Proteolysis and tyrosine phosphorylation of p34cdc2/cyclin B. The role of MCM2 and initiation of DNA replication to allow tyrosine phosphorylation of p34cdc2

Previously, it has been shown that Aspergillus cells lacking the function of nimQ and the anaphase-promoting complex (APC) component bimEAPC1 enter mitosis without replicating DNA. Here nimQ is shown to encode an MCM2 homologue. Although mutation of nimQMCM2 inhibits initiation of DNA replication, a few cells do enter mitosis. Cells arrested at G1/S by lack of nimQMCM2 contain p34(cdc2)/cyclin B, but p34(cdc2) remains tyrosine dephosphorylated, even after DNA damage. However, arrest of DNA replication using hydroxyurea followed by inactivation of nimQMCM2 and bimEAPC1 does not abrogate the S phase arrest checkpoint over mitosis. nimQMCM2, likely via initiation of DNA replication, is therefore required to trigger tyrosine phosphorylation of p34(cdc2) during the G1 to S transition, which may occur by inactivation of nimTcdc25. Cells lacking both nimQMCM2 and bimEAPC1 are deficient in the S phase arrest checkpoint over mitosis because they lack both tyrosine phosphorylation of p34(cdc2) and the function of bimEAPC1. Initiation of DNA replication, which requires nimQMCM2, is apparently critical to switch mitotic regulation from the APC to include tyrosine phosphorylation of p34(cdc2) at G1/S. We also show that cells arrested at G1/S due to lack of nimQMCM2 continue to replicate spindle pole bodies in the absence of DNA replication and can undergo anaphase in the absence of APC function.     

A biomechanical comparison of intramedullary nail and crossed lag screw fixation for tibiotalocalcaneal arthrodesis

A series of Pyrazolo[1,5-a]pyrido[3,4-e]pyrimidin-6-ones (4a-p) was prepared by a simple synthetic procedure based on the reaction of hydroxylamine or methoxyamine with 2,3-substituted ethyl 7-dimethylaminovinyl pyrazolo[1,5-a]pyrimidin-6-carboxylates (3a-p). The antimicrobial activity of the obtained compounds was evaluated on a series of standard strains of Gram positive, Gram negative bacteria and fungi. None of the tested compounds showed significant activity.     

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.     

Effects of postruminal protein on fatty acid digestibility in dairy cows

OBJECTIVES: We investigated the relation between insulin and coronary atherosclerosis and restenosis of the coronary arteries, by performing elective percutaneous transluminal coronary angioplasty (PTCA). BACKGROUND: Insulin is known to promote atherosclerosis of the arteries and has been implicated in the development of restenosis after PTCA. METHODS: Of 210 angina patients who underwent PTCA, newly detected lesions in 35 consecutive nondiabetic subjects without previous intervention on the same main coronary arteries were analyzed after a 75-g oral glucose tolerance test (OGTT) and follow-up coronary angiography. Atherosclerotic lesions were evaluated by pattern, severity and extent. Restenosis was defined as loss of gain, the percentage of loss of the initial gain in the coronary diameter achieved by PTCA > or = 50%. RESULTS: Patients with restenosis had a significantly higher extent index (a marker of atherosclerosis), insulin area, ratio of insulin area to glucose area, insulinogenic index and minimal lumen diameter after PTCA than those without restenosis (p=0.001, 0.011, 0.002, 0.016 and 0.041, respectively). Simple regression analysis revealed that only the ratio of insulin area to glucose area (a relative marker of enhanced insulin secretion) significantly correlated with the extent index (p=0.035). Extent index, insulin area, the ratio of insulin area to glucose area and insulinogenic index significantly correlated with loss of gain (p=0.001, 0.010, 0.002 and 0.032, respectively). Stepwise multiple regression analyses revealed that extent index and the ratio of insulin area to glucose area significantly correlated with loss of gain. CONCLUSIONS: Enhanced secretion of insulin during the OGTT might be useful as a predictor of coronary atherosclerosis and of restenosis after elective PTCA in nondiabetic patients with effort angina.     

The dynamics of an infectious disease in a population with birth pulses

In December 1996, a questionnaire about farm management and parasite control measures in calves was sent to 956 randomly chosen dairy cattle farmers in The Netherlands. Another 150 farmers in the vicinity of Deventer who had vaccinated their calves in 1995 against lungworm were approached with the same questions. Our object was to investigate the consequences on worm control of the withdrawal of the lungworm vaccine from the market for reasons of possible BSE contamination of the vaccine. OF the returned questionnaires, 411 (43%) of the 'at random' group and 89 (59.3%) of the 'Deventer' group were valid. The most important data with regard to the farms of the 'at random' group (41) were: mean area 31.6 ha, mean number of calves 23, heifers 23 and milking cows 53. Sheep (mean 37) were present on 18.3% of the farms. With regard to management: 74.5% of the farmers turned the calves in their first year onto pasture, 25.5% kept them indoors. The average time on pasture was ca. 5 months. Rational grazing was practise on 81.4% of the farms, on 18.6% calves were set stocked. The first pasture of the calves was mown before turn-out on 72.9% of the farms. On 48.2% of these farms, calves were always moved to mown pastures. With regard to treatments: 33.8% of the farmers vaccinated their calves against lungworm in the years 1993, 1994 and 1995. Despite the withdrawal of the vaccine from the market in 1996, 7.2% of the farmers vaccinated their calves as recommended, with two doses, and 13.1% with a single dose. At turn-out, 41.5% of the farmers gave the calves a preventive anthelmintic treatment. Of these treatments, 66.9% were sustained of pulse release long acting device. During the grazing season, 36.6% of the farmers treated their calves. After housing 50.3% of the farmers gave a treatment. Signs of lungworm infection were noticed on 18.6% of the farms. Of the 'Deventer' group (89 farmers), 96.6% turned the calves out, Of these farmers, 86.0% had used the lungworm vaccine in 1995. In 1996, 52.7% of the farmers had vaccinated the calves:36.5% with a single dose and 16.2% with the double dose. Of the 35 farmers who did not vaccinate in 1996, 62.9% gave a preventive treatment at turn-out. Clinical signs of lungworm infection were not observed on the 12 farms which vaccinated the calves twice. On 11% of the farms which vaccinated once and on 14% of the farms which did not vaccinate, signs of lungworm infection were observed. It is concluded that more than 80% of Dutch dairy cattle farmers take appropriate measures to control gastrointestinal nematode and lungworm infections in calves in their first grazing season by grazing on aftermath, rotational grazing on mown pastures combined or not with preventive anthelmintic treatments. However, combinations of aftermath grazing and preventive treatment occurred on 30% of the farms. This may be overprotective and may prevent sufficient build up of immunity, causing worm problems at a later age. The withdrawal of the lungworm vaccine from the market did not cause a rise in lungworm problems. Some farmers did vaccinate, despite the withdrawal. The majority used other preventive treatment measures, mainly the application of long acting boli.     

Genetics of the hydrophilic surfactant proteins A and D

The use of candidate genes has increased the ability to identify genetic factors involved in diseases with complex and multifactorial etiology. The surfactant proteins (SP) A and D are involved in host defense and inflammatory processes of the lung, which are often components of pulmonary disease. Therefore, the SP-A and SP-D genes make particularly good candidates to study factors contributing to pulmonary disease etiopathogenesis. Moreover, SP-A also plays a role in the surface tension lowering abilities of pulmonary surfactant, which is essential for normal lung function. Although genetic variability at the SP-D locus may exist among humans, allelic variants have not yet been characterized. On the other hand, the human SP-A genes (SP-A1 and SP-A2) are characterized by genetically dependent splice variants at the 5' untranslated region and allelic variants. The polymorphisms that give rise to SP-A1 and SP-A2 alleles are contained within coding regions, potentially having an effect on protein function. There appears to be a correlation between SP-A genotype and SP-A mRNA content. Furthermore, one SP-A2 allele (1A0) shown to associate with low SP-A mRNA levels is found with higher frequency in a subgroup with respiratory distress syndrome. The evidence gathered thus far indicates that SP-A, possibly by interacting with other surfactant components, may play a role (e.g. be a susceptibility factor) in the development of respiratory disease.     

Biochemical and morphometric analyses show that myelination in the insulin-like growth factor 1 null brain is proportionate to its neuronal composition

To elucidate the role of insulin-like growth factor 1 (IGF1) in the normal development of brain myelination, we used behavioral, biochemical, and histological analyses to compare the myelination of brains from Igf1(-/-) and wild-type (WT) littermate mice. The studies were conducted at postnatal day 40, at which time the Igf1(-/-) mice weighed approximately 66% less than wild-type mice. However, the Igf1(-/-) brain weight was only reduced by approximately 34%. Formal neurological testing showed no sign of central or peripheral myelinopathy in Igf1(-/-) mice. Myelin composition was not significantly different, and myelin concentration, normalized to brain weight or protein, was equal in Igf1(-/-) and WT mice. Likewise, concentrations of myelin-specific proteins (MBP, myelin proteolipid protein, MAG, and 2',3'-cyclic nucleotide,3'-phosphodiesterase) were not significantly different in Igf1(-/-) and WT mice. The myelin-associated lipids galactocerebroside and sulfatide were modestly reduced in Igf1(-/-) brains. Regional oligodendrocyte populations and myelin staining patterns were comparable in Igf1(-/-) and WT brains, with the notable exception of the olfactory system. The Igf1(-/-) olfactory bulb was profoundly reduced in size and was depleted of mitral neurons and oligodendrocytes, and its efferent tracts were depleted of myelin. In summary, this study shows that myelination of the Igf1(-/-) brain is proportionate to its neuronal composition. Where projection neurons are preserved despite the deletion of IGF1, as in the cerebellar system, oligodendrocytes and myelination are indistinguishable from wild type. Where projection neurons are depleted, as in the olfactory bulb, oligodendrocytes are also depleted, and myelination is reduced in proportion to the reduced projection neuron mass. These data make a strong case for the primacy of axonal factors, not including IGF1, in determining oligodendrocyte survival and myelination.     

Maintenance of myonuclear domain size in rat soleus after overload and growth hormone/IGF-I treatment

The purpose of this study was to determine the effects of functional overload (FO) combined with growth hormone/insulin-like growth factor I (GH/IGF-I) administration on myonuclear number and domain size in rat soleus muscle fibers. Adult female rats underwent bilateral ablation of the plantaris and gastrocnemius muscles and, after 7 days of recovery, were injected three times daily for 14 days with GH/IGF-I (1 mg/kg each; FO + GH/IGF-I group) or saline vehicle (FO group). Intact rats receiving saline vehicle served as controls (Con group). Muscle wet weight was 32% greater in the FO than in the Con group: 162 +/- 8 vs. 123 +/- 16 mg. Muscle weight in the FO + GH/IGF-I group (196 +/- 14 mg) was 59 and 21% larger than in the Con and FO groups, respectively. Mean soleus fiber cross-sectional area of the FO + GH/IGF-I group (2,826 +/- 445 microm2) was increased compared with the Con (2,044 +/- 108 microm2) and FO (2,267 +/- 301 microm2) groups. The difference in fiber size between the FO and Con groups was not significant. Mean myonuclear number increased in FO (187 +/- 15 myonuclei/mm) and FO + GH/IGF-I (217 +/- 23 myonuclei/mm) rats compared with Con (155 +/- 12 myonuclei/mm) rats, although the difference between FO and FO + GH/IGF-I animals was not significant. The mean cytoplasmic volume per myonucleus (myonuclear domain) was similar across groups. These results demonstrate that the larger mean muscle weight and fiber cross-sectional area occurred when FO was combined with GH/IGF-I administration and that myonuclear number increased concomitantly with fiber volume. Thus there appears to be some mechanism(s) that maintains the myonuclear domain when a fiber hypertrophies.     

Histopathologic validation of the sentinel lymph node hypothesis for breast carcinoma

BACKGROUND AND OBJECTIVE: The sentinel node hypothesis assumes that a primary tumor drains to a specific lymph node in the regional lymphatic basin. To determine whether the sentinel node is indeed the node most likely to harbor an axillary metastasis from breast carcinoma, the authors used cytokeratin immunohistochemical staining (IHC) to examine both sentinel and nonsentinel lymph nodes. METHODS: From February 1994 through October 1995, patients with breast cancer were staged with sentinel lymphadenectomy followed by completion level I and II axillary dissection. If the sentinel node was free of metastasis by hematoxylin and eosin staining (H&E), then sentinel and nonsentinel nodes were examined with IHC. RESULTS: The 103 patients had a median age of 55 years and a median tumor size of 1.8 cm (58.3% T1, 39.8% T2, and 1.9% T3). A mean of 2 sentinel (range, 1-8) and 18.9 nonsentinel (range, 7-37) nodes were excised per patient. The H&E identified 33 patients (32%) with a sentinel lymph node metastasis and 70 patients (68%) with tumor-free sentinel nodes. Applying IHC to the 157 tumor-free sentinel nodes in these 70 patients showed an additional 10 tumor-involved nodes, each in a different patient. Thus, 10 (14.3%) of 70 patients who were tumor-free by H&E actually were sentinel node-positive, and the IHC lymph node conversion rate from sentinel node-negative to sentinel node-positive was 6.4% (10/157). Overall, sentinel node metastases were detected in 43 (41.8%) of 103 patients. In the 60 patients whose sentinel nodes were metastasis-free by H&E and IHC, 1087 nonsentinel nodes were examined at 2 levels by IHC and only 1 additional tumor-positive lymph node was identified. Therefore, one H&E sentinel node-negative patient (1.7%) was actually node-positive (p < 0.0001), and the nonsentinel IHC lymph node conversion rate was 0.09% (1/1087; p < 0.0001). CONCLUSIONS: If the sentinel node is tumor-free by both H&E and IHC, then the probability of nonsentinel node involvement is <0.1%. The true false-negative rate of this technique using multiple sections and IHC to examine all nonsentinel nodes for metastasis is 0.97% (1/103) in the authors' hands. The sentinel lymph node is indeed the most likely axillary node to harbor metastatic breast carcinoma.     

Identification of ligand binding sites on integrin alpha4beta1 through chemical cross-linking

We have used chemical cross-linking to identify sequences in integrin alpha4beta1 that are involved in its interactions with ligands. A recently described leucine-aspartic acid-valine (LDV)-based small molecule inhibitor of alpha4beta1 (BIO-1494), that contained a single reactive amino group for targeting the cross-linking, was used for these studies. The specificity of the interaction was defined by (i) the ability to block the interaction with a competitive inhibitor lacking the reactive group, (ii) the absolute requirement of divalent cations for cross-linking, and (iii) the lack of cross-linking to the functionally related integrin alpha4beta7. With ANB-NOS as the cross-linker, only the beta1 chain was labeled with BIO-1494, while with the more flexible cross-linker DSS both the alpha4 and beta1 chains were modified. Similar results were obtained when cross-linking was performed on K562 cells expressing alpha4beta1 but not on K562 cells expressing alpha2beta1. The site of cross-linking on the beta1 chain was localized by CNBr peptide mapping within residues 130-146, a region that contains the putative metal binding site DXSXS and for which analogous data had been generated with RGD binding to integrin alphaIIbbeta3. The striking similarity between the data we generated for an LDV ligand and published data for the RGD family supports the notion of a common ligand binding pocket formed by both integrin chains. The cross-linking strategy developed here should serve as a useful tool for studying alpha4beta1 function.