deletion of ROX1. These results indicate the existence of a parallel regulatory system which produces an identical response to oxygen by a different mechanism than that controlling the hypoxic regulon.

An evaluation of operator preference of diamond burs in coronal tooth preparation.

Three postgraduate prosthodontic students served as clinicians/evaluators in a study rating their preferences for three different diamond cutting instruments from three manufacturers. Each evaluator prepared the axial walls of complete veneer crowns on extracted molar teeth and then ranked their preference of the instruments. To prepare nine teeth, each of the three instruments was used in random order and without knowledge of the specific manufacturer. The methodology for analyzing the evaluators' preferences and the results are discussed.

The relationship between human epidermal growth-like factor receptor expression and cellular transformation in NIH3T3 cells.

A collection of cell lines expressing each human epidermal growth factor receptor (HER) family member alone or in all pairwise combinations in a clone of NIH3T3 cells (3T3-7d) devoid of detectable epidermal growth factor receptor family members has been generated. Transformation, as measured by growth in soft agar, occurred only in the presence of appropriate ligand and only in cells expressing two different HER family members. Transfection of oncogenic neu (Tneu), conferred ligand-independent transformation only in cells which co-expressed HER1, HER3, or HER4, but not when expressed alone or with HER2. Cell lines were also tested for their ability to form tumors in animals. None of the cell lines expressing single HER family members was able to form tumors in animals with the exception of HER1, which was weakly tumorigenic. Although unable to form tumors when expressed alone, HER2 was tumorigenic when expressed with HER1 or HER3, but not HER4. Of all complexes analyzed, cells expressing HER1 + HER2 were the most aggressive. The relationship between HER1 activation, intracellular calcium fluxes, and phospholipase Cgamma1 activation is well established. We found that activation of HER1 was required for the induction of a calcium flux and the phosphorylation of phospholipase Cgamma1. These activities were independent of, and unaffected by, the co-expression of any other family member. Further, heregulin stimulation of all cell lines including those containing HER1 did not demonstrate any effect on intracellular calcium levels or phospholipase Cgamma1 phosphorylation. This demonstrates that heregulin induced cellular transformation by activating HER3- and HER4-containing complexes does not require the activation of either phospholipase Cgamma1 or the mobilization of intracellular calcium.

HER4-mediated biological and biochemical properties in NIH 3T3 cells. Evidence for HER1-HER4 heterodimers.

The EGF receptor family of tyrosine kinase growth factor receptors is expressed in a variety of cell types and has been implicated in the progression of certain human adenocarcinomas. The most recent addition to this family of receptors, HER4, was expressed in NIH 3T3 cells to determine its biological and biochemical characteristics. Cells expressing HER4 were responsive to heregulin beta2 as demonstrated by an increase in HER4 tyrosine phosphorylation and ability to form foci on a cell monolayer. HER4 exhibited in vitro kinase activity and was able to phosphorylate the regulatory subunit of phosphatidylinositol 3-kinase and SHC. Peptide competition studies identified tyrosine 1056 of HER4 as the phosphatidylinositol 3-kinase binding site and tyrosines 1188 and 1242 as two potential SHC binding sites. Interestingly, transfection of HER4 into NIH 3T3 cells conferred responsiveness to EGF with respect to colony formation in soft agar. It was also found that in response to heregulin beta2, endogenous murine HER1 or transfected human HER1 became phosphorylated when HER4 was present. This demonstrates that HER1 and HER4 can exist in a heterodimer complex and likely activate each other by transphosphorylation.

HER4 expression correlates with cytotoxicity directed by a heregulin-toxin fusion protein.

We have constructed, expressed, and purified a fusion protein, HAR-TX beta 2, consisting of heregulin-beta 2 fused to a binding-defective form of Pseudomonas exotoxin A, PE40. The fusion protein was found to induce receptor tyrosine phosphorylation in CEM cells transfected with HER4 alone or in combination with HER2 but not in cells transfected with HER2 or HER1 alone. The phosphorylation of receptor tyrosines was both dose-dependent and saturable in amounts similar to those shown to be active for native heregulin. HAR-TX beta 2 was specifically cytotoxic toward a variety of carcinoma cell lines in the ng/ml range. However, some tumor cell lines were found to be insensitive to the cytotoxic action of the fusion protein even at > 2 micrograms/ml. Relative amounts of HER4, HER3, and HER2 were determined on seven cell lines sensitive and four cell lines insensitive to HAR-TX beta 2. All lines that express HER4 were killed by HAR-TX beta 2, while none lacking HER4 were affected. HAR-TX beta 2 was able to bind to and signal via tyrosine phosphorylation in cell lines that co-express HER2 and HER3 in the absence of HER4 without inducing cytotoxicity. Thus HAR-TX beta 2 may prove to be a useful reagent for the targeting and elimination of HER4-positive tumor cells.

The use of orthodontics before fixed prosthodontics in restorative dentistry.

For a variety of reasons, orthodontic intervention is often overlooked as a viable modality to correct occlusal, axial, rotational, and space discrepancies before undertaking fixed prosthetic rehabilitation. However, patient treatment is being enhanced as never before by such intervention. This valuable treatment option facilitates tooth preparation, path of insertion, optimum oral hygiene, and a better pontic and abutment design, while occlusal forces can be directed against the long axes of the teeth for a more predictable prognosis. Moreover, this interdisciplinary approach can be cost-effective to patients and their treating dentists from the standpoint of producing more stable, durable, and esthetic restorations.

Pulp response to a novel adhesive calcium hydroxide based cement.

This study compares pulp responses to 3 formulations of calcium hydroxide, namely: a) An experimental adhesive calcium hydroxide cement containing polyacrylic acid, b) Dycal (L.D> Caulk Co, Milford, Delaware) Batch Nos 176970/176990, c) "Analar" calcium hydroxide mixed with sterile distilled water. After 28 days dentine bridges were present in 77% of teeth capped with the test material, 64% of teeth treated with Dycal and in 62% of teeth capped with calcium hydroxide and water. Inflammatory infiltrates were observed in a number of teeth remote from the bridges. Bacteria were detected in these specimens. Exposed rat molar pulp responses to an experimental adhesive calcium hydroxide cement were similar to to those observed with 2 other calcium hydroxide formulations.