Cysteine in the triple helical domain of the proα2(I) chain of type-I collagen in nonlethal forms of osteogenesis imperfecta

Summary To determine if some individuals with deforming varieties of osteogenesis imperfecta (OI) carry point mutations in the COL1A2 gene of type-I collagen, we examined collagens synthesized by cell strains from affected individuals for the presence of cysteine in the triple helical domain of the 2(I) chain, a domain from which it is normally excluded. We identified 4 individuals out of 60 whose cells synthesized a population of 2(I) chains with a cysteine residue in the triple helix. The clinical differences among the affected individuals and the heterogeneity in the locations of the cysteine residues suggest that the position of the substitution within the chain is important in determining the clinical phenotype. These data confirm that individuals with nonlethal OI may commonly harbor defects in the COL1A2 gene, and suggest that many of the defects are substitutions for glycine residues in the 2(I) triple helical domain.     

Evidence for the involvement of (Cu-ATP)2− in the inhibition of human erythrocyte (Ca2+ + Mg2+-ATPase by copper

The effects of copper on the activity of erythrocyte $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ have been tested on membranes stripped of endogenous calmodulin or recombined with purified calmodulin. The interactions of copper with Ca²⁺, calmodulin and (Mg-ATP)^2? were determined by kinetic studies. The most striking result is the potent competitive inhibition exerted by (Cu-ATP)^2? against (Mg-ATP)^2?$\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 2.8 μ}\text{M}$), while free copper gives no characteristic inhibition. Our results also demonstrate that copper does not compete with calcium either on the enzyme or on calmodulin. The fixation of calmodulin on the enzyme is not altered in the presence of copper as shown by the fact that the dissociation constant remains unaffected. It may be speculated that (Cu-ATP)^2? is the active form of copper, which could plausibly be at the origin of some of the pathological features of erythrocytes observed in conditions associated with excess copper.     

Analysis of the meiosis in the F_1 hybrids of Longiflorum × Asiatic(LA) of lilies(Lilium) using genomic in situ hybridization

Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modern lily cultivars.One of the main trends of lily breeding is to realize introgression between these groups.With cut style pollination and embryo rescue,distant hybrids between the two groups have been obtained.However,the F1 hybrids are highly sterile or some of them could produce a small number of 2n gametes,and their BC1 progenies are usually triploids.Dutch lily breeders have selected many cultivars...     

Four variants of human plasma α2-glycoprotein (ZAG) in the Japanese population

Summary Zn-₂-glycoprotein (ZAG) of plasma from the general Japanese adult population (n=1224) was studied by polyacrylamide gel isoelectric focusing (IEF) followed by immunoblotting with specific antiserum to ZAG. Most of the plasmas showed a common band pattern, while 16 samples showed four other patterns. These ZAG band patterns were easily differentiated by desialyzing the samples prior to IEF. The asialo form of ZAG commonly showed a single band. The 16 plasma samples presenting double bands were classified into four types containing the common single band. The differences in ZAG phenotypes may be suggested to be due to amino acid substitutions of the ZAG molecule. The statistical frequencies of five alleles, which we proposed to designate ZAG^*1, ZAG^*2, ZAG^*3, ZAG^*4, and ZAG^*5, were 0.9935, 0.0025, 0.0016, 0.0004, and 0.0020, respectively. The genetic transmission of the rare alleles ZAG^*3 and ZAG^*4 was confirmed by two family studies.     

2-(2-Benzofuranyl)-2-Imidazoline Attenuates the Disruption of the Blood–Brain Barrier in EAE via NMDAR

Blood–brain barrier (BBB) disruption has been recognized as an early hallmark of multiple sclerosis (MS) pathology. Our previous studies have shown that 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) protected against experimental autoimmune encephalomyelitis (EAE), a classic animal model of MS. However, the potential effects of 2-BFI on BBB permeability have not yet been evaluated in the context of EAE. Herein, we aimed to investigate the effect of 2-BFI on BBB permeability in both an animal model and an in vitro BBB model using TNF-α to imitate the inflammatory damage to the BBB in MS. In the animal model, 2-BFI reduced neurological deficits and BBB permeability in EAE mice compared with saline treatment. The Western blot results indicated that 2-BFI not only alleviated the loss of the tight junction protein occludin caused by EAE but also inhibited the activation of the NR1-ERK signaling pathway. In an in vitro BBB model, 2-BFI (100 μM) alleviated the TNF-α-induced increase in permeability and reduction in expression of occludin in monolayer bEnd.3 cells. Similar protective effects were also observed after treatment with the NMDAR antagonist MK801. The Western blot results showed that the TNF-α-induced BBB breakdown and increase in NMDAR subunit 1 (NR1) levels and ERK phosphorylation could be blocked by pretreatment with 2-BFI or MK801. However, no additional effect was observed on BBB permeability or the expression of occludin and p-ERK after pretreatment with both 2-BFI and MK801. Our study indicates that 2-BFI alleviates the disruption of BBB in the context of inflammatory injury similar to that of MS by targeting NMDAR1, as well as by likely activating the subsequent ERK signaling pathway. These results provide further evidence for 2-BFI as a potential drug for the treatment of MS.

     

Ecological Monitoring of the Water Area of Port Vostochnyi (Wrangel Bay) in the Sea of Japan (1995–2002)

Benthic surveys carried out in 1989 (literature data), 1995, and 2001 (original data) revealed changes in the structure of soft bottom communities of Wrangel Bay (Peter the Great Bay, Sea of Japan), where a deep-water port of Vostochnyi is situated. The functioning of the port has caused no significant environmental pollution by heavy metals, hydrocarbons, and other pollutants. Bottom dredging and the siltation induced by it have drastically lowered the benthos biomass, changed benthic communities, and altered their trophic structure. Sestonophages have disappeared nearly altogether being replaced by detrivores. The ecological state of the benthos is characterized as unstable.     

Rapid accumulation of polymorphonuclear neutrophils in the Corpus luteum during prostaglandin F(2α)-induced luteolysis in the cow

Prostaglandin F(2α) (PGF(2α)) induces luteolysis within a few days in cows, and immune cells increase in number in the regressing corpus luteum (CL), implying that luteolysis is an inflammatory-like immune response. We investigated the rapid change in polymorphonuclear neutrophil (PMN) numbers in response to PGF(2α) administration as the first cells recruited to inflammatory sites, together with mRNA of interleukin-8 (IL-8: neutrophil chemoattractant) and P-selectin (leukocyte adhesion molecule) in the bovine CL. CLs were collected by ovariectomy at various times after PGF(2α) injection. The number of PMNs was increased at 5 min after PGF(2α) administration, whereas IL-8 and P-selectin mRNA increased at 30 min and 2 h, respectively. PGF(2α) directly stimulated P-selectin protein expression at 5-30 min in luteal endothelial cells (LECs). Moreover, PGF(2α) enhanced PMN adhesion to LECs, and this enhancement by PGF(2α) was inhibited by anti-P-selectin antibody, suggesting that P-selectin expression by PGF(2α) is crucial in PMN migration. In conclusion, PGF(2α) rapidly induces the accumulation of PMNs into the bovine CL at 5 min and enhances PMN adhesion via P-selectin expression in LECs. It is suggested that luteolytic cascade by PGF(2α) may involve an acute inflammatory-like response due to rapidly infiltrated PMNs.     

Characterization of the Distal Polyadenylation Site of the ?-Adducin (Add2) Pre-mRNA

Most genes have multiple polyadenylation sites (PAS), which are often selected in a tissue-specific manner, altering protein products and affecting mRNA stability, subcellular localization and/or translability. Here we studied the polyadenylation mechanisms associated to the beta-adducin gene (Add2). We have previously shown that the Add2 gene has a very tight regulation of alternative polyadenylation, using proximal PAS in erythroid tissues, and a distal one in brain. Using chimeric minigenes and cell transfections we identified the core elements responsible for polyadenylation at the distal PAS. Deletion of either the hexanucleotide motif (Hm) or the downstream element (DSE) resulted in reduction of mature mRNA levels and activation of cryptic PAS, suggesting an important role for the DSE in polyadenylation of the distal Add2 PAS. Point mutation of the UG repeats present in the DSE, located immediately after the cleavage site, resulted in a reduction of processed mRNA and in the activation of the same cryptic site. RNA-EMSA showed that this region is active in forming RNA-protein complexes. Competition experiments showed that RNA lacking the DSE was not able to compete the RNA-protein complexes, supporting the hypothesis of an essential important role for the DSE. Next, using a RNA-pull down approach we identified some of the proteins bound to the DSE. Among these proteins we found PTB, TDP-43, FBP1 and FBP2, nucleolin, RNA helicase A and vigilin. All these proteins have a role in RNA metabolism, but only PTB has a reported function in polyadenylation. Additional experiments are needed to determine the precise functional role of these proteins in Add2 polyadenylation.     

Cloning and expression of the α–L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae

 First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abf B) was amplified with the polymerase chain reaction technique. The abf B DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1 _P ) and terminator (PGK1 _T ) sequences on a multicopy episomal plasmid. The resulting construct PGK1 _P -abf B-PGK1 _T was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf 2) that is 94% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively. Received: 29 January 1996/Received revision: 3 May 1996/Accepted: 9 May 1996     

Combination of Azidothymidine (AZT) and (E)-5-(2-Bromovinyl)-2′-deoxyuridine (BVDU) Inhibits the Replication of Herpes Simplex Virus Type 1 (HSV-1) and Type 2 (HSV-2) and Varicella Zoster Virus (VZV) Strains That Are Deficient in the Expression of the Viral Thymidine Kinase (tk)

Abstract

Combinations of high concentrations of AZT with BVDU, acyclovir (ACV) or ganciclovir (GCV) show antagonism against TK⁺ HSV-1, but not TK⁺ VZV strains, in cell cultures. When BVDU and AZT were used in combination against TK^? HSV-1, TK^? HSV-2 and TK^? VZV strains, a pronounced inhibition of viral replication was observed. This potentiating effect was not seen if AZT was combined with ACV or GCV.     

Acidification of lakes in Šumava (Bohemia) and in the High Tatra Mountains (Slovakia)

Acidification of lakes takes place when pH of rainwater is less than 4.5 and the catchments lie on sensitive geology. Both conditions are met for most lakes in Bohemia and Slovakia. Since 1978 we have studied mountain lakes in the Sumava and in the High Tatra Mountains. In Šumava the three lakes under study are of glacial origin. The catchments are small, with steep sides covered by spruce. The bedrocks are biotite-rich paragneiss, together with gneiss, quartzite and granite. In summer 1936 surface pH was 5.7–6.9 in the Lake Čertovo and 6.9–7.0 in the Lake Černé. Now the pH values are 4.3–4.8 in the two lakes and in the Lake Prášilské as well. Old reports on zooplankton are from the years 1871, 1892–96, 1935–37, 1947 and 1960. Since 1979 we have not found any planktonic Crustacea in the lakes Černé and Čertovo. Lake Prášilské is inhabited by Daphnia longispina and Cyclops abyssorum. In July 1989 the pH values were 4.4, 4.7 and 4.7, concentrations of labile monomeric Al were 0.83, 0.68 and 0.24 mg l^-1 in the lakes Čertovo, Černé and Prašilské, respectively. High levels of toxic Al compounds might be responsible for the extinction of planktonic Crustacea in the lakes Čertovo and Černé. All the three lakes are void of fish at present. In the High Tatra Mts. we examined more than 40 lakes above timberline in altitudes 1612–2145 m. They are all clearwater, naturally fishless lakes. The bedrock is granite. Owing to different levels of calcium the lakes are now in different stages of acidification. According to recent changes in the zooplankton they can be divided into three groups: (1) Species composition of planktonic Crustacea has not changed. (2) Planktonic Crustacea were present until 1973 but are absent now. (3) From the original species of Crustacea only Chydorus sphaericus is present. The three groups are well separated along the gradients of calcium, ANC and pH. They can be identified with the Henriksen's bicarbonate (our group 1), intermediate (our group 2) and acid (our group 3) lakes. We suppose that in the process of acidification the lakes of the group (2) have been shifted from oligotrophy to ultraoligotrophy.     

A two-site immunometric assay for the detection of the expression level of α2(C2)-adrenergic receptor produced in Escherichia coli

Human ₂(C2)-adrenergic receptor was expressed in Escherichia coli as a fusion protein with Bacillus circulans var. alcalophilus cyclomaltodextrin glucanotransferase. For the determination of the expression level (0.6 mg of solubilized fusion protein l^–1 of E. coli culture), a two-site immunometric assay based on two monoclonal antibodies with different epitopes was developed.     

Role of the guanidine group in the N-terminal fragment of PTH(1–11)

A series of PTH hybrids containing a diamine [NH₂(CH₂) _n NH₂; n = 4, 5, 6] in the C-terminal position was synthesized based on the H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH₂ (Har = homoarginine) template. The compounds were pharmacologically characterized at PTH1R receptors for agonist activity.     

Residues 240–250 in the C-Terminus of the Pirh2 Protein Complement the Function of the RING Domain in Self-Ubiquitination of the Pirh2 Protein

Pirh2 is a p53 inducible gene that encodes a RING-H2 domain and is proposed to be a main regulator of p53 protein, thus fine tuning the DNA damage response. Pirh2 interacts physically with p53 and promotes its MDM2-independent ubiquitination and subsequent degradation as well as participates in an auto-regulatory feedback loop that controls p53 function. Pirh2 also self-ubiquitinates. Interestingly, Pirh2 is overexpressed in a wide range of human tumors. In this study, we investigated the domains and residues essential for Pirh2 self-ubiquitination. Deletions were made in each of the three major domains of Pirh2: the N-terminal domain (NTD), Ring domain (RING), and C-terminal domain (CTD). The effects of these deletions on Pirh2 self-ubiquitination were then assessed using in vitro ubiquitination assays. Our results demonstrate that the RING domain is essential, but not sufficient, for Pirh2 self-ubiquitination and that residues 240–250 of the C-terminal domain are also essential. Our results demonstrate that Pirh2 mediated p53 polyubiquitination occurs mainly through the K48 residue of ubiquitin in vitro. Our data further our understanding of the mechanism of Pirh2 self-ubiquitination and may help identify valuable therapeutic targets that play roles in reducing the effects of the overexpression of Pirh2, thus maximizing p53''s response to DNA damage.     

The role of food in the population dynamics of Orchesella cincta (Linné) (Collembola)

Summary The feeding activity of the collembolan species Orchesella cincta (Linné) was studied in the laboratory and in the field. In the laboratory it was shown that at any one time only about 50–60% of the animals were feeding; the remainder were occupied with molting activities. Most of the time similar values were obtained in the field but during winter and during dry summer periods high percentages of empty guts could be demonstrated, indicating an absence or inaccessability of food. When favorable weather returned, a high feeding activity was again observed. Laboratory experiments and field observations indicate that temporary starvation periods are followed by synchronization of the molting and reproductive rhythms and an apparent increase in longevity. These phenomena are important for the restoration of the population after unfavorable periods but reduce the stabilizing force of food scarcity.