Miller-Fisher Syndrome in the Setting of Influenza A Infection.

Miller-Fisher syndrome (MFS) is a rare, and milder, variant of Guillain-Barre syndrome (GBS) that is characterized by ophthalmoplegia, areflexia, and ataxia, with the additional possibility of limb weakness. There is not a specific demographic or common situation in which MFS is usually seen. This paper details a suspected case of MFS in a 59-year-old male with a concurrent influenza infection. He had been experiencing progressive flu-like symptoms a few days prior to the onset of his neurological symptoms, presenting to the hospital with diplopia and paresthesias of his extremities. His physical exam on admission revealed areflexia and gait instability, as well as oculomotor nerve palsies that were causing his diplopia. After running tests to rule out other possible causes of his presentation, along with having a positive influenza A test, he was diagnosed with MFS and started on intravenous immunoglobulin (IVIG). His symptoms resolved by the end of the treatment course. Based on his presentation and resolution of symptoms, this would be one of the few reported cases of MFS following influenza A infection.

New USEPA water quality criteria by 2012: GOMA concerns and recommendations.

The Gulf of Mexico Alliance (GOMA) was tasked by the five Gulf State Governors to identify major issues affecting the Gulf of Mexico (GoM) and to set priorities for ameliorating these problems. One priority identified by GOMA is the need to improve detection methods for water quality indicators, pathogens and microbial source tracking. The United States Environmental Protection Agency (USEPA) is tasked with revising water quality criteria by 2012; however, the locations traditionally studied by the USEPA are not representative of the GoM and this has raised concern about whether or not the new criteria will be appropriate. This paper outlines a number of concerns, including deadlines associated with the USEPA Consent Decree, which may prevent inclusion of research needed to produce a well-developed set of methods and criteria appropriate for all regulated waters. GOMA makes several recommendations including ensuring that criteria formulation use data that include GoM-specific conditions (e.g. lower bather density, nonpoint sources), that rapid-testing methods be feasible and adequately controlled, and that USEPA maintains investments in water quality research once the new criteria are promulgated in order to assure that outstanding scientific questions are addressed and that scientifically defensible criteria are achieved for the GoM and other regulated waterbodies.

Left ventricular myocardial deformation as measure of hemodynamic burden in congenital valvular aortic stenosis.

BACKGROUND: Changes in 2D echocardiography (2DE) speckle tracking imaging (STI) derived left ventricular (LV) strain (S) and strain rate (SR) precedes diminution of LV ejection fraction (LVEF) in adult valvular aortic stenosis (AS). We prospectively examined whether 2DE-STI derived multidirectional LV S and SR correlate with AS severity in children using LV mass index (MI) as the principal outcome variable. METHODS: 52 children (10.4 ± 7.3 years) with isolated congenital AS were included; 13 mild (2.5 m/s < Vmax < 3.0 m/s), 25 moderate (3.0 m/s < Vmax < 4.0 m/s), and 14 severe (Vmax > 4.0 m/s). 2DE including Doppler and STI longitudinal strain (LS), strain rate (LSR), circumferential strain (CS), and strain rate (CSR) were measured. Univariate and multivariable linear regressions identified correlations between LVMI and strain indices. RESULTS: Three clinical and 2DE variables, and four strain indices were independently associated with LVMI. LVMI correlated positively with systolic blood pressure and aortic regurgitation, and negatively with LVEF. LVMI correlated positively with LSR (four-chamber) and CSR (basal), and negatively with segmental CS in the inferior (basal) and anteroseptal (distal) segments. LVMI showed significant inverse association with LS (P = .05), LSR (P < .001), CS (P < .005), and CSR (P < .0001), independent of AS severity. CONCLUSIONS: Independent of clinical and 2DE findings including contemporaneous Doppler estimates of AS gradient, both longitudinal and circumferential strain indices correlate with LVMI as a measure of cumulative hemodynamic burden. This association implies subclinical LV dysfunction.

Binding of RFX2 and NF-Y to the testis-specific histone H1t promoter may be required for transcriptional activation in primary spermatocytes.

The testis-specific linker histone H1t is transcribed exclusively in pachytene spermatocytes during spermatogenesis. The H1t promoter contains two imperfect inverted repeats which together comprise the X-box motif that is known to bind the transcription factor regulatory factor X (RFX). Out of all the histone H1 family promoters this motif appears only in the H1t promoter and may contribute to H1t tissue-specific expression. We show by Western blotting, EMSA, ChIP analysis, and real-time RT-PCR that the rat H1t X-box is bound by RFX2 in vivo in spermatocytes. We demonstrate that transcription factor NF-Y binds to the CCAAT-box motif that is located downstream and adjacent to the X-box and that testis NF-Y interacts either directly or indirectly with RFX2. Furthermore, we show that both the X-box and CCAAT-box are required for promoter activity and that co-expression of RFX2 greatly enhances testis histone H1t promoter activity in the GC-1spg germinal cell line.

Transcription factor RFX4 binding to the testis-specific histone H1t promoter in spermatocytes may be important for regulation of H1t gene transcription during spermatogenesis.

The X-box binding protein RFX4 is highly expressed in testis in contrast with other tissues, but its function there is unknown. Another family member abundant in testis, RFX2, has been shown to bind to the X-Box elements in the promoter of the testis specific histone H1t, which is expressed only in pachytene spermatocytes. RFX proteins are known to dimerize, and there is the possibility that the abundant testis RFX4, which is also expressed in pachytene spermatocytes as shown by RT-PCR and Western blotting, may interact with RFX2 in these cells. In EMSA anti-RFX2 polyclonal antibodies produce a supershifted complex with testis extracts and an X-Box probe. On the other hand, RFX4 polyclonal antibodies do not supershift the complex but appear to enhance formation of the complex. RFX4 appears to co-precipitate with RFX2 in immunoprecipitation, and to co-purify with RFX2 in an affinity purification using a biotinylated X-box affinity probe. In ChIP assays RFX4 also binds to the H1t promoter in vivo. These data suggest a possible regulatory role for RFX4 in transcription of the histone H1t gene during spermatogenesis.

Transcriptional repression of the testis-specific histone H1t gene mediated by an element upstream of the H1/AC box.

The testis-specific histone H1t gene is transcribed exclusively in primary spermatocytes and may be important for chromatin structure, transcription, and DNA repair during this stage of spermatogenesis. Transcriptional repression of the gene in other cell types is mediated in part by specific proximal and distal promoter elements and in some cell types by methylation of CpG dinucleotides within the promoter. Our laboratory identified a distal promoter element located between 948 and 780 bp upstream from the transcription initiation site and another laboratory identified a GC-rich region between the TATA box and transcription initiation site that contribute to repression. In this article we address transcriptional repression of the histone H1t gene by an element within the proximal promoter. We report discovery of an element designated H1t promoter repressor element (RE) located between -130 and -106 bp that contributes to repression. The findings support the hypothesis that multiple mechanisms are involved in transcriptional repression of the H1t gene. Transcriptional repression mediated by the RE element in NIH 3T3 cells appears to differ significantly from the mechanism mediated by the GC-rich region. Furthermore, binding proteins that form the RE complex are not present in rat testis where the gene is actively transcribed. Our findings provide a molecular basis for histone H1t gene repression.

Upregulation of prostate specific membrane antigen/folate hydrolase transcription by an enhancer.

Prostate specific membrane antigen (PSMA), also known as folate hydrolase (FOLH1), is a 100 kDa glycoprotein with elevated expression in prostate epithelial tissue. Expression of PSMA is upregulated as prostate tumor grade increases and is found in the vasculature of many tumors, with no presence in benign tissues. Due to the potential of the regulatory elements of the PSMA promoter and enhancer to be used in gene therapy and as biomarkers for prostate cancer under conditions of androgen ablation during treatment, we sequenced and analyzed the ability of 5.5 kb of PSMA promoter/leader region to promote transcription. A recently discovered enhancer, found in the third intron of the PSMA gene, FOLH1, was also studied. The promoter/leader region sequence provided basal expression in transcription assays, while addition of the enhancer activated transcription 41-fold in transient transfections and 144-fold in stable transfections of the LNCaP prostate cell line. This enhancement of transcription was not found in nonprostate cell lines or prostate cell lines that do not express PSMA. An analysis of the ability of androgens to act via the PSMA promoter/leader region and enhancer to activate transcription in transiently transfected LNCaP cells revealed no significant androgen response using the FOLH1 promoter/leader region and a downregulation of 42% with addition of the enhancer. In stably transfected LNCaP cells, the FOLH1 promoter/leader region produced a 21% downregulation in response to androgens, while addition of the enhancer resulted in a 45% downregulation. These results demonstrate significant upregulation of transcription by the PSMA promoter/enhancer, with specificity for the LNCaP prostate cell line, and downregulation of transcription in response to physiological levels of androgen.

A colorimetric method for monitoring tryptic digestion prior to shotgun proteomics.

Tryptic digestion is an important preanalytical step in shotgun proteomics because inadequate or excessive digestion can result in a failed or incomplete experiment. Unfortunately, this step is not routinely monitored before mass spectrometry because methods available for protein digestion monitoring either are time/sample consuming or require expensive equipment. To determine if a colorimetric method (ProDM Kit) can be used to identify the extent of tryptic digestion that yields the best proteomics outcome, plasma and serum digested for 8 h and 24 h were screened with ProDM, Bioanalyzer, and LC/MS/MS, and the effect of digestion on the number of proteins identified and sequence coverage was compared. About 6% and 16% less proteins were identified when >50% of proteins were digested in plasma and serum, respectively, compared to when ~46% of proteins were digested. Average sequence coverage for albumin, haptoglobin, and serotransferrin after 2 h, 8 h, and 24 h digestion was 52%, 45%, and 45% for serum and 54%, 47%, and 42% for plasma, respectively. This paper reiterates the importance of optimizing the tryptic digestion step and demonstrates the extent to which ProDM can be used to monitor and standardize protein digestion to achieve better proteomics outcomes.

Subcutaneously administered ofatumumab in rheumatoid arthritis: a phase I/II study of safety, tolerability, pharmacokinetics, and pharmacodynamics.

OBJECTIVE: To investigate the safety and tolerability of a single subcutaneous (SC) dose of ofatumumab, a fully human anti-CD20 monoclonal antibody, in patients with rheumatoid arthritis (RA) taking background methotrexate (MTX). Secondary objectives included characterizing pharmacokinetics and pharmacodynamics. METHODS: In this single-blind, phase I/II study, 35 patients with RA were randomized in 5 cohorts to receive a single subcutaneous (SC) ofatumumab dose ranging from 0.3 to 100 mg, or placebo, following premedication with oral acetaminophen and antihistamine. Patients were followed for 24 weeks with extended followup to monitor B cell and immunoglobulin recovery for up to 2 years if required. RESULTS: Thirty-five patients received the following treatment: 0.3 mg, n = 4; 3 mg, n = 6; 30 mg, n = 8; 60 mg, n = 6; 100 mg, n = 3; placebo, n = 8. The most common adverse events in the combined ofatumumab groups were headache, nausea, and upper respiratory tract infection. Because of tolerability concerns, only 3 patients were given 100 mg. For the 30-100 mg doses, median maximum plasma concentration values ranged from 4.02 to 4.49 days. Mean elimination half-life values ranged from 5.20 to 6.83 days. Increasing peripheral median B cell depletion was observed from 0.3 mg up to 30 mg, and full target B cell depletion was achieved with 30 mg, 60 mg, and 100 mg. CONCLUSION: Treatment of RA patients with SC ofatumumab doses of 30 mg or higher resulted in profound and prolonged B cell depletion in blood. Single doses up to 60 mg were tolerated without glucocorticoid premedication. (ClinicalTrials.gov identifier NCT00686868).

Influence of post-traumatic stress disorder on neuroinflammation and cell proliferation in a rat model of traumatic brain injury.

Long-term consequences of traumatic brain injury (TBI) are closely associated with the development of severe psychiatric disorders, such as post-traumatic stress disorder (PTSD), yet preclinical studies on pathological changes after combined TBI with PTSD are lacking. In the present in vivo study, we assessed chronic neuroinflammation, neuronal cell loss, cell proliferation and neuronal differentiation in specific brain regions of adult Sprague-Dawley male rats following controlled cortical impact model of moderate TBI with or without exposure to PTSD. Eight weeks post-TBI, stereology-based histological analyses revealed no significant differences between sham and PTSD alone treatment across all brain regions examined, whereas significant exacerbation of OX6-positive activated microglial cells in the striatum, thalamus, and cerebral peduncle, but not cerebellum, in animals that received TBI alone and combined TBI-PTSD compared with PTSD alone and sham treatment. Additional immunohistochemical results revealed a significant loss of CA3 pyramidal neurons in the hippocampus of TBI alone and TBI-PTSD compared to PTSD alone and sham treatment. Further examination of neurogenic niches revealed a significant downregulation of Ki67-positive proliferating cells, but not DCX-positive neuronally migrating cells in the neurogenic subgranular zone and subventricular zone for both TBI alone and TBI-PTSD compared to PTSD alone and sham treatment. Comparisons of levels of neuroinflammation and neurogenesis between TBI alone and TBI+PTSD revealed that PTSD did not exacerbate the neuropathological hallmarks of TBI. These results indicate a progressive deterioration of the TBI brain, which, under the conditions of the present approach, was not intensified by PTSD, at least within our time window and within the examined areas of the brain. Although the PTSD manipulation employed here did not exacerbate the pathological effects of TBI, the observed long-term inflammation and suppressed cell proliferation may evolve into more severe neurodegenerative diseases and psychiatric disorders currently being recognized in traumatized TBI patients.

The effect of selenium enrichment on baker's yeast proteome.

The use of regular yeast (RY) and selenium-enriched yeast (SEY) as dietary supplement is of interest because the Nutritional Prevention of Cancer (NPC) trial revealed that SEY but not RY decreased the incidence of prostate cancer (PC). Using two-dimensional difference in gel electrophoresis (2D-DIGE)-tandem mass spectrometry (MS/MS) approach, we performed proteomic analysis of RY and SEY to identify proteins that are differentially expressed as a result of selenium enrichment. 2D-DIGE revealed 96 candidate protein spots that were differentially expressed (p≤0.05) between SEY and RY. The 96 spots were selected, sequenced by LC/MS/MS and 37 proteins were unequivocally identified. The 37 identified proteins were verified with ProteinProphet software and mapped to existing Gene Ontology categories. Furthermore, the expression profile of 5 of the proteins with validated or putative roles in the carcinogenesis process, and for which antibodies against human forms of the proteins are available commercially was verified by western analysis. This study provides evidence for the first time that SEY contains higher levels of Pyruvate Kinase, HSP70, and Elongation factor 2 and lower levels of Eukaryotic Translation Initiation Factor 5A-2 and Triosephosphate Isomerase than those found in RY.

Protocol for a randomised controlled trial to improve cognitive functioning in older adults: the Iowa Healthy and Active Minds Study.

OBJECTIVES: Gradual age-related cognitive deteriorations are common and are hypothesised to be partially attributable to declines in information-processing speed. The Iowa Healthy and Active Minds Study will evaluate the efficacy and effectiveness of a computerised visual processing speed training programme (Road Tour, Posit Science Corporation, San Francisco, California). METHODS AND ANALYSIS: Using a 3:3:4:4 ratio within two age strata (50-64 vs ≥ 65 years old), 681 men and women attending family care clinics were randomised to four treatment groups: 10 h of on-site Road Tour training, 10 h of on-site Road Tour training with 4 h of booster training at 11 months postrandomisation, 10 h of on-site attention control using computerised crossword puzzles (Boatload of Crosswords, Boatload Puzzles, LLC, Yorktown Heights, New York) and 10 h of at-home Road Tour training using the participant's personal computer. The primary outcome, visual processing speed, was assessed at randomisation and post-training (6-8 weeks postrandomisation), and is being reassessed at 1-year postrandomisation using the Useful Field of View test. Five secondary outcomes (Symbol Digit Modalities Test, Trail Making Tests A and B, Controlled Oral Word Association Test, Digit Vigilance Test, and the Stroop Colour and Word Test) were assessed at randomisation and will be reassessed at 1-year postrandomisation. Seven hypotheses will be tested using intent-to-treat analyses involving multiple linear, logistic, Poisson and negative binomial regression. ETHICS AND DISSEMINATION: Ethics approval was provided by the University of Iowa Institutional Review Board (IRB-03 protocol 200908789). All participants completed signed informed consent prior to enrollment. Road Tour is commercially available from Posit Science Corporation, which provided it to Iowa Healthy and Active Minds Study at no cost. All participants will receive a free copy of Road Tour for unlimited perpetual use at study completion. Clinical Trial Registration Number NCT01165463.

Cytoreductive surgery and intraperitoneal hyperthermic chemotherapy in the treatment of peritoneal carcinomatosis.

PURPOSE: Cytoreductive surgery (CS) and intraperitoneal hyperthermic chemotherapy (IPHC) in the treatment of peritoneal carcinomatosis (PC) in 15 patients are described. SUMMARY: Fifteen patients with PC who were treated with CS and IPHC were retrospectively identified between January 2002 and December 2006. All patients underwent cytoreduction immediately followed by IPHC with mitomycin or cisplatin. The time between undergoing CS and IPHC and the date of the last follow-up appointment or the date of death was used to calculate survival data for each patient. Nine patients had complete cytoreduction, and all but one patient had evidence of invasive disease at the time of surgery. Eleven patients required concomitant bowel resection at the time of debulking. Thirteen patients required blood transfusions during the perioperative period. Nine patients were discharged home, and four were discharged to a rehabilitation facility. Two patients died during the perioperative hospital admission, both of whom had a preoperative Eastern Cooperative Oncology Group (ECOG) performance status score of 2. The median survival time was 8.4 months, similar to the findings of previously published studies. Further studies are needed to see if tumor type, ECOG performance status score, degree of cytoreduction, and the chemotherapy agent used in IPHC can be correlated to quality of life and survival in patients with heterogeneous primary sources of intraabdominal malignancies. CONCLUSION: Combination treatment with CS followed by IPHC in 15 patients with heterogeneous primary sources of intraabdominal malignancies resulted in a median survival time of 8.4 months.

Transient receptor potential vanilloid type 4 channel expression in chronic rhinosinusitis.

BACKGROUND: Transient receptor potential (TRP) channels are a novel class of nonvoltage gated membrane cation channels that can be activated by mechanical stimulation and temperature change. Recently, TRP vanilloid type 4 (TRPV4) has been implicated in detecting viscosity changes in fallopian tube epithelial cells and inducing a compensatory response in ciliary activity and, as such, represents a possible molecular trigger for modulating respiratory ciliary activity. Thus, the goal of this study was to establish the expression pattern of TRPV4 in human sinonasal mucosa and determine whether expression is altered in chronic rhinosinusitis (CRS). METHODS: Sinus mucosal biopsy specimens were obtained from patients with CRS, CRS with nasal polyps (NPs), and healthy controls. TRPV4 mRNA and protein expression were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot analysis, respectively. TRPV4 gene expression was measured next using quantitative RT-PCR. Immunofluorescence was performed on sinus mucosal explants and respiratory epithelial air-liquid interface cultures to localize cellular expression. RESULTS: TRPV4 mRNA and protein were expressed in all samples. There was a statistically significant increase (p < 0.05) in TRPV4 gene expression in nonpolypoid CRS patients, but no difference in CRS with NP. Dual label immunofluorescence showed TRPV4 expression to be mutually exclusive of ciliated cells. CONCLUSION: Although TRPV4 represents an ideal molecular trigger for ciliary modulation, absent expression of the channel in ciliated cells precludes this function. However, altered expression of the channel in CRS and presumed expression of TRPV4 in secretory cells of the mucosa indicate a potential role in mucus homeostasis and CRS pathogenesis.

Transcription factor RFX2 is abundant in rat testis and enriched in nuclei of primary spermatocytes where it appears to be required for transcription of the testis-specific histone H1t gene.

Previous work in our laboratory revealed upregulated transcription of the testis-specific linker histone H1t gene in pachytene primary spermatocytes during spermatogenesis. Using the H1t X-box as an affinity chromatography probe, we identified Regulatory Factor X2 (RFX2), a member of the RFX family of transcription factors, as a nuclear protein that binds the probe. We also showed that RFX2 activated the H1t promoter in transient expression assays. However, other RFX family members have the same DNA-binding domain and they also may regulate H1t gene expression. Therefore, in this study we examined the distribution of RFX2 and other RFX family members in rat testis germinal cells and in several tissues. Among tissues examined, RFX2 is most abundant in testis. Testis RFX2 is most abundant in spermatocytes where transcription of the H1t gene is upregulated and the steady-state H1t mRNA level is high. RFX2 levels decrease but RFX1 levels increase in early spermatids where H1t gene transcription is downregulated. Antibodies against RFX2 generate a shifted band in electrophoretic mobility shift assays (EMSA) using H1t or testisin X-box DNA probes with nuclear proteins from spermatocytes. These data support the hypothesis that RFX2 expression is upregulated in spermatocytes where it participates in activating transcription of the H1t gene and other testis genes. These data also support the possibility that other RFX family members may bind to the H1t promoter in other testis germinal cell types and in nongerminal cells to downregulate H1t gene transcription.

Transcriptional activation of the testis-specific histone H1t gene by RFX2 may require both proximal promoter X-box elements.

The rat testis-specific linker histone H1t gene is transcribed in pachytene primary spermatocytes during spermatogenesis. Our previous work using transgenic mice demonstrated that spermatocyte-specific transcription of the H1t gene is dependent upon a proximal promoter element designated the TE element. TE is composed of two adjacent and inverted imperfect repeat sequences designated TE1 and TE2 and both of these palindromic elements are similar in sequence to the X-box, a DNA consensus sequence that binds regulatory factor X (RFX). RFX2 is the major enriched protein derived from rat testis nuclear extracts when using the TE1 element as an affinity chromatography probe. Co-expression of RFX2 together with an H1t promoted reporter vector in transient expression assays activates the H1t promoter in the GC-2spd germinal cell line, and mutation of either X-box significantly represses activity. However, RFX2 partially reactivates the promoter when either of the X-box elements is independently mutated. In order to totally block reactivation by RFX2, it is necessary to mutate both X-boxes simultaneously. Therefore, RFX2 appears to be able to bind to either X-box independently to partially activate the promoter of the testis-specific histone H1t gene, but simultaneous binding of RFX2 to both X-box elements may be required for maximal promoter activation.

Specific binding of nuclear proteins to a bifunctional promoter element upstream of the H1/AC box of the testis-specific histone H1t gene.

The testis-specific histone H1t gene is transcribed exclusively in primary spermatocytes during spermatogenesis. Studies with transgenic mice show that 141 base pairs (bp) of the H1t proximal promoter accompanied with 800 bp of downstream sequence are sufficient for tissue-specific transcription. Nuclear proteins from testis and pachytene spermatocytes produce footprints spanning the region covering the repressor element (RE) from 100 to 125 nucleotides upstream of the H1t transcriptional initiation site. Only testis nuclear proteins bind to the 5'-end of the element and produce a unique, low-mobility complex in electrophoretic mobility shift assays. This testis complex is distinct from the complex formed by a repressor protein derived from several cell lines that binds to the 3'-end of the element. The testis complex band is formed when using nuclear proteins from primary spermatocytes, where the H1t gene is transcribed, and band intensity drops 70%-80% when using nuclear proteins from early spermatids, where H1t gene transcription ceases. Protein-DNA cross-linking experiments using testis nuclear proteins produce electrophoretic bands of 59, 52, and 50 kDa on SDS/PAGE gels.

TE2 and TE1 sub-elements of the testis-specific histone H1t promoter are functionally different.

The testis-specific linker histone H1t gene is transcribed exclusively in pachytene primary spermatocytes. Tissue specific expression of the gene is mediated in part by transcriptional factors that bind elements located within the proximal and distal promoter. A 40 bp promoter element, designated H1t/TE, that is located within the proximal promoter between the CCAAT-box and AC-box, is known to be essential for H1t gene transcription in transgenic animals. In the present study, we show by SDS-PAGE analysis of UV crosslinked protein and DNA and by electrophoretic mobility shift assays (EMSA) of testis nuclear proteins separated on a non-denaturing glycerol gradient that the TE1 sub-element is bound by a protein complex. Mutation of TE1 leads to a drop in H1t promoter activity in germinal GC-2spd cells as well as in nongerminal Leydig, NIH3T3, and C127I cell lines. Although TE1 and TE2 sub-elements have similar sequences, mutation of the TE2 sub-element causes an increase in promoter activity in C127I and Leydig cells. The rat TE1 but not TE2 contains a CpG dinucleotide and this cytosine is methylated in liver but not in primary spermatocytes. Methylation of the cytosine at this site almost eliminates nuclear protein binding. Thus, there are significant functional differences in the TE2 and TE1 sub-elements of the H1t promoter with TE1 serving as a transcriptional activator binding site and TE2 serving as a repressor binding site in some cell lines.

H1t/GC-box and H1t/TE1 element are essential for promoter activity of the testis-specific histone H1t gene.

The testis-specific linker histone H1t gene is transcribed exclusively in mid to late pachytene primary spermatocytes. Tissue-specific expression of the gene is mediated primarily through elements located within the proximal promoter. Previous work in transgenic animals identified a unique 40-base pair promoter element designated H1t/TE that is essential for spermatocyte-specific expression. The H1t/TE element contains three subelements designated TE2, GC-box, and TE1 based on in vitro footprinting and electrophoretic mobility shift assays. Because GC-box is a consensus site for binding of Sp transcription-factor family members, experiments were performed demonstrating that two Sp family members, Sp1 and Sp3, were present in testis cells from 9-day-old and adult rats and in pachytene primary spermatocytes and early spermatids. A 95- to 105-kDa form of Sp1 is most abundant in the tissues and cell lines examined, but a 60-kDa form of Sp1 is the most abundant species in spermatocytes and early spermatids. Further examination of Sp1 and Sp3 from adult testis, primary spermatocytes, and early spermatids showed that they can bind to the H1t/TE element. In order to determine the contributions of the subelements to H1t transcription, we mutated each of them in H1t promoter luciferase reporter vectors. Mutation of the GC-box and TE1 subelement reduced expression 77% and 49%, respectively, compared with the wild-type H1t promoter in transient expression assays in a testis GC-2spd cell line that was derived from germinal cells. These studies suggest that Sp transcription factors may be involved in transcription of the H1t gene and the GC-box and the TE1 subelement are required for activation of the H1t promoter.