The biotechnology and applications of antibody engineering

The exquisite specificity of monoclonal antibodies (MAb) has long provided the potential for creating new reagents for the in vivo delivery of therapeutic drugs or toxins to defined cellular target sites or improved methods of diagnosis. However, many difficulties associated with their production, affinity, specificity, and use in vivo have largely confined their application to research or in vitro diagnostics. This situation is beginning to change with the recent developments in the applied molecular techniques that allow the engineering of the genes that encode antibodies rather than the manipulation of the intact antibodies themselves. Techniques, such as the polymerase chain reaction, have provided essential methods with which to generate and modify the genetic constituents of antibodies, allow their conjugation to toxins or drugs, provide ways of humanizing murine antibodies, and allow discrete modular antigen binding components to be produced. More recent developments of in vitro expression systems and powerful phage surface display technologies will without doubt play a major role in future antibody engineering and in the successful development of new diagnostic and therapeutic antibody-based reagents.     

Distributions of the use frequencies of amino acids in the hypervariable regions of immunoglobulins

Two types of distributions for the frequencies of occurrence of amino acids in each position of hypervariable regions CDR-1 and CDR-2 were obtained for 2,000 immunoglobulins. The results show that some positions fit an inverse power-law distribution, while others fit an exponential-type distribution. As a result of comparison with structural data in the literature it is proposed that sites in which the frequency distribution fits the inverse power law are critical to maintaining canonical shapes of the recognition regions or are involved in modulating these canonical conformations, while those sites where the distribution fits the exponential law are those which should be exclusively involved in the recognition mechanism. Correspondence to: F. Lara-Ochoa     

Stem growth,flower formation,and endogenous gibberellins in a normal and a dwarf strain of Silene armeria

The physiological basis of dwarfism in a single-gene, recessive mutant of Silene armeria L. was investigated through comparison with a normal strain. Exposure of the normal strain to long days led to stem growth and flower formation while similar exposure of the dwarf strain led only to flowering, with very little stem growth. Application of gibberellin A₃ or A₄₊₇ in short days promoted stem elongation in the normal strain, but had a much lesser effect in the dwarf strain. Upon extraction and chromatographic fractionation of the endogenous gibberellins (GAs) in the normal strain of S. armeria, three zones of GA activity were found. An increase in one zone of activity was found in both strains after 1 long day. Neither the quality nor the quantity of the extractable GAs differed greatly between the dwarf and the normal strain. Vegetative dwarf scions, grafted onto fully induced, normal stocks formed flowers, but their growth habit was not changed. Thus, the lack of stem growth in response to long days in the dwarf strain appears to result from a lack of GA sensitivity in the stem tissue of these plants. However, during flower formation dwarf plants did exhibit elongation of the peduncles. This response was suppressed by the growth retardant 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride (AMO-1618), and applied GA₃ could partially overcome this inhibition. Thus, peduncle elongation in the dwarf strain appears to be regulated by endogenous GAs.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - LD long day(s) - SD short day(s)     

Human heat shock gene expression and the modulation of plasma membrane Na+, K+-ATPase activity

Benzodiazepine receptor solubilized from bovine cortical membranes was bound to a new benzodiazepine affinity column, the synthesis of which is described. Bio-specific elution with the benzodiazepine compound chlorazepate resulted in the elution of fractions highly enriched in specific binding for the GABA receptor agonist muscimol. Specific activity for [³H]muscimol binding was >1.3 nmol/mg protein. It is shown that [³H]flunitrazepam binding activity can be recovered by removal of chlorazepate from the purified fraction. These results strongly support a model which suggests that the 2 binding sites reside on the same physical entity.     

Studies of vegetative compatibility-incompatibility in higher plants

Summary In order to elucidate the events that lead to cellular autolysis, and thus better understand the mechanism of cellular incompatibility betweenSedum telephoides andSolanum pennellii stems, we have followed the appearance and fate of the hydrolytic enzyme acid phosphatase in both the compatibleSedum autograft and the incompatibleSedum/Solanum heterograft. Acid phosphatase was localized by a modified Gomori-type reaction. Following an initial association with the endoplasmic reticulum and dictyosomes by 6–10 hours after grafting, acid phosphatase activity in the compatibleSedum autograft was associated primarily with the plasmalemma, tonoplast, and vacuole. This strict compartmentation in membranes or organelles and absence of enzyme from the cytosol was maintained throughout the development of the compatible autograft inSedum. Although acid phosphatase activity in the incompatible heterograft betweenSedum andSolanum was initially similar to the compatible autograft inSedum, a marked difference in enzyme localization occurred in the two graft partners over time.Solanum cells accumulated increased amounts of acid phosphatase, but the enzyme remained sequestered in the plasmalemma, tonoplast, and vacuole. In comparableSedum cells, however, there was a dramatic increase in acid phosphatase activity in the cytosol, often without any prior compartmentation within the vacuole. This high activity of acid phosphatase in theSedum cytosol was correlated with cellular autolysis, death, and eventual cell collapse to form the characteristic necrotic layer that insulates the stock from the scion. These results suggest that the lethal cellular senescence associated withSedum cells of the incompatible heterograft is correlated with a cytoplasmic release of acid phosphatase. A similar release of the enzyme does not occur in theSolanum stock or in the compatibleSedum autograft. Thus, while acid phosphatase synthesis and/or activation is induced in both the compatible and incompatible grafts, incompatibility betweenSedum andSolanum involves a failure ofSedum cells to isolate hydrolytic enzymes from the cytosol, which subsequently leads to cellular necrosis.Supported in part by grants from the Academic Senate of UCLA, Sigma Xi, the American Philosophical Society, and the URC of Baylor University.     

Adrenal medullary cyclic nucleotide phosphodiesterase: lack of activation by the calcium-dependent regulator.

Calcium-dependent regulator, a calcium-binding protein isolated from brain and adrenal medulla, has been shown to activate a brain calcium-sensitive cyclic nucleotide phosphodiesterase. To determine if this protein has the same role in the adrenal medulla, the cyclic nucleotide phosphodiesterase of adrenal medulla was characterized. Neither crude nor partially purified adrenal medullary phosphodiesterase was inhibited by EGTA or stimulated by calcium and the calcium-dependent regulator, whereas similar brain preparations displayed sensitivity to these agents. As the calcium-dependent regulator does not appear to stimulate adrenal medullary cyclic nucleotide phosphodiesterase activity, alternate roles of this protein in adrenal medulla are suggested.     

Diagnostic and prognostic value of circular RNA CDR1as/ciRS‐7 for solid tumours: A systematic review and meta‐analysis

The circular RNA, CDR1as/ciRS‐7, functions as a vital regulator in various cancers; however, the predictive value of CDR1as remains controversial. Therefore, a comprehensive analysis for clarifying the precise diagnostic and prognostic value of CDR1as in solid tumours is needed. A literature review of several databases was conducted for identifying potential studies. Pooled odds ratios (ORs) and hazard ratios (HRs) were used for evaluating the diagnostic accuracy variables and survival. Overall, 15 studies (1787 patients) and 11 studies (1578 patients) were included for diagnostic and prognostic outcome syntheses, respectively. Up‐regulated CDR1as expression was found to be correlated with worse clinicopathological characteristics, including the T status, N status, histological grade, TNM stage and distant metastasis. The synthesized sensitivity was 0.72 (95% confidence interval [CI], 0.65‐0.79), and the specificity was 0.80 (95% CI, 0.74‐0.86). The positive likelihood ratio (LR), negative LR and diagnostic odds ratio (DOR) were 3.70, 0.34 and 10.80, respectively. The area under the receiver operator characteristic curve was 0.84 (95% CI, 0.80‐0.87). In the pooled prognostic analysis, patients with high CDR1as expression had worse overall survival (HR = 2.40, P < 0.001) and disease‐free survival (HR = 1.74, P < 0.001). These results suggest that CDR1as is a reliable diagnostic and prognostic biomarker with high accuracy and efficiency, which may potentially facilitate clinical decisions on solid tumours in the future.     

Structures composing protein domains

This review summarizes available data concerning intradomain structures (IS) such as functionally important amino acid residues, short linear motifs, conserved or disordered regions, peptide repeats, broadly occurring secondary structures or folds, etc. IS form structural features (units or elements) necessary for interactions with proteins or non-peptidic ligands, enzyme reactions and some structural properties of proteins. These features have often been related to a single structural level (e.g. primary structure) mostly requiring certain structural context of other levels (e.g. secondary structures or supersecondary folds) as follows also from some examples reported or demonstrated here. In addition, we deal with some functionally important dynamic properties of IS (e.g. flexibility and different forms of accessibility), and more special dynamic changes of IS during enzyme reactions and allosteric regulation. Selected notes concern also some experimental methods, still more necessary tools of bioinformatic processing and clinically interesting relationships.     

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.     

Inhibition of circular RNA CDR1as increases chemosensitivity of 5‐FU‐resistant BC cells through up‐regulating miR‐7

This study aims to explore the mechanism of Circular RNA CDR1as implicating in regulating 5‐fluorouracil (5‐FU) chemosensitivity in breast cancer (BC) by competitively inhibiting miR‐7 to regulate CCNE1. Expressions of CDR1as and miR‐7 in 5‐FU‐resistant BC cells were determined by RT‐PCR. CCK‐8, colony formation assay and flow cytometry were applied to measure half maximal inhibitory concentration (IC50), 5‐Fu chemosensitivity and cell apoptosis. Western blot was used to detect the expressions of apoptosis‐related factors. CDR1as was elevated while miR‐7 was inhibited in 5‐FU‐resistant BC cells. Cells transfected with si‐CDR1as or miR‐7 mimic had decreased IC50 and colony formation rate, increased expressions of Bax/Bcl2 and cleaved‐Caspase‐3/Caspase‐3, indicating inhibition of CDR1as and overexpression of miR‐7 enhances the chemosensitity of 5‐FU‐resistant BC cells. Targetscan software indicates a binding site of CDR1as and miR‐7 and that CCNE1 is a target gene of miR‐7. miR‐7 can gather CDR1as in BC cells and can inhibit CCNE1. In comparison to si‐CDR1as group, CCNE1 was increased and chemosensitivity to 5‐Fu was suppressed in si‐CDR1as + miR‐7 inhibitor group. When compared with miR‐7 mimic group, CDR1as + miR‐7 mimic group had increased CCNE1 and decreased chemosensitivity to 5‐Fu. Nude mouse model of BC demonstrated that the growth of xenotransplanted tumour in si‐CDR1as + miR‐7 inhibitor group was faster than that in si‐CDR1as group. The tumour growth in CDR1as + miR‐7 mimic group was faster than that in miR‐7 mimic group. CDR1as may regulate chemosensitivity of 5‐FU‐resistant BC cells by inhibiting miR‐7 to regulate CCNE1.