Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily

A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca2+, undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (Kd ≈ 0.37 μM), the apparent Hill coefficient for Ca2+-induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca2+ binding N-terminal half of the transporter faces the cytosol.

Catalytically inactive lipoprotein lipase expression in muscle of transgenic mice increases very low density lipoprotein uptake: Direct evidence that lipoprotein lipase bridging occurs in vivo

Lipoprotein lipase (LPL) is the central enzyme in plasma triglyceride hydrolysis. In vitro studies have shown that LPL also can enhance lipoprotein uptake into cells via pathways that are independent of catalytic activity but require LPL as a molecular bridge between lipoproteins and proteoglycans or receptors. To investigate whether this bridging function occurs in vivo, two transgenic mouse lines were established expressing a muscle creatine kinase promoter-driven human LPL (hLPL) minigene mutated in the catalytic triad (Asp156 to Asn). Mutated hLPL was expressed only in muscle and led to 3,100 and 3,500 ng/ml homodimeric hLPL protein in post-heparin plasma but no hLPL catalytic activity. Less than 5 ng/ml hLPL was found in preheparin plasma, indicating that proteoglycan binding of mutated LPL was not impaired. Expression of inactive LPL did not rescue LPL knock-out mice from neonatal death. On the wild-type (LPL2) background, inactive LPL decreased very low density lipoprotein (VLDL)-triglycerides. On the heterozygote LPL knock-out background (LPL1) background, plasma triglyceride levels were lowered 22 and 33% in the two transgenic lines. After injection of radiolabeled VLDL, increased muscle uptake was observed for triglyceride-derived fatty acids (LPL2, 1.7×; LPL1, 1.8×), core cholesteryl ether (LPL2, 2.3×; LPL1, 2.7×), and apolipoprotein (LPL1, 1.8×; significantly less than cholesteryl ether). Skeletal muscle from transgenic lines had a mitochondriopathy with glycogen accumulation similar to mice expressing active hLPL in muscle. In conclusion, it appears that inactive LPL can act in vivo to mediate VLDL removal from plasma and uptake into tissues in which it is expressed.

Solution to Darwin's dilemma: Discovery of the missing Precambrian record of life

In 1859, in On the Origin of Species, Darwin broached what he regarded to be the most vexing problem facing his theory of evolution—the lack of a rich fossil record predating the rise of shelly invertebrates that marks the beginning of the Cambrian Period of geologic time (≈550 million years ago), an “inexplicable” absence that could be “truly urged as a valid argument” against his all embracing synthesis. For more than 100 years, the “missing Precambrian history of life” stood out as one of the greatest unsolved mysteries in natural science. But in recent decades, understanding of life's history has changed markedly as the documented fossil record has been extended seven-fold to some 3,500 million years ago, an age more than three-quarters that of the planet itself. This long-sought solution to Darwin's dilemma was set in motion by a small vanguard of workers who blazed the trail in the 1950s and 1960s, just as their course was charted by a few pioneering pathfinders of the previous century, a history of bold pronouncements, dashed dreams, search, and final discovery.

Evolutionary relationships of the coelacanth, lungfishes, and tetrapods based on the 28S ribosomal RNA gene.

The origin of land vertebrates was one of the major transitions in the history of vertebrates. Yet, despite many studies that are based on either morphology or molecules, the phylogenetic relationships among tetrapods and the other two living groups of lobe-finned fishes, the coelacanth and the lungfishes, are still unresolved and debated. Knowledge of the relationships among these lineages, which originated back in the Devonian, has profound implications for the reconstruction of the evolutionary scenario of the conquest of land. We collected the largest molecular data set on this issue so far, about 3,500 base pairs from seven species of the large 28S nuclear ribosomal gene. All phylogenetic analyses (maximum parsimony, neighbor-joining, and maximum likelihood) point toward the hypothesis that lungfishes and coelacanths form a monophyletic group and are equally closely related to land vertebrates. This evolutionary hypothesis complicates the identification of morphological or physiological preadaptations that might have permitted the common ancestor of tetrapods to colonize land. This is because the reconstruction of its ancestral conditions would be hindered by the difficulty to separate uniquely derived characters from shared derived characters in the coelacanth/lungfish and tetrapod lineages. This molecular phylogeny aids in the reconstruction of morphological evolutionary steps by providing a framework; however, only paleontological evidence can determine the sequence of morphological acquisitions that allowed lobe-finned fishes to colonize land.

Aldehyde dehydrogenase class 3 expression: Identification of a cornea-preferred gene promoter in transgenic mice

Aldehyde dehydrogenase class 3 (ALDH3) constitutes 20–40% of the total water-soluble proteins in the mammalian cornea. Here, we show by Northern blot analysis that ALDH3 expression in the mouse is at least 500-fold higher in the cornea than in any other tissue examined, with very low levels of expression detected in the stomach, urinary bladder, ocular lens, and lung. Histochemical localization reveals that this exceptional level of expression in the mouse cornea occurs in the anterior epithelial cells and that little ALDH3 is present in the keratocytes or corneal endothelial cells. A 13-kbp mouse ALDH3 promoter fragment containing >12 kbp of the 5′ flanking sequence, the 40-bp untranslated first exon, and 29 bp of intron 1 directed cat reporter gene expression to tissues that express the endogenous ALDH3 gene, except that transgene promoter activity was higher in the stomach and bladder than in the cornea. By contrast, when driven by a 4.4-kbp mouse ALDH3 promoter fragment [1,050-bp 5′ flanking region, exon 1, intron 1 (3.4 kbp), and 7 bp of exon 2] expression of the cat reporter gene was confined to the corneal epithelial cells, except for very low levels in the liver, effectively reproducing the corneal expression pattern of the endogenous ALDH3 gene. These results indicate that tissue-specific expression of ALDH3 is determined by positive and negative elements in the 5′ flanking region of the gene and suggests putative silencers located in intron 1. We demonstrate regulatory sequences capable of directing cornea-specific gene expression, affording the opportunity for genetic engineering in this transparent tissue.

Cytotoxic analogs of luteinizing hormone-releasing hormone containing doxorubicin or 2-pyrrolinodoxorubicin, a derivative 500-1000 times more potent.

Doxorubicin (DOX) and its daunosamine-modified derivative, 2-pyrrolino-DOX, which is 500-1000 times more active than DOX, were incorporated into agonistic and antagonistic analogs of luteinizing hormone-releasing hormone (LH-RH). The conjugation of DOX with LH-RH analogs was performed by using N-(9-fluorenylmethoxycarbonyl)-DOX-14-O-hemiglutarate, a dicarboxylic acid ester derivative of DOX. Coupling this derivative covalently to the epsilon-amino group of the D-Lys side chain of agonist [D-Lys6]LH-RH or antagonistic analog AC-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys-Leu-Arg-Pro-D-Ala-NH 2 [where Nal(2) = 3-(2-naphthyl)alanine, Pal(3) = 3-(3-pyridyl)alanine, and Phe(4CI) = 4-chlorophenylalanine] was followed by the removal of the 9-fluorenylmethoxycarbonyl protective group to yield cytotoxic derivatives of LH-RH analogs containing DOX. From these DOX containing LH-RH hybrids, intensely potent analogs with daunosamine-modified derivatives of DOX can be readily formed. Thus, cytotoxic LH-RH agonist containing DOX (AN-152) can be converted in a 66% yield by a reaction with a 30-fold excess of 4-iodobutyraldehyde in N,N-dimethylformamide into a derivative having 2-pyrrolino-DOX (AN-207). Hybrid molecules AN-152 and AN-207 fully preserve the cytotoxic activity of their radicals, DOX or 2-pyrrolino-DOX, respectively, in vitro, and also retain the high binding affinity of the peptide hormone portion of the conjugates to rat pituitary receptors for LH-RH. These highly potent cytotoxic analogs of LH-RH were designed as targeted anti-cancer agents for the treatment of various tumors that possess receptors for the carrier peptide. Initial in vivo studies show that the hybrid molecules are much less toxic than the respective cytotoxic radicals incorporated and significantly more active in inhibiting tumor growth.

High yield conversion of doxorubicin to 2-pyrrolinodoxorubicin, an analog 500-1000 times more potent: structure-activity relationship of daunosamine-modified derivatives of doxorubicin.

A convenient, high yield conversion of doxorubicin to 3'-deamino-3'-(2''-pyrroline-1''-yl)doxorubicin is described. This daunosamine-modified analog of doxorubicin is 500-1000 times more active in vitro than doxorubicin. The conversion is effected by using a 30-fold excess of 4-iodobutyraldehyde in anhydrous dimethylformamide. The yield is higher than 85%. A homolog of this compound, 3'-deamino-3'-(1'',3''-tetrahydropyridine-1''-yl)doxorubicin, was also synthesized by using 5-iodovaleraldehyde. In this homolog, the daunosamine nitrogen is incorporated into a six- instead of a five-membered ring. This analog was 30-50 times less active than its counterpart with a five-membered ring. A similar structure-activity relationship was found when 3'-deamino-3'-(3''-pyrrolidone-1''-yl)doxorubicin (containing a five-membered ring) and 3'-deamino-3'-(3''-piperidone-1''-yl)doxorubicin (with a six-membered ring) were tested in vitro, the former being 5 times more potent than the latter. To further elucidate structure-activity relationships, 3'-deamino-3'-(pyrrolidine-1''-yl)doxorubicin, 3'-deamino-3'-(isoindoline-2''-yl)doxorubicin, 3'-deamino-3'-(2''-methyl-2''-pyrroline-1''-yl)doxorubicin, and 3'-deamino-3'-(3''-pyrroline-1''-yl)doxorubicin were also synthesized and tested. All the analogs were prepared by using reactive halogen compounds for incorporating the daunosamine nitrogen of doxorubicin into a five- or six-membered ring. These highly active antineoplastic agents can be used for incorporation into targeted cytotoxic analogs of luteinizing hormone-releasing hormone intended for cancer therapy.