Results and Prospects of Using Activator of Hematopoietic Stem Cell Differentiation in Complex Therapy for Patients with COVID-19.

The paper presents the results of a standard and complex treatment method using the peptide drug thymus thymalin in patients with COVID-19. One of the mechanisms of the immunomodulatory effect of thymalin is considered to be the ability of this peptide drug to influence the differentiation of human hematopoietic stem cells (HSCs). It was found that, as a result of standard treatment, patients in the control group showed a decrease in the concentration of the pro-inflammatory cytokine IL-6, C-reactive protein, D-dimer. The addition of thymalin to standard therapy accelerated the decline in both these indicators and the indicators of the T cell system. This has helped reduce the risk of blood clots in COVID-19 patients. The revealed properties of the thymus peptide preparation are the rationale for its inclusion in the complex treatment of coronavirus infection. Peptideswith potential biological activity against SARS-CoV-2 virus [29]. Note: Nitrogen atoms are shown in blue, oxygen atoms - in red, carbon atoms - in gray, hydrogen atoms - in white, and phosphorus atoms - in yellow.

Clinicopathologic correlation of up-regulated genes identified using cDNA microarray and real-time reverse transcription-PCR in human colorectal cancer.

PURPOSE: We hypothesize that changes in the transcription of up-regulated genes are biologically meaningful and may be linked to variations in tumor behavior and clinical features. This study aimed to find individual up-regulated genes responsible for clinicopathologic variations in human colorectal cancer. EXPERIMENTAL DESIGN: Genes up-regulated concurrently in four microarray experiments were taken as candidate genes; 20 candidate genes were verified using real-time reverse transcription-PCR in these four experiments, along with 27 new samples. The presence or absence of up-regulation of these genes was correlated with 10 clinicopathologic variables from 31 patients. The mRNA transcript levels of these 20 candidate genes in the 31 paired samples were also correlated with each other to disclose any expression relationship. RESULTS: Forty percent (8/20) of the candidate genes were verified by real-time reverse transcription-PCR to have a tumor/normal expression ratio > 2. Up-regulation of THY1 and PHLAD1 was associated with the presence of anemia in colon cancer patients (P = 0.036 and 0.009, respectively). Up-regulation of HNRPA1 was more significant in cancer growing in the right-sided colon than the left side (P = 0.027). Up-regulated GPX2 was related to a higher degree of tumor differentiation (P = 0.019). c-MYC was significantly over-expressed in specimens from male compared with female colon cancer patients (P = 0.012). GRO1 was significantly up-regulated in patients younger than 65 years old (P = 0.010) and was found to be frequently over-expressed when cancers were less invasive. In addition, we found that normalized transcript levels of HNRPA1 were tightly associated with that of c-MYC (r = 0.948). CONCLUSIONS: Validation of microarray data using another independent laboratory approach is mandatory and statistical correlation between gene expression status and the patient's clinical features may reveal individual genes relevant to tumor behavior and clinicopathologic variations in human colorectal cancer.

Human UP1 as a model for understanding purine recognition in the family of proteins containing the RNA recognition motif (RRM).

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is a prototype for the family of eukaryotic RNA processing proteins containing the common RNA recognition motif (RRM). The region consisting of residues 1-195 of hnRNP A1 is referred to as UP1. This region has two RRMs and has a high affinity for both single-stranded RNA and the human telomeric repeat sequence d(TTAGGG)(n). We have used UP1's novel DNA binding to investigate how RRMs bind nucleic acid bases through their highly conserved RNP consensus sequences. Nine complexes of UP1 bound to modified telomeric repeats were investigated using equilibrium fluorescence binding and X-ray crystallography. In two of the complexes, alteration of a guanine to either 2-aminopurine or nebularine resulted in an increase in K(d) from 88nM to 209nM and 316nM, respectively. The loss of these orienting interactions between UP1 and the substituted base allows it to flip between syn and anti conformations. Substitution of the same base with 7-deaza-guanine preserves the O6/N1 contacts but still increases the K(d) to 296nM and suggests that it is not simply the loss of affinity that gives rise to the base mobility, but also the stereochemistry of the specific contact to O6. Although these studies provide details of UP1 interactions to nucleic acids, three general observations about RRMs are also evident: (1) as suggested by informatic studies, main-chain to base hydrogen bonding makes up an important aspect of ligand recognition (2) steric clashes generated by modification of a hydrogen bond donor-acceptor pair to a donor-donor pair are poorly tolerated and (3) a conserved lysine position proximal to RNP-2 (K(106)-IFVGGI) orients the purine to allow stereochemical discrimination between adenine and guanine based on the 6-position. This single interaction is well-conserved in known RRM structures and appears to be a broad indicator for purine preference in the larger family of RRM proteins.

Altered subcellular localization of suppressin, a novel inhibitor of cell-cycle entry, is an independent prognostic factor in colorectal adenocarcinomas.

PURPOSE: Suppressin (SPN), a novel inhibitor of the entry into the cell cycle, has properties of a tumor suppressor gene; however, its role in the development and progression of a human malignancy is not studied. Therefore, we evaluated the status of spn and its prognostic value in human colorectal adenocarcinoma (CRC). EXPERIMENTAL DESIGN: Inhibition of cell proliferation by exogenous/extracellular SPN was assessed by [(3)H]thymidine incorporation. The genetic status of spn in two colon cancer cell lines (LS180 and WiDr) and in a human CRC was determined using direct cDNA sequencing techniques. Phenotypic expression of SPN was evaluated in 105 CRC archival tissues using immunohistochemical methods. Univariate Kaplan-Meier and multivariate Cox proportional hazards models were used to determine the prognostic significance of SPN expression. RESULTS: Exogenous SPN inhibited the proliferation of the LS180 cell line, which also has a mutation in one allele of the spn gene. The spn gene was also mutated in the primary CRC. Expression of SPN was primarily cytoplasmic in nonmucinous CRCs and nuclear in mucinous CRCs. However, the evaluation of 85 nonmucinous CRCs demonstrated that nuclear localization of SPN, nuclear accumulation of p53, and nodal status were independent prognostic indicators with hazard ratios of 2.34, 2.33, and 3.04, respectively. Nuclear localization of SPN plus nuclear accumulation of p53 formed a stronger prognostic indicator (hazard ratios = 5.45) than local nodal status. CONCLUSIONS: This is the first report of genetic alterations in the spn gene in a human malignancy and suggests that genetic alterations in spn and the resulting immunohistochemical phenotypes based on SPN subcellular localization in CRCs may be useful in determining prognosis of patients with subtypes of CRC.

Molecular cloning, sequence analysis, expression, and tissue distribution of suppressin, a novel suppressor of cell cycle entry.

Suppressin (SPN) is an inhibitor of cell proliferation that was originally identified and purified to homogeneity from bovine pituitaries (LeBoeuf, R. D., Burns, J. N., Bost, K. L., and Blalock, J. E. (1990) J. Biol. Chem. 265, 158-165). In this report we have cloned the full-length cDNA encoding rat SPN and have identified the tissue distribution of SPN expression. The cDNA of SPN is 1882 nucleotides with a 1488-base coding region and 55 and 339 nucleotides of 5'- and 3'-untranslated sequences, respectively. Northern gel analysis of rat pituitary mRNA showed a single hybridizing species at approximately 2 kilobases. Sequence analyses showed that the nucleotide and deduced amino acid sequences of SPN are novel and unrelated to any known vertebrate inhibitors of proliferation. However, the deduced amino acid sequence of SPN contains two domains that have extensive sequence identity with a recently cloned transcription activator in Drosophila, deformed epidermal autoregulatory factor-1 (DEAF-1, see Gross, C. T., and McGinnis, W. (1996) EMBO J. 15, 1961-1970) suggesting that SPN represents a vertebrate cognate of deformed epidermal autoregulatory factor-1. Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses showed that the SPN mRNA and the SPN protein are expressed in every tissue examined including testis, spleen, skeletal muscle, liver, kidney, heart, and brain suggesting that SPN may be involved in the control of proliferation in a variety of cell types.

Negative regulatory molecules in the neuroendocrine and immune systems: suppressin as an example.

The early foundations of both neuroendocrinology and immunology were established by studies that linked the production, secretion, and action of circulating factors to the physiological state of an organism. These studies ultimately identified the cells of the neuroendocrine and immune systems as a rich source of such homeostatic regulatory molecules, and currently they are referred to as neuroendocrine hormones, peptides, and cytokines. More recently, two additional concepts have been added to this model. The first was that immune cells produce neuroendocrine hormones and peptides and that neuroendocrine cells produce cytokines. The second was the notion that both positive and negative factors control a variety of physiological processes. Recently, we have identified a new polypeptide negative regulator of cell proliferation that we have named suppressin (SPN). This negative regulatory molecule is also produced by both neuroendocrine and immune cells. The objective of this article is to provide an example of the biochemical, cellular, and molecular approaches used to characterize SPN and that could be used to characterize similar molecules from neuroendocrine and immune sources.

Cloning and direct sequencing from lambda cDNA libraries using the polymerase chain reaction: suppressin and the vasopressin receptor as models.

A strategy using the polymerase chain reaction (PCR) to screen a lambda gt11 pituitary cDNA library for cDNAs encoding suppressin, a putative anti-proliferative protein, and a putative vasopressin receptor is described. The use of this technique will facilitate the demonstration of e.g. the presence of "neuropeptide receptors" on cells of the lymphoid system, confirming the concept of "shared ligands and receptors" by the neuroendocrine and the immune system. Neither of the genes encoding the proteins of the present study have previously been cloned. The PCR-screening procedure requires sequence information from the gene of interest which permits the generation of complementary primers. These primers are then used in combination with lambda phage primers complementary to regions flanking the cloning site in a PCR to amplify cDNAs derived from the gene of interest. This novel screening procedure yields cDNA related to the gene of interest, including the largest clone present in the library. To confirm the utility of this technique for cDNA libraries, the library was also screened using traditional cDNA hybridization techniques. The largest clone obtained by screening the cDNA library with PCR was the same as that obtained by the conventional technique. Thus, the results of these studies show that the PCR method can be used instead of more conventional means to screen cDNA libraries. Lastly, we describe a protocol for directly sequencing PCR-amplified DNA using the same primers that are used for amplification. The combined use of these two strategies permits cloning and sequencing of cDNAs from lambda cDNA libraries in a fraction of the time required using traditional screening techniques, but with identical results.

Partial nucleotide sequence of the parvalbumin from chicken thymus designated "avian thymic hormone".

The incomplete amino acid sequence of the protein identified as avian thymic hormone was recently reported [Brewer et al. (1989), Biochem. Biophys. Res. Commun. 160, 11555-1161], and a very high degree of homology to the parvalbumins was apparent. Using mixed oligonucleotide primers based on the reported protein sequence, we have succeeded in amplifying and cloning a 188 bp fragment of the coding region for this protein, beginning with double-stranded cDNA prepared from chicken thymus mRNA. The translated nucleotide sequence of this fragment and the reported amino acid sequence display substantial disagreement. Most notably, the nucleotide sequence indicates that the CD site of avian thymic hormone is a typical parvalbumin CD site.

Amino acid sequence of thymopoietin isolated from skin.

The isolation of thymopoietin-reactive material in fetal bovine skin was monitored by means of a radioimmunoassay to thymopoietin. The amino acid sequence of this material was determined to be identical with that of thymopoietin isolated from the thymus. Experimental evidence suggests that thymopoietin in the circulation derives from the thymus and not from the skin, suggesting that the thymopoietin in keratinocytes has a local function, either apocrine and/or immunoregulatory.

In vitro synthesis of thymosin beta 4 encoded by rat spleen mRNA.

Thymosin beta 4, containing 43 amino acids and acetylated at the NH2 terminus, is synthesized in vitro in a rabbit reticulocyte lysate or in a yeast protein-synthesis system in the presence of mRNA from rat spleen. The product formed was identified as beta 4 by immunoprecipitation by a specific anti-beta 4 antiserum, comigration with authentic beta 4 in NaDodSO4/polyacrylamide gel electrophoresis and in HPLC, and identity of peptide fragments. The immunoprecipitable product generated in the wheat germ protein-synthesizing system emerged slightly ahead of beta 4 in HPLC and appeared to lack the NH2-terminal acetyl group. There was no evidence for formation of a larger polypeptide precursor of beta 4 in any of the three systems used. In sucrose density gradient centrifugation, the mRNA coding for beta 4 was recovered in the 7-8S mRNA fraction.

Synthesis of thymosin alpha 1 precursor cDNA and purification of active mRNA by affinity chromatography.

1. Thymosin alpha 1 precursor [3H]cDNA-cellulose synthesis was carried out by reverse transcription with RNA-dependent DNA polymerase from avian myeloblastosis virus using oligo(dT)-bound to cellulose as a primer. 2. Unlabelled thymosin alpha 1 precursor cDNA-cellulose was synthetized in a preparative scale to be used in affinity chromatography. 3. Poly(A)-mRNA from calf thymus was subjected to cDNA-cellulose affinity chromatography and an active messenger obtained in a yield of 3% respect to the total thymus poly(A)-mRNA chromatographied. 3. The analysis of the translation products of the purified mRNA have shown it as the thymosin alpha 1 precursor messenger.

Purification of thymus mRNA coding for a 16,000-dalton polypeptide containing the thymosin alpha 1 sequence.

A mRNA fraction purified by preparative polyacrylamide disc gel electrophoresis from calf thymus polysomes codes for a polypeptide(s) having a mass of 16,000-17,000 daltons. This polypeptide contains amino acid sequences corresponding to residues 11-18 and 19-25 of thymosin alpha 1. The yield of the octapeptide indicates that the 16,000-dalton peptide is the major product formed in the cell-free synthesis system containing the purified mRNA.

Translation of mRNA from calf thymus in the wheat germ system: evidence for a precursor of thymosin alpha1.

When translated in the wheat germ system, mRNA from fresh calf thymus stimulates incorporation of radioactive amino acids into an acid-insoluble product, and 10--20% of the total radioactivity incorporated is precipitated with antisera to active thymosin fractions. In sodium dodecyl sulfate disc gel electrophoresis, radioactivity was recovered mainly in two peptides, corresponding to 16,000 and 11,000 daltons; the latter probably represents incomplete chains. Tryptic digests of each of these peptides yielded fragments corresponding to the sequence of residues 15--19 of thymosin alpha1; these peptides were characterized by cochromatography with digests of synthetic thymosin alpha1 and by Edman degradation. Thus, the 16,000-dalton peptide synthesized in the cell-free system appears to be q precursor of thymosin alpha1 and possibly of other peptides in the fractions isolated from calf thymus. The results support the conclusion that this peptide is synthesized in the thymus gland.