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A hybrid M-ary spread spectrum (SS) communication scheme which combined with orthogonal SS and CPSK technique is proposed. Based on dual orthogonal SS, every SS code is treated as original code to adopt CPSK modulation, the unattached synchronous code is chosen to achieve system synchronization. At the receiver, A circular correlator based on transform domain processing is employed to de-spread the received signals. Compared with M-ary SS and CPSK, the proposed scheme is able to obtain higher bandwidth efficiency, stronger anti-intercept capability, and better synchronous performance. The Monte-Carlo simulation results demonstrate that the proposed scheme can achieve better synchronous and bit error rate (BER) performance under additional white gauss noise channel models.

Towards understanding the IGF system during cancer growth and progression, progressive prostate cancer models, such as SV40 large T antigen immortalized human prostate epithelial cells (P69, M2182, M2205, and M12) and LNCaP sublines (C4, C4-2, and C4-2B4), were used. IGF-II mRNA levels progressively increase as prostate cancer cells become more tumorigenic and metastatic, suggesting that IGF-II contributes in part to prostate cancer progression. The role of IGF-II in cancer cell growth was evaluated in LNCaP, PC3, and M12 prostate cancer cell lines and MCF-7 breast cancer cell line by ribozyme/antisense strategies which were previously shown to suppress endogenous IGF-II expression and cell growth in PC-3 cells [Xu et al., Endocrinol 140 (1999) 2134]. Retroviral mediated transient expression of IGF-II-specific ribozyme (RZ) caused extensive cell death. In stably cloned cell lines, both RZ and mutant ribozyme (MRZ) inhibited cancer cell growth, suggesting that antisense effects of MRZ may be sufficient for cell growth inhibition. These results confirm an important role of IGF-II in cancer cell growth and progression, and support further development of gene therapy targeting IGF-II.

Upregulation of insulin-like growth factor (IGF)-II expression has been reported for a variety of childhood and adulthood tumors. We determined IGF-II gene promoter usage in human cancerous and benign tissues by semiquantitative RT-PCR using P1–P4-specific primers. Although the human IGF-II gene structure is commonly thought to consist of nine exons and four promoters, we detected substantial utilization of a previously reported exon 4b, which is downstream of exon 4. Thus, exon 4b was intensively studied using 4b-specific primers. IGF-II gene promoter usage is highly variable in malignant and benign breast, prostate, and bladder tissues. While a majority of samples utilized P2–P4 promoters in a variety of combinations, when quantitated, P3 and P4 promoters were much more active than P2 promoter. This study not only demonstrated that IGF-II gene promoter usage is highly variable in malignant and benign tissues, but suggested that alternatively spliced exon 4b should be recognized as a 10th exon.

Phosphodiesterase type 3 isoforms, PDE3A and 3B, are expressed primarily in cardiovascular and adipose tissues, respectively. We previously reported a shorter transcript of 4.4-kb PDE3A which is predominantly transcribed in human placenta, whereas a full-length 7. 6-kb transcript corresponding to the cardiac PDE3A cDNA has not been characterized. Due to unfortunate circumstances created by changes in PDE3 nomenclature, PDE3B gene structure previously reported used PDE3A in its title. Here, we describe PDE3A gene structure, which comprises 16 exons spanning over 130 kb on chromosome 12p12. Two PDE3 isoforms share similar gene organization, but localize to different chromosomes. The most distal transcription initiation site of the PDE3A gene is approximately 1071 bases upstream of the ATG site, suggesting that exon 1 consists of 1071 and 960 bp of untranslated and translated sequences, respectively. The proximal 5'-flanking region, which does not contain TATA-like sequences, exhibited weak but significant promoter activity. Results suggest potential involvement of distal promoter/enhancer and translational regulation for expression of the 7.6-kb transcript.

We have previously expressed the human insulin receptor beta subunit domain containing transmembrane and cytoplasmic domains (IRTMTPK) in insect cells, and showed that the purified IRTMTPK was highly active (Li, S. L., Yan, P.-F., Pax, I. B., and Fujita-Yamaguchi, Y. (1992) Biochemistry 31, 12455-12462). To investigate the role of COOH-terminal and acidic domains of the insulin receptor kinase, we have expressed deletion mutants IRTMTPK delta CT (delta 76 amino acids) and IRTMTPK delta Acid (delta 19 amino acids). Both enzymes were purified by a one-step method using the same immunoaffinity column as used for IRTMTPK. While Km and Vmax for prephosphorylated IRTMTPK and delta Acid mutant enzyme determined using poly(Glu, Tyr)(4:1) were similar, catalytic efficiency of the delta CT mutant enzyme was significantly lower than those of IRTMTPK and delta Acid mutant enzyme as judged by Km and Vmax. Experiments for thermostability and susceptibility to proteases revealed that Tm of delta CT mutant enzyme was 3.5 degrees C lower than that of IRTMTPK enzyme (= 33.3 degrees C) and that delta CT mutant enzyme was digested by either trypsin or Lys-C into a 28,000 core domain much faster than IRTMTPK. Activation of delta CT mutant enzyme by polylysine was less significant than that of IRTMTPK and delta Acid mutant enzyme, approximately 4-versus approximately 17-fold. These studies suggested that the COOH-terminal domain plays important roles in both catalytic efficiency and stability of the insulin receptor kinase, and that the acidic domain by itself is not responsible for kinase activation by polylysine.

Ultraviolet irradiation of carboxymethylated D-glyceraldehyde-3-phosphate dehydrogenase leads to the formation of a fluorescent NAD derivative. The structure of the enzyme from Palinurus versicolor carrying this derivative has been determined by molecular replacement and refined using the restrained least-squares method to 2.7 A with a final crystallographic R-factor of 0.205. The polypeptide chain folding and subunit arrangement closely resemble the known structure of Homarus americanus GAPDH. The structure at the modified active site confirms that the photochemical reaction is a half-of-the-sites reaction and occurs in the red and yellow subunit pair. The stereochemical relationship between the Trp residues at the active site shows that Trp 310 is most probably involved in a radiationless energy transfer to the fluorophore. A large solvent channel connecting the catalytic and NAD(+)-binding sites was found parallel to the crystallographic axis alpha from packing analysis, suggesting that this crystal form may be suitable for a kinetic crystallographic study of the apoenzyme.

The activation of phosphodiesterase 3B (PDE3B) reduces free fatty acid output from adipocytes. A reduced PDE3B gene expression could lead to insulin resistance. To determine whether there are polymorphisms associated with type 2 diabetes in PDE3B gene promoter, this 5 ′ flanking region was isolated. The transcription initiation site was located 206 bp upstream from the translation start site. Sequences of 2 kb of the 5 ′ flanking region for 24 type 2 diabetic Japanese subjects were initially analyzed using PCR direct sequencing, and the regions including the identified polymorphisms were then examined. In 98 controls and 98 type 2 diabetic subjects, −1947T > C, −567G > A, −465G > T, −458T > C, and −1727_−1726insTCAATT were found. Only −465G > T and this insertion had more than 5% frequencies. Since a complete linkage disequilibrium existed between them, −465G > T was further analyzed, along with a previously identified +1389G > A in the coding region, in a total of 200 controls and 207 type 2 diabetic subjects. These allele frequencies were not significantly different between these two groups (controls vs. cases; −465G > T, 12.0% vs. 10.1%, P =0.435; +1389G > A, 30.3% vs. 33.3%, P =0.408). These genotype distributions were not significantly different between these two groups. The T/T genotype at −465 was rare although this frequency could be higher in type 2 diabetes (4/207 subjects) than controls (0/200 subjects). The linkage disequilibrium existed between −465G > T and +1389G > A, and the estimated haplotype frequencies defined by these SNPs were not significantly different between the cases and controls. Thus, the identified polymorphisms are unlikely to have major effects on susceptibility to Japanese type 2 diabetes.

Insulin-like growth factors (IGF) I and II are potent mitogens for a variety of cancer cells. The proliferative and anti-apoptotic actions of IGF are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and IGF-II bind with high affinity. To investigate the mitogenic and anti-apoptotic activities of IGF-IR and to achieve better inhibition of IGF-IR function, single-chain antibodies against human IGF-IR (alphaIGF-IR scFvs) were constructed and expressed. IgG cDNA encoding variable regions of light and heavy chains (VL and VH) from mouse IgG were cloned from a hybridoma producing the 1H7 alphaIGF-IR monoclonal antibody [Li et al., Biochem Biophys Res Commun 196: 92-98 (1993)]. The splice-overlap extension polymerase chain reaction was used to assemble a gene encoding the alphaIGF-IR scFv, including the N-terminal signal peptide, VL, linker peptide, VH, and C-terminal DYKD tag. Two types of soluble alphaIGF-IR scFvs, a prototype alphaIGF-IR scFv and its alternative type alphaIGF-IR scFv-Fc, were constructed and expressed in murine myeloma cells. alphaIGF-IR scFv-Fc, containing the human IgG1 Fc domain, was stably expressed in NS0 myeloma cells, using a glutamine synthase selection system, and purified from the conditioned medium of stable clones by protein-A--agarose chromatography. Levels of alphaIGF-IR scFv-Fc expression ranged from 40 mg/l to 100 mg/l conditioned medium. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis under reducing and nonreducing conditions indicated that alphaIGF-IR scFv-Fc is a dimeric antibody. alphaIGF-IR scFv-Fc retained general characteristics of the parental 1H7 monoclonal antibody except that its binding affinity for IGF-IR was estimated to be approximately 10(8) M(-1), which was one-order of magnitude lower than that of 1H7 monoclonal antibody. Injection of alphaIGF-IR scFv-Fc (500 microg/mouse, twice a week) significantly suppressed MCF-7 tumor growth in athymic mice. These results suggest that the alphaIGF-IR scFv-Fc is a first-generation recombinant alphaIGF-IR for the potential development of future alphaIGF-IR therapeutics.

Previously, we showed by in situ hybridization that insulin-like growth factor (IGF)-II is upregulated in approximately 50% of prostate, breast, and bladder tumours. In this study, a quantitative competitive reverse transcription and polymerase chain reaction (QC RT-PCR) assay was established and used to quantify human IGF-II mRNA levels in cells and tissues. In this QC RT-PCR assay, a competitor IGF-II RNA, prepared from a newly constructed plasmid encoding the human IGF-II sequence with a 110-bp fragment inserted, was added to RNA samples prior to RT-PCR. The human IGF-II specific QC RT-PCR assay has allowed us to readily compare the levels of IGF-II mRNA in human tissues and cultured cells. Consistent with our previous observations by in situ hybridization, IGF-II mRNA was up-regulated in 50% of cancerous breast tissues examined as compared to the matching benign tissues, and IGF-II mRNA levels were higher in bladder tumours than breast and prostate tumours. In summary, we present here quantitative data confirming that a subclass of breast cancer samples has elevated levels of IGF-II transcripts by the new competitive RT-PCR assay.

During denaturation by GuHCl, papain shows a rapid decrease in activity with increasing concentrations of the denaturant followed by an intermediate stage of relatively little change from 1 to 2 M before complete inactivation at 4 M GuHCl. At GuHCl concentrations lower than 2 M, enzyme activity is more sensitive to GuHCl than noticeable conformation changes as followed by fluorescence and CD measurements. Kinetics of GuHCl inactivation were studied by following the substrate reaction in the presence of denaturant and the apparent rate constants thus obtained were found to be only slightly higher than those for conformational changes. However, apparent inactivation rate constants obtained in the presence of saturating concentration of substrate are actually inactivation constants for the ES complex. The inactivation rates at different substrate concentrations were, therefore, followed and the microscopic inactivation rate constants for the free enzyme obtained (Tsou, C.L. (1988) Adv. Enzymol. 61, 381-436). It was found that substrate protects strongly against inactivation and at the same GuHCl concentration, the inactivation rate of the free enzyme is 100-fold higher than that of unfolding. The above results show that the activity of papain is more sensitive to GuHCl than its overall conformation and like the enzymes previously studied in this laboratory, its active site is more flexible than the enzyme molecule as a whole.

The inactivation of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) (GAPDH) during guanidine hydrochloride (GdnHCl) denaturation has been compared with its state of aggregation and unfolding, by light scattering and fluorescence measurements. The enzyme first dissociates at low concentrations of GdnHCl, followed by the formation of a highly aggregated state with increasing denaturant concentrations, and eventually by complete unfolding and dissociation to the monomer at concentrations of greater than 2 M GdnHCl. The aggregation and final dissociation correspond roughly with the two stages of fluorescence changes reported previously (Xie, G.-F. and Tsou, C.-L. (1987) Biochim. Biophys. Acta 911, 19-24). Rate measurements show a very rapid inactivation, the extents of which increase with increasing concentrations of GdnHCl. This initial rapid phase of inactivation which takes place before dissociation and unfolding of the molecule is in agreement with the results obtained with other enzymes, that the active site is affected before noticeable conformational changes can be detected for the enzyme molecule as a whole. A scheme for the steps leading to the final denaturation, and dissociation of the enzyme to the inactive and unfolded monomer, is proposed.

The inactivation of d -glyceraldehyde-3-phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating) EC 1.2.1.12) (GAPDH) during thermal denaturation has been compared to its dissociation-aggregation measured by light scattering and changes in secondary structure measured by CD in the far ultraviolet. The inactivation at 38.5°C consists of two stages. The rate of the first stage is too fast to be followed by conventional methods. The extent of this fast stage inactivation increases with increasing temperature and, more markedly, with increasing pH. At this stage, the inactivation is reversible and no appreciable dissociation or change in secondary structure can be detected. The secondary structure of the enzyme is relatively heat stable, showing no appreciable change at 38.5°C. At this temperature, the enzyme first dissociates within several minutes probably into dimers and with prolonged heating, it becomes irreversibly aggregated. The above results are in accord with the earlier suggestion, based on results obtained during denaturation of a number of enzymes by guanidine hydrochloride (GdnHCl) and urea, that for some enzymes the active site is situated in a region more susceptible to perturbation than the molecule as a whole (Tsou, C.-L. (1986) Trends Biochem. Sci. 11, 427).

Three classes of hepatic alcohol dehydrogenase (ADH), analogous to those of human liver, are present in Macaca nemestrina. Their functional, compositional, and structural features have been established with isozymes purified to homogeneity by affinity and conventional ion-exchange chromatography. One unusual molecular form of M. nemestrina ADH is electrophoretically indistinguishable as it comigrates with one of the cathodic class I isozymes on starch gel electrophoresis. While its substrate and inhibitor specificity, a high Km value for ethanol (50 mM at pH 10), and lack of binding to the pyrazole affinity resin are consistent with the kinetics of class II ADH, the physiochemical and compositional properties are virtually identical with all other known mammalian alcohol dehydrogenases. The unexpected presence of this previously unknown ADH variant in livers of M. nemestrina demonstrates the need for prudence in assignment of ADH isozymes. Classification based solely on electrophoretic position in starch gels and enzymatic properties of human ADH but without isolation and characterization of individual isozymes may prove insufficient and inadequate. The genetic or phenotypic nature of this isozyme remains to be demonstrated.