End-To-End Automated Intact Protein Mass Spectrometry for High-Throughput Screening and Characterization of Bispecific and Multispecific Antibodies.

Bispecific antibodies (bsAbs) and multispecific antibodies (msAbs) represent a promising frontier in therapeutic antibody development, offering unique capabilities not achievable with traditional monoclonal antibodies. Despite their potential, significant challenges remain due to their increased molecular complexity. One prominent challenge is the correct assembly of light and heavy chains, as improper pairing leads to mispaired or incompletely assembled species that lack therapeutic efficacy and possess undesired properties, impairing the developability, manufacturability, and safety.

Far-upstream elements are dispensable for tissue-specific proenkephalin expression using a Cre-mediated knock-in strategy.

Several cis-regulatory DNA elements are present in the 5' upstream regulatory region of the enkephalin gene (ENK) promoter. To determine their role in conferring organ-specificity of ENK expression in mice and to circumvent the position effects from random gene insertion that are known to often frustrate such analysis in transgenic mice, we used a Cre-mediated gene knock-in strategy to target reporter constructs to a "safe haven" loxP-tagged locus in the hypoxanthine phosphoribosyltransferase (HPRT) gene. Here we report reliable and reproducible reporter gene expression under the control of the 5' upstream regulatory region of the mouse ENK gene in gene-modified mice using this Cre-mediated knock-in strategy.

Alternative non-coding exons support serotonin transporter mRNA expression in the brain and gut.

A number of studies in recent years have linked polymorphisms within the serotonin transporter (5HTT) gene to affective disorders and anxiety traits. The human 5HTT mRNA is alternatively spliced, and the splice variants are equally expressed in the human placental cell line and dorsal raphe. In this study, using 5' rapid amplification of cDNA ends, we show that the rat 5HTT mRNA is alternatively spliced, leading to three distinct mRNAs differing in the 5' untranslated region.

Platelet-activating factor evokes Ca2+ transients after the blockade of ryanodine receptor by dantrolene in RAW 264.7 macrophages.

In the present study we studied platelet-activating factor (PAF)-, and ATP-induced increases in intracellular Ca2+ concentration ([Ca2+]i) using RAW 264.7 macrophages filled with fura-2/AM and imaged with fluorescence video microscopy. We found that the prevalence of detectable [Ca2+]i responses to PAF application was significantly higher in the presence of dantrolene.

Hydrophilic, pro-drug analogues of T138067 are efficacious in controlling tumor growth in vivo and show a decreased ability to cross the blood brain barrier.

The novel anticancer compound T138067 is an irreversible inhibitor of tubulin polymerization. Amides 3-6 were synthesized using standard methodologies and determined to be significantly less lipophilic than T138067 based on logP calculations. Tubulin polymerization and [(3)H]-T138067 competition assays revealed that these amides are pro-drugs for parent aniline 2.

Low temperature prevents potentiation of norepinephrine release by phenylephrine.

The effect of 1-phenylephrine (1-PE), an alpha(1)-receptor agonist, was investigated on the release of tritiated norepinephrine ([3H]NE). Pairs of guinea pig vasa deferentia were loaded with [3H]NE, superfused continuously, and stimulated electrically. 1-PE (10, 100 microM) enhanced the basal release of tritium in concentration-dependent manner.

GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus.

Cannabinoids have been shown to disrupt memory processes in mammals including humans. Although the CB1 neuronal cannabinoid receptor was identified several years ago, neuronal network mechanisms mediating cannabinoid effects are still controversial in animals, and even more obscure in humans. In the present study, the localization of CB1 receptors was investigated at the cellular and subcellular levels in the human hippocampus, using control post mortem and epileptic lobectomy tissue.

Multiple cellular mechanisms mediate the effect of lobeline on the release of norepinephrine.

The complex effect of lobeline on [(3)H]norepinephrine ([(3)H]NE) release was investigated in this study. Lobeline-induced release of [(3)H]NE from the vas deferens was strictly concentration-dependent. In contrast, electrical stimulation-evoked release was characterized by diverse effects of lobeline depending on the concentration used: at lower concentration (10 microM), it increased the release and at high concentration (100 and 300 microM), the evoked release of [(3)H]NE was abolished.

Modulation of norepinephrine release by ATP-dependent K(+)-channel activators and inhibitors in guinea-pig and human isolated right atrium.

Objective: The aim of this study was to show, whether ATP sensitive K+ channels (KATP channels), are involved in the modulation of norepinephrine (NE) release from the sympathetic nerves innervating the guinea-pig and human right atrium.

Methods: The resting and stimulation-evoked release of [3H]norepinephrine ([3H]NE) was measured from the isolated guinea-pig and human right atrium and the effect of activators and inhibitors of ATP sensitive K+ channels was studied.

Results: Cromakalim (30-300 microM), a KATP channel-agonist decreased concentration-dependently the stimulation-evoked release of NE from the guinea-pig atrium, an effect, antagonized by glibenclamide, a KATP channel-antagonist (30 microM).

Isolation and structural and genetic analysis of the mouse enkephalin gene and its d(AC/TG)n repeats.

Enkephalins, the endogenous opioids, mediate a wide variety of intercellular communications through ontogeny and their involvement has been suggested in drug addiction and alcohol abuse as well as in various neuropsychiatric disorders. In order to generate a genetic model, we have isolated the mouse enkephalin (mENK) gene, analyzed its regulatory region and compared its structure to the well characterized rat ENK (rENK) gene. We analyzed 2600 bp and found 3 highly homologous regions: The highest level (98%) of positional and sequence homology between mice and rats was in the TATA/proximal regulatory region.

Nitric oxide (NO) increases acetylcholine release from and inhibits smooth muscle contraction of guinea-pig gastric fundus.

Experiments were carried out to investigate the interaction between nitric oxide (NO) and cholinergic neurotransmission in smooth muscle strips of guinea-pig gastric fundus. Electrical field stimulation (2 Hz, 1 ms, 360 shocks) evoked atropine-sensitive contractions. Dimethylphenylpiperazinium (DMPP) (100 microM), a nicotinic receptor agonist, reversed the stimulation-evoked contraction and resulted in relaxation.

Selective, covalent modification of beta-tubulin residue Cys-239 by T138067, an antitumor agent with in vivo efficacy against multidrug-resistant tumors.

Microtubules are linear polymers of alpha- and beta-tubulin heterodimers and are the major constituents of mitotic spindles, which are essential for the separation of chromosomes during mitosis. Here we describe a synthetic compound, 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (T138067), which covalently and selectively modifies the beta1, beta2, and beta4 isotypes of beta-tubulin at a conserved cysteine residue, thereby disrupting microtubule polymerization. Cells exposed to T138067 become altered in shape, indicating a collapse of the cytoskeleton, and show an increase in chromosomal ploidy.

[Active movement therapy after flexor tendon suture using a new dynamic control splint].

The authors constructed a new dynamic guiding splint assisting the active mobilisation after flexor tendon repair distal to the wrist. In these cases, the "inverse" wrist position seems to be the best position for mobilisation. This means that finger flexion should be carried out during wrist extension, and finger extension during wrist flexion.

Differential mechanisms involved in the effect of nicotinic agonists DMPP and lobeline to release [3H]5-HT from rat hippocampal slices.

In the present study we investigated the effect of different nicotinic agonists (dimethylphenyl-piperazinium-iodide (DMPP), (-)nicotine, cytisine, (-)-lobeline, and (-)epibatidine) and antagonists (mecamylamine and dihydro-beta-erythroidine) on the release of [3H]5-HT from hippocampal slices. The nicotinic agonists DMPP and lobeline and electrical field stimulation, released [3H]5-HT from the hippocampus; other nicotinic agonists, such as (-)-nicotine, cytisine, and (-)-epibatidine had no effect. Unlike lobeline-induced release of [3H]5-HT, the effect of DMPP (10 and 40 microM) was antagonized by mecamylamine (20 and 10 microM).

Protein-DNA interactions during phenotypic differentiation.

We have been studying the molecular mechanism of neuronal differentiation through which the multipotent precursor becomes limited to the final transmitter phenotype. Here we focused on the role of the 5' proximal regulatory cassette (-190; +53 bp) of the rat enkephalin (rENK) gene in the developmental regulation of the enkephalin phenotype. Several well characterized cis-elements, including AP2, CREB, NF1, and NFkB, reside on this region of the rENK gene.

[Does the flexed position of the wrist releave the stress on the suture in the flexor tendon?].

Authors have examined the optimal position of the wrist from the viewpoint of active motion during the postoperative care, after the suture of the flexor tendons of the hand. Based on clinical and cadaveric experiences a motion of the wrist (inverse) in contrary direction to the motion of the fingers is suggested i. e.

[Immediate full-range active movement after suturing of the flexor tendon. A new dynamic guiding splint].

Immediate full range active movement after suture of the flexor tendon. New dynamic guiding splintAuthors emphasize based on literary data, the advantages of the active postoperative movements. A technique for suture and mobilization protocol are offered.

[Tensile strength of new and modified flexor tendon sutures].

Authors tested modified and new sutures and different sewing materials on the deep flexor tendons of the hand in cadavera. Tensile and pull strength examinations were performed. The results were compared with the Kirchmayr-Kessler suture.

[Measurement of the tensile force exerted on the flexor tendons of the hand].

Immediate postoperative active motion after flexor tendon repair is a recurring theme in hand surgery. In the present study the authors determined the tensile stress of the sutured tendon subjected to active motion by the use of cadaveric hands and a tensiometer. The force necessary to draw forth the proximal stump was also measured.

[Primary tensile strength of newer and modified tendon sutures. A comparative in-vitro study].

Modified with new methods of flexor tendon sutures compared with the Kessler technique on cadaveric tendons are reported. The authors offer some suturing methods which allow active movement of the reconstructed flexor tendons.

[Direct measurement of the pulling force affecting the flexor tendons of the hand].

A thought, returning again and again in hand surgery, is the immediate introduction of active motion therapy after the reconstructive operations of the flexor tendons. To the active motion an adequately strong tendon suture, to the definition of "adequately strong tendon suture" however the measurement of the pulling force, acting actually on the flexor tendons is necessary. Authors attempted to measure this pulling force during flexor tendon operations on cadaver's hand.

[Modification of the Matti-Russe operation].

The essential feature is the curved formation of the bone groove and of the bone graft. The field of indication of the Matti-Russe's operation is widened and the mechanical stability increased.