Tracer and electrical coupling of rat suprachiasmatic nucleus neurons

Whole-cell recording from single neurons of the suprachiasmatic nucleus with an electrode containing the tracer neurobiotin resulted in the staining of multiple neurons in 30% of the cases. Typically, one neuron was darkly stained with dendritic processes and an axon clearly visible while other neurons were lightly stained. The darkly-stained cells were identified as the recorded neuron and tracer-coupled to one to five lightly stained neurons. The resting membrane potential, input membrane conductance, membrane capacitance, the decay time constant and the maximum H-current amplitude of the recorded neurons with tracer-coupled cells were not significantly different from those of neurons not showing tracer coupling. Stimulation of the preoptic area activated an antidromic action potential or an all-or-none small slow inward current in some neurons when the synaptic transmission was blocked by a calcium-free/Mn2+ solution. The small slow inward current did not "collide" with an orthodromically activated action spike suggesting that the current represents the signal from an electrotonically-coupled neuron. In addition, the frequency of biphasic field currents from a neighbouring cell firing were increased by depolarization and decreased by hyperpolarization of the recorded cell. These data demonstrate a chemical and electrical low-resistance coupling of suprachiasmatic nucleus neurons, which could be important in synthesizing the suprachiasmatic nucleus circadian rhythm.     

Identification of genes expressed in human CD34(+) hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning

Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34(+) HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34(+) cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2, 603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5' ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation.     

Autoimmune hepatitis (history of the research, current aspects)

Important achievement in the study of autoimmune hepatitis are shown, particularly in establishing the role of hepatitis viruses, other hepatotropic viruses and some medicines. The last classification of chronic hepatitis based on the etiology, autoimmune hepatitis considers autoimmune hepatitis as a disease of unknown etiology. It is thought valid that investigation of autoimmune reactions produced by hepatitis viruses and other agents will enable a revision of classification of chronic hepatitis.     

An analysis of preliminary results in screening for prostatic cancer]

450 males aged over 50 years free of urological symptoms were screened for prostatic cancer using three techniques; finger rectal examination (FRE), transrectal ultrasound investigation (TUI), assay for prostatic specific antigen in the serum (SPSA). SPSA quantities under 4 ng/ml, 4-10 ng/ml, 10-20 ng/ml, over 20 ng/ml were registered in 206(45.8%), 135(30%), 69(15.4%) and 40(8.8%) patients, respectively. Detectability of prostatic cancer increases by 33,37.9, 45.5, 69.2% due to TUI, FRE, TUI + FRE, all the three methods, respectively. Prostatic biopsy was needed in 102 (22.7%) cases. From the 450 examinees, prostatic cancer was diagnosed in 25 (5.6%). SPSA was high in all of them, higher than 10 ng/ml in 92%. 20 (80%) of 25 patients with cancer had early stages of the disease (TI-2). The study is going on.     

Label-retaining cells are preferentially located in fornical epithelium: implications on conjunctival epithelial homeostasis

PURPOSE: To determine the cell kinetic properties of epithelial cells from various zones of the conjunctiva. METHODS: The morphology and cell kinetics of bulbar, fornical, and palpebral conjunctival epithelium were studied in neonatal and adult SENCAR mice. To examine the proliferative rate of the conjunctival epithelium, a single administration of tritiated thymidine (3H-TdR) was used to detect cells in "S" phase. Proliferative rates were also assessed by determining mitotic activity after an intraperitoneal injection of colchicine to arrest cells in mitosis. To detect slow-cycling cells, mice received 3H-TdR continuously for 1 week. After a 4-week chase, animals were sacrificed and eyes were surgically removed. All tissues were immediately fixed in formalin and processed for histology and autoradiography. RESULTS: Slow-cycling cells, detected as label-retaining cells (LRCs), were identified in bulbar, fornical, and palpebral epithelia, as well as in limbal epithelium. The greatest number of LRCs was found in fornical epithelium. In addition, we found a number of label-retaining goblet cells. This cell population was shown to incorporate 3H-TdR after a single pulse administration, and mitotic figures were seen in goblet cells after colchicine treatment, indicating that conjunctival goblet cells have proliferative capabilities. CONCLUSIONS: These findings are consistent with earlier in vitro data that the fornical epithelium may be a zone enriched in conjunctival epithelial stem cells. This has important implications in conjunctival epithelial development and is relevant in wound repair. Furthermore, the concept that goblet cells are slow-cycling cells with proliferative capabilities provides new insights into the area of conjunctival homeostasis.     

Porcine pyridoxal kinase c-DNA cloning, expression and confirmation of its primary sequence

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.     

Hematological investigations in conditions of long-term space flights

In the nearly 15-month mission aboard MIR the cosmonaut-physician and members of three crews (MIR-15, -16, and -17) carried out a program of hematological investigations. Most of the changes related to the red blood system and included reduction in hemoglobin and hematocrit. Erythrocytes had decreased concentration and took on abnormal forms. There were also signs of altered metabolism of erythrocytes. Of interest are phase-by-phase variations in the levels of erythrocytes in the course of long-term stay in microgravity, and absence of a convincing correlation between hemoglobin, erythrocyte and hematocrit levels. But for lymphocytosis that returned to the norm already on the first day of recovery, no material changes occurred to the leukocyte profile. Investigations at the landing site displayed erythropenia, decreased reticulocytes and ensuing reticulocyte reaction, and gradual regain of the erythrocyte number that can be viewed as a normal physiological reaction of the blood system to the set of factors of spaceflight and early readaptation. Besides, the investigations showed a large individually of blood reactions to prolonged stay in space flight.