Apoptosis Susceptibility Genes on Mouse Chromosome 9 (Rapop2) and Chromosome 3 (Rapop3)

The strain distribution pattern of susceptibility to thymocyte apoptosis induced by ionizing radiation in 20 CcS/Dem recombinant congenic (RC) strains derived from the strains BALB/cHeA (susceptible) and STS/A (resistant) indicates that this trait is controlled by several genes. Recently, we mapped a novel apoptosis susceptibility gene Rapop1 (radiation-induced apoptosis 1) to chromosome 16 (N. Mori et al., 1995, Genomics 25: 604-614). In the present study, the analysis of F2 crosses between the resistant RC strain CcS-8 and the susceptible strain BALB/cHeA or the highly susceptible RC strain CcS-10 demonstrated two additional apoptosis susceptibility genes, Rapop2 and Rapop3, located in the proximal region of chromosome 9 and the telomeric region of chromosome 3, respectively. The possible candidate genes for these loci are discussed.     

The Tight Skin (Tsk) Mutation in the Mouse, a Model for Human Fibrotic Diseases, Is Tightly Linked to the β2-Microglobulin (B2m) Gene on Chromosome 2

The Tsk mutation in the mouse is characterized by the excessive accumulation of collagen in skin and various internal organs, including the heart and lungs. These connective tissue abnormalities are similar to those present in human systemic sclerosis or scleroderma. The Tsk mutation provides an opportunity to investigate, at the molecular level, the pathogenesis of tissue fibrosis. As a first step to cloning the Tsk gene, we report the localization of the Tsk mutation with respect to known molecular markers on mouse chromosome 2. N2 progeny carrying the Tsk mutation were obtained from an intersubspecific backcross of [(C57BL/6-pa +/+ Tsk × Mus castaneus)F1 × M. castaneus ] mice. Genomic DNA from each N2 mouse was subjected to Southern and PCR analyses to identify restriction fragment length polymorphisms and simple sequence length polymorphisms, respectively. Our results refine the location of Tsk to a 3-cM region, eliminate several genes from consideration as the Tsk mutation, identify molecular probes tightly linked with Tsk, and suggest candidate genes responsible for the Tsk phenotype.     

η-Allyl-cobalt (II) complexes

(η³-Cyclooctenyl)Co(bisphosphine) compounds react with HBF₄ in the presence of alkenes with oxidation of the metal to give the novel, paramagnetic organocobalt(II) species [(η³-cyclooctenyl)Co(bisphosphine)]⁺BF₄⁻, (η³-2-RC₃H₄)Co(bisphosphine) complexes react similarly. The Co(II) compounds form adducts with CO and NO (the latter being diamagnetic) and undergo facile chemical and electrochemical reduction.     

Separation and concentration of Δ-6-keto-PGF1α using monoclonal antibody to ω3-olefin structure of trienoic prostanoids

A monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate Δ¹⁷-6-keto-prostaglandin F_1α (PGF_1α) in the human sera. cis-3-Hexen-1-ol was conjugated with the human serum albumin (HSA) according to the N-succinimidylester method and hyperimmunized to BALB/c mouse. The monoclonal afntibodies were obtained from hybridoma clones established by a fusion between SP2/0-Ag14-k13 mouse myeloma cells and splenocytes of a mouse. A monoclonal antibody, named 4G9-12B, recognized the epitope characteristic for ω3-olefin structure. The 4G9-12B antibody became more specific for Δ¹⁷-6-keto-PGF_1α than 6-keto-PGF_1α by applying inhibition ELISA using amino-residue coating plates. Using the prepared immunoaffinity columns of this antibody, Δ¹⁷-6-keto-PGF_1α was clearly detected in 6 pg/ml of the human blood sera by GC/MS analysis. These results suggest that the monoclonal antibody to the partial structure of trienoic prostanoid, ω3-olefin unit, and that its immunoaffinity columns are useful in separating and concentrating Δ¹⁷-6-keto-PGF_1α in the human blood or urine.     

TheCmv1Host Resistance Locus Is Closely Linked to theLy49Multigene Family within the Natural Killer Cell Gene Complex on Mouse Chromosome 6

Natural killer (NK) cells play important roles in controlling tumor cells and against a range of infectious organisms. Recent studies of mouse NK cell surface receptors, which may be involved in the specificity of NK cells, have shown that many of these molecules are encoded by theLy49andLy55(Nkrp1) multigene families that map to distal mouse chromosome 6. Also mapping to this NK cell gene complex (NKC) is the resistance locus,Cmv1,which is involved in genetically determined resistance to murine cytomegalovirus (MCMV). The aim of this study was to localizeCmv1more precisely in relation to other NKC loci by generating a high-resolution genetic map of the region. We have analyzed 1250 backcross mice comprising panels of 700 (BALB/c × C57BL/6J)F₁× BALB/c and 550 (A/J × C57BL/6J)F₁× A/J progeny. A total of 25 polymorphic genes or microsatellite markers were analyzed over a region of 10 map units fromD6Mit134toD6Mit59.TheCmv1phenotypes of mice recombinant in this interval were tested by infection with MCMV. The results obtained indicate that the functionally important NKC region is a tightly linked cluster of loci spanning at least 0.4 map units. Furthermore,Cmv1maps distal to, but very closely linked to, theLy49multigene family (<0.2 map units), suggesting that MCMV resistance may be conferred by MHC class I-specific NK cell receptors.     

Bis(η-allyl) (η-pentalene) zirconium: preparation, structure and structural dynamics

The preparation of a novel mononuclear complex of zirconium having an η⁸-bonded pentalene ligand and two η³-allyl groups is described. Its structure has been determined by ¹H and ¹³C NMR spectroscopy. At room temperature some of the NMR signals are broadened, revealing that the compound is structurally dynamic. It is shown that the compound has C₂ symmetry with the enantiomeric forms undergoing racemisation.     

Neuro-steroids: 3β-hydroxy-Δ5-derivatives in the rodent brain

Pregnenolone, dehydroepiandrosterone and their sulfate esters have been characterized in the rat brain. Their formation or accumulation depend on in situ mechanisms unrelated to the peripheral endocrine glands. Although their functions are still poorly understood, they may affect the brain by metabolism to sex steroid hormones and they may be functionally related to sexual behavior, possibly through direct modulations of the firing rates of neurons.     

A Novel Putative Transporter Maps to the Osteosclerosis (oc) Mutation and Is Not Expressed in theocMutant Mouse

The phenotype of mice homozygous for the osteosclerosis (oc) mutation includes osteopetrosis, and a variety of studies demonstrate that osteoclasts in these mice are present but nonfunctional. We have identified a novel gene that has homology to a family of 12-transmembrane domain proteins with transport functions and maps to proximal mouse chromosome 19, in a region to which theocmutation has been previously assigned. The putative transporter is abundant in normal kidney, but its expression is markedly reduced in kidneys fromoc/ocmice when tested using Northern and Western analyses. Southern analysis of this gene, which we callRoct(reduced inoctransporter), demonstrates that it is intact and unrearranged inoc/ocmice.In situstudies show thatRoctis expressed in developing bone. We propose that the absence ofRoctexpression results in an osteopetrosis phenotype in mice.     

Dominant Lethality of the Mouse Skeletal Mutation Tail-short (Ts) Is Determined by theTsAllele from Mating Partners

Mice with the Tail-short (Ts) mutation have a short, kinky tail and numerous skeletal abnormalities, including a homeotic anteroposterior patterning problem involving the axial skeleton. The viability ofTsheterozygotes varies dramatically, depending on the mouse strain crossed with the mutant strain. At the extremes, the heterozygotes are viable or lethal prenatally. In this study, we found that laboratory mouse strains could be divided into two groups. A cross with strains from the first group yielded viableTsheterozygotes, whereas a cross with the second group resulted in dominant lethalityin utero.We planned to map the gene(s) that controls strain differences in the viability of theTsheterozygotes. The result clearly indicated that a single chromosomal region, genetically inseparable from theTslocus, is responsible for these differences. This suggests that allelism at theTslocus generates variable manifestation of the mutant phenotype. Morphological and histological analyses indicated that embryos from the lethal cross exhibit severe developmental defects from the gastrulation stage through the early fetal stage. In particular, the umbilical vein does not develop properly. All of these results suggest that the phenotype of theTsmutant is modified by theTsalleles of the mating partners.     

ILSMRs (intensifier of β-lactam-susceptibility in methicillin-resistant Staphylococcus aureus) from Tara [Caesalpinia spinosa (Molina) Kuntze]

Four quinic acid gallates were isolated from the dried pods of Tara [Caesalpinia spinosa (Molina) Kuntze]. These compounds intensified the susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) to oxacillin. 3,4,5-Tri-O-galloylquinic acid methyl ester (2) was the most effective compound of them.     

Assignment of the βB1 Crystallin Gene (CRYBB1) to Human Chromosome 22 and Mouse Chromosome 5

By using primers complementary to the rat βB1 crystallin gene sequence, we amplified exons 5 and 6 of the orthologous human gene (CRYBB1). The amplified human segments displayed greater than 88% sequence homology to the corresponding rat and bovine sequences.CRYBB1was assigned to the group 5 region in 22q11.2–q12.1 by hybridizing the exon 6 PCR product to somatic cell hybrids containing defined portions of human chromosome 22. The exon 5 and exon 6 PCR products ofCRYBB1were used to localize, by interspecific backcross mapping, the mouse gene (Crybb1) to the central portion of chromosome 5. Three other β crystallin genes (βB2(−1), βB3, and βA4) have previously been mapped to the same regions in human and mouse. We demonstrate that the βB1 and βA4 crystallin genes are very closely linked in the two species. These assignments complete the mapping and identification of the human and mouse homologues of the major β crystallins genes that are expressed in the bovine lens.     

Preparation of poly(ε-caprolactone)/poly(trimethylene carbonate) blend nanofibers by electrospinning

Poly(ε-caprolactone) (PCL)/poly(trimethylene carbonate) (PTMC) blend nanofibers have been prepared for the first time using an electrospinning process. The mixed dichloromethane (DCM) and N,N-dimethylformamide (DMF) (75/25, v/v) was found to be the most suitable solvent for electrospinning. Various blends of PCL/PTMC solutions were investigated for the formation of nano-scale fibers and it was found that the average diameter of the fibers was reduced and the morphology became finer when PTMC content was increased. FT-IR and DSC analysis indicated that the molecular interactions between PCL and PTMC were weak and they were phase-separated in the fibers. Due to the biocompatible properties of PCL and PTMC, the spun nanofibers developed here could have applications in the biomedical field.     

Mapping of the Gracile Axonal Dystrophy (gad) Gene to a Region between D5Mit197 and D5Mit113 on Proximal Mouse Chromosome 5

The gracile axonal dystrophy (gad) mouse, which shows hereditary sensory ataxia and motor paresis, has been morphologically characterized by the dying back type of axonal degeneration in the nerve terminals of dorsal root ganglion cells and motor neurons. In the present study, using an intraspecific backcross between gad and C57BL/6J mice, the gracile axonal dystrophy (gad) gene was mapped to a region between D5Mit197 and D5Mit113. Estimated distances between gad and D5Mit197 and between gad and D5Mit113 are 0.4 ± 0.3 and 5.0 ± 1.0 cM, respectively. The gene order was defined: centromere- D5Mit81-D5Mit233-D5Mit184/D5Mit254-D5Mit256-D5Mit197-gad-D5Mit113-D5Mit7 . The mouse map location of the gad locus appears to be in a region homologous to human 4p15-p16. Our present data suggest that the nearest flanking marker D5Mit197 provides a useful anchor for the isolation of the gad gene in a yeast artificial chromosome contig.     

Differential steric effects in the axial ligation of pyridine and imidazole to α- and β-alkylcobinamides

One subclass of B₁₂-requiring enzymes is now known to bind their B₁₂ coenzymes “base-off,” with a histidine residue from the protein supplying an imidazole ligand to the cobalt center. Recent results from Sirovatka and Finke (J.M. Sirovatka and R.G. Finke, J.Am. Chem. Soc. 119, (1997) 3057) show that imidazole has an extraordinary trans effect on the mode of carbon–cobalt bond cleavage in coenzyme B₁₂ analogs, compared to pyridine or the natural 5,6-dimethylbenzimidazole ligand, and it was suggested that a differential steric effect could, in part, account for the uniqueness of the imidazole ligand. Such a differential steric effect for imidazole and pyridine is now demonstrated by studies of the thermodynamics of ligation of these ligands to the α and β diastereomers of two alkylcobinamides (RCbi⁺s, derivatives of cobalamins which lack the normal axial nucleotide) based on the known differences in steric crowding of the α (“lower”) and β (“upper”) axial ligand positions of cobalt corrinoids. Imidazole binds more tightly than pyridine to both diastereomers of NCCH₂Cbi⁺ and CF₃Cbi⁺, in all cases due to a more favorable entropy change, which is the result of lowered steric interference with corrin side chain thermal motions.     

Antibacterial activity of α-mangostin against vancomycin resistant Enterococci (VRE) and synergism with antibiotics

alpha-Mangostin, isolated from the stem bark of Garcinia mangostana L., was found to be active against vancomycin resistant Enterococci (VRE) and methicillin resistant Staphylococcus aureus (MRSA), with MIC values of 6.25 and 6.25 to 12.5 microg/ml, respectively. Our studies showed synergism between alpha-mangostin and gentamicin (GM) against VRE, and alpha-mangostin and vancomycin hydrochloride (VCM) against MRSA. Further studies showed partial synergism between alpha-mangostin and commercially available antibiotics such as ampicillin and minocycline. These findings suggested that alpha-mangostin alone or in combination with GM against VRE and in combination with VCM against MRSA might be useful in controlling VRE and MRSA infections.     

Preparation of trimethylsilyl alkyne complexes of bis(pentamethylcyclopentadienyl)zirconium, (η-C5Me5)2Zr(RC≡CSiMe3), and their regioselective reactions with nitrous oxide

Benzene solutions of Cp^*₂ZrCl₂ (1) (Cp^* = η⁵-C₅Me₅) react with the alkynes Me₃SiC≡CPh, Me₃SiC≡C(c-C₅H₉) and Me₃SiC≡CCMe₃ in the presence of Na/Hg amalgam to afford high yields of the respective alkyne complexes Cp^*₂Zr(Me₃SiC≡CPh) (2), Cp^*₂Zr{Me₃SiC≡C(c-C₅H₉)} (3) and Cp^*₂Zr(Me₃SiC≡CCMe₃) (4) as crystalline compounds. Complex 2 crystallizes in the triclinic space group with a = 9.791(6), b = 10.466(6), c = 15.756(12) Å, = 86.09 (5), β = 72.09(5), γ = 72.06(4)° and Z = 2. The least-squares refinement converged to R(F) = 0.0604 and R(wF) = 0.0628 for the 3655 unique data with F_o > 4σ (F_o). Salient metrical parameters of the bound alkyne include the following: C(30)-C(31) = 1.340(9) Å; Zr-C(30) = 2.178(6) Å; Zr-C(31) = 2.219(5) Å; C(30)-C(31)-Si = 141.0(5)°; C(31)-C(30)-C(26) = 135.5(5)°. Nitrous oxide reacts with 2 or 3 to afford ((5) R = Ph; (6) R = c-C₅H₉) and 1 equiv. of N₂ via an intermediate, , which is unstable with respect to loss of dinitrogen to give the oxametallacyclobutene derivatives 5 and 6. The oxygen-atom insertion is regiospecific for the Zr-C bond that is attached to the carbyl (Ph or c-C₅H₉) substituent. Under similar conditions, complex 4, in which the alkyne is particularly labile, gives a myriad of products in its reaction with N₂O.     

β-secretase (BACE1)-inhibiting stilbenoids from Smilax Rhizoma

In the course of searching for BACE1 (beta-secretase) inhibitors from natural products, the ethyl acetate soluble fraction of Smilax Rhizoma (the dried rhizomes of Smilax china L.) showed potent inhibitory activity. The active compounds were identified as a trans/cis-resveratrol mixture, oxyresveratrol, veraphenol, and cis-scirpusin A. They were shown to non-competitively inhibit BACE1 with the Ki values of 5.4 x 10(-6), 5.4 x 10(-6), 3.4 x 10(-6), and 5.4 x 10(-6)M and IC(50) values of 1.5 x 10(-5), 7.6 x 10(-6), 4.2 x 10(-6), and 1.0 x 10(-5)M, respectively. The active compounds were less inhibitory to alpha-secretase (TACE) and other serine proteases such as chymotrypsin, trypsin, and elastase, suggesting that they were relatively specific inhibitors of BACE1.     

Isolation of Two Novel WNT Genes, WNT14 and WNT15, One of Which (WNT15) Is Closely Linked to WNT3 on Human Chromosome 17q21

TheWntgene family consists of at least 15 structurally related genes that encode secreted extracellular signaling factors. Wnt proteins function in a range of critical developmental processes in both vertebrates and invertebrates and are implicated in regulation of cell growth and differentiation in certain adult mammalian tissues, including the mammary gland. We have isolated a number of WNT sequences from human genomic DNA, two of which, designated WNT14 and WNT15, represent novel members of theWntgene family. We also isolated WNT sequences from human mammary cDNA and present evidence that WNT13 is expressed in human breast tissue, in addition to those previously described. WNT14 and WNT15 appear to have originated from an ancestral branch of theWntgene family that also includes theWnt9sequences found in jawless and cartilaginous fishes. AWnt14cDNA was also isolated from chicken and a partialWnt15sequence from mouse. We show that human WNT14 maps to chromosome 1 and that WNT15 maps distal to BRCA1 on chromosome 17q21, where it lies within 125 kb of another WNT family member, WNT3.