Somatic Proteins Research Articles

The role of potential splicing factors including RBMY, RBMX, hnRNPG‐T and STAR proteins in spermatogenesis*

Investigations into the RBM gene family are uncovering networks of protein interactions which regulate RNA processing, and which might operate downstream of signal transduction pathways. Similar pathways likely operate in germ cells and somatic cells, with RBMY, hnRNPGT and T-STAR proteins providing germ cell-specific components. These pathways may be important for normal germ cell development, and might be compromised in men with Y chromosome deletions affecting RBMY gene expression. The STAR proteins have multiple functions in pre-mRNA splicing, signalling and cell cycle control. These processes might have to be very finely regulated during germ cell development, which involves both two sequential meiotic divisions (meiosis I and II) as well as mitotic (spermatogonial) cell divisions, and which is controlled by paracrine signalling within the testis from Sertoli cells.

Somatic embryogenesis in Solanum tuberosum from cell suspension cultures: histological analysis and extracellular protein patterns

An embryogenic cell suspension, continuously grown in Murashige and Skoog (MS) medium with 0.5 mg/L of 2,4-dichlorophenoxyacetic acid, was established from friable callus of Solanum tuberosum internode sections. The cell suspension was predominantly composed of cell masses and free embryogenic cells. When transferred to an auxin-free medium with zeatin, somatic embryos (SEs) developed and converted to complete plants when cultured on solid MS medium without growth regulators. The system produced approximately 600 SEs per 50 mL of medium. In this investigation, accumulation of extracellular proteins (EPs) of different molecular weights were found associated to different phases of the embryogenic process. At the initiation of the cell suspension, cell clusters and free cells present in the culture (phase "A") secreted a 78kDa EP, unique to this phase. In phase "B", which is related to embryonic cell determination process, proteins (7-14kDa) were secreted mainly by embryogenic cells. In phase "C", SEs in different developmental stages secreted protein of 32 kDa, which appeared as a particular feature of the phase. EPs of phase "D", secreted by torpedo and mature embryos, had molecular weights between 20 and 50 kDa. Further studies will be necessary to identify these proteins and link them to previously identified somatic embryogenesis-related proteins. Histological analysis of the potato embryogenesis in liquid media showed unicellular origin of the SE.

Histone H1 and the origin of protamines.

We present evidence that chordate protamines have evolved from histone H1. During the final stages of spermatogenesis, the compaction of DNA in many organisms is accomplished by the replacement of histones with a class of arginine-rich proteins called protamines. In other organisms, however, condensation of sperm DNA can occur with comparable efficiency in the presence of somatic-type histones or, alternatively, an intermediate class of proteins called protamine-like proteins. The idea that the highly specialized sperm chromosomal proteins (protamines) and somatic chromosomal proteins (histones) could be related dates back almost to the discovery of these proteins. Although this notion has frequently been revisited since that time, there has been a complete lack of supporting experimental evidence. Here we show that the emergence of protamines in chordates occurred very quickly, as a result of the conversion of a lysine-rich histone H1 to an arginine-rich protamine. We have characterized the sperm nuclear basic proteins of the tunicate Styela montereyensis, which we show consists of both a protamine and a sperm-specific histone H1 with a protamine tail. Comparison of the genes encoding these proteins to that of a sister protochordate, Ciona intestinalis, has indicated this rapid and dramatic change is most likely the result of frameshift mutations in the tail of the sperm-specific histone H1. By establishing an evolutionary link between the chromatin-condensing histone H1s of somatic tissues and the chromatin-condensing proteins of the sperm, these results provide unequivocal support to the notion that vertebrate protamines evolved from histones.

Two-Dimensional Protein Pattern Analysis of Extracellular Proteins Secreted by Embryogenic and Non-Embryogenic Suspension Cultures of Dactylis Glomerata L.

ABSTRACTProteins from the medium of orchardgrass (Dactylis glomerata L.) embryogenic suspension culture during defined stages of somatic embryogenesis were compared with those of non-embryogenic suspension culture during unorganized cell proliferation. After separation on two-dimensional polyacrylamide gel electrophoresis, we were able to classify 40 proteins in each of 6 groups: a group common to both embryogenic and non-embryogenic suspension cultures, a group specific only for embryogenic, respectively non-embryogenic suspension cultures and 3 groups of microcluster-, proembryogenic masses- and somatic embryo-specific proteins. One of the groups is particularly interesting as it corresponds to polypeptides that are related to the earliest stages of somatic embryo-genesis-the transition of microclusters into proembryogenic masses when no morphological changes are visible yet. Using this experimental approach, we identified the major extracellular proteins involved in somatic embryo development and we discuss on the possibility for their use as early markers for somatic embryogenesis.

The effect of convection on the nutritional status of haemodialysis patients.

Malnutrition, defined as insufficient protein-calorie intake, is highly prevalent in haemodialysis (HD) patients. It is commonly associated with decreased body weight, depleted energy stores (fat tissue) and loss of somatic proteins. It has been suggested that there may be at least two fundamentally different types of malnutrition in HD patients. The first is related to low protein and energy intake. This type of malnutrition may be amenable to adequate nutritional and dialysis support. In contrast, the second type of malnutrition is associated with inflammation and atherosclerotic cardiovascular disease (MIA syndrome). This type of malnutrition is much more difficult to reverse with nutritional support and dialysis therapy, unless the underlying co-morbid conditions and chronic inflammatory response are treated adequately. There is no single measurement which can be used to determine the presence of malnutrition. Therefore, a panel of measurements is recommended, including a measure of body composition, a measure of dietary protein intake and at least one measure of serum protein status. Therapeutic interventions in malnutrition of HD patients include dietary prescription, correction of metabolic acidosis, therapeutic intervention for co-morbidities, an adequate dose of dialysis and finally interventions on dialysis techniques, membrane and dialysate quality. It is a widespread belief that convective treatments give a clinical advantage over standard diffusive HD, when considering the physiological outcomes, such as haemodynamic instability. Since convective treatments are usually performed utilizing synthetic biocompatible membranes, it is very difficult to separate the effects of convection from those of biocompatibility on a given outcome, e.g. the nutritional status of HD patients. There are, to date, no prospective randomized clinical trials with sufficient power in which the effects of convective therapies on the nutritional status of HD patients were compared with those of other dialysis modalities. Morbidity and mortality of HD patients are strictly associated, but not necessarily linked by a cause-effect relationship to some nutritional parameters, notably serum albumin levels. Thus, as the presence of malnutrition is one of the strongest predictors of mortality, the crucial point is to demonstrate the superiority of convective treatments over standard diffusive HD on hard outcomes: morbidity and mortality. Even though almost all available studies support the hypothesis that convection and high-flux biocompatible membranes are associated with reduced morbidity and mortality risks, there is, to date, no absolute proof showing a cause-effect relationship between convection and high-flux biocompatible membranes and the outcome of HD patients. In conclusion, convective therapies offer the opportunity for as yet unrivalled small and/or middle molecular clearance. Regarding short-term complications, there is ample evidence for the fact that haemodynamic stability is better maintained during haemofiltration than during standard HD. Although a beneficial effect of convective therapies was suggested mainly in uncontrolled studies, as far as the nutritional status, the morbidity and the mortality of HD patients are concerned, there appears to be a shortage of well-controlled prospective randomized clinical trials with sufficient power assessing the potential merits of convection.

Predictors of serum creatinine in haemodialysis patients: a cross-sectional analysis.

C-reactive protein (CRP) levels, an acute phase response index, predict cardiovascular outcome and are inversely related to visceral proteins, including albuminaemia in haemodialysis patients. Less definite is the relationship between inflammation and markers of somatic proteins such as serum creatinine in such patients. To explore these questions, a cross-sectional analysis of potential predictors of serum creatinine was performed. One hundred and seventy-nine prevalent haemodialysis patients as of June 2001 were included in the cohort. Midweek pre-dialysis blood samples were collected during the months of June, September through to December 2001 inclusive, and determinations of serum urea (urease method), creatinine (alkaline picrate method) and CRP levels by means of a high sensitivity immunonephelometric method were performed. Furthermore, pre- and post-dialysis body weights were recorded and 2 min post-dialysis serum urea levels were determined three times. They were utilized for the calculation of single pool Kt/V and of normalized protein catabolic rate (nPCR). Each of the data represents the mean of three determinations made every 3 months in the study period. The analysis of multivariate linear regression was able to validate our model characterized by a dependent variable, serum creatinine and four independent variables (age, CRP, Kt/V and nPCR) (R(2)=0.60; F=24.10; P<0.00001; SE=1.94). Age (-0.08 mg/dl decrease in serum creatinine per 1-year increase in age), Kt/V (-0.25 mg/dl decrease in serum creatinine per 0.1 increase in Kt/V) and nPCR (0.10 mg/dl increase in serum creatinine per 0.1 g protein/kg/day increase in nPCR) were independently predictive of serum creatinine (P<0.00001). CRP and dialysis vintage did not predict serum creatinine. Stratifying the patients for the effects of CRP, only CRP values </=4 mg/l were directly predictive of serum creatinine (P<0.00001), whereas CRP values >4 mg/l were not. A further insight was given by the stratification of the patients for the effects of the interquartile ranges of CRP: it showed a progressive and statistically significant reduction of beta-coefficient inversely related to the increasing CRP values (P=0.003). Thus, the nature of the correlation between CRP and serum creatinine changes with increasing CRP values: from being a direct one, it shows a trend towards a transformation into an indirect one with beta=0 at a CRP value of approximately 9 mg/l. However, this indirect relationship does not reach statistical significance. The present cross-sectional study suggests that the activation of acute phase response does not influence creatinine metabolism in haemodialysis patients; in contrast, age, Kt/V and nPCR predict serum creatinine levels. Larger prospective trials are needed to achieve a definitive answer about the relationship between somatic proteins, acute phase response activation and nutrition in dialysis patients.

Identification of excretory-secretory products of larval and adult Ostertagia ostertagi by immunoscreening of cDNA libraries.

Excretory-secretory (ES) products of Ostertagia ostertagi, an abomasal nematode of cattle, are considered to be important for the development and survival of the parasite within the host. To gain insight in the composition of these ES products of both larval (L3, L4) and adult life stages of Ostertagia cDNA libraries of the parasite were immunoscreened with polyclonal rabbit serum raised against these ES products. This approach led to the identification of 41 proteins, amongst which are structural proteins such as actin, kinesin and vitellogenin, housekeeping proteins such as those involved in protein folding, different metabolic pathways or mitochondrial functioning and proteins associated with stress (heat shock protein) or antioxidantia (thioredoxin peroxidase). A large number of the isolated proteins were similar to hypothetical proteins of the model nematode Caenorhabditis elegans. Because somatic proteins can be non-specifically released during in vitro culturing as nematodes deteriorate, it was checked if the isolated proteins are genuinely secreted. The amino acid sequences of the translated cDNAs were investigated for signal peptides and monospecific antibodies against the isolated proteins were purified and used to develop Western blots of ES and somatic extracts. In this manner it could be proven that 15 cDNAs code for genuine secreted proteins. The identification of these ES antigens allows to select proteins with potential protective capacities, which are targets for vaccine development.

Onset of serum antibodies to Pasteurella (Mannheimia) haemolytica following vaccination with five commercial vaccines

The purpose of this study was to compare the antibody responses of cattle vaccinated with one of five commercial non-living P. haemolytica vaccines during the first two weeks after a single vaccination. Antibodies to surface antigens (whole cells) and to the leukotoxin (LKT) were quantified at Days 7 and 14 after vaccination. In addition, antibodies to specific P. haemolytica whole cell protein antigens were determined by immunoblots using sera from vaccinated and control calves. Three hundred pre-weaned, mixed beef breed steers and heifers, greater than three months old, were equally divided among five vaccinated and one placebo groups. Cattle were subcutaneously injected with one of the following commercial P. haemolytica vaccines: One Shot, One Shot Ultra 8, Pyramid 4+Presponse SQ, Pulmo-guard PH-M or Poly-Bac B 1. Antibody responses to both LKT and whole cells were highest on Day 7 for One Shot and One Shot Ultra. By Day 14, Pulmo-Guard stimulated serum antibodies to both antigen preparations that were equivalent to those for One Shot and One Shot Ultra. Serum antibodies to LKT and whole cells were detected in the Pyramid/Presponse- and Poly-Bac 1 - vaccinated groups; however, those responses were not significantly different from the placebo group. Each vaccine, except for the Pyramid 4/Presponse - vaccinated group, stimulated antibodies to similar P. haemolytica somatic proteins, particularly at 200, 100, 60-70, 45, 35 and 25 kDa. Sera from the Pyramid 4/Presponse-vaccinated group recognized fewer protein bands, mainly those at 200, 70-75, 48 and 35 kDa. In conclusion, various commercial vaccines induced differences in the rapidity and the intensity of serum antibody responses to P. haemolytica whole cells and LKT.

Human sperm telomere-binding complex involves histone H2B and secures telomere membrane attachment.

Telomeres are unique chromatin domains located at the ends of eukaryotic chromosomes. Telomere functions in somatic cells involve complexes between telomere proteins and TTAGGG DNA repeats. During the differentiation of germ-line cells, telomeres undergo significant reorganization most likely required for additional specific functions in meiosis and fertilization. A telomere-binding protein complex from human sperm (hSTBP) has been isolated by detergent treatment and was partially purified. hSTBP specifically binds double-stranded telomeric DNA and does not contain known somatic telomere proteins TRF1, TRF2, and Ku. Surprisingly, the essential component of this complex has been identified as a specific variant of histone H2B. Indirect immunofluorescence shows punctate localization of H2B in sperm nuclei, which in part coincides with telomeric DNA localization established by fluorescent in situ hybridization. Anti-H2B antibodies block interactions of hSTBP with telomere DNA, and spH2B forms specific complex with this DNA in vitro, indicating that this protein plays a role in telomere DNA recognition. We propose that hSTBP participates in the membrane attachment of telomeres that may be important for ordered chromosome withdrawal after fertilization.

Three major somatic embryogenesis related proteins in Cichorium identified as PR proteins

In Cichorium hybrid clone '474' (C. intybus L., var. sativum x C. endivia L., var. latifolia), the direct somatic embryogenesis process in leaf tissues is accompanied by an overall increase in the amount of proteins secreted into the culture medium. Amongst these, three major protein bands of 38 kDa, 32 kDa and 25 kDa were found in the conditioned media. These extracellular protein bands accumulated in the medium of the embryogenic Cichorium hybrid up to 8-fold compared with those in the medium of a nonembryogenic variety. 32 and 25 kDa proteins were purified from the medium and their identities were determined as already described for 38 kDa beta-1,3-glucanases. To investigate their possible function in somatic embryogenesis, peptide sequences, serological relationships or biochemical properties revealed that there were at least two acidic chitinases of 32 kDa and one glycosylated osmotin-like protein of 25 kDa in the embryogenic culture medium. Comparing the amounts of the 38 kDa glucanases, the 32 kDa chitinases, and the 25 kDa osmotin-like protein present in the conditioned media of the embryogenic '474' hybrid and of a non-embryogenic variety, a 2-8-fold higher accumulation of these proteins was observed in the embryogenic hybrid culture medium. This may suggest that part of the accumulation of these three pathogenesis-related (PR) proteins could be correlated with the somatic embryogenesis process. Their possible involvement in this developmental process is discussed.

Three major somatic embryogenesis related proteins in Cichorium identified as PR proteins

In Cichorium hybrid clone ‘474’ ( C . intybus L., var. sativum× C . endivia L., var. latifolia), the direct somatic embryogenesis process in leaf tissues is accompanied by an overall increase in the amount of proteins secreted into the culture medium. Amongst these, three major protein bands of 38 kDa, 32 kDa and 25 kDa were found in the conditioned media. These extracellular protein bands accumulated in the medium of the embryogenic Cichorium hybrid up to 8‐fold compared with those in the medium of a non‐embryogenic variety. 32 and 25 kDa proteins were purified from the medium and their identities were determined as already described for 38 kDa β‐1,3‐ glucanases. To investigate their possible function in somatic embryogenesis, peptide sequences, serological relationships or biochemical properties revealed that there were at least two acidic chitinases of 32 kDa and one glycosylated osmotin‐like protein of 25 kDa in the embryogenic culture medium. Comparing the amounts of the 38 kDa glucanases, the 32 kDa chitinases, and the 25 kDa osmotin‐like protein present in the conditioned media of the embryogenic ‘474’ hybrid and of a non‐embryogenic variety, a 2–8‐fold higher accumulation of these proteins was observed in the embryogenic hybrid culture medium. This may suggest that part of the accumulation of these three pathogenesis‐related (PR) proteins could be correlated with the somatic embryogenesis process. Their possible involvement in this developmental process is discussed.

Identification and characterization of a bovine sperm protein that binds specifically to single-stranded telomeric deoxyribonucleic acid.

Telomere DNA at the physical termini of chromosomes forms a single-stranded 3' overhang. In lower eukaryotes, e.g., ciliated protozoa, this DNA extension is capped by specific proteins that have been structurally and functionally characterized. Much less is known about single-stranded telomere DNA-binding proteins in vertebrates. Here we describe a new protein from bovine sperm designated bsSSTBP that specifically interacts with single-stranded (TTAGGG)(N) DNA. The bsSSTBP was extracted from nuclei by 0.6 M KCl. The native size of this protein, estimated by gel filtration, was 20-40 kDa. SDS-PAGE of the UV cross-linked complex between bsSSTBP and telomere DNA indicated that several polypeptides are involved in complex formation. Bovine sSSTB had high specificity toward nucleotide sequence, since single nucleotide substitutions in the (TTAGGG)(4) substrate suppressed binding. The minimal number of (TTAGGG) repeats required for binding of bsSSTBP was 3, and the protein recognized linear but not folded DNA structures. We propose that the bsSSTBP participates in telomere-telomere interactions and the telomere membrane localization observed in mature sperm. In mammals, somatic telomere-binding proteins are apparently substituted by sperm-specific ones that may lead to a structural reorganization of telomere domains to fulfill functions important during meiosis and fertilization.

Production of granulocyte-macrophage colony stimulating factor (GM-CSF) by high cell density fermentation of secretory recombination Escherichia Coli

High cell density fermentation production of recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) was studied in an Escherichia coli secretory expression system. By using glycerol instead of glucose as a carbon source and feeding in a specific fed-batch mode and by controlling the temperature at about 30°C and maintaining the dissolved oxygen level at 20∼25% saturation, cell density increased from 30 to 91.8 g l −1, the production of recombinant GM-CSF remained high and the fermentation time was reduced from 45 to 30 h. Recombination GM-CSF was purified by hydrophobic column-molecular sieve-ion exchange column chromatography. The expression of recombinant GM-CSF was not affected by fermentation methods, the production of GM-CSF reached 28.04% (about 1.68 g l −1) and 27.51% (about 1.64 g l −1) of total somatic proteins for high cell density and normal fermentation, respectively. The corresponding value for specific activity of GM-CSF was 2.6×10 7, and 2.4×10 7, respectively.

Presence of antibodies specific for proteins ofContracaecum osculatum(Rudolphi, 1908) in plasma of several Antarctic teleosts

The immunoglobulin fraction was purified from the plasma of an Antarctic teleost,Chionodraco hamatus(Lönnberg, 1906) by thiophilic adsorption chromatography and size-exclusion FPLC. Somatic proteins ofContracaecum osculatumlarvae were recovered by boiling whole nematodes in phosphate buffer containing 2% SDS and 10% β-mercaptoethanol. Immunoglobulins fromC. hamatusplasma recognisedC. osculatumsomatic antigens either blotted onto nitrocellulose or immobilised on a HiTrap NHS-activated column. The relative molecular mass of the majorC. osculatumantigen was 91kDa. Plasma from specimens ofC. hamatus,Notothenia coriiceps(Richardson, 1844),Notothenia gibberifrons(Lönnberg, 1905),Chaenocephalus aceratus(Lönnberg, 1906),Pseudochaenichthys georgianus(Norman, 1937),Trematomus bernacchii(Boulenger, 1902) were tested in double immunodiffusion againstC. osculatumproteins. Immunoprecipitates were observed with individual plasma of five out of six species. The results demonstrate the presence of nematode-specific antibodies in Antarctic fish plasma.

The mouse histone H1 genes: gene organization and differential regulation

There are six mouse histone H1 genes present in the histone gene cluster on mouse chromosome 13. These genes encode five histone H1 variants expressed in somatic cells, H1a to H1e, and the testis-specific H1t histone. Two of the genes that have not been assigned previously to the five somatic H1 subtypes have been identified as encoding the H1b and H1d subtypes. Three of the H1 genes, H1a, H1c and H1t, are present on an 80 kb segment of DNA that contains nine core histone genes. Two others, H1d and H1e, are present in a second patch, while the H1b gene is at least 500 kb away in a patch containing 14 core histone genes. The histone H1 genes are differentially expressed. All five genes for the somatic histone H1 proteins are expressed in exponentially growing cells. However, the levels of H1a, H1b and H1d mRNAs are greatly reduced in cells that are terminally differentiated or arrested in G0, while the H1c and H1e mRNAs continue to be expressed. In addition to the major RNA that ends at the stem-loop, the H1c gene expresses a longer, polyadenylated mRNA in differentiated cells, although in varying amounts. None of the other histone H1 genes encodes detectable amounts of polyadenylated mRNAs.

The effect of γ-radiation and heat shock on protein synthesis and antioxidant enzymes in the gastrointestinal parasite, Heligmosomoides polygyrus

Protein synthesis and antioxidant enzyme activities were investigated in γ-irradiated (300 Gy) and heat shocked (42°C) larval stages of the gastrointestinal parasite, Heligmosomoides polygyrus bakeri (H. polygyrus). No qualitative or quantitative differences were observed in the incorporation of ( 35S)-methionine into somatic proteins of unirradiated or irradiated exsheathed third-stage (L3) larvae at either 37°C or 42°C. The rate of protein synthesis doubled in L3 stages maintained at 42°C compared with 37°C, irrespective of whether the larvae had been irradiated or not. The composition of excretory/secretory (ES) proteins varied between unirradiated and irradiated exsheathed L3 larvae maintained under identical conditions. Prominent heat-inducible proteins of 26 and 17 kDa were synthesised and excreted at 42°C by both unirradiated and irradiated L3 stages. No major differences in protein synthesis could be detected between unirradiated and irradiated fourth-stage (L4) larvae. Temperature elevation significantly reduced protein synthesis in L4 stages, most notably in unirradiated parasites. Heat-inducible proteins were not detected in response to either irradiation or temperature elevation in L4 larvae. Immune sera recognised a similar spectrum of antigens in both unirradiated and irradiated L4 somatic and ES preparations and reacted with antigens from irradiated L4 parasites with less intensity than with antigens from unirradiated L4 larvae. Catalase was the only antioxidant enzyme examined with activity that changed significantly in irradiated parasites, being reduced to approximately 36% of normal levels in irradiated L4 stages. No significant difference existed between irradiated and unirradiated parasites in the levels of activity of superoxide dismutase and glutathione reductase.

Somatic antigens of adult Dicrocoelium dendriticum recognised by bile antibodies of naturally infected cattle

Bile samples, from slaughtered cattle harbouring between 120 and 280 adult lancet flukes, were used to investigate the range of somatic proteins inducing local antibody responses in naturally infected animals. Lancet fluke infections induced local (bile) antibody responses against Tris-buffered saline (TBS) soluble, sodium dodecyl sulphate (SDS) soluble and 2-mercaptoethanol (2-Me) soluble somatic proteins of adult Dicrocoelium dendriticum. IgA antibody isotypes predominated in the response against buffer-soluble somatic antigens, whereas SDS-soluble and 2-Me-soluble proteins induced similar level of both IgA and IgG1 antibodies. Analysis of the antigens recognised by particular isotype-specific bile antibodies suggests that different antigens preferentially induce isotype restricted antibody responses. The bile antibody response was highly species specific, only one antigen from somatic protein extracts of Fasciola hepatica being precipitated by bile samples showing the highest reactivity against D. dendriticum.

Metabolic labelling of wild and laboratory subspecies of the trichostrongyle nematode Heligmosomoides polygyrus

The immunological, biochemical and taxonomic relationship between wild and laboratory subspecies of Heligmosomoides polygyrus was studied by metabolically labelling parasite proteins with [ 35S]-methionine. Much variability, both in content and synthesis of proteins was observed between the two subspecies. Laboratory female worms had a higher protein content and incorporated more radioactive label into somatic proteins than their wild counterparts. Incorporation of radioactivity into excretory/secretory (ES) proteins, predicted to contain important antigens, demonstrates a major reduction in synthesis of proteins with molecular weights 66, 55, 43, 41, 40, 39, 37, 28 and 16 kDa by laboratory females. These differences in protein synthesis might explain the differing infectivities of the two subspecies when passaged in inbred laboratory ( Mus musculus) and wild ( Apodemus sylvaticus) mice.

Antigen-specific T-cell responses during primary and secondary Listeria monocytogenes infection

Although murine listeriosis is a widely used experimental model for the analysis of cell-mediated immunity, there is little information about individual T-cell antigens of Listeria monocytogenes which are recognized during primary and secondary infection. To study the antigen responses of L. monocytogenes-reactive T cells, somatic and secreted listerial proteins were separated by two-dimensional gel electrophoresis and subsequently divided into 480 liquid fractions. Antigen-specific T cells isolated from mice at different times of primary and secondary listeriosis were tested for their capacity to proliferate with distinct protein fractions. Supernatants of these cultures were screened for the production of gamma interferon, interleukin-4 (IL-4), and IL-10. Proliferation of antigen-specific T cells correlated with the production of high concentrations of gamma interferon, whereas IL-4 and IL-10 production in response to listerial protein fractions could not be detected. During both primary and secondary listeriosis, T cells recognized a multitude of somatic and secreted proteins rather than one or a few dominant antigens. Secreted proteins were recognized before somatic proteins, and T cells recognized different fractions in secreted and somatic proteins.

Structure and expression of the mouse testicular H1 histone gene (H1t)

A mouse genomic library was screened with a human testicular H1 (H1t) gene fragment. One phage containing the testis specific mouse H1t histone gene and its flanking regions was isolated. Northern blot analysis showed that the mouse H1t gene is expressed only in mouse testis at the stage of pachytene spermatocytes and that the H1t mRNA is not polyadenylated. This mouse H1t gene encodes a protein wich differs from the somatic mouse H1 proteins, but is similar to the known H1t proteins from rat [1], and man [2].