Cyclin D1 expression and cell cycle response in DNA mismatch repair-deficient cells upon methylation and UV-C damage

We have evaluated cell survival, apoptosis, and cell cycle responses in a panel of DNA mismatch repair (MMR)-deficient colon and prostate cancer cell lines after alkylation and UV-C damage. We show that although these MMR-deficient cells tolerate alkylation damage, they are as sensitive to UV-C-induced damage as are the MMR-proficient cells. MMR-proficient cells arrest in the S-G2 phase of the cell cycle and initiate apoptosis following alkylation damage, whereas MMR-deficient cells continue proliferation. However, two prostate cancer cell lines that are MMR-deficient surprisingly arrest transiently in S-G2 after alkylation damage. Progression through G1 phase initially depends on the expression of one or more of the D-type cyclins (D1, D2, and/or D3). Analysis of cyclin D1 expression shows an initial MMR-independent decrease in the protein level after alkylation as well as UV-C damage. At later time points, however, only DNA damage-arrested cells showed decreased cyclin D1 levels irrespective of MMR status, indicating that reduced cyclin D1 could be a result of a smaller fraction of cells being in G1 phase rather than a result of an intact MMR system. Finally, we show that cyclin D1 is degraded by the proteasome in response to alkylation damage.     

Distribution of neuropeptide Y Y1 and Y2 receptors in the postmortem human heart

In the present study, we present for the first time the presence and distribution of neuropeptide Y (NPY) receptors Y1 and Y2 in the human postmortem heart using specific antibodies raised against extracellular parts of the receptors. A more intensive staining against the Y2 than against the Y1 receptors was detected on both atrial and ventricular cardiomyocytes. Immunoreactivity against both receptors was identified on both conducting fibers and cardiac nerves. More vessels stained positively for the Y2 than for the Y1 receptor, but the Y1 receptors were more abundant in subendocardial than subepicardial vessels of the left ventricular wall.     

Merlin links to the cAMP neuronal signaling pathway by anchoring the RIbeta subunit of protein kinase A

The cAMP-protein kinase A (PKA) pathway, important in neuronal signaling, is regulated by molecules that bind and target PKA regulatory subunits. Of four regulatory subunits, RIbeta is most abundantly expressed in brain. The RIbeta knockout mouse has defects in hippocampal synaptic plasticity, suggesting a role for RIbeta in learning and memory-related functions. Molecules that interact with or regulate RIbeta are still unknown. We identified the neurofibromatosis 2 tumor suppressor protein merlin (schwannomin), a molecule related to the ezrin-radixin-moesin family of membrane-cytoskeleton linker proteins, as a binding partner for RIbeta. Merlin and RIbeta demonstrated a similar expression pattern in central nervous system neurons and an overlapping subcellular localization in cultured hippocampal neurons and transfected cells. The proteins were coprecipitated from brain lysates by cAMP-agarose and coimmunoprecipited from cellular lysates with specific antibodies. In vitro binding studies verified that the interaction is direct. The interaction appeared to be under conformational regulation and was mediated via the alpha-helical region of merlin. Sequence comparison between merlin and known PKA anchoring proteins identified a conserved alpha-helical PKA anchoring protein motif in merlin. These results identify merlin as the first neuronal binding partner for PKA-RIbeta and suggest a novel function for merlin in connecting neuronal cytoskeleton to PKA signaling.     

Immunoneutralization of endogenous glucagon-like peptide-2 reduces adaptive intestinal growth in diabetic rats

Supraphysiological doses of glucagon-like peptide-2 (GLP-2) have been shown to induce intestinal growth by increasing villus height and crypt depth and by decreasing apoptosis, but a physiological effect of GLP-2 has not yet been demonstrated. Earlier, we found elevated levels of endogenous GLP-2 in untreated streptozotocin diabetic rats associated with marked intestinal growth. In the present study, we investigated the role of endogenous GLP-2 for this adaptive response. We included four groups of six rats: (1) diabetic rats treated with saline, (2) diabetic rats treated with non-specific antibodies, (3) diabetic rats treated with polyclonal GLP-2 antibodies and (4) non-diabetic control rats treated with saline. All animals were treated with once daily intraperitoneal injections for 13 days and killed on day 14. Diabetic rats treated with saline or non-specific antibodies had a significantly (P<0.01) increased area of mucosa (13.00+/-0.64 and 13.37+/-0.60 mm(2), respectively) in the proximal part of the small intestine compared with non-diabetic controls (7.97+/-0.70 mm(2)). In contrast, diabetic rats treated with GLP-2 antibodies had a significantly (P<0.01) smaller increase in area of mucosa in the proximal part of the small intestine (10.84+/-0.44 mm(2)). Antibody treatment had no effect on body weight, blood glucose concentrations and food intake. Thus, blocking of endogenous GLP-2 in a model of adaptive intestinal growth reduces the growth response, providing strong evidence for a physiological growth factor function of GLP-2.     

Light/dark regulation of higher plant nitrate reductase related to hysteresis and calcium/magnesium inhibition

Examination of nitrate reductase (NR, EC 1.6.6.1) activity in crude extracts made from squash leaves before and after a light/dark transition, indicates the existence of two different forms of nitrate reductase; a 'light form' with a pH optimum of 7.8 that is not inhibited by calcium or magnesium, and a 'dark form' with a pH optimum of 7.6 that is strongly inhibited by calcium or magnesium. The same properties also characterise purified NR. The 'light and dark forms' of NR correspond to the two kinetically different forms of purified NR showing (1) linear product formation and (2) delayed product formation, i.e. hysteretic behaviour.     

Genotypic Diversity among Bacillus cereus and Bacillus thuringiensis Strains

Twenty-four strains of Bacillus cereus were analyzed by pulsed-field gel electrophoresis (PFGE) and compared with 12 Bacillus thuringiensis strains. In addition, the 36 strains were examined for variation in 15 chromosomal genes encoding enzymes (by multilocus enzyme electrophoresis [MEE]). The genome of each strain had a distinct NotI restriction enzyme digestion profile by PFGE, and the 36 strains could be assigned to 27 multilocus genotypes by MEE. However, neither PFGE nor MEE analysis could distinguish between the two species. Two of the B. cereus strains contained extrachromosomal DNA that hybridized to a cryIA insecticidal toxin probe, and seven strains contained DNA with homology to a Tn4430 transposon probe derived from B. thuringiensis. The results strongly indicate that B. cereus and B. thuringiensis should be regarded as one species.     

Targeting properties of an anti-CD16/anti-CD30 bispecific antibody in an in vivo system

Bispecific antibodies are currently being used in clinical trials in increasing numbers in the areas of breast cancer, prostate cancer, non-Hodgkin's lymphoma and Hodgkin's lymphoma. We have previously performed two clinical trials in patients with Hodgkin's disease with an anti-CD30/anti-CD16 bispecific antibody and demonstrated a 30% response rate in a cohort of patients otherwise resistant to standard therapeutic modalities. However, no surrogate marker could be defined in these trials indicative of optimal antibody dosing/scheduling or predictive for favorable response. In order to evaluate accurately the potential biodistribution properties of bispecific antibody in patients, we have performed a detailed analysis of the binding properties and animal model in vivo characteristics of these constructs. For this purpose, the parental antibodies (anti-CD30 and anti-CD16) and the bispecific antibody (anti-CD30/anti-CD16) were radiolabeled with either ¹²⁵I or ¹¹¹In. Antibody integrity and binding properties after labeling were confirmed by Scatchard plot and Lindmo analysis. ¹¹¹In-labeled antibodies revealed superior targeting properties in a standard SCID mouse tumor model. Both the bivalent parental anti-CD30 monoclonal antibody and the monovalent anti-CD30/anti-CD16 bispecific antibody showed excellent uptake in CD30⁺ tumors which did not differ significantly between the two (maximum uptake 16.5% ± 4.2% vs. 18.4% ± 3.8% injected dose/gram tissue). The equivalent targeting properties of the bispecific antibody compared with the parental anti-CD30 antibody encourages the further clinical development of this bispecific antibody, and might help to explain the clinical responses seen with this antibody so far in patients suffering from Hodgkin's disease. Received: 26 October 2000 / Accepted: 15 December 2000     

Human and mouse mitochondrial orthologs of bacterial ClpX

We have determined the cDNA sequence and exon/intron structure of the human CLPX gene encoding a human ortholog of the E. coli ClpX chaperone and protease subunit. The CLPX gene comprises 14 exons and encodes a 633-amino acid-long precursor polypeptide. The polypeptide contains an N-terminal putative mitochondrial transit peptide, and expression of a full-length ClpX cDNA tagged at its C-terminus (Myc-His) shows that the polypeptide is transported into mitochondria. FISH analysis localized the CLPX gene to human Chromosome (Chr) 15q22.1-22.32. This localization was refined by radiation hybrid mapping placing the CLPX gene 4.6 cR distal to D15S159. Murine ClpX cDNA was sequenced, and the mouse Clpx locus was mapped to a position between 31 and 42 cM offset from the centromere on mouse Chr 9. Experimental observations indicate the presence of a pseudogene in the mouse genome and sequence variability between mouse ClpX cDNAs from different strains. Alignment of the human and mouse ClpX amino acid sequences with ClpX sequences from other organisms shows that they display the typical modular organization of domains with one AAA⁺ domain common to a large group of ATPases and several other domains conserved in ClpX orthologs linked by non-conserved sequences. Notably, a C-4 zinc finger type motif is recognized in human and mouse ClpX. This motif of so far unknown function is present only in a subset of the known ClpX sequences. Received: 5 April 2000 / Accepted: 14 June 2000