Inhibition of apoptosis exacerbates fatigue-damage tendon injuries in an in vivo rat model.

Tendinopathy is a common and progressive musculoskeletal disease. Increased apoptosis is an end-stage tendinopathy manifestation, but its contribution to the pathology of the disease is unknown. A previously established in vivo model of fatigue damage accumulation shows that increased apoptosis is correlated with the severity of induced tendon damage, even in early onset of the disease, supporting its implication in the pathogenesis of the disease. Consequently, this study aimed to determine: (1) whether apoptosis could be inhibited after fatigue damage and (2) whether its inhibition could lead to remodeling of the extracellular matrix (ECM) and pericellular matrix (PCM), to ultimately improve the mechanical properties of fatigue-damaged tendons. The working hypothesis was that, despite the low vascular nature of the tendon, apoptosis would be inhibited, prompting increased production of matrix proteins and restoring tendon mechanical properties. Rats received 2 or 5 d of systemic pan-caspase inhibitor (Q-VD-OPh) or dimethyl sulfoxide (DMSO) carrier control injections starting immediately prior to fatigue loading and were sacrificed at days 7 and 14 post-fatigue-loading. Systemic pan-caspase inhibition for 2 d led to a surprising increase in apoptosis, but inhibition for 5 d increased the population of live cells that could repair the fatigue damage. Further analysis of the 5 d group showed that effective inhibition led to an increased population of cells producing ECM and PCM proteins, although typically in conjunction with oxidative stress markers. Ultimately, inhibition of apoptosis led to further deterioration in mechanical properties of fatigue-damaged tendons.

Chronic corticosterone-induced impaired cognitive flexibility is not due to suppressed adult hippocampal neurogenesis.

Hippocampal neurogenesis has been implicated in the etiology of depression. Recent studies suggest new neurons add flexibility to hippocampal-dependent learning and memory. We hypothesized that suppressed hippocampal neurogenesis may contribute to impaired cognitive flexibility associated with depression. The chronic corticosterone (CORT)-induced animal model of depression was used. In Experiment 1, rats received either CORT (40mg/kg) or vehicle injections for 21days and were subjected to Water maze during the last six days of drug treatment. No group differences were found during the spatial learning phase; however, cognitive flexibility, measured by reversal training, was significantly impaired in the CORT-treated rats. The probe test revealed enhanced memory of the new platform location for the CORT-treated rats. Given the time newborn neurons require to mature, we presumed if impaired cognitive flexibility seen in Experiment 1 were due to suppressed neurogenesis, terminating CORT treatment 3days prior to behavioural testing should still induce the impairment. Therefore, Experiment 2 was similar to Experiment 1, except that CORT injections were terminated 3days prior to behavioural assessment. However, not only was spatial learning significantly enhanced in the CORT-treated rats, but there were also no group differences during reversal or probe tests. Bromodeoxyruidine, administered a day after the first drug treatments in both experiments, was quantified and revealed the number of new neurons were the same in both groups in both experiments. Results suggest cognitive flexibility is impaired in the CORT-induced animal model of depression; an effect that is reversible and independent of suppressed hippocampal neurogenesis.

Psychosomatic Medicine for Non-Psychiatric Residents: Video Education and Incorporation of Technology.

OBJECTIVES: Psychiatric education for non-psychiatric residents varies between training programs, and may affect resident comfort with psychiatric topics. This study's goals were to identify non-psychiatric residents' comfort with psychiatric topics and to test the effectiveness of a video intervention. METHODS: Residents in various departments were given a survey. They were asked to rank their comfort level with multiple psychiatric topics, answer questions about medical decision making capacity (MDMC), watch a 15-min video about MDMC, and answer a post-test section. RESULTS: In total, 91 Internal Medicine, General Surgery, and Obstetrics and Gynecology residents responded to the study. Of the 91 residents, 55 completed the pre- and post-test assessments. There was no significant difference in correct responses. Residents' comfort levels were assessed, and a significant improvement in comfort level with MDMC was found. CONCLUSIONS: This study highlights potential opportunities for psychiatric education, and suggests brief video interventions can increase resident physicians' comfort with a psychiatric topic.

Delayed exercise promotes remodeling in sub-rupture fatigue damaged tendons.

Tendinopathy is a common musculoskeletal injury whose treatment is limited by ineffective therapeutic interventions. Previously we have shown that tendons ineffectively repair early sub-rupture fatigue damage. In contrast, physiological exercise has been shown to promote remodeling of healthy tendons but its utility as a therapeutic to promote repair of fatigue damaged tendons remains unknown. Therefore, the objective of this study was to assess the utility of exercise initiated 1 and 14 days after onset of fatigue damage to promote structural repair in fatigue damaged tendons. We hypothesized that exercise initiated 14 days after fatigue loading would promote remodeling as indicated by a decrease in area of collagen matrix damage, increased procollagen I and decorin, while decreasing proteins indicative of tendinopathy. Rats engaged in 6-week exercise for 30 min/day or 60 min/day starting 1 or 14 days after fatigue loading. Initiating exercise 1-day after onset of fatigue injury led to exacerbation of matrix damage, particularly at the tendon insertion. Initiating exercise 14 days after onset of fatigue injury led to remodeling of damaged regions in the midsubstance and collagen synthesis at the insertion. Physiological exercise applied after the initial biological response to injury has dampened can potentially promote remodeling of damaged tendons.

PPARδ binding to heme oxygenase 1 promoter prevents angiotensin II-induced adipocyte dysfunction in Goldblatt hypertensive rats.

OBJECTIVE: Renin-angiotensin system (RAS) regulates adipogenic response with adipocyte hypertrophy by increasing oxidative stress. Recent studies have shown the role of peroxisome proliferator-activated receptor-δ (PPARδ) agonist in attenuation of angiotensin II-induced oxidative stress. The aim of this study was to explore a potential mechanistic link between PPARδ and the cytoprotective enzyme heme oxygenase-1 (HO-1) and to elucidate the contribution of HO-1 to the adipocyte regulatory effects of PPARδ agonism in an animal model of enhanced RAS, the Goldblatt 2 kidney 1 clip (2K1C) model. METHOD: We first established a direct stimulatory effect of the PPARδ agonist (GW 501516) on the HO-1 gene by demonstrating increased luciferase activity in COS-7 cells transfected with a luciferase-HO-1 promoter construct. Sprague-Dawley rats were divided into four groups: sham-operated animals, 2K1C rats and 2K1C rats treated with GW 501516, in the absence or presence of the HO activity inhibitor, stannous mesoporphyrin (SnMP). RESULTS: 2K1C animals had increased visceral adiposity, adipocyte hypertrophy, increased inflammatory cytokines, increased circulatory and adipose tisssue levels of renin and Ang II along with increased adipose tissue gp91 phox expression (P<0.05) when compared with sham-operated animals. Treatment with GW 501516 increased adipose tissue HO-1 and adiponectin levels (P<0.01) along with enhancement of Wnt10b and ß-catenin expression. HO-1 induction was accompanied by the decreased expression of Wnt5b, mesoderm specific transcript (mest) and C/EBPα levels and an increased number of small adipocytes (P<0.05). These effects of GW501516 were reversed in 2K1C animals exposed to SnMP (P<0.05). CONCLUSION: Taken together, our study demonstrates, for the first time, that increased levels of Ang II contribute towards adipose tissue dysregulation, which is abated by PPARδ-mediated upregulation of the heme-HO system. These findings highlight the pivotal role and symbiotic relationship of HO-1, adiponectin and PPARδ in the regulation of metabolic homeostasis in adipose tissues.

Local drug delivery agents as adjuncts to endodontic and periodontal therapy.

In the treatment of intracanal and periodontal infections, the local application of antibiotics and other therapeutic agents in the root canal or in periodontal pockets may be a promising approach to achieve sustained/controlled drug release, high antimicrobial activity and low systemic side effects. The conventional method for the elimination of subgingival microbial infection includes mechanical debridement, irrigation with antimicrobial agents or surgical access. But, the effectiveness of conventional nonsurgical treatment is limited by lack of accessibility to bacteria in deeper periodontal pockets, and/or does not completely eliminate intracanal microorganisms. Surgical intervention may be beneficial but cannot be done in all cases, medically compromised cases and also in patients not willing to be subjected to surgical therapy. Development of local drug delivery systems provides an answer to all such difficulties. This comprehensive review tries to cover the detailed information about the latest advances in the various local drug delivery systems, their indications, contraindications and their advantages over systemic drug therapy.

Tendon fatigue in response to mechanical loading.

Tendinopathies are commonly attributable to accumulation of sub-rupture fatigue damage from repetitive use. Data is limited to late stage disease from patients undergoing surgery, motivating development of animal models, such as ones utilizing treadmill running or repetitive reaching, to investigate the progression of tendinopathies. We developed an in vivo model using the rat patellar tendon that allows control of the loading directly applied to the tendon. This manuscript discusses the response of tendons to fatigue loading and applications of our model. Briefly, the fatigue life of the tendon was used to define low, moderate and high levels of fatigue loading. Morphological assessment showed a progression from mild kinks to fiber disruption, for low to high level fatigue loading. Collagen expression, 1 and 3 days post loading, showed more modest changes for low and moderate than high level fatigue loading. Protein and mRNA expression of Ineterleukin-1ß and MMP-13 were upregulated for moderate but not low level fatigue loading. Moderate level (7200 cycles) and 100 cycles of fatigue loading resulted in a catabolic and anabolic molecular profile respectively, at both 1 and 7 days post loading. Results suggest unique mechanisms for different levels of fatigue loading that are distinct from laceration.

Giant condyloma acuminatum of Buschke and Lowenstein: successful surgical treatment.

The Buschke-Löwenstein tumour is an extremely rare, slow-growing, locally destructive, cauliflower-like mass, also known as giant condyloma acuminatum. We report a case of a 42-year-old man who presented to the department of surgery with a two-year history of a perineal tumour. The mass was painless initially but had become painful more recently. After histopathological confirmation, the tumour was removed surgically, as it was resistant to medical treatment. There has been considerable debate regarding the exact nature, aetiology and treatment of these lesions.

Vulval lymphoedema following pulmonary tuberculosis.

Acquired lymphoedema of the vulva is induced by impaired lymph flow. We present the case of a 35-year-old woman having lymphoedema of the vulva following pulmonary tuberculosis, which she had developed four years back for which she had taken a full course of antitubercular treatment for nine months from the Chest and Tuberculosis department. The biopsy taken from the perianal swellings showed hyperkeratosis and acanthosis with multiple dilated lymph specs.

An upregulation of DNA-methyltransferase 1 and 3a expressed in telencephalic GABAergic neurons of schizophrenia patients is also detected in peripheral blood lymphocytes.

Several lines of schizophrenia (SZ) research suggest that a functional downregulation of the prefrontal cortex GABAergic neuronal system is mediated by a promoter hypermethylation, presumably catalyzed by an increase in DNA-methyltransferase-1 (DNMT-1) expression. This promoter hypermethylation may be mediated not only by DNMT-1 but also by an entire family of de novo DNA-methyltransferases, such as DNA-methyltransferase-3a (DNMT-3a) and -3b (DNMT-3b). To verify the existence of an overexpression of DNMT-3a and DNMT-3b in the brain of schizophrenia patients (SZP), we compared their mRNA expression in Brodmann's area 10 (BA10) and in the caudate nucleus and putamen obtained from the Harvard Brain Tissue Resource Center (Belmont, MA) from both nonpsychiatric subjects (NPS) and SZP. Our results demonstrate that DNMT-3a and DNMT-1 are expressed and co-localize in distinct GABAergic neuron populations whereas DNMT-3b mRNA is virtually undetectable. We also found that unlike DNMT-1, which is frequently overexpressed in telencephalic GABAergic neurons of SZP, DNMT-3a mRNA is overexpressed only in layer I and II GABAergic interneurons of BA10. To ascertain whether these DNMT expression differences observed in brain tissue could also be detected in peripheral tissues, we studied whether DNMT-1 and DNMT-3a mRNAs were overexpressed in peripheral blood lymphocytes (PBL) of SZP. Both DNMT-1 and DNMT-3a mRNAs are expressed in the PBL and although DNMT-3a mRNA levels in the PBL are approximately 1/10 of those of DNMT-1, the comparison of the PBL content in NPS and SZP showed a highly significant 2-fold increase of both DNMT-1 and DNMT-3a mRNA in SZP. These changes were unaffected by the dose, the duration, or the type of antipsychotic treatment. The upregulation of DNMT-1 and to a lesser extent that of DNMT-3a mRNA in PBL of SZP supports the concept that this readily available peripheral cell type can express an epigenetic variation of specific biomarkers relevant to SZ morbidity. Hence, PBL studies may become useful to investigate a diagnostic epigenetic marker of SZ morbidity.

Topical lidocaine for the treatment of postherpetic neuralgia.

BACKGROUND: The cause of postherpetic neuralgia is damage to peripheral neurons, dorsal root ganglia, and the dorsal horn of the spinal cord, secondary to herpes zoster infection (shingles). In postherpetic neuralgia, peripheral neurons discharge spontaneously and have lowered activation thresholds, and exhibit an exaggerated response to stimuli. Topical lidocaine dampens peripheral nociceptor sensitisation and central nervous system hyperexcitability, and may benefit patients with postherpetic neuralgia. OBJECTIVES: To examine the efficacy and safety of topical lidocaine in the treatment of postherpetic neuralgia. SEARCH STRATEGY: We searched the Cochrane Pain, Palliative and Supportive Care Group Trials Register, The Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, and LILACS, SIGLE for conference proceedings, Citation Index, the reference lists of all eligible trials, key textbooks, and previous systematic reviews. We also wrote to authors of all identified trials. SELECTION CRITERIA: Randomised or quasi-randomised trials comparing all topical applications of lidocaine, including gels and patches in patients of all ages with postherpetic neuralgia (pain persisting at the site of shingles at least one month after the onset of the acute rash). DATA COLLECTION AND ANALYSIS: Two review authors extracted data, and a third checked them. We obtained some missing data from the US Food and Drugs Administration. MAIN RESULTS: Three trials involving 182 topical lidocaine treated participants and 132 control participants were included. Two trials gave data on pain relief, and the remaining study provided data on secondary outcome measures. The largest trial published as an abstract compared topical lidocaine patch to a placebo patch and accounted for 150 of the 314 patients (48%).A meta-analysis combining two of the three studies identified a significant difference between the topical lidocaine and control groups for the primary outcome measure: a mean improvement in pain relief according to a pain relief scale. Topical lidocaine relieved pain better than placebo (P = 0.003). There was a statistical difference between the groups for the secondary outcome measure of mean VAS score reduction (P = 0.03), but this was only for a single small trial. There were a similar number of adverse skin reactions in both treatment and placebo groups. The highest recorded blood lidocaine concentration varied between 59 ng/ml and 431 ng/ml between trials. The latter figure is high and the authors of the study suggest that the sample had been contaminated during the assay procedure. AUTHORS' CONCLUSIONS: There is insufficient evidence to recommend topical lidocaine as a first-line agent in the treatment of postherpetic neuralgia with allodynia. Further research should be undertaken on the efficacy of topical lidocaine for other chronic neuropathic pain disorders, and also to compare different classes of drugs (e.g. topical anaesthetics versus anti-epileptics).

The ins and outs of RNAi in mammalian cells.

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. RNAi in mammalian cells is achieved through introduction or expression of 21-23 bp small interfering RNAs (siRNAs) in cells or animals. Currently, there are six ways of producing siRNAs. siRNAs can be produced by chemical synthesis, in vitro transcription, or RNase III/Dicer digestion of long dsRNAs. Alternatively, they can be expressed in vivo from plasmids, PCR cassettes, or viral vectors that include a CMV or polymerase III (pol III) transcription unit. So far, these approaches have been used to create siRNAs for use in loss-of-function studies. However, it is clear that siRNAs also hold great promise as therapeutic tools. First, their activity seems to be very sequence-specific. Moreover, siRNAs could be modified in order to increase their stability and potency in vivo. Here, we will review the issues and findings related to siRNA design and production. Moreover, we will summarize new findings on siRNA specificity, modification, and delivery, which are critical to their use as therapeutic agents.

Myelinated and unmyelinated primary afferent axons form contacts with cholinergic interneurons in the spinal dorsal horn.

Cholinergic interneurons in laminae III/IV of the dorsal horn contain co-localised gamma-aminobutyric acid (GABA) and frequently form axoaxonic synapses with terminals of primary afferents. They are therefore probably last-order interneurons involved in presynaptic inhibition. The purpose of the present investigation was to determine if these cells receive direct input from primary afferents. Relationships between primary afferents and interneurons were investigated in adult rats. Myelinated primary afferents were labelled with the B-subunit of cholera toxin (CTb). Unmyelinated afferents were labelled with IB4 lectin and an antibody to identify calcitonin-gene-related peptide (CGRP). Cholinergic neurons were labelled with an antibody raised against choline acetyltransferase and examined with a confocal microscope. Cells were reconstructed with NeuroLucida for Confocal and afferent contacts plotted. Interneurons (N=30) received an average of 20.2+/-11.9 (SD) contacts from CTb-labelled primary afferents, which were preferentially distributed on proximal and intermediate dendrites. Interneurons with dendrites which extended into lamina II (N=20) received an average of 27.4+/-19.0 IB4 contacts (on intermediate and distal dendrites) and 9.2+/-6.8 CGRP contacts. It is concluded that cholinergic interneurons receive contacts from both myelinated and unmyelinated primary afferents and different classes of afferent target particular dendritic domains. Cholinergic interneurons are likely to be components of an inhibitory feedback pathway that is monosynaptically activated by primary afferents.

Targeted gene knockout by 2'-O-aminoethyl modified triplex forming oligonucleotides.

Triplex forming oligonucleotides (TFOs) are of interest because of their potential for facile gene targeting. However, the failure of TFOs to bind target sequences at physiological pH and Mg(2+) concentration has limited their biological applications. Recently, pyrimidine TFOs with 2'-O-aminoethyl (AE) substitutions were shown to have enhanced kinetics and stability of triplex formation (Cuenoud, B., Casset, F., Husken, D., Natt, F., Wolf, R. M., Altmann, K. H., Martin, P., and Moser H. E. (1998) Angew. Chem. Int. Ed. 37, 1288--1291). We have prepared psoralen-linked TFOs with varying amounts of the AE-modified residues, and have characterized them in biochemical assays in vitro, and in stability and HPRT gene knockout assays in vivo. The AE TFOs showed higher affinity for the target in vitro than a TFO with uniform 2'-OMe substitution, with relatively little loss of affinity when the assay was performed in reduced Mg(2+). Once formed they were also more stable in "physiological" buffer, with the greatest affinity and stability displayed by the TFO with all but one residue in the AE format. However, TFOs with lesser amounts of the AE modification formed the most stable triplexes in vivo, and showed the highest HPRT gene knockout activity. We conclude that the AE modification can enhance the biological activity of pyrimidine TFOs, but that extensive substitution is deleterious.

The last exon of SNAP-23 regulates granule exocytosis from mast cells.

SNAP-25 and its ubiquitous homolog SNAP-23 are members of the SNARE family of proteins that regulate membrane fusion during exocytosis. Although SNAP-23 has been shown to participate in a variety of intracellular transport processes, the structural domains of SNAP-23 that are required for its interaction with other SNAREs have not been determined. By employing deletion mutagenesis we found that deletion of the amino-terminal 18 amino acids of SNAP-23 (encoded in the first exon) dramatically inhibited binding of SNAP-23 to both the target SNARE syntaxin and the vesicle SNARE vesicle-associated membrane protein(VAMP). By contrast, deletion of the carboxyl-terminal 23 amino acids (encoded in the last exon) of SNAP-23 does not affect SNAP-23 binding to syntaxin but profoundly inhibits its binding to VAMP. To determine the functional relevance of the modular structure of SNAP-23, we overexpressed SNAP-23 in cells possessing the capacity to undergo regulated exocytosis. Expression of human SNAP-23 in a rat mast cell line significantly enhanced exocytosis, and this effect was not observed in transfectants expressing the carboxyl-terminal VAMP-binding mutant of SNAP-23. Despite considerable amino acid identity, we found that human SNAP-23 bound to SNAREs more efficiently than did rat SNAP-23. These data demonstrate that the introduction of a "better" SNARE binder into secretory cells augments exocytosis and defines the carboxyl terminus of SNAP-23 as an essential regulator of exocytosis in mast cells.

Adjuvancy enhancement of muramyl dipeptide by modulating its release from a physicochemically modified matrix of ovalbumin microspheres. I. In vitro characterization.

The weak immunogenicity of subunit vaccines has necessitated research into the development of novel adjuvants and methods to enhance the adjuvancy associated with vaccine delivery systems. The purpose of the present study was to modulate the release of muramyl dipeptide (MDP) from a physicochemically modified matrix of ovalbumin microspheres (OVA-MSs). A two-component MS vaccine delivery system was fabricated, which utilized OVA as the antigen and delivery matrix, and MDP as the adjuvant. The MSs were prepared from OVA using a water/oil emulsion method, followed by suspension cross-linking using glutaraldehyde. The MS matrix was modified with respect to the degree of cross-linking by varying the concentration of glutaraldehyde and matrix density, a function of disulfide-bond formation. The modifications in the MS matrix were characterized using SDS-PAGE, scanning electron microscopy, differential scanning calorimetry, and thin layer wicking (TLW). The in vitro release of MDP and OVA from the various preparations of OVA-MSs exhibited triphasic and biphasic profiles, respectively. The degree of cross-linking and the matrix density were found to be significant physicochemical parameters that affected the release profiles of MDP and OVA through two mechanisms: controlled surface erosion and bulk degradation of the MSs.

Adjuvancy enhancement of muramyl dipeptide by modulating its release from a physicochemically modified matrix of ovalbumin microspheres. II. In vivo investigation.

In the present study, sustaining the release of adjuvants was investigated using microspheres as a means to increase the immune response (i.e. efficacy) and, ultimately, to reduce adverse effects to vaccine components. To date, most attempts have focused on sustaining the release of antigens. The utility of currently used vaccine adjuvants may be improved by sustaining their release. The development, modification and characterization of a two-component microsphere vaccine delivery system was demonstrated in our previous report [Puri et al., J. Control. Release (2000) in press]. Briefly, ovalbumin (OVA) was utilized as the model antigen (Ag) and delivery matrix and MDP or threonyl-MDP served as the model adjuvants. The release pattern of MDP was modulated from a physicochemically modified matrix of OVA microspheres (OVA-MSs). The purpose of the present study was to evaluate the adjuvancy of MDP in mice by modulating its release from OVA-MSs. Mice were immunized intradermally (i.d.) with various preparations of OVA-MSs, using a single-shot-immunization technique. Positive and negative control preparations were evaluated as well. An inverse relationship was observed between the in vitro release rate of MDP and the in vivo OVA-specific IgG antibody (Ab) immune response in mice. These results demonstrated that modulating the release pattern of MDP or threonyl-MDP enhanced their adjuvant effect. In conclusion, the current results demonstrate that the sustained and controlled release of adjuvants is extremely important for inducing a high level and prolonged period of immunostimulation while potentially minimizing therapy-limiting adverse effects.

The underwhite (uw) locus acts autonomously and reduces the production of melanin.

The mouse has provided several significant models for hypopigmentation disorders, including the major forms of albinism. Mutations at the mouse underwhite locus confer one of the most severe hypopigmentation phenotypes, similar to mutations at the pink-eyed dilution locus that is a model for type 2 oculocutaneous albinism. A melanocyte cell line established from underwhite mutant mice failed to pigment under conditions that support pigment production in wild-type melanocytes and melanoblasts from underwhite skin graft transplants failed to produce melanin in normal skin, demonstrating that the action of the gene encoded by the underwhite locus is intrinsic to melanocytes. Mice with mutations at the underwhite locus and either the pink-eyed dilution locus or the melanocortin receptor 1 locus exhibited more severe hypopigmentation than either mutation alone, suggesting that the actions of these genes are independent. These results demonstrate that the underwhite locus is a major determinant of mammalian pigmentation.

Aberrant pH of melanosomes in pink-eyed dilution (p) mutant melanocytes.

In past studies, we cloned the mouse p gene and its human homolog P, which is associated with oculocutaneous albinism type 2. Both mouse and human genes are expressed in melanocytes and encode proteins predicted to have 12 membrane-spanning domains with structural homology to known ion transporters. We have also demonstrated that the p protein is localized to the melanosomal membrane and does not function as a tyrosine transporter. In this study, immunohistochemistry and confocal microscopy were used to show that the p protein plays an important role in the generation or maintenance of melanosomal pH. Melanosomes (and their precursor compartments) were defined by antiserum directed against the melanosomal marker tyrosinase related protein 1. Acidic vesicles were identified by 3-(2, 4-dinitroanilino)-3'-amino-N-methyldipropylamine incorporation, visualized with anti-dinitrophenol. In C57BL/6+/+ (wild-type) melanocytes, 94.2% of vesicles demonstrated colocalization of tyrosinase related protein 1 and 3-(2, 4-dinitroanilino)-3'-amino-N-methyldipropylamine, indicating that almost all melanosomes or their precursors were acidic. By contrast, only 7%-8% of the staining vesicles in p mutant cell lines (pJ/pJ and pcp/p6H) showed colocalization of tyrosinase related protein 1 and 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine. Thus, without a functional p protein, most melanosomes and their precursors are not acidic. As mammalian tyrosinase activity in situ is apparently dependent on low pH, we postulate that in the absence of a low pH environment brought about by ionic transport mediated by the p protein, tyrosinase activity is severely impaired, leading to the minimal production of melanin that is characteristic of p mutants. Additionally (or alternatively), an abnormal pH may also impair the assembly of the normal melanogenic complex.